FIGL1 prevents aberrant chromosome associations and fragmentation and limits crossovers in polyploid wheat meiosis.

Meiotic crossovers (COs) generate genetic diversity and are crucial for viable gamete production. Plant COs are typically limited to 1-3 per chromosome pair, constraining the development of improved varieties, which in wheat is exacerbated by an extreme distal localisation bias. Advances in wheat genomics and related technologies provide new opportunities to investigate, and possibly modify, recombination in this important crop species. Here, we investigate the disruption of FIGL1 in tetraploid and hexaploid wheat as a potential strategy for modifying CO frequency/position. We analysed figl1 mutants and virus-induced gene silencing lines cytogenetically. Genetic mapping was performed in the hexaploid. FIGL1 prevents abnormal meiotic chromosome associations/fragmentation in both ploidies. It suppresses class II COs in the tetraploid such that CO/chiasma frequency increased 2.1-fold in a figl1 msh5 quadruple mutant compared with a msh5 double mutant. It does not appear to affect class I COs based on HEI10 foci counts in a hexaploid figl1 triple mutant. Genetic mapping in the triple mutant suggested no significant overall increase in total recombination across examined intervals but revealed large increases in specific individual intervals. Notably, the tetraploid figl1 double mutant was sterile but the hexaploid triple mutant was moderately fertile, indicating potential utility for wheat breeding.

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.

Development and characterisation of interspecific hybrid lines with genome-wide introgressions from Triticum timopheevii in a hexaploid wheat background.

BACKGROUND: Triticum timopheevii (2n = 4x = 28; AtAtGG), is an important source for new genetic variation for wheat improvement with genes for potential disease resistance and salt tolerance. By generating a range of interspecific hybrid lines, T. timopheevii can contribute to wheat's narrow gene-pool and be practically utilised in wheat breeding programmes. Previous studies that have generated such introgression lines between wheat and its wild relatives have been unable to use high-throughput methods to detect the presence of wild relative segments in such lines. RESULTS: A whole genome introgression approach, exploiting homoeologous recombination in the absence of the Ph1 locus, has resulted in the transfer of different chromosome segments from both the At and G genomes of T. timopheevii into wheat. These introgressions have been detected and characterised using single nucleotide polymorphism (SNP) markers present on a high-throughput Axiom® Genotyping Array. The analysis of these interspecific hybrid lines has resulted in the detection of 276 putative unique introgressions from T. timopheevii, thereby allowing the generation of a genetic map of T. timopheevii containing 1582 SNP markers, spread across 14 linkage groups representing each of the seven chromosomes of the At and G genomes of T. timopheevii. The genotyping of the hybrid lines was validated through fluorescence in situ hybridisation (FISH). Comparative analysis of the genetic map of T. timopheevii and the physical map of the hexaploid wheat genome showed that synteny between the two species is highly conserved at the macro-level and confirmed the presence of inter- and intra-genomic translocations within the At and G genomes of T. timopheevii that have been previously only detected through cytological techniques. CONCLUSIONS: In this work, we report a set of SNP markers present on a high-throughput genotyping array, able to detect the presence of T. timopheevii in a hexaploid wheat background making it a potentially valuable tool for marker assisted selection (MAS) in wheat pre-breeding programs. These valuable resources of high-density molecular markers and wheat-T. timopheevii hybrid lines will greatly enhance the work being undertaken for wheat improvement through wild relative introgressions.

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).

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.

High-density SNP genotyping array for hexaploid wheat and its secondary and tertiary gene pool.

In wheat, a lack of genetic diversity between breeding lines has been recognized as a significant block to future yield increases. Species belonging to bread wheat's secondary and tertiary gene pools harbour a much greater level of genetic variability, and are an important source of genes to broaden its genetic base. Introgression of novel genes from progenitors and related species has been widely employed to improve the agronomic characteristics of hexaploid wheat, but this approach has been hampered by a lack of markers that can be used to track introduced chromosome segments. Here, we describe the identification of a large number of single nucleotide polymorphisms that can be used to genotype hexaploid wheat and to identify and track introgressions from a variety of sources. We have validated these markers using an ultra-high-density Axiom(®) genotyping array to characterize a range of diploid, tetraploid and hexaploid wheat accessions and wheat relatives. To facilitate the use of these, both the markers and the associated sequence and genotype information have been made available through an interactive web site.

Discovery and development of exome-based, co-dominant single nucleotide polymorphism markers in hexaploid wheat (Triticum aestivum L.).

Globally, wheat is the most widely grown crop and one of the three most important crops for human and livestock feed. However, the complex nature of the wheat genome has, until recently, resulted in a lack of single nucleotide polymorphism (SNP)-based molecular markers of practical use to wheat breeders. Recently, large numbers of SNP-based wheat markers have been made available via the use of next-generation sequencing combined with a variety of genotyping platforms. However, many of these markers and platforms have difficulty distinguishing between heterozygote and homozygote individuals and are therefore of limited use to wheat breeders carrying out commercial-scale breeding programmes. To identify exome-based co-dominant SNP-based assays, which are capable of distinguishing between heterozygotes and homozygotes, we have used targeted re-sequencing of the wheat exome to generate large amounts of genomic sequences from eight varieties. Using a bioinformatics approach, these sequences have been used to identify 95 266 putative single nucleotide polymorphisms, of which 10 251 were classified as being putatively co-dominant. Validation of a subset of these putative co-dominant markers confirmed that 96% were true polymorphisms and 65% were co-dominant SNP assays. The new co-dominant markers described here are capable of genotypic classification of a segregating locus in polyploid wheat and can be used on a variety of genotyping platforms; as such, they represent a powerful tool for wheat breeders. These markers and related information have been made publically available on an interactive web-based database to facilitate their use on genotyping programmes worldwide.

Targeted re-sequencing of the allohexaploid wheat exome.

Bread wheat, Triticum aestivum, is an allohexaploid composed of the three distinct ancestral genomes, A, B and D. The polyploid nature of the wheat genome together with its large size has limited our ability to generate the significant amount of sequence data required for whole genome studies. Even with the advent of next-generation sequencing technology, it is still relatively expensive to generate whole genome sequences for more than a few wheat genomes at any one time. To overcome this problem, we have developed a targeted-capture re-sequencing protocol based upon NimbleGen array technology to capture and characterize 56.5 Mb of genomic DNA with sequence similarity to over 100 000 transcripts from eight different UK allohexaploid wheat varieties. Using this procedure in conjunction with a carefully designed bioinformatic procedure, we have identified more than 500 000 putative single-nucleotide polymorphisms (SNPs). While 80% of these were variants between the homoeologous genomes, A, B and D, a significant number (20%) were putative varietal SNPs between the eight varieties studied. A small number of these latter polymorphisms were experimentally validated using KASPar technology and 94% proved to be genuine. The procedures described here to sequence a large proportion of the wheat genome, and the various SNPs identified should be of considerable use to the wider wheat community.