基于SNP分子标记的华东地区松材线虫种群遗传分化研究

【目的】华东地区的苏浙皖鲁是中国松材线虫病发生最早的4个省区,华东地区也是松材线虫最适生长区,研究该区域松材线虫种群的遗传分化情况,可为建立我国松材线虫病的疫源追溯体系提供重要的基础信息。【方法】收集华东地区安徽(AH)、福建(FJ)、江苏(JS)、江西(JX)、山东(SD)和浙江(ZJ)6个省份的60个县级行政区松材线虫病疫木样本,经过分离纯化获得虫株,提取虫株DNA并进行高通量全基因组重测序,运用生物信息学软件分析各区域松材线虫虫株的SNP位点数量和种类,依此遗传标记采用聚类分析方法比较各区域不同虫株间的遗传分化情况,后采用Treemix分析群体间的基因渗入路线。【结果】共分离收集到华东地区松材线虫虫株67个,对所有虫株进行全基因组测序。经序列比对分析,67个虫株中共有SNP基因型12种,其中A→G、C→G、C→T、G→A、G→C、T→C这6种基因型出现的频率明显高于其他6种基因型。从67个虫株中共获得6 531 684个SNP位点,不同虫株间的SNP位点数量存在差异。不同地理区域松材线虫的SNP总数、纯合子数量、缺失SNP数量以及特有SNP数量在省际均未表现显著差异,与入侵时间也无特别显著的相关性。主成分分析结果表明,67个虫株可以分为3个类群,各类群与地理来源存在着一定的相关性。大多数虫株属于类群1,它包括了所有的浙江虫株和江西虫株,以及其他4个省份的大多数虫株;类群2涉及江苏、安徽、山东和福建的14个虫株;类群3仅涉及安徽、福建和山东的7个虫株。通过Treemix检测得到2条基因迁移路线,分别为类群2的江苏(2-JS)向类群1的安徽(1-AH)迁移、类群3的山东(3-SD)向类群2的安徽(2-AH)迁移。【结论】华东地区松材线虫虫株存在较为丰富的SNP位点,不同虫株间SNP特征存在较大差异,SNP多样性变化与入侵时间没有呈现明显规律性。总体上华东地区松材线虫虫株可以分为3个类群,不同类群与地理区域间呈现一定的相关性,在1-AH和2-AH所在区域可能存在被其他区域的其他类群虫株再次入侵的情况。

Genetic differentiation of Bursaphelenchus xylophilus in east China based on single nucleotide polymorphisms (SNP) markers

【Objective】 Jiangsu, Zhejiang, Anhui and Shandong, these four provinces in east China, are recognized as the initial occurrence of pine wilt disease in China. And east China is also the most suitable growth area for Bursaphelenchus xylophilus. Studying the genetic differentiation of the B. xylophilus population in those regions will provide important basic genetic information to establish a B. xylophilus epidemic source tracing system in China. 【Method】 Samples of B. xylophilus were collected from pine trees affected by pine wilt disease in east China, and strains were isolated and purified. The genomic deoxyribonucleic acid (DNA) of strains was extracted and sequenced. The number and types of single nucleotide polymorphism (SNP) loci and genotypes in each region were analyzed using bioinformatics software. Cluster analysis was used to compare the genetic differentiation among different strains based on SNP markers, while Treemix was used to detect gene penetration routes. 【Result】 A total of 67 B. xylophilus strains were isolated and collected in 60 county-level administrative regions of Anhui (AH), Fujian (FJ), Jiangsu (JS), Jiangxi (JX), Shandong (SD) and Zhejiang Province (ZJ) in East China. All strains were sequenced through the whole genome; genome calling analysis indicated 12 genotypes, of the 67 strains, where six types (i.e., A→G, C→G, C→T, G→A, G→C and T→C) occurred at a higher frequency than others. A total of 6 531 684 SNP loci were obtained and the number of SNP was different between strains. The number of SNP counts, homozygotes, missing SNPs, and private SNPs showed no significant regional differences among the six provinces, and there was no significant correlation with invasion time. Principal component analysis showed that 67 strains of B. xylophilus may be divided into three groups; each group had a specific correlation with a geographic origin. Most strains belonged to group 1, which included all strains from Zhejiang and Jiangxi, as well as most of strains from the other four provinces. Group 2 included 14 strains from Jiangsu, Anhui, Shandong and Fujian, and group 3 included only seven strains from Anhui, Fujian and Shandong provinces. Two gene migration routes detected by Treemix: ① group 2 of Jiangsu to group 1 of Anhui; and ② group 3 of Shandong to group 2 of Anhui. 【Conclusion】 There were abundant SNP loci in B. xylophilus in east China, where the SNP characteristics of different strains were relatively disparate, and there was no clear regularity between SNP diversity and invasion time. Overall, the B. xylophilus strains in east China may be divided into three groups, and there was a specific correlation between different groups and geographic areas. In the area where the 1-AH and 2-AH groups were located, there may have been re-invasion by strains from groups in other areas.