基于COI基因的广西广地龙(Amynthas aspergillum)种群遗传多样性分析

Analysis of Genetic Diversity of Amynthas aspergillum Population in Guangxi Based on COI Gene

关键词:  广地龙  COI基因  遗传多样性  桂十味  道地药材

Key words:  Amynthas aspergillum  COI gene  genetic diversity  Genuine medicinal material  medicinal materials

摘要:

广地龙是参状远盲蚓(Amynthas aspergillum)的俗名，是我国广东与广西地区广泛分布的蚯蚓物种，有着重要的药用价值。近几年，由于中药材市场对广地龙需求旺盛，野生的广地龙种群与数量急剧下降。因此，深入研究广地龙的遗传多样性与群体结构，对推动种质资源保护、人工养殖及种群选育具有重要意义。本研究根据蚯蚓形态，同时基于COI基因的分子分类方法鉴定获取广西广地龙样品，并基于COI基因序列开展单倍型分析、遗传多样性评估、系统发育树构建、群体结构分析以及MK检验等，以探讨广地龙在野外和养殖群体中的遗传多样性及分布格局。实验共获得103条广地龙样品；广地龙主要分布于广西南部(南宁、玉林、钦州、防城港等地)，少数分布于广西西部(百色、河池)和北部(柳州、桂林、贺州)，且与其他远盲属(Amynthas)的蚯蚓物种共享栖息地。在103条广地龙COI基因序列中共检测到98个变异位点和25种单倍型，整体单倍型多样性指数为0.919，核苷酸多样性指数为0.050。种群间遗传分化固定系数(Fixation index，F_st)分析显示不同地理群体间的遗传分化指数为0-0.08，部分单倍型如Hap1、Hap2、Hap4和Hap15在多个地理群体中共享。基于COI基因构建的系统发育树揭示广地龙存在两个高度分化的主要分支(F_st=0.943)，且MK检验显示结果差异显著(P<0.001)，但种群分化与地理位置无显著对应关系，即同一采集点常存在多个线粒体谱系。此外，在广地龙养殖群体中仅发现7种单倍型，且彼此聚类在一起，表明当前养殖群体的遗传多样性较低，对广地龙遗传资源保护有限。广西南部地区凭借适宜的降水和温度条件，成为广地龙最适宜的栖息与繁殖区域，并拥有最高的单倍型多样性，因此具有重要的生态与保护价值。广地龙群体中存在线粒体基因组深度分化的现象，且当前养殖群体中遗传多样性较低，因此有必要划定一些核心保护区域，并加强就地保护及长期监测工作，以促进该药用资源的可持续利用。

Abstract:

Guang Dilong is the common name of Amynthas aspergillum,which is a widely distributed earthworm species in Guangdong and Guangxi regions of China,and has important medicinal value.In recent years,due to the high demand for A.aspergillum in the Chinese herbal medicine market,the population and quantity of wild A.aspergillum have declined sharply.Therefore,in-depth study of the genetic diversity and population structure of A.aspergillum is of great significance to promote the protection of germplasm resources,artificial breeding and population breeding.In this study,according to the morphology of earthworms,and based on the molecular classification method of COI gene,the samples of A.aspergillum in Guangxi were obtained.Then,based on the COI gene sequence,haplotype analysis,genetic diversity assessment,phylogenetic tree construction,population structure analysis and MK test were carried out to explore the genetic diversity and distribution pattern of A.aspergillum in wild and cultured populations.A total of 103 samples of A.aspergillum were obtained.It was mainly distributed in southern Guangxi (Nanning,Yulin,Qinzhou,Fangchenggang,etc.),and a few was distributed in western Guangxi (Baise,Hechi) and northern Guangxi (Liuzhou,Guilin,Hezhou).It shared habitats with other earthworm species of the genus Amynthas.A total of 98 variation sites and 25 haplotypes were detected in 103 COI gene sequences.The overall haplotype diversity index was 0.919,and the nucleotide diversity index was 0.050.F_st analysis showed that the genetic differentiation index among different geographic populations was 0-0.08,and some haplotypes such as Hap1,Hap2,Hap4 and Hap15 were shared among multiple geographic populations.The phylogenetic tree based on COI gene revealed that there were two highly differentiated main branches (F_st=0.943),and the MK test showed that the results were significantly different (P<0.001),but there was no significant correspondence between population differentiation and geographical location,that is,there were often multiple mitochondrial lineages at the same sampling site.In addition,only 7 haplotypes were found in the cultured population of A.aspergillum and clustered together,indicating that the genetic diversity of the current breeding population was low and the protection of the genetic resources of A.aspergillum was limited.With suitable precipitation and temperature conditions,southern Guangxi had become the most suitable habitat and breeding area for A.aspergillum,and had the highest haplotype diversity,so it had important ecological and conservation value.There is a deep differentiation of the mitochondrial genome in the A.aspergillum population,and the genetic diversity in the current breeding population is low.Therefore,it is necessary to delineate some core protection areas and strengthen in-situ protection and long-term monitoring to promote the sustainable utilization of the medicinal resources.