Next Generation Sequencing Enabled Genetics in Hexaploid Wheat
Abstract Next Generation Sequencing (NGS) is providing new methodologies to improve and complement traditional genetic approaches. These strategies, collectively termed NGS-enabled genetics, consist of identifying variation in bulks of plants that have been assembled based on a specific phenotype of interest. We examined NGS-enabled genetics in hexaploid wheat by using near isogenic lines (NIL) differing across a specific disease resistance locus. RNA-Seq of NILs allowed the identification of SNPs across this locus and helped distinguish allelic SNPs from homoeologous variants. F2 bulks were assembled based on opposing disease resistance phenotypes and the frequency of the informative allelic SNPs was examined across bulks using RNA-Seq. Variants enriched in the corresponding bulks are expected to be most closely linked to the phenotype of interest and were prioritized for validation. Recent advances in cereal genomics in the form of wheat gene models, sequenced diploid progenitors, and the advances in the Chromosome-based Survey Sequencing Project enabled us to develop a pipeline to automatically design SNP-based markers. These high-throughput assays were used to genotype the original individuals used to assemble the bulks and to generate a genetic map across the target locus. Linked markers are now being incorporated into marker assisted selection programs by breeders.
Keywords Bulk frequency ratio • Bulked segregant analysis • Genotyping • KASP • Marker assisted selection • Near isogenic lines • Next generation sequencing • RNA-Seq
Wheat is among the most important crops in the world providing over 20 % of the world's calorie and protein intake (FAO 2012). Genetic improvement and breeding, however, are hampered by the large proportion of repetitive elements (>80 %, Flavell et al. 1974) across its large genomes and the polyploid nature of hexaploid wheat which often leads to functional redundancy among homoeologous genes (Avni et al. 2014). The advances in next generation sequencing (NGS) technologies are creating new opportunities to exploit the variation in the wheat genome for crop improvement, both in repetitive regions (insertion site-based polymorphism (ISBP) markers, Paux et al. 2010) and in low copy regions (gene based single nucleotide polymorphisms (SNPs), Allen et al. 2013).
The challenges that are thus emerging are how to prioritize the large number of SNPs available both in public databases, for example Cerealsdb.uk.net (Wilkinson et al. 2012), and those obtained with Next Generation Sequencing (NGS), as well as how to convert these into high-throughput (HTP) markers for breeding. One option is to exploit synteny between grass species (Moore et al. 1995), although this approach is limited to gene-based SNPs. The recent release of the Wheat Chromosome-based Survey Sequencing Project coordinated by the International Wheat Genome Sequencing Consortium (IWGSC) allows broad scale localisation to a particular homoeologous chromosome arm, although resolution is limited.
A complementary approach is to exploit genetics to decrease the background complexity of the wheat genome. Near isogenic lines (NILs) that only differ across a specific target region can be a powerful resource for this purpose. A great example is the set of Avocet-S NILs for major disease resistance (R) genes against yellow rust (Yr) developed by Colin Wellings and colleagues at the University of Sydney. We have used the Avocet S-Yr15 NILs (Fig. 22.1a) to develop a segregating BC7F2 population that was phenotyped through virulence assays to the wheat yellow rust pathogen (Puccinia striiformis). Resistant plants, composed of both homozygous and heterozygous individuals across the Yr15 locus, were pooled into a resistant bulk. In an analogous manner, susceptible plants were grouped into a susceptible bulk (Fig. 22.1b). This strategy, first published as bulked segregant analysis (BSA) in 1991 (Michelmore et al. 1991), can now be further exploited with NGS approaches. An initial complexity reduction is achieved with the Avocet NILs whose main difference is across the R gene locus. This list of SNPs can then be further refined by the BSA approach. In this study we highlight how the combination of genetic and genomic resources, along with methods such as BSA, can be used to leverage new biological research and develop HTP markers for breeding.
Fig. 22.1 Segregating population for Yr15. (a) Resistant and susceptible phenotypes of Avocet S and Avocet S + Yr15 challenged with the yellow rust pathogen (Puccinia striiformis), respectively. Scale bar; 1 cm. (b) Segregating lines were developed by crossing a homozygous resistant NIL, Avocet S + Yr15, to Avocet S. Heterozygous plants were then self-pollinated to produce a segregating F2 population with an expected 3:1 segregation ratio for resistant and susceptible individuals, respectively. The resistant and susceptible plants were grouped by phenotype into the corresponding bulks. The resistant bulk includes both homozygous and heterozygous individuals across the Yr15 locus as exemplified by the red squares on the wheat chromosomes