山白树微卫星特征分析及分子标记开发

目的:山白树是中国特有珍稀濒危植物。分析微卫星特征,开发其微卫星分子标记。方法:采用Illumina二代测序技术建立山白树基因组文库和微卫星文库,分析微卫星组成类型,设计山白树引物,用5个山白树种群进行扩增和检测以分析其多态性。结果:二代测序共返回38,942,660条100 bp配对序列,通过质量修剪及拼接得到200,386条拼接序列,其中长度 335 bp的为7 614条;在7 614条序列中检测出微卫星位点694个,其中单核苷酸重复最多,单核苷酸重复中A/T重复数量最多;二核苷酸长度变异最大,其中AG/CT重复数量最多,重复长度变异情况与微卫星丰度呈正相关。为36条山白树微卫星重复次数高的序列设计引物,经聚合酶链式反应(PCR)扩增和聚丙烯酰胺凝胶电泳检测,20对引物多态性丰富且条带清晰,等位基因数(NA)在3~6之间,平均为4,多态性信息含量(PIC) 0. 535 5~0. 754 0,平均值0. 615 5。对5个山白树种群的群体遗传分析发现,该物种遗传多样性较高(h=0. 697 5,I=1. 436 8,HE=0. 702 2),种群遗传分化显著(Fst=0. 374)。结论:通过部分山白树的群体遗传也可以说明本次开发的引物可用性较好。该研究开发了山白树微卫星分子标记引物,为山白树分子遗传学奠定了基础。

Development of Novel Microsatellite Markers and Characteristic Analysis for Sinowilsonia henryi

Objective: To analyze the microsatellite characteristics and develop microsatellite markers for Sinowilsonia henryi, a rare and endangered plant endemic to China.Method: The Illumina second generation sequencing technology was used to establish the genome library and the microsatellite library of S. henryi. The compositions of the microsatellite were analyzed,and the primers of S. henryi were designed. Its polymorphism was analyzed by the amplification and detection of 5 S. henryi populations.Result: A total of 38,942,and 660 matched sequences of 100 bp were returned by the second generation sequencing. Among them,200,386 splice sequences were obtained by mass pruning and splicing; the number of sequences with length greater than 335 bp was 7 614, and 694 microsatellite loci were detected in the 7 614 sequences,in which the single nucleotide repeats were most and the number of A/T repeated in the single nucleotide repeats was highest. Dinucleotide showed the greatest length variation; the number of AG/CT repeats was highest, and the variation in repeat length was positively correlated with microsatellite abundance. The primers were designed for 36 S. henryi microsatellite sequences with high repetition. By PCR amplification and polyacrylamide gel electrophoresis,20 pairs of primers showed rich polymorphism and clear bands. The number of alleles (N_A) ranged from 3 to 6,with average of 4;the polymorphism information content (PIC) ranged from 0.535 5 to 0.754 0,with average of 0.615 5.The population genetic analysis of 5 S. henryi populations showed that the genetic diversity of the species was high (h=0.697 5,I=1.436 8,H_E=0.702 2),and the population genetic differentiation was significant (F_st=0.374).Conclusion: The population genetic analysis of S. henryi also showed that the primers developed by this study had a high usability. In this study,the primers of microsatellite markers of S. henryi were established,laying the foundation for the molecular genetics of S. henryi.