Development and Application of a Whole-Genome Simple Sequence Repeat Panel for High-Throughput Genotyping in Soybean

DNA RESEARCH 18, 107–115, (2011) Advance Access Publication: 30 March 2011 doi:10.1093/dnares/dsr003 Development and Application of a Whole-Genome Simple Sequence Repeat Panel for High-Throughput Genotyping in Soybean TAKASHI Sayama1,2,†, TAE-YOUNG Hwang2,†, KUNIHIKO Komatsu2, YOSHITAKE Takada3, MASAKAZU Takahashi4, SHIN Kato5, HIROKO Sasama1,2, AYAKO Higashi2, YUMI Nakamoto2, HIDEYUKI Funatsuki2,‡, and MASAO Ishimoto1,2,* National Institute of Agrobiological Sciences, 2-1-2 Kannondai, Tsukuba, Ibaraki 305-8602, Japan1; National Agricultural Research Center for Hokkaido Region, 1 Hitsujigaoka, Toyohira, Sapporo, Hokkaido 062-8555, Japan2; National Agricultural Research Center for Western Region, 1-3-1 Senyuu, Zentsuuji, Kagawa 765-8508, Japan3; National Agricultural Research Center for Kyushu Okinawa Region, 2421 Suya, Koshi, Kumamoto 8611192, Japan4 and National Agricultural Research Center for Tohoku Region, 297 Uenodai, Kariwano, Daisen, Akita 019-2112, Japan5 *To whom correspondence should be addressed. Tel./Fax. þ81 29-838-7452. Email: Edited by Kazuo Shinozaki (Received 19 December 2010; accepted 16 February 2011) Abstract Among commonly applied molecular markers, simple sequence repeats (SSRs, or microsatellites) possess advantages such as a high level of polymorphism and codominant pattern of inheritance at individual loci. To facilitate systematic and rapid genetic mapping in soybean, we designed a genotyping panel comprised 304 SSR markers selected for allelic diversity and chromosomal location so as to provide wide coverage. Most primer pairs for the markers in the panel were redesigned to yield amplicons of 80– 600 bp in multiplex polymerase chain reaction (PCR) and fluorescence-based sequencer analysis, and they were labelled with one of four different fluorescent dyes. Multiplex PCR with sets of six to eight primer pairs per reaction generated allelic data for 283 of the 304 SSR loci in three different mapping populations, with the loci mapping to the same positions as previously determined. Four SSRs on each chromosome were analysed for allelic diversity in 87 diverse soybean germplasms with four-plex PCR. These 80 loci showed an average allele number and polymorphic information content value of 14.8 and 0.78, respectively. The high level of polymorphism, ease of analysis, and high accuracy of the SSR genotyping panel should render it widely applicable to soybean genetics and breeding. Key words: SSR marker; fluorescent primer; multiplex PCR; polymorphic information content; highthroughput genotyping 1. Introduction A comprehensive genetic linkage map is fundamental to modern plant genetics and breeding because it † ‡ These authors contributed equally to this article. Present address: National Agricultural Research Center for Western Region, 6-12-1 Nishifukatsu, Fukuyama, Hiroshima 721-8514, Japan. allows the identification and utilization of agronomic trait loci, such as qualitative and quantitative trait loci, as well as the evaluation of genetic diversity and genomic structure of genetic resources. It also serves as a scaffold for construction of a physical map. In the case of soybean [Glycine max (L.) Merrill], a grain legume of global importance, many useful agronomic trait loci associated with growth, product quality, tolerance to biotic and abiotic stresses, and # The Author 2011. Published by Oxford University Press on behalf of Kazusa DNA Research Institute. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http:// creativecommons.org/licenses/by-nc/2.5), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. 108 SSR Panel for High-Throughput Genotyping in Soybean other characteristics have been identified in genetic resources and deposited in public databases such as Soybase (http://soybase.org). The first soybean linkage map was constructed on the basis of phenotypic traits,1 but it did not contain sufficient information for application to the above-mentioned purposes. Since the 1990s, various types of molecular markers, including restriction fragment length polymorphism, random amplification of polymorphic DNA, amplified fragment length polymorphism, simple sequence repeat (SSR, or microsatellite), and single nucleotide polymorphism (SNP) markers, have been developed and applied to soybean, and enriched marker information has enabled genetic analyses of qualitative and quantitative traits.2 – 4 Among such molecular markers, SSRs in particular have contributed to the construction of a genomewide linkage map for soybean with a converged linkage group (LG) number equal to the chromosome number. As previously described,5 these microsatellite markers have several advantages, including (i) a codominant manner of inheritance at each locus, (ii) a high level of polymorphism in the form of multiple alleles, (iii) a non-biased distribution in the genome, and (iv) ease of detection of polymorphism by the polymerase chain reaction (PCR) and subsequent electrophoresis.6 – 9 It is thus possible to detect allelic differences at highly polymorphic loci among genetic resources as well as among many segregating populations derived from the hybridization of any given genotypes.10 – 12 Furthermore, given that most PCR primer pairs for SSR markers were designed to yield a single amplification product for each allele in spite of the complex chromosomal structure of soybean,13 – 15 each SSR marker localizes a definite site in the genome, unlike other molecular markers. These features also allow the simultaneous detection of multiple SSR loci with the use of multiplex PCR analysis.11 In addition, high-throughput and repetitive genotyping can be performed by semiautomated methods with a combination of fluorescently labelled SSR markers.11,12,16 This strategy has been successfully adapted and improved for analysis of genetic diversity and high-throughput mapping in various species, including human,17,18 mouse,19 rat,20 rice,21,22 and sunflower.23 However, in spite of the public availability of many SSR markers for soybean,24 no comprehensive SSR genotyping panel has yet been developed for whole-genome coverage. A high-throughput genotyping system for soybean that is based on an SNP array capable of high multiplexing and which discriminates up to 384 or 1536 mapped SNPs in one reaction has been described.25,26 Although the high multiplexing capacity and continued improvement of the SNP array may make this a standard technique in the foreseeable future, the [Vol. 18 quantity and quality of SNP loci in soybean are still not sufficient for application of this system to many genotypes. Moreover, the present panel system for SNPs is not sufficiently flexible for modification of marker selection and is not a cost-effective solution. In the latest extensive molecular linkage map, nearly 2000 SSR markers (...truncated)