Development of genome-wide InDel markers and their integration with SSR, DArT and SNP markers in single barley map

Zhou et al. BMC Genomics (2015) 16:804 DOI 10.1186/s12864-015-2027-x RESEARCH ARTICLE Open Access Development of genome-wide InDel markers and their integration with SSR, DArT and SNP markers in single barley map Gaofeng Zhou1, Qisen Zhang2, Cong Tan3, Xiao-qi Zhang4 and Chengdao Li4* Abstract Background: Development of molecular markers such as SSR (simple sequence repeat), DArT (diversity arrays technology) and SNP (single nucleotide polymorphism) is fundamental for linkage map construction and QTL mapping. However, DArT and SNP genotyping require special tools, and detection of SSR polymorphisms requires time-consuming polyacrylamide electrophoresis. Furthermore, many markers have been mapped in different populations such that their genetic positions are inconsistent. Recently, InDel (insertion and deletion) markers have become popular in genetic map construction and map-based cloning. Results: Aligning genomic DNA sequences in two barley cultivars (Morex and Barke) identified 436,640 InDels. We designed 1140 InDel markers across the barley genome with an average genetic distance of 1 cM, each having a unique location in the barley genome. High-resolution melting (HRM) technology was used to genotype 55 InDel markers; those PCR amplicons with melting temperature differences >0.3 °C were ideal for HRM genotyping. The 1140 InDel markers together with 383 SSRs, 3909 gene-based SNPs and 1544 DArT markers were integrated into single barley genetic map according to their physical map positions. Conclusions: High-density InDel markers with specific genome locations were developed, with 6976 molecular markers (SSRs, DArTs, SNPs and InDels) integrated into single barley genetic map. HRM genotyping of the InDel markers each with single PCR band will facilitate quick map construction and gene fine-mapping. Keywords: InDel, Barley, High-resolution melting, HRM, SNP, DArT, Genetic map Background Traditional markers have played a pivotal role in genetic map construction and marker-assisted selection (MAS) in breeding programs. Genetic maps consist of several types of molecular markers including RFLP (restriction fragment length polymorphism), AFLP (amplified fragment length polymorphism), SSR (simple sequence repeat), STS (sequence-tagged site), DArT (diversity arrays technology) and SNP (single nucleotide polymorphism). RFLP markers have been used to construct first generation genetic maps [1, 2], but such hybridization-based markers have practical disadvantages. This led to interest in PCR-based markers, in particular those based on SSRs. SSR markers were derived from sequences held in public databases including * Correspondence: 4 Western Barley Genetics Alliance/Western Australian State Agricultural Biotechnology Centre, Murdoch University, Murdoch, WA 6150, Australia Full list of author information is available at the end of the article genome sequences and EST sequences [3–5], and small insert genomic libraries [4, 6, 7]. A high-density consensus genetic map containing 775 SSR loci has been constructed in barley [8]. DArT can detect and genotype DNA variations at several hundred genomic loci in parallel without relying on sequence information. DArT markers have been successfully applied to genetic maps and diversity analyses of barley germplasm. The first genetic map consisting of 385 DArT markers was constructed in a population derived from a cross between Steptoe and Morex [9]. These genetic maps were used to construct consensus maps which included RFLPs, SSRs, STSs and DArTs [10, 11]. The first sets of SNP markers were developed by resequencing the European barley gene pool in elite barley genotypes and exploring EST sequences. Based on this information, a pilot oligo nucleotide pool assay containing SNPs in 1524 barley unigenes was developed for use with Illumina Golden Gate Bead Array technology [12, 13]. Later, 3072 SNPs markers © 2015 Zhou et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Zhou et al. BMC Genomics (2015) 16:804 were developed based on barley ESTs and sequenced amplicons [14]. A total of 2383 markers including SNP, DArT, SSR and STS markers were mapped in single population [15]. Recently, the genotyping by sequencing (GBS) approach has provided low-cost, highdensity genotype information. High-density markers have been mapped in maize, wheat and barley using this technology [16–19]. GBS is a powerful method for developing high-density markers in species without a sequenced genome while providing a genome shotgun sequence. However, there are disadvantages of these types of markers. DArT genotyping requires special equipment which is unavailable in most research institutions. For SNP markers, although recent advances in molecular techniques have enabled high-throughput SNP genotyping including microarray hybridization, allele-specific PCR detection and primer extension [20–22], and lower-throughput and less equipment dependence including cleaved amplified polymorphic sequence (CAPS) markers [23] and allele-specific PCR primers [24], they are either costly or low-throughput. GBS is high throughput, but costly for barley with its large genome. SSR markers are extensively used in QTL mapping and MAS, however some are nonspecific, or very weak. Furthermore, due to minor differences between genotypes for some SSR markers, laborious sequence-grade high-resolution gels or costly capillary electrophoresis systems are required to genotype these markers. In contrast to DArT, SSR and SNP markers, InDel markers with moderate polymorphism differences can be amplified using regular PCR instruments and genotyped using an agarose gel electrophoresis system or HRM (high-resolution melting) technology. InDel markers have been successfully used for genetic studies in rice [25] and Arabidopsis [26]. HRM curve analysis is a post-PCR analysis method for characterizing nucleic acid samples based on DNA strand dissociation behaviour during transition from double-stranded DNA to single-stranded DNA with increasing temperature. It uses intercalating dyes, highly accurate melt curves and application of specific statistical analyses of genetic variations in PCR amplicons. The amplicon differences are reflected in the melting temperatures. HRM has been used for SNP genotyping and InDel genotyping in wheat [27]. Although InDel markers are advantageous for genetic studies, genome-wide InDel markers have not been explored in barley. The rece (...truncated)