Development of a next generation SNP genotyping array for wheat.

High-throughput genotyping arrays have provided a cost-effective, reliable and interoperable system for genotyping hexaploid wheat and its relatives. Existing, highly cited arrays including our 35K Wheat Breeder's array and the Illumina 90K array were designed based on a limited amount of varietal sequence diversity and with imperfect knowledge of SNP positions. Recent progress in wheat sequencing has given us access to a vast pool of SNP diversity, whilst technological improvements have allowed us to fit significantly more probes onto a 384-well format Axiom array than previously possible. Here we describe a novel Axiom genotyping array, the 'Triticum aestivum Next Generation' array (TaNG), largely derived from whole genome skim sequencing of 204 elite wheat lines and 111 wheat landraces taken from the Watkins 'Core Collection'. We used a novel haplotype optimization approach to select SNPs with the highest combined varietal discrimination and a design iteration step to test and replace SNPs which failed to convert to reliable markers. The final design with 43 372 SNPs contains a combination of haplotype-optimized novel SNPs and legacy cross-platform markers. We show that this design has an improved distribution of SNPs compared to previous arrays and can be used to generate genetic maps with a significantly higher number of distinct bins than our previous array. We also demonstrate the improved performance of TaNGv1.1 for Genome-wide association studies (GWAS) and its utility for Copy Number Variation (CNV) analysis. The array is commercially available with supporting marker annotations and initial genotyping results freely available.

A wheat chromosome segment substitution line series supports characterization and use of progenitor genetic variation.

Genome-wide introgression and substitution lines have been developed in many plant species, enhancing mapping precision, gene discovery, and the identification and exploitation of variation from wild relatives. Created over multiple generations of crossing and/or backcrossing accompanied by marker-assisted selection, the resulting introgression lines are a fixed genetic resource. In this study we report the development of spring wheat (Triticum aestivum L.) chromosome segment substitution lines (CSSLs) generated to systematically capture genetic variation from tetraploid (T. turgidum ssp. dicoccoides) and diploid (Aegilops tauschii) progenitor species. Generated in a common genetic background over four generations of backcrossing, this is a base resource for the mapping and characterization of wheat progenitor variation. To facilitate further exploitation the final population was genetically characterized using a high-density genotyping array and a range of agronomic and grain traits assessed to demonstrate the potential use of the populations for trait localization in wheat.

The Use and Limitations of Exome Capture to Detect Novel Variation in the Hexaploid Wheat Genome.

The bread wheat (Triticum aestivum) pangenome is a patchwork of variable regions, including translocations and introgressions from progenitors and wild relatives. Although a large number of these have been documented, it is likely that many more remain unknown. To map these variable regions and make them more traceable in breeding programs, wheat accessions need to be genotyped or sequenced. The wheat genome is large and complex and consequently, sequencing efforts are often targeted through exome capture. In this study, we employed exome capture prior to sequencing 12 wheat varieties; 10 elite T. aestivum cultivars and two T. aestivum landrace accessions. Sequence coverage across chromosomes was greater toward distal regions of chromosome arms and lower in centromeric regions, reflecting the capture probe distribution which itself is determined by the known telomere to centromere gene gradient. Superimposed on this general pattern, numerous drops in sequence coverage were observed. Several of these corresponded with reported introgressions. Other drops in coverage could not be readily explained and may point to introgressions that have not, to date, been documented.

The role of gene flow and chromosomal instability in shaping the bread wheat genome.

Bread wheat (Triticum aestivum) is one of the world's most important crops; however, a low level of genetic diversity within commercial breeding accessions can significantly limit breeding potential. In contrast, wheat relatives exhibit considerable genetic variation and so potentially provide a valuable source of novel alleles for use in breeding new cultivars. Historically, gene flow between wheat and its relatives may have contributed novel alleles to the bread wheat pangenome. To assess the contribution made by wheat relatives to genetic diversity in bread wheat, we used markers based on single nucleotide polymorphisms to compare bread wheat accessions, created in the past 150 years, with 45 related species. We show that many bread wheat accessions share near-identical haplotype blocks with close relatives of wheat's diploid and tetraploid progenitors, while some show evidence of introgressions from more distant species and structural variation between accessions. Hence, introgressions and chromosomal rearrangements appear to have made a major contribution to genetic diversity in cultivar collections. As gene flow from relatives to bread wheat is an ongoing process, we assess the impact that introgressions might have on future breeding strategies.

Interactions between two QTLs for time to anthesis on spike development and fertility in wheat.

Earliness per se (Eps) genes are reported to be important in fine-tuning flowering time in wheat independently of photoperiod (Ppd) and vernalisation (Vrn). Unlike Ppd and Vrn genes, Eps have relatively small effects and their physiological effect along with chromosomal position are not well defined. We evaluated eight lines derived from crossing two vernalisation insensitive lines, Paragon and Baj (late and early flowering respectively), to study the detailed effects of two newly identified QTLs, Eps-7D and Eps-2B and their interactions under field conditions. The effect of both QTLs was minor and was affected by the allelic status of the other. While the magnitude of effect of these QTLs on anthesis was similar, they are associated with very different profiles of pre-anthesis development which also depends on their interaction. Eps-7D affected both duration before and after terminal spikelet while not affecting final leaf number (FLN) so Eps-7D-early had a faster rate of leaf appearance. Eps-2B acted more specifically in the early reproductive phase and slightly altered FLN without affecting the leaf appearance rate. Both QTLs affected the spike fertility by altering the rate of floret development and mortality. The effect of Eps-2B was very small but consistent in that -late allele tended to produce more fertile florets.

Genetic variation in wheat grain quality is associated with differences in the galactolipid content of flour and the gas bubble properties of dough liquor.

Lipids affect the quality of wheat flour for breadmaking. One possible mechanism is stabilization of the gas cells which are formed during dough mixing and expanded during fermentation, leading to a greater loaf volume and evenness of texture. We therefore compared the lipidomic profiles of flour and dough liquor fractions (which contain surface-active components present at the gas bubble interface) from two sets of wheat lines differing in allelic variation at a QTL for loaf volume. Analyses of fractions from three field trials showed consistent increases in the contents of galactolipids (monogalactosyl diglyceride and digalactosyl diglyceride) in flour and dough liquor of the lines with the increasing (good quality) allele. Biophysical analysis showed that this was associated with greater elasticity of the dough liquor fraction. This is consistent with published studies reporting a relationship between galactolipids and breadmaking quality and suggests a mechanism of action for the QTL.

Historical changes in the contents and compositions of fibre components and polar metabolites in white wheat flour.

Thirty-nine UK adapted wheat cultivars dating from between 1790 and 2012 were grown in replicated randomised field trials for three years, milled, and white flour analysed for the contents of dietary fibre components (arabinoxylan and ß-glucan) and polar metabolites (sugars, amino acids, organic acids, choline and betaine) to determine whether the composition had changed due to the effects of intensive breeding. The concentrations of components varied between study years, indicating strong effects of environment. Nevertheless, some trends were observed, with the concentrations of arabinoxylan fibre and soluble sugars (notably sucrose, maltose and fructose) increasing and most amino acids (including asparagine which is the precursor of acrylamide formed during processing) decreasing between the older and newer types. The concentration of betaine, which is beneficial for cardio-vascular health, also increased. The study therefore provided no evidence for adverse effects of intensive breeding on the contents of beneficial components in wheat flour.

Exploiting the genome of Thinopyrum elongatum to expand the gene pool of hexaploid wheat.

KEY MESSAGE: One hundred and thirty four introgressions from Thinopyrum elongatum have been transferred into a wheat background and were characterised using 263 SNP markers. Species within the genus Thinopyrum have been shown to carry genetic variation for a very wide range of traits including biotic and abiotic stresses and quality. Research has shown that one of the species within this genus, Th. elongatum, has a close relationship with the genomes of wheat making it a highly suitable candidate to expand the gene pool of wheat. Homoeologous recombination, in the absence of the Ph1 gene, has been exploited to transfer an estimated 134 introgressions from Th. elongatum into a hexaploid wheat background. The introgressions were detected and characterised using 263 single nucleotide polymorphism markers from a 35 K Axiom® Wheat-Relative Genotyping Array, spread across seven linkage groups and validated using genomic in situ hybridisation. The genetic map had a total length of 187.8 cM and the average chromosome length was 26.8 cM. Comparative analyses of the genetic map of Th. elongatum and the physical map of hexaploid wheat confirmed previous work that indicated good synteny at the macro-level, although Th. elongatum does not contain the 4A/5A/7B translocation found in wheat.

Segregation distortion: Utilizing simulated genotyping data to evaluate statistical methods.

Segregation distortion is the phenomenon in which genotypes deviate from expected Mendelian ratios in the progeny of a cross between two varieties or species. There is not currently a widely used consensus for the appropriate statistical test, or more specifically the multiple testing correction procedure, used to detect segregation distortion for high-density single-nucleotide polymorphism (SNP) data. Here we examine the efficacy of various multiple testing procedures, including chi-square test with no correction for multiple testing, false-discovery rate correction and Bonferroni correction using an in-silico simulation of a biparental mapping population. We find that the false discovery rate correction best approximates the traditional p-value threshold of 0.05 for high-density marker data. We also utilize this simulation to test the effect of segregation distortion on the genetic mapping process, specifically on the formation of linkage groups during marker clustering. Only extreme segregation distortion was found to effect genetic mapping. In addition, we utilize replicate empirical mapping populations of wheat varieties Avalon and Cadenza to assess how often segregation distortion conforms to the same pattern between closely related wheat varieties.

Developing a High-Throughput SNP-Based Marker System to Facilitate the Introgression of Traits From Aegilops Species Into Bread Wheat (Triticum aestivum).

The genus Aegilops contains a diverse collection of wild species exhibiting variation in geographical distribution, ecological adaptation, ploidy and genome organization. Aegilops is the most closely related genus to Triticum which includes cultivated wheat, a globally important crop that has a limited gene pool for modern breeding. Aegilops species are a potential future resource for wheat breeding for traits, such as adaptation to different ecological conditions and pest and disease resistance. This study describes the development and application of the first high-throughput genotyping platform specifically designed for screening wheat relative species. The platform was used to screen multiple accessions representing all species in the genus Aegilops. Firstly, the data was demonstrated to be useful for screening diversity and examining relationships within and between Aegilops species. Secondly, markers able to characterize and track introgressions from Aegilops species in hexaploid wheat were identified and validated using two different approaches.

Genome Targeted Introgression of Resistance to African Stem Rust from Aegilops sharonensis into Bread Wheat.

Many accessions of the wheat wild relative Sharon goatgrass ( Eig., ) are resistant to African races of the stem rust pathogen (i.e., Ug99 group races), which currently threaten wheat production worldwide. A procedure was designed to introgress the respective resistances to specific bread wheat genomes by producing plants homozygous for the A and B genomes and hemizygous for the D and S genomes or homozygous for the A and D genomes and hemizygous for the B and S genomes. In these genotypes, which lack the allele, homeologous pairing was expected mainly between chromosomes of the D and S genomes or B and S genomes, respectively. An antigametocidal (AG) wheat mutant () was used to overcome gametocidal effects. Wheat lines initially found resistant at the seedling stage were also highly resistant at the adult plant stage in rust nurseries established in the field. DNA of 41 selected homozygous resistant lines, analyzed by the Axiom wheat 820K SNP array, showed alien chromatin mainly in wheat chromosomes 1B, 1D, and 5B. This work suggests that, in most cases, it is possible to target introgressions into the homeologous chromosome of a selected genome of bread wheat.