Brachypodium Chromosome Research Articles

Reconstruction of evolutionary trajectories of chromosomes unraveled independent genomic repatterning between Triticeae and Brachypodium

BackgroundAfter polyploidization, a genome may experience large-scale genome-repatterning, featuring wide-spread DNA rearrangement and loss, and often chromosome number reduction. Grasses share a common tetraploidization, after which the originally doubled chromosome numbers reduced to different chromosome numbers among them. A telomere-centric reduction model was proposed previously to explain chromosome number reduction. With Brachpodium as an intermediate linking different major lineages of grasses and a model plant of the Pooideae plants, we wonder whether it mediated the evolution from ancestral grass karyotype to Triticeae karyotype.ResultsBy inferring the homology among Triticeae, rice, and Brachpodium chromosomes, we reconstructed the evolutionary trajectories of the Triticeae chromosomes. By performing comparative genomics analysis with rice as a reference, we reconstructed the evolutionary trajectories of Pooideae plants, including Ae. Tauschii (2n = 14, DD), barley (2n = 14), Triticum turgidum (2n = 4x = 28, AABB), and Brachypodium (2n = 10). Their extant Pooidea and Brachypodium chromosomes were independently produced after sequential nested chromosome fusions in the last tens of millions of years, respectively, after their split from rice. More frequently than would be expected by chance, in Brachypodium, the ‘invading’ and ‘invaded’ chromosomes are homoeologs, originating from duplication of a common ancestral chromosome, that is, with more extensive DNA-level correspondence to one another than random chromosomes, nested chromosome fusion events between homoeologs account for three of seven cases in Brachypodium (P-value≈0.00078). However, this phenomenon was not observed during the formation of other Pooideae chromosomes.ConclusionsNotably, we found that the Brachypodium chromosomes formed through exclusively distinctive trajectories from those of Pooideae plants, and were well explained by the telomere-centric model. Our work will contribute to understanding the structural and functional innovation of chromosomes in different Pooideae lineages and beyond.

Chromosome identification and reconstruction of evolutionary rearrangements in Brachypodium distachyon, B. stacei and B. hybridum.

Background and AimsThe Brachypodium genus represents a useful model system to study grass genome organization. Palaeogenomic analyses (e.g. Murat F, Armero A, Pont C, Klopp C, Salse J. 2017. Reconstructing the genome of the most recent common ancestor of flowering plants. Nature Genetics49: 490–496) have identified polyploidization and dysploidy as the prime mechanisms driving the diversity of plant karyotypes and nested chromosome fusions (NCFs) crucial for shaping grass chromosomes. This study compares the karyotype structure and evolution in B. distachyon (genome Bd), B. stacei (genome Bs) and in their putative allotetraploid B. hybridum (genomes BdBs).Methods Brachypodium chromosomes were measured and identified using multicolour fluorescence in situ hybridization (mcFISH). For higher resolution, comparative chromosome barcoding was developed using sets of low-repeat, physically mapped B. distachyon-derived bacterial artificial chromosome (BAC) clones.Key ResultsAll species had rather small chromosomes, and essentially all in the Bs genome were morphometrically indistinguishable. Seven BACs combined with two rDNA-based probes provided unambiguous and reproducible chromosome discrimination. Comparative chromosome barcoding revealed NCFs that contributed to the reduction in the x = 12 chromosome number that has been suggested for the intermediate ancestral grass karyotype. Chromosome Bd3 derives from two NCFs of three ancestral chromosomes (Os2, Os8, Os10). Chromosome Bs6 shows an ancient Os8/Os10 NCF, whilst Bs4 represents Os2 only. Chromosome Bd4 originated from a descending dysploidy that involves two NCFs of Os12, Os9 and Os11. The specific distribution of BACs along Bs9 and Bs5, in both B. stacei and B. hybridum, suggests a Bs genome-specific Robertsonian rearrangement.ConclusionsmcFISH-based karyotyping identifies all chromosomes in Brachypodium annuals. Comparative chromosome barcoding reveals rearrangements responsible for the diverse organization of Bd and Bs genomes and provides new data regarding karyotype evolution since the split of the two diploids. The fact that no chromosome rearrangements were observed in B. hybridum compared with the karyotypes of its phylogenetic ancestors suggests prolonged genome stasis after the formation of the allotetraploid.

Sequencing flow-sorted short arm of Haynaldia villosa chromosome 4V provides insights into its molecular structure and virtual gene order

BackgroundHaynaldia villosa (H. villosa) has been recognized as a species potentially useful for wheat improvement. The availability of its genomic sequences will boost its research and application.ResultsIn this work, the short arm of H. villosa chromosome 4V (4VS) was sorted by flow cytometry and sequenced using Illumina platform. About 170.6 Mb assembled sequences were obtained. Further analysis showed that repetitive elements accounted for about 64.6% of 4VS, while the coding fraction, which is corresponding to 1977 annotated genes, represented 1.5% of the arm. The syntenic regions of the 4VS were searched and identified on wheat group 4 chromosomes 4AL, 4BS, 4DS, Brachypodium chromosomes 1 and 4, rice chromosomes 3 and 11, and sorghum chromosomes 1, 5 and 8. Based on genome-zipper analysis, a virtual gene order comprising 735 gene loci on 4VS genome was built by referring to the Brachypodium genome, which was relatively consistent with the scaffold order determined for Ae. tauschii chromosome 4D. The homologous alleles of several cloned genes on wheat group 4 chromosomes including Rht-1 gene were identified.ConclusionsThe sequences provided valuable information for mapping and positional-cloning genes located on 4VS, such as the wheat yellow mosaic virus resistance gene Wss1. The work on 4VS provided detailed insights into the genome of H. villosa, and may also serve as a model for sequencing the remaining parts of H. villosa genome.

Collinearity Analysis and High-Density Genetic Mapping of the Wheat Powdery Mildew Resistance Gene Pm40 in PI 672538.

The wheat powdery mildew resistance gene Pm40, which is located on chromosomal arm 7BS, is effective against nearly all prevalent races of Blumeria graminis f. sp tritici (Bgt) in China and is carried by the common wheat germplasm PI 672538. A set of the F1, F2 and F2:3 populations from the cross of the resistant PI 672538 with the susceptible line L1034 were used to conduct genetic analysis of powdery mildew resistance and construct a high-density linkage map of the Pm40 gene. We constructed a high-density linkage genetic map with a total length of 6.18 cM and average spacing between markers of 0.48 cM.Pm40 is flanked by Xwmc335 and BF291338 at genetic distances of 0.58 cM and 0.26 cM, respectively, in deletion bin C-7BS-1-0.27. Comparative genomic analysis based on EST-STS markers established a high level of collinearity of the Pm40 genomic region with a 1.09-Mbp genomic region on Brachypodium chromosome 3, a 1.16-Mbp genomic region on rice chromosome 8, and a 1.62-Mbp genomic region on sorghum chromosome 7. We further anchored the Pm40 target intervals to the wheat genome sequence. A putative linear index of 85 wheat contigs containing 97 genes on 7BS was constructed. In total, 9 genes could be considered as candidates for the resistances to powdery mildew in the target genomic regions, which encoded proteins that were involved in the plant defense and response to pathogen attack. These results will facilitate the development of new markers for map-based cloning and marker-assisted selection of Pm40 in wheat breeding programs.

The use of SNP hybridisation arrays and cytogenetics to characterise deletions of chromosome 4B in hexaploid wheat (Triticum aestivum L.).

Many deletions of the wheat Della ( Rht - B1 ) gene and its flanking regions were isolated in a simple phenotypic screen, and characterised by modified analysis of SNP hybridisation data and cytogenetics. In a dwarf wheat suppressor screen, many tall 'revertants' were isolated following mutagenesis of a severely dwarfed (Rht-B1c) hexaploid wheat. About 150 lines were identified as putative deletions of Rht-B1c, based on the PCR analysis. Southern blot hybridisation established that most of them lacked the Rht-B1 gene, but retained the homoeologues Rht-A1 and Rht-D1. PCR assays were developed for orthologues of two genes that flank Rht-1/Della in the genomes of the model species Brachypodium and rice. Deletion of the B-genome-specific homoeologues of these two genes was confirmed in the Rht-B1 deletion lines, indicating loss of more than a single gene. SNP chip hybridisation analysis established the extents of deletion in these lines. Based on the synteny with Brachypodium chromosomes 1 and 4g, and rice chromosomes 3g and 11g, notional deletion maps were established. The deletions ranged from interstitial deletions of 4BS through to loss of all 4BS markers. There were also instances, where all 4BS and 4BL markers were lost, and these lines had poor fertility and narrow stems and leaves. Cytogenetic studies on selected lines confirmed the loss of portions of 4BS in lines that lacked most or all 4BS markers. They also confirmed that lines lacking both 4BS and 4BL markers were nullisomics for 4B. These nested deletion lines share a common genetic background and will have applications in assigning markers to regions of 4BS as well as to 4BL. The potential for this type of analysis in other regions of the wheat genome is discussed.

Toward a better understanding of the genomic region harboring Fusarium head blight resistance QTL Qfhs.ndsu-3AS in durum wheat.

New molecular markers were developed and mapped to the FHB resistance QTL region in high resolution. Micro-collinearity of the QTL region with rice and Brachypodium was revealed for a better understanding of the genomic region. The wild emmer wheat (Triticum dicoccoides)-derived Fusarium head blight (FHB) resistance quantitative trait locus (QTL) Qfhs.ndsu-3AS previously mapped to the short arm of chromosome 3A (3AS) in a population of recombinant inbred chromosome lines (RICLs). This study aimed to attain a better understanding of the genomic region harboring Qfhs.ndsu-3AS and to improve the utility of the QTL in wheat breeding. Micro-collinearity of the QTL region with rice chromosome 1 and Brachypodium chromosome 2 was identified and used for marker development in saturation mapping. A total of 42 new EST-derived sequence tagged site (STS) and simple sequence repeat (SSR) markers were developed and mapped to the QTL and nearby regions on 3AS. Further comparative analysis revealed a complex collinearity of the 3AS genomic region with their collinear counterparts of rice and Brachypodium. Fine mapping of the QTL region resolved five co-segregating markers (Xwgc1186/Xwgc716/Xwgc1143/Xwgc501/Xwgc1204) into three distinct loci proximal to Xgwm2, a marker previously reported to be closely linked to the QTL. Four other markers (Xwgc1226, Xwgc510, Xwgc1296, and Xwgc1301) mapped farther proximal to the above markers in the QTL region with a higher resolution. Five homozygous recombinants with shortened T. dicoccoides chromosomal segments in the QTL region were recovered by molecular marker analysis and evaluated for FHB resistance. Qfhs.ndsu-3AS was positioned to a 5.2cM interval flanked by the marker Xwgc501 and Xwgc510. The recombinants containing Qfhs.ndsu-3AS and new markers defining the QTL will facilitate utilization of this resistance source in wheat breeding.

Mapping of SrTm4, a Recessive Stem Rust Resistance Gene from Diploid Wheat Effective to Ug99.

Race TTKSK (or Ug99) of Puccinia graminis f. sp. tritici, the causal agent of wheat stem rust, is a serious threat to wheat production worldwide. Diploid wheat, Triticum monococcum (genome Am), has been utilized previously for the introgression of stem rust resistance genes Sr21, Sr22, and Sr35. Multipathotype seedling tests of biparental populations demonstrated that T. monococcum accession PI 306540 collected in Romania contains a recessive resistance gene effective to all P. graminis f. sp. tritici races screened, including race TTKSK. We will refer to this gene as SrTm4, which is the fourth stem rust resistance gene characterized from T. monococcum. Using two mapping populations derived from crosses of PI 272557×PI 306540 and G3116×PI 306540, we mapped SrTm4 on chromosome arm 2AmL within a 2.1 cM interval flanked by sequence-tagged markers BQ461276 and DR732348, which corresponds to a 240-kb region in Brachypodium chromosome 5. The eight microsatellite and nine sequence-tagged markers linked to SrTm4 will facilitate the introgression and accelerate the deployment of SrTm4-mediated Ug99 resistance in wheat breeding programs.

Fine mapping of <i>Hch1</i>, the causal D-genome gene for hybrid chlorosis in interspecific crosses between tetraploid wheat and <i>Aegilops tauschii</i>

Hybrid chlorosis, one of the reproductive barriers between tetraploid wheat and its D-genome progenitor, Aegilops tauschii, inhibits normal growth of synthetic wheat hexaploids. Hybrid chlorosis appears to be due to an epistatic interaction of two loci from the AB and D wheat genomes. Our previous study assigned the causal D-genome gene for hybrid chlorosis, Hch1, to the short arm of chromosome 7D. Here, we constructed a fine map of 7DS near Hch1 using 280 F2 individuals from a cross of two wheat synthetic lines, one showing normal growth and the other showing hybrid chlorosis. The hybrid chlorosis phenotype was controlled by a single dominant allele of the Hch1 locus in the synthetic hexaploids. Hch1 was closely linked to four new markers within 0.2 cM, and may be localized near or within the two Ae. tauschii scaffolds containing the linked markers on 7DS. Comparative analysis of the Hch1 chromosomal region for Ae. tauschii, barley and Brachypodium showed that a local inversion occurred in the region proximal to Hch1 during the divergence between barley and Ae. tauschii, and that the Hch1 region on wheat 7DS is syntenic to Brachypodium chromosome 1. These observations provide useful information for further studies toward map-based cloning of Hch1.

Chromosomal location and comparative genomics analysis of powdery mildew resistance gene Pm51 in a putative wheat-Thinopyrum ponticum introgression line.

Powdery mildew (PM) is a very destructive disease of wheat (Triticum aestivum L.). Wheat-Thinopyrum ponticum introgression line CH7086 was shown to possess powdery mildew resistance possibly originating from Th. ponticum. Genomic in situ hybridization and molecular characterization of the alien introgression failed to identify alien chromatin. To study the genetics of resistance, CH7086 was crossed with susceptible genotypes. Segregation in F2 populations and F2:3 lines tested with Chinese Bgt race E09 under controlled conditions indicated that CH7086 carries a single dominant gene for powdery mildew resistance. Fourteen SSR and EST-PCR markers linked with the locus were identified. The genetic distances between the locus and the two flanking markers were 1.5 and 3.2 cM, respectively. Based on the locations of the markers by nullisomic-tetrasomic and deletion lines of ‘Chinese Spring’, the resistance gene was located in deletion bin 2BL-0.89-1.00. Conserved orthologous marker analysis indicated that the genomic region flanking the resistance gene has a high level of collinearity to that of rice chromosome 4 and Brachypodium chromosome 5. Both resistance specificities and tests of allelism suggested the resistance gene in CH7086 was different from previously reported powdery mildew resistance genes on 2BL, and the gene was provisionally designated PmCH86. Molecular analysis of PmCH86 compared with other genes for resistance to Bgt in the 2BL-0.89-1.00 region suggested that PmCH86 may be a new PM resistance gene, and it was therefore designated as Pm51. The closely linked flanking markers could be useful in exploiting this putative wheat-Thinopyrum translocation line for rapid transfer of Pm51 to wheat breeding programs.

Common Wheat Chromosome 5B Composition Analysis Using Low‐Coverage 454 Sequencing

The sequencing of individual chromosomes of common wheat is in progress. The molecular size of wheat chromosome 5B is nearly 870 Mb (5BL = 580 Mb and 5BS = 290 Mb). We produced the first low coverage 454‐sequencing of the long and short arms of wheat chromosome 5B (110,793 and 39,695 reads, which compose 8 and 6% of total 5BL and 5BS length, respectively) and calculated the ratios of the different families of repetitive sequences, including transposable elements (TEs), satellite repeats (Afa, pSc119.2, 5S rDNA and 45S rDNA), and microsatellites, as well as direct and inverted repeat motifs. The TEs accounted for 70% of the total analyzed nucleotide sequences. The content of the Cereba retrotransposon family differed between the two arms of chromosome 5B. Comparing the reads of chromosome 5B with the data from chromosome 5A, we found the retrotransposons Fatima and Sakura and DNA transposon Jorge were prevalent in 5B. The hypothetical coding sequences accounted for 2.0% of the short arm and 2.07% of the long arm. Using in silico mapping, we identified the regions of synteny with rice and Brachypodium chromosomes (1,073,526 and 1,767,298 bp aligned, respectively), and the result was consistent with the data from the expressed sequence tag (EST) mapping of wheat 5B chromosome to the genomes of these grasses. Thus, these results show that low coverage survey sequencing can provide useful information about the composition and evolution of wheat chromosome 5B.

Fine physical and genetic mapping of powdery mildew resistance gene MlIW172 originating from wild emmer (Triticum dicoccoides).

Powdery mildew, caused by Blumeria graminis f. sp. tritici, is one of the most important wheat diseases in the world. In this study, a single dominant powdery mildew resistance gene MlIW172 was identified in the IW172 wild emmer accession and mapped to the distal region of chromosome arm 7AL (bin7AL-16-0.86-0.90) via molecular marker analysis. MlIW172 was closely linked with the RFLP probe Xpsr680-derived STS marker Xmag2185 and the EST markers BE405531 and BE637476. This suggested that MlIW172 might be allelic to the Pm1 locus or a new locus closely linked to Pm1. By screening genomic BAC library of durum wheat cv. Langdon and 7AL-specific BAC library of hexaploid wheat cv. Chinese Spring, and after analyzing genome scaffolds of Triticum urartu containing the marker sequences, additional markers were developed to construct a fine genetic linkage map on the MlIW172 locus region and to delineate the resistance gene within a 0.48 cM interval. Comparative genetics analyses using ESTs and RFLP probe sequences flanking the MlIW172 region against other grass species revealed a general co-linearity in this region with the orthologous genomic regions of rice chromosome 6, Brachypodium chromosome 1, and sorghum chromosome 10. However, orthologous resistance gene-like RGA sequences were only present in wheat and Brachypodium. The BAC contigs and sequence scaffolds that we have developed provide a framework for the physical mapping and map-based cloning of MlIW172.

Genetic control of rhizomes and genomic localization of a major-effect growth habit QTL in perennial wildrye

Rhizomes are prostrate subterranean stems that provide primitive mechanisms of vegetative dispersal, survival, and regrowth of perennial grasses and other monocots. The extent of rhizome proliferation varies greatly among grasses, being absent in cereals and other annuals, strictly confined in caespitose perennials, or highly invasive in some perennial weeds. However, genetic studies of rhizome proliferation are limited and genes controlling rhizomatous growth habit have not been elucidated. Quantitative trait loci (QTLs) controlling rhizome spreading were compared in reciprocal backcross populations derived from hybrids of rhizomatous creeping wildrye (Leymus triticoides) and caespitose basin wildrye (L. cinereus), which are perennial relatives of wheat. Two recessive QTLs were unique to the creeping wildrye backcross, one dominant QTL was unique to the basin wildrye backcross, and one additive QTL was detectable in reciprocal backcrosses with high log odds (LOD = 31.6) in the basin wildrye background. The dominant QTL located on linkage group (LG)-2a was aligned to a dominant rhizome orthogene (Rhz3) of perennial rice (Oryza longistamina) and perennial sorghum (Sorghum propinquum). Nonparametric 99 % confidence bounds of the 31.6-LOD QTL were localized to a distal 3.8-centiMorgan region of LG-6a, which corresponds to a 0.7-Mb region of Brachypodium Chromosome 3 containing 106 genes. An Aux/IAA auxin signal factor gene was located at the 31.6-LOD peak, which could explain the gravitropic and aphototropic behavior of rhizomes. Findings elucidate genetic mechanisms controlling rhizome development and architectural growth habit differences among plant species. Results have possible applications to improve perennial forage and turf grasses, extend the vegetative life cycle of annual cereals, such as wheat, or control the invasiveness of highly rhizomatous weeds such as quackgrass (Elymus repens).

Barley and Wheat Share the Same Gene Controlling the Short Basic Vegetative Period

Basic vegetative period (BVP) is an important trait for determining flowering time and adaptation to variable environments. A short BVP barley mutant is about 30 d shorter than its wild type. Genetic analysis using 557 F2 individuals revealed that the short BVP is governed by a single recessive gene (BVP-1) and was further validated in 2 090 F3 individuals. The BVP-1 gene was first mapped to barley chromosome 1H using SSR markers. Comparative genomic analysis demonstrated that the chromosome region of BVP-1 is syntenic to rice chromosome 5 and Brachypodium chromosome 2. Barley ESTs/genes were identified after comparison with candidate genes in rice and Brachypodium; seven new gene-specific markers were developed and mapped in the mapping populations. The BVP-1 gene co-segregated with the Mot1 and Ftsh4 genes and was flanked by the gene-specific markers AK252360 (0.2 cM) and CA608558 (0.5 cM). Further analysis demonstrated that barley and wheat share the same short BVP gene controlling early flowering.

Development of 5123 Intron-Length Polymorphic Markers for Large-Scale Genotyping Applications in Foxtail Millet

Generating genomic resources in terms of molecular markers is imperative in molecular breeding for crop improvement. Though development and application of microsatellite markers in large-scale was reported in the model crop foxtail millet, no such large-scale study was conducted for intron-length polymorphic (ILP) markers. Considering this, we developed 5123 ILP markers, of which 4049 were physically mapped onto 9 chromosomes of foxtail millet. BLAST analysis of 5123 expressed sequence tags (ESTs) suggested the function for ∼71.5% ESTs and grouped them into 5 different functional categories. About 440 selected primer pairs representing the foxtail millet genome and the different functional groups showed high-level of cross-genera amplification at an average of ∼85% in eight millets and five non-millet species. The efficacy of the ILP markers for distinguishing the foxtail millet is demonstrated by observed heterozygosity (0.20) and Nei's average gene diversity (0.22). In silico comparative mapping of physically mapped ILP markers demonstrated substantial percentage of sequence-based orthology and syntenic relationship between foxtail millet chromosomes and sorghum (∼50%), maize (∼46%), rice (∼21%) and Brachypodium (∼21%) chromosomes. Hence, for the first time, we developed large-scale ILP markers in foxtail millet and demonstrated their utility in germplasm characterization, transferability, phylogenetics and comparative mapping studies in millets and bioenergy grass species.

High mature grain phytase activity in the Triticeae has evolved by duplication followed by neofunctionalization of the purple acid phosphatase phytase (PAPhy) gene.

The phytase activity in food and feedstuffs is an important nutritional parameter. Members of the Triticeae tribe accumulate purple acid phosphatase phytases (PAPhy) during grain filling. This accumulation elevates mature grain phytase activities (MGPA) up to levels between ~650 FTU/kg for barley and 6000 FTU/kg for rye. This is notably more than other cereals. For instance, rice, maize, and oat have MGPAs below 100 FTU/kg. The cloning and characterization of the PAPhy gene complement from wheat, barley, rye, einkorn, and Aegilops tauschii is reported here. The Triticeae PAPhy genes generally consist of a set of paralogues, PAPhy_a and PAPhy_b, and have been mapped to Triticeae chromosomes 5 and 3, respectively. The promoters share a conserved core but the PAPhy_a promoter have acquired a novel cis-acting regulatory element for expression during grain filling while the PAPhy_b promoter has maintained the archaic function and drives expression during germination. Brachypodium is the only sequenced Poaceae sharing the PAPhy duplication. As for the Triticeae, the duplication is reflected in a high MGPA of ~4200 FTU/kg in Brachypodium. The sequence conservation of the paralogous loci on Brachypodium chromosomes 1 and 2 does not extend beyond the PAPhy gene. The results indicate that a single-gene segmental duplication may have enabled the evolution of high MGPA by creating functional redundancy of the parent PAPhy gene. This implies that similar MGPA levels may be out of reach in breeding programs for some Poaceae, e.g. maize and rice, whereas Triticeae breeders should focus on PAPhy_a.

High-density mapping and marker development for the powdery mildew resistance gene PmAS846 derived from wild emmer wheat (Triticum turgidum var. dicoccoides)

Powdery mildew, caused by Blumeria graminis f. sp. tritici, is an important foliar disease of wheat worldwide. The dominant powdery mildew resistance gene PmAS846 was transferred to the hexaploid wheat lines N9134 and N9738 from wild emmer wheat (Triticum dicoccoides) in 1995, and it is still one of the most effective resistance genes in China. A high resolution genetic map for PmAS846 locus was constructed using two F(2) populations and corresponding F(2:3) families developed from the crosses of N9134/Shaanyou 225 and N9738/Huixianhong. Synteny between wheat and Brachypodium distachyon and rice was used to develop closely linked molecular markers to reduce the genetic interval around PmAS846. Twenty-six expressed sequence tag-derived markers were mapped to the PmAS846 locus. Five markers co-segregated with PmAS846 in the F(2) population of N9134/Shaanyou 225. PmAS846 was physically located to wheat chromosome 5BL bin 0.75-0.76 within a gene-rich region. The markers order is conserved between wheat and Brachypodium distachyon, but rearrangements are present in rice. Two markers, BJ261635 and CJ840011 flanked PmAS846 and narrowed PmAS846 to a region that is collinear with 197 and 112kb genomic regions on Brachypodium chromosome 4 and rice chromosome 9, respectively. The genes located on the corresponding homologous regions in Brachypodium, rice and barley could be considered for further marker saturation and identification of potential candidate genes for PmAS846. The markers co-segregating with PmAS846 provide a potential target site for positional cloning of PmAS846, and can be used for marker-assisted selection of this gene.

Comparative DNA Sequence Analysis Involving Wheat, Brachypodium and Rice Genomes Using Mapped Wheat ESTs

Comparative genomics is a powerful approach to transfer genomic information from a sequenced genome to closely related species. We conducted a comparative genomics study between wheat and brachypodium genomes, using 8 210 mapped wESTs for BLASTn against the high-quality genome sequence of brachypodium. As many as 5 208 wESTs spread over all the 21 wheat chromosomes showed significant similarity with genomic regions in all the five brachypodium chromosomes (Bd1 to Bd5). Wheat ESTs belonging to seven homoeologous groups were also used to construct seven consensus maps (WC1 to WC7) for wheat chromosomes. Each consensus wheat chromosome matched only one or two brachypodium chromosomes. Following are the broad syntenic relationship that were observed between brachypodium and wheat chromosomes: WC1-Bd2/Bd3, WC2-Bd1/Bd5, WC3-Bd2, WC4-Bd1, WC5-Bd4/Bd1/Bd3, WC6-Bd3 and WC7-Bd1/Bd3. Eighty two (82) conserved syntenic blocks were also identified between wheat and brachypodium genomes. Ka/Ks analysis was performed on 153 sequence pairs, of which 98 had Ka/Ks ratio <1 suggesting their evolution under purifying selection involving no divergence in encoded amino acids. Results of this study suggested that after divergence from a common ancestral genome the common syntenic features maintained between genomes of wheat and brachypodium.

First Survey of the Wheat Chromosome 5A Composition through a Next Generation Sequencing Approach

Wheat is one of the world's most important crops and is characterized by a large polyploid genome. One way to reduce genome complexity is to isolate single chromosomes using flow cytometry. Low coverage DNA sequencing can provide a snapshot of individual chromosomes, allowing a fast characterization of their main features and comparison with other genomes. We used massively parallel 454 pyrosequencing to obtain a 2x coverage of wheat chromosome 5A. The resulting sequence assembly was used to identify TEs, genes and miRNAs, as well as to infer a virtual gene order based on the synteny with other grass genomes. Repetitive elements account for more than 75% of the genome. Gene content was estimated considering non-redundant reads showing at least one match to ESTs or proteins. The results indicate that the coding fraction represents 1.08% and 1.3% of the short and long arm respectively, projecting the number of genes of the whole chromosome to approximately 5,000. 195 candidate miRNA precursors belonging to 16 miRNA families were identified. The 5A genes were used to search for syntenic relationships between grass genomes. The short arm is closely related to Brachypodium chromosome 4, sorghum chromosome 8 and rice chromosome 12; the long arm to regions of Brachypodium chromosomes 4 and 1, sorghum chromosomes 1 and 2 and rice chromosomes 9 and 3. From these similarities it was possible to infer the virtual gene order of 392 (5AS) and 1,480 (5AL) genes of chromosome 5A, which was compared to, and found to be largely congruent with the available physical map of this chromosome.

Leymus EST linkage maps identify 4NsL–5NsL reciprocal translocation, wheat-Leymus chromosome introgressions, and functionally important gene loci

Allotetraploid (2n=4x=28) Leymus triticoides and Leymus cinereus are divergent perennial grasses, which form fertile hybrids. Genetic maps with n=14 linkage groups (LG) comprised with 1,583 AFLP and 67 heterologous anchor markers were previously used for mapping quantitative trait loci (QTLs) in these hybrids, and chromosomes of other Leymus wildryes have been transferred to wheat. However, identifications of the x=7 homoeologous groups were tenuous and genetic research has been encumbered by a lack of functional, conserved gene marker sequences. Herein, we mapped 350 simple sequence repeats and 26 putative lignin biosynthesis genes from a new Leymus EST library and constructed one integrated consensus map with 799 markers, including 375 AFLPs and 48 heterologous markers, spanning 2,381 centiMorgans. LG1b and LG6b were reassigned as LG6b* and LG1b*, respectively, and LG4Ns and LG4Xm were inverted so that all 14 linkage groups are aligned to the x=7 Triticeae chromosomes based on EST alignments to barley and other reference genomes. Amplification of 146 mapped Leymus ESTs representing six of the seven homoeologous groups was shown for 17 wheat-Leymus chromosome introgression lines. Reciprocal translocations between 4L and 5L in both Leymus and Triticum monococcum were aligned to the same regions of Brachypodium chromosome 1. A caffeic acid O-methyltransferase locus aligned to fiber QTL peaks on Leymus LG7a and brown midrib mutations of maize and sorghum. Glaucousness genes on Leymus and wheat chromosome 2 were aligned to the same region of Brachypodium chromosome 5. Markers linked to the S self-incompatibility gene on Leymus LG1a cosegregated with markers on LG2b, possibly cross-linked by gametophytic selection. Homoeologous chromosomes 1 and 2 harbor the S and Z gametophytic self-incompatibility genes of Phalaris, Secale, and Lolium, but the Leymus chromosome-2 self-incompatibility gene aligns to a different region on Brachypodium chromosome 5. Nevertheless, cosegregation of self-incompatibility genes on Leymus presents a powerful system for mapping these loci.

Fine mapping and comparative genomics integration of two quantitative trait loci controlling resistance to powdery mildew in a Spanish barley landrace

The intervals containing two major quantitative trait loci (QTL) from a Spanish barley landrace conferring broad spectrum resistance to Blumeria graminis were subjected to marker saturation. First, all the available information on recently developed marker resources for barley was exploited. Then, a comparative genomic analysis of the QTL regions with other sequenced grass model species was performed. As a result of the first step, 32 new markers were added to the previous map and new flanking markers closer to both QTL were identified. Next, syntenic integration revealed that the barley target regions showed homology with regions on chromosome 6 of rice (Oryza sativa), chromosome 10 of Sorghum bicolor and chromosome 1 of Brachypodium distachyon. A nested insertion of ancestral syntenic blocks on Brachypodium chromosome 1 was confirmed. Based on sequence information of the most likely candidate orthologous genes, 23 new barley unigene-derived markers were developed and mapped within the barley target regions. The assessment of colinearity revealed an inversion on chromosome 7HL of barley compared to the other three grass species, and nearly perfect colinearity on chromosome 7HS. This two-step marker enrichment allowed for the refinement of the two QTL into much smaller intervals. Inspection of all predicted proteins for the barley unigenes identified within the QTL intervals did not reveal the presence of resistance gene candidates. This study demonstrates the usefulness of sequenced genomes for fine mapping and paves the way for the use of these two loci in barley breeding programs.