- Top of page
- Materials and Methods
The study of apomixis (asexual reproduction through seeds) is of great interest for both practical and theoretical reasons. The harnessing of apomixis for agricultural purposes could be of great economic and humanitarian benefit, because it would enable the propagation of hybrid genotypes indefinitely (Spillane et al., 2004). For evolutionary biologists, apomictic systems allow for testing of hypotheses of the persistence of sexual reproduction despite its twofold cost (Charlesworth, 1990; West et al., 1999). Although biologists have long grappled with apomictic mechanisms, their control is still relatively poorly understood at the genetic and molecular levels (Bicknell & Koltunow, 2004). It has been hypothesized that apomixis is directly derived from normal sexual pathways (Nogler, 1984a; Holsinger, 2000; Koltunow & Grossniklaus, 2003). This bypassing of normal sexuality may have resulted from gene-expression changes during polyploidization and/or hybridization, as most apomictic species are allopolyploids (Carman, 1997). A number of model species have been well developed for the study of apomixis, each with their own advantages and disadvantages (Bicknell & Koltunow, 2004). Genetic studies with these systems suggest that the trait is under the control of either two or several genes (van Dijk et al., 1999; Noyes & Rieseberg, 2000; Albertini et al., 2001; Matzk et al., 2005), or by a single dominant locus (Leblanc et al., 1995; Bicknell et al., 2000), which can be located on supernumerary or hemizygous chromatin (Ozias-Akins et al., 1998; Roche et al., 2001; Sharbel et al., 2004).
In comparison with other apomictic complexes, apomictic reproduction in Boechera has several factors making it more akin to normal sexual reproduction. These factors combine to make Boechera a compelling and tractable system for genetic studies of the control of apomixis. Perhaps the most promising and rare characteristic of Boechera is that apomixis can occur at the diploid level (Böcher, 1951, 1969; Sharbel & Mitchell-Olds, 2001). Evidence of diploid apomictic Boechera lines has been gathered through cytological investigations (Böcher, 1951; Naumova et al., 2001); the occurrence of unreduced gametes and heterozygous genotypes (Dobešet al., 2004b; Sharbel et al., 2004, 2005); and the persistence of heterozygosity in progeny (Roy, 1995; Schranz et al., 2005). Additionally, these diploid lines often contain heterochromatic (Het) and/or supernumerary chromosomes that could be responsible for the apomictic phenotype (Böcher, 1954; Sharbel et al., 2004; Kantama, 2005; Sharbel et al., 2005). Another major advantage is the close relationship of Boechera to the model plant Arabidopsis thaliana (Koch et al., 2001). Boechera is the only documented case of natural apomixis in the Brassica family (Brassicaceae). In addition to A. thaliana having its complete genome sequenced, there is also an unrivalled understanding of normal sexual reproduction and a variety of apomixis-like mutations that have been identified (Koltunow & Grossniklaus, 2003), which should provide an excellent framework for comparison.
Several aspects of the Boechera breeding system simplify genetic and molecular investigations, compared with other apomictic lineages. First, apomictic Boechera accessions have probably evolved from self-compatible and highly self-fertilizing sexual types (Roy, 1995), whereas most other apomictic groups are derived from self-incompatible and out-crossing taxa (Asker & Jerling, 1992). Second, as in normal sexual taxa, the embryo is derived from the megaspore mother cell (MMC). The MMC generally enters meiosis I, but fails to complete the reductional phase (apomeiosis), and then undergoes normal meiosis II to form a nonreduced restitution nucleus (Taraxacum-type diplospory) (Böcher, 1951; Naumova et al., 2001; Taskin et al., 2004). In other forms of apomixis, the embryo either develops from the nucellus (apospory) or else forgoes meiosis (Antennaria-type diplospory) (Crane, 2001). Third, Boechera apomicts are pseudogamous, meaning that fertilization of the central cell is still required for normal endosperm development (Böcher, 1951; Naumova et al., 2001; Taskin et al., 2004). Pseudogamy is usually only found in conjunction with apospory (Richards, 1986; Asker & Jerling, 1992), and typically occurs in lineages that descend from outbreeding taxa (Mogie, 1992). Finally, apomixis in Boechera is incomplete (facultative apomixis), with sexual reproduction still occurring at an often high frequency (Böcher, 1951; Schranz et al., 2005). Both the formation of viable pollen and facultative apomixis allow for potential hybridization with sexual Boechera lineages.
The ability to cross sexual and asexual lineages of Boechera is critical for the establishment of a genetically tractable research system. Many of the major advances in our understanding of the genetic control of apomixis have come from analysis of the progeny from such crosses (Ozias-Akins et al., 1998; van Dijk et al., 1999; Noyes & Rieseberg, 2000). However, there are several important caveats for analysis of the inheritance of apomixis from such studies, including the problems of making interploidy and/or interspecific crosses (discussed by Bicknell & Koltunow, 2004; Matzk et al., 2005). In Boechera we can partially mitigate these complications. The presence of diploid apomicts means that we can avoid making interploidy crosses (Schranz et al., 2005). Also, a robust phylogenetic hypothesis exists for the relationship of sexual and apomictic lineages, with direct evidence of past hybridization events (Sharbel & Mitchell-Olds, 2001; Koch et al., 2003; Dobešet al., 2004a, 2004b; Schranz et al., 2005). Specifically, the sexual species Boechera stricta is almost exclusively diploid, sexual, and forms a well supported monophyletic clade (lineage II of Dobešet al., 2004b; Schranz et al., 2005). Boechera holboellii is diploid or triploid, often apomictic (Sharbel et al., 2004, 2005), paraphyletic (I. A. Al-Shehbaz, pers. comm.), and is scattered between two other lineages with other species of Boechera (lineages I and III of Dobešet al., 2004b; Schranz et al., 2005). The interspecific hybrid species Boechera divaricarpa has repeated independent origins by hybridization of B. stricta and B. holboellii-like plants (Koch et al., 2003; Dobešet al., 2004a, 2004b). These B. divaricarpa lineages are diploid or triploid, and are often apomictic (Schranz et al., 2005). Thus apomictic lineages of B. divaricarpa are known to have a mixed dosage of B. stricta and B. holboellii-like chromosomes. By making crosses between diploid B. stricta and B. divaricarpa, we are mirroring the evolutionary steps that have probably happened within the group.
We reported previously on reciprocal crosses between a sexual B. stricta line (SAD12 = ES6) and a number of diploid apomictic B. divaricarpa lines, including individuals from the Vipond Park site (VP9 = ES9) (Schranz et al., 2005). Analysis of the reciprocal crosses of B. stricta SAD12 and B. divaricarpa VP9 found that all F1 progeny were diploid and apomictically derived when VP9 was the maternal parent, whereas all F1 progeny were triploid and sexually derived when SAD12 was the maternal parent (Schranz et al., 2005).
In this work, we continue our genetic analysis of these genotypes. First, we provide additional information about the diploid apomictic parent (VP9), including analysis of male meiosis, pollen analysis showing nonreduced gamete production, cytology documenting the presence of a heterochromatic (Het) chromosome, microsatellite analysis of progeny derived from self-pollination demonstrating persistent heterozygosity, and genomic in situ hybridization (GISH) analysis showing its hybrid origin. From the cross we analysed the F1, F2 progeny, selected F3 and test-cross (TC) progeny using a combination of flow cytometry, karyotype analysis, genome painting (GISH), and seed and pollen grain analyses. In particular, we were interested in testing the hypothesis that the Het chromosome would act as a dominant locus and confer the apomictic phenotype on transmission. Considering Boechera's great potential for studies of apomixis, this study serves as a first genetic investigation into the control and transmission of apomixis in the group, and has important consequences for our understanding of the evolutionary history of apomixis and hybridization.
- Top of page
- Materials and Methods
Here we present the first genetic analysis of the mechanisms and inheritance of apomictic reproduction in Boechera. First, we characterized more fully the paternal diploid apomictic line VP9. Genome painting revealed its hybrid origin, whereas the analysis of male meiosis demonstrated that the chromosomes are fully synaptic, including a heterochromatic (Het) chromosome that fully pairs with one of the other chromosomes. Male meiosis produced both reduced and unreduced gametes, suggesting first- or second-division restitution. Heterozygosity was also maintained in self-pollinated progeny, thus supporting its apomictic nature. We wondered if this Het chromosome carries the genetic elements controlling apomixis in a manner similar to several other systems where supernumerary or hemizygous chromosomes carry an apomixis factor (Ozias-Akins et al., 1998; Bicknell et al., 2000; Roche et al., 2001; Labombarda et al., 2002; Akiyama et al., 2005). Therefore we analysed the progeny derived from a cross between a diploid sexual and this diploid apomictic line. The resulting triploid F1 was largely sterile, producing limited numbers of viable pollen and seed. When the few seeds were grown, they produced plants that were mostly aneuploid or near-tetraploid. Hence they were not apomictically derived replicates of the three maternal genomes, but rather the product of a reduced or unreduced meiosis. Two of the recovered lines were fertile, sexual and near-tetraploid. Genome painting (GISH) analysis of one of these fertile lines found that it had a nearly balanced chromosome complement of 14 B. stricta and 13 B. holboellii chromosomes, suggesting that genome balance may play an important role in establishing the sexual phenotype. However, future work will be needed to establish if these correspond to complete chromosomal complements. Below we discuss in greater detail some of the implications of our results for understanding the inheritance, control and evolution of apomixis in Boechera, and the potential importance of the generation of sexual tetraploid lineages.
Control of apomixis in Boechera
Asexual seed formation is a complex trait resulting from modification of the sexual life cycle (reviewed by Koltunow & Grossniklaus, 2003; Bicknell & Koltunow, 2004). The normal sexual pathway can be altered or deregulated by the inheritance of a single dominant gene, hemizygous genomic region, and/or supernumerary element (Nogler, 1984b; Sherwood et al., 1994; Bicknell et al., 2000; Roche et al., 2001). Our F1 plants were derived from the union of a reduced egg cell from the diploid sexual maternal plant and a nonreduced pollen grain from the apomictic paternal plant, as shown by GISH analysis. Hence the F1 progeny probably inherited the complete genome of the apomictic parent. However, there may have been cross-over events during the failed meioses of the paternal VP9 plant that could have produced recombinant pollen grains. Future segregation analyses of molecular markers will be necessary to examine this possibility.
Our chromosome analyses showed that the diploid apomictic paternal plant had an aberrant chromosome resembling the heterochromatic (Het) chromosome in other 14- and 15-chromosome Boechera apomicts (Kantama, 2005), but lacked the extra chromosome found in the 15-chromosome apomicts (Sharbel et al., 2004, 2005). The offspring individuals in the F2 progeny displayed varying aneuploid and polyploid chromosome numbers, suggesting that apomixis in Boechera is not regulated by the simple inheritance of a single dominant locus or factor (e.g. the Het chromosome). If it were, then the F1 line, which contains the Het chromosome, would be apomictic with all or most derived F2 progeny being identical to the maternal plant.
Various authors have pointed to polyploidy as being indispensible in the regulation and transmission of the apomixis trait (Quarin et al., 2001). But in Boechera, where naturally occurring triploids are indeed apomictic, they can be highly facultative with many progeny derived sexually (Schranz et al., 2005). The results of our crossing have also shown that an increase in ploidy from diploidy to triploidy is not necessarily associated with the transfer or expression of the apomictic phenotype.
It has also been postulated that the hybrid constitution of apomictic lineages, rather than polyploidy, will lead to expression of the apomictic phenotype (Bicknell & Koltunow, 2004). Specifically, the hybridization-derived floral asynchrony hypothesis postulates that apomixis is caused by the differential expression and temporal regulation of genes in normal sexual reproductive pathways (Carman, 1997; Koltunow & Grossniklaus, 2003). In agreement with this hypothesis is our demonstration by GISH that the diploid apomictic parent (VP9) is of hybrid origin, with seven B. stricta and seven B. holboellii-like chromosomes. In addition, all diploid and triploid lines that were apomictic and/or produced nonreduced gametes were also highly heterozygous, attesting to their likely hybrid origin (Schranz et al., 2005). Similarly, individuals found to be heterozygous at microsatellite loci (Dobešet al., 2004b) and at a sequenced molecular marker (Sharbel et al., 2004, 2005) tended to produce nonreduced gametes, and were concluded to be apomictic.
However, hybridization alone cannot explain apomixis in Boechera. Our triploid F1, which contained a complete haploid genome from the highly inbred maternal B. stricta plant and the potentially complete diploid B. divaricarpa genome from the apomictic paternal plant, is not apomictic. One possible explanation for this failure in transmission of the apomixis phenotype could be segregation of the apomixis gene(s) caused by recombination during the production of the 2n pollen grains in the apomictic paternal plant. Chromosome analysis, however, showed that all or part of the heterochromatic (Het) chromosome was transmitted to the F1 plant. Another explanation for the discrepancy may lie in the relative dosage of B. stricta and B. holboellii-like chromosomes. GISH analyses of the diploid apomictic showed there is a 1 : 1 genome ratio, but in the F1 triploid there is a ratio of 2 : 1 in favour of the sexual B. stricta genome. The higher dose of B. stricta might allow for the correct, and disruptive, expression of sexuality in the triploid. Naturally occurring triploid apomictic lineages may have two doses of B. holboellii-like chromosomes, or a dosage closer to 1 : 1 if the triploid apomictic line is derived from the union of two aneuploid gametes. However, one of the recovered F2 near-tetraploid lines has a nearly 1 : 1 genome ratio, yet is sexual. Sexuality at the tetraploid level may be feasible because of an interaction of dosage and polyploidization, or alternatively because of the segregation of a gamete-lethality factor.
The results of our pollen analysis may shed light on the possible segregation of a gamete-lethal factor or gene. In all samples, except the diploid sexual line, there a significant fraction of nonviable pollen grains were produced, which were of approximately the same size as viable haploid pollen. In contrast, viable pollen grains in all samples, except for the sexual diploid, were of approximately diploid size. No viable haploid-sized pollen was observed for any of these lines. Some of the nonviable pollen grains might be caused simply by chromosomal imbalances, particularly those generated from aneuploid F2 lines where the percentages of nonviable pollen grains were as high as 98%. However, both the apomictic diploid parent and the recovered sexual tetraploids produced, on average, 25% nonviable gametes and 75% viable diploid-sized pollen grains. This result would be consistent with the recessive lethal gametophytic selection hypothesis of (Nogler, 1984b), in which an apomixis gene or factor is lethal when it occurs in a haploid pollen grain in the absence of a wild-type allele. Future studies examining the patterns of inheritance of molecular and/or cytological markers will be critical for resolving models for the control of apomixis in Boechera.
Escape from apomixis, low fertility and return to sexuality
Traditionally, apomictic lineages were thought to have limited evolutionary potential and to be doomed to extinction (Darlington, 1939; Stebbins, 1950). Later, researchers realized that the maintenance of male function and facultative apomixis provided mechanisms for perpetuating apomictic lineages by transferring apomixis genes via hybridization with sexual relatives (van Dijk, 2003). The transfer of apomixis genes into new genetic backgrounds would allow the purging or masking of deleterious mutations that may have accumulated in the apomict (Muller's ratchet), and would generate new genetic diversity, allowing escape from parasitism. Another possibility is that the hybridization of asexuals with sexuals allows genes from the apomictic parent to re-enter the sexual gene pool (Chapman et al., 2003), undergo recombination, and potentially go on to form new apomictic lineages (de Wet, 1968).
We have detected such a shift in breeding system, from apomict diploid to low-fertility sexual triploid to fertile and sexual tetraploid, in our experimental cross of Boechera. In many ways our cross follows the classic model of the formation of autotetraploids via a triploid bridge, and the inherent problems of meiosis in triploids and the frequent endosperm developmental problem of the triploid block (Ramsey & Schemske, 1998; Husband, 2004; Henry et al., 2005).
There are two routes from apomictic genotypes to the formation of sexual tetraploids in Boechera. In this work we demonstrated that, by crossing the two diploids, we could generate sexual near-tetraploids in only two generations. In our earlier work, we frequently derived de novo tetraploids directly from the crossing of a sexual diploid and an apomictic triploid line. These sexual tetraploid lines can then undergo all the classic advantages of sex, such as recombination and independent assortment. There could also be chances for homologous pairing, translocations and epigenetic modifications often seen in new polyploid lines (Osborn et al., 2003), all generating new phenotypic diversity. Also, it is important to note that these tetraploid Boechera lines could potentially express apomixis, or some partial component of apomixis, at some frequency, caused by the penetration or expressiveness of the traits. The partial expression of apomixis, especially parthenogenesis, may be particularly important for the generation of new diploid apomictic lineages. The establishment of diploid hybrid apomictic haplotypes in Boechera could be caused by homoploid hybridization, or alternatively by base chromosome number reductions (Asker & Jerling, 1992). If reduced 2n egg cells from tetraploid Boechera lineages develop parthenogenically, they could establish new diploid lineages that could be either sexual or apomictic. Such cycles of ploidy have been described for other apomictic complexes, such as Potentilla argentea and the Bothriochloa–Dichanthium complex, and are known as the ‘diploid–tetraploid–dihaploid cycle’ (de Wet, 1968; Asker & Jerling, 1992). The independent assortment and recombination of chromosomes derived from the parental genotypes or species of the tetraploid means that there could be unusual constellations of B. stricta and B. holboellii chromosomes or chromosome regions in dihaploids produced parthenogenically.
Interestingly, few naturally occurring tetraploid Boechera lineages have been detected (reviewed by Dobešet al., 2006), but when they do occur, they are likely to be sexual (Böcher, 1969; Johnson, 1970; Schranz et al., 2005). The low frequency of tetraploids could be explained by a potential propensity to produce dihaploid offspring. Alternatively they may be selected against, possibly because of a fitness disadvantage. It is interesting to note that sexual tetraploids existing at low frequency in populations of dandelions have been postulated to be critical for the creation of new triploid apomictic cytotypes, by the production of reduced 2n gametes that recombine with reduced n gametes from sexual diploids (Verduijn et al., 2004). Overall, the historical occurrence of hybridization shifts in breeding systems and alterations of ploidy have profound implications for our understanding of the inheritance, transmission and evolution of genetic and quantitative variation for Boechera.