洛阳地区5种嗜尸性麻蝇分子鉴定研究

Molecular identification of five common Sarcophagidae species of necrophagous flies from Luoyang

目的应用经典DNA条形码和28S r RNA基因鉴定洛阳地区5种常见嗜尸性麻蝇种属,评价其在常见嗜尸性麻蝇种属鉴定中应用的可行性。方法收集洛阳地区常见嗜尸性麻蝇类标本18只,经形态学分类鉴定后,Chelex-100法提取足部DNA,扩增并测序线粒体细胞色素C氧化酶亚基I(COI)和28S核糖体核糖核酸(28S r RNA)基因片段,BLAST比对后,与来自DNA条形码数据库(Barcode of Life Data,BOLD)的中国和韩国地区20条相应蝇种序列进行比对,用MEGA 7.0软件整理分析所得序列,计算碱基组成、种内及种间进化分歧率,并构建系统发育树。结果形态学鉴定18只嗜尸性麻蝇归于3属5种。每个样本获得COI和28S r RNA的扩增长度分别为646bp和721bp。在线BLAST比对结果显示样本COI序列相似度99%以上,邻近法构建发育树Bootstrap值为1 000,5种麻蝇可以较好聚类,与形态学鉴定结果一致。38个样本COI种内范围为0~0.022,棕尾别麻蝇、酱亚麻蝇和急钩(亚)麻蝇种间范围在0.057~0.090;赤尾麻蝇和红尾粪麻蝇种间范围在0~0.086。5种麻蝇的28S r RNA序列发育树显示,棕尾别麻蝇和急钩(亚)麻蝇明显各自聚类,其余3种成一类。结论对于本研究中的5种嗜尸性麻蝇,基于COI基因的DNA条形码可以有效区分棕尾别麻蝇、酱亚麻蝇和急钩(亚)麻蝇,28S r RNA基因只可以区分棕尾别麻蝇,二者可作为形态学鉴定之外的重要方法补充。

Objective To identify the common Sarcophagidae species of necrophagous flies in Luoyang by DNA barcoding and 28 S ribosomal RNA（28 S rRNA） gene and evaluate its effectiveness for forensic practice. Methods Eighteen Sarcosaprophagous flies were collected and classified by entomologists with traditional morphological characteristics. The DNA of flies was extracted with Chelex-100 method. The fragments of mitochondrial cytochromec oxidase subunit I（COI） and 28 S rRNA gene were amplified and sequenced. Twenty corresponding species（China and South Korea） were loaded from Barcode of Life Data System（BOLD） and added to the alignment. All the sequences were analyzed by MEGA 7.0 software package for nucleotide composition, genetic distance computation and phylogenetic tree construction. Results Eighteen Sarcosaprophagous flies were classified into 5 species of 3 genera. The result of amplification with 18 samples showed that length of the obtained COI and 28 S rRNA gene sequences were 646 bp and 721 bp, respectively. And the result of alignment on BLAST online showed that index of similarity of the same species was above 99%. The thirty-eight COI sequences of Sarcosaprophagous flies were clustered into five groups by a neighbor-joining（NJ） tree on value of Bootstrap 1000. The intraspecific difference in COI was 0 to 0.022 while the interspecific difference ranged from 0.057 to 0.090 excluding Sarcophaga Africa and Sarcophaga haemorrhoidalis, which was 0~0.086. The NJ tree of 28 S rRNA showed Sarcophaga peregrine and Sarcophaga portschinskyi sequences were obviously clustered into two groups and the others a group. Conclusion For the five sarcophagous flies in this study, the DNA barcoding based on COI gene were able to effectively identify the Sarcophaga peregrine, Sarcophaga dux and Sarcophaga portschinskyi, while 28 S rRNA gene can only differentiate Sarcophaga peregrine from others. DNA barcoding based on COI gene and 28 S rRNA gene can be used as supplemental molecular markers for identifying these species.