大口黑鲈微卫星多重PCR体系及3个群体的遗传分析_傅建军.pdf

doi:10.7541/2023.2022.0296大口黑鲈微卫星多重PCR体系及3个群体的遗传分析傅建军1 龚雅婷2 朱文彬1 强 俊1 张林兵3 王兰梅1 罗明坤1 董在杰1,2(1.中国水产科学研究院淡水渔业研究中心农业农村部淡水渔业和种质资源利用重点实验室,无锡 214081;2.南京农业大学无锡渔业学院,无锡 214128;3.安徽张林渔业有限公司,铜陵 244100)摘要:为开展大口黑鲈(Micropterus salmoides)种质评估和遗传分析提供便捷的研究手段,试验筛选了扩增效果好、具有多态性的18个微卫星位点,构建了6组3重PCR体系。随后将多重PCR体系应用于大口黑鲈3个群体(美国群体USA、“优鲈1号”YLO和杂交子代HYB)的遗传分析。结果显示,各位点的等位基因数(Na)、有效等位基因数(Ne)、观测杂合度(Ho)、期望杂合度(He)和多态性信息含量(PIC)分别介于314、1.6225.841、0.3330.806、0.3850.833和0.3610.810,其均值分别为7.722、3.056、0.577、0.626和0.579。剔除2个偏离Hardy-Weinberg平衡位点的分析显示,YLO的遗传多样性水平最低,HYB的平均观测杂合度最高(Ho=0.743),其余多态性指标均在USA中呈现最高值。USA与YLO间的Neis遗传距离最远(0.362),HYB与USA和YLO间的Neis遗传距离分别为0.112和0.179。两两群体间均存在极显著的遗传分化(P0.05),2个位点(P3L3和P5L2)极显著偏离HW平衡(P0.05);而P5L2位点在USA和HYB群体内符合HW平衡(P0.05),在YLO群体内表现为单态(纯合)。为了避免可能存在无效等位基因对遗传多样性参数估算带来的影响,在后续的群体遗传分析中将剔除P3L3和P5L2位点的相关数据。如表 3所示,USA群体具有最高的等位基因数(6.625)、有效等位基因数(3.544)、期望杂合度(0.663)和多态性信息含量(0.611);HYB群体具有最高的观测杂合度(0.743);而YLO群体的各项遗传多样性参数均低于USA群体和HYB群体。2.32.3 群体遗传分化和遗传结构分析如表 4所示,USA群体与YLO群体的遗传距离最远(0.362),而HYB群体与亲本群体间的遗传距离相近,与USA群体和YLO群体间的遗传距离分别为0.112和0.179。同时,3个群体间均存在极显著遗传分化(P0.01);基于Balloux等34对遗传分化水平的界定,USA群体与HYB群体间存在较低遗传分化(FST0.5),说明能为大口黑鲈群体遗传研究提供有效的遗传信息。这些标记在整体上呈现了较高的杂合度水平,所有位点的平均观测杂合度和期望杂合度分别为0.577和0.626。目前,基于微卫星标记针对大口黑鲈群体开展了较多的遗传多样性研究36,24,25。本研究筛选和检测的18个位点的多态性信息含量和杂合度等参数普遍高于梁素娴等3、樊佳佳等4、孙成飞等5和Bai等24的报道,而与苏胜彦6和Wang等25的报道相近。在不同的研究报道中,由于所用标记、检测方法、群体遗传背景和样本量存在差异,往往导致在分析结果上存在差异,这在一定程度上降低了相关研究间的可比性。鉴于此,开发或筛选多态性高的微卫星标记,同时采用通用性好的检测方法,有助于不同试验间的数据共享和比较。此外,多重PCR体系将为试验提供便利,并可有效降低检测成本,现已被较多应用于水产遗传育种研究领域1318。本研究筛选了18个具有较高多态性的大口黑鲈微表 4 大口黑鲈3个群体间Neis遗传距离(对角线下)和遗传固定指数FST(对角线上)Tab.4 Neis genetic distances(below diagonal)and geneticfixation indexes FST(above diagonal)among three populations inMicropterus salmoides群体Population 美国群体USA 优鲈1号YLO 杂交群体HYB美国群体USA0.209*0.044*优鲈1号YLO0.3620.132*杂交群体HYB0.1120.179注:*表示极显著(P0.01),下同Note:*indicate extremely significant(P0.01),the sameapplies below表 5 大口黑鲈3个群体分子遗传变异方差分析(AMOVA)结果Tab.5 Analysis of molecular variance of three populations inMicropterus salmoides变异来源Source ofvariation变异方差Sum ofsquares方差组分Variancecomponents百分比Percentagevariation(%)固定指数Fixationindex群体间Amongpopulations106.5510.67612.8650.129*群体内Withinpopulations975.6944.58187.135总和Total1082.2455.2571.00abK=2K=30.800.600.40聚类比率Proportion of clusters聚类比率Proportion of clusters0.2001.000.800.600.400.200美国群体 USA优鲈1号 YLO杂交群体 HYB美国群体 USA优鲈1号 YLO杂交群体 HYB图 2 大口黑鲈个体在不同K值下的遗传结构图Fig.2 Genetic structure assumed under different inferred K values for Micropterus salmoides individuals1518水 生 生 物 学 报47 卷卫星标记,并成功构建了6组多重PCR体系,这可为大口黑鲈群体遗传分析提供高效便捷的研究工具。本研究中2个微卫星位点(P3L3和P5L2)在整体水平偏离HW平衡且存在较高无效等位基因频率,这可能会对遗传多样性参数的估算带来影响35,因此在后续的分析中剔除了这2个位点的数据。就群体属性而言,本研究中群体材料包含原产地引进的USA群体、经历长期人工选择的YLO群体和人工配组的HYB群体,3个群体所经历人工选择强度和历程存在不同,会导致其在等位基因频率上的差异。在整体水平的遗传多样性参数估算中,由于种群结构的存在,会引起Wahlund效应(混合群体期望杂合度偏高),进而对位点HW平衡和无效等位基因分析产生影响。在对P3L3和P5L2位点在3个群体内等位基因频率的分析中,发现P5L2位点在YLO群体中表现为纯合,而P3L3和P5L2在其余单个群体中均未发现偏离HW平衡现象。其中,YLO群体的选育基础群体均来自于国内养殖群体且经历了高强度的人工选择,这可能导致该群体在某些位点上已经(或高度)纯合;在统计分析中,易被判定是存在无效等位基因导致的杂合度缺失。因此,本研究中发现部分位点在整体水平偏离HW平衡并存在较高无效等位基因频率的现象,推测是3个研究群体间的遗传结构差异所导致。在本研究中,YLO群体的遗传多样性水平最低,与孙成飞等5和苏胜彦等6基于相同检测技术对国内养殖群体的检测结果相似。在长期人工定向选择和繁育进程中,可能导致“优鲈1号”遗传多样性水平的降低;罗相忠等16和董在杰等36分别在对鲢(Hypophthalmichthys molitrix)和鲤(Cyprinuscarpio)的群体遗传分析中均有类似发现。本研究中美国引进的USA群体,相较于国内养殖和选育群体5,6,10,其多项遗传多样性参数均呈现较高水平,这与其引进后经历繁育和选育世代较短有关,说明该群体保留了丰富的遗传变异信息,存在开展选育利用的潜力。此外,HYB群体呈现较高的遗传多样性和杂合度水平,尤其是观测杂合度大于其亲本USA群体和YLO群体,类似结果在周家辉等10和王宏玉等37分别开展的鱼类群体间和种间杂交试验中均有发现。用于杂交配组的亲本群体在等位基因频率上往往存在差异,杂交过程会伴随不同等位基因的重组,进而提高杂交子代的观测杂合度,这也为杂交子代的表型变异提供遗传基础。正如李江涛等11研究发现大口黑鲈群体间杂交子代的生长性状和体型指标在变异系数上均有所提高。因此,在开展鱼类群体选育的过程中,通常会利用不同群体开展杂交配组来构建基础群体,为人工选择提供充足的遗传变异。在本研究中,USA群体和YLO群体间存在较大遗传分化(FST=0.209,P0.01),反映了2群体在等位基因频率上的差异,这可能与YLO群体经历长期定向选育对其等位基因频率的影响有关。同时,这也预示着利用上述群体开展杂种优势利用的潜力,正如周家辉等10、李江涛等11和龚雅婷等12利用大口黑鲈群体开展配组试验,均发现杂交子代具有生长优势。在对本研究3个大口黑鲈群体的遗传结构和主成分判别分析中,同样发现USA群体和YLO群体间存在明显的遗传结构差异,进一步验证了通过微卫星标记开展种质鉴定(或判别)的应用效果;其分析结果的可视化水平与傅建军等13和Fang等14在种间鉴定中的实验结果相当。可见,本研究所筛选并构建的微卫星多重PCR体系,可为大口黑鲈群体遗传评估和鉴定提供有效手段。此外,考虑到大口黑鲈是引进物种,其在国内的种群资源有限,加之繁殖周期较短,这容易导致养殖和育种群体遗传多样性降低和近交衰退,进而加剧群体间的遗传分化进程。因此,非常有必要鼓励国内养殖和育种单位从原产地更多引进优质群体资源,用于开展遗传改良和(或)杂种优势利用,为国内大口黑鲈养殖业健康可持续发展提供种质支撑。参考文献:National Characteristic Freshwater Fish Industry 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Wen-Bin1,QIANG Jun1,ZHANG Lin-Bing3,WANG Lan-Mei1,LUO Ming-Kun1 and DONG Zai-Jie1,2(1.Key Laboratory of Freshwater Fisheries and Germplasm Resources Utilization,Ministry of Agriculture and Rural Affairs,Freshwater Fisheries Research Center,Chinese Academy of Fishery Sciences,Wuxi 214081,China;2.Wuxi Fisheries College,Nanjing Agricultural University,Wuxi 214128,China;3.Zhanglin Fisheries Company,Tongling 244100,China)Abstract:Largemouth bass(Micropterus salmoides)has been introduced approximately for forty years,and it be-comes an economically important cultured fish in China.However,there is a lack of natural population supply in breed-ing and a risk of genetic degradation in the utilization progress.The imbalance between large industrial demand andshortage of improved varieties of M.salmoides are arisen in China.Genetic evolution is the baseline for genetic im-provement and selection breeding programs.The microsatellites with multiplex PCR method is popularity applied forgenetic analyses in fish species.In this study,we developed six multiplex PCR panels based on 18 polymorphic mi-crosatellites,and subsequently used in genetic analyses for three populations of M.salmoides,i.e.,original populationof American(USA),“Youlu No.1”variety(YLO),and their hybrid population(HYB).The results showed that,theparameters of number of alleles(Na),effective number of alleles(Ne),observed heterozygosity(Ho),expected heterozy-gosity(He)and polymorphism information content(PIC)for 18 loci were ranged from 3 to 14,1.622 to 5.841,0.333 to0.806,0.361 to 0.810,mean with 7.722,3.056,0.577 and 0.579,respectively.Excluding two loci deviated from Hardy-Weinberg equilibrium,the lowest values of all diversity parameters were observed in YLO,while the highest Ho wasdetected in HYB(0.743),and the highest values of other diversity parameters were detected in USA.The farthest Neisgenetic distance was evaluated between US