Abstract：Aconitum carmichaeli is a perennial herb of Aconitum in family Ranunculace, and it can be radix aconiti as medicine. Reports about Aconitum drug poisoning incidents have been very common, which are mainly caused by failure to identify species of Aconitum effectively. Through the research on development of Aconitum microsatellite primer and genetic diversity, the research realized fast and accurate identification of genetic resources and fake A. carmichaeli, which provided scientific guarantee for protection, development and successive research on superior genetic resources of A. carmichaeli. By extracting DNA of A. carmichaeli, establishing genomic library of A. carmichaeli, and sequencing A. carmichaeli with Illumina high-throughput sequencing technology, the research acquired plenty of 100 bp short-sequence microsatellite fragments. After screening the microsatellite fragments, microsatellite library of A. carmichaeli was established. According to the results, through second-generation high-throughput sequencing technology of Illumina, 38 942 660 strips of 125 bp pairing sequence were acquired. Through quality trim, 200 386 strips of splicing sequence were obtained after assembly. After filtration and screening, 12 095 strips of sequence fragments being larger than 299 bp were acquired finally. The fragments with repeat segment being larger than 100 bp were selected from microsatellite library of A. carmichaeli for primer design of microsatellite, and 55 pairs of microsatellite primers were obtained. By the use of these 55 pairs of microsatellite primers, amplification and polypropylene gel electrophoresis were carried out for 80 individuals from 4 wild Aconitum populations to detect the polymorphism of microsatellite primers and genetic diversity of population. After detection, 14 pairs of Aconitum microsatellite primers with clear stripe and rich polymorphism were acquired. For the 14 pairs of Aconitum microsatellite primers, average number of allele (A) was 4.85, average observation heterozygosity (Ho) was 0.613, average expected heterozygosity (He) was 0.583 and PIC was 0.528. Four sibling species of A. carmichaeli were used for universality detection of microsatellite primers. It was found that the stripe of 11 pairs of microsatellite primers were clear, and they occupied 78.57% of total microsatellite primers. According to the results of universality detection of Aconitum microsatellite primers, most of the microsatellite primers were proved amplifiable in sibling species of A. carmichaeli. Through analysis of genetic diversity of 4 A. carmichaeli populations, it was found that A. carmichaeli showed high genetic diversity at species level. Besides, for the Aconitum populations, A was 3.25, HO was 0.612, He was 0.493, Shannon's information index (I) was 0.851 and Nei's genetic diversity index (h) was 0.505. Genetic differentiation between populations showed that certain genetic differentiation (FST=0.149) existed in 4 populations of A. carmichaeli and gene flow between populations (NM)=1.432 was limited. According to research results, 14 pairs of microsatellite primers developed by the research showed high polymorphism and good universality, which could be used for identification of genetic resources, analysis of genetic diversity and construction of DNA fingerprint of A. carmichaeli. From the perspective of species level, A. carmichaeli had rich genetic diversity, which verifies abundant heritable variation ability of A. carmichaeli from DNA level and explained strong evolution potentiality of A. carmichaeli. The study will offer scientific guarantee for identification of genetic resources and fake A. carmichaeli, protection, development and successive research on superior genetic resources of A. carmichaeli.
[1] 中国科学院中国植物志编辑委员会，中国植物志(第65卷)[M]. 北京:科学出版社,1979，(第27卷)：264-265 [2] 中国药典[S]. 一部, 2015：39-40 [3] 王峰峰，宋兆辉，张兰兰，等. 乌头碱、新乌头碱、次乌头碱水解和醇解产物的研究[M]. 中国中药杂志，2012，37(11)：1564-1568 [4] 肖培根，王锋鹏，高峰，等. 中国乌头属植物药用价值亲缘研究[J]. 植物分类学报，2006，44(1)：1-46 [5] 罗艳，杨亲二. 四川乌头属的修订[J]. 植物分类学报，2005，43(4)：289-386 [6] 胡平，夏燕莉，周先建，等. 乌头种质资源形态学多态性研究初报[J]. 资源开发与场，2008，24(5)：448-449 [7］Dong Y C，Cao Y S，Zhang X Y，et al. Establishment of candidate core collections in Chinese common wheat germplasm[J]. J Plant Genet Res, 2003, 26: 41-48 [8]刘峰，冯雪梅，钟文，等.适合棉花品种鉴定的SSR核心引物的筛选[J]. 分子植物育种，2009，7(6)：1160-1168 [9]谢明璐，侯北伟，韩丽，等. 珍稀铁皮石斛SSR标记的开发及种质纯度鉴定[J]. 药学学报，2010，45(5)：667?672 [10]魏丽娟，钱伟， 付东辉，等. 甘蓝型油菜种质资源遗传多样性SSR分析及芸薹属核心SSR引物筛选[D]. 重庆：西南大学，2012：27-32 [11]叶占洋，王兆山，李云晓，等.中国特有濒危植物翅果油树的SSR引物开发及特性[J]. 西北植物学报，2016，36(2)：0274-0279 [12]刘丽，张金智，梅丽，等. 兼性无融合生殖龙须草SSR引物开发及杂交后代检测[J]. 西北植物学报，2008，8(10)：1947-1953 [13]尹国英，杨晓燕，何其波，等. 鉴定烟草种质资源SSR核心引物筛选和验证[J]. 植物遗传资源学报，2013，14(5)：960-965 [14]韩瑞霞，张宗文，吴斌，等. 苦荞SSR引物开发及其在遗传多样性分析中的应用[J]. 植物遗传资源学报，2012，13(5)：759-764 [15]邓绍勇，温强. 大青属植物通用性SSR引物筛选及应用[J]. 中草药，2014，45(22)：3317-3322 [16]王小童，周天华，陈征，等. 开口箭种质资源的SSR遗传多样性分析[J]. 陕西理工学院学报，2016，01(2016)：1673－2944 [17]朱大强，李存. 四种常用高通量测序拼接软件的应用比较[J]. 生物信息学，2012，(02) [18]李彩娟. 基于第二代测序的大鳞副泥鳅微卫星分子标记的开发与应用[D]. 苏州大学，2014 [19] Tian-Hua Zhou, Shan-Shan Dong, Li Shan, et al. Genetic Structure Within and Among Populations of Saruma henryi, an Endangered Plant Endemic to China[J]. Biochemical Genetics. 2012, 50: 146-158. [20] Tian-Hua Zhou, Gui-Fang Zhao, Zengqian Qian,et al. Characterization of EST-derived microsatellite loci in Saruma henryi Oliv., an endangered Chinese endemic herb[J]. Conservation Genet Resour. 2009. 1: 67-70. [21] Tian-Hua Zhou, Zengqian Qian, Shan Li, et al.. Genetic diversity of the endangered Chinese endemic herb Saruma henryi Oliv. (Aristolochiaceae) and its implications for conservation[J]. Population Ecology. 2010. 52:223-231. [22] 侯宁，张妍，鲁翠云，等. 微卫星DNA标记分析德国锦鲤的遗传潜力[J]. 遗传，2007，29(12)：1509-1518. [23] Nei M.Molecular evolutonary[M]. New York:Columbia University press, 1987: 121-134 [24] Wright S.Evolution and the genetics of populations//Variability within and among natural populations[M]. Chicago: University of chicago press, 1978.4 [[25] Xiao Hu, Ying Diao, Isolation and characterization of simple sequence repeat loci in Miscanthus floridulus and their potential use as markers in related species[J]. Biomass and Bioenergy 2012, (18)I-4. [26] Solomon B, Mengyu M.Assessment of genetic variation in tomato(Solanum lycopersicum L.)inbred lines using SSR molecular markers[J]. Genettics and Genomics 2008, (35)373?379. [27] 李忠虎，刘晓东，王小琦，等. 党参微卫星引物筛选及群体遗传多样性研究[J]. 中草药，2013，44(2)：210-214.
