Natural Genetic Variation Underlying Tiller Development in Barley (Hordeum vulgare L).

In barley (Hordeum vulgare L.), lateral branches called tillers contribute to grain yield and define shoot architecture, but genetic control of tiller number and developmental rate are not well characterized. The primary objectives of this work were to examine relationships between tiller number and other agronomic and morphological traits and identify natural genetic variation associated with tiller number and rate, and related traits. We grew 768 lines from the USDA National Small Grain Collection in the field and collected data over two years for tiller number and rate, and agronomic and morphological traits. Our results confirmed that spike row-type and days to heading are correlated with tiller number, and as much as 28% of tiller number variance was associated with these traits. In addition, negative correlations between tiller number and leaf width and stem diameter were observed, indicating trade-offs between tiller development and other vegetative growth. Thirty-three quantitative trait loci (QTL) were associated with tiller number or rate. Of these, 40% overlapped QTL associated with days to heading and 22% overlapped QTL associated with spike row-type, further supporting that tiller development is associated with these traits. Some QTL associated with tiller number or rate, including the major QTL on chromosome 3H, were not associated with other traits, suggesting that some QTL may be directly related to rate of tiller development or axillary bud number. These results enhance our knowledge of the genetic control of tiller development in barley, which is important for optimizing tiller number and rate for yield improvement.

Single nucleotide polymorphisms in a regulatory site of VRN-A1 first intron are associated with differences in vernalization requirement in winter wheat.

Winter wheats require a long exposure to cold temperatures (vernalization) to accelerate flowering. However, varieties differ in the length of the period of cold required to saturate the vernalization response. Here we show that single nucleotide polymorphisms (SNP) at the binding site of the GRP2 protein in the VRN-A1 first intron (henceforth, RIP3) are associated with significant differences in heading time after a partial vernalization treatment. The ancestral winter VRN-A1 allele in 'Triple Dirk C' has one SNP in the RIP3 region (1_SNP) relative to the canonical RIP3 sequence, whereas the derived 'Jagger' allele has three SNPs (3_SNPs). Both varieties have a single VRN-A1 copy encoding identical proteins. In an F2 population generated from a cross between these two varieties, plants with the 3_SNPs haplotype headed significantly earlier (P < 0.001) than those with the 1_SNP haplotype, both in the absence of vernalization (17 days difference) and after 3-weeks of vernalization (11 days difference). Plants with the 3_SNPs haplotype showed higher VRN-A1 transcript levels than those with the 1_SNP haplotype. The 3_SNPs haplotype was also associated with early heading in a panel of 127 winter wheat varieties grown in three separate controlled-environment experiments under partial vernalization (36 to 54 days, P < 0.001) and one experiment under field conditions (21 d, P < 0.0001). The RIP3 polymorphisms can be used by wheat breeders to develop winter wheat varieties adapted to regions with different duration or intensity of the cold season.

Development and Genetic Characterization of an Advanced Backcross-Nested Association Mapping (AB-NAM) Population of Wild × Cultivated Barley.

The ability to access alleles from unadapted germplasm collections is a long-standing problem for geneticists and breeders. Here we developed, characterized, and demonstrated the utility of a wild barley advanced backcross-nested association mapping (AB-NAM) population. We developed this population by backcrossing 25 wild barley accessions to the six-rowed malting barley cultivar Rasmusson. The 25 wild barley parents were selected from the 318 accession Wild Barley Diversity Collection (WBDC) to maximize allelic diversity. The resulting 796 BC2F4:6 lines were genotyped with 384 SNP markers, and an additional 4022 SNPs and 263,531 sequence variants were imputed onto the population using 9K iSelect SNP genotypes and exome capture sequence of the parents, respectively. On average, 96% of each wild parent was introgressed into the Rasmusson background, and the population exhibited low population structure. While linkage disequilibrium (LD) decay (r(2) = 0.2) was lowest in the WBDC (0.36 cM), the AB-NAM (9.2 cM) exhibited more rapid LD decay than comparable advanced backcross (28.6 cM) and recombinant inbred line (32.3 cM) populations. Three qualitative traits: glossy spike, glossy sheath, and black hull color were mapped with high resolution to loci corresponding to known barley mutants for these traits. Additionally, a total of 10 QTL were identified for grain protein content. The combination of low LD, negligible population structure, and high diversity in an adapted background make the AB-NAM an important tool for high-resolution gene mapping and discovery of novel allelic variation using wild barley germplasm.

Genome-wide comparative diversity uncovers multiple targets of selection for improvement in hexaploid wheat landraces and cultivars.

Domesticated crops experience strong human-mediated selection aimed at developing high-yielding varieties adapted to local conditions. To detect regions of the wheat genome subject to selection during improvement, we developed a high-throughput array to interrogate 9,000 gene-associated single-nucleotide polymorphisms (SNP) in a worldwide sample of 2,994 accessions of hexaploid wheat including landraces and modern cultivars. Using a SNP-based diversity map we characterized the impact of crop improvement on genomic and geographic patterns of genetic diversity. We found evidence of a small population bottleneck and extensive use of ancestral variation often traceable to founders of cultivars from diverse geographic regions. Analyzing genetic differentiation among populations and the extent of haplotype sharing, we identified allelic variants subjected to selection during improvement. Selective sweeps were found around genes involved in the regulation of flowering time and phenology. An introgression of a wild relative-derived gene conferring resistance to a fungal pathogen was detected by haplotype-based analysis. Comparing selective sweeps identified in different populations, we show that selection likely acts on distinct targets or multiple functionally equivalent alleles in different portions of the geographic range of wheat. The majority of the selected alleles were present at low frequency in local populations, suggesting either weak selection pressure or temporal variation in the targets of directional selection during breeding probably associated with changing agricultural practices or environmental conditions. The developed SNP chip and map of genetic variation provide a resource for advancing wheat breeding and supporting future population genomic and genome-wide association studies in wheat.

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.

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

BACKGROUND: Genetic 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. RESULTS: Based 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. CONCLUSIONS: The 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.

Microsatellite-based deletion bin system for the establishment of genetic-physical map relationships in wheat (Triticum aestivum L.).

Because of polyploidy and large genome size, deletion stocks of bread wheat are an ideal material for physically allocating ESTs and genes to small chromosomal regions for targeted mapping. To enhance the utility of deletion stocks for chromosome bin mapping, we characterized a set of 84 deletion lines covering the 21 chromosomes of wheat using 725 microsatellites. We localized these microsatellite loci to 94 breakpoints in a homozygous state (88 distal deletions, 6 interstitial), and 5 in a heterozygous state representing 159 deletion bins. Chromosomes from homoeologous groups 2 and 5 were the best covered (126 and 125 microsatellites, respectively) while the coverage for group 4 was lower (80 microsatellites). We assigned at least one microsatellite in up to 92% of the bins (mean 4.97 SSR/bin). Only a few discrepancies concerning marker order were observed. The cytogenetic maps revealed small genetic distances over large physical regions around the centromeres and large genetic to physical map ratios close to the telomeres. As SSRs are the markers of choice for many genetic and breeding studies, the mapped microsatellite loci will be useful not only for deletion stock verifications but also for allocating associated QTLs to deletion bins where numerous ESTs that could be potential candidate genes are currently assigned.

Inheritance of Resistance to Stagonospora nodorum Leaf Blotch in Kansas Winter Wheat Cultivars.

Stagonospora nodorum blotch can cause serious yield and quality losses of wheat (Triticum aestivum) in many countries worldwide. Although there are other control methods, host resistance is the most desirable. Three recent Kansas winter wheat cultivars (Betty, Heyne, and 2163) have been developed with moderate levels of resistance to the leaf phase of Stagonospora nodorum blotch. To determine inheritance of resistance and allelism, these cultivars were crossed with one of three susceptible lines (Larned, KS96WGRC39, or Newton) and intercrossed in all possible combinations, including reciprocals. The parents, F1, F2, and F3 generations were tested for resistance to S. nodorum in the greenhouse as 4-week-old seedlings. Cytoplasmic effects were not detected in any cross. The mean levels of infection in the F1s of the two crosses Betty × Larned and Heyne × KS96WGRC39 indicated resistance was dominant. The observed phenotypic ratios of F2 plants for both crosses were not significantly different from the expected ratio for a single dominant gene. The ratio observed for F3 lines in the Betty × Larned cross fit that expected for a single dominant gene. However, the observed ratio of the F3 lines from the cross Heyne × KS96WGRC39 did not fit the ratio expected for a single dominant gene. The allelism test for Betty and Heyne indicated that they have different resistance genes. The F1 mean rating of the cross 2163 × Newton was intermediate between the two parents, indicating the absence of dominance for resistance in 2163. The phenotypic ratio observed in the F2 plants from this cross did not fit the ratio expected for a single dominant gene. The simple genetic control of resistance in cv. Betty makes it a useful source of resistance for wheat breeding programs.

Assessment of Aegilops tauschii for resistance to biotypes of wheat curl mite (Acari: Eriophyidae).

Aegilops tauschii, the wild diploid D-genome progenitor of wheat, Triticum aestivum L., is an important source of resistance to several arthropod pests and pathogens. A total of 108 Ae. tauschii accessions from different geographic regions were evaluated for resistance to biotypes of the wheat curl mite, Aceria tosichella Keifer, from Kansas, Nebraska, and Montana. The wheat curl mite is the only vector known to transmit wheat streak mosaic virus. Wheat curl mite resistance was detected in germplasm from all the geographic locations represented. The highest percentage of resistant accessions originated from Turkey, followed by Afghanistan and the Caspian Sea region of Iran. Sixty-seven percent of the accessions exhibited resistance to at least one wheat curl mite biotype and 19% were resistant to all the three biotyopes. Resistance to the accessions tested occurred more frequently in the Nebraska and Kansas biotypes (69% and 64%, respectively) than did resistance to the Montana biotype (42%), although the frequency of resistance was not significant. The differential reactions of accessions to the different wheat curl mite biotypes suggests that Ae. tauschii has at least five different genes for resistance to mite colonization. Ae. tauschii continues to be a very useful source for wheat curl mite resistance genes for bread wheat improvement.