Rebekah E. Oliver

Model SNP development for complex genomes based on hexaploid oat using high-throughput 454 sequencing technology

BackgroundGenetic markers are pivotal to modern genomics research; however, discovery and genotyping of molecular markers in oat has been hindered by the size and complexity of the genome, and by a scarcity of sequence data. The purpose of this study was to generate oat expressed sequence tag (EST) information, develop a bioinformatics pipeline for SNP discovery, and establish a method for rapid, cost-effective, and straightforward genotyping of SNP markers in complex polyploid genomes such as oat.ResultsBased on cDNA libraries of four cultivated oat genotypes, approximately 127,000 contigs were assembled from approximately one million Roche 454 sequence reads. Contigs were filtered through a novel bioinformatics pipeline to eliminate ambiguous polymorphism caused by subgenome homology, and 96 in silico SNPs were selected from 9,448 candidate loci for validation using high-resolution melting (HRM) analysis. Of these, 52 (54%) were polymorphic between parents of the Ogle1040 × TAM O-301 (OT) mapping population, with 48 segregating as single Mendelian loci, and 44 being placed on the existing OT linkage map. Ogle and TAM amplicons from 12 primers were sequenced for SNP validation, revealing complex polymorphism in seven amplicons but general sequence conservation within SNP loci. Whole-amplicon interrogation with HRM revealed insertions, deletions, and heterozygotes in secondary oat germplasm pools, generating multiple alleles at some primer targets. To validate marker utility, 36 SNP assays were used to evaluate the genetic diversity of 34 diverse oat genotypes. Dendrogram clusters corresponded generally to known genome composition and genetic ancestry.ConclusionsThe high-throughput SNP discovery pipeline presented here is a rapid and effective method for identification of polymorphic SNP alleles in the oat genome. The current-generation HRM system is a simple and highly-informative platform for SNP genotyping. These techniques provide a model for SNP discovery and genotyping in other species with complex and poorly-characterized genomes.

SNP Discovery and Chromosome Anchoring Provide the First Physically-Anchored Hexaploid Oat Map and Reveal Synteny with Model Species

A physically anchored consensus map is foundational to modern genomics research; however, construction of such a map in oat (Avena sativa L., 2n = 6x = 42) has been hindered by the size and complexity of the genome, the scarcity of robust molecular markers, and the lack of aneuploid stocks. Resources developed in this study include a modified SNP discovery method for complex genomes, a diverse set of oat SNP markers, and a novel chromosome-deficient SNP anchoring strategy. These resources were applied to build the first complete, physically-anchored consensus map of hexaploid oat. Approximately 11,000 high-confidence in silico SNPs were discovered based on nine million inter-varietal sequence reads of genomic and cDNA origin. GoldenGate genotyping of 3,072 SNP assays yielded 1,311 robust markers, of which 985 were mapped in 390 recombinant-inbred lines from six bi-parental mapping populations ranging in size from 49 to 97 progeny. The consensus map included 985 SNPs and 68 previously-published markers, resolving 21 linkage groups with a total map distance of 1,838.8 cM. Consensus linkage groups were assigned to 21 chromosomes using SNP deletion analysis of chromosome-deficient monosomic hybrid stocks. Alignments with sequenced genomes of rice and Brachypodium provide evidence for extensive conservation of genomic regions, and renewed encouragement for orthology-based genomic discovery in this important hexaploid species. These results also provide a framework for high-resolution genetic analysis in oat, and a model for marker development and map construction in other species with complex genomes and limited resources.

Molecular cytogenetic characterization of four partial wheat-Thinopyrum ponticum amphiploids and their reactions to Fusarium head blight, tan spot, and Stagonospora nodorum blotch

Four wheat (Triticum aestivum L.)-Thinopyrum ponticum derivatives SS5 (PI604926), SS156 (PI604947), SS363 (PI604970), and SS660 (PI604879), were identified as resistant to Fusarium head blight (FHB), a serious fungal disease of wheat worldwide. Seedling reactions to tan spot and Stagonospora nodorum blotch (SNB), two important foliar diseases of wheat, suggest that these four derivatives are resistant to tan spot and two of them (SS5 and SS156) are resistant to SNB. Fluorescent genomic in situ hybridization (FGISH) patterns of mitotic chromosomes indicate that these four derivatives are partial wheat-Th. ponticum amphiploids, each with a total of 56 chromosomes, though with different amounts of Th. ponticum chromatin. These four amphiploids were hybridized with each other to determine homology between the Th. ponticum genomes in each of the amphiploids. Analysis of chromosome pairing in the F1 hybrids using FGISH suggests that each amphiploid carries a similar set of Th. ponticum chromosomes. These wheat-Th. ponticum amphiploids represent a potential novel source of resistance to FHB, tan spot, and SNB for wheat breeding.

Utilization of alien genes to enhance Fusarium head blight resistance in wheat – A review

Fusarium head blight (FHB) is a destructive disease of wheat worldwide. Sources of resistance to FHB are limited in wheat. Search for novel sources of effective resistance to this disease has been an urgent need in wheat breeding. Fusarium head blight resistance has been identified in relatives of wheat. Alien chromatin carrying FHB resistance genes has been incorporated into wheat through chromosome addition, substitution, and translocation. Relatives of wheat demonstrate a great potential to enhance resistance of wheat to FHB.

A SNP Genotyping Array for Hexaploid Oat

Recognizing a need in cultivated hexaploid oat (Avena sativa L.) for a reliable set of reference single nucleotide polymorphisms (SNPs), we have developed a 6000 (6K) BeadChip design containing 257 Infinium I and 5486 Infinium II designs corresponding to 5743 SNPs. Of those, 4975 SNPs yielded successful assays after array manufacturing. These SNPs were discovered based on a variety of bioinformatics pipelines in complementary DNA (cDNA) and genomic DNA originating from 20 or more diverse oat cultivars. The array was validated in 1100 samples from six recombinant inbred line (RIL) mapping populations and sets of diverse oat cultivars and breeding lines, and provided approximately 3500 discernible Mendelian polymorphisms. Here, we present an annotation of these SNPs, including methods of discovery, gene identification and orthology, population‐genetic characteristics, and tentative positions on an oat consensus map. We also evaluate a new cluster‐based method of calling SNPs. The SNP design sequences are made publicly available, and the full SNP genotyping platform is available for commercial purchase from an independent third party.

A consensus map in cultivated hexaploid oat reveals conserved grass synteny with substantial subgenome rearrangement

We constructed a hexaploid oat consensus map from 12 populations representing 19 parents. The map represents the most common physical chromosome arrangements in oat. Deviations from the consensus map may indicate physical rearrangements. Large chromosomal translocations vary among different varieties. There is regional synteny with rice but considerable subgenome rearrangement.

Homoeology of Thinopyrum junceum and Elymus rectisetus chromosomes to wheat and disease resistance conferred by the Thinopyrum and Elymus chromosomes in wheat

Thirteen common wheat “Chinese Spring” (CS)-Thinopyrum junceum addition lines and three common wheat “Fukuhokomuji”(Fuku)-Elymus rectisetus addition lines were characterized and verified as disomic additions of a Th. junceum or E. rectisetus chromosome in the wheat backgrounds by fluorescent genomic in situ hybridization. Another Fuku-E. rectisetus addition line, A1048, was found to contain multiple segregating E. rectisetus chromosomes. Seven partial CS-Th. junceum amphiploids were identified to combine 12–16 Th. junceum chromosomes with CS wheat chromosomes. The disomic addition lines AJDAj5, 7, 8, 9, and HD3508 were identified to contain a Th. junceum chromosome in homoeologous group 1. Two of them, AJDAj7 and AJDAj9, had the same Th. junceum chromosome. AJDAj2, 3, and 4 contained a Th. junceum chromosome in group 2, HD3505 in group 4, AJDAj6 and AJDAj11 in group 5, and AJDAj1 probably in group 6. The disomic addition lines A1026 and A1057 were identified to carry an E. rectisetus chromosome in group 1 and A1034 in group 5. E. rectisetus chromosomes in groups 1–6 were detected in A1048. The homoeologous group of the Th. junceum chromosome in HD3515 could not be determined in this study. Several Th. junceum and E. rectisetus chromosomes in the addition lines were found to contain genes for resistance to Fusarium head blight, tan spot, Stagonospora nodorum blotch, and stem rust (Ug99 races). Understanding of the homoeology of the Th. junceum and E. rectisetus chromosomes with wheat will facilitate utilization of the favorable genes on these alien chromosomes in wheat improvement.

Comparative Systems Biology Reveals Allelic Variation Modulating Tocochromanol Profiles in Barley (Hordeum vulgare L.)

Tocochromanols are recognized for nutritional content, plant stress response, and seed longevity. Here we present a systems biological approach to characterize and develop predictive assays for genes affecting tocochromanol variation in barley. Major QTL, detected in three regions of a SNP linkage map, affected multiple tocochromanol forms. Candidate genes were identified through barley/rice orthology and sequenced in genotypes with disparate tocochromanol profiles. Gene-specific markers, designed based on observed polymorphism, mapped to the originating QTL, increasing R2 values at the respective loci. Polymorphism within promoter regions corresponded to motifs known to influence gene expression. Quantitative PCR analysis revealed a trend of increased expression in tissues grown at cold temperatures. These results demonstrate utility of a novel method for rapid gene identification and characterization, and provide a resource for efficient development of barley lines with improved tocochromanol profiles.

Genome-Wide Association Mapping of Barley Yellow Dwarf Virus Tolerance in Spring Oat (Avena sativa L.)

Barley yellow dwarf viruses (BYDVs) are responsible for the disease barley yellow dwarf (BYD) and affect many cereals including oat (Avena sativa L.). Until recently, the molecular marker technology in oat has not allowed for many marker-trait association studies to determine the genetic mechanisms for tolerance. A genome-wide association study (GWAS) was performed on 428 spring oat lines using a recently developed high-density oat single nucleotide polymorphism (SNP) array as well as a SNP-based consensus map. Marker-trait associations were performed using a Q-K mixed model approach to control for population structure and relatedness. Six significant SNP-trait associations representing two QTL were found on chromosomes 3C (Mrg17) and 18D (Mrg04). This is the first report of BYDV tolerance QTL on chromosome 3C (Mrg17) and 18D (Mrg04). Haplotypes using the two QTL were evaluated and distinct classes for tolerance were identified based on the number of favorable alleles. A large number of lines carrying both favorable alleles were observed in the panel.