SNP-based pool genotyping and haplotype analysis accelerate fine-mapping of the wheat genomic region containing stripe rust resistance gene Yr26
NGS-assisted super pooling emerging as powerful tool to accelerate gene mapping and haplotype association analysis within target region uncovering specific linkage SNPs or alleles for marker-assisted gene pyramiding.
Conventional gene mapping methods to identify genes associated with important agronomic traits require significant amounts of financial support and time. Here, a single nucleotide polymorphism (SNP)-based mapping approach, RNA-Seq and SNP array assisted super pooling analysis, was used for rapid mining of a candidate genomic region for stripe rust resistance gene Yr26 that has been widely used in wheat breeding programs in China. Large DNA and RNA super-pools were genotyped by Wheat SNP Array and sequenced by Illumina HiSeq, respectively. Hundreds of thousands of SNPs were identified and then filtered by multiple filtering criteria. Among selected SNPs, over 900 were found within an overlapping interval of less than 30 Mb as the Yr26 candidate genomic region in the centromeric region of chromosome arm 1BL. The 235 chromosome-specific SNPs were converted into KASP assays to validate the Yr26 interval in different genetic populations. Using a high-resolution mapping population (> 30,000 gametes), we confined Yr26 to a 0.003-cM interval. The Yr26 target region was anchored to the common wheat IWGSC RefSeq v1.0 and wild emmer WEWSeq v.1.0 sequences, from which 488 and 454 kb fragments were obtained. Several candidate genes were identified in the target genomic region, but there was no typical resistance gene in either genome region. Haplotype analysis identified specific SNPs linked to Yr26 and developed robust and breeder-friendly KASP markers. This integration strategy can be applied to accelerate generating many markers closely linked to target genes/QTL for a trait of interest in wheat and other polyploid species.
The authors are grateful to Prof. R.A. McIntosh, Plant Breeding Institute, University of Sydney, for critical review of this manuscript; Prof. Peidu Chen and Prof. Aizhong Cao, Cytogenetics Institute, Nanjing Agricultural University, for providing Yr26 germplasms and genetic populations. This study was financially supported by International S&T Cooperation Program of China (2015DFG32340), National Natural Science Foundation of China (31371924), the National Key Research and Development Program of China (Grant no. 2016YFE0108600), the earmarked funds for Modern Agro-industry Technology Research System (No. CARS-3-1-11) and National Natural Science Foundation for Young Scientists of China (Grant 31701421).
Author contribution statement
JHW designed and conducted the experiments, analyzed the data, and wrote the manuscript. QDZ analyzed the data, prepared the figures for the manuscript and contributed to writing the RNA-Seq sections; QLW participated in creating the genetic populations and analyzed the SNP array data. SJL, JMM and SH participated in greenhouse and field experiments and contributed to the genotyping experiment. SZY assisted in analyzing the data and prepared the figures for the manuscript. HS and AD analyzed the data with the wild emmer genome. LLH participated in revising the manuscript. DJH and ZSK conceived and directed the project and revised the manuscript.
Compliance with ethical standards
Conflict of interest
The authors have declared that no competing interests exist.
I declare on behalf of my co-authors that the work described is original, previously unpublished research, and not under consideration for publication elsewhere. The experiments in this study comply with the current laws of China.
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