›› 2011, Vol. 54 ›› Issue (12): 1423-1432.

• 研究论文 • 上一篇    下一篇

火红熊蜂微卫星标记的筛选及种特异性分析

 安建东, 黄家兴, 董捷, 周冰峰   

  1.  福建农林大学蜂学学院, 福州 350002
  • 收稿日期:2011-05-06 修回日期:2011-11-21 出版日期:2011-12-20 发布日期:2011-12-20
  • 通讯作者: 周冰峰 E-mail:bingfengfz@126.com
  • 作者简介:安建东, 男, 1975年生, 甘肃天水人, 博士研究生, 副研究员, 研究方向为昆虫授粉, E-mail: anjiandong@yahoo.com.cn
  • 基金资助:

    国家自然科学基金项目(30800805, 30901055, 31072092); 国家“948”项目(2011-S1); 国家蜜蜂产业技术体系建设专项经费项目(CARS-45)

Isolation and characterization of microsatellite markers from Bombus pyrosoma (Hymenoptera, Apidae), a bumblebee species endemic to China

 AN  Jian-Dong, HUANG  Jia-Xing, DONG  Jie, ZHOU  Bing-Feng   

  1.  College of Bee Science, Fujian Agriculture and Forestry University, Fuzhou 350002, China
  • Received:2011-05-06 Revised:2011-11-21 Online:2011-12-20 Published:2011-12-20
  • Contact: ZHOU Bing-Feng E-mail:bingfengfz@126.com
  • About author:anjiandong@yahoo.com.cn

摘要: 火红熊蜂Bombus pyrosoma是中国特有的熊蜂资源, 在华北地区分布十分丰富, 是众多野生植物和农作物的重要传粉者。为了探究火红熊蜂的遗传结构及其进化关系, 首次开展了该种熊蜂卫星标记的筛选及其种特异性研究。本研究采用生物素-磁珠吸附分离法从火红熊蜂基因组中筛选微卫星标记, 并选取黑熊蜂亚属Melanobombus其他7种熊蜂进行了这些标记的种特异性验证。结果表明: 不同探针与磁珠杂交产生的微卫星标记阳性克隆率存在很大差异, TC探针的微卫星阳性克隆率最高为82%; TG探针次之, 为28%; AT和GC探针分离未获得微卫星标记克隆。根据获得的阳性克隆的序列设计引物进行筛选验证, 共获得31对熊蜂微卫星序列特异性引物。微卫星标记分析发现, 完美型(perfect)18个, 占58.1%; 非完美型(imperfect)10个, 占32.3%; 混合完美型(compound perfect)1个, 占3.2%; 混合非完美型(compound imperfect)2个, 占6.4%。不同探针的微卫星核心序列重复数不同, TC探针和TG探针的微卫星核心序列最高重复数分别为28和15个, 最低重复数分别为7和11个。利用所获得的31对微卫星引物对黑熊蜂亚属其他7种熊蜂进行检测, 有26对引物可以扩增所测试的7种熊蜂, 其他5对引物只能扩增部分熊蜂种类, 其中BPM5是火红熊蜂种特异性引物。本研究从火红熊蜂基因组中筛选的微卫星标记, 不仅可用于火红熊蜂的遗传结构、 分子进化和资源保护等方面的研究, 而且可进一步用于黑熊蜂亚属其他种群的遗传特性分析。

关键词:  , 熊蜂, 火红熊蜂, 分子标记, 微卫星标记, 磁珠富集, 引物设计, 种特异性

Abstract: Bombus pyrosoma, a bumblebee species endemic to China, plays a vital role as one of the most abundant pollinators for many wild flowers and crops in North China. We report for the first time  the isolation and characterization of microsatellite markers within this population in order to support investigations into its genetic structure and evolution. Magnetic bead-based enrichment, PCR and bioinformatics were used to isolate microsatellite markers from B. pyrosoma. The species specificity of the isolated primers was confirmed through tests with seven other bumblebee species of the subgenus Melanobombus also known from China. Our results show that four dinucleotide probes have different hybridization rates to the microsatellite DNA sequence. The highest hybridization rate (82%) was obtained with the probe TC, the moderate hybridization rate (28%) with TG, and the lowest hybridization rates (0%) with AT and GC. For 31 polymorphic microsatellite loci isolated from B. pyrosoma, four types of microsatellite can be found by sequence analysis, i.e., 18 perfect types (58.1%), 10 imperfect types (32.3%), 1 compound perfect type (3.2%) and 2 compound imperfect types (6.4%). The repeat unit of the microsatellite core sequence is different in each probe. The highest repeat unit for TC and TG probes is 28 and 15, respectively, and the lowest repeat unit for TC and TG probes is 7 and 11, respectively. The primer specificity test on 31 primer pairs showed that products can be amplified from all seven bumblebee species with 26 primer pairs, and the other 5 primer pairs were found to be specific for some of the seven species, of which one primer (BPM5) was found to be specific for B. pyrosoma. These novel microsatellite markers will be useful not only for future studies on the genetic structure, molecular evolution and resource conservation of this endemic species, but also for population genetic studies of other Melanobombus species.

Key words:  Bumblebee, Bombus pyrosoma, molecular marker, microsatellite markers, magnetic bead enrichment, primer design, species specificity