摘要
粗脚粉螨是我国常见的粉螨,是仓储粮食、经济作物和食用菌的重要害螨,其经济重要性受到国内外的广泛重视。本研究采用ISSR分子标记探讨了不同居群粗脚粉螨的遗传多样性及其居群遗传结构。主要研究内容及结果如下。
(1)探索并确定了粗脚粉螨基因组DNA提取的最佳方案。SDS(10%m/v)40μl,蛋白酶K(10mg/mL)6μl,STE 204μl;消化温度:45℃;消化时间:10-12h;粗脚粉螨个体数:50只;总体积250μl。
(2)建立了ISSR-PCR最佳反应体系。通过正交实验方法,建立了粗脚粉螨ISSR-PCR最佳反应体系:Mg~(2+)浓度2.0mmol/L、Taq DNA聚合酶0.75U、dNTPs浓度0.25μmol/L、引物浓度0.3μmol/L、模版DNA量20ng,总体积为25μl。
(3)ISSR-PCR扩增结果:共筛选出18条条带清晰、扩增稳定、多态性高的引物,分别对江西南昌(NC)、九江(JJ)及安徽巢湖两个不同寄主(AH-1、AH-2)共4个不同居群进行ISSR-PCR扩增,共获得136条带,其中多态性条带107条,多态带百分比(PPB)为78.68%。
(4)遗传多样性分析:在居群水平上,平均期望杂合度(He),Shannon信息指数(Ⅰ)和多态带百分比分别为0.1302,0.1875和30.15%;在物种水平上,期望杂合度(He),Shannon指数(Ⅰ)和多态带百分率(PPB)分别为0.3586,0.5314和78.68%。4个居群的遗传多样性由高到低为:JJ＞NC＞AH-1＞AH-2,九江居群遗传多样性丰富度最高。
(5)群体间遗传变异分析:各居群间的基因分化系数Gst为0.6283,说明四个居群间表现出较高水平的遗传分化,这与AMOVA分析结果(F_(ST)=0.5824,P＜0.001)接近。遗传结构分析表明居群间基因流(Nm=0.2957)水平较低。基于遗传距离的UPMGA聚类分析显示安徽粗脚粉螨两居群亲缘关系最为紧密,九江居群与其他居群亲缘关系较远。分析结果可得:不同居群的粗脚粉螨发生了一定的分化,地理居群遗传差异大于不同寄主居群遗传差异,各居群遗传距离与地理距离无关,说明地理隔离并不是形成遗传差异的主导因素。
Acarus siro Linnaeus,a common acaroid mite in China,is one of the main pest mites damaging the stored products,economic crops and edible fungi.The mite is widely studied due to its economic importance.In this paper,the genetic structure and genetic diversity of A.siro in different area of China were studied by using ISSR marker.The main results were as follows:
(1)The discussion and establishment of the optimum protocol extracting DNA of A. siro:SDS(10%m/v) 40μl,proteinase K(10mg/mL) 6μl,STE 204μl,total 250μl of digesting system,and the samples each consisting of 50 individuals were digested at 45℃for 10-12h.
(2)Optimization of ISSR-PCR reaction system:the optimal ISSR-PCR reaction system was established with L_(16)(4~5) orthogonal design:10×PCR Buffer 2.5mmol/L, Mg~(2+) 2.0mmol/L,dNTPs 0.25μmol/L,ISSR primers 0.3μmol/L,DNA 20ng,Taq DNA polymerase 0.75U,total 25μl.
(3)The results of amplification:18 primers were selected for their ability to produce clear and reproducible patterns of polymorphic bands.Those primers were then used for analysing genetic diversity within and among populations of A.siro collected in Nanchang,Jiujiang and another two populations sampled in two sorts of feedstuff in Anhui province(NC,JJ,AH-1,AH-2).A total of 136 DNA bands were yielded,107 of which were polymorphic and the percentage of polymorphic bands(PPB) was 78.68%.
(4)Evaluation of genetic diversity:At population level,the value of expected heterozygosity(He) was 0.1302,Shannon information index(I) 0.1875 and the percentage of polymorphic bands(PPB) 30.15%;at species level,the values of He,I, PPB were 0.3586 0.5314 and 78.68%,respectively.The levels of genetic diversity could be ordered in abundance as JJ＞NC＞AH-1＞AH-2 and the JJ population possessed the highest level.
(5) Analysis to genetic variation:the value of Gst(coefficient of gene differentiation) was 0.6283,which was consistent with AMOVA analysis(Fst=0.5824,P＜0.001), indicating high level of genetic differentiation among populations.Nei's genetic diversity analysis showed that the level of gene flow(Nm=0.3717) was low between populations.Dendrogram constructed using the UPMGA method and Nei's unbias genetic distance showed the relationships between two Anhui populations was closest. And the populations of A.siro strongly differentiated between Jiujiang and others. The results indicated that there was a certain genetic differentiation between these different populations;Geographic difference caused stronger differentiation than habitat difference.Moreover,there was no significant correlation between the genetic distance and geographic distance,indicating that geographic isolation was not the main factors inducing genetic difference.
引文
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