Construction of High-Density Genetic Map in Barley through Restriction-Site Associated DNA Sequencing

RESEARCH ARTICLE Construction of High-Density Genetic Map in Barley through Restriction-Site Associated DNA Sequencing Gaofeng Zhou1, Qisen Zhang2, Xiao-qi Zhang3, Cong Tan3, Chengdao Li1,2,3* 1 Department of Agriculture and Food, South Perth, WA, Australia, 2 Australian Export Grains Innovation Centre, South Perth, WA, Australia, 3 Western Australian State Agricultural Biotechnology Centre, Murdoch University, Murdoch, WA, Australia a11111 * Abstract Published: July 16, 2015 Genetic maps in barley are usually constructed from a limited number of molecular markers such as SSR (simple sequence repeat) and DarT (diversity arrays technology). These markers must be first developed before being used for genotyping. Here, we introduce a new strategy based on sequencing progeny of a doubled haploid population from Baudin × AC Metcalfe to construct a genetic map in barley. About 13,547 polymorphic SNP tags with >93% calling rate were selected to construct the genetic map. A total of 12,998 SNP tags were anchored to seven linkage groups which spanned a cumulative 967.6 cM genetic distance. The high-density genetic map can be used for QTL mapping and the assembly of WGS and BAC contigs. The genetic map was evaluated for its effectiveness and efficiency in QTL mapping and candidate gene identification. A major QTL for plant height was mapped at 105.5 cM on chromosome 3H. This QTL with LOD value of 13.01 explained 44.5% of phenotypic variation. This strategy will enable rapid and efficient establishment of high-density genetic maps in other species. Copyright: © 2015 Zhou et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Introduction OPEN ACCESS Citation: Zhou G, Zhang Q, Zhang X-q, Tan C, Li C (2015) Construction of High-Density Genetic Map in Barley through Restriction-Site Associated DNA Sequencing. PLoS ONE 10(7): e0133161. doi:10.1371/journal.pone.0133161 Editor: Tongming Yin, Nanjing Forestry University, CHINA Received: February 26, 2015 Accepted: June 23, 2015 Data Availability Statement: Deep sequencing data is available from the NCBI via the accession number SRP057861. SNP marker accession numbers are also available from the NCBI, and the relevant accession numbers are located in S1 File of the Supporting Information. Funding: This work was supported by the fund from Grains Research and Development Corporation (GRDC) of Australia (DAW00233.), Department of Agriculture and Food Western Australia, and Western Australian State Agricultural Biotechnology Centre (SABC). Barley is the fourth most-abundant cereal in the world (http://faostat.fao.org), with uses ranging from food, feed, malting and brewing to being a model organism in molecular research. Genetic maps play a pivotal role in QTL mapping for agronomic traits. Several types of molecular markers have been developed for linkage map construction in barley, with the two most popular being SSR (simple sequence repeat) and DarT (diversity arrays technology). Recently, high-density SNP markers were designed in barley based on cDNA polymorphisms [1]. The downside of these markers is that they must be ready before conducting genetic map construction. Advances in high-throughput DNA sequencing technology make it possible to construct de novo genetic maps. Recently, a number of species have been sequenced. For instance, genomes of two main crops, wheat [2] and barley [3], were sequenced using whole-genome shotgun PLOS ONE | DOI:10.1371/journal.pone.0133161 July 16, 2015 1/9 Construction of High-Density Genetic Map in Barley Competing Interests: The authors have declared that no competing interests exist. sequencing technology. Furthermore, genotyping-by-sequencing (GBS), a low cost, reduced representation sequencing method, is becoming a common approach for whole-genome marker profiling in many species. Analysis of a recombination population allows the construction of de novo genetic maps. There are limitations, however, to those species with large genome size, such as wheat and barley; so several targeted complex DNA reduction methods have been applied to produce high-quality polymorphism data at a relatively low per sample cost. Yang et al. [4] applied NGS (next-generation sequencing)-based RAD (restriction-site associated DNA)-sequencing technology to construct a lupin genetic map. A total of 94 recombinant inbred lines and their parental lines were sequenced and 8,244 sequence-tagged markers were integrated into linkage groups [4]. In barley, Poland et al. [5] used a novel two-enzyme approach for complexity reduction to genotype bi-parental DH population and anchor over 34,000 SNPs into a reference barley genetic map. The high-density genetic map will facilitate QTL mapping and fine mapping. Furthermore, high-density genetic maps help in the construction of physical maps, assembly of BAC contigs and whole-genome shotgun sequence contigs. Next-generation wholegenome shotgun sequencing is popular and sequence assembly can be achieved by software, but the difficulty is to link the nearby sequence contigs to each other and provide a linear order of contigs along each chromosome. BAC physical maps, BAC-end sequences and fully sequenced BAC sequences provide a framework for the assembly of barley whole-genome shotgun sequences [3]. However, the development of these resources requires substantial time and labour. Recently, a new method called POPSEQ was introduced to anchor NGS contigs assemblies. Instead of constructing BAC physical maps, high-density genetic maps from population sequencing allow de novo production of genetically-anchored linear shotgun sequence contigs [6]. Baudin and AC Metcalfe are the two international benchmark varieties for malting quality from Australia and Canada, which have been used worldwide as parents for commercial barley breeding. They differ in plant height, malting quality and disease resistance. Genetic map construction is the basis for QTL mapping for these traits and eventual identification of underlying genes. The objective of this work was to construct a high-density genetic map using NGS-based RAD-sequencing technology. The high-density markers will facilitate exploration of more genetic markers within QTL regions and conduct fine mapping work. The effectiveness and efficiency of this genetic map was evaluated by mapping QTL for plant height in the population. In addition, some barley BAC contigs (cv. Morex) were anchored to the high-resolution genetic map based on their sequence homology. The genetically linear BAC contigs orders will assist physical map construction in barley. Materials and Methods Whole-genome shotgun sequencing Illumina paired-end libraries were generated from genomic DNA of 94 individuals from the AC Metcalfe and Baudin DH population and their parental lines. R (...truncated)