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.

CerealsDB: A Whistle-Stop Tour of an Open Access SNP Resource.

The CerealsDB website, created by members of the Functional Genomics Group at the University of Bristol, provides access to a database containing SNP and genotyping data for hexaploid wheat and, to a lesser extent, its progenitors and several of its relatives. The site is principally aimed at plant breeders and research scientists who wish to obtain information regarding SNP markers; for example, obtain primers used for their identification or the sequences upon which they are based. The database underpinning the website contains circa one million putative varietal SNPs of which several hundreds of thousands have been experimentally validated on a range of common genotyping platforms. For each SNP marker, the site also hosts the allelic scores for thousands of elite wheat varieties, landrace cultivars, and wheat relatives. Tools are available to help negotiate and visualize the datasets. The website has been designed to be simple and straightforward to use and is completely open access.

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.

CerealsDB-new tools for the analysis of the wheat genome: update 2020.

CerealsDB (www.cerealsdb.uk.net) is an online repository of mainly hexaploid wheat (Triticum aestivum) single nucleotide polymorphisms (SNPs) and genotyping data. The CerealsDB website has been designed to enable wheat breeders and scientists to select the appropriate markers for research breeding tasks, such as marker-assisted selection. We report a large update of genotyping information for over 6000 wheat accessions and describe new webtools for exploring and visualizing the data. We also describe a new database of quantitative trait loci that links phenotypic traits to CerealsDB SNP markers and allelic scores for each of those markers. CerealsDB is an open-access website that hosts information on wheat SNPs considered useful for both plant breeders and research scientists. The latest CerealsDB database is available at https://www.cerealsdb.uk.net/cerealgenomics/CerealsDB/indexNEW.php.

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.

Stability of wheat grain yields over three field seasons in the UK.

Ensuring food security in a changing climate is a major contemporary challenge and requires development of climate-resilient crops that perform well under variable environments. The hypothesis that yield stability in suboptimal conditions is linked to yield penalties in optimal conditions was investigated in field-grown wheat in the UK. The phenotypic responses, rate of wheat crop development, and final grain yield to varying sowing date, rainfall, air temperature, and radiation patterns were studied for a panel of 61 elite commercial wheat cultivars grown in the UK in 2012, 2013, and 2014. Contrasting climatic patterns, particularly rainfall accumulation and distribution over the season, influenced the relative performance of the cultivars affecting the duration of grain development stage and impacting on productivity. Indices for crop productivity, yield stability, and performance under suboptimal conditions revealed four cultivars with a combination of stable and high relative grain yields over the three seasons: Gladiator, Humber, Mercato, and Zebedee. Genetic similarity between cultivars partially explained yield performance in the contrasting seasons. The year of release of the cultivars correlated with grain yield but not with yield stability, supporting the contention that breeding for yield potential does not select for climate resilience and yield stability of crops. Further analysis of the outstanding cultivars may unravel target traits for breeding efforts aimed at increasing wheat yield potential and stability in the changing climate.

Conversion of array-based single nucleotide polymorphic markers for use in targeted genotyping by sequencing in hexaploid wheat (Triticum aestivum).

Wheat breeders and academics alike use single nucleotide polymorphisms (SNPs) as molecular markers to characterize regions of interest within the hexaploid wheat genome. A number of SNP-based genotyping platforms are available, and their utility depends upon factors such as the available technologies, number of data points required, budgets and the technical expertise required. Unfortunately, markers can rarely be exchanged between existing and newly developed platforms, meaning that previously generated data cannot be compared, or combined, with more recently generated data sets. We predict that genotyping by sequencing will become the predominant genotyping technology within the next 5-10 years. With this in mind, to ensure that data generated from current genotyping platforms continues to be of use, we have designed and utilized SNP-based capture probes from several thousand existing and publicly available probes from Axiom® and KASP™ genotyping platforms. We have validated our capture probes in a targeted genotyping by sequencing protocol using 31 previously genotyped UK elite hexaploid wheat accessions. Data comparisons between targeted genotyping by sequencing, Axiom® array genotyping and KASP™ genotyping assays, identified a set of 3256 probes which reliably bring together targeted genotyping by sequencing data with the previously available marker data set. As such, these probes are likely to be of considerable value to the wheat community. The probe details, full probe sequences and a custom built analysis pipeline may be freely downloaded from the CerealsDB website (http://www.cerealsdb.uk.net/cerealgenomics/CerealsDB/sequence_capture.php).

Introgression of Aegilops speltoides segments in Triticum aestivum and the effect of the gametocidal genes.

Background and Aims: Bread wheat (Triticum aestivum) has been through a severe genetic bottleneck as a result of its evolution and domestication. It is therefore essential that new sources of genetic variation are generated and utilized. This study aimed to generate genome-wide introgressed segments from Aegilops speltoides. Introgressions generated from this research will be made available for phenotypic analysis. Methods: Aegilops speltoides was crossed as the male parent to T. aestivum 'Paragon'. The interspecific hybrids were then backcrossed to Paragon. Introgressions were detected and characterized using the Affymetrix Axiom Array and genomic in situ hybridization (GISH). Key Results: Recombination in the gametes of the F1 hybrids was at a level where it was possible to generate a genetic linkage map of Ae. speltoides. This was used to identify 294 wheat/Ae. speltoides introgressions. Introgressions from all seven linkage groups of Ae. speltoides were found, including both large and small segments. Comparative analysis showed that overall macro-synteny is conserved between Ae. speltoides and T. aestivum, but that Ae. speltoides does not contain the 4A/5A/7B translocations present in wheat. Aegilops speltoides has been reported to carry gametocidal genes, i.e. genes that ensure their transmission through the gametes to the next generation. Transmission rates of the seven Ae. speltoides linkage groups introgressed into wheat varied. A 100 % transmission rate of linkage group 2 demonstrates the presence of the gametocidal genes on this chromosome. Conclusions: A high level of recombination occurs between the chromosomes of wheat and Ae. speltoides, leading to the generation of large numbers of introgressions with the potential for exploitation in breeding programmes. Due to the gametocidal genes, all germplasm developed will always contain a segment from Ae. speltoides linkage group 2S, in addition to an introgression from any other linkage group.

High-density genotyping of the A.E. Watkins Collection of hexaploid landraces identifies a large molecular diversity compared to elite bread wheat.

The importance of wheat as a food crop makes it a major target for agricultural improvements. As one of the most widely grown cereal grains, together with maize and rice, wheat is the leading provider of calories in the global diet, constituting 29% of global cereal production in 2015. In the last few decades, however, yields have plateaued, suggesting that the green revolution, at least for wheat, might have run its course and that new sources of genetic variation are urgently required. The overall aim of our work was to identify novel variation that may then be used to enable the breeding process. As landraces are a potential source of such diversity, here we have characterized the A.E. Watkins Collection alongside a collection of elite accessions using two complementary high-density and high-throughput genotyping platforms. While our results show the importance of using the appropriate SNP collection to compare diverse accessions, they also show that the Watkins Collection contains a substantial amount of novel genetic diversity which has either not been captured in current breeding programmes or which has been lost through previous selection pressures. As a consequence of our analysis, we have identified a number of accessions which carry an array of novel alleles along with a number of interesting chromosome rearrangements which confirm the variable nature of the wheat genome.

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.

High-Density SNP Genotyping Array for Hexaploid Wheat and Its Relatives.

A lack of genetic diversity between wheat breeding lines has been recognized as a significant block to future yield increases. Wheat breeding and prebreeding strategies are increasingly using material from wheat ancestors or wild relatives to reintroduce diversity. Where molecular markers are polymorphic between the host and introgressed material, they may be used to track the size and location of the introgressed material through generations of backcrossing. To generate markers for this purpose, sequence capture targeted resequencing was carried out for a range of wheat varieties, wheat relatives, and wheat progenitors. From these sequences, putative SNPs were identified and used to generate the Axiom® Wheat HD array. A selection of varieties representing a selection of elite wheat breeding material, progenitor species, and wild relatives were used to validate the array. The procedures used are described here in detail.

Quantifying rooting at depth in a wheat doubled haploid population with introgression from wild emmer.

Background and Aims: The genetic basis of increased rooting below the plough layer, post-anthesis in the field, of an elite wheat line (Triticum aestivum 'Shamrock') with recent introgression from wild emmer (T. dicoccoides), is investigated. Shamrock has a non-glaucous canopy phenotype mapped to the short arm of chromosome 2B (2BS), derived from the wild emmer. A secondary aim was to determine whether genetic effects found in the field could have been predicted by other assessment methods. Methods: Roots of doubled haploid (DH) lines from a winter wheat ('Shamrock' × 'Shango') population were assessed using a seedling screen in moist paper rolls, in rhizotrons to the end of tillering, and in the field post-anthesis. A linkage map was produced using single nucleotide polymorphism markers to identify quantitative trait loci (QTLs) for rooting traits. Key Results: Shamrock had greater root length density (RLD) at depth than Shango, in the field and within the rhizotrons. The DH population exhibited diversity for rooting traits within the three environments studied. QTLs were identified on chromosomes 5D, 6B and 7B, explaining variation in RLD post-anthesis in the field. Effects associated with the non-glaucous trait on RLD interacted significantly with depth in the field, and some of this interaction mapped to 2BS. The effect of genotype was strongly influenced by the method of root assessment, e.g. glaucousness expressed in the field was negatively associated with root length in the rhizotrons, but positively associated with length in the seedling screen. Conclusions: To our knowledge, this is the first study to identify QTLs for rooting at depth in field-grown wheat at mature growth stages. Within the population studied here, our results are consistent with the hypothesis that some of the variation in rooting is associated with recent introgression from wild emmer. The expression of genetic effects differed between the methods of root assessment.

Wheat Landrace Genome Diversity.

Understanding the genomic complexity of bread wheat (Triticum aestivum L.) is a cornerstone in the quest to unravel the processes of domestication and the following adaptation of domesticated wheat to a wide variety of environments across the globe. Additionally, it is of importance for future improvement of the crop, particularly in the light of climate change. Focusing on the adaptation after domestication, a nested association mapping (NAM) panel of 60 segregating biparental populations was developed, mainly involving landrace accessions from the core set of the Watkins hexaploid wheat collection optimized for genetic diversity. A modern spring elite variety, "Paragon," was used as common reference parent. Genetic maps were constructed following identical rules to make them comparable. In total, 1611 linkage groups were identified, based on recombination from an estimated 126,300 crossover events over the whole NAM panel. A consensus map, named landrace consensus map (LRC), was constructed and contained 2498 genetic loci. These newly developed genetics tools were used to investigate the rules underlying genome fluidity or rigidity, e.g., by comparing marker distances and marker orders. In general, marker order was highly correlated, which provides support for strong synteny between bread wheat accessions. However, many exceptional cases of incongruent linkage groups and increased marker distances were also found. Segregation distortion was detected for many markers, sometimes as hot spots present in different populations. Furthermore, evidence for translocations in at least 36 of the maps was found. These translocations fell, in general, into many different translocation classes, but a few translocation classes were found in several accessions, the most frequent one being the well-known T5B:7B translocation. Loci involved in recombination rate, which is an interesting trait for plant breeding, were identified by QTL analyses using the crossover counts as a trait. In total, 114 significant QTL were detected, nearly half of them with increasing effect from the nonreference parents.

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.