Chen Jingfang, Li Ronghua, Xia Yanshi, Bai Guihua, Guo Peiguo, Wang Zhiliang, Zhang Hua, Siddique Kadambot H M
International Crop Research Center for Stress Resistance, College of Life Sciences, Guangzhou University, Guangzhou, China.
Hard Winter Wheat Genetics Research Unit, United States Department of Agriculture-Agricultural Research Service, Manhattan, Kansas, United States of America.
PLoS One. 2017 Sep 13;12(9):e0184736. doi: 10.1371/journal.pone.0184736. eCollection 2017.
Flowering Chinese cabbage is one of the most important vegetable crops in southern China. Genetic improvement of various agronomic traits in this crop is underway to meet high market demand in the region, but the progress is hampered by limited number of molecular markers available in this crop. This study aimed to develop EST-SSR markers from transcriptome sequences generated by next-generation sequencing. RNA-seq of eight cabbage samples identified 48,975 unigenes. Of these unigenes, 23,267 were annotated in 56 gene ontology (GO) categories, 6,033 were mapped to 131 KEGG pathways, and 7,825 were assigned to clusters of orthologous groups (COGs). From the unigenes, 8,165 EST-SSR loci were identified and 98.57% of them were 1-3 nucleotide repeats with 14.32%, 41.08% and 43.17% of mono-, di- and tri-nucleotide repeats, respectively. Fifty-eight types of motifs were identified with A/T, AG/CT, AT/AT, AC/GT, AAG/CTT and AGG/CCT the most abundant. The lengths of repeated nucleotide sequences in all SSR loci ranged from 12 to 60 bp, with most (88.51%) under 20 bp. Among 170 primer pairs were randomly selected from a total of 4,912 SSR primers we designed, 48 yielded unambiguously polymorphic bands with high reproducibility. Cluster analysis using 48 SSRs classified 34 flowering Chinese cabbage cultivars into three groups. A large number of EST-SSR markers identified in this study will facilitate marker-assisted selection in the breeding programs of flowering Chinese cabbage.
菜心是中国南方最重要的蔬菜作物之一。为满足该地区对市场的高需求，正在对这种作物的各种农艺性状进行遗传改良，但这一进程因该作物可用分子标记数量有限而受阻。本研究旨在从下一代测序产生的转录组序列中开发EST-SSR标记。对8个菜心样本进行RNA测序，鉴定出48,975个单基因。在这些单基因中，23,267个被注释到56个基因本体（GO）类别中，6,033个被映射到131条KEGG通路中，7,825个被分配到直系同源群（COG）簇中。从单基因中鉴定出8,165个EST-SSR位点，其中98.57%为1-3核苷酸重复，单核苷酸、二核苷酸和三核苷酸重复分别占14.32%、41.08%和43.17%。鉴定出58种基序类型，其中A/T、AG/CT、AT/AT、AC/GT、AAG/CTT和AGG/CCT最为丰富。所有SSR位点中重复核苷酸序列的长度范围为12至60 bp，大多数（88.51%）在20 bp以下。从我们设计的总共4,912个SSR引物中随机选择170对引物，其中48对产生了具有高重复性的明确多态性条带。使用48个SSR进行聚类分析，将3个菜心品种分为3组。本研究中鉴定出的大量EST-SSR标记将有助于菜心育种计划中的标记辅助选择。
