水泡带绦虫海南株的分子鉴定及系统发育分析

Molecular identification and phylogenetic analysis of Taenia hydatigena Hainan isolate

目的以COI基因和ND1基因作为DNA条形码对水泡带绦虫海南株进行分子鉴定和系统发育分析。方法PCR扩增COI基因和ND1基因片段,GenBank数据库中进行BLAST多序列比对确定虫种。使用MEGA11软件构建基于邻接法的COI基因和ND1基因系统发育树,并计算基于K2P模型的种内遗传距离和种间遗传距离;分析核苷酸组成,计算碱基转换率、碱基颠换率及碱基转换与颠换比值。使用DNAsp软件分析COI基因和ND1基因的单倍型多样性。登录ABGD网站检测COI基因和ND1基因是否存在明显的DNA barcoding gap,以评估COI基因和ND1基因作为DNA条形码进行物种分子鉴定的有效性。利用DAMBE软件检测碱基替换饱和度,以明确COI基因和ND1基因构建系统发育树的准确性。结果COI基因的单倍型多样性为0.8276,含有81个可变位点,碱基转换率(si)为10%,碱基颠换率为(sv)13%,转换与颠换比值为0.77。ND1基因的的单倍型多样性为0.9381,含有94个可变位点,碱基转换率(si)为10%,碱基颠换率(sv)为11%,转换与颠换比值为0.91。COI基因和ND1基因均具有明显的DNA barcoding gap,可作为有效的DNA条形码进行带绦虫属内物种的分子鉴别。COI基因、ND1基因Iss<Iss.c,且P<0.05,两者均不存在碱基替换饱和,包含了足够的系统发育信息,适合进行系统发育分析。结论单基因作为DNA条形码能够快速、简便的鉴别物种,而多重DNA条形码能够提高分辨率和计算效率,是分析系统发育关系和探索生物多样性的有效识别工具。

Objective Molecular identification and phylogenetic analysis of Taenia hydatigena Hainan isolate were implemented using the COI gene and the ND1 gene as DNA barcodes.Methods Morphological characteristics of mature segments were treated with ethanol and then in xylene,and observed by optical microscope.Total genomic DNA from the sample tissue was extracted and then used as a PCR template.The COI and ND1 gene fragments were amplified by PCR,and multiple sequence alignments were performed by BLAST of the GenBank database to determine species.The phylogenetic trees were constructed based on the COI gene and the ND1 gene using the neighbor-joining algorithm.The intraspecific genetic distance and interspecific genetic distance were calculated using the K2 P model.The nucleotide composition was analyzed,and the transitional substitution rates,the transversional substitution rates,and the transitional/transversional ratios were estimated in the MEGA version 11 software.Haplotype diversity was evaluated using DNAsp software.The DNA barcoding gaps of the COI and ND1 gene sequences were detected on the ABGD website to assess the effectiveness of the COI gene and the ND1 gene as DNA barcodes for species molecular identification.The substitution saturation was tested by the DAMBE software to clarify the accuracy of constructing phylogenetic trees based on the COI gene and ND1 gene.Results Taenia hydatigena is 122.4 cm long,consisting of 250-300 segments.An ovary and 600-700 testes exist in each mature segment.Each gravid segment contains a uterus that is filled with oval eggs.The COI sequence of Taenia hydatigena Hainan isolate obtained by PCR amplification was 422 bp with a G+C content of 33.1%,found to have a close phylogenetic relationship with Taenia hydatigena of Turkey(JN827307).The ND1 sequence of Taenia hydatigena Hainan isolate obtained by PCR amplification was 471 bp with a G+C content of 28.8%,found to have a close phylogenetic relationship with Taenia hydatigena of Sichuan Ya’an,China(JN831281).Genetic diversity analysis demonstrated that the ND1 gene with haplotype diversity of 0.9381 revealed higher genetic diversity than the COI gene with haplotype diversity of 0.8276,with 15 different haplotypes from the 21 ND1 and 29 COI sequences,respectively.The ND1 gene contained 94 variable sites,which was higher than the 81 variable sites contained by the COI gene.The ratio of transitional to transversional pairs in the COI gene was 0.77,with the transitional rate of 10%and the transversional rate of 13%,separately.Besides,the ratio of transitional to transversional pairs in the ND1 gene was 0.91,with the transitional rate of 10%and the transversional rate of 11%,separately.Obvious barcoding gaps were observed in the COI gene and the ND1 gene,effectively discriminating Taenia species.Substitution saturation tests for the COI,ND1 sequences indicated little saturation(Iss<Iss.c;P<0.05),and contained sufficient phylogenetic information to be suitable for phylogenetic analysis.Conclusion A single gene used as a DNA barcode marker could quickly and easily distinguish between species,whilst the multiple DNA barcodes contribute to improving resolution and computational efficiency and are effective identification tools for analyzing phylogenetic relationships and exploring biodiversity.