Martin Mau

Asexual reproduction through seeds in plants (i.e., apomixis) is a heritable trait, and apomixis- linked loci have been identified in multiple species. However, direct identification of genomic elements is typically hindered as apomixis-linked loci and are commonly found in recombination-suppressed and repetitive regions. Heterochromatinized elements, such as B chromosomes and other supernumerary chromosomal DNA fragments have long been known to be associated with asexuality in both plants and animals and are prime candidate regions for the evolution of multiple apomixis factors controlling the individual elements of apomixis. Here, we examined molecular evolution, gene regulation, and chromosomal location of a male apomeiosis factor (UPG2), a long noncoding RNA gene, in sexual and apomictic Boechera with and without male apomeiosis (i.e., balanced and unbalanced apomicts). We revealed the origin of the gene in the apomixis genome on an apomixis-specific, supernumerary heterochromatic Boechera chromosome (Boe1). The UPG2 is active in the tapetum at male meiosis. We found allele classes specific to apomictic and sexual Boechera accessions and a third class that shares the features of both and points to a convergent transition state. Sex alleles are found only in some of the sexual accessions and have higher nucleotide divergence and lower transcriptional activity compared to apo alleles. These data demonstrate selective pressure to maintain the function of UPG2 for unreduced pollen formation in apomicts as the occasional transmission of the allele from unbalanced apomicts into sexual organisms that lead to pseudogenization and functional decay of copies in sexual organisms.

Summary: The mechanisms of initiation and transmission of apomixis (asexual reproduction through seeds) in natural plant populations are important for understanding the evolution of reproductive variation. Here, we used the phylogenetic diversity of the genus Boechera (Brassicaceae), together with natural diversity in pollen types produced by apomictic lines, to test whether hybridization triggers the transition to asexuality, and whether a ‘triploid bridge’ is required for the formation of polyploid apomicts. We performed crosses between diploid sexual recipient and diploid apomictic donor lines and tested whether the mating system (interspecific hybridization vs intraspecific outcrossing) or pollen type (haploid vs diploid) influenced the transmission of apomixis from diploid apomictic donors into sexual recipients. We used genetic markers and flow cytometric analyses of embryo and endosperm in seeds to infer the reproductive mode. Within a single generation, initiation of both diploid and polyploid apomixis in sexual Boechera can occur. Diploid apomixis is transmitted through haploid pollen (infectious asexuality) and polyploids can form through multiple pathways. The three functional elements of apomixis occasionally segregate. Variation in pollen ploidy and the segregation of apomixis elements drive reproductive diversity of hybrids and outcrosses and can be utilized for apomixis initiation in crop breeding programs.

Significance: Ecological-niche differentiation in diploid sexual–polyploid asexual complexes has been observed within and among many taxa, yet the relative contributions of reproductive system and ploidy are not fully understood. Here, we assess niche characteristics of sexual diploid, apomictic (asexual) diploid, and triploid Boechera (Brassicaceae) lineages. We find strong evidence for widespread hybridization and, to a lesser degree, ploidy variation as factors that together overcome the adaptive disadvantages of apomictic (i.e., asexual) reproduction. When controlling for ploidy, we find only modest evidence for putatively asexually driven ecological-niche divergence among reproductive systems, a finding that contradicts the well-supported patterns of geographic parthenogenesis.

In apomictic Boechera spp., meiotic diplospory leads to the circumvention of meiosis and the suppression of recombination to produce unreduced male and female gametes (i.e. apomeiosis). Here, we have established an early flower developmental staging system and have performed microarray-based comparative gene expression analyses of the pollen mother cell stage in seven diploid sexual and seven diploid apomictic genotypes to identify candidate factors for unreduced pollen formation. We identified a transcript unique to apomictic Boechera spp. called UPGRADE2 (BspUPG2), which is highly up-regulated in their pollen mother cells. BspUPG2 is highly conserved among apomictic Boechera spp. genotypes but has no homolog in sexual Boechera spp. or in any other taxa. BspUPG2 undergoes posttranscriptional processing but lacks a prominent open reading frame. Together with the potential of stably forming microRNA-like secondary structures, we hypothesize that BspUPG2 functions as a long regulatory noncoding messenger RNA-like RNA. BspUPG2 has apparently arisen through a three-step process initiated by ancestral gene duplication of the original BspUPG1 locus, followed by sequential insertions of segmentally duplicated gene fragments, with final exonization of its sequence structure. Its genesis reflects the hybridization history that characterizes the genus Boechera.

The genetic mechanisms causing seed development by gametophytic apomixis in plants are predominantly unknown. As apomixis is consistently associated with hybridity and polyploidy, these confounding factors may either (a) be the underlying mechanism for the expression of apomixis, or (b) obscure the genetic factors which cause apomixis. To distinguish between these hypotheses, we analyzed the population genetic patterns of diploid and triploid apomictic lineages and their sexual progenitors in the genus Boechera (Brassicaceae). We find that while triploid apomixis is associated with hybridization, the majority of diploid apomictic lineages are likely the product of intra-specific crosses. We then show that these diploid apomicts are more likely to sire triploid apomictic lineages than conspecific sexuals. Combined with flow cytometric seed screen phenotyping for male and female components of apomixis, our analyses demonstrate that hybridization is an indirect correlate of apomixis in Boechera.

Pollen size is often used as a biological parameter to estimate the ploidy and viability of mature pollen grains. In general, pollen size quantification is performed one- or two-dimensionally using image-based diameter measurements. As these approaches are elaborate and time consuming, alternative approaches that enable a quick, reliable analysis of pollen size are highly relevant for plant research. In this study, we present the volume-based particle size analysis technique as an alternative method to characterize mature pollen. Based on a comparative assay using different plant species (including tomato, oilseed rape, kiwifruit, clover, among others), we found that volume-based pollen size measurements are not biased by the pollen shape or position and substantially reduce non-biological variation, allowing a more accurate determination of the actual pollen size. As such, volume-based particle size techniques have a strong discriminative power in detecting pollen size differences caused by alterations in the gametophytic ploidy level and therefore allow for a quick and reliable estimation of the somatic ploidy level. Based on observations in Arabidopsis thaliana gametophytic mutants and differentially reproducing Boechera polyantha lines, we additionally found that volume-based pollen size analysis provides quantitative and qualitative data about alterations in male sporogenesis, including aneuploid and diploid gamete formation. Volume-based pollen size analysis therefore not only provides a quick and easy methodology to determine the somatic ploidy level of flowering plants, but can also be used to determine the mode of reproduction and to quantify the level of diplogamete formation.

Apomixis, a natural form of asexual seed production in plants, is considered to have great biotechnological potential for agriculture. It has been hypothesised that de-regulation of the sexual developmental pathway could trigger apomictic reproduction. The genus Boechera represents an interesting model system for understanding apomixis, having both sexual and apomictic genotypes at the diploid level. Quantitative qRT-PCR is the most extensively used method for validating genome-wide gene expression analyses, but in order to obtain reliable results, suitable reference genes are necessary. In this work we have evaluated six potential reference genes isolated from a 454 (FLX) derived cDNA library of Boechera. RNA from live microdissected ovules and anthers at different developmental stages, as well as vegetative tissues of apomictic and sexual Boechera, were used to validate the candidates.