Genus Boechera Research Articles

Sex- versus apomixis-specific polymorphisms in the 5'UTR of APOLLO from Boechera shift gene expression from somatic to reproductive tissues in Arabidopsis.

Among candidate genes underlying the control components of apomixis, APOLLO is known for its strong linkage to apomeiosis in the genus Boechera. The gene has "apo alleles," which are characterized by a set of linked apomixis-specific polymorphisms, and "sex alleles." All apomictic Boechera genotypes are heterozygous for the apo/sex alleles, whereas all sexual genotypes are homozygous for sex alleles. In this study, native and synthetic APOLLO promoters were characterized by detecting the expression level of the β-glucuronidase (GUS) gene in Arabidopsis. Comparing various flower developmental stages in transgenic lines containing different constructs with 2-kb native transgenic lines revealed that changes to the APOLLO promoter causes shifts in tissue and developmental stage specificity of GUS expression. Importantly, several apomixis-specific polymorphisms in the 5'UTR change the timing and location of GUS activity from somatic to reproductive tissues. These synthetic data simulate a plausible evolutionary process, whereby apomixis-specific gene activity can be achieved.

Differential expression and evolutionary diversification of RNA helicases in Boechera sexual and apomictic reproduction.

In higher plants, sexual reproduction is characterized by meiosis of the first cells of the germlines, and double fertilization of the egg and central cell after gametogenesis. In contrast, in apomicts of the genus Boechera, meiosis is omitted or altered and only the central cell requires fertilization, while the embryo forms parthenogenetically from the egg cell. To deepen the understanding of the transcriptional basis underlying these differences, we applied RNA-seq to compare expression in reproductive tissues of different Boechera accessions. This confirmed previous evidence of an enrichment of RNA helicases in plant germlines. Furthermore, few RNA helicases were differentially expressed in female reproductive ovule tissues harboring mature gametophytes from apomictic and sexual accessions. For some of these genes, we further found evidence for a complex recent evolutionary history. This included a homolog of Arabidopsis thaliana FASCIATED STEM4 (FAS4). In contrast to AtFAS4, which is a single-copy gene, FAS4 is represented by three homologs in Boechera, suggesting a potential for subfunctionalization to modulate reproductive development. To gain first insights into functional roles of FAS4, we studied Arabidopsis lines carrying mutant alleles. This identified the crucial importance of AtFAS4 for reproduction, as we observed developmental defects and arrest during male and female gametogenesis.

DEVELOPMENT OF FEMALE REPRODUCTIVE STRUCTURES IN <i>BOECHERA</i> (BRASSICACEAE) SPECIES UNDER SEXUAL AND APOMICTIC REPRODUCTIVE MODES

The results of comparative embryological study of the two species from the genus Boechera (B. falcata and B. stricta), which are characterized by a sexual mode of reproduction, and the apomictic M4B accession, an ancient natural hybrid of B. stricta × B. retrofracta, are presented. A detailed description of the ovule development of the studied plants is described. A significant similarity of the ovule development has been shown; a general plan of the ovules structure is ortho-campylotropic, bitegmal, medionucellate. The development of the embryo sac in B. falcata and B. stricta corresponds to the monosporic Polygonum type; in the ovules of the M4B accession, an apomictic unreduced female gametophyte is predominantly formed as a result of Taraxacum-type diplospory. However, in this accession apomixis is facultative, since a small amount of the ovules in the ovary forms a reduced embryo sac with participation of meiosis, but unlike B. falcata and B. stricta, after the second meiotic division, a triad of megaspores is formed, of which the chalazal one is functional. In the ovules of the apomictic M4B accession, underdevelopment of the inner integument was often observed, which, apparently, correlates with arrest of the megasporocyte and embryo sac development, since destruction was often observed in ovules with a short inner integument. This abnormality is likely due to the hybrid nature of the M4B accession rather than apomixis. In this accession, the embryo is formed parthenogenetically, but its development requires endosperm, which apparently develops by pseudogamy, only after triple fusion (fertilization of the fused polar nuclei by sperm). In the absence of endosperm, the embryo perishes, and seed development is arrested.

Seed size, endosperm and germination variation in sexual and apomictic Boechera.

Asexual reproduction results in offspring that are genetically identical to the mother. Among apomictic plants (reproducing asexually through seeds) many require paternal genetic contribution for proper endosperm development (pseudogamous endosperm). We examined phenotypic diversity in seed traits using a diverse panel of sexual and apomictic accessions from the genus Boechera. While genetic uniformity resulting from asexual reproduction is expected to reduce phenotypic diversity in seeds produced by apomictic individuals, pseudogamous endosperm, variable endosperm ploidy, and the deviations from 2:1 maternal:paternal genome ratio in endosperm can all contribute to increased phenotypic diversity among apomictic offspring. We characterized seed size variation in 64 diploid sexual and apomictic (diploid and triploid) Boechera lineages. In order to find out whether individual seed size was related to endosperm ploidy we performed individual seed measurements (projected area and mass) using the phenoSeeder robot system and flow cytometric seed screen. In order to test whether individual seed size had an effect on resulting fitness we performed a controlled growth experiment and recorded seedling life history traits (germination success, germination timing, and root growth rate). Seeds with triploid embryos were 33% larger than those with diploid embryos, but no average size difference was found between sexual and apomictic groups. We identified a maternal effect whereby chloroplast lineage 2 had 30% larger seeds than lineage 3, despite having broad and mostly overlapping geographic ranges. Apomictic seeds were not more uniform in size than sexual seeds, despite genetic uniformity of the maternal gametophyte in the former. Among specific embryo/endosperm ploidy combinations, seeds with tetraploid (automomous) endosperm were on average smaller, and the proportion of such seeds was highest in apomicts. Larger seeds germinated more quickly than small seeds, and lead to higher rates of root growth in young seedlings. Seed mass is under balancing selection in Boechera, and it is an important predictor of several traits, including germination probability and timing, root growth rates, and developmental abnormalities in apomictic accessions.

Phylogenetic and Expression Analysis of CENH3 and APOLLO Genes in Sexual and Apomictic Boechera Species.

Apomictic plants (reproducing via asexual seeds), unlike sexual individuals, avoid meiosis and egg cell fertilization. Consequently, apomixis is very important for fixing maternal genotypes in the next plant generations. Despite the progress in the study of apomixis, molecular and genetic regulation of the latter remains poorly understood. So far APOLLO gene encoding aspartate glutamate aspartate aspartate histidine exonuclease is one of the very few described genes associated with apomixis in Boechera species. The centromere-specific histone H3 variant encoded by CENH3 gene is essential for cell division. Mutations in CENH3 disrupt chromosome segregation during mitosis and meiosis since the attachment of spindle microtubules to a mutated form of the CENH3 histone fails. This paper presents in silico characteristic of APOLLO and CENH3 genes, which may affect apomixis. Furthermore, we characterize the structure of CENH3 by bioinformatic tools, study expression levels of APOLLO and CENH3 transcripts by Real-Time Polymerase Chain Reaction RT-PCR in gynoecium/siliques of the natural diploid apomictic and sexual Boechera species at the stages of meiosis and before and after fertilization. While CENH3 was a single copy gene in all Boechera species, the APOLLO gene have several polymorphic alleles associated with sexual and apomictic reproduction in the Boechera genera. Expression of the APOLLO apo-allele during meiosis was upregulated in gynoecium of apomict B. divaricarpa downregulating after meiosis until the 4th day after pollination (DAP). On the 5th DAP, expression in apomictic siliques increased again. In sexual B. stricta gynoecium and siliques APOLLO apo-allele did not express. Expression of the APOLLO sex-allele during and after meiosis in gynoecium of sexual plants was several times higher than that in apomictic gynoecium. However, after pollination the sex-allele was downregulated in sexual siliques to the level of apomicts and increased sharply on the 5th DAP, while in apomictic siliques it almost did not express. At the meiotic stage, the expression level of CENH3 in the gynoecium of apomicts was two times lower than that of the sexual Boechera, decreasing in both species after meiosis and keep remaining very low in siliques of both species for several days after artificial pollination until the 4th DAP, when the expression level raised in sexual B. stricta siliques exceeding 5 times the level in apomictic B. divaricarpa siliques. We also discuss polymorphism and phylogeny of the APOLLO and CENH3 genes. The results obtained may indicate to a role of the CENH3 and APOLLO genes in the development of apomixis in species of the genus Boechera.

The spread of infectious asexuality through haploid pollen.

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.

Family Cruciferae Juss. (Brassicaceae Burnett) of SAKHA (Yakutia) Republic

The paper offers a new revision of the species composition of the Cruciferae family in Yakutia, which is supplemented by information on the geographical distribution and taxonomic composition. In the present work, the crucifers of Yakutia are represented by 39 genera and 102 species. Among them, there are two endemic species (Boechera falcata (Turcz.) Al-Shehbaz and Smelowskia jacutica (Botsch. et Karav.) Al-Shehbaz et Warwick), and one subendemic (Isatis jacutensis (N. Busch) N. Busch). Traditionally, the genus Draba is the richest in the number of species in the territories reaching into the Arctic. At present, taking the latest processing of the genus by V. V. Petrovsky (Petrovsky, 2018) into account, it numbers 32 species, which amounts to about 1/3 of the total species richness of this family in Yakutia. The paper summarizes the nomenclature changes that have affected a number of representatives of this family in recent years, and proposes new combinations in the genus Boechera (B. pendula (L.) V. I. Dorof. Comb. Nov.) and Thellungiella (T. bursifolia (DC.) V. I. Dorof. Comb. Nov.). An analysis of the diversity of crucifers made it possible to assert that the flora of Yakutia is obviously insufficiently studied in its northwest and northeast reaches, and to suggest that the floristic zoning of Yakutia should be supplemented by outlining three supra-regions, namely the Verkhoyansk (eastern), Vilui (western) and Arctic (northern) supra-regions.

Chromosomal Evolution and Apomixis in the Cruciferous Tribe Boechereae.

The mustard family (Brassicaceae) comprises several dozen monophyletic clades usually ranked as tribes. The tribe Boechereae plays a prominent role in plant research due to the incidence of apomixis and its close relationship to Arabidopsis. This tribe, largely confined to western North America, harbors nine genera and c. 130 species, with >90% of species belonging to the genus Boechera. Hundreds of apomictic diploid and triploid Boechera hybrids have spurred interest in this genus, but the remaining Boechereae genomes remain virtually unstudied. Here we report on comparative genome structure of six genera (Borodinia, Cusickiella, Phoenicaulis, Polyctenium, Nevada, and Sandbergia) and three Boechera species as revealed by comparative chromosome painting (CCP). All analyzed taxa shared the same seven-chromosome genome structure. Comparisons with the sister Halimolobeae tribe (n = 8) showed that the ancestral Boechereae genome (n = 7) was derived from an older n = 8 genome by descending dysploidy followed by the divergence of extant Boechereae taxa. As tribal divergence post-dated the origin of four tribe-specific chromosomes, it is proposed that these chromosomal rearrangements were a key evolutionary innovation underlaying the origin and diversification of the Boechereae in North America. Although most Boechereae genera exhibit genomic conservatism, intra-tribal cladogenesis has occasionally been accompanied by chromosomal rearrangements (particularly inversions). Recently, apomixis was reported in the Boechereae genera Borodinia and Phoenicaulis. Here, we report sexual reproduction in diploid Nevada, diploid Sandbergia, and tetraploid Cusickiella and aposporous apomixis in tetraploids of Polyctenium and Sandbergia. In sum, apomixis is now known to occur in five of the nine Boechereae genera.

Gene Function Rather than Reproductive Mode Drives the Evolution of RNA Helicases in Sexual and Apomictic Boechera.

In higher plants, sexual and asexual reproductions through seeds (apomixis) have evolved as alternative strategies. Evolutionary advantages leading to coexistence of both reproductive modes are currently not well understood. It is expected that accumulation of deleterious mutations leads to a rapid elimination of apomictic lineages from populations. In this line, apomixis originated repeatedly, likely from deregulation of the sexual pathway, leading to alterations in the development of reproductive lineages (germlines) in apomicts as compared with sexual plants. This potentially involves mutations in genes controlling reproduction.Increasing evidence suggests that RNA helicases are crucial regulators of germline development. To gain insights into the evolution of 58 members of this diverse gene family in sexual and apomictic plants, we applied target enrichment combined with next-generation sequencing to identify allelic variants from 24 accessions of the genus Boechera, comprising sexual, facultative, and obligate apomicts. Interestingly, allelic variants from apomicts did not show consistently increased mutation frequency. Either sequences were highly conserved in any accession, or allelic variants preferentially harbored mutations in evolutionary less conserved C- and N-terminal domains, or presented high mutation load independent of the reproductive mode. Only for a few genes allelic variants harboring deleterious mutations were only identified in apomicts. To test if high sequence conservation correlates with roles in fundamental cellular or developmental processes, we analyzed Arabidopsis thaliana mutant lines in VASA-LIKE (VASL), and identified pleiotropic defects during ovule and reproductive development. This indicates that also in apomicts mechanisms of selection are in place based on gene function.

The Boechera Genus as a Resource for Apomixis Research.

The genera Boechera (A. Löve et D. Löve) and Arabidopsis, the latter containing the model plant Arabidopsis thaliana, belong to the same clade within the Brassicaceae family. Boechera is the only among the more than 370 genera in the Brassicaceae where apomixis is well documented. Apomixis refers to the asexual reproduction through seed, and a better understanding of the underlying mechanisms has great potential for applications in agriculture. The Boechera genus currently includes 110 species (of which 38 are reported to be triploid and thus apomictic), which are distributed mostly in the North America. The apomictic lineages of Boechera occur at both the diploid and triploid level and show signs of a hybridogenic origin, resulting in a modification of their chromosome structure, as reflected by alloploidy, aneuploidy, substitutions of homeologous chromosomes, and the presence of aberrant chromosomes. In this review, we discuss the advantages of the Boechera genus to study apomixis, consider its modes of reproduction as well as the inheritance and possible mechanisms controlling apomixis. We also consider population genetic aspects and a possible role of hybridization at the origin of apomixis in Boechera. The molecular tools available to study Boechera, such as transformation techniques, laser capture microdissection, analysis of transcriptomes etc. are also discussed. We survey available genome assemblies of Boechera spp. and point out the challenges to assemble the highly heterozygous genomes of apomictic species. Due to these challenges, we argue for the application of an alternative reference-free method for the comparative analysis of such genomes, provide an overview of genomic sequencing data in the genus Boechera suitable for such analysis, and provide examples of its application.

Ecological differentiation facilitates fine-scale coexistence of sexual and asexual Boechera.

Ecological differentiation (ED) between sexual and asexual organisms may permit the maintenance of reproductive polymorphism. Several studies of sexual/asexual ED in plants have shown that the geographic ranges of asexuals extend beyond those of sexuals, often in areas of higher latitude or elevation. But very little is known about ED at fine scales, wherein coexistence of sexuals and asexuals may be permitted by differential niche occupation. We used 149 populations of sexual and apomictic lineages in the genus Boechera (rock cress) collected across a portion of this mustard's vast range. We characterized reproductive mode, ploidy, and species identity or hybrid parentage of each individual, and then used a multipronged statistical approach to (1) identify ED between sexuals and asexuals; (2) investigate the impacts of two confounding factors, polyploidy and hybridization, on ED; and (3) determine the environmental variables underlying ED. We found that sexuals and asexuals are significantly ecologically differentiated across the landscape, despite fine-scale interdigitation of these two reproductive forms. Asexual reproduction was strongly associated with greater disturbance, reduced slope, and greater environmental variability. Although ploidy had little effect on the patterns observed, hybridization has a unique impact on the relationships between asexual reproduction and specific environmental variables. Ecological differentiation along the axes of disturbance, slope, and climatic variability, as well as the effects of heterozygosity, may contribute to the maintenance of sexuality and asexuality across the landscape, ultimately impacting the establishment and spread of asexual lineages.

Assembly and annotation of genomes of some species from the apomictic genus Boechera and evolutionary analysis of apomixis-associated genes

Assembly and annotation of genomes of some species from the apomictic genus Boechera and evolutionary analysis of apomixis-associated genes

Establishing the cell biology of apomictic reproduction in diploid Boechera stricta (Brassicaceae).

Background and aimsIn the Brassicaceae family, apomictic development is characteristic of the genus Boechera. Hybridization, polyploidy and environmental adaptation that arose during the evolution of Boechera may serve as (epi)genetic regulators of apomictic initiation in this genus. Here we focus on Boechera stricta, a predominantly diploid species that reproduces sexually. However, apomictic development in this species has been reported in several studies, indicating non-obligate sexuality.MethodsA progressive investigation of flower development was conducted using three accessions to assess the reproductive system of B. stricta. We employed molecular and cyto-embryological identification using histochemistry, transmission electron microscopy and Nomarski and epifluorescence microscopy.Key ResultsData from internal transcribed spacer (ITS) and chloroplast haplotype sequencing, in addition to microsatellite variation, confirmed the B. stricta genotype for all lines. Embryological data indicated irregularities in sexual reproduction manifested by heterochronic ovule development, longevity of meiocyte and dyad stages, diverse callose accumulation during meiocyte-to-gametophyte development, and the formation of triads and tetrads in several patterns. The arabinogalactan-related sugar epitope recognized by JIM13 immunolocalized to one or more megaspores. Furthermore, pollen sterility and a high frequency of seed abortion appeared to accompany reproduction of the accession ES512, along with the initiation of parthenogenesis. Data from flow cytometric screening revealed both sexual and apomictic seed formation.ConclusionThese results imply that B. stricta is a species with an underlying ability to initiate apomixis, at least with respect to the lines examined here. The existence of apomixis in an otherwise diploid sexual B. stricta may provide the genomic building blocks for establishing highly penetrant apomictic diploids and hybrid relatives. Our findings demonstrate that apomixis per se is a variable trait upon which natural selection could act.

Admixture, evolution, and variation in reproductive isolation in the Boechera puberula clade

BackgroundHybridization is very common in plants, and the incorporation of new alleles into existing lineages (i.e. admixture) can blur species boundaries. However, admixture also has the potential to increase standing genetic variation. With new sequencing methods, we can now study admixture and reproductive isolation at a much finer scale than in the past. The genus Boechera is an extraordinary example of admixture, with over 400 hybrid derivates of varying ploidy levels. Yet, few studies have assessed admixture in this genus on a genomic scale.ResultsIn this study, we used Genotyping-by-Sequencing (GBS) to clarify the evolution of the Boechera puberula clade, whose six members are scattered across the western United States. We further assessed patterns of admixture and reproductive isolation within the group, including two additional species (B. stricta and B. retrofracta) that are widespread across North America. Based on 14,815 common genetic variants, we found evidence for some cases of hybridization. We find evidence of both recent and more ancient admixture, and that levels of admixture vary across species.ConclusionsWe present evidence for a monophyletic origin of the B. puberula group, and a split of B. puberula into two subspecies. Further, when inferring reproductive isolation on the basis of presence and absence of admixture, we found that the accumulation of reproductive isolation between species does not seem to occur linearly with time since divergence in this system. We discuss our results in the context of sexuality and asexuality in Boechera.

Assembly of the Boechera retrofracta Genome and Evolutionary Analysis of Apomixis-Associated Genes.

Closely related to the model plant Arabidopsis thaliana, the genus Boechera is known to contain both sexual and apomictic species or accessions. Boechera retrofracta is a diploid sexually reproducing species and is thought to be an ancestral parent species of apomictic species. Here we report the de novo assembly of the B. retrofracta genome using short Illumina and Roche reads from 1 paired-end and 3 mate pair libraries. The distribution of 23-mers from the paired end library has indicated a low level of heterozygosity and the presence of detectable duplications and triplications. The genome size was estimated to be equal 227 Mb. N50 of the assembled scaffolds was 2.3 Mb. Using a hybrid approach that combines homology-based and de novo methods 27,048 protein-coding genes were predicted. Also repeats, transfer RNA (tRNA) and ribosomal RNA (rRNA) genes were annotated. Finally, genes of B. retrofracta and 6 other Brassicaceae species were used for phylogenetic tree reconstruction. In addition, we explored the histidine exonuclease APOLLO locus, related to apomixis in Boechera, and proposed model of its evolution through the series of duplications. An assembled genome of B. retrofracta will help in the challenging assembly of the highly heterozygous genomes of hybrid apomictic species.

Depletion of Key Meiotic Genes and Transcriptome-Wide Abiotic Stress Reprogramming Mark Early Preparatory Events Ahead of Apomeiotic Transition.

Molecular dissection of apomixis – an asexual reproductive mode – is anticipated to solve the enigma of loss of meiotic sex, and to help fixing elite agronomic traits. The Brassicaceae genus Boechera comprises of both sexual and apomictic species, permitting comparative analyses of meiotic circumvention (apomeiosis) and parthenogenesis. Whereas previous studies reported local transcriptome changes during these events, it remained unclear whether global changes associated with hybridization, polyploidy and environmental adaptation that arose during evolution of Boechera might serve as (epi)genetic regulators of early development prior apomictic initiation. To identify these signatures during vegetative stages, we compared seedling RNA-seq transcriptomes of an obligate triploid apomict and a diploid sexual, both isolated from a drought-prone habitat. Uncovered were several genes differentially expressed between sexual and apomictic seedlings, including homologs of meiotic genes ASYNAPTIC 1 (ASY1) and MULTIPOLAR SPINDLE 1 (MPS1) that were down-regulated in apomicts. An intriguing class of apomict-specific deregulated genes included several NAC transcription factors, homologs of which are known to be transcriptionally reprogrammed during abiotic stress in other plants. Deregulation of both meiotic and stress-response genes during seedling stages might possibly be important in preparation for meiotic circumvention, as similar transcriptional alteration was discernible in apomeiotic floral buds too. Furthermore, we noted that the apomict showed better tolerance to osmotic stress in vitro than the sexual, in conjunction with significant upregulation of a subset of NAC genes. In support of the current model that DNA methylation epigenetically regulates stress, ploidy, hybridization and apomixis, we noted that ASY1, MPS1 and NAC019 homologs were deregulated in Boechera seedlings upon DNA demethylation, and ASY1 in particular seems to be repressed by global DNA methylation exclusively in the apomicts. Variability in stress and transcriptional response in a diploid apomict, which is geographically distinct from the triploid apomict, pinpoints both common and independent features of apomixis evolution. Our study provides a molecular frame-work to investigate how the adaptive traits associated with the evolutionary history of apomicts co-adapted with meiotic gene deregulation at early developmental stage, in order to predate meiotic recombination, which otherwise is thought to be favorable in stress and low-fitness conditions.

Searching for Diamonds in the Apomictic Rough: A Case Study Involving <I>Boechera lignifera</I> (Brassicaceae)

Abstract The genus Boechera is one of the most difficult species complexes in North America, with about 70 sexual diploids and hundreds of apomictic taxa representing diverse combinations of nearly every known sexual genome. In this study, we set out to clarify the taxonomy of Boechera lignifera, which currently includes a small number of sexual diploid populations in addition to the widespread apomictic diploid upon which the name is based. Using data from cytological studies, microsatellite DNA analyses, geography, and morphology, we demonstrate that the apomictic populations are genetically quite divergent from the sexual diploids. We propose the name Boechera kelseyana to accommodate the sexual diploid taxon, which occurs entirely south of the geographic range of B. lignifera. Boechera kelseyana is consistently separable from B. lignifera based on pollen and seed morphology, the length and proximal orientation of fruiting pedicels, differences in the branching and orientation of trichomes on the lower...

Karyotype evolution in apomictic Boechera and the origin of the aberrant chromosomes.

Chromosome rearrangements may result in both decrease and increase of chromosome numbers. Here we have used comparative chromosome painting (CCP) to reconstruct the pathways of descending and ascending dysploidy in the genus Boechera (tribe Boechereae, Brassicaceae). We describe the origin and structure of three Boechera genomes and establish the origin of the previously described aberrant Het and Del chromosomes found in Boechera apomicts with euploid (2n = 14) and aneuploid (2n = 15) chromosome number. CCP analysis allowed us to reconstruct the origin of seven chromosomes in sexual B. stricta and apomictic B. divaricarpa from the ancestral karyotype (n = 8) of Brassicaceae lineage I. Whereas three chromosomes (BS4, BS6, and BS7) retained their ancestral structure, five chromosomes were reshuffled by reciprocal translocations to form chromosomes BS1-BS3 and BS5. The reduction of the chromosome number (from x = 8 to x = 7) was accomplished through the inactivation of a paleocentromere on chromosome BS5. In apomictic 2n = 14 plants, CCP identifies the largely heterochromatic chromosome (Het) being one of the BS1 homologues with the expansion of pericentromeric heterochromatin. In apomictic B. polyantha (2n = 15), the Het has undergone a centric fission resulting in two smaller chromosomes - the submetacentric Het' and telocentric Del. Here we show that new chromosomes can be formed by a centric fission and can be fixed in populations due to the apomictic mode of reproduction.

A Conserved Apomixis-Specific Polymorphism Is Correlated with Exclusive Exonuclease Expression in Premeiotic Ovules of Apomictic Boechera Species

Apomixis (asexual seed production) is characterized by meiotically unreduced egg cell production (apomeiosis) followed by its parthenogenetic development into offspring that are genetic clones of the mother plant. Fertilization (i.e. pseudogamy) of the central cell is important for the production of a functional endosperm with a balanced 2:1 maternal:paternal genome ratio. Here, we present the APOLLO (for apomixis-linked locus) gene, an Aspartate Glutamate Aspartate Aspartate histidine exonuclease whose transcripts are down-regulated in sexual ovules entering meiosis while being up-regulated in apomeiotic ovules at the same stage of development in plants of the genus Boechera. APOLLO has both "apoalleles," which are characterized by a set of linked apomixis-specific polymorphisms, and "sexalleles." All apomictic Boechera spp. accessions proved to be heterozygous for the APOLLO gene (having at least one apoallele and one sexallele), while all sexual genotypes were homozygous for sexalleles. Apoalleles contained a 20-nucleotide polymorphism present in the 5' untranslated region that contains specific transcription factor-binding sites for ARABIDOPSIS THALIANA HOMEOBOX PROTEIN5, LIM1 (for LINEAGE ABNORMAL11, INSULIN1, MECHANOSENSORY PROTEIN3), SORLIP1AT (for SEQUENCES OVERREPRESENTED IN LIGHT-INDUCED PROMOTERS IN ARABIDOPSIS THALIANA1), SORLIP2AT, and POLYA SIGNAL1. In the same region, sexalleles contain transcription factor-binding sites for DNA BINDING WITH ONE FINGER2, DNA BINDING WITH ONE FINGER3, and PROLAMIN BOX-BINDING FACTOR. Our results suggest that the expression of a single deregulated allele could induce the cascade of events leading to asexual female gamete formation in an apomictic plant.

The Conserved Chimeric Transcript UPGRADE2 Is Associated with Unreduced Pollen Formation and Is Exclusively Found in Apomictic Boechera Species

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.