DNA barcoding of 4 species of cheyletid mites based on COI and 18S rRNA gene sequences

Abstract

Objective To analyze the sequences of mitochondrial cytochrome C oxidase subunit I gene ( COI ) and 18S ribosomal RNA gene ( 18S rRNA ), so as to identify the feasible DNA barcodes for 4 species of cheyletid mites and improve the DNA barcoding database for cheyletid mites. Methods Cheyletid mite samples were collected from small-scale flour mills in Fuyang, Wuhu and Tongling cities of Anhui Province from May 2018 to July 2019, extracted and morphologically identified. Then, genomic DNA was extracted from a single cheyletid mite, and the COI and 18S rRNA gene sequences were obtained by PCR amplification, cloning and sequencing. The obtained sequences were aligned using the BLAST software. Multiple sequence alignment was done using the software ClustalX version 1.83 using the known gene sequences from cheyletid mites. The genetic distance was calculated using the software MEGA X, and the phylogenetic tree was created using the maximum likelihood method. Results The DNA barcoding results of Cheyletus malaccensis , C. carnifex and Cheletomorpha lepidopterorum were consistent with the morphological identification, while no sequences pertaining to Eucheyletia reticulate were retrieved in the GenBank database. The proportions of A + T were 69.6% and 55.1% in the COI and 18S rRNA sequences of 4 cheyletid mites species, respectively, and the numbers of base substitutions were 137 and 46, respectively. There were 154 to 321 and 58 to 99 inter-species variation loci in the COI and 18S rRNA gene sequences of 4 cheyletid mites species, respectively, and the intra-species genetic distance was all 0.020 or less in the COI and 18S rRNA gene sequences of 4 cheyletid mites species, with inter-species genetic distance of 0.235 to 0.583 and 0.078 to 0.114, respectively. Phylogenetic analysis based on COI and 18S rRNA genes showed that all four species of cheyletid mites were clustered into a branch with a 100% supportive rate, which was consistent with the morphological identification. Conclusion Mitochondrial COI gene is superior to 18S rRNA gene as DNA barcodes for 4 species of cheyletid mites, which is more suitable to be used to investigate the phylogenetic relationship of at genus and species levels. ［摘要］ 目的 基于线粒体细胞色素C氧化酶亚基 (I COI ) 与核糖体 18S 小亚基 ( 18S rRNA ) 基因序列, 分析确定 4 种肉食 螨合适的 DNA 条形码, 从而进一步完善肉食螨DNA条形码数据库。 方法 2018 年 5 月—2019 年 7 月于安徽省阜阳、芜 湖、铜陵等 3 市小型面粉作坊采集分离肉食螨样本, 经形态学鉴定后, 提取单个螨基因组 DNA, 经PCR扩增、克隆和测序 获得 COI 与 18S rRNA 基因序列, 所获序列进行BLAST比对。结合已报道肉食螨序列, 用 ClustalX 1.83 软件进行多序列比 对, 基于MEGA X软件进行序列分析并计算遗传距离, 采用最大似然法构建系统发育树。 结果 马六甲肉食螨、转开肉 食螨、鳞翅触足螨分子鉴定结果与形态学一致, 而GenBank中缺少网真扇毛螨相关序列。4 种肉食螨 COI 与 18S rRNA 基 因序列 (A + T) 分别为 69.6% 和 55.1%, 碱基替换数分别为 137 对和 46 对。基于 COI 与 18S rRNA 基因序列变异位点分析, 发现 4 种肉食螨种间变异位点分别为 154 ~ 321 个和 58 ~ 99 个。4 种肉食螨 COI 与 18S rRNA 基因序列种内遗传距离均 ≤ 0.020, 种间遗传距离分别为 0.235 ~ 0.583 和 0.078 ~ 0.114。基于 COI 与 18S rRNA 基因序列的系统发育树显示, 4 种肉 食螨均能各自聚为一支, 支持率均达 100%, 与形态学鉴定结果一致。 结论 线粒体 COI 基因序列作为 4 种肉食螨DNA 条形码优于 18S rRNA 基因序列, 更适用于探索肉食螨的属、种低阶元亲缘关系。