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  • American Society of Plant Biologists
[h=1]Sex-Determining Mechanisms in Land Plants[/h]
+ Author Affiliations

  • Purdue Genetics Program and Department of Botany and Plant Pathology, Purdue University, West Lafayette, Indiana 47906



  1. Published online before print April 2004, doi: http:/​/​dx.​doi.​org/​10.​1105/​tpc.​016667 The Plant Cell June 2004 vol. 16 no. suppl 1 S61-S71






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[h=2]INTRODUCTION[/h] Sex determination is a process that leads to the physical separation of male and female gamete-producing structures to different individuals of a species. Even though sexually reproducing species have only three possible options—to relegate the two sexes to separate individuals, to keep them together on the same individual, or to have a combination of both—plants in particular display a great variety of sexual phenotypes. In angiosperms, a sex-determining process is manifest in species that are monoecious, in which at least some flowers are unisexual but the individual is not, or dioecious, in which unisexual plants produce flowers of one sex type. In plants that produce no flowers and are homosporous, sex determination is manifest in the gametophyte generation with the production of egg- and sperm-forming gametangia on separate individual gametophytes. The determinants of sexual phenotype in plants are diverse, ranging from sex chromosomes in Marchantia polymorpha and Silene latifolia to hormonal regulation in Zea mays and Cucumis sativa to pheromonal cross-talk between individuals in Ceratopteris richardii. Here, we highlight recent efforts aimed at understanding the genetic and molecular mechanisms responsible for sex determination in several plant species that separate their sexes into two individuals or flowers. Representatives of all major land plant lineages are included to give an evolutionary perspective, which is important in understanding how different sex-determining mechanisms evolved and their consequences in plant development and evolution. Although great progress has been made in genetically identifying the genes that regulate sex expression in these species, few of them have been cloned. Because a “one-size-fits-all” mechanism of sex determination will not account for the variety of sexual systems in plants, future efforts at cloning these genes in several well-chosen model systems will be necessary to understand these processes at the molecular level. There are several excellent recent reviews of sex determination that describe species that have not been included to which the reader is directed (Ainsworth, 1999, 2000; Geber et al., 1999; Matsunaga and Kawano, 2001; Negrutiu et al., 2001; Barrett, 2002; Charlesworth, 2002). We begin with the most basal lineage of the land plants.

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[h=2]THE BRYOPHYTES[/h] The bryophytes are a group of plants that includes the modern liverworts, hornworts, and mosses. In this group, the haploid gametophyte is the dominant phase of the life cycle, which is illustrated in Figure 1. Their diploid spore-producing sporophytes are very small, short-lived, and parasitic upon the independent gamete-producing gametophyte generation. All bryophytes are homosporous, producing only one type of spore, yet all three groups of extant bryophytes are represented by species that are homothallic, with one individual gametophyte producing both male and female sex organs (gametangia), and species that are heterothallic, with one individual producing only male or female gametangia (Smith, 1955). Sexual dimorphism in heterothallic species can be extreme, as exemplified by members of the genus Micromitrium, in which the dwarf male gametophyte grows on the leaves of the markedly larger female plant (Smith, 1955).
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Figure 1. The Life Cycle of the Liverwort Marchantia polymorpha.
Haploid gametophytes develop gametangia in antheridiophores and archegoniophores that produce the sperm and egg, respectively. Upon fertilization, which is facilitated by raindrops, the diploid sporophyte remains attached to the archegoniophore and produces yellow sporangia, in which haploid spores are formed after meiosis. The spores are liberated and germinate to form a new gametophyte thallus. The sex of the thallus depends on which sex chromosome it inherits. Photographs courtesy of Katsuyuki Yamato, Kyoto University.


In many species of bryophytes, heterothallism (unisexuality) has been correlated with the presence of sex chromosomes (Smith, 1955). Although the extent of heterothallism and sex chromosomes in the bryophytes has not been assessed systematically, this is the only known group of homosporous plants that uses sex chromosomes in sex determination. To date, studies of bryophyte sex determination have focused on the heterothallic liverwort Marchantia polymorpha. In this species, the male and female thalli (vegetative gametophytes) look alike, although males and females can be distinguished easily by differences in the morphology of the sexual structure each produces. A gametophyte bears gametangia on stalked branches called antheridiophores (if male) or archegoniophores (if female) that arise from the upper surface of the thallus (Figure 1). Antheridiophores produce sperm-forming antheridia, and archegoniophores produce egg-forming archegonia. The sex of each haploid gametophyte is determined by cytologically distinct sex chromosomes, with males having one very small Y chromosome and no X chromosome and females having one X chromosome and no Y chromosome (Lorbeer, 1934). In addition to its rapid growth (it is often an invasive weed in greenhouses), its ability to be propagated vegetatively by gemma cups (Figure 1), and its ability to be transformed (Takenaka et al., 2000), Marchantia has a relatively small genome size of 280 Mbp distributed among eight autosomes plus one sex chromosome (Okada et al., 2000), making it a worthy model organism amenable to genomics-style investigations.
Working on the assumption that sex-determining factors exist on the Marchantia sex chromosomes, Okama and colleagues (2000) set out to identify these factors by constructing separate male and female P1-derived artificial chromosome (PAC) libraries and identifying clones specific to either the male or the female genome. Their screen resulted in 70 male-specific PAC clones that hybridized only the Y chromosome by fluorescence in situ hybridization. No female-specific clones were found, indicating that the X chromosome does not harbor long stretches of unique sequences, as does the Y chromosome. To date, two male-specific PAC clones with insert sizes totaling 126 kb have been sequenced (Okada et al., 2001; Ishizaki et al., 2002). This and other analyses have revealed that approximately one-fourth to one-third of the 10-Mb Y chromosome of Marchantia consists of an estimated 600 to 15,000 copies of an element of variable length (0.7 to 5.2 kb) that contains other smaller repetitive elements (Okada et al., 2001; Ishizaki et al., 2002). Of the six putative protein-encoding genes found embedded within the repeats, all are present in multiple copies on the Y chromosome based on DNA gel blot hybridization. Two of these genes, named ORF162 (Okada et al., 2001) and M2D3.5 (Ishizaki et al., 2002), are unique to the Y chromosome; the remaining four genes are present in low copy number on the X chromosome or the autosomes. ORF162 encodes a putative protein with a RING finger domain; M2D3.5 is a member of the same gene family. ORF162 transcripts are detectable only in the male sexual organs, indicating that the gene family represented by ORF162 and M2D3.5 may be important in the development of the antheridiophore. Of the four genes also present on the X chromosome or the autosomes, only one (M2D3.4) is restricted in its expression to the male gametophyte, indicating that the M2D3.4 X or automosmal homolog might be a pseudogene. M2D3.4 encodes a putative protein similar to a Lilium longiflorum gene that is expressed exclusively in the male gametic cells. The remaining three genes are not sex specific in their expression. The functions of the Y chromosome–encoded genes are as yet unknown.
Although only a small portion of the Marchantia Y chromosome has been sequenced, it is sufficient to make meaningful comparisons with the euchromatic male-specific region (MSY) of the human Y chromosome, the sequence of which was published recently (Skaletsky et al., 2003; see also Hawley, 2003). The sequence of the human MSY region and limited comparative sequencing of the MSY regions in great apes (Rozen et al., 2003) have added new insights to our understanding of how the mammalian testis gene families on the Y chromosome have been maintained over the course of evolution. As will be shown, the similarities between the human and liverwort Y chromosomes are striking and may reflect a common mechanism underlying the evolution of the Y chromosome in these two disparate organisms. The MSY region of the human Y chromosome is made up of three classes of sequences: X transposed, X degenerate, and ampliconic, the latter representing ∼30% of the MSY euchromatin. Because the Marchantia X chromosome has not been sequenced, it is only possible to make comparisons between the human ampliconic sequences, which are Y specific, and the Marchantia Y chromosome sequences. Like the Marchantia Y chromosome sequences, the ampliconic regions of the MSY consist of highly repetitive sequences unique to the Y chromosome, although the sizes, sequences, and stoichiometries of the repetitive elements very considerably between the two species. Protein-encoding genes or gene families (six genes in Marchantia and nine gene families in human) occur within repetitive elements, and all are present in multiple copies on the Y chromosome. In both organisms, homologs also may be present on the X or autosomal chromosomes in low copy number. Although all protein-encoding genes found within the human ampliconic sequences are expressed only in the testis, at least some of the protein-encoding genes identified in Marchantia are male organ specific in their expression.
According to the prevailing theory of mammalian sex chromosome evolution (Graves and Schmidt, 1992; Jegalian and Page, 1998; Lahn and Page, 1999), the X and Y chromosomes are derived from an ancient autosomal pair of chromosomes. The Y chromosome acquired genes, especially those that enhance male fertility, by a series of autosomal transpositions that were then amplified on the Y chromosome, whereas the X chromosome maintained its ancestral genes. Other genes present on the Y chromosome (i.e., X homologs) were lost, probably aided by a lack of X-Y recombination, leading to a mostly degenerate Y chromosome. The recent sequencing results suggest that the uniformity of the ampliconic repetitive sequences of the human Y chromosome is maintained from generation to generation by intrachromosomal Y-Y gene conversion. This occurs at relatively high rates: ∼600 nucleotides per newborn human male are estimated to have undergone Y-Y gene conversion (Rozen et al., 2003). Although it is not known if there are higher order palindromic repeated sequences in Marchantia as there are in humans (these palindromic sequences may be necessary for gene conversion), the homogeneity of repetitive sequences in the Marchantia Y chromosome and the relatively high frequency of male-specific genes within these repetitive elements suggest gene conversion playing a role in maintaining the repetitive elements of the Marchantia Y chromosome.
One important distinction between Marchantia and humans is that X-X recombination cannot occur in Marchantia because all diploid sporophytes are X-Y. This suggests that forces other than homologous interchromosomal recombination are responsible for preventing the degeneration of the X chromosome, although size alone may not be an indicator of chromosome degeneracy. Future efforts to sequence the entire Y and X chromosomes in Marchantia will be very important in understanding how sex chromosomes specify sexual phenotype in this species and how both the X and Y chromosomes evolved in this ancient lineage of plants.

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[h=2]THE LYCOPHYTES[/h] Another group of land plants deserving attention from an evolutionary perspective is the lycophyte lineage, which includes the modern Lycopodiales genera Selaginella and Isoetes. This lineage is most closely related to the earliest vascular plants that first appeared on land ∼250 to 400 million years ago (Kenrick and Crane, 1997). Although the earliest lycophytes and extant members of the Lycopodiales are homosporous and produce only one type of spore, Selaginella and Isoetes are heterosporous, with their sporophytes producing free-living megaspores and microspores that give rise to the female and male gametophytes, respectively. Of the lycophytes, Selaginella has great potential as a useful comparative system for the study of sex determination in plants, in part because many species have very small genome sizes (J.A. Banks, unpublished observations). Selaginella produces microsporangia and megasporangia that are born on the same strobilus or in different strobili of the same plant, depending on the species; strictly unisexual species have not been reported. In Selaginella moellendorfii, for example, each strobilis bears both kinds of sporangia, with microsporangia at the bottom and macrosporangia toward the top of each strobilis. In such plants, sex determination would be viewed as the process that regulates the sexual identity of the sporangia in the strobilis, a mechanism that has clear parallels with floral organ identity in angiosperms that produce perfect flowers. Although we know virtually nothing about this group of plants beyond descriptive biology, this lineage holds important clues for understanding the evolution of heterospory from homospory, a switch that occurred many times during land plant evolution (Stewart and Rothwell, 1993) and that has had a major impact on the timing of sex determination from the gametophyte to sporophyte generation in plants (Sussex, 1966). The question of how heterospory evolved from homospory is difficult to study in the heterosporous angiosperm lineage because their homosporous progenitors are probably extinct.

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[h=2]THE HOMOSPOROUS FERNS[/h] Recent phylogenetic analyses of vascular seed–free plants group the leptosporangiate and eusporangiate ferns and members of Equisetum and Psilotum into a monophyletic clade that is sister to the seed plants (Pryer et al., 2001). With few exceptions, they are homosporous plants. The one plant for which a sex-determining pathway has been genetically well defined is the leptosporangiate fern Ceratopteris richardii. Like Marchantia, Ceratopteris is homosporous and produces only one type of haploid spore. Although the sex of the Marchantia gametophyte is determined genetically by sex chromosomes, the sex of the Ceratopteris gametophyte (male or hermaphroditic) is determined epigenetically by the pheromone antheridiogen. Since their discovery by Dopp (1950) in the fern Pteridium aquilinum, antheridiogens have been identified and characterized from many species of leptosporangiate ferns (reviewed by Naf, 1979; Yamane, 1998), suggesting that it is a common mode of regulating sexual phenotypes in this group of plants. Although the structure of the Ceratopteris antheridiogen is unknown, all other fern antheridiogens characterized to date are mostly novel gibberellins (Yamane, 1998).
The Ceratopteris male and hermaphroditic gametophytes are easy to distinguish not only by the type of gamete produced but also by the presence or absence of a multicellular meristem. As illustrated in Figure 2, the hermaphrodite forms a single meristem and meristem notch that gives the hermaphrodite its heart-shaped appearance. Cells of this meristem differentiate as egg-forming archegonia, sperm-forming antheridia, or simply enlarge, adding to the growing sheet of photosynthetic parenchyma cells that make up most of the hermaphrodite prothallus. A Ceratopteris spore grown in isolation always develops as a hermaphrodite. A male gametophyte develops from a spore only if the spore is placed in medium that had previously supported the growth of a hermaphrodite. Lacking a multicellular meristem, almost all cells of the male gametophyte terminally differentiate as antheridia. The male-inducing pheromone that is secreted by the Ceratopteris hermaphrodite is called A[SUB]CE[/SUB] for antheridiogen Ceratopteris. Based on physiological studies in Ceratopteris (Banks et al., 1993), A[SUB]CE[/SUB] is not secreted by the hermaphrodite until after it loses the competence to respond to its male-inducing effects, which corresponds to the initiation of the meristem. A gametophyte will develop as a male only if it is exposed continuously to A[SUB]CE[/SUB] from a very young age, between 2 to 4 days after spore inoculation. Thus, in a population of spores, those that germinate first become A[SUB]CE[/SUB]-secreting meristic hermaphrodites, whereas those that germinate later become ameristic males under the influence of A[SUB]CE[/SUB] secreted by its neighboring hermaphrodites.
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Figure 2. The Sex-Determining Mutants of Ceratopteris richardii.
The her1 (hermaphroditic) mutant and the wild-type hermaphrodite are indistinguishable, as are the tra1 (transformer) mutant and the wild-type males, except that the her1 and tra1 mutants are insensitive to the absence or presence of A[SUB]CE[/SUB]. The A[SUB]CE[/SUB]-insensitive fem1 (feminization) gametophyte produces no antheridia. The man1 (many antheridia) mutant produces ∼10 times more antheridia than hermaphrodites, whereas the not1 (notchless) mutant rarely produces antheridia. The meristem notch normally present on the hermaphrodite often is missing in the not1 mutant, giving it a cup-shaped appearance. The novel phenotypes of the fem1 tra1 and fem1 not1 tra1 mutants are shown. an, antheridia; ar, archegonia; mn, meristem notch.


To understand how A[SUB]CE[/SUB] represses the development of female traits (i.e., archegonia and meristem) and promotes the development of male traits (i.e., antheridia) in Ceratopteris, a genetics approach has been used to identify the genes involved in this response (Banks, 1998; Strain et al., 2001). To date, five phenotypic classes of mutants have been identified; representatives of each class are illustrated in Figure 2. In addition to those that are always hermaphroditic (the hermaphroditic mutants), always male (the transformer [tra] mutants), or always female (the feminization [fem] mutants) regardless of the absence or presence of A[SUB]CE[/SUB], there are mutants that produce excessive antheridia (the many antheridia mutants) as well as the feminizing mutants that often lack a meristem notch (the notchless mutants). By comparing the phenotypes of double mutant gametophytes to each single mutant gametophyte parent, the epistatic interactions among these genes have been assessed. One particularly informative phenotype is the fem1 tra1 double mutant, also illustrated in Figure 2. Unlike other double mutant combinations, this one has a novel phenotype unlike that of either parent. This finding suggests that these two genes (FEM1 and TRA1) define two separate pathways, one specifying male development and the other female development. The sex-determining mutants have been ordered into a genetic sex-determining pathway, illustrated in Figure 3, that is most consistent with the genetic data. In this pathway, the sex of the gametophyte ultimately depends on the activities of two genes, one specifying the development of male traits (FEM1) and the other specifying the development of female traits (TRA). These genes also repress each other, so that when TRA is active, FEM1 is not and visa versa. What determines which of these two genes is expressed in the gametophyte (and thus its sex) is A[SUB]CE[/SUB], which ultimately represses the TRA genes, as described in the legend to Figure 3.
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Figure 3. The Genetic Sex-Determining Pathways in the Fern Ceratopteris, the Fly Drosophila melanogaster, and the Nematode Caenorhabditis elegans.
The genetic model of sex determination in Ceratopteris (Strain et al., 2001) is dependent on two genes, FEM1 and TRA (there are at least two TRA genes), which promote the differentiation of male (antheridia) and female (meristem and archegonia) traits, respectively. FEM1 and TRA also antagonize each other such that if FEM1 is active, TRA is not, and vice versa. What determines which of these two genes prevails in the gametophyte and thus its sex is the pheromone A[SUB]CE[/SUB], which activates the HER genes, of which there are at least five, and sets into motion a series of switches that ultimately result in male development (i.e., FEM1 on and TRA off). These switches are thrown in the opposite direction when spores germinate in the absence of A[SUB]CE[/SUB]. Although FEM1 represses TRA and TRA represses FEM1, they do not do so directly. TRA activates MAN1, which represses FEM1, and FEM1 activates NOT1, which represses TRA. Because TRA and FEM1 are the primary regulators of sex, NOT1 and MAN1 are considered regulators of the regulators. The sex determination pathway in C. elegans (Hodgkin, 1987; Villeneuve and Meyer, 1990) is linear and consists of a series of negative control switches. The state of the initial switch gene (xol-1) is dependent on the ratio of X to autosomal (A) chromosomes. If the ultimate downstream gene in this pathway (TRA-1) is high, the nematode develops as a hermaphrodite. If TRA-1 is low, it develops as a male. The linear sex determination pathway shown for Drosophila is from the mid-1980s (Baker and Ridge, 1980; Cline, 1983). There are actually other sex-determining pathways that account for most aspects of sexual phenotype (reviewed by Oliver, 2002); the pathway shown is the somatic pathway. In the soma, Sxl is the key regulator of sex, and its state of activity is determined by the X:A ratio. The dsx gene is the downstream regulatory gene that ultimately determines whether male or female genes are expressed in the soma.


In comparing mechanisms of gametophytic sex determination in homosporous bryophytes and ferns, one obvious question that arises is what drove Marchantia to an X-Y chromosomal mechanism of sex determination and Ceratopteris to an epigenetically regulated mechanism dependent on pheromonal cross-talk between individuals? The answer to this question probably lies in the different ratios of males and females or hermaphrodites that occur in the populations of each species. In Marchantia, the segregation of X and Y sex chromosomes during meiosis in the sporophyte ensures that each gametophyte progeny has an equal probability of being either male or female, barring selection. In Ceratopteris, the A[SUB]CE[/SUB] response allows the ratio of males to hermaphrodites to vary depending on the density of the population, such that as the population density increases, the proportion of males also increases. Although the underlying sex-determining mechanism is inflexible in Marchantia, it is flexible enough in Ceratopteris to allow each individual to determine its sex according to the size of the population in which it resides and the speed at which it germinates relative to its neighbors. The flexibility of the Ceratopteris sex-determining mechanism is reflected in its sex-determining pathway, and this becomes especially apparent compared with the sex-determining pathways known from other organisms, including Drosophila melanogaster and Caenorhabditis elegans, which are illustrated in Figure 3. In both of these animals, an individual's sex (male or female in D. melanogaster and male or hermaphrodite in C. elegans) is determined genetically by the ratio of X to autosomal chromosomes. This ratio is read and either activates or represses the activities of downstream genes in each pathway. In both animals, the sex ultimately depends on the state of the terminal gene in each linear pathway, TRA1 in the case of C. elegans. The Ceratopteris sex-determining pathway is distinctly different from those of D. melanogaster and C. elegans in that it is not linear and there are two sex-determining genes, one for male and one for female development. Their ability to repress each other endows each Ceratopteris spore with the flexibility to determine its sex upon germination based on environmental cues.
So why would a flexible mechanism of sex determination that allows sex ratios to vary be adaptive in ferns but not in bryophytes? The answer to this question may lie in the ephemeral nature of the fern gametophyte. Although the gametophytes of bryophytes are persistent, the gametophytes of ferns are not. In Ceratopteris, for example, gametophytes reach sexual maturity only 14 days after spore inoculation and die once they are fertilized. The limited time that a fern gametophyte is able to be crossed by another might favor a sex-determining system that would promote outcrossing by increasing the proportion of males when population densities are high and ensuring a high proportion of hermaphrodites capable of self-fertilization when population densities are low. Because there are a variety of sex-determining mutants available in Ceratopteris, the hypothesis related to the consequences of variable versus fixed sex ratios can be tested easily, at least under defined laboratory conditions.
Future studies to clone the sex-determining genes in Ceratopteris will be necessary to understand their biochemical functions and to test their interactions predicted by the genetic model. Although the size of the Ceratopteris genome is probably very large (n = 37), the ability to inactivate genes in the Ceratopteris gametophyte by RNA interference (Stout et al., 2003; G. Rutherford, M. Tanurdzic, and J.A. Banks, unpublished observations) provides an important means to study the effects of inactivating potential sex-determining genes in the Ceratopteris gametophyte.

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[h=2]THE FLOWERING PLANTS[/h] Although unisexuality is very common in animals, hermaphroditism is the rule in angiosperms. Approximately 90% of all angiosperm species have perfect flowers with specialized organs producing microspores or megaspores from which the male or female gametophytes develop. Of the remaining species, approximately half are monoecious, producing unisexual flowers of both sexes on the same individual, and the other half are dioecious, with unisexual male and female flowers arising on separate individuals (Yampolsky and Yampolsky, 1922). The distribution of dioecy and monoecy within the angiosperm phylogenetic tree strongly favors the evolutionary scenario in which unisexual flowers evolved from perfect flowers multiple times in the angiosperm lineage (Lebel-Hardenack and Grant, 1997; Charlesworth, 2002). Not surprisingly, there are a variety of sex determination mechanisms in the angiosperms. For organizational purposes only, sex determination in monoecious and dioecious species are treated separately in this review.
[h=3]The Monoecious Angiosperms[/h] There are numerous terms to describe the variety of sexual phenotypes observed in monoecious plant species (defined and compiled by Sakai and Weller, 1999). Although this rich nomenclature is appropriate, it tends to confound the problem of sex determination in this group of plants. For simplicity, monoecious species here are grouped into two categories: those that produce only unisexual male and female flowers on the same plant, and those that produce both unisexual and perfect flowers on the same plant.
Zea mays (maize) is an example of a monoecious species that produces only unisexual flowers in separate male and female inflorescences, referred to as the tassel and ear, respectively. Unisexuality in maize occurs through the selective elimination of stamens in ear florets (flowers) and by the elimination of pistils in tassel florets (reviewed by Irish, 1999). Two general classes of sex-determining mutants have been identified in maize, including those that masculinize ears and those that feminize tassels. The anther ear (an1) and dwarf (d1, d2, d3, and d5) mutants of maize are recessive and masculinize ears by preventing stamen abortion in the female florets (Wu and Cheung, 2000). The dominant dwarf mutation, d8, has a similar phenotype. The D1, D3, and AN1 genes encode enzymes involved in the biosynthesis of the plant hormone gibberellin (GA) (Bensen et al., 1995; Winkler and Helentjaris, 1995; Spray et al., 1996). The D8 gene encodes the maize homolog of GIBBERELLIN INSENSITIVE/REPRESSOR OF gal-3 (Peng et al., 1999), a family of transcription factors that negatively regulate GA responses in plants (Richards et al., 2001; reviewed by Olszewski et al., 2002). GA signaling is thought to be derepressed in the d8 mutant, resulting in a dominant phenotype. The molecular identity of these genes provides direct evidence that endogenous GAs have a feminizing role in sex determination in maize.
The tasselseed1 (ts1) and ts2 mutants of maize feminize male florets in the tassel, as illustrated in Figure 4. In addition to the tassel phenotype, the second floret of the ear spikelet, which normally fails to develop, develops normally in the ts mutants, leading to a double-kerneled spikelet in the ear (Dellaporta and Calderon-Urrea, 1994; Irish, 1999). The TS2 gene has been cloned and shown to encode a putative short-chain alcohol dehydrogenase with signature motifs of steroid dehydrogenases (DeLong et al., 1993). TS2 mRNA is expressed in the subepidermal layer of the gynoecium before its abortion, which correlates well with the timing and location of cell death responsible for the pistil abortion in wild-type male florets (Calderon-Urrea and Dellaporta, 1999). The expression of ts2 requires the wild-type TS1 gene. Although the pistils of ear florets also express TS1 and TS2, they are protected from a deathly fate by the action of another gene, SILKLESS1, that interacts directly or indirectly with TS2 (Calderon-Urrea and Dellaporta, 1999). Although the cloning of the sex-determining genes in maize demonstrates that GAs and possibly other steroid-like hormones play a pivotal role in stamen abortion and feminization of flowers, the spatial distribution of these molecules could have an effect on the sex determination process, as exemplified by a steep gradient in GA abundance along the maize shoot (Rood et al., 1980), which correlates well with the male-suppressing and female-promoting phenotypic effects of GA. How the synthesis or transport of these molecules is regulated, and the identity of the downstream targets of these hormones, remain to be discovered.
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Figure 4. The ts4 Mutant of Maize.
The wild-type male tassel (left) produces only male florets, whereas plants homozygous for the ts4 allele (right) form hermaphroditic flowers with functional pistils, allowing seeds to form in the tassel. Photographs courtesy of Erin Irish, University of Iowa.


Another monoecious plant, Cucumis sativus (cucumber), has served as a model system for sex determination studies since the 1950s and early 1960s, driven by breeding programs for hybrid seed production. Cucumber plants are mostly monoecious but can be dioecious or hermaphroditic, depending on the genotype. Regardless of their sex, all floral buds are initially hermaphroditic, and it is the arrest of stamen or pistil development that leads to unisexual flowers. In monoecious cucumbers, male flowers form at the bottom and female flowers form at the top of each shoot. There are three major genes that affect the arrangement of unisexual flowers or their sex; they are designated the F, A, and M genes (following the nomenclature of Pierce and Werner, 1990). The F gene is semidominant and affects the expression of femaleness along the plant, causing it to extend the gradient of femaleness toward the bottom of the plant. The A gene is epistatic to F, and it too is required for the expression of femaleness. The M gene is required for maleness in that it is not required for the establishment of the gender gradient along the shoot but rather for the selective abortion of pistils and stamens in female and male flowers, respectively (Perl-Treves, 1999). With respect to the F and M genes, M-ff plants are monoecious, M-F- plants are female, mmF- plants are hermaphroditic, and mmff plants are andromonoecious (with male and hermaphroditic flowers).
In addition to the sex-determining genes, plant hormones have long been implicated in the sex-determining process in cucumber. GA and ethylene application and the use of GA and ethylene inhibitors can subvert the genotypic constitution of the plant, with GA acting mainly as a masculinizing agent and ethylene acting as a feminizing agent (Perl-Treves, 1999). By treating monoecious and andromonoecious cucumber plants with various combinations of GA and ethrel or GA and ethylene inhibitors, Yin and Quinn (1995) demonstrated that ethylene is the main regulator of sex determination, with GA functioning upstream of ethylene, possibly as a negative regulator of endogenous ethylene production. These findings led them to propose a model for how sex determination might occur (Yin and Quinn, 1995), with ethylene serving both as a promoter of the female sex and an inhibitor of the male sex. The basic tenets of the model are that the F gene should encode a molecule that would determine the range and gradient of ethylene production along the shoot, thereby acting to promote femaleness, whereas the M gene should encode a molecule that perceives the ethylene signal and inhibits stamen development above threshold ethylene levels. This model is consistent with how unisexual flowers might arise very early and very late during shoot development; however, the model also predicts an entire range of intermediate types rarely or never seen in cucumber. As suggested by Perl-Treves (1999), variations in the model of Yin and Quinn and the incorporation of additional factors in the sex-determining process in cucumber could account for the observed lack of intermediate types.
Recent results from several laboratories have provided molecular evidence in favor of the ethylene theory of sex determination in cucumber. Two 1-aminocyclopropane-1-carboxylic acid synthase genes, CS-ACS1 and CS-ACS2, have been identified in cucumber, and one of them (CS-ACS1) maps to the F locus (Trebitsh et al., 1997). The monoecious cucumber genome has only one copy (Cs-ACS1), whereas the gynoecious genome has both copies. The expression of both genes correlates with sexual phenotype, with gynoecious plants accumulating more transcript than monoecious or andromonoecious plants (Kamachi et al., 1997; Yamasaki et al., 2001). Although these studies are consistent with the female-promoting effects of ethylene, they do not address the question of how ethylene inhibits stamen abortion in gynoecious and not andromonoecious plants. Yamasaki et al. (2001) provided evidence suggesting that the product of the M locus mediates the inhibition of stamen development by ethylene (i.e., M affects sensitivity to ethylene). This finding indicates that ethylene concentration, which is likely to be dependent on the F locus, and the differential sensitivity of males and females to ethylene, which is likely to be dependent on the M locus, are both important in regulating sexual phenotype in cucumber. Definitive cloning of the M and F genes will allow these hypotheses to be tested directly.
Because sex determination in cucumber and most other angiosperm species occurs via selective abortion of flower organs, Kater et al. (2001) set out to establish whether this abortion is based on organ identity or positional information within the flower. The availability of cucumber homologs of the MADS box ABC homeotic genes and the ability to express them ectopically in cucumber allowed these authors to show that the sex determination machinery in cucumber selectively aborts sex organs based on their position rather than their identity (i.e., in male flowers, carpels are aborted only in the fourth whorl, and in female flowers, stamens abort only in the third whorl). In addition, because nonreproductive organs that develop in the inner whorls of a C-class homeotic mutant are not aborted, Kater et al. (2001) speculated that C-class gene products might be targets of the sex-determining process. Even though this is a tempting speculation, it leaves the question of abortion timing unresolved.
Several studies have addressed the role of the MADS box floral homeotic genes in the sex determination process in many monoecious and dioecious plants, including Asparagus officinalis (Park et al., 2003), Betula pendula (Elo et al., 2001), Gerbera hybrida (Yu et al., 1999), Populus deltoides (Sheppard et al., 2000), Rumex acetosa (Ainsworth et al., 1995), Silene latifolia (Hardenack et al., 1994), and maize (Heuer et al., 2001). In all cases, the expression of B- or C-function floral homeotic genes in carpels or stamens was shown to decline in organs targeted for abortion in the unisexual flower. However, it is not clear if the changes in expression of these genes are a cause or a consequence of organ abortion. Furthermore, evidence that sex-determining mutants cosegregate with MADS box genes is lacking. That plants may not use MADS box genes for sex determination would not be surprising given that the unisexual flowers of most monoecious and dioecious plants are derived from bisexual flowers with all sex organ primordia present.
Another plant within the monoecious group of angiosperms that has been studied is Carica papaya (papaya). Papaya is a polygamous species with three sexes—females, males, and hermaphrodites. In this species, sexual phenotype is controlled by a single gene with three alleles: the dominant M (for male), the dominant M[SUP]h[/SUP] (for hermaphrodite), and the recessive m (for female) alleles (Storey, 1938). The progeny of self-fertilized hermaphrodites (genotypically M[SUP]h[/SUP]m) segregate hermaphrodites and females in a 2:1 ratio. The lack of male progeny indicate that the MM genotype is lethal, perhaps because of a lethal gene closely linked to the M locus. This and other crosses led early workers to the conclusion that all males are genotypically M[SUP]h[/SUP]M. Thus, females are the homogametic and males the heterogametic sex. Papaya flowers of different sexes also display secondary sexual characteristics that cosegregate with the M allele. The apparent lack of recombination between the loci responsible for primary and secondary sex traits indicates that both loci are tightly linked and inherited en bloc, much like a sex chromosome (Storey, 1953), although heteromorphic chromosomes do not exist (Kumar et al., 1945).
The economical importance of papaya has driven sex determination research in this species, because the pyriform fruits from hermaphroditic trees are preferred by consumers more than the spherical fruits produced by the female trees. Because it is only upon flowering that the sex of the individual papaya tree can be determined, molecular markers that cosegregate with the M alleles have been sought intensively. To date, two groups have reported randomly amplified polymorphic DNA markers that are highly specific for males and hermaphrodites but absent in females (Deputy et al., 2002; Urasaki et al., 2002). One randomly amplified polymorphic DNA marker was also shown by DNA gel blot hybridization to be completely absent from the female genome (Urasaki et al., 2002), providing the first molecular evidence for genomic differentiation between the sexes. Although papaya is a tropical tree and not considered a model plant system, it has a small genome of 371 Mbp (Arumuganathan and Earle, 1991) and may be transformable (Fitch et al., 1992). These facts, coupled with its economic importance in tropical and subtropical regions of the world, make it is a species worthy of further study.

[h=3]The Dioecious Angiosperms[/h] Silene latifolia is a dioecious species with individual plants producing either all female or all male flowers. As it is by far the best characterized dioecious species to date, our review of sex-determining mechanisms in dioecious plants will focus on this species. In male and female S. latifolia flowers, the gynoecium and androecium initiate but arrest development prematurely, leading to functionally unisexual flowers (Grant et al., 1994). The sexual phenotype of individuals is determined by sex chromosomes; males are heterogametic (XY) and females are homogametic (XX). Early cytogenetic studies of sex-determining mutants in S. latifolia led Westergaard (1946, 1958) to conclude that the Y chromosome is divided into three regions relevant to sex expression: one required for the suppression of female development and two required for the promotion of male development. None of these regions would be necessary for the development of female reproductive organs, because these functions would reside on the X or autosomal chromosomes. Additional sex-determining mutants have been generated recently by x-ray mutagenesis of pollen and selecting both hermaphrodites and asexual F1 progeny (Farbos et al., 1999; Lardon et al., 1999; Lebel-Hardenack et al., 2002). These mutants verify the earlier work of Westergaard (1946, 1958; his lines were apparently lost) and have resulted in the identification of two additional classes of mutants, those that are not Y linked and hermaphroditic and those that are Y linked and asexual. The hermaphroditic deletion mutants are likely to contain a gene(s) necessary for the female-suppressing function, whereas the asexual deletion mutants likely contain the male-promoting gene(s). Genetic screens to identify mutant XX hermaphrodites or asexuals have not been reported. Although these sex-determining genes have not been cloned, the construction of an amplified fragment length polymorphism map of the Y chromosome using lines deleted for overlapping regions of the Y chromosome will be useful for genetic and physical mapping of the sex-determining mutants (Lebel-Hardenack et al., 2002) and may ultimately lead to their cloning.
Another approach to identify sex-determining genes in S. latifolia has been to clone genes that are expressed specifically in the male flowers and determine their linkage to the Y chromosome (reviewed by Charlesworth, 2002). Of the >50 isolated genes that have been correlated with sex expression in S. latifolia to date, only the four listed in Table 1 have been shown to be linked to the Y chromosome. Genes linked exclusively to the Y chromosome have not been found, because all of the Y-linked genes have X or autosomal homologs. These data indicate that the S. latifolia Y chromosome most closely resembles the X-degenerate class of sequences of the human Y chromosome. Genes within this class have an X homolog, the X and Y homologs encode similar but nonidentical isoforms, many of them are ubiquitously expressed, and all are present in low copy number in the genome (Skaletsky et al., 2003). As shown in Table 1, many of these features are shared with the Y-linked genes of S. latifolia.
View this table:


Table 1. Sex Chromosome–Linked Genes in S. latifolia


Although molecular approaches have not yet succeeded in identifying the major regulatory sex-determining genes in S. latifolia, this work has and will continue to test theories of how Y chromosomes evolved from an ancestral pair of autosomes in plants (Charlesworth, 1996, 2002; Negrutiu et al., 2001). These theories state that once mutations that result in genetically determined males (where female genes are repressed) and females (where male genes are repressed) occur, recombination between the sex-determining genes must be suppressed to avoid recombinant asexual or hermaphroditic offspring. Another consequence of nonrecombination is the certainty that unisexual male and female offspring will be produced in equal ratios. Recombination between homologous chromosomes often is suppressed by the accumulation of chromosomal inversions on one homolog (in this case, the Y). The lack of X-Y recombination would eventually lead to degeneracy and loss of gene function on the Y chromosome, with the exception of genes required for male fertility and those necessary to suppress female fertility. The S. latifolia Y chromosome appears not to fit this paradigm for several reasons. First, the Y chromosome is 1.4 times larger than the X chromosome and is largely euchromatic, indicating that it may not be degenerate (Ciupercescu et al., 1990; Grabowska-Joachimiak and Joachimiak, 2002). Second, measurements of DNA polymorphism in sex-linked gene pairs have revealed that although the DNA polymorphism of S4Y-1 is greater than that of S4X-1, the DNA polymorphism of SlY-1 is 20-fold lower than that of SlX-1 (Filatov et al., 2000, 2001; Filatov and Charlesworth 2002). Given that the S. latifolia sex chromosomes diverged less than ∼20 million years ago (Desfeux et al., 1996; Charlesworth, 2002), which is much less than the 240- to 320-million-year timeline for human sex chromosome evolution (Lahn and Page, 1999), it is likely that the S. latifolia Y chromosome is at a relatively young stage of evolution (Charlesworth, 2002). Comparative sequencing of the S. latifolia sex chromosomes will be important in understanding the evolution of the Y chromosome in plants, especially compared with the Y chromosome of Marchantia and the sex-determining chromosome region of papaya.


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[h=2]FUTURE DIRECTIONS[/h] Sex determination in plants is a fundamental developmental process that is particularly important for economic reasons, because the sexual phenotypes of commercially important crops dictate how they are bred and cultivated. Although most crop plants are not considered model systems—and sex determination is not a problem that can be addressed in the model angiosperm Arabidopsis—the economic value in manipulating the sexual phenotypes of crop plants should continue to drive interest in this area of research. Recent studies of sex-determining mechanisms have demonstrated clearly that angiosperms, including crop plants, have evolved a variety of sex-determining mechanisms that involve a number of different genetic and epigenetic factors, from sex chromosomes to plant hormones. Although the determinants of sexual phenotype are diverse, determining whether the downstream master sex-regulatory genes that specify male or female development are held in common or not will require cloning the sex-determining genes from a variety of plant species.
Choosing to study sex determination in plants representing other major land plant lineages will allow several broader developmental and evolutionary questions to be addressed. One unresolved question is how heterospory evolved from homospory. By identifying the sex-determining genes in homosporous plants such as Ceratopteris and examining the expression of possible homologous genes in closely related heterosporous species, one can test the hypothesis that the switch from homospory to heterospory involved a heterochronic shift in the timing of expression of these genes from the gametophyte to the sporophyte generation. Other questions to be resolved are how sex chromosomes evolved in plants and whether similar processes led to distinct sex chromosomes in plants and animals. Comparing the Y chromosome sequences of Marchantia and S. latifolia, for example, will be invaluable in understanding how male-promoting genes and female-suppressing genes became localized to a Y chromosome and how recombination between the Y and its homolog was and continues to be suppressed.

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[h=2]Acknowledgments[/h] Support was provided by the National Science Foundation (MCB-9723154). This is journal paper 17271 of the Purdue University Agricultural Experiment Station.

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[h=2]Footnotes[/h]


  • Received August 25, 2003.
  • Accepted January 19, 2004.
  • Published April 14, 2004.
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[h=1]Sex chromosomes in flowering plants1[/h]
+ Author Affiliations

  • [SUP]2[/SUP]Department of Plant Biology, University of Illinois at Urbana–Champaign, Illinois 61801 USA;
  • [SUP]3[/SUP]USDA-ARS, Pacific Basin Agricultural Research Center, Hilo, Hawaii 96720 USA; and
  • [SUP]4[/SUP]Plant Genome Mapping Laboratory, University of Georgia, Athens, Georgia 30602 USA

  • Received for publication 8 August 2006.
  • Accepted for publication 21 November 2006.


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[h=2]Abstract[/h] Sex chromosomes in dioecious and polygamous plants evolved as a mechanism for ensuring outcrossing to increase genetic variation in the offspring. Sex specificity has evolved in 75% of plant families by male sterile or female sterile mutations, but well-defined heteromorphic sex chromosomes are known in only four plant families. A pivotal event in sex chromosome evolution, suppression of recombination at the sex determination locus and its neighboring regions, might be lacking in most dioecious species. However, once recombination is suppressed around the sex determination region, an incipient Y chromosome starts to differentiate by accumulating deleterious mutations, transposable element insertions, chromosomal rearrangements, and selection for male-specific alleles. Some plant species have recently evolved homomorphic sex chromosomes near the inception of this evolutionary process, while a few other species have sufficiently diverged heteromorphic sex chromosomes. Comparative analysis of carefully selected plant species together with some fish species promises new insights into the origins of sex chromosomes and the selective forces driving their evolution.

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Sexual reproduction is a prominent feature of the life cycle in most animals and plants. The enormous diversity of life forms and their wide range of genome complexity, from the single, circular chromosome of prokaryotes to the 640 chromosomes of the angiosperm species Sedum suaveolens, are made possible partly by sex. Weismann, (1889)⇓ proposed that sexual reproduction increases genetic diversity and provides various combinations of alleles for natural selection. However, several difficulties with this explanation have arisen, such as recombination breaking up assemblies of favorable genes and the two-fold cost of sex (only one of the two parents of sexual reproductive system is capable of bearing offspring) (Williams, 1975⇓; Smith, 1978⇓). Two recent reports support Weismann's hypothesis; faster evolutionary adaptation was shown in a sexual strain than in an asexual strain of yeast (Goddard et al., 2005⇓), and a reduction of deleterious mutations was detected in a sexually reproducing species compared to an asexually reproducing species of water fleas (Paland and Lynch, 2006⇓).
Dioecism has arisen independently from hermaphroditic ancestors in many plant families and genera. Flowering plants began appearing in fossil records at a minimum of 124.6 million year ago (MYA) (Sun et al., 2002⇓), and the crown-group angiosperms were estimated to have evolved 158–179 MYA based on DNA sequence data (Wikström et al., 2001⇓). The ancestor of flowering plants was likely hermaphroditic (Takhtajan, 1969⇓). Although the vast majority of extant flowering plants are hermaphrodites, monoecious and dioecious species occur in 75% of angiosperm families, including all six dicotyledonous and all five monocotyledonous subclasses (Yampolsky and Yampolsky, 1922⇓; Renner and Ricklefs, 1995⇓). Dioecious species accounted for 6% (14 620) of the species in 7.1% (959) of genera and 38% (167) of families of flowering plants (Renner and Ricklefs, 1995⇓). Interestingly, early flower development is similar in many unisexual and hermaphroditic species (reviewed by Dellaporta and Calderon-Urrea, 1993⇓; Matsunaga and Kawano, 2001⇓). Flower ontogeny and the broad phylogenetic distribution of unisexual species suggest that dioecism has arisen independently from hermaphroditic ancestors on many occasions in different plant families and genera. In the majority of cases, the evolution of dioecism has taken place at the species level (Westergaard, 1958⇓).
Dioecism accompanied by sex chromosome dimorphism is common in animals but less prevalent in plants. Appearance of heteromorphic sex chromosomes is the consequence of the evolution of sex to ensure dioecy and to enforce a 1:1 segregation ratio of male and female individuals. Sex chromosomes have evolved in a limited number of dioecious angiosperms, and only a few have been characterized at the cytological and/or molecular levels (Charlesworth, 2002⇓). The intensively studied heteromorphic sex chromosomes in Silene might have originated about 5–10 MYA based on the molecular clock calibration from the nuclear genes Chs and Adh in the family Brassicaceae (Koch et al., 2000⇓; Nicolas et al., 2005⇓), although substitution rates for some plant Adh genes could be 10 times slower (Gaut et al., 1996⇓). The homomorphic papaya sex chromosomes evolved more recently as revealed by a study of X and Y gene pairs (Q. Yu, P. Moore, J, Jiang, A. Paterson, R. Ming, unpublished data). Their recent origin is consistent with the primitive nature of most plant sex chromosomes compared with the 300 million-year-old sex chromosomes in mammals (Lahn and Page, 1999⇓).
Dioecious plants provide a particularly interesting system in which to study the genetics and evolution of sex chromosomes (Westergaard, 1958⇓). First, plants with unisexual flowers have evolved independently and multiple times from bisexual progenitors, which provides the opportunity to study different mechanisms of sex determination. Second, many dioecious plant species have fertile bisexual relatives, which makes possible comparative studies of sex determination mechanisms and the origins of sex chromosomes from autosomes. Third, in plants it is possible to follow both forward and reverse evolution between dioecism and hermaphroditism because dioecious mutants from hermaphrodites and hermaphrodite mutants from the dioecious plants are each occasionally found. Finally, different plant species at various stages of sex chromosome evolution provide a time series for examining the forces driving Y chromosome degeneration. The recent origin of sex chromosomes in Silene and papaya offers the opportunity to access the very early stages of sex chromosome evolution (Westergaard, 1958⇓; Filatov et al., 2000⇓; Negrutiu et al., 2001⇓; Filatov and Charlesworth, 2002⇓; Liu et al., 2004⇓).
Sex chromosomes were discovered more than a century ago (McClung, 1901⇓). Theoretical, cytogenetic, and classical genetic studies of sex chromosome evolution dominated the decades following this discovery. In the past two decades, genomic and molecular biology techniques have made it possible to clone the sex determination genes and to sequence sex chromosomes, as done for mammals (Sinclair et al., 1990⇓; Skaletsky et al., 2003⇓; Ross et al., 2005⇓). In plants, sex determination genes were cloned for monoecious maize (DeLong et al., 1993⇓; Bensen et al., 1995⇓). Genetic maps were constructed for sex chromosomes of asparagus (Reamon-Büttner and Jung, 2000⇓), hops (Seefelder et al., 2000⇓), and papaya (Ma et al., 2004⇓). Sex chromosome-specific genes were cloned and analyzed in Silene (Delichère et al., 1999⇓; Atanassov et al., 2001⇓; Moore et al., 2003⇓; Nicolas et al., 2005⇓). Sequencing of the male-specific region of the Y chromosome and corresponding region of the X chromosome is underway in papaya. These recently available genomic, molecular, and genetic data are shedding light on sex chromosome evolution and will allow us to look into how sex chromosomes evolve from autosomes. In this review, we will summarize our current understanding on the evolutionary origin and consequence of sex chromosomes in angiosperm with an emphasis on early stages of sex chromosome evolution.

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[h=2]SEX DETERMINATION IN FLOWERING PLANTS[/h] The hypothesis that angiosperms originated from a hermaphroditic ancestor is supported by the prevalence of hermaphrodites among about 72% of all species (Takhtajan, 1969⇓). Diverse mechanisms that evolved to promote outcrossing include temporal separation of the maturation of male or female organs, gametophytic or sporophytic self-incompatibility, structural avoidance, monoecy, and ultimately dioecy. The first step toward sex chromosome evolution is the occurrence of male sterile or female sterile mutations leading to the development of unisexual flowers. Such mutations occur frequently and repeatedly in plant species, as demonstrated by the presence of unisexual species in 75% of angiosperm families and numerous male sterile mutants in domesticated crop plants (Yampolsky and Yampolsky, 1922⇓; Renner and Ricklefs, 1995⇓). Also, stamen and carpel development involve large numbers of genes at various developmental stages, and mutations of any of the many regulatory genes could trigger abortion or loss of function of male and/or female organs (Wellmer et al., 2004⇓). In dioecious flowers, male sterile mutation can occur from the early stages of the first appearance of the stamen primordia until the late stages of microspore formation; likewise, female sterility can also occur from early stages of inception of the carpel until the late stage of the formation of microspores (Matsunaga and Kawano, 2001⇓).
Unisexual species have sex determination mechanisms ranging from genetic to environmental, but genetic factors play far greater roles. Genetic mechanisms range from a single locus on an autosome to heteromorphic sex chromosomes containing multiple genes involved in sex determination. In some unisexual species, such as hemp, cucumber, and maize, male or female sterility is influenced by environmental factors such as light intensity, day length, temperature, mineral nutrition, and plant hormones (Frankel and Galun, 1977⇓; Chailakhyan, 1979⇓). Cytoplasmic male sterility caused by mutations on the mitochondrial genome is widely used in production of crops that benefit greatly from heterosis, but only nuclear male sterility can trigger sex chromosome evolution.
Chromosomal sex determination systems may be an evolutionary consequence of natural selection in favor of dioecy. Sex chromosomes do not appear suddenly in animals or plants. Rather, it is the pair of autosomes bearing the sex determination genes that have evolved specialized features, i.e., the degeneration of the Y chromosome, resulting in heteromorphy that became the hallmark of sex chromosomes. Two major sex chromosome systems have evolved. One is the active Y chromosome system, or the XY system (or ZW in a heterogametic female system), in which females have two of the same kind of sex chromosome (XX), while males have two distinct sex chromosomes (XY). The other is the X-to-autosome (A) balance system, in which the ratio of X:A chromosomes determines sex by an X chromosome counting system and the Y chromosome is dispensable.
Among the many dioecious plant species, only a few have evolved sex chromosomes (Westergaard, 1958⇓; Renner and Ricklefs, 1995⇓; Charlesworth and Guttman, 1999⇓) (Table 1). As in mammals, some dioecious flowering species have an active-Y system of sex determination with heterogametic males (XY) and homogametic females (XX), such as white campion (Silene latifolia), papaya (Carica papaya), and asparagus (Asparagus officianalis). Several dioecious species have the X: A dosage compensation system for sex determination, for example, sorrel (Rumex) and hops (Humulus). An X-to-autosome ratio of 1.0 or higher results in a female and 0.5 or lower defines a male (Ono, 1935⇓; Westergaard, 1958⇓), which is similar to the situation in Drosophila and Caenorhabditis elegans (reviewed by Mittwoch, 1996⇓). These two sex chromosome systems may reflect two different stages of sex chromosome evolution that will be discussed further in the following sections.
View this table:


Table 1. List of plant species with homomorphic or heteromorphic sex chromosomes


In some species, sex expression is under epigenetic control, mediated by chromatin modifications of the sex determining regions, including DNA methylation and nucleosomal histone acetylation (reviewed by Vyskot, 1999⇓). In white campion, hypomethylation induced sex reversal of genetically male plants (XY) to form a perfect flower (Finnegan et al., 2000⇓). Sex determination is not the simple hierarchical chain reaction once envisioned but is the result of interactions among a complex network of genes as learned from the study of the sex-determining region Y (Sry) gene pathway that controls human sex determination (Koopman, 1999⇓). The epigenetic influence over sex determination might be the consequence of the regulation of the expression of a gene or genes in this network.

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[h=2]SUPPRESSION OF RECOMBINATION AT THE SEX DETERMINATION LOCUS[/h] Suppression of recombination is a pivotal event in sex chromosome evolution. Without suppression of recombination, the male sterile or female sterile mutations could revert to hermaphroditism. The typical features of young sex chromosomes are suppression of recombination at and around the sex determination locus and moderate degeneration of the male-specific region.
Suppression of recombination in specific chromosomal regions is a widespread phenomenon in sexually reproducing organisms. It is often triggered by rearrangement or modification of DNA sequences. Direct evidence was documented for suppression of recombination within two pericentric inversions on human chromosomes 1 and 8 (Jaarola et al., 1998⇓). Inversions were also the cause of suppression of recombination that led to the degeneration of the Y chromosome in human (Lahn and Page, 1999⇓). Although the contents of recombination-suppressed regions evolve rapidly, the regions themselves appear to remain in the same location over long periods (Bowers et al., 2005⇓). Gradual reduction of recombination rates might account in part for reducing recombination of the X and Y chromosomes in Silene (Nicolas et al., 2005⇓). DNA methylation is another mechanism for suppression of recombination as reported in the fungus Ascobolus immersus (Maloisel and Rossignol, 2006⇓).
Suppression of genetic recombination has been documented for primitive and advanced sex chromosomes (Westergaard, 1958⇓; Ohno, 1967⇓; Ma et al., 2004⇓). The papaya sex locus has been genetically mapped to linkage group 1 (Sondur et al., 1996⇓). In a recently constructed high density map, a total of 225 markers, which is 66% of the 342 markers on linkage group 1, co-segregated with the sex locus, indicating severe suppression of recombination at this region (Ma et al., 2004⇓). A similar result has been shown on a linkage map of the medaka fish. Two large clusters of loci consisting of 19 and 13 markers, respectively, flanked a region of 2.6 cM containing the male-determining region (Naruse et al., 2000⇓). In asparagus, the sex locus is located on linkage group 5 where 20 random markers have been mapped to a total of 55.3 cM, averaging one marker in 2.6 cM of this linkage group (Reamon-Büttner and Jung, 2000⇓; Jamsari et al., 2004⇓). However, nearer the sex locus, four markers, excluding the newly integrated sex-linked STS markers, were mapped in a region of 0.5 cM. Recombination suppression at and near the sex locus was further confirmed by physical mapping of the male-specific region in papaya (Liu et al., 2004⇓) and by genetic mapping using male and female meioses in medaka (Kondo et al., 2001⇓).
In the XY system, suppression of recombination and DNA sequence degeneration occur on the Y chromosome, while X chromosomes in females recombine normally. In addition to the large clusters of sex co-segregating markers, suppression of recombination in the male-specific region of the Y chromosome can be evaluated by comparing X and Y linkage maps. In hops (Humulus lupulus), the two markers flanking the male sex determination locus were 3.7 cM apart on the Y chromosome linkage map, while the genetic distance between the same two markers was 14.3 cM on the X chromosome linkage map, an almost four-fold reduction of recombination on the Y chromosome (Seefelder et al., 2000⇓). A similar reduction of genetic distance is found in the male meiotic map of the three-spined stickleback, a fish species with an emerging Y chromosome (Peichel et al, 2004⇓). The sex determination gene of sticklebacks is located at the end of linkage group 19. A pair of markers next to the sex determination gene was 6.4 cM apart based on male meiosis (i.e., the primitive Y chromosome) but 25.7 cM apart based on female meiosis (the X chromosome); again recombination on the Y chromosome was reduced approximately four-fold.
Multiple genes in the suppressed male-specific region of the Y chromosome may be specialized in male-specific functions. In white campion, three dispersed male determining loci were located on the Y chromosome, with two on one arm containing genes controlling carpel suppression and early stamen promoting and one on the other arm conferring late anther fertility (Westergaard, 1958⇓; Farbos et al., 1999⇓; Lardon et al., 1999⇓; Lebel-Hardenack et al., 2002⇓). Because these genes are physically located on distant segments of the Y chromosome, they could potentially segregate, resulting in hermaphroditic and sterile individuals unless recombination between them was suppressed.

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[h=2]EMERGENCE OF PRIMITIVE SEX CHROMOSOMES[/h] Sex chromosomes are usually heteromorphic. However, a number of dioecious flowering plant species appear to have an active Y system for sex determination without any cytological evidence of heteromorphism. For example, in papaya, it has been controversial for several decades whether sex determination is controlled by a single gene, a complex of genes, or sex chromosomes (Storey, 1953⇓, 1976⇓). Recently developed genomic and molecular techniques make it possible to examine the molecular basis of sex determination in papaya and other dioecious plant species to resolve such issues.
Sex determination in papaya was first thought to be controlled by a single gene with three alleles, M, M[SUP]h[/SUP], and m, with classical Mendelian segregation in populations generated from crosses among three sex types (Hofmeyr, 1938⇓; Storey, 1938⇓). However, male individuals (Mm) and hermaphrodite individuals (M[SUP]h[/SUP]m) are heterozygous, whereas female individuals (mm) are homozygous recessive. The genotypes with homozygous dominant alleles, MM, M[SUP]h[/SUP]M[SUP]h[/SUP], and MM[SUP]h[/SUP], are lethal, resulting in a 2:1 segregation of hermaphrodite to female from self-pollinated hermaphroditic seeds and a 1:1 segregation of male to female or hermaphrodite to female from cross-pollinated female seeds. On the basis of co-segregation of long peduncles of flowers with males and the lethal factor with hermaphrodites and males, Storey (1953)⇓ proposed that sex determination in papaya is controlled by a group of closely linked genes that are confined to a small region on the sex chromosome within which recombination is suppressed. Influenced by the sex determination system in Drosophila, Hofmeyr (1939⇓, 1967⇓) suggested that the symbols M[SUB]1[/SUB] (M) and M[SUB]2[/SUB] (M[SUP]h[/SUP]) represent inactivated regions of slightly different lengths from which vital genes are missing. On the basis of interspecific hybridization in Caricaceae and the fact that homozygous dominant genotypes were not viable, Horovitz and Jiménez (1967)⇓ proposed that sex determination in papaya is the XX-XY type. A more recent modification of the model proposed to explain papaya sex expression is that the three alleles encode different trans-acting factors to activate or inhibit stamen and carpel development (Sondur et al., 1996⇓).
As a step toward cloning the sex determination gene in papaya, a high-density genetic mapping of the papaya genome was constructed using 1494 amplified fragment length polymorphism (AFLP) markers, five sequence-characterized amplified region (SCAR) markers, and two morphological markers. It is clear from this high-density map that recombination was suppressed in the region containing the sex determination locus as 225 markers co-segregated with sex (Ma et al., 2004⇓). The initial physical map of the non-recombining region resulted in an estimated size of 4–5 Mbp or 10% of the chromosome (Liu et al., 2004⇓). Our most current physical map consists of five contigs spanning a combined 6 Mbp length, suggesting that the non-recombining region is about 10–15% of the chromosome (Q. Yu, P. Moore, J, Jiang, A. Paterson, R. Ming, unpublished data). Considering that 225 (66%) of the 342 markers on linkage group 1 were in the non-recombining region, the polymorphism rate in this region was increased 14-fold compared with the markers on the remainder of the chromosome. The differences in recombination indicate a high sequence divergence between the X and Y homologs in this region. Selective sequencing of the BACs (bacterial artificial clones) identified by male-specific markers has revealed a decreased gene density and an increased transposable element density in the non-recombinant Y region. These findings led to the conclusion that a pair of incipient sex chromosomes has formed in papaya (Liu et al., 2004⇓). Two additional lines of evidence supported the existence of sex chromosomes in papaya: (1) Embryo development stops in 20–50 days after pollination on genotypes with two Y chromosomes (or homozygous dominant alleles as called previously) (Chiu et al., 2000⇓). (2) A pair of chromosomes separated precociously in 60–70% of pollen mother cells (Kumar et al., 1945⇓; Storey, 1953⇓), which is likely due to the lack of homology over the 10–15% of the male specific (non-recombining) region (Liu et al., 2004⇓).
Papaya is a major fruit crop in tropical and subtropical regions. The lethal effect of two Y chromosomes has precluded the development of true-breeding hermaphroditic cultivars. The lack of true-breeding hermaphrodites creates a problem of segregating sex types, which requires growing multiple seedlings per hill and then manually thinning out undesirable sex types or excess trees when the plants are old enough to flower. By that time, the plants have undergone considerable resource competition with their undesirable neighbors. Because of this situation and its economic significance, papaya has received considerable efforts to discover DNA markers to clone the sex determination genes that might be used to identify and eliminate undesirable sex types at the seed or early seedling stage. It is conceivable that similar incipient sex chromosomes exist in other angiosperm species that have not undergone intensive research.
A primitive Y chromosome, recently reported in stickleback fish (Gasterosteus aculeatus), is morphologically indistinguishable from the X chromosome (Peichel et al., 2004⇓). It is evident that the sex-determining genes in some species are not located on classical heteromorphic sex chromosomes but rather within non-recombining regions on apparently homomorphic sex chromosomes. These primitive Y chromosomes share common features with degenerated Y chromosomes in plants and animals: heterozygosity in males or females, suppression of recombination, accumulation of repetitive sequences, and substantial X–Y nucleotide divergence. These common properties reveal similar, if not the same, evolutionary processes affecting the emergence and progress of sex chromosomes across life kingdoms.

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[h=2]DEGENERATION OF THE Y CHROMOSOME[/h] The forces driving Y chromosome degeneration were proposed by evolutionary biologists and validated by molecular evidence obtained from model species. Once recombination has been suppressed in a chromosomal region, the proto-type Y chromosome will gradually accumulate deleterious mutations by a process known as Muller's ratchet (Muller, 1964⇓; Felsenstein, 1974⇓). Selection for favorable alleles may drag along some of these deleterious mutations, and thus “hitchhiking” may add to the degeneration of the suppressed region (Rice, 1987⇓). Other factors involved in Y chromosome degeneration may include background selection (Charlesworth et al., 1993⇓; Charlesworth, 1994⇓), which accelerates the fixation of mildly deleterious alleles and delays the fixation of mildly advantageous alleles, and the Hill–Robertson effect (McVean and Charlesworth, 2000⇓), which inhibits the spread of favorable alleles and the elimination of deleterious ones from the interference of closely linked alleles under selection. Accumulated deleterious mutations may cause the Y chromosome to degenerate in both size and gene content and to diverge from the X chromosome (Charlesworth and Charlesworth, 2000⇓), as a result of recombination suppression around the male-specific region of the Y chromosome (MSY).
The precocious separation of the primitive sex chromosomes in pollen mother cells (Storey, 1953⇓) and the lethal effect of the YY genotype are clear indications of Y chromosome degeneration in papaya. Despite its small MSY, a mosaic arrangement of X- and Y-like sequences was detected in this small region (Liu et al., 2004⇓). Evidence of Muller's ratchet is abundant and extremely low gene density was detected from the MSY sequence (Q. Yu, P. Moore, J, Jiang, A. Paterson, R. Ming, unpublished data). Most frequently, these deleterious mutations are caused by transposable element insertions because over-abundant retroelements were found in the papaya MSY as in the Drosophila and human Y chromosomes (Steinmann and Steinmann, 1992⇓; Skaletsky et al., 2003⇓; Liu et al., 2004⇓). Duplications, frequently detected by DNA markers and direct DNA sequencing in papaya MSY (Liu et al., 2004⇓; Q. Yu, P. Moore, J, Jiang, A. Paterson, R. Ming, unpublished data), could potentially play a role in protecting essential genes from degeneration, as is the case of the nine giant palindrome structures of the human MSY (Skaletsky et al., 2003⇓). Extensive sequence divergence between the primitive X and Y chromosomes has been detected (Liu et al., 2004⇓). Furthermore, the hermaphrodite Y[SUP]h[/SUP] and male Y in papaya share nearly identical DNA sequences in most parts of the MSY, yet sequence divergence did occur on these two Y chromosomes. These results indicate that the MSY in papaya hermaphrodites and males is derived from a common ancestral chromosome much more recently than the divergence of the X and Y chromosomes. This observation supports the hypothesis that males are modified hermaphrodites (or vice versa) in papaya (Storey, 1976⇓).
In sticklebacks, extensive sequence divergence has been found between the X and Y chromosomes with an average of only 64% identity based on comparison of 250-kb sequences. As in papaya, duplicated sequences are present, and repetitive DNA has accumulated in the evolving Y chromosome. Interestingly, the Y region is 87 kb longer than its corresponding region on X and had large gaps in the sequence alignment because of insertions on the Y chromosome. This result may indicate that the sex determination region on the stickleback Y chromosome has been slightly enlarged compared to X, even though there are no cytologically visible differences between these two sex chromosomes. In other words, the primitive stickleback Y chromosome may be on an evolutionary path toward becoming significantly larger, like the enormous Y chromosome of the white campion, most likely through transposable element insertions and intrachromosomal duplication.
The medaka male-specific region is about 260 kb and represents only 1% of the chromosome. This region has accumulated repetitive sequences, and some genes have degenerated (Nanda et al., 2002⇓). A male determination gene, MDY, in the medaka male-specific region has been characterized in detail. MDY is required and sufficient for male sex expression (Matsuda et al., 2002⇓). No homologue has been found on the X chromosome, but there is an autosomal homologue, dmrt1. The MDY gene seems to have originated by a recent duplication of the autosomal gene, dmrt1 (Nanda et al., 2002⇓), which is present only in close relatives of medaka (Kondo et al., 2004⇓).
Evidence for genetic degeneration of the Y chromosome has emerged with the accumulation of DNA sequence data of the classic flowering plant model for sex chromosome research, the white campion. The Y chromosome of white campion is largely non-recombining with the X chromosome. However, the Y chromosome looks euchromatic. Microdissected Y chromosome DNA of white campion competes with female genomic DNA to hybridize to the Y chromosome by fluorescent in situ hybridization (FISH) resulting in similar signal patterns on the X and Y chromosomes and autosomes (Matsunaga et al., 1999⇓). To date, only two lines of evidence suggest genetic degeneration of its Y chromosome. One is that the YY genotype is not viable (Ye et al., 1990⇓), and the other is that a functional X-linked male reproductive organ-specific gene 3 (MROS3) has a degenerated Y-linked copy (Guttman and Charlesworth, 1998⇓). Five additional sex-linked housekeeping genes with intact X- and Y-linked copies have been isolated in white campion, including Silene latifolia X-gene 1/Y-gene 1 (SlX1/SlY1) (Delechère et al., 1999), SlX3/SlY3 (Nicolas et al., 2005⇓), SlX4/SlY4 (Atanassov et al., 2001⇓), Differential Display 44 X- and Y-linked allele (DD44X/Y) (Moore et al., 2003⇓), and Silene latifolia spermidine synthase X- and Y-linked allele (SlssX/Y) (Filatov, 2005a⇓), all with functional Y-linked genes. Collectively, this suggests that the degeneration of genes on the white campion Y is at a very early stage. Gene sequences have somehow diverged from their respective X homologs. The synonymous (silent mutation) divergence between the gene copies on the X and Y chromosome is 1.7%, 16%, 8%, and 7% for SlX1/SlY1, SlX4/SlY4, DD44X/Y, and SlssX/Y, respectively.
A comparative genetic map for these X-linked genes was recently constructed (Filatov, 2005b⇓). The gene order corresponds to what would be expected from the evolutionary strata model proposed for human sex chromosomes in that the least diverged sequence is closest to the pseudo-autosomal region (PAR) (Lahn and Page, 1999⇓). The least diverged (SlX1/SlY1) and the most diverged (SlX4/SlY4) genes are at opposite ends of the map, while the other two genes (DD44X/Y and SlssX/Y) with intermediate divergence are in between. The SlX1/SlY1 gene is the closest to the PAR. According to Filatov (2005a)⇓, all amino acid replacements between SlssX and SlssY occurred in the Y-linked gene. Some of these mutations affect highly conserved amino acid residues and are likely to disrupt the function of the SlssY gene, even though it is actively transcribed. The SlssY gene has an elevated synonymous substitution rate, compared with SlssX, suggesting that the Y chromosome has a higher mutation rate than the X chromosome (Filatov, 2005a⇓).
The sequence of the human MSY provided new insights into our understanding of the structure and evolution of the Y chromosome (Skaletsky et al., 2003⇓). The human MSY is made up of three classes of sequences, X-transposed, X-degenerate, and ampliconic (that exist within multiple, repeated palindromic segments) sequences. In addition to the genes in the ampliconic regions, most other genes in the MSY are found in the X-degenerate regions, which were once identical to the X sequence but have now diverged extensively from it. These genes display 60–96% sequence identity to their X-linked homologues and seem to be remnants of their ancient autosomes. Half of the genes in X-degenerate regions are pseudogenes, with sequence similarity to functional X homologues, while hundreds of other X-homologous genes were lost in the process of Y chromosome degeneration. The Y chromosome has acquired male fertility genes and lost many other genes, whereas the X chromosome has maintained its ancestral genes. Sequence divergence between X and Y chromosomes shows a clear pattern in relation to their chromosomal positions. The sequences of genes closest to the PAR have the least synonymous site divergence, while loci further away have extensively diverged (Lahn and Page, 1999⇓; Skaletsky et al., 2003⇓).
These data demonstrate that degenerative processes have occurred in the non-recombining region of the primitive Y chromosome, even though sex chromosomes of papaya, white campion, and stickleback fish have originated recently. As evolution proceeds, the Y chromosome may be predicted to change as a result of transposable element insertions, duplications, inversions, and translocations. The ancient human Y chromosome shrank to about one-sixth the size of the corresponding X chromosome during its 300 million years of evolutionary history. The human Y chromosome is an excellent case to reveal what could happen during the degeneration process. Conversely, the primitive Y chromosomes in plants are ideal models to study the mechanisms underlying the initial stages of sex chromosome evolution.

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[h=2]STAGES OF SEX CHROMOSOME EVOLUTION[/h] Westergaard (1958)⇓ grouped plant sex chromosome into three types illustrating different evolutionary stages. In the first, the most primitive sex chromosome was characterized by the viability of the YY genotype, in which Y differs from X only in the sex determination genes; this condition is represented by Ecballium. The second type is that the YY genotype is inviable, while the Y chromosome plays a decisive role in sex determination; this condition is represented by papaya and white campion. The third type is that the Y chromosome is irrelevant to sex determination and sex is determined through the X–autosome balance; this condition is represented by sorrel (Rumex acetosa) and Japanese hop (Humulus japonicus).
Recent extensive genetic and genomic studies on the male-specific region in these species have led to refined models (Jablonka and Lamb, 1990⇓; Charlesworth, 1996⇓; Charlesworth and Charlesworth, 2000⇓; Charlesworth et al., 2005⇓). Based on the most recent data, sex chromosome evolution might be divided into five stages (Fig. 1). (1) A male sterile or a female sterile mutation occurs on a chromosome and recombination is suppressed at this locus and its immediate neighboring region leading to initiation of the degeneration process, but the YY genotype is viable. Asparagus is a good example for this stage. (2) Suppression of recombination spreads to additional linked loci that lead to the degeneration of a small chromosomal region and the formation of a male-specific region on the primitive Y chromosome. The loss of gene content is sufficiently extensive to cause lethality of the YY genotype even though the primitive sex chromosomes still appear to be homomorphic at the cytological level. The papaya sex chromosomes are at this early stage. (3) The accumulation of transposable elements and the duplication within the male-specific region cause the expansion of DNA content on the Y chromosome. The non-recombining region spreads to the majority of the Y chromosome and further degeneration occurs. At this stage, the X and Y chromosomes are heteromorphic, and the Y chromosome is physically larger than the X chromosome. The Silene sex chromosomes possess these properties. (4) Severe degeneration of the Y chromosome causes the loss of function for most genes and this enables deletions of the non-functioning Y chromosome sequences to result in shrinking the Y chromosome in size. There are no known plant sex chromosomes at this stage, but the sex chromosomes in mammals are good examples. It is also possible that some sex chromosome systems would not have this phase of shrinking in size but would continue to expand and degenerate until stage 5 when the Y chromosome got lost. In either case, a small portion of the sex chromosomes is still recombining to keep the X and Y chromosome pair together. (5) Suppression of recombination spreads to the entire Y chromosome. Further reduction in size of the Y chromosome and complete loss of the recombining pseudo-autosomal region occur. The Y chromosome is totally lost and sex determination is controlled by X to autosome ratio. A new Y chromosome might evolve, but it would have no effect on sex determination. Sorrel, Japanese hop, Drosophila, and nematode sex chromosomes are at this stage.
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Fig. 1. The five stages of sex chromosome evolution based on the size of the nonrecombining region, degree of degeneration, and size of Y chromosome. Stage 1: Suppression of recombination at the sex determination locus and its neighboring regions led to mild degeneration of the suppressed region. YY genotype is viable. Stage 2: Suppression of recombination continues to spread, and a small MSY region evolved. YY genotype is not viable. Stage 3: The MSY expands in size and degenerates in gene content by accumulation of transposable element insertions and intrachromosomal rearrangements. The X and Y chromosomes become heteromorphic. Stage 4: Severe degeneration of the Y chromosome causes loss of function for most genes. Deletion of nonfunctional DNA sequences results in shrinking of the Y chromosome in size. Stage 5: Suppression of recombination spreads to the entire Y chromosome. The Y chromosome is lost, and X-to-autosome ratio sex determination system has evolved


Sex chromosomes and maternal inheritance of organelle genomes are two evolutionary processes that are comparable between animals and plants. Plant sex chromosomes are mostly at early stages of evolution, but sorrel and Japanese hop appear to be exceptions. Although the sorrel and Japanese hop sex chromosomes evolved much more recently (angiosperm evolved about 158–179 MYA) than the 300 million years old mammal sex chromosomes, they appear to be at a later stage of sex chromosome evolution because the two Y chromosomes do not contain sex determination genes. This might imply that the rate of Y chromosome degeneration varies among species or that some species-specific chromosomal events could dramatically alter the course of sex chromosome evolution.

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[h=2]MODEL SPECIES FOR THE STUDY OF PLANT SEX CHROMOSOMES[/h] Sex chromosomes of plants were first described in white campion and hop (Blackburn, 1923⇓; Winge, 1923⇓), sorrel (Kihara and Ono, 1923⇓), and Elodea (Santos, 1923⇓). Heteromorphic sex chromosomes have been convincingly demonstrated in species of four families (Table 1) (Westergaard, 1958⇓; Parker, 1990⇓; Charlesworth and Guttman, 1999⇓; Matsunaga and Kawano, 2001⇓). Among the species with heteromorphic sex chromosomes, white campion has distinctive X and Y chromosomes, which are the largest and second largest chromosomes, respectively, in the male plants. Sorrel has one large X and a pair of different small Y chromosomes in male plants.
Among the species with homomorphic sex chromosomes, papaya has a clearly defined MSY region, while asparagus has an M locus with evidence of suppression of recombination (Reamon-Büttner and Jung, 2000⇓; Jamsari et al., 2004⇓; Liu et al., 2004⇓). A female heterozygous sex chromosome system might exist among Fragaria species in Rosaceae (Kihara, 1930⇓).
Sex determination of asparagus is under the control of an active-Y chromosome system. The genome size of diploid Asparagus officinalis is 1323 Mbp/1C for 10 chromosomes, 3.6× the papaya genome of 372 Mbp/1C for nine chromosomes (Arumuganathan and Earle, 1991⇓). The large genome of asparagus allows the separation of its 10 pairs of chromosomes by size and morphology. The chromosome pair 5 of Asparagus officinalis was identification as the sex chromosomes by trisomic analysis, although the X and Y chromosomes are homomorphic and contain only a small amount of constitutive heterochromatin (Loptien, 1979⇓). The Y chromosome contains two tightly linked genes, a male activator (M) and a female suppressor (F) supported by the appearance of rare hermaphrodite and sterile plants in the asparagus population (Marks, 1973⇓). The viability of the YY genotype indicates that the Y chromosome degeneration is mild and at an early stage of sex chromosome evolution.
Papaya is trioecious with three sex types: male, female, and hermaphrodite. Classical genetic and cytogenetic studies led to several hypotheses for papaya sex determination (Hofmeyr, 1938⇓, 1939⇓; Storey, 1953⇓; Horovitz and Jiménez, 1967⇓). Based on high density genetic mapping, physical mapping, and DNA sequence data, sex determination in papaya is controlled by a pair of homomorphic primitive sex chromosomes with a small MSY that account for 10–15% of the Y chromosome (Ma et al., 2004⇓; Liu et al., 2004⇓; Q. Yu, P. Moore, J, Jiang, A. Paterson, R. Ming, unpublished data). The available genomic resources and the small MSY region opened the door for sequencing the MSY and the corresponding region of the X chromosome and the eventual cloning of the sex determination genes.
In white campion, the Y chromosome is decisive in determining sex as shown by three observations: (1) application of hormones does not convert the sex, (2) the presence of a single Y chromosome can suppress female development when three X chromosomes are present, and (3) autosome ratios have no effect on the sex determining factors on the Y chromosome (Westergaard, 1958⇓). In other words, as is true in mammals, the sex of the individual is determined entirely by the presence or absence of the Y chromosome, which is called the active-Y system.
In sorrel, male plants have one X and two different Y chromosomes (2n = 15, XY[SUB]1[/SUB]Y[SUB]2[/SUB]), and females have two X chromosomes (2n = 14, XX). The two Y chromosomes are highly heterochromatic. Both Y chromosomes in sorrel are required for pollen fertility but not for stamen development. In contrast to white campion, Y chromosomes in sorrel do not suppress female gynoecium development and do not contain male determining genes, because plants with 2A + 2X + 1 or 2Y were female. Instead of an active-Y system, sorrel sex is determined by an X-to-autosome balance system (Ono, 1935⇓; Westergaard, 1958⇓). The Y chromosome deletion caused the merger of the pollen mother cells and sterile pollen. The centromeres of the two Y chromosomes are variable, which may involve the expression of the sequences on Y chromosome (Ainsworth et al., 1999⇓).

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[h=2]PROSPECTS[/h] To understand the origin and the process of sex chromosome evolution, it is necessary to study a series of organisms at various stages in the evolutionary process. Flowering plants provide such a series of incipient, early stage, and late stage sex chromosomes. Cloning of the sex determination genes from multiple species is a critical step toward elucidating the sex determination pathways and unraveling the mysteries of sex chromosome evolution in flowering plants. However, the Y chromosomes have evolved from the suppression of recombination at the sex determination locus, and map-based cloning is not an option. Sequencing the MSY region from Y chromosomes at different evolutionary stages would facilitate the cloning of the sex determination genes in addition to revealing the sequence features and evolutionary history of the Y chromosomes. Specifically, the complete sequence of the MSY provides the necessary genomic resources to locate the deleted region(s) of induced sex-reversal Y deletion lines generated via irradiation (Farbos et al., 1999⇓) and subsequently the candidate genes for sex determination. The small MSY region of papaya is being sequenced, and whole-genome shotgun sequencing of the female papaya genome is well underway. Knowing the genomic sequences of the sex chromosomes and/or the entire genome will remedy difficulties with drawing conclusions based on fragmented DNA markers and/or sequence data and will naturally expedite identifying the sex determining genes. The resulting genomic tools and resources would also foster investigations of interactions between genotype and environment in sex determination and sex chromosome evolution.

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[h=2]Footnotes[/h]
  • ↵1 The authors thank M. Moore for editing the manuscript. This work was supported by a grant from the National Science Foundation to R.M., Q.Y., P.H.M., J.J., and A.H.P. (DBI-0553417) and with startup funds from the University of Illinois at Urbana–Champaign to R.M.
  • ↵5  Author for correspondence (rming@life.uiuc.edu ), phone: (217) 333-1221

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[h=2]Sexual Reproduction[/h] Reproduction is a biological process by which living organisms produce more individuals of their own kind. There are two modes of plant reproduction: asexual reproduction and sexual reproduction. Sexual reproduction in plants consists of alternating, multicellular haploid and diploid generations. In angiosperms, the female gametophyte is the embryo sac and the male gametophyte is the pollen. The haploid egg and sperm fuse to form diploid zygotes, from which new sporophytes develop. In asexual reproduction, offspring are produced without meiosis or fusion of gametes and the plant multiplies through tubers, bulbs, corms and other vegetative parts. Sometimes a third mode of reproduction, apomixis, may be distinguished. Apomixis is the formation of new individuals from the sexual organs of a plant, without fertilization (Fryxell, 1957).
Knowledge of the mode of reproduction of a given species is essential for a plant breeder to accomplish crop improvement in that species. A plant breeder needs to know both how the plant reproduces naturally, and which possible methods of reproduction can be employed for artificial breeding (Fryxell, 1957). Knowledge of the natural mode of reproduction of a species helps the breeder to predict its behavior in field conditions, and knowledge of the possible methods helps to determine the potential manipulation available to accomplish crop improvement (Fryxell, 1957). For hybrid development in a naturally self-pollinated plant, the breeder needs to emasculate the female parent and artificially pollinate it with desired pollen to obtain a particular cross. For obtaining successful emasculation and artificial pollination, prior knowledge of floral biology, that includes time of anthesis and period of stigma receptivity of the species is required. The choice of selection method in breeding also depends on the natural mode of reproduction of a species. Mass selection, pure line selection, pedigree method, bulk population breeding and backcross breeding methods are all commonly used in self-pollinated crops whereas mass selection for intra-population improvement, and recurrent selection methods for inter-population improvement, are used in cross pollinated crops (Chahal and Gosal, 2002).
[h=3]Flower Morphology[/h] Flowers are the reproductive organs of a plant and the knowledge of various parts of a typical flower is necessary to understand plant sexual reproduction. A flower consists of different floral whorls, each with a different function. The outermost whorl is called calyx and consists of sepals. Sepals are usually green and they enclose and protect the developing bud. The whorl next to calyx is the corolla, which consists of petals, which usually help to attract the pollinators. In some species such as tulips, the sepals and petals look very much alike and act together to provide the color attracting pollinators. Together, the calyx and corolla make up the perianth. The whorl next to the corolla is androecium, which consists of male organs called stamens. Each stamen typically consists of a slender stalk or filament attached to the flower at its base and carrying on its free, upper end, a structure called an anther, which contains the pollen. Finally, the innermost whorl of the flower, the gynoecium, consists of the female organs called carpels. Each carpel consists of a basal ovary containing the ovules, a slender column-shaped structure, the style, and on the end of the style the stigma, the function of which is to receive the pollen grains. Additional whorls, such as the epicalyx, consisting of bracts, which occur outside the calyx, may be present in some flowers such as Cotton.


Fig.1. Longitudinal section of a typical flower. The four main floral whorls, calyx of sepals, corolla of petals, androecium of stamens, and gynoecium of pistils of a typical flower have been labeled in the figure.




The floral morphology discussed above is very typical, but in the real world this logical regular pattern of flower parts is not always so obvious. In atypical flowers, some parts may appear similar or some parts may be missing or some parts or groups of whorls may be coalesced. The most common instance of similarity of parts is resemblance between the sepals and the petals, which has already been mentioned as occurring in tulips. Similarly, brightly colored leaves and bracts surrounding the flower may also be confused with the petals (e.g. Bougainvillea). Many species have evolutionarily lost some parts of the flowers. The most obvious situation is that in which a plant or a species has different male and female flowers. In this case, flowers have lost one sexual function, allowing them to specialize in the other. Cohesion and fusion are common both within and among flowers. The petals may be fused to make a tube, as in a petunia flower. Flowers may combine to form what is called an inflorescence as in Brassicas.
[h=3]Hermaphrodity and Unisexuality[/h] In a flower, androecium and gynoecium are called the essential floral parts as they are directly involved in reproduction. All the other floral parts are known as the non-essential whorls as they contribute indirectly to reproduction, i.e. by protecting the developing bud or attracting pollinators etc. The flowers, in which one of the essential parts is missing, are called unisexual flowers. Unisexual flowers are subcategorized as pistillate/ female flowers, when only gynoecium is present or staminate/ male flowers when only androecium is present. Different families have different types of flowers, legumes have bisexual flowers with petals modified into banner petal, wing petals, and keel (Fig.1), cucurbits usually have unisexual flowers but may sometimes have bisexual flowers.Fig.2 shows a female watermelon flower.


Fig.2. Longitudinal section of a legume flower. Legumes have perfect flowers. A typical legume flower has a corolla modified into banner, wing petals and keel.





Fig.3. Longitudinal section of a female Watermelon flower. This is an example of unisexual flowers in cucurbits. Among the essential floral whorls, only gynoecium is present, so it is a pistillate/female flower.





Fig.4.1.Terminology. Pink flower = female flower; Blue flower = male flower; Bicolored flower = bisexual/hermaphrodite flower. The first two plants show the dioecious condition in which male and female flowers are borne on separate plants while the fourth plant exhibits monoecy, where male and female flowers are borne on the same plant. The third plant shows the bisexual/hermaphroditic condition with male and female parts in the same flower on the plant.




There exists a specific terminology for plants based on what type of flowers they bear and which type of flowers exist on each plant. The plant is called bisexual/hermaphrodite if it bears only bisexual/hermaphrodite flowers. The plants bearing unisexual flowers are further subcategorized as monoecious if both the male and female flowers occur on the same plant and dioecious if male and female flowers occur on different plants.
Another condition called subdioecy may sometimes occur. Under subdioecy, the plants are subcategorized as andromonoecious if both the male and hermaphrodite flowers; gynomonoecious if both female and hermaphrodite flowers; trimonoecious if female, male and hermaphrodite flowers are borne on the same plant.


Fig.4.2. Subdioecy. Pink flower = female flower; Blue flower = male flower; Bicolored flower = bisexual/hermaphrodite flower. The first plant is gynomonoecious i.e. it bears both the hermaphrodite and female flowers. The second plant is andromonoecious i.e. it bears both the male and hermaphrodite flowers and the third plant is trimonoecious i.e. it bears all three types of flowers, male, female and hermaphrodite.





[h=3]Self-pollination and Cross-pollination[/h] Sexually reproducing plants can be subcategorized based on the source of the pollen that pollinates the plant. Self-pollination occurs when the pollen from a flower pollinates the stigma of the same flower or another flower on the same plant. A species is said to be cross-pollinated if the pollen from a flower on one plant pollinates the stigma of a flower on another plant. Stebbins (1950) observed that there is a relationship between the length of lifecycle of a plant and its reproduction mode. Since annual plants have fewer opportunities for genetic recombination in their short life span, self-pollination is the key to reproductive assurance (Duvick, 1966). On the other hand, perennials mostly tend to outcross because they have more opportunities to genetically recombine in a life span spread over many years (Duvick, 1966). The terms self-pollinating and cross pollinating crops just mean that one method of pollination is more predominant than the other in that crop because some amount of outcrossing in self-pollinating crops and selfing in cross-pollinating crops commonly occurs.Table1 gives information about the common agricultural crops and their mode of pollination.







[h=4]Mechanisms Influencing the Mode of Pollination[/h] a. Morphological mechanisms
i. Mechanisms promoting self-pollination
Monoecy, the presence of male and female organs in the same flower or on the same plant, facilitates self-pollination (e.g. wheat). Cleistogamy, or flowers opening only after pollination has occurred, is also called bud pollination, as the pollination takes place when the flower is still unopened. In this case there is some chance of cross-pollination, as the flower finally opens. However, cleistogamy ensures self-pollination as the flower never opens (e.g. basal inflorescences of California oat grass). Sometimes the morphology of the flower is such that the pistil is enclosed in the staminal cone (e.g. tomato). In such flowers, as soon as the male and female organs reach sexual maturity self-pollination occurs.
ii. Mechanisms promoting cross pollination
In dioecious species, those with different male and female plants, the only possibility is cross pollination (e.g. papaya). Sometimes, in a perfect flower, stamens and pistils attain maturity at different times, such condition is called dichogamy. Dichogamy ensures cross pollination due to lack of synchronization of maturity in the reproductive parts of a flower. Protandry is the condition of a flower if male matures first (e.g. maize), and protogyny if female matures first (e.g. pearl millet).
b. Genetically controlled pollination systems
Male sterility is a condition that occurs when a plant produces non-functional pollen whereas self-incompatibility is a condition in which the plant produces functional pollen that cannot fertilize the female gamete of the same genotype. Self-pollination cannot occur in any of these systems, so the default mode of pollination is cross-pollination. The male sterility and self-incompatibility systems are explained in detail later in this chapter.
[h=4]Homozygosity and Heterozygosity [/h] The genetic structure of a plant species is largely determined by its reproductive system. In asexually reproducing species, offspring are genetically identical to their parents. Any variation in the asexual progeny is attributable to the environmental effects or a rare genetic mutation. Vegetatively reproducing plants are heterozygous and their heterozygosity is fixed through clonal propagation because no recombination occurs and all the progeny essentially arise from the same plant. In sexually reproducing species, two kinds of mating are possible: self-pollination and cross-pollination. There is no opportunity for gene recombination in self-pollination, except the occasional events of outcrossing. In self-pollinating species, variation is more common among populations than within populations. This trend has been reported in Leavenworthia of the Brassicaceae family (Charlesworth, 1998; Liu et al., 1999). This variation among populations in a self-pollinating species is greater than that observed in a cross-pollinating species. The genetic structure of a species further influences the adaptability of that species. The wider genetic base of the cross-pollinating species gives them better buffering capacity to survive various biotic and abiotic stresses as compared to the self-pollinating species. This idea is supported by the experiment conducted by Stevens (1948) when he estimated the crop losses due to disease in different reproduction systems. The results suggested that maximum disease loss occurs in asexually propagating species, followed by self-pollinating, and finally the cross-pollinating species. However, in cross pollinated crops, continuous (artificial) self-pollination has an adverse effect in the form of inbreeding depression. This occurs due to the accumulation of deleterious recessive alleles, which express in the homozygous state in the selfed plants of a cross pollinated species. The self-pollinated plants do not face this problem because due to continuous selfing over many generations, the deleterious recessive alleles get purged.
The mode of reproduction also influences the genetic structure of the population. Self-pollination increases homozygosity due to accumulation of similar alleles resulting from selfing over several generations, whereas cross-pollination increases heterozygosity due to frequent recombination and segregation. So the genetic structure of a self-pollinated population is heterogeneous with homozygous individuals, and that of a cross pollinated population is homogeneous with heterozygous individuals. The influence of selfing on heterozygisity is demonstrated in Fig. 5.1, 5.2 and 5.3.







Fig.5.1 & 5.2. Effect of self-pollination on heterozygosity. Fig.5.1 and fig. 5.2 are different ways of demonstrating the effect of self-pollination on heterozygosity. Aa is a single locus with two alleles in a F1 hybrid between two inbred lines with genotypes AA and aa. After the first generation of selfing, the proportion of homozygotes and heterozygotes in the population is same i.e. 50%. But on further selfing, the proportion of heterozygotes decreases while that of homozygotes increases.





Fig.5.3. Effect of self-pollinating on heterozygosity. This figure provides a graphical representation of the effect of self-pollination on heterozygosity. The starting population S0 is 100% heterozygous and after 1 generation of selfing, in S1 the level of heterozygosity falls to 50%. In subsequent generations of selfing, from S2 to S5 , the heterozygosity goes on decreasing and the homozygosity keeps on increasing. Thus, the general trend being: selfing increases homozygosity and decreases heterozygosity.




a. Inbreeding
Self-pollinated crops and cross-pollinated crops response differently to inbreeding. In general, inbreeding is the natural mode of breeding in self-pollinated crops and it produces desirable results by increasing homozygosity and uniformity of the plants. There is no adverse effect of inbreeding on the self-pollinated plants because due to continuous selfing over the generations, the population has been purged of the recessive deleterious alleles. However, enforced inbreeding in naturally cross-pollinating species may lead to drastic consequences. The adverse effects of inbreeding can be illustrated by the results of independent inbreeding experiments in corn, conducted by East (1908) and Shull (1909). Allard (1960) summarized the most important effects of continued inbreeding reported by these investigators as follows: It starts with the appearance of several lethal and subvital types in early generations of selfing, followed by separation of population into distinct lines, which become increasingly uniform within and increasingly distinct from other lines over the generations of selfing. Many of these lines show general decrease in their vigor and fertility and are difficult to maintain even in the most favorable cultural conditions. In the end, even the lines that survive exhibit decreased size and vigor.
b. Hybrid vigor/Heterosis
It is the phenomenon of increased vigor in the hybrids as compared to both of its parents. This phenomenon came to light in the 20th century in corn F1 hybrids. The resulting plant had a higher growth rate, was phenotypically superior and had increased yield as compared to the parents. Because one hybrid could not adapt to the whole country, different hybrids had to be developed in order to adapt to specific areas. The basic mechanisms proposed to be involved in the heterosis are dominance and overdominance. Dominance means that the dominant allele masks the effect of recessive allele. Overdominance means that the combination of genotypes from two different parents leads to supplementing the effect of each other; therefore, the effects lead to increased vigor (Chahal and Gosal, 2002). Most heterosis studies have been done on corn as heterosis has been exploited commercially in corn more than any other crop.
[h=3]Self Incompatibility[/h] Self-incompatibility (SI) in flowering plants is thought to be an evolutionary advantage due to its effectiveness in avoiding inbreeding and encouraging outcrossing. In recent years, many vegetable and fruit hybrid cultivars have been created by means of SI. One advantage is the possibility to produce F1 hybrids using two SI lines as parental components in order to eliminate laborious emasculation of the female parent. In modern plant breeding, F1 hybrids are one of the most important objectives of breeders. The first F1 hybrid breeding system was established in corn, as ‘hybrid corn’ in the USA in 1921. One problem of this system was high seed costs due to laborious emasculation of the male flower (tassel) (Franklin-Tong, 2008). Self-Incompatibility (SI) is the mechanism utilized by flowering plants (angiosperms) to prevent self-pollination (Silva and Goring, 2001). Therefore, breeders started to utilize SI to achieve a more efficient F1 hybrid breeding system. Because SI prevents self-fertilization and promotes outcrossing, F1 hybrid seeds can be produced readily from two parental SI lines with the help of pollinators.
[h=4]Mechanisms of Self-Incompatibility[/h] Flowering plants have evolved several unique mechanisms for SI. Some species of flowering plants produce unisexual flowers, which are either male/staminate or female/pistillate thereby acting as natural barrier to self-fertilization (McCubbin and Kao, 2000). However, the vast majority of flowering plants have perfect/hermaphrodite/bisexual flowers, containing both male and female reproductive organs within close proximity on the same flower (Kao and McCubbin, 1996).
SI is broadly categorized into heteromorphic SI and homomorphic SI. Heteromorphic self-incompatibility refers to SI due to some morphological barriers in flowers which occurs in some hermaphroditic flowering plants that produce structurally distinct reproductive organs, for instance: thrum flowers with long stamens and a short style or pin flowers with short stamens and a long style (e.g Primula, Oxalis). Relative positions of reproductive organs pose a topological barrier to self-fertilization (Ebert et al., 1989). Another form of SI is homomorphic SI, in which avoidance of self-fertilization depends on genetic mechanisms. Based on the type of genetic mechanisms involved, it is subcategorized into gametophytic SI (GSI) and sporophytic SI (SSI).
Gametophytically controlled SI is the most widespread SI system. SI is usually controlled by a single S locus that has multiple S-alleles (Franklin-Tong and Franklin, 2003). In some species (e.g. the grasses), there are two loci, S and Z (Franklin-Tong and Franklin, 2003). In the GSI system, the SI phenotype of the pollen is determined by its own haploid (gametophytic) genome. In the SSI system, the pollen SI phenotype is determined by the diploid genome of its parent (sporophyte) (Silva and Goring, 2001) (Fig.6).


Fig.6. Self-incompatibility systems. (A) Gametophytic self-incompatibility: The SI phenotype of the pollen is determined by its own haploid (gametophytic) genome. (B) Sporophytic self-incompatibility: The pollen SI phenotype is determined by the diploid genome of its parent (sporophyte) (Adapted from Silva and Goring, 2001)





[h=4]Self-Incompatibility as an Important Trait in Plant Breeding[/h] The advantage of SI in plant breeding is that heterozygosity is promoted by outcrossing. In modern plant breeding, F1-hybrids are of great economic importance in a number of crops on account of their uniformity and hybrid vigor (heterosis) (Franklin-Tong, 2008). One efficient method of producing F1-hybrid seed on a large scale is utilizing SI. The SI trait is essential to avoid contamination by self-pollinated seeds (Franklin-Tong, 2008). The main problem of utilizing SI is establishment of two pure line parents in SI plants. To resolve this problem, breeders choose the SI lines from a large number of seed stocks and use physiological or genetic breakdown of SI to produce pure lines because SI is not always stable (Franklin-Tong, 2008). SI can be easily overcome under various external and physiological conditions such as treatment with CO2 gas, irradiation and pistil grafting (Nettancourt, 2001).
In the SSI system, receptor protein S-receptor kinase (SRK) covers the stigmatic surface just prior to anthesis and acts as a barrier for penetration of the stigma by germinating pollen grains (Silva and Goring, 2001). Seed sets of pure line parents can be obtained if pollen is applied after buds opening and before the SRK protein barrier formed (Franklin-Tong, 2008). This procedure is called bud pollination. By using bud pollination, SI can be overcome. Thus, self-pollinated seeds can be produced and SI parental lines can be maintained (Franklin-Tong, 2008). However, efficacy of production of a large number of parental seeds of F1-hybrid by bud pollination needs to be enhanced (Franklin-Tong, 2008). Unfortunately, none of the major field crop plants has the SSI system. Brassica SSI is the most extensively studied SI system (Franklin-Tong, 2008). The well-known genetic and molecular mechanisms of Brassica SSI make Brassica an important subject in plant breeding. The first successful F1-hybrid variety of cabbage (cv. Suteki kanran) by employing the SI trait was produced in a Japanese seed company, Sakata Seed Co. in 1940 (Franklin-Tong, 2008). Later, in 1950, another Japanese seed company, Takii & Co. Ltd., released F1-hybrid varieties of cabbage (cv. Choko-1c) and Chinese cabbage (cv. Choko-1cc) (Franklin-Tong, 2008).
[h=4]Breakdown of Self-Incompatibility[/h] When hybridization involves species that have SI, this barrier to self-pollination must be overcome or lost in order to establish parental pure lines. Different types of modifications can lead to breakdown of SI. The physiological modifications are often temporary and cannot be transmitted from one generation to the next (Nettancourt, 2001). Genetic modifications may or may not be permanent and result in various effects (Nettancourt, 2001). Recently, hybrid self-fertility resulting from epigenetic changes in expression of the S-locus genes has been demonstrated (Nasrallah et al., 2007). Epigenetic mechanism for breakdown of SI in hybrids is reversible and noteworthy in facilitating hybrid in the future.
a. Physiological breakdown of self-incompatibility
There are various physiological factors and environmental circumstances that can prevent SI. Because SI phenotype of the pollen and pistil is fully determined in the mature flower, selfing can be induced by using immature material in which the S phenotype is not yet expressed (bud pollination) or by using old flowers and aged pollen, in which SI are getting weaker (Mable, 2008). Several studies also point out that heat treatment at a temperature ranging from 32°C to 60°C can lead to breakdown of SI in the pistil during the first two days following pollination in many plant genera (Lilium, Trifolium, Lycopersicum, Raphanus and Brassica) (Nettancourt, 2001). The mechanism of heat treatment is still not clear (Nettancourt, 2001). Another applicable method for breakdown of SI is treatment with CO2 gas. The most active concentrations of CO2 gas range between 3% and 5% (Franklin-Tong, 2008). The timing of the CO2 gas treatment is critical, which is usually right after pollen grains are germinated on the stigma papilla cells (Nettancourt, 2001). Although the mechanism of the CO2 gas effect has not been elucidated and genetic variations exist in the reaction to treatment of CO2 gas, CO2 gas treatment has replaced bud pollination with honey bees on large-scale propagation of the parental seeds of F1-hybrid in Brassica (Nettancourt, 2001). A special method of breakdown of SI is the “mentor effect”, which results from pollination with mixtures of compatible (mentor) and incompatible pollen (Nettancourt, 2001). The mentor effect can be enhanced by using inactivated or dead pollen (Nettancourt, 2001). The mentor effect has been applied successfully to a wide range of different plant genera, such as Citrus, Cola, Crocus, Lotus, Paspalum and Theobroma (Nettancourt, 2001). However, the understanding of the effects of mentor pollen is limited and mentor effects are not always reproducible (Nettancourt, 2001). Other methods of physiological breakdown include irradiation, hormones, pistil grafting (Nettancourt, 2001).
b. Genetic breakdown of self-incompatibility
Genetic breakdown of SI occurs in nature and can be induced by breeders artificially. Tetraploid relatives (or induced tetraploids) derived from GSI diploid progenitors usually display a self-compatible (SC) phenotype (Horandl, 2010). Breakdown of SI occurs in pollen, whereas the pistil maintains the function of identifying incompatible pollen and then rejects it. Self-fertilization results from a loss of the incompatible phenotype in the diploid pollen produced by the tetraploid plant (Nettancourt, 2001). For example, pollen from SC Petunia axillaris tetraploid (S1S1S2S2) can grow in both SI progenitor (S1S2) diploid and SC tetraploid (S1S1S2S2) pistils (Nettancourt, 2001). Remarkably, SI breakdown functions only in diploid heteroallelic pollen (S1S2), which is called competitive interaction (Nettancourt, 2001). The mechanism of a competitive interaction that does not occur between identical alleles but take place between different ones is not known (Nettancourt, 2001). On the other hand, modern biotechnologies including site-directed mutagenesis, the use of anti-sense DNA, exploitation of the silencing effects of certain duplications or the swapping of S-locus DNA sequences can be applied to make irreversible switches from SI to self-compatibility (Horandl, 2010).
[h=4]Applications of Self-Incompatibility in Plant Breeding[/h] The most important application of SI is in Brassica breeding. Vegetable Brassicas are an important and highly diversified group of crops grown world-wide. In western countries, cultivated Brassica crops include cabbage, broccoli, cauliflower and brussel sprouts (Kole, 2007a). In Asia, chinese cabbage is the most important Brassica crop (Kole, 2007). In recent years, most of the vegetables cultivated over a large part of the world have been F1 hybrid varieties. However, F1 hybrids of Brassica have progressed slowly owing to instability and complex inheritance of the SI (Kole, 2007a). Two major seed production methods employing the SI system are used in Brassica crops. One method is the single cross, in which SI is overcome in pure line parents by treating them with 4-5% CO2 gas (Franklin-Tong, 2008). The other method is a double cross, in which hybrid seeds are produced by two near isogenic lines for each parent (self incompatible, but cross compatible).
Cauliflower is one of the most attractive crops within the Brassica species. The advantages of F1 hybrids in cauliflower have been demonstrated in uniform maturity, early and high yield and biotic and abiotic stresses (Kucera, 2006). An interesting study compared two mechanisms used in commercial hybrid seed production of cauliflower, SI and cytoplasmic male sterility (CMS) (Kucera, 2006). In this study, hybrid seed production of SI lines derived from the cultivar Montano were achieved by spraying with 3% NaCl solution in the evening and using bumblebees as pollinators (Kucera, 2006). Hybridization experiment in two CMS line cultivars (Brilant and Fortuna) achieved seed set via honeybee pollination with their fertile analogues respectively (Kucera, 2006). The conclusion generated from this study was that both SI and CMS lines are suitable for hybrid breeding of cauliflower. In regards to seed production, SI appears to be more effective than CMS (Kucera, 2006).
[h=3]Male Sterility[/h] Male sterility occurs in plants where pollen or anthers fail to function properly. Male sterility was first described by Kölreuter in 1763 when he observed abortion of anthers in specific hybrids (Vinod, 2005). Male sterility can be caused by many factors, including mutations, diseases, or unfavorable environmental and growth conditions (Budar and Pelletier, 2001). The effects of male sterility can vary greatly depending on the species and environment. Some specific effects include absence of stamens in bisexual species, no male flower production in dioecious species, and failure to produce pollen-forming tissues in anthers (Vinod, 2005). Other effects include viable pollen produced in non-dehiscent anthers, deformed or non-viable pollen grains, and abnormal pollen maturation (Vinod, 2005).
Male sterility systems have been exploited in the plant breeding world to help breeders produce F1 hybrid cultivars. The first system using male sterility was developed in 1943 for onions, and soon after, systems were developed for corn, beet, sorghum, rice, sunflower, carrot, and rapeseed (Budar and Pelletier, 2001). Male sterility also plays an important role in keeping newly-introduced plant species from becoming invasive (Gardner et al., 2009). Many of the plant species categorized as invasive today started out as introduced ornamental plants, and male sterility can help prevent unwanted gene flow between invasive and native species. Ornamental crops can also benefit from male sterility by redirecting plant resources away from seed production, allowing for more vegetative and flower growth (Gardner et al., 2009).
[h=4]Types of Male Sterility[/h] Three types of male sterility exist: genetic male sterility (GMS), cytoplasmic male sterility (CMS), and cytoplasmic-genetic male sterility (CGMS). Genetic male sterility is controlled by a single recessive gene (ms) in the nucleus (Edwardson, 1970). Effects from this type of sterility include reduced pollen production, reduced anther size, and total pollen abortion (Poehlman and Sleper, 1995). GMS is quite common in nature, but it is not very useful to plant breeders because it cannot be used to maintain a pure line of male-sterile crops (Acquaah, 2007). Because male-sterile plants are homozygous recessive (msms), they must be crossed with a heterozygous (Msms) source of pollen, then the male-sterile offspring can be selected (Acquaah, 2007). Because only half of the seeds that result from crossing the male-sterile with the male-fertile plants can be used for future breeding (Poehlman and Sleper, 1995), genetic sterility is not the most optimal method for inducing male sterility in plant breeding. GMS has been observed in sunflower, tomato, cucurbits, wheat and barley.
CMS is a maternally-inherited sterility caused by expression of a mitochondrial gene which causes the production of non-viable pollen without affecting other functions of the plant (Budar and Pelletier, 2001). CMS plants still maintain normal female fertility and normal vegetative growth (Mihr et al., 2001). Two types of cytoplasm behavior can be seen, normal (N) and sterile (S) (Vinod, 2005). CMS is often induced by inter- or intra-specific crosses which combine different nuclear genes with different cytoplasm (Mihr et al., 2001). This sterility system is quite common in nature and has been observed in over 150 plant species (Schnable and Wise, 1998). Some plants naturally carry recessive male sterile genes in their cytoplasms which can be accidentally maintained in hybrid breeding lines, and these male sterile genes may eventually express as a result of a natural mutation (Schnable and Wise, 1998). Effects of CMS include abnormalities such as production of non-viable pollen, absence of stamens, and abnormal production of the cadastral (boundary) patterns in flowers (Pelletier and Budar, 2007). Anther and pollen development may be inhibited at different stages in plant development, either before or after meiosis (Mihr et al., 2001). CMS has been observed in maize, cotton, rice, and sorghum.
Cytoplasmic-genetic male sterility is influenced by both nucleic and mitochondrial genes and is very common in the plant world (Vinod, 2005). There are three different scenarios for CGMS system (Fig. 7), including the crossing of plants with normal/sterile cytoplasm with the plants carrying different fertility restorer genotypes i.e. RfRf, Rfrf, and rfrf. In the first case, sterile cytoplasm is paired with Rfrf, the outcome of this system will always be restoration of male fertility. In the second case, sterile cytoplasm is paired with restorer genes rfrf or Rfrf. Even though sterile cytoplasm is included, the presence of the heterozygous restorer gene mandates the outcome of this system to be male-fertile while plants with the homozygous recessive nuclear genotype will be sterile. In the third case, sterile cytoplasm is partnered with the homozygous recessive restorer genotype, rfrf. Fertility is not restored to the breeding line in this system because of the presence of both sterile cytoplasm and the homozygous recessive restorer genotype (Edwardson, 1970). CGMS has been used in commercial hybrid seed production of sorghum, maize and pearl millet.


Fig. 7 Cytoplasmic-genetic male sterility system: In case (A) sterile cytoplasm is paired with restorer genes Rfrf and even though sterile cytoplasm is included, the outcome of this system will be male-fertile if the restorer genotype is heterozygous dominant Rfrf and male-sterile if the restorer genotype is homozygous recessive rfrf. In case (B) sterile cytoplasm paired with rfrf restorer genes will yield male sterile plants, while sterile cytoplasm paired with Rfrf will yield male fertile plants. In case (C), sterile cytoplasm is partnered with the homozygous recessive restorer genotype, rfrf, yielding sterile plants (Adapted from Allard ,1960).





[h=4]Mechanism of Fertility Restoration in CGMS hybrid lines[/h] Plant breeders use CMS systems to produce hybrid seeds by developing female lines carrying CMS cytoplasm and by breeding male lines that carry the restorer genes (Schnable and Wise, 1998). Crossing these lines yields fertile progeny because the male maintainer lines contribute nuclear restoring genes (Schnable and Wise, 1998). The first crops that used the CMS fertility-restoring gene system as a method for commercially producing hybrid seed were sorghum and corn (Poehlman and Sleper, 1995).
The main CMS model used for hybrid lines utilizes a system with three lines. The A-line consists of lines that are seed-bearing and are used as the female parent line. A-lines contain the homozygous recessive non-restorer genes (rf1, rf2). They are then backcrossed with inbred lines which do not contain restorer genes and are a source of CMS. Five to seven backcrosses are usually necessary until the wanted genotype from the A-line is recovered in the male-sterile cytoplasm. A male-fertile line, the B-line, is used to pollinate the A-line and maintain its male-sterility. The B-line has the same genotype as the A-line and the same non-restorer genes (rf1, rf2), but with normal cytoplasm (Poehlman and Sleper, 1995).
A third line, called the R-line, is used as the fertile pollen-giving parent in a hybrid cross. R-lines are also used to restore fertility to progeny of the hybrid cross (Poehlman and Sleper, 1995). R-lines are chosen based on their ability to produce large amounts of pollen and anthers that are suitable for proper pollen dissemination .The R-lines are able to restore fertility to the hybrid progeny because they contain the nuclear restorer genes Rf1, Rf2. They may have either normal or sterile cytoplasm, but it is advantageous for breeders to use R-lines with sterile cytoplasm because presence of restorer genes can be more easily conferred (Poehlman and Sleper, 1995). R-lines are also expected to cross with A-lines to produce very vigorous hybrid progeny (Poehlman and Sleper, 1995). This superior combining ability is an important aspect of R-lines that plant breeders look for when trying to obtain higher yielding crops (Poehlman and Sleper, 1995). One study showed that R-lines with higher ability to restore fertility can produce hybrid cotton plants with higher heterosis levels (Zhang et al., 2010).
The usable hybrid seed is produced from the A-line after it has been crossed with the R-line. In commercial production, where large amounts of seeds need to be produced, R-lines are planted in alternating rows between A-lines. Pollen from the R-line is blown by wind and naturally pollinates the A-line plants. This method is a much easier and more efficient tool for pollinating hybrid lines than some other, more labor-intensive methods, such as hand emasculation and hand pollination (Poehlman and Sleper, 1995).
A genetic-cytoplasmic male sterility model was discovered in the 1940s in corn (Zea mays) and utilized until the 1970s (Vinod, 2005). This system, called Texas cytoplasmic male sterility (CMS-T), was used in 85% of all US corn in the 1970s (Vinod, 2005). During the peak of CMS-T use, a new strain of the southern corn leaf blight pathogen, Bipolaris maydis, developed and wiped out all of the CMS-T corn in the Eastern US (Vinod, 2005). The CMS-T corn carried a specific gene, T-urf13, which caused chimeric sequences to be expressed in mitochondria, thus causing male sterility (Budar and Pelletier, 2001). However, this gene also caused the corn crop to be highly susceptible to two fungal pathogens, Bipolaris maydis and Phylostica maydis (Budar and Pelletier, 2001). The T-urf13 gene was highly susceptible to a specific toxin (T-toxin) produced by the T-strain of Bipolaris maydis (Vinod, 2005).
Cytoplasmic male sterility is an important area of plant breeding and crop research. The ability to control the fertility of hybrid progeny has many advantages to farmers and breeders. Male-sterile plants make the breeder’s job easier by eliminating the risk of unwanted cross-pollination and gene flow. Eliminating risk of gene flow also greatly assists farmers. They expect all hybrid crops to turn out similar in size and quality because that is what the consumer market demands. Male sterility also allows F1 hybrid seed production companies to obtain more consistent profit because farmers cannot save seed. The current research and future outlook of engineered male-sterile crops looks promising. More research needs to be done on finding applicable systems within each specific crop, but the end benefits should be worth the effort.
[h=3]Interspecific Hybridization[/h] Interspecific hybridization is also called wide hybridization as it involves crossing distantly related or sometimes unrelated species. Many problems are encountered in intercrossing these species. Sometimes the plants can be easily crossed but the hybrid formed is sterile. In other cases, the hybrid resulting from such crosses get aborted at embryonic stages. In the worst situations, there are some morphological reproductive barriers which do not even allow the crossing and fertilization to occur easily. Various strategies such as bridge crossing, somatic hybridization, induced polyploidy, embryo rescue and use of plant growth regulators can be employed to overcome these problems and accomplish successful interspecific hybridization in widely related species. When two species cannot be intercrossed to produce a fertile hybrid, a third species which can be easily crossed with both the parental types can be used to produce a bridge cross. R.C Buckner and his colleagues have used this method to cross Italian ryegrass (Lolium multiflorum) with tall fescue (Festuca arundinacea) using meadow fescue (Fescue pratensis) as the bridging species (Aquaah,2007).
Somatic hybridization is a technique that can be used to bypass the mating barriers between widely related species. The individual somatic cell protoplasts from both the species are fused using polyethylene glycol (PEG) or electric fusion and a somatic cell hybrid is formed. This somatic hybrid is later regenerated into a full hybrid plant. Solanum brevidens and S. phureja have been used in somatic hybridization technique to introduce disease resistance for Potato leaf roll virus and Potato virus X into the S. tuberosum species. The nuclear genome can be rendered inactive by treating with Iodoacetate to get the desired hybrids (Chahal and Gosal, 2002). Embryo rescue can be employed in situations when there are risks of embyo abortion or loss of viability of the hybrid seeds. In research regarding the production of hybrids between Cicer arietinum and C. bijugum (wild variety), it was demonstrated that the hybrid produced from the cross did not yield viable seeds. However, if the embryos are cultured on the suitable media they can be regenerated into plantlets and then transferred to soil (Clarke et al., 2006). Plant growth regulators such as auxins and gibberlins can be used to save the weak hybrid plants from the crosses that do not survive under natural conditions. Induced polyploidy is helpful to partially or completely overcome the sterility in wide intercross hybrids (Fehr et al., 1980).
[h=2]Asexual Reproduction[/h] Asexual reproduction in plants is of two types: Vegetative propagation and apomixis. Some plants naturally reproduce vegetatively through tubers (e.g. potato), rhizomes (e.g. ginger), bulbs (e.g. onion) etc. Others are artificially propagated vegetatively using various methods like grafting, layering, budding etc.. Vegetative reproduction is extremely common in perennial plants, especially in grasses and aquatic plants. However, there are very few species that rely solely on vegetative reproduction. Mostly the species that reproduce vegetatively also reproduce sexually through seed (e.g. Trifolium repens)(Burdon,1980).
Apomixis is the process by which plants reproduce asexually through seed (Nogler, 1984). Nogler (1984) divided apomixis into three main groups according to the origin and development of the maternal embryos: apospory, diplospory and adventitious embryony. In aposporous species, embryo sacs are formed from the nucellar cells while in diplospory, the megaspore develops from the reproductive tissue but meiosis fails partially. In adventitious embryony, embryo develops directly from a somatic cell of the megagametophyte. Apomixis may be facultative or obligate. It is said to be facultative when some progeny from a plant may result from a normal meiosis and/or normal fertilization in addition to the apomictic progeny. Apomixis is obligate when all the progeny are maternal and there is no chance of developing a progeny from sexual reproduction in that plant. A major potential application is “hybrid crops that clone themselves” (Carman et al., 1985). Because the hybrids formed by wide hybridization may be sterile, they can only be asexually propagated and apomixis will facilitate their asexual propagation through seed. Other advantages of apomixis include uniformity of plants and virus free propagation because viruses are usually not transmitted through seeds. However, apomictic breeding has not realized its potential because there are very few economically important apomictic crops. Apomixis is less widespread than vegetative reproduction, although it has been reported from at least 30 families of flowering plants (Grant, 1981), and it is especially common in grasses.  
[h=2]References[/h] Acquaah, G. 2007. Principles of plant genetics and breeding Blackwell Pub., Malden, MA ; Oxford.
Allard, R.W. 1960. Principles of Plant Breeding Wiley, New York.
Budar, F., and G. Pelletier. 2001. Male sterility in plants: occurrence, determinism, significance and use. Comptes Rendus de l'Académie des Sciences - Series III - Sciences de la Vie 324:543-550.
Burdon J., and J. Harper.1980. Relative growth rates of individual members of a plant population. The Journal of Ecology:953-957.
Carman J.G., C.F. Crane , J.E. Hughes.1985. Hybrid crops that clone themselves. Utah Sci 46:90-94.
Chahal, G.S., and S.S. Gosal. 2002. Principles and Procedures of Plant Breeding, Alpha Science International Ltd., Pangbourne.
Clarke H.J., J.G.Wilson, I.Kuo , M.M. Lulsdorf, N., Mallikarjuna , J. Kuo, K.H.M Siddique. .2006. Embryo rescue and plant regeneration in vitro of selfed chickpea .Cicer arietinum L.. and its wild annual relatives. Plant Cell Tissue and Organ Culture 85:197-204.
Cramer C.S., T.C. Wehner.1999. Little heterosis for yield and yield components in hybrids of six cucumber inbreds. Euphytica 110:99-108.
Duvick, D.N. 1966. Influence of morphology and sterility on breeding methodology., Plant Breeding Univ. Press, Ames, Iowa. 85-139.
East E. .1908. Inbreeding in corn. Rep. Conn. Agric. Exp. Stn 1907:419-428.
Ebert P.R., M.A Anderson, R. Bernatzky ,M .Altschuler, A.E Clarke. .1989. Genetic polymorphism of self-incompatibility in flowering plants. Cell 56:255-62.
Edwardson, J.R. 1970. Cytoplasmic male sterility. Bot. Rev. 36:341-420.
Fehr W.R., and H.H.Hadley ,American Society of Agronomy., Crop Science Society of America. 1980. Hybridization of crop plants American Society of Agronomy : Crop Science Society of America, Madison, Wis.
Franklin-Tong N.V., F.C. Franklin.2003. Gametophytic self-incompatibility inhibits pollen tube growth using different mechanisms. Trends Plant Sci 8:598-605.
Franklin-Tong V.E.E. .2008. Self incompatibility in flowering plants evolution diversity and mechanisms Spinger-Verlag, Berlin/Heidelberg.
Fryxell, P.A. 1957. Mode of Reproduction of Higher Plants. Botanical Review 23:135-233.
Gardner, N., R .Felsheim, and A.G.Smith. 2009. Production of male- and female-sterile plants through reproductive tissue ablation. J. Plant Physiol. 166:871-881.
Grant V. .1981. Plant speciation New York: Columbia University Press xii, 563p.-illus., maps, chrom. nos.. En 2nd edition. Maps, Chromosome numbers. General .KR, 198300748..
Gu Y.H., W.H. Ko. .2001. Creation of hybrid vigor through nuclear transplantation in Phytophthora. Canadian Journal of Microbiology 47:662-666.
Holsinger, K.E. 2000. Reproductive systems and evolution in vascular plants. Proceedings of the National Academy of Sciences of the United States of America 97:7037-42.
Horandl E. .2010. The evolution of self-fertility in apomictic plants. Sex Plant Reprod 23:73-86.
Kao T.H., A.G. McCubbin.1996. How flowering plants discriminate between self and non-self-pollen to prevent inbreeding. Proc Natl Acad Sci U S A 93:12059-65.
Kole C. .2007. Genome Mapping and Molecular Breeding in Plants - Vegetables. 1st ed. Springer, New York.
Kucera V.C., V. , M.Vyvadilova, M. Klima.2006. Hybrid breeding of cauliflower using self-incompatibility and cytoplasmic male sterilit. Horticultural Science - UZPI 33:4.
Liu, F., D. Charlesworth, and M. Kreitman. 1999. The Effect of Mating System Differences on Nucleotide Diversity at the Phosphoglucose Isomerase Locus in the Plant Genus Leavenworthia. Genetics 151:343-357.
Mable B.K. .2008. Genetic causes and consequences of the breakdown of self-incompatibility: case studies in the Brassicaceae. Genet Res 90:47-60.
McCubbin A.G., and T. Kao. .2000. Molecular recognition and response in pollen and pistil interactions. Annu Rev Cell Dev Biol 16:333-64.
Mihr C., M. Baumgärtner, J.H. Dieterich, U.K. Schmitz, and H.P. Braun. 2001. Proteomic approach for investigation of cytoplasmic male sterility .CMS. in Brassica. J. Plant Physiol. 158:787-794.
Nasrallah J.B., Liu P., S. Sherman-Broyles ,R.Schmidt, and M.E. Nasrallah. .2007. Epigenetic mechanisms for breakdown of self-incompatibility in interspecific hybrids. Genetics 175:1965-73.
Nettancourt D.D. .2001. Incompatibility and Incongruity in Wild and Cultivated Plants. 2nd ed. Springer, Berlin/Heidelberg/New York.
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Orians C.M. .2000. The effects of hybridization in plants on secondary chemistry: Implications for the ecology and evolution of plant-herbivore interactions. American Journal of Botany 87:1749-1756.
Ortega E., and F. Dicenta.2003. Inheritance of self-compatibility in almond: breeding strategies to assure self-compatibility in the progeny. Theor Appl Genet 106:904-11.
Pelletier, G., and F. Budar. 2007. The molecular biology of cytoplasmically inherited male sterility and prospects for its engineering. Curr. Opin. Biotechnol. 18:121-125.
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Schatz B., A.Geoffroy, B. Dainat , J.M. Bessiere, B. Buatois, M. Hossaert-McKey, M.A. Selosse.2010. A Case Study of Modified Interactions with Symbionts in a Hybrid Mediterranean Orchid. American Journal of Botany 97:1278-1288.
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Ha great thread. If I remember, Weedman had the quotes from the DJ Short book ready. Mine is hard copy and kindle but my iPad isn't handy.

Paraphrased, there are true female (won't self-pollinate no mater what Soma does, haha), female with male traits (stress nanners), hermie (full on calyx with pistils as well as full ballsacks developing during flower from the same flower nodes), males with female traits (an occasional pistil, or trichomes for example), and true males. He suggests that true males and true females are possibly nonexistent in tropical sativas.
 
I'll post the DJ Short direct quote if i'm still here later and Weedman doesn't beat me to it ;)

Love ya, GoAuto
 
Good show--GoAuto.

Great resource.

This grow I attempted to trim only during veg after third week and a week before 12/12.

However--lack of light penetration and a torrent of undercarriage immature limps/buds--compelled me to hack away.

Two Blueberry crosses (freebies from questionable source) from same seed pack--hermied at week four. This seems to be a critical week before most strains start heavy flowering--including autos at week four from seedling.

They are well structured/topped plants and already have a pleasant blueberry aroma. With such low bud density and a big stretch decided to pluck and watch. The male flowers did not look to be in great health. Have heard of some being sterile.

Been checking daily. The tent is simply too full with so many strains. Several are hermie prone--one has 50% Thai genetics and have other berries. So, grow will be interesting.

Most of the hermies I grow are sneaky. For I have often not seen any evidence early on. Most of the seeded bud appears to, generally, be late pollination. Little specks of seeds in back of buds. Only had one in second grow--go full hermie that was years ago--relative to this site. Even then I thought--wow--what an aromatic hashy aroma. Started harvesting and chalk full of seeds. Environmental stress--hot closet, little air circulation and a haphazard grower--:grin:.

Now, I have had several photos pop bananas when ripe near harvest. This is not a problem, unless, one has immature females.

When we grow different strains or add a plant during the grow we have to watch for the late nanners. Suspect when we get immature seeds it is from an early blooming female. May have a photo somewhere.

Below is a photo of limb cut a few days ago from the Berry cross photo. It is a younger branch. The few male flowers found were located on nodes next to and under existing flowers. I consider this kind of deal to be like a partial hermie. I have never had a full blown top to bottom hermie--which showed its true colors--that is excepting the one described above. It did not reveal any visible male flowers, of course, I was a rookie.

P1010294.jpg

Still most of us do find a mature seed or several from time to time. About to grind and there is a seed--what!

Cannabis is designed to keep on truckin--one way or another.

Cheers
 
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breaking the myth that hermies breed fem seeds "always"

a local grower in my area of the world bred seeds for himself and only himself, many years went by and he worked with only one single strain, one he created himself, and which i have no idea what strains were involved in the making

he named the strain brain warp or mind warp, i never knew exactly which name was right because the dealer i got the bud from would use both names and when asked, even he didnt know for sure which was right

so back on topic, hermies and the seeds they produce.

when growing and breeding indoors this person was careful and watched for hermies, the weed we got at that time never had any seeds in it, when he moved to a farm or farm land area and started growing huge crops outdoors it was very difficult for him to inspect the vast number of plants and hermies were slipping past him

each year his weed became more and more full of seed

finally a day came when the dealer brought me some of that growers seeds, a few weeks later he asked for them back because the grower mentioned (late) he didnt want the dealer to share those seeds (they were meant for him only)

well it was too late, i had planted them the day i got them and already had 2-3 week old plants, i refused to kill them, they were hermies unfortunately, and i continued to grow out the seeds i got each crop, seeds that were produced due to hermie pollen, i grew these seeds going on almost 3 years, and let me be very clear here, they produced males and females, most of which were also hermie prone, the few that didnt show any hermie traits were cloned and used in breeding projects, the hermie trait even though not showing in the plants used, did in fact pass on the hermie trait within the new crosses

some accidental crosses were done due to the hermie strain pollinating other strains in the room, and some of those did produce 100% females, but some did not

early fems were made by some so called breeders who used late nanner pollen, the rodelization method, my spelling might be wrong, any one who has grown long enough has or has heard of those early fems producing males, that is something we do not hear of from modern properly made fems

so PLEASE do not continue spreading the myth that hermies breed fems "always" because that simply is not true, i see members on this site and even a staff member recently claim to get feminized seeds from hermies "always" and yes the staff member did use the word always, or a similar word with a definite meaning

the chance for feminized seed is great, but it is not guaranteed

edit; please dont any one mention dj shorts idea that breeding with a hermie male is a good thing, that claim has been proven false by many people, some professional breeders, some hobby breeders, but the claim has been proven as completely false many MANY times, most long time growers and breeders figure dj was simply very stoned at the time and it sounded good in his head so he shared the idea as fact, when he did not actually do the work needed to prove it either way

also, a lot of people figure dj short as well as others like nevil were just at the right place at the right time and got lucky to be handed quality genetics to work with, remember they didnt have a lot of competition back in the day, many people, including a lot of people on this site could have been dj short back then, the plants he was given to work with were special, but they were also common, what wasnt common was the number of actual breeders working with those strains back then

ask dj short why he cant reproduce blue berry from scratch today, if he was as good as he and his worshipers claim he is then he would be able to do it, i mean he did it originally right? he should know the recipe right? which brings me back to the fact that he was simply at the right place at the right time, because he has been unable to recreate blue berry, hence all his new "blue" strains
 
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There are some articles online discussing the career of DJ as an early breeder and how he discovered the Berry phenotypes.

If anyone finds a good one--please link it or them for us.

Cheers
 
Really informative thread I will come back to page 2 when able!:smoking: Thanks GA!
 
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