Genome size and base chromosome number vary greatly among domesticated members of the family Poaceae. For example, the genomes of wheat (Triticum aestivum L.), maize (Zea mays L.), and rice (Oryza sativa L.) have base chromosome numbers of 7, 10, and 12, respectively, and genomes size ranging from 389 (rice) to approximately 16,000 Mbp (wheat) (IRGSP, 2005; Moolhuijzen et al., 2007). Despite such differences, early comparative genetic studies relying on mapped molecular markers revealed significant synteny (physical colocalization of genetic loci) across diverse members of the grass family (Ahn et al., 1993; Devos et al., 1994; Moore et al., 1995; Moore et al., 1997). Early studies also suggested that conservation of gene order (collinearity) within syntenic blocks is frequently disrupted (Bennetzen and Ramakrishna, 2002; Feuillet and Keller, 2002; Sorrells et al., 2003). With the emergence of whole genome sequences (WGSs), more detailed comparisons have confirmed both significant macro-scale conservation of collinearity among grass species (Salse and Feuillet 2007, Salse et al., 2008, 2009; IBI, 2010) and frequent loss of microcollinearity (Wicker et al., 2010).
Oat (Avena sativa L.) is a member of the Poaceae, subfamily Pooideae, tribe Poeae, and subtribe Aveninae. Species of the Poaeae together with those of the tribes Triticeae and Bromeae encompass the vast majority of economically important temperate cereal, turf, and forage crops. Cultivated oat is an allohexaploid (2n = 6x = 42) that derives from three ancestral diploid Avena genomes (A, C, and D), with the C genome more distinct from the others (Fu and Williams, 2008). Oat is considered an important animal fodder crop in many areas of the world and produces grain that is valuable for human health, cosmetics, and other uses (Lee-Manion et al., 2009; Meydani, 2009; Baumann et al., 2009). Nevertheless, oat production globally is less than 4 and 17% that of the related cool season cereal grains wheat and barley (Hordeum vulgare L.), respectively (FAO, 2008). This disparity in economic importance has generated a gap in oat genome research when compared to wheat and barley as well as other important grasses such as rice and maize. In addition, the polyploid nature of the oat genome and the large amount of repetitive DNA present therein (recently reviewed by Rines et al., 2006), also pose obstacles to genome research.
Many genetic linkage maps exist for hexaploid oat, the first of which was developed from a cross between cultivars Kanota and Ogle (K×O) (O'Donoughue et al., 1995). The K×O map has become a reference for cultivated oat studies and new versions have been released with the addition of new markers (Wight et al., 2003; Tinker et al., 2009). Additional linkage maps have been developed in other populations, and comparisons between them have revealed inconsistencies in marker order and infrequent resolution of homologous linkage groups (LGs) across maps (Jin et al., 2000; Portyanko et al., 2001; Kremer et al., 2001; Groh et al., 2001; Badaeva et al., 2010). As a result, a resolution of the hexaploid oat genome into 21 LGs representing each of its chromosomes has not yet been published. In contrast, diploid Avena species (2n = 14) possess a simpler genome and so mapping with diploid Avena spp. circumvents complexities posed by a polyploid genome (Wight et al., 2006; Mugford et al., 2009). Only a few mapping efforts have been conducted on diploid Avena spp. (O'Donoughue et al., 1992; Rayapati et al., 1994; Van Deynze et al., 1995; Yu and Wise, 2000; Kremer et al., 2001). In three of these studies, seven LGs, presumed to represent each of the seven chromosomes, were delineated (O'Donoughue et al., 1992; Van Deynze et al., 1995; Yu and Wise, 2000).
Use of linkage maps for comparative genome analysis identified significant synteny between diploid Avena spp., a consensus map of Triticeae, rice, and maize (Van Deynze et al., 1995). Surprisingly, however, alignment of linkage maps of hexaploid oat and diploid Avena spp. has resolved only a small number of apparently highly fragmented syntenic blocks (O'Donoughue et al., 1995; Portyanko et al., 2001; Kremer et al., 2001; Wight et al., 2003). It has been postulated that extensive fragmentation of diploid groups and subsequent rearrangements occurred to form hexaploid groups (Hayasaki et al., 2000; Linares et al., 2000), to the point that assigning hexaploid LGs to single ancestral diploid chromosomes may not be possible.
The model grass Brachypodium distachyon (L.) P. Beauv. possesses a compact 272 Mbp genome that has been sequenced (IBI, 2010). Because it is a member of the grass subfamily Pooideae and belongs to a tribe (Brachypodieae) that is a sister group to the core pooids (Kellogg, 2001), B. distachyon has potential value for genomics research in many domesticated grasses in the Pooideae (Garvin, 2007; Garvin et al., 2008). Indeed, whole-genome comparisons with barley and wheat revealed extensive blocks of synteny covering more than 99% of the B. distachyon genome (IBI, 2010). The objective of this study was to employ the B. distachyon genome sequence as a resource to gain new insights into genome evolution in the genus Avena and to expand on our knowledge of homeologous and homologous relationships between LGs in oat.
MATERIALS AND METHODS
The B. distachyon WGS was downloaded from Brachypodium.org (2010). A diploid Avena spp. genetic linkage map developed from the cross Avena atlantica B. R. Baum & Fedak × A. hirtula Lag. published by Van Deynze et al. (1995) was chosen because of its good marker coverage, because its seven major LGs that likely correspond to the seven diploid chromosomes, and because many mapped markers represent complementary DNA (cDNA) clones. This map is an expansion of the one previously published (O'Donoughue et al., 1992). Both parental lines have an A type of Avena spp. genome (subgroup A). The most updated version of the map has 367 restriction fragment length polymorphism (RFLP) markers spanning a total genetic distance of 737.4 cM (see GrainGenes, 2010).
Two hexaploid cultivated oat genetic maps that share one parent in common were used for the analysis. One of these was developed from the cross A. sativa (syn. A. byzantina K. Koch) cv. Kanota × A. sativa cv. Ogle. The K×O map was the first hexaploid oat map constructed (O'Donoughue et al., 1995). It was updated by Wight et al. (2003) and most recently by Tinker et al. (2009). The K×O map is the most complete molecular linkage map of cultivated oat published to date. It has a total of 2491 mapped markers, mostly diversity arrays technology (DArT), RFLP, amplified fragment length polymorphism (AFLP), random amplified polymorphic DNA (RAPD), and a small number of other markers, grouped in 34 LGs. In our analysis, only LGs with a length of 8 cM or more and comprising at least five markers were included (29 LGs total). The second hexaploid oat map employed was developed from a cross between Ogle and A. byzantina cv. TAM O-301 (O×T). The map contains 441 markers on 34 LGs with a total map length of 2049 cM (Portyanko et al., 2001). Some of the O×T LGs are very short and contained only a few markers and thus were not included in the analysis.
For clarity, we followed the LG nomenclature that was first described in several previous publications (Van Deynze et al., 1995; Portyanko et al., 2001; Tinker et al., 2009). Oat marker sequence information was retrieved from GrainGenes (2010) and the National Center for Biotechnology Information (NCBI, 2010) or kindly provided by S. J. Molnar (personal communication, 2010) and aligned against the B. distachyon genome using the BLAST nucleotide (blastn) algorithm (Altschul et al., 1990, 1997) with an E-value threshold of 1 × 10−15. Following blastn searches, the matched homologous B. distachyon sequences were displayed in the Brachypodium Genome Browser v.1.68 (Brachypodium.org, 2010) to record their genome positions.
CIRCOS v.0.52 (Krzywinski et al., 2009) was used for alignment display. A syntenic block was defined when the combination of these three criteria was met: (i) a proposed “bundle” of five or more orthologous loci equal or below the E-value threshold was identified, (ii) no orthologous locus in a bundle was more than 10 Mbp from its nearest orthologous neighbor in the B. distachyon genome, and (iii) no orthologous locus in a bundle was more than 25 cM from its nearest orthologous neighbor in the oat genetic linkage map being analyzed. The number of bundled orthologous loci was relaxed to three, maximum physical distance to 20 Mbp, and maximum genetic distance to 50 cM in O×T alignments because of the lower number of informative markers. For relative genome size representation, the following values of 1C (pg) were considered: 4.4 (A. hirtula), 13.7 (A. byzantina), and 13.23 (A. sativa) (Bennett and Smith, 1976). These were converted into physical distances by using the estimate of one picogram of DNA per 1000 Mbp (1 Gbp) to calculate the cM:bp ratio for each map. For the sake of figure clarity, twists were eliminated from ribbons joining syntenic blocks with inverted marker orientations. The overall bundling strategy for syntenic block identification thus eliminates outlier orthologous alignments that might be products of molecular map errors, alignments to members of gene families with conserved domains, or small translocations, all of which can confound identification of broader embedded patterns of synteny.
Diploid Avena Species–Brachypodium distachyon Alignment
The most recent version of the Avena atlantica × A. hirtula map has 367 molecular markers, and sequence data was available for 291 of them. BLAST nucleotide comparisons of the sequences to the B. distachyon genome sequence (IBI, 2010) identified 254 (87%) that exhibited sequence similarity with at least one B. distachyon locus at or below our established threshold level of 1 × 10−15. The average length of the filtered oat marker sequences employed for alignments to the B. distachyon genome sequence was 407 nucleotides. Six of these markers aligned to three or more B. distachyon loci and 41 (14%) mapped to two loci. The rest of the oat markers (207 total) detected a single presumed ortholog in the B. distachyon genome. Oat markers that matched to three or more B. distachyon loci were not further considered because of uncertain orthology or paralogy relationships.
The remaining markers with one or two hits identified 289 pairs of orthologous loci. These were grouped into bundles that define syntenic blocks as described in Materials and Methods. These syntenic blocks covered an estimated 83.2% of the B. distachyon genome and 87% of the diploid Avena spp. linkage map. The seven diploid LGs each had syntenic blocks corresponding to no more than two B. distachyon chromosomes (Fig. 1A). The relationship between these two genomes is thus defined by a total of 18 blocks of synteny (Fig. 1A; Supplemental Fig. S1). Interesting patterns of rearrangement that disrupt synteny are seen. For example, the entire LGs A and D are inserted into the centromeric regions of B. distachyon chromosomes 2 (Bd2) and 1 (Bd1), respectively, similar to the nested insertions of entire chromosomes described for sorghum [Sorghum bicolor (L.) Moench], barley, and rice relative to B. distachyon chromosomes (IBI, 2010). The number of pairs of orthologous loci was adequate to examine whether both genomes exhibit conserved gene order at a macro-genome scale (macro-collinearity). Results indicate significant large-scale conservation of collinearity between diploid Avena spp. and B. distachyon syntenic blocks (Fig. 2H and 2I), with nearly 70% of the aligned markers preserving the order between genomes. Thus, the B. distachyon and diploid Avena spp. genomes appear largely syntenic, with a modest number of major structural rearrangements having occurred since their divergence.
The B. distachyon–diploid Avena spp. alignments were used to develop ancestral chromosome models for the subtribe Aveninae to extend on the grass chromosome evolution model proposed by Salse et al. (2009). This was done by assigning segments of each diploid LG to common ancestral chromosomes based on their synteny with B. distachyon (Fig. 3). For instance, the first segment of diploid LG A aligns to the central portion of Bd3, which according to the evolutionary model corresponds to a segment of proto-chromosome A8. The central segment of LG A aligns to the first half of Bd2, which originates from proto-chromosome A1. The last segment of LG A is syntenic to the first portion of Bd3, which traces back to a segment of ancestral chromosome A10. Similar putative chromosome evolution maps were developed for the rest of the diploid oat LGs (Fig. 3).
Hexaploid Oat–Brachypodium distachyon Alignments
The latest K×O update (Tinker et al., 2009) added DArT markers to the existing map, and with its 2491 markers it is the largest published map for cultivated oat. BLAST nucleotide homology searches with the total of 1298 markers for which sequence data was available produced alignments at or below the cutoff value (1 × 10−15) in 533 cases. These markers defined syntenic blocks covering 87% of the B. distachyon genome and 76.6% of the hexaploid oat linkage map. The average length of the markers with blastn scores exceeding the threshold was 488 nucleotides. Twenty-one of the oat markers mapped to three or more B. distachyon loci and so were not included in the analysis. Another 106 (11%) identified two different B. distachyon loci. Interestingly, of the 844 DArT markers used for sequence comparisons, 500 (59%) had no homology (>1 × 10−01) to the B. distachyon genome and only 188 (22%) had BLAST values above our threshold cutoff value (1 × 10−15). After bundling orthologous loci according the criteria described in Materials and Methods, a total of 50 syntenic blocks were identified, with most K×O groups exhibiting some synteny with B. distachyon (Fig. 1B; Supplemental Fig. S2). Five LGs (KO_1_3_38_X4, KO_39, KO_50, KO_51, and KO_52) did not have enough orthologous marker relationships to meet the minimum criteria for establishing syntenic blocks (see Materials and Methods).
A similar analysis was completed with the O×T recombinant inbred line (RIL) population (Portyanko et al., 2001). The current version of the O×T map has 411 markers, 236 of which have associated sequence information (GrainGenes, 2010). BLAST nucleotide alignments with these markers against the B. distachyon genome yielded 204 (86.4%) with values at or below the established threshold and thus were regarded as potential orthologous pairs for the genome comparison analysis. Nine of the E-value filtered markers (4%) mapped to three or more loci and were omitted from the analysis, and 39 (16%) identified two different B. distachyon loci. The average length of the filtered oat aligned markers was 502 nucleotides. Because of the lower number of informative sequences compared to the K×O map, the criteria for grouping homologous pairs into bundles for the O×T map were relaxed. After grouping, the percentage of genomic coverage of the syntenic blocks was 50.7% for the B. distachyon genome sequence and 51.9% for the O×T map. Just 18 syntenic blocks were identified versus the 50 blocks found for the K×O map, presumably due to the significantly lower numbers of informative markers in the O×T population. Sixteen O×T LGs did not have an identified syntenic block with B. distachyon (Supplemental Fig. S3).
Diploid Brachypodium distachyon–Hexaploid Oat Alignments
While blocks of syntenic markers have been previously reported between diploid and hexaploid oat (O'Donoughue et al., 1995; Kremer et al., 2001; Wight et al., 2003), a full resolution of diploid and hexaploid LG relationships has not been completed. We performed a three-way comparison between B. distachyon and both diploid and hexaploid maps to identify syntenic blocks from different linkage maps that colocalize to the same chromosome intervals of B. distachyon. Such colocalization provides evidence for a homologous relationship between LGs. Further, it helps identify homeologous LGs in hexaploid maps. Many syntenic blocks from diploid and hexaploid linkage maps aligned to common B. distachyon genome regions (Fig. 2). Table 1 condenses the homologies that could be inferred between diploid Avena spp. and K×O hexaploid oat LGs. From this analysis it is clear that, compared to diploid LGs, hexaploid homologous LGs are fragmented, suggesting that translocations have taken place in most cultivated oat LGs compared to our proposed ancestral diploid Aveninae chromosome structural model. When compared to previous proposed diploid–hexaploid oat homologous relationships (O'Donoughue et al., 1995; Groh et al., 2001; Wight et al., 2003), our results were largely in agreement (Table 1), which lends strong support to the methodology we employed. Our results also expand significantly on our knowledge of homologous relationships between diploid and hexaploid oat by proposing 16 additional homologous relationships.
|Diploid LG||K×O Linkage Groups|
Hexaploid segments homologous to each diploid LG were used to postulate homeologous relationships in hexaploid oat. For example, diploid LG F has two blocks of synteny with B. distachyon, one to a terminal region of Bd1 and another to a terminal portion of Bd4. Two different segments of the hexaploid LGs KO_5_30 and KO_16_23 showed synteny to the same two regions of the B. distachyon genome (Fig. 2F). A segment of LG KO_21+49_31+40 is syntenic to the distal region of Bd1 but not Bd4, but it is the only other hexaploid linkage map region that showed synteny to that region of Bd1. Thus, we hypothesize that these three hexaploid LG segments probably constitute a homeologous series homologous to diploid chromosome F. Proposed homeologous relationships between the other hexaploid LGs were inferred in the same manner (Fig. 4). For establishing potential homeologous relationships, we also employed evidence from other sources summarized in Table 1. It is noteworthy that a series of K×O segments homologous to LG C, comprising KO_1_3_38, KO_4_12_13, and KO_15 (Fig. 4C), each have two segments of synteny to the same two distinct regions of Bd2. The LG KO_1_3_38 was intentionally separated into five different LGs, since they were postulated to be involved as quadrivalent arms in a translocation event, and designated with the suffixes _X1, _X2, _X3, _X4, and _break in the last version of the K×O map (Tinker et al., 2009). This was done to improve the quality of mapping within the four proposed quadrivalent arms. Our analysis supports rejoining at least _X1, _X3, and _break. If the fragmentation of this map region by Tinker et al. (2009) is in fact correct, then rejoining these groups would be relevant for only one configuration of the two parent genomes involved in the translocation. Although more unjoined LGs must exist in the K×O map, our results corroborate the notion that extensive fragmentation and chromosomal segmental rearrangements have occurred in the diploid genomes of hexaploid oat.
Homology between the Kanota × Ogle and Ogle × TAM O-301 Groups
An additional use of the genome alignments is that they permitted an indirect comparison between two principal hexaploid oat maps through the genome of B. distachyon without the need for markers shared in common between maps. In principle, if one LG from O×T aligns to the same B. distachyon region with a K×O LG, they are likely to be homologous LGs. Accordingly, 11 homologies between LGs of the two maps could be established through B. distachyon (Fig. 5; Table 2). We compared the homologous relationships found through B. distachyon alignments with those reported by Wight et al. (2003) and found agreement for four LGs (Table 2). We could not corroborate four of the previously reported homologies. In addition, we added seven new putative homologies.
Oat genetic linkage mapping efforts face many obstacles, including a large genome replete with repetitive elements (Molnar et al., 2011), intergenomic translocations (Jellen et al., 1994; Hayasaki et al., 2000; Linares et al., 2000; Sanz et al., 2010), and occasional homeologous pairing during meiosis (Singh and Kolb, 1991) that can confound correct ordering of markers. We conducted a series of genome comparisons between selected oat genetic linkage maps and the B. distachyon WGS to assess the suitability of the latter to facilitate oat genetics research. By using B. distachyon as an anchor genome, our approach precluded the need for common markers to undertake comparisons between oat linkage maps and also provided an opportunity to examine potential genome relationships between Avena species with different ploidy levels.
In the absence of more extensive information on genomic sequence organization such as physical maps, genetic linkage maps can be a useful tool to evaluate the degree of structural conservation between genomes of related species (Salse et al., 2004). The primary drawback of marker-based comparative genomics of Avena species is the lack of sufficient numbers of common markers with which to compare different maps. The use of different marker technologies and mapping populations has generated many markers unique to particular maps, which hinders their comparison and integration. In this study, three Avena spp. molecular maps were anchored to the recently released B. distachyon WGS. Potential orthologous relationships and total genomic coverage in the alignments to B. distachyon were greatest for the diploid Avena spp. map. The K×O hexaploid oat map alignment to B. distachyon also revealed a significant level of coverage, while the O×T alignments covered only approximately half of the map. The disparity reflects the nature of the markers employed from each map and marker density. The diploid and O×T maps employed cDNA-derived probes representing exonic regions of genes, while the K×O map contains many DArT markers that are not necessarily exonic or even genic. In fact, only 19% of the DArT markers aligned to the B. distachyon genome at the threshold we employed.
In some instances alignments identified two or three comparable highly significant BLAST hits between oat marker sequences and the B. distachyon genome, suggesting the detection of paralogous genes. It can be difficult to differentiate the ortholog from paralogs in such instances for a number of reasons. For instance, an intervening fragment of less conserved intron sequence in the bona fide ortholog could lead to a false conclusion of paralogy. Because of the limited number of available markers, we included markers with one or two best matches for genome comparisons. Comparative genomics can be undertaken by different approaches depending on the depth of genome sequence information. Our criteria for marker grouping relied on both the density and colocalization of orthologous loci between oat and B. distachyon. Only orthologous pairs that were adjacent to each other in segments of each genome were grouped. This minimizes the chance for the inclusion of solitary alignments that might be a product of mapping errors or multiple hits due to repetitive motifs.
For most B. distachyon chromosomes, the length covered by syntenic blocks with oat was substantial, frequently restricting the nonsyntenic parts only to the regions surrounding the centromeres. In general, alignments were located in gene-rich regions of the B. distachyon genome (IBI, 2010), reflecting the genic nature of many of the oat markers. There is a remarkable dearth of alignments to the short arm of B. distachyon chromosome 5 (Bd5S) for all three oat genome comparisons. This is consistent with a previous study that showed Bd5S to have a gene density of approximately half of the rest of the B. distachyon genome, a higher proportion of long terminal repeat retrotransposons, and the lowest level of collinear genes compared to rice and sorghum (IBI, 2010).
Comparison between the Kanota × Ogle and Ogle × TAM O-301 Maps
The K×O and O×T molecular maps were compared to each other using B. distachyon as an anchor genome. Nine O×T LGs were found to share homology with at least one K×O LG, with a total of 11 blocks of synteny (Table 2). Notably, our alignments confirmed several previous proposed homologous relationships and added many new ones. In an earlier study comparing these same two maps, Wight et al. (2003) described eight homologous relationships when employing the criterion of at least four shared markers. Four of these relationships were among those identified in the present study. However, four other homologous relationships proposed by Wight and colleagues were not detected in our comparisons. The lack of corroboration for them may simply reflect regions of homology between oat maps that lack useful sequence information such as might be encountered with non-genic markers, no sequence information, or no identified B. distachyon orthology. Additionally, seven new proposed homologous relationships between LGs were found thus demonstrating that even with modest marker numbers and low or even no common markers it is possible to employ the B. distachyon genome sequence to further resolve LG homologies between maps.
While a number of new homologous relationships were found between the K×O and O×T maps, the number is perhaps lower than might be expected given that they share a parent in common and have the same number of LGs. Several factors could underlie this result. First, both mapping populations originated from the cross between two different ecotypes, a winter type (A. byzantina cv. Kanota or cv. TAM O-301) and a spring type (A. sativa cv. Ogle), with the goal of creating a population that would provide a high level of sequence polymorphism for mapping efforts. However, at least three major intergenomic translocations are known to differentiate Ogle and Kanota (Jellen et al., 1994). Other genome rearrangements such as paracentric inversions may lead to reduced recombination or irregular gametes; these have been also described for this cross and could cause some of the reported anomalous segregation (Jellen et al., 1994; Fedak et al., 1999). Further, multivalent formation and chromosomal interchanges appear to be common even in hybrids between A. sativa cultivars (Singh and Kolb, 1991). These intervarietal chromosome structural rearrangements therefore might explain the surprisingly low level of homology revealed even among A. sativa × A. sativa maps (Zhu and Kaeppler, 2003). Lastly, mapping errors could be present in one or both maps, which would also affect the accuracy of the alignments. Our results suggest that while alignments through B. distachyon overcome the need for shared markers between different maps and thereby increase the number of homologies identified, chromosomal rearrangements might be present in intervarietal crosses of hexaploid Avena spp. more frequently than previously thought. That would explain the lack of homologous LGs found among maps that had a considerable number of common markers (Jin et al., 2000; Groh et al., 2001; Zhu and Kaeppler, 2003).
Evolution from a Diploid Ancestor
Because the divergence of B. distachyon and oat was more recent than their divergence from other sequenced grass species, oat is expected to exhibit more synteny with B. distachyon. This appears to be true for diploid Avena spp., in which we identified 18 syntenic blocks with B. distachyon versus the 22 and 24 described for sorghum and rice, respectively (IBI, 2010). In contrast, alignment of expressed sequence tag (EST) maps from barley and wheat to B. distachyon revealed 21 and 20 blocks of synteny, respectively (IBI, 2010). In an earlier comparative marker-based study Van Deynze et al. (1995) found 18 conserved blocks between diploid Avena spp. and a Triticeae consensus map. However, that number was increased to 20 when compared to rice. Notwithstanding the fact that different methodologies and varying levels of input information may affect inferences of synteny, our results suggest that oat may share a degree of genome conservation with B. distachyon similar to that for members of the Triticeae. This would be consistent with the current models of the evolution of the Pooideae, in which the tribe Brachypodieae is a sister group to both the Triticeae tribe and subtribe Aveninae (formerly tribe Aveneae) (Bouchenak-Khelladi et al., 2008).
Diverse fossil pollen records and recent whole genome and large genome map comparisons situate a common ancestor of the different grass subfamilies 45 to 60 million years ago (Kellogg, 2001, Salse et al., 2008, 2009; IBI, 2010). A currently well accepted evolutionary model proposes that this ancestor had five proto-chromosomes that were subsequently subjected to a series of whole genome and segmental duplications, translocations, and chromosome fusions that transformed the number of chromosomes from 5 to 10 and then the 12 of the paleo-tetraploid, which preceded the divergence of the grasses (Paterson et al., 2004; Salse et al., 2008). Additional ancestral chromosomal translocations, fusions, and nested chromosome insertions further modeled each species’ genome. The divergence of Brachypodieae from other important tribes and subtribes with a base chromosome number of seven, including the Triticeae and by default the Aveninae, has been estimated to have been between 32 and 39 million y ago (IBI, 2010). Our genome alignments permitted a postulation of the evolution of Aveninae chromosome structure from ancestral chromosomes by relating oat genomic segments to the proposed chromosome evolutionary model for B. distachyon (IBI 2010). For instance, if a segment of a diploid oat LG is syntenic to a section of the B. distachyon genome, it is presumed to derive from a common ancestral chromosome segment. Duplicated regions within the B. distachyon genome can also assist in identifying ancestral common chromosomes before divergence, if they can be tracked in diploid Avena spp. LGs. For instance, LG C aligns with two ancient internal duplications in B. distachyon chromosome 2 (Bd2) (Fig. 2C), which suggests that both LG C and Bd2 originated from the same proto-chromosome (A5) after the ancestral whole genome duplication. Likewise, the LG D showed synteny with the central part of Bd1 and the two telomeric regions of Bd3 (Fig. 2D), which accurately reflects another interchromosomal duplication in B. distachyon. Subsequently, we adopted the proposed evolutionary model of monocots (Salse et al., 2009; IBI, 2010) to develop an ancestral Aveninae genome structure (Fig. 3). Completely relating the seven diploid Avena spp. chromosomes to the proposed five grass progenitor proto-chromosomes would require similar depths of genome alignment as available for other members of the family.
Hexaploid Oat Genome Rearrangements Compared to Diploid Avena Species
Intricate patterns of duplications and rearrangement appear to have occurred and shaped the genome of cultivated oat from its diploid progenitors (Jellen et al., 1995; Hayasaki et al., 2000; Wight et al., 2003). Our comparative approach using the B. distachyon genome as a bridge for comparisons provided additional evidence of diploid chromosomes not being highly conserved in the hexaploid genome and instead appearing frequently fragmented and disrupting chromosome orthologous relationships. Based on our results and those of previous studies, we traced segments of different hexaploid LGs to each putative diploid chromosome and tentatively assigned homeologous sets. Some diploid LGs such as B, F, and G appeared to have suffered limited fragmentation in the hexaploid oat genome as only regions of three or four hexaploid LGs were found homeologous. Nevertheless, there is a generally low level of large-scale (e.g., large LG) conservation of genome structure between diploid Avena spp. and hexaploid oat.
These series of chromosomal rearrangements are presumed to have occurred both in diploid progenitors before polyploidization and after the polyploid genome was established. Indeed, the A, C, and D progenitor genomes possess a series of chromosomal differences that still can be detected in related hexaploid oat species (Linares et al., 2000; Badaeva et al., 2010; Sanz et al., 2010). As well, cultivated oat might have also undergone additional rearrangement after both tetraploidy and hexaploidy, as is known to occur in other species (for reviews, see Liu et al., 2009 and Feldman and Levy, 2009). This includes gain or loss of parental segments, gene suppression and activation, structural rearrangements, and epigenetic modifications. These structural and functional modifications can occur immediately after genomes have combined to form a polyploid and are presumably required to stabilize combined genomes and permit fertility. Once diploidization has occurred, the genomic plasticity of the polyploid oat genome might confer adaptation to diverse environmental conditions and thus an evolutionary advantage, similar to what has been postulated for wheat (Dubcovsky and Dvorak, 2007; Feldman and Levy, 2009).
Brachypodium distachyon as a Potential Guide for Linkage Map Construction
Our results indicate that B. distachyon–oat alignments may provide some guidance to ongoing efforts seeking to correctly coalesce oat LGs. For instance, contiguous synteny of different oat LGs when aligned to B. distachyon may be suggestive of the LGs residing on the same chromosome. For example, we found that the short KO_8 group aligns to a region of the B. distachyon genome that is contiguous to a region that aligns to KO_14, which might indicate that both are part of the same oat chromosome. Similarly, KO_9 and KO_2 align to a collinear segment in B. distachyon. Other putative combinations were also considered likely. Thus, patterns of synteny between oat and B. distachyon revealed in this analysis may be a useful resource for the oat genetics community as it seeks to construct enhanced linkage maps.
Our study shows that the B. distachyon genome shares large syntenic blocks with that of oat, especially with diploid Avena spp., and corroborates previous observations that hexaploid oat LGs exhibit significant fragmentation compared to diploid Avena spp. The construction of a complete and integrated genetic linkage map for cultivated oat is highly desired; however, this has been problematic due to genome rearrangements and other possible chromosome alterations. Our genome alignments and inferences of homology and homeology within and between different oat linkage maps should assist ongoing efforts to develop and improve oat linkage maps. Further, the analytical strategy we employed to use B. distachyon as an anchor genome to help resolve homologous and homeologous relationships in oat should be applicable to other cool-season grasses for which genetic maps are limited.
Supplemental Information Available
Supplemental material is available free of charge at http://www.crops.org/publications/tpg.