基于18S和28S rDNA基因的房舍甲螨DNA条形码比较研究

Comparative study on DNA barcoding of house oribatid mites based on the 18S and 28S ribosomal DNA gene

目的比较18S和28S rDNA基因应用于房舍甲螨物种鉴定的有效性及适用性。方法2021年2—10月在安徽省部分地区(亳州、阜阳、淮南、滁州、合肥、芜湖、铜陵、安庆和黄山等9市)的居室、农舍及仓储等房舍采集灰尘并分离甲螨,利用形态学特征鉴定螨种,提取单只甲螨基因组DNA,PCR扩增甲螨18S rDNA,28S rDNA D3和D8基因片段并测序。从GenBank下载房舍甲螨基因序列,用MEGA X软件进行序列特征分析和遗传距离计算,并通过ABGD网站进行DNA条形码间隙分析。采用邻接法构建甲螨的系统进化树。结果共采集甲螨53只,经形态学鉴定属于3科5属5种,分别为滑菌甲螨(15只)、盛若甲螨(9只)、新小奥甲螨(10只)、棒梳枝奥甲螨(10只)和间新美奥甲螨(9只)。从该5种甲螨扩增获得18S rDNA、28S rDNA D3和D8基因单倍型分别为7、5和11条;从GenBank中共下载房舍甲螨序列42条。序列分析结果显示,18S rDNA、28S rDNA D3和D8基因长度分别为442~590、247~331、270~283 bp,GC含量分别为45.6%、52.2%和57.5%,变异率分别为17.6%、21.4%和21.7%。18S rDNA、28S rDNA D3和D8基因种内遗传距离平均值分别为0.001、0.001和0.005,种间遗传距离平均值分别为0.064、0.115和0.109,种间遗传距离平均值均大于种内遗传距离平均值(10倍以上)。DNA条形码间隙分析结果显示,18S rDNA和28S rDNA D3基因种内和种间遗传距离存在重叠区,28S rDNA D8基因则存在明显DNA条形码间隙。系统进化树分析结果显示,房舍甲螨科属分类阶元聚类结果与形态学聚类结果一致,金黄缝甲螨和淡红缝甲螨在18S rDNA基因系统进化树显示聚集在一起,无法分开;28S rDNA D3基因系统进化树呈现出交叉类聚。结论18S rDNA、28S rDNA D3和D8基因能够有效地用于房舍甲螨的物种鉴定,28S rDNA D8基因适合于低分类阶元螨种的鉴定,18S rDNA和28S rDNA D3基因适合于高阶元螨种。

Objective To compare the validity and applicability of 18S and 28S rDNA genes in species identification of house oribatid mites.Methods Oribatid mite were isolated from the dust samples collected from residential houses,farmhouses and warehouses in 9 citiess of Anhui Province(Bozhou,Fuyang,Huainan,Chuzhou,Hefei,Wuhu,Tongling,Anqing,Huangshan)from February 2021 to October 2021,and the mite species were identified morphologically.The genomic DNA was extracted from each individual oribatid mite,and the 18S rDNA,28S rDNA D3 and D8 genes were obtained by PCR,and sequenced.The sequences of house oribatid mites genes were downloaded from GenBank.The sequence feature analysis and genetic distance calculation were carried out based on MEGA X software,and the DNA barcoding gap analysis was performed through ABGD website.The phylogenetic tree was created using neighbor joining method.Results A total of 53 mites were collected and morphologically identified of species,which belongs to three families,five genera and five species,particularly for Scheloribates laevigatus(15),Oribatula sakamorii(9),Oppiella nova(10),Ramusella clavipectinata(10),Neoamerioppia interrogate(9).The 18S rDNA,28S rDNA D3 and D8 gene haplotypes were obtained from these five mite species with 7,5 and 11 haplotypes,respectively.A total of 42 sequences of house oribatid mites were downloaded from GenBank.Sequence analysis showed that the lengths of 18S rDNA,28S rDNA D3 and D8 genes were 442-590,247-331 and 270-283 bp,respectively,with GC contents of 45.6%,52.2%and 57.5%,and variation rates of 17.6%,21.4%and 21.7%,respectively.The mean intraspecies genetic distances of 18S rDNA,28S rDNA D3 and D8 gene were 0.001,0.001 and 0.005,respectively.The mean interspecies genetic distances were 0.064,0.115 and 0.109,respectively,with the mean interspecies genetic distances greater than the mean intra-species genetic distances(more than 10-fold).The results of DNA barcode gap analysis showed that there were overlapping regions between the intraspecific and interspecific genetic distances of 18S rDNA and 28S rDNA D3 gene sequences,and there was an significant DNA barcode gap in 28S rDNA D8 gene sequences.The phylogenetic tree showed that the taxonomic order clustering of house oribatid mite family are consistent with the morphological clustering results,but Hypochthonius luteus and H.rufulus showed clustering together in the 18S rDNA gene systematic evolution tree and could not be separated.The 28S rDNA D3 gene systematic evolution tree showed cross-class clustering.Conclusion 18S rDNA and 28S rDNA D3 and D8 genes can be effectively used for species identification of house oribatid mites,of them,28S rDNA D8 gene is suitable for the species identification of low-classification taxa,while 18S rDNA and 28S rDNA D3 genes are suitable for the species of high-classification taxa.