大口黑鲈微卫星DNA指纹图谱的构建和遗传结构分析

摘要

以国内目前养殖的大口黑鲈[ Micropterus salmoides (Lacépède)]群体(CH)、2009年引进的佛罗里达亚种(FL-09)、2010年引进的佛罗里达亚种(FL-10)、2010年引进的北方亚种(NT-10)为实验材料,应用43个微卫星DNA标记对这4个大口黑鲈群体进行遗传检测,构建了各群体的微卫星DNA指纹图谱,并对其遗传结构进行分析.结果显示:CH、FL-09、FL-10和NT-10群体的平均等位基因数(A)分别为2.58、3.74、3.70和4.21,平均期望杂合度(He)分别为0.4549、0.4896、0.5010和0.6138,平均多态信息量(PIC)分别为0.3786、0.4443、0.4566和0.5546,表明国内目前养殖的大口黑鲈群体遗传多样性水平远远低于国外新引进的大口黑鲈群体.利用UPGMA法对4个群体进行聚类,结果FL-09和FL-10聚为一支,遗传距离为0.0506; NT-10和CH聚为另一支,遗传距离为0.4244,推测FL-09和FL-10两个佛罗里达亚种属于相同的群体,而NT-10和CH两个北方亚种来自不同的群体,甚至不同的水系.同时从指纹图谱中,筛选到5个特异的微卫星标记(JZL114、MiSaTPW11、Lma120、Mdo6和Msa121),可以鉴别FL、NT-10和CH群体,其中MiSaTPW11和Msa121这两个标记组合可以完全鉴别这3个群体.将5个特异性微卫星标记的图谱数据转化成计算机可以识别的数码指纹,可以方便应用于大口黑鲈不同群体及其杂交种的鉴定.研究结果可以为我国大口黑鲈种质资源保存、品种鉴定和良种选育提供理论依据.%DNA fingerprinting offers great potential in aquaculture and in fisheries as a tool for identification of individuals, construction of pedigrees and population genetic analysis. Microsatellites, also called simple sequence repeats (SSR), are widely dispersed along and among chromosomes, and each locus is characterized by a known DNA sequence. Due to a tendency for hypervariability and because only small amounts of tissue are required for typing, microsatellites have supplanted allozymes in recent years as the genetic markers of choice for many biological problems including parentage assessment, genomic mapping and establishment DNA fingerprinting. Largemouth bass (Micropterus sal-moides) was introduced from Taiwan to Guangdong province of China in 1983, because of delicious taste, fast growth, and wide temperature tolerance, it has been bred in many region of China as a main cultured fish species with annual production of 100,000 tons. The largemouth bass consists of two subspecies, the Florida largemouth bass (M. s. floridanus), which is native to peninsular Florida, and the northern largemouth bass (M. s. salmoides), whose native range extends throughout the central and eastern U.S. as well as northeastern Mexico and southeastern Canada. Research shows that largemouth bass cultured in china attribute to the northern largemouth bass (M .s. salmoides). In this paper, the microsatellite DNA fingerprinting of four largemouth bass populations was established, including cultured stock of M. salmoides in China (CH), M. s. floridanus was introduced in 2009 (FL-09), M. s. floridanus was introduced in 2010 (FL-10) and M. s. salmoides was introduced in 2010 (NT-10), and the genetic structure of them was analyzed by 43 microsatellite markers. The results showed that a total of 246 alleles were obtained from the four largemouth bass populations, and the alleles ranged from 2-13 in each locus. The mean value of number on alleles (A) and the mean value of expected heterozygosity (He) were calculated by popgene 3.2 software. The result was that the mean value of number on alleles {A) of CH, FL-09, FL-10 and NT-10 was 2.58, 3.74, 3.70 and 4.21, the mean value of expected heterozygosity (He) was 0.4549, 0.4896, 0.5010 and 0.6138, and the mean value of polymorphism information content (PIC) was 0.3786, 0.4443, 0.4566 and 0.5546, respectively. It indicated that the genetic diversity of cultured stock on largemouth bass in China was lower than that of introduced largemouth bass populations. Using Unweighted pair-group method with arithmetic means method (UPGMA) based on their genetic distances, the cluster analysis in four populations showed that FL-09 and FL-10 were grouped together, genetic distance was 0.0506; The NT-10 and CH were grouped together, genetic distance was 0.4244. The results showed that the FL-09 and FL-10 of M. s. floridanus belonged to same population, but NT-10 and CH of M. s. salmoides came from different populations, and even different river system. Then five pairs of specific microsatellite markers (JZL114, MiSaTPWll, Lmal20, Mdo6 and Msal21) were screened out from microsatellite DNA fingerprinting which could be used to identify FL, NT-10 and CH populations. Especially microsatellite markers (MiSaTPWll and Msal21) combination could completely identify FL, NT-10 and CH populations. The amplified data from five pairs of special markers were transformed to the application platform of digital microsatellite DNA fingerprinting, which could be used to identify largemouth bass species and hybrid identification. These results could provide a theoretical basis for germplasm conservation, species identification and breeding of largemouth bass in China.