《植物生理学报》 2018, 54(5): 863-871

龙眼顶芽转录组简单重复序列(SSR)标记信息分析及分子标记开发

郭栋梁, 王静, 韩冬梅, 潘学文, 李建光^*

广东省农业科学院果树研究所, 农业部南亚热带果树生物学与遗传资源利用重点实验室, 广州510641

通信作者：李建光；E-mail: 550777196@qq.com

摘　要：

随着新一代测序技术的发展, 大量的转录组数据和表达序列标签(EST)成为开发简单重复序列(SSR)标记的可利用资源。本研究利用MISA软件筛选龙眼(Dimocarpus longan)顶芽转录组数据库序列, 从114 445条龙眼转录组unigene序列中发现11 546个SSR位点, SSR出现频率为10.09%。其中1 975条unigene含有两个或两个以上EST-SSR位点, 占所有SSR位点的比例为17.10%, SSR出现的平均距离为7.52 kb。从龙眼转录组SSR核苷酸基序类型来看, 二核苷酸(52.11%)和三核苷酸(46.15%)出现频率最高, 占所有核苷酸出现频率的99.26%。在龙眼转录组SSR中二核苷酸重复基元出现频率最高的是AG/CT (4 250个, 占36.81%), 三核苷酸重复基元出现频率最高的是AAG/CTT (1 109个, 占9.61%)。对含SSR位点的9 571条unigene序列进行引物设计, 共设计出了8 347对SSR位点特异引物。随机挑选合成50对EST-SSR引物, 以‘石硖’、‘储良’、‘古山2号’、‘立冬本’等四份龙眼材料的基因组DNA为模板对这批引物进行PCR扩增、筛选, 结果表明, 其中21对引物能产生理想的PCR产物, 有效扩增率为42%; 16对引物扩增条带具有多态性, 占有效引物的76.2%; 16对多态性引物共扩增获得50个条带, 其中多态性片段21个, 每对引物平均产生1.31个多态性片段。

关键词：龙眼; 转录组; SSR

收稿：2018-03-02   修定：2018-03-29

资助：国家自然科学基金青年基金(31301741)和广东省农业科学院院长基金(201309)。

Analysis on simple sequence repeat (SSR) information in apical transcriptome and development of molecular markers in Dimocarpus longan

GUO Dong-Liang, WANG Jing, HAN Dong-Mei, PAN Xue-Wen, LI Jian-Guang^*

Institute of Fruit Tree Research, Guangdong Academy of Agricultural Sciences; Key Laboratory of South Subtropical Fruit Biology and Genetic Resource Utilization, Ministry of Agriculture, Guangzhou 510641, China

Corresponding author: LI Jian-Guang; E-mail: 550777196@qq.com

Abstract:

With the development of next-generation sequencing technologies, a large number of transcriptome data and EST sequences have become available resources for developing simple sequence repeat (SSR) markers. Transcriptome of Dimocarpus longan terminal buds was screened using MISA software. A total of 11 546 SSRs that occurred in 114 445 unigenes was identified and the frequency of these SSRs was 10.09%. 1 975 unigenes contain two and more SSRs, and the frequency of two and more SSRs was 17.10%. The mean distance of SSRs was 7.52 kb in the unigenes. Among longan transcriptome SSRs, dinucleotide and trinucleotide were major repeat types, accounting for 52.11% and 46.15%, respectively. Furthermore, AG/CT and AAG/CTT were the most frequent motifs in dinucleotide and trinucleotide repeats, accounting for 36.81% and 9.61%, respectively. 8 347 pairs of SSR primers were designed for 9 571 SSR loci using Primer 5.0 software and 50 pairs of primers were randomly selected to evaluate their polymorphism across four varities, including ‘Shixia’, ‘Chuliang’, ‘Gushan No.2’ and ‘Lidongben’. The results show that the PCR products of 21 primers were clear and effective and the effective ratio was 42%. Sixteen pairs of primers were polymorphic, and their polymorphic percentage reached 76.2%. Among 50 amplification bands, 21 polymorphic bands were obtained by using 16 pairs polymorphic primers, and the average polymorphic amplification band of each EST-SSR primer was 1.31.

Key words: longan; transcriptome; SSR