基于线粒体COI基因和D-loop区联合序列的杂交鲫与野生鲫群体遗传特征研究

【目的】分析杂交起源的合方鲫品系和新型同源二倍体类鲫(NCRC)品系、实验红鲫品系及野生鲫群体的遗传特征,揭示杂交鲫群体与野生鲫群体的种质资源现状,为开展鲫育种工作提供科学依据。【方法】以2个杂交品系(合方鲫和NCRC)、1个实验室养殖品系(实验红鲫)和3个野生鲫群体(分别采自湖南省长沙市望城区、长沙市长沙县和常德市)为研究对象,采用PCR扩增6个鲫群体的线粒体COI基因和D-loop区序列,使用DnaSP 6.12分析群体遗传多样性,利用MEGA 7.0.26计算群体内和群体间的遗传距离,运用TCS Network算法绘制单倍型网络,再以Structure 2.3.4推测群体遗传结构;利用Arlequin 3.5.2.2计算群体间的遗传分化系数(F_ST),并以邻接法(NJ)、最大似然法(ML)和贝叶斯推断法(BI)分别构建系统发育进化树;通过Tajima’s D和Fu’s Fs中性检验结合核苷酸错配分布分析评估群体历史动态。【结果】基于线粒体COI基因和D-loop区联合序列,从6个鲫群体中共鉴定出25个单倍型(Hap1~Hap25);且3个野生鲫群体的单倍型多样性(h,0.582~0.877)和核苷酸多样性(π,0.00227~0.00532)明显高于2个杂交品系和实验红鲫品系。构建的单倍型网络结构图和系统发育进化树均显示,合方鲫品系可形成独立分支,且与日本白鲫聚在一起。同时,合方鲫品系与其他群体间的遗传距离(0.0539~0.0628)和遗传分化系数(F_ST,0.95147~0.98331)均较大,其次为NCRC品系和实验红鲫品系,说明养殖群体与野生鲫群体间已产生明显的遗传分化。在Structure聚类分析的基础上进行AMOVA分析,结果表明可将6个鲫群体划分为合方鲫品系、NCRC品系、实验红鲫品系和野生鲫群体共4组,且组间遗传变异占绝大部分(90.14%)。从群体历史动态检验结果来看,6个鲫群体在近期历史上保持相对稳定,未曾经历过明显的群体扩张。【结论】合方鲫品系、NCRC品系及实验红鲫品系3个养殖群体与3个野生鲫群体间已产生明显的遗传分化,且存在一定程度的遗传多样性降低等问题。因此,在后续的鲫育种工作中应适当扩大繁育亲本数量,避免近亲繁殖和瓶颈效应,注重保护养殖群体种质资源的遗传多样性。

Population genetic characteristics of hybrid and wild crucian carps in China based on joint sequences of mitochondrial COI gene and D-loop region

【Objective】To analyze the genetic characteristics of hybrid crucian carp derived from Carassius cuvieri(♀)×C. cuvieri red var.(♂) (HFJ),new homodiploid crucian carp-like species derived from Cyprinus carpio L.(♀)×Megalobrama amblycephala(♂) (NCRC),laboratory red crucian carp(RCC) and wild crucian carp population strains,to reveal current situation of hybrid and wild population germplasm resources,thus providing scientific evidence for crucian carp breeding.【Method】With 2 hybrid strains(HFJ and NCRC),1 laboratory strain(RCC),and 3 wild populations(collected from Wangcheng District,Changsha County,Changsha City and Changde City of Hunan Province) as research objects,mitochondrial COI gene and D-loop region of these 6 populations were amplified with PCR. DnaSP 6.12 was used to analyze the genetic diversity;MEGA 7.0.26 was used to calculate the genetic distance within and between populations;TCS Network algorithm was used to plot haplotype network and Structure 2.3.4 was used to predict the population genetic structure. Arlequin 3.5.2.2 was used to calculate genetic differentiation coefficient(F_ST) and the phylogenetic trees were constructed by Neighbor-joining method(NJ),Maximum likelihood method(ML) and Bayesian inference method(BI). Demographic history of the populations were evaluated by Tajima's D and Fu's Fs neutrality test combined with nucleotide mismatch distribution analysis.【Result】Based on joint sequences of mitochondrial DNA COI gene and D-loop,a total of 25 haplotypes(Hap1-Hap25) were identified from the 6 populations;haplotype diversity(h,0.582-0.877) and nucleotide diversity(π,0.00227-0.00532) of the 3 wild populations were significantly higher than those of2 hybrid strains and RCC strain. Haplotype network structure chart and phylogenetic tree showed that HFJ strain,which could form an independent branch,clustered with Japanese white crucian carp. Meanwhile,the genetic distance(0.0539-0.0628) and genetic differentiation coefficient(F_ST,0.95147-0.98331) between HFJ strain and other populations were the largest,followed by NCRC and RCC,indicating an obvious genetic differentiation between cultured and wild populations. AMOVA analysis based on Structure clustering analysis showed that the 6 populations were divided into 4 groups:HFJ,NCRC,RCC,and wild crucian carp,with between-group genetic variation accounted for the most(90.14%). Demographic history test of the populations showed that Neutrality tests and mismatch distribution analysis indicated that all of the 6 crucian carp populations remained stable in near history without any obvious expansion.【Conclusion】The results indicated that obvious genetic differentiation has been seen among HFJ,NCRC,and RCC strains,with problems such as declining genetic diversity. Therefore,in subsequent breeding,the number of breeding parents should be properly increased to avoid inbreeding and bottleneck effect,and more attention should be paid to protect genetic diversity of germplasm resources of cultured populations.