Genome wide characterization of simple sequence repeats in watermelon genome and their application in comparative mapping and genetic diversity analysis

Zhu et al. BMC Genomics (2016) 17:557 DOI 10.1186/s12864-016-2870-4 RESEARCH ARTICLE Open Access Genome wide characterization of simple sequence repeats in watermelon genome and their application in comparative mapping and genetic diversity analysis Huayu Zhu1†, Pengyao Song1†, Dal-Hoe Koo2, Luqin Guo1, Yanman Li1, Shouru Sun1, Yiqun Weng2,3* and Luming Yang1* Abstract Background: Microsatellite markers are one of the most informative and versatile DNA-based markers used in plant genetic research, but their development has traditionally been difficult and costly. The whole genome sequencing with next-generation sequencing (NGS) technologies provides large amounts of sequence data to develop numerous microsatellite markers at whole genome scale. SSR markers have great advantage in cross-species comparisons and allow investigation of karyotype and genome evolution through highly efficient computation approaches such as in silico PCR. Here we described genome wide development and characterization of SSR markers in the watermelon (Citrullus lanatus) genome, which were then use in comparative analysis with two other important crop species in the Cucurbitaceae family: cucumber (Cucumis sativus L.) and melon (Cucumis melo L.). We further applied these markers in evaluating the genetic diversity and population structure in watermelon germplasm collections. Results: A total of 39,523 microsatellite loci were identified from the watermelon draft genome with an overall density of 111 SSRs/Mbp, and 32,869 SSR primers were designed with suitable flanking sequences. The dinucleotide SSRs were the most common type representing 34.09 % of the total SSR loci and the AT-rich motifs were the most abundant in all nucleotide repeat types. In silico PCR analysis identified 832 and 925 SSR markers with each having a single amplicon in the cucumber and melon draft genome, respectively. Comparative analysis with these crossspecies SSR markers revealed complicated mosaic patterns of syntenic blocks among the genomes of three species. In addition, genetic diversity analysis of 134 watermelon accessions with 32 highly informative SSR loci placed these lines into two groups with all accessions of C.lanatus var. citorides and three accessions of C. colocynthis clustered in one group and all accessions of C. lanatus var. lanatus and the remaining accessions of C. colocynthis clustered in another group. Furthermore, structure analysis was consistent with the dendrogram indicating the 134 watermelon accessions were classified into two populations. (Continued on next page) * Correspondence: ; † Equal contributors 2 Horticulture Department, University of Wisconsin, Madison, WI 53706, USA 1 College of Horticulture, Henan Agricultural University, 63 Nongye Road, Zhengzhou 450002, China Full list of author information is available at the end of the article © 2016 The Author(s). 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. Zhu et al. BMC Genomics (2016) 17:557 Page 2 of 17 (Continued from previous page) Conclusion: The large number of genome wide SSR markers developed herein from the watermelon genome provides a valuable resource for genetic map construction, QTL exploration, map-based gene cloning and marker-assisted selection in watermelon which has a very narrow genetic base and extremely low polymorphism among cultivated lines. Furthermore, the cross-species transferable SSR markers identified herein should also have practical uses in many applications in species of Cucurbitaceae family whose whole genome sequences are not yet available. Keywords: SSR, Watermelon, Comparative genomics, Synteny, Cucurbits, Genetic diversity Background Watermelon (Citrullus lanatus) is an important horticultural crop and one of the most consumed fresh fruits globally. It belongs to the genus Citrullus, which contains four diploid species: Citrullus lanatus (Thunb.) Mat-sum. & Nakai, C. colocynthis (L.) Schrad, C. ecir rhosus Cogn. and C. rehmii De Winter [1, 2]. Among these four species, Citrullus lanatus includes the cultivated watermelon (C. lanatus var. lanatus) which thrives in West Africa and has been cultivated widely worldwide (also called ‘egusi’ melon) and the preserving melon (C. lanatus var. citroides) that is grown in Southern Africa (also called ‘tsamma’ melon) [3, 4], and C. colocynthi (‘bitter apple’) is a perennial species grown in sandy areas throughout northern Africa, south-western Asia, and the Mediterranean [2, 5]. The long term domestication and selection for desirable horticultural qualities has made the cultivated watermelon with a narrow genetic base and susceptibility to a large number of diseases and pests [6]. Evaluating the phylogenetic relationships among different species in Citrullus genus will help us for improving watermelon cultivars in diseases resistance [1]. Watermelon has a small genome of 425 Mb, and the genome of the elite Chinese watermelon line 97103 [7] and the American heirloom watermelon cultivar Charleston Gray have been sequenced and released in cucurbit genomics database (www.icugi.org). The availability of these genomic resources of watermelon have greatly promoted the fundamental researches including the development of molecular markers and genetic map construction [8, 9], gene/QTL mapping [10, 11], molecular breeding, and comparative genomics [12]. Microsatellites or simple sequence repeats (SSRs), are one of the most commonly used marker in many genetic applications since the early 1990s including mapping, fingerprinting, genetic diversity and population structure analysis [13–16]. Because of their reproducibility, multiallelism, co-dominance, relative abundance, good genome coverage and versatile platforms to genotype, the use of microsatellites is likely to continue to be used for some years to come. Furthermore, they are comparatively cheap to genotype and provide more population genetic information per marker than bi-allelic markers such as single nucleotide polymorphisms (SNP) [17, 18]. A single set of microsatellite markers can be used to genotype several related species, but SNP markers in general lack crossspecies utility, and are therefore only suitable for population and paternity studies in a single species [19–21]. The microsatellite loci can be detected both in genomic sequences and expressed sequence tag (EST), which were named genomic SSRs and EST-SSR. EST-SSRs are useful for genetic analysis, but their relati (...truncated)