《植物生理学报》 2017, 53(7): 1267-1278

普通油茶转录组EST-SSR分子标记开发

李海波¹, 王珊², 丁红梅³, 陈友吾¹, 胡传久¹, 李楠¹, 魏海龙^1,*

¹浙江省林业科学研究院, 杭州310023; ²浙江农林大学林业与生物技术学院, 浙江临安311300; ³浙江中医药大学分析测试中心, 杭州310053

通信作者：魏海龙；E-mail: 1178646251@qq.com

摘　要：

通过转录组测序技术鉴别普通油茶(Camellia oleifera) EST-SSR位点, 开发筛选适用于普通油茶种质资源评价与利用的多态性SSR标记, 对普通油茶的遗传变异研究和油茶良种的分子标记辅助育种具有重要意义。利用 MISA 工具从普通油茶转录组67 434条Unigenes (63.39 MB)中筛选得到26 751个包含1~6个核苷酸重复类型的SSR位点, SSR的分布频率为39.67%, 发生频率为1/2.33 kb; SSR位点中的主导类型是二核苷酸重复, 占总SSR的44.28%, 其次是单核苷酸(35.28%)和三核苷酸重复(18.27%)。通过SSR标记的琼脂糖凝胶电泳和荧光标记毛细管电泳, 从143对SSR引物中遴选出了20对扩增效率高、稳定性好的多态性SSR引物, 并对来自浙江产区的57个普通油茶品种进行遗传关系分析。结果显示: 20对SSR引物共检测出129个等位基因, 每对引物2~17个, 平均6.45个; 多态性信息量(PIC)为0.0499~0.8635, 平均0.4355。57个普通油茶品种在Dice遗传相似系数大约0.47时聚为一类, 在大约0.60时可分为七个大类。本研究印证了利用普通油茶转录组数据开发SSR标记的可行性, 也证明了茶属树种SSR标记的可转移性; 不仅为普通油茶遗传多样性分析和遗传图谱构建提供了有价值的候选标记, 也为普通油茶良种的鉴别与分子选育提供了分子技术手段。

关键词：油茶; 转录组; EST-SSR; 遗传关系; 多态性

收稿：2017-04-17   修定：2017-06-08

资助：浙江省科研院所扶持专项(2014F30002和2013F50012)和浙江省重大科技专项(2010C02005-1)。 * 通讯作者(E-mail: 1178646251@qq.com)。

Development of EST-SSR molecular markers based on transcriptome sequencing of Camellia oleifera

LI Hai-Bo¹, WANG Shan², DING Hong-Mei³, CHEN You-Wu¹, HU Chuan-Jiu¹, LI Nan¹, WEI Hai-Long^1,*

¹Zhejiang Academy of Forestry, Hangzhou 310023, China; ²School of Forestry and Bio-technology, Zhejiang Agriculture and Forestry University, Lin’an, Zhejiang 311300, China; ³Analysis and Test Center, Zhejiang Chinese Medical University, Hangzhou 310053, China

Corresponding author: WEI Hai-Long; E-mail: 1178646251@qq.com

Abstract:

Identificaiton of EST-SSR loci based on transcriptome data and development of polymorphic SSR markers suitable for evaluation and application of germplasm resources on Camellia oleifera are critical for the study on genetic variation of C. oleifera and molecular marker assistant breeding in superior cultivars of C. oleifera. A total of 26 751 SSR loci, including the types of 1–6 nucleotide repeats with occurring frequency of 1/2.33 kb, were identified from 67 434 unigenes (63.39 MB) in C. oleifera transcriptome by using MISA software. The distribution frequency of SSRs was 39.67%. Dinucleotide repeat was the main type, accounted for as much as 44.28% of all SSRs, followed by mononucleotide repeat (35.28%) and trinucleotide repeat (18.27%). Using agarose electrophoresis and capillary electrophoresis, 143 SSR markers were detected, among which 20 can produce clear, reproducible and polymorphic bands. Genetic relationship analysis among 57 C. oleifera cultivars from Zhejiang production areas was perfomed with the 20 fluorescent labeled SSR primer pairs. The results showed 129 alleles were detected from the 20 SSR loci, the number of alleles detected by each SSR primer pairs ranged from 2 to 17, with an average of 6.45. Polymorphism information content (PIC) ranged from to 0.0499–0.8635, with an average of 0.4355. Fifty-seven C. oleifera cultivars were clustered into one group at a genetic similarity coefficients (GSC) level by Dice of 0.47, seven groups at 0.60. Our present study verified that using C. oleifera transcriptome data to develop SSR markers was feasible, also proved that the transferability of SSR markers across species of Camellia. The SSR markers obtained in the study could be valuable candidate markers for genetic diversity analysis and genetic mapping construction in C. oleifera, also be used as a molecular technical tool for identification and molecular marker assistant breeding in superior cultivars of C. oleifera.

Key words: Camellia oleifera; transcriptome; EST-SSR; polymorphism; genetic relationship