Genome-wide analysis of microsatellite markers based on the sequenced database of Chinese spring wheat (Triticum aestivum L.)

Microsatellites or simple sequence repeats (SSRs) are distributed across both prokaryotic and eukaryotic genomes and have been widely used for genetic studies and molecular marker assisted breeding of crops. Although a draft sequence for hexaploid bread wheat is available, to date, there is no published systemic analysis of SSRs in wheat. In the present study, we identified 364,347 SSRs from among 10,603,760 sequences of the Chinese spring wheat (CSW) genome, which were present at a density of 36.68 SSR/Mb. In total, we detected 488 types of motifs ranging from di- to hexanucleotides, among which dinucleotide repeats dominated, accounting for approximately 42.52% of the genome. The density of tri- to hexanucleotide repeats was 24.97%, 4.62%, 3.25% and 24.65%, respectively. AG/CT, AAG/CTT, AGAT/ATCT, AAAAG/CTTTT and AAAATT/AATTTT were the most frequent repeats among di- to hexanucleotide repeats. Among the 21 chromosomes of CSW, the density of repeats was highest on chromosome 2D and lowest on chromosome 3A. The proportions of di-, tri-, tetra-, penta- and hexanucleotide repeats on each chromosome, and even on the whole genome, were almost identical. In addition, 295,267 SSR markers were successfully developed from the 21 chromosomes of CSW, which cover the entire genome at a density of 29.73 per Mb. All of the SSR markers were validated by reverse electronic-Polymerase Chain Reaction (re-PCR); 70,564 (23.9%) were found to be monomorphic and 224,703 (76.1%) were found to be polymorphic. A total of 45 monomorphic markers were selected randomly for validation purposes; 24 (53.3%) amplified one locus, 8 (17.8%) amplified multiple identical loci, and 13 (28.9%) did not amplify any fragments from the genomic DNA of CSW. Then a dendrogram was generated based on the 24 monomorphic SSR markers among 20 wheat cultivars and three species of its diploid ancestors showing that monomorphic SSR markers represented a promising source to increase the number of genetic markers available for the wheat genome. The results of this study will be useful for investigating the genetic diversity and evolution of wheat and related species. At the same time, the results will facilitate comparative genomic studies and marker-assisted breeding (MAS) in plants.

微卫星（simple sequence repeats，SSRs）广泛分布于原核与真核生物基因组中，已被广泛应用于作物的遗传研究与分子标记辅助育种。尽管六倍体普通小麦的草图基因组序列已发布，但截至目前，尚未有针对小麦中SSRs的系统性分析研究发表。本研究从中国春小麦（Chinese spring wheat，CSW）基因组的10603760条序列中，共鉴定出364347个SSRs，密度为36.68个SSRs每兆碱基（SSR/Mb）。本研究共检测到488种基序类型，跨度从二核苷酸到六核苷酸重复，其中二核苷酸重复占比最高，约占全基因组的42.52%。三核苷酸、四核苷酸、五核苷酸和六核苷酸重复的密度分别为24.97%、4.62%、3.25%和24.65%。在二至六核苷酸重复中，AG/CT、AAG/CTT、AGAT/ATCT、AAAAG/CTTTT以及AAAATT/AATTTT为最常见的重复基序。在中国春小麦的21条染色体中，重复序列密度最高的为2D染色体，最低的为3A染色体。每条染色体乃至全基因组范围内，二、三、四、五、六核苷酸重复的占比均几乎一致。此外，本研究从中国春小麦的21条染色体中成功开发出295267个SSR标记，覆盖全基因组，密度为每兆碱基29.73个标记。所有SSR标记均通过反向电子聚合酶链反应（reverse electronic-Polymerase Chain Reaction，re-PCR）进行了验证：其中70564个（占比23.9%）为单态标记，224703个（占比76.1%）为多态标记。随机选取45个单态标记进行验证：其中24个（占比53.3%）可扩增出单一位点，8个（占比17.8%）可扩增出多个相同位点，剩余13个（占比28.9%）未能从中国春小麦基因组DNA中扩增出任何片段。基于这24个单态SSR标记，对20个小麦品种及其3个二倍体祖先物种进行聚类分析并构建聚类树，结果表明单态SSR标记可作为提升小麦基因组遗传标记数量的宝贵资源。本研究结果可为解析小麦及其近缘物种的遗传多样性与进化历程提供支撑，同时也将推动植物比较基因组学研究与分子标记辅助育种（marker-assisted breeding，MAS）的发展。