基于线粒体DNA Cyt b、12S rRNA和D-loop序列的铜鱼养殖群体遗传多样性分析

    Analysis of genetic diversity of the cultured population of Coreius heterodon based on mitochondrial DNA Cyt b, 12S rRNA, and D-loop sequence

    • 摘要:
      目的 本研究分别利用线粒体DNA Cyt b、12S rRNA和D-loop序列对铜鱼(Coreius heterodon)养殖群体进行遗传多样性分析,初步了解其遗传多样性。
      方法 采用已报道的特异性引物对线粒体DNA Cyt b、12S rRNA和D-loop序列进行聚合酶链式反应(PCR)扩增,并采用双脱氧测序法(Sanger)对扩增产物进行测序。遗传多样性参数由DNASP 6.12软件计算;采用MEGA 11.0 软件分析DNA序列的碱基组成和变异位点;利用 Kimura双参数模型计算单倍型间的遗传距离,采用邻接法(NJ)和最大似然法(ML)构建D-loop全序列单倍型系统进化树。
      结果 用于分析的线粒体DNA Cyt b、12S rRNA和D-loop序列长度分别为1110~1 147、425~445和972~1 023 bp,平均长度分别为1 122.77、430.00和1 000.47 bp,中位数长度分别为1 122、429和1 001 bp。在线粒体DNA Cyt b、12S rRNA和D-loop序列中,分别检测出8、1和14个变异位点,以及22、19和30种单倍型,单倍型间遗传距离分别为0.000~0.004、0.000~0.005和 0.000~0.007。基于线粒体DNA Cyt b、12S rRNA和D-loop序列的平均单倍型多样性分别为0.547±0.0105、0.186±0.007 8和0.885±0.001 3;平均核苷酸多样性分别为0.000 76、0.000 44和0.002 58;平均核苷酸差异数为0.830、0.186和2.432。同时,歧点分布分析图谱呈现单峰型,在中性检验中Tajima’s D(D=−1.040,P>0.01)为负值,但统计结果为不显著(P>0.01)。
      结论 铜鱼养殖群体遗传多样性相对较低,且单倍型间存在广泛的基因交流,但铜鱼养殖群体发生了种群数量下降。因此,建立起遗传多样性丰富的铜鱼人工繁殖的基础亲鱼群体,解决铜鱼亲本培育问题,持续壮大铜鱼养殖群体规模是首要任务。

       

      Abstract:
      Objective In this study, the genetic diversity of the cultured population of Coreius heterodon was analyzed by using the sequences of mitochondrial DNA Cyt b, 12S rRNA and displacement loop region (D-Loop), and the genetic diversity and genetic structure were preliminarily understood.
      Methods Using the specific primers designed by previous researchers based on the sequences of mitochondrial DNA Cyt b, 12S rRNA and D-Loop, polymerase chain reaction (PCR) amplification was carried out, and Sanger sequencing was performed. The genetic diversity parameters were calculated by the DNASP 6.12 software. The MEGA 11.0 software was used to count the base composition and variable sites of the DNA sequences. The genetic distances among haplotypes were calculated by the Kimura two-parameter model. The haplotype phylogenetic trees of the complete sequences of the mitochondrial DNA control region were constructed by neighbor-joining (NJ) method and the maximum likelihood (ML) method.
      Results The sequences of mitochondrial DNA Cyt b, 12S rRNA, and D-loop used for analysis were 1 110—1 147 bp, 425—445 bp, and 972—1 023 bp, with average lengths 1 122.77, 430.00, and 1 000.47 bp, and median lengths of 1 122, 429, and 1 001 bp, respectively. A total of 8, 1, 14 variable sites , and 22, 19, 30 haplotypes were identified in the mitochondrial DNA Cyt b, 12SRNA and D-loop, respectively, respectively, with genetic distance between haplotypes ranging from 0.000 to 0.004, 0.000 to 0.005 and 0.000 to 0.007, respectively. The average haplotype diversities based on mitochondrial DNA Cyt b, 12S rRNA, and D-loop were 0.547±0.010 5, 0.186±0.007 8, and 0.885±0.001 3, respectively; the average nucleotide diversities were 0.000 76, 0.000 44 and 0.002 58, respectively; and the average number of nucleotide differences was 0.830, 0.186, and 2.432, respectively. Mismatch-distribution showed a unimodal pattern, and Tajima’s D value was negative (D=-1.04, P>0.01) in the neutrality test, but the statistical result was not significant.
      Conclusion The genetic diversity of the cultured C. heterodon population is relatively low, and there is extensive gene flow between haplotypes, with no genetic differentiation of the population, but the cultured C. heterodon population has undergone a population quantity decline. Therefore, establishing a broodstock population with rich genetic diversity for artificial breeding of C. heterodon, solving the problem of broodstock cultivation, and continuously expanding the scale of C. heterodon farming population is the primary task.

       

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