苦瓜MSAP反应体系的优化及其种质资源表观遗传多样性

Optimization of MSAP Reaction System and Epigenetic Diversity of Germplasm Resources in Bitter Gourd(Momordica charantia L.)

为研究苦瓜表观遗传多样性,通过正交设计方法优化了MSAP技术中预扩增和选择性扩增系统的关键因素,构建了适用于苦瓜的MSAP反应体系,并利用其对52份苦瓜种质资源的表观遗传多样性进行分析。结果表明:30μL MSAP双酶切反应体系中,加入Eco RⅠ和MspⅠ/HpaⅡ各10 U,于37℃水浴酶切4 h时使酶切完全;最佳预扩增反应体系20μL,包含连接产物4μL,10×PCR buffer 2.0μL(25 mmol/L),d NTPs0.3μL(2.5 mmol/μL),Taq酶0.1μL(5 U/μL),10 mmol/L上下游引物各1.0μL;最佳选择性扩增反应体系20μL,包含4μL稀释150倍的预扩增产物。从110对引物中选出扩增效果好,条带清晰的12对引物组合利用最佳反应体系对52份苦瓜种质资源进行MSAP扩增,共得到430条条带,其中多态性条带218条,多态性比率为50.70%。对DNA甲基化模式进行分析,其中类型Ⅰ(非甲基化类型)3109条,类型Ⅱ(半甲基化类型)633条,类型Ⅲ(全甲基化类型)606条,类型Ⅳ(外部甲基化)1250条,苦瓜甲基化模式主要为外部甲基化为主。Nei's基因多样性指数和Shannon信息指数分别为0.226 5、0.372 6,该结果表明试验中所用的苦瓜种质资源具有丰富的遗传多样性,遗传变异和进化水平较高。UPGMA聚类分析结果表明12个不同地区苦瓜种质资源在相似性系数0.88处可分为4大类,大部分种质被聚集在第1类中,又可以分为5个亚群,亚群Ⅰ:中国海南、中国广东、中国福建和日本;亚群Ⅱ:中国云南;亚群Ⅲ:泰国和格林纳达;亚群Ⅳ:中国北京;亚群Ⅴ:中国台湾。本研究通过利用正交优化建立MSAP反应体系,并对苦瓜种质资源甲基化表观遗传多样性进行研究,这种基因组结构水平上的研究将为苦瓜种质资源的种群分化及物种进化的进一步研究提供依据。

In order to study the apparent genetic diversity of Momordica charantia, the orthogonal design method was used to optimize the key factors and selective pre amplification system in MSAP technology. Then, the MSAP reaction system suitable for M. charantia was constructed, which was used for the analysis of apparent genetic diversity of 52 M. charantia germplasm resources. The results showed that： in 30 p,L of MSAP double digestion reaction system, 10 U of EcoR Ⅰ and Msp ⅠHpa Ⅱ were entirely digested at 37℃ for 4 h. The optimal pre-amplification reaction system was 20μL, containing 4 p,L of ligation product, 10xPCR buffer 2.0μL （25 mmol/L）, dNTPs 0.3 μL （2.5 mmol/p,L）, Taq polymer 0.1 μL （5 U/μL）, 1.0 μL upstream and downstream primers （10 mmol/L）.In 20 μL of the optimal selective reaction system, there were 4 μL ofpre-amplification products which was diluted 150 times. 12 pairs of primers with good amplification effect and clear bands were selected from 110 pairs of primers, and the MSAP amplification of 52 accessions of M. charantia germplasm was carried out by the best reaction system. A total of 430 bands were obtained, in which there were 218 polymorphic bands, with the polymorphism ratio of 50.70%. The analysis of DNA methylation patterns showed that there were 3109 of type Ⅰ（non-methylated type）, 633 of type Ⅱ（semi-methylation type）, 606 of type Ⅱ （full methylation type）, type Ⅳ （external Methylation） 1250, and the methylation model of M. charantia was mainly for external methylation. Nei＇s gene diversity index and Shannon information index were 0.2265, and 0.3726, respectively. These results indicated that the germplasm resources ofM. charantia used in the experiment were rich in genetic diversity, and the genetic variation and evolution level were higher： UPGMA cluster analysis implied that germplasm resources from 12 different regions could be divided into 4 categories at the similarity coefficient of 0.88. Most of the germplasm was clustered in the first category and could be divided into five subgroups. Subgroup Ⅰ ： Hainan, China, Guangdong, China, Fujian China and Japan; subgroup Ⅱ ： Yurman, China; subgroup Ⅱ ： Thailand and Grenada; subgroup Ⅳ ： Beijing, China; subgroup Ⅴ ： Taiwan, China. In this study, the MSAP reaction system was established by using orthogonal optimization, and the apparent genetic diversity of the germplasm resources of M. charantia was studied. This study could provide a basis for further studies on population differentiation and species evolution of M. charantia germplasm resources.