摘要
【目的】筛选适合夏蜡梅(Sinocalycanthus chinensis)遗传多样性分析的SSR(simple sequence repeats)引物并进行遗传多样性分析。【方法】从夏蜡梅转录组数据库组装的125 014条unigene序列中,随机选取了344对SSR引物,以夏蜡梅6个不同种群的DNA为模板,运用聚丙烯酰胺凝胶电泳法进行引物筛选,根据扩增产物带型的丰富度,筛选出具有多态性的SSR引物;挑选扩增带型数在4以上的SSR引物,进一步验证这些引物在夏蜡梅种群间和种群内的适用性;利用已验证的引物对6个夏蜡梅种群的遗传多样性和遗传结构进行分析。【结果】共有148对引物能够扩增出清晰条带,占选取引物总数的43.02%,其中29对引物具有多态性,占扩增总数的19.60%;有15对引物扩增带型数在4以上,这些引物具有更高的多态性。对这15对高多态性引物进一步分析发现,根据扩增的带型数和遗传参数,这些引物均可用于种群间遗传多样性分析;同时,这15对引物可进一步分为3类,所揭示的遗传多样性程度依次降低。在种群内遗传多样性分析时,根据带型数及其分布和遗传参数,第1类、第2类及第3类中的P004和P061在各个种群内扩增的带型数较多且分布较为均匀,具有更高的多态性;而P021、P075、P095、P098、P018等5对引物在部分种群内扩增的带型数相对较少且分布较为集中,多态性相对较低。利用15对引物分析6个种群的遗传多样性得知,夏蜡梅的总基因多样性指数(Ht)为0.625 0,各种群的基因多样性指数为0.251 8~0.400 2,Shannon’s指数(I)为0.419 7~0.668 1,平均基因多样性指数(Hs)为0.3230。【结论】此次实验结果说明夏蜡梅物种水平具有较高的遗传多样性,但各种群内遗传多样性较低。聚类分析将6个种群分为3个分支,且各分支之间遗传距离较远,种群间遗传分化系数(Gst)为0.437,种群内近交系数(Fis)为0.430,种群间基因流(Nm)为0.309 4,基于AMOVA分子方差分析,56%的变异来自种群间,44%的变异来自种群内,这说明夏蜡梅种群间遗传分化显著,种群内存在显著的近交现象,各种群间基因交流受阻。
【Objective】Our objective was to screen out the primers to analysis the intra-population and inter-population genetic diversity of Sinocalycanthus chinensis and genetic diversity analysis.【Methods】From the 125 014 unigene sequences assembled from the S. chinensis transcriptome database,344 pairs of SSR（ simple sequence repeats） primers were randomly selected. Firstly,six different populations of DNA were used as templates for polyacrylamide gel electrophoresis. Primers were screened to screen for polymorphic SSR primers based on the richness of amplified product bands; SSR primers with an amplification band number of 4 or more were selected to further verify that these primers were suitable within populations and among populations of S. chinensis.The applicability of the use of the validated primers was used to analyze the genetic diversity and genetic structure of six populations of S. chinensis.【Results】Our results showed that 148（ 43.02%） SSR primer pairs could amplify clear bands,and among them,29（ 19.60%） pairs had polymorphisms. There were 15 primer pairs that could amplify more than 4 bands,and these primers had a higher polymor-phism. Further analysis of the 15 primer pairs revealed that these primers all could be used for genetic diversity analysis among populations based on the number of bands amplified and genetic parameters. Additionally,the 15 primer pairs could be further divided into three groups,and the degree of genetic diversity in turn decreased. In the intra-population genetic diversity analysis,the amplified bands of P004 and P061 in the first,second and third class were high and had a uniform distribution,with higher polymorphism in each population according to the number of bands,their distribution,and genetic parameters. However,the genetic diversity of P021,P075,P095,P98 and P018 was a bit lower than that of the others. The diversity of species level（ Ht） was 0.625 0. The genetic diversity index of each population ranged from 0.251 8 to 0.400 2. The Shannon index（ I） ranged from 0. 419 7 to 0. 668 1,and the average genetic diversity index（ Hs） was0. 323 0. 【Conclusion】The results indicated that the species level had high genetic diversity,but the genetic diversity intra-population is low. Cluster analysis divided the six populations into three groups,and the genetic distance was far between the groups. The genetic differentiation coefficient（ Gst） was 0.437,the inbred coefficient（ Fis） within the population was 0.430,and the inter-population gene flow（ Nm） was 0.309 4. Based on the AMOVA molecular variance analysis,56%of the variation was inter-population and 44% of the variation was intra-population. The above shows that there was significant genetic differentiation among populations of S. chinensis,there was a significant inbreeding phenomenon within populations,and gene exchange between various populations was blocked. In this study,we first published the SSR primer information of S. chinensis and provided the support for research on the mating system of S. chinensis in the future.
作者
吴静
黄耀辉
董彬
张超
付建新
周莉花
赵宏波
WU Jing;HUANG Yaohui;DONG Bin;ZHANG Chao;FU Jianxin;ZHOU Lihua;ZHAO Hongbo(School of Landscape Architecture, Zhejiang A ＆ F University, Lin＇an 311300, China)
出处
《南京林业大学学报（自然科学版）》
CAS
CSCD
北大核心
2018年第3期58-66,共9页
Journal of Nanjing Forestry University：Natural Sciences Edition
基金
国家自然科学基金项目(31101571
31401902)
关键词
夏蜡梅
SSR分子标记
引物筛选
适用性验证
遗传多样性
Sinocalycanthus chinensis
SSR molecular marker
primer screening
applicability verification
genetic diversity
