New DArT markers for oat provide enhanced map coverage and global germplasm characterization

BACKGROUND: Genomic discovery in oat and its application to oat improvement have been hindered by a lack of genetic markers common to different genetic maps, and by the difficulty of conducting whole-genome analysis using high-throughput markers. This study was intended to develop, characterize, and apply a large set of oat genetic markers based on Diversity Array Technology (DArT). RESULTS: Approximately 19,000 genomic clones were isolated from complexity-reduced genomic representations of pooled DNA samples from 60 oat varieties of global origin. These were screened on three discovery arrays, with more than 2000 polymorphic markers being identified for use in this study, and approximately 2700 potentially polymorphic markers being identified for use in future studies. DNA sequence was obtained for 2573 clones and assembled into a non-redundant set of 1770 contigs and singletons. Of these, 705 showed highly significant (Expectation < 10E-10) BLAST similarity to gene sequences in public databases. Based on marker scores in 80 recombinant inbred lines, 1010 new DArT markers were used to saturate and improve the 'Kanota' × 'Ogle' genetic map. DArT markers provided map coverage approximately equivalent to existing markers. After binning markers from similar clones, as well as those with 99% scoring similarity, a set of 1295 non-redundant markers was used to analyze genetic diversity in 182 accessions of cultivated oat of worldwide origin. Results of this analysis confirmed that major clusters of oat diversity are related to spring vs. winter type, and to the presence of major breeding programs within geographical regions. Secondary clusters revealed groups that were often related to known pedigree structure. CONCLUSION: These markers will provide a solid basis for future efforts in genomic discovery, comparative mapping, and the generation of an oat consensus map. They will also provide new opportunities for directed breeding of superior oat varieties, and guidance in the maintenance of oat genetic diversity

Population Genomics Related to Adaptation in Elite Oat Germplasm

Six hundred thirty five oat ( L.) lines and 4561 single-nucleotide polymorphism (SNP) loci were used to evaluate population structure, linkage disequilibrium (LD), and genotype–phenotype association with heading date. The first five principal components (PCs) accounted for 25.3% of genetic variation. Neither the eigenvalues of the first 25 PCs nor the cross-validation errors from = 1 to 20 model-based analyses suggested a structured population. However, the PC and = 2 model-based analyses supported clustering of lines on spring oat vs. southern United States origin, accounting for 16% of genetic variation ( < 0.0001). Single-locus -statistic () in the highest 1% of the distribution suggested linkage groups that may be differentiated between the two population subgroups. Population structure and kinship-corrected LD of = 0.10 was observed at an average pairwise distance of 0.44 cM (0.71 and 2.64 cM within spring and southern oat, respectively). On most linkage groups LD decay was slower within southern lines than within the spring lines. A notable exception was found on linkage group Mrg28, where LD decay was substantially slower in the spring subpopulation. It is speculated that this may be caused by a heterogeneous translocation event on this chromosome. Association with heading date was most consistent across location-years on linkage groups Mrg02, Mrg12, Mrg13, and Mrg24

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

Polymorphism in the Barley Granule Bound Starch Synthase 1 (<i>Gbss1</i>) Gene Associated with Grain Starch Variant Amylose Concentration

Granule bound starch synthase 1 (GBSS1) accumulation within starch granules and structure of <i>Gbss1</i> alleles were determined for nine barley (Hordeum vulgare L.) genotypes producing amylose-free (undetectable), near-waxy (1.6–4.5%), normal (25.8%), and increased (38.0–40.8%) amylose grain starches. Compared to normal starch granules, GBSS1 accumulation was severely reduced in three near-waxy, slightly reduced in two waxy, and slightly elevated in three increased amylose starches. <i>Gbss1</i> nucleotide sequence analysis for the nine genotypes distinguished them into three <i>Gbss1</i> groups with several single-nucleotide polymorphisms. A new unique Q312H substitution within GBSS1 was discovered in near-waxy genotype SB94912 with reduced amylose (1.6%) concentration relative to the other two near-waxy lines, CDC Rattan and CDC Candle (4.5%). The two waxy genotype GBSS1 showed a previously described D287V change for CDC Alamo and a new G513W change for CDC Fibar. Both amino acid alterations are conserved residues within starch synthase domains involved in glucan interaction. The increased amylose genotypes showed several unique nucleotide changes within the second and fourth <i>Gbss1</i> introns, but only SB94893 GBSS1 showed a unique amino acid substitution, A250T in exon 6. The <i>Gbss1</i> nucleotide differences were used to design genetic markers to monitor <i>Gbss1</i> alleles in genotypes with various amylose grain starches

Development of Barley (<i>Hordeum vulgare</i> L.) Lines with Altered Starch Granule Size Distribution

Microscope analysis of starches prepared from 139 barley genotypes identified a Japanese genotype, Kinai Kyoshinkai-2 (KK-2), with altered starch granule size distribution. Compared to normal barley starch, KK-2 produced consistently higher volumes of starch granules with 5–15 μm diameter and reduced volumes of starch granules with >15 μm diameter when grown in different environments. A cross between KK-2 and normal starch cultivar CDC Kendall was made and led to the production of 154 F₅ lines with alterations to the normal 7:3:1 distribution for A-:B-:C-type starch granule volumes. Three F₅ lines showed unimodal starch granule size distribution due to apparent lack of very small (<5.0 μm diameter) C-type starch granules, but the phenotype was accompanied by reduced grain weight and total starch concentration. Five F₅ lines produced a significantly larger population of large (>15 μm diameter) A-type starch granules as compared to normal starch and showed on average a 10:4:1 distribution for A-:B-:C-type starch granule volumes. The unusual starch phenotypes displayed by the F₅ lines confirm starch granule size distribution in barley can be genetically altered