论著

基于18S和28S rDNA基因的房舍甲螨DNA条形码比较研究

  • 陈慧林 ,
  • 朱永航 ,
  • 范海页 ,
  • 赵薇 ,
  • 刘璐瑶 ,
  • 曹玉祥 ,
  • 叶长江 ,
  • 孙恩涛
展开
  • 皖南医学院卫生检验与检疫学教研室,安徽芜湖 241002
陈慧林(2001-),女,本科生,从事媒介生物检验检疫研究。E-mail:chl2218721585@163.com
*孙恩涛(1980-),男,博士,教授,从事媒介生物检验检疫工作。E-mail:asdentao@126.com

收稿日期: 2022-06-22

  修回日期: 2022-10-10

  网络出版日期: 2023-04-10

基金资助

国家自然科学基金(31870352);安徽省大学生创新创业教育训练计划(S202110368044);皖南医学院2021年度校大学生科研资助金(WK2021XS20)

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

  • CHEN Huilin ,
  • ZHU Yonghang ,
  • FAN Haiye ,
  • ZHAO Wei ,
  • LIU Luyao ,
  • CAO Yuxiang ,
  • YE Changjiang ,
  • SUN Entao
Expand
  • Laboratory of Health Inspection and Quarantine, Wannan Medical College, Wuhu 241002, Anhui, China

Received date: 2022-06-22

  Revised date: 2022-10-10

  Online published: 2023-04-10

Supported by

National Natural Science Foundation of China(31870352);Anhui Province University Students Innovation and Entrepreneurship Education and Training Program Project(S202110368044);2021 University Students Scientific Research Grant Program of Wannan Medical College(WK2021XS20)

摘要

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。

本文引用格式

陈慧林 , 朱永航 , 范海页 , 赵薇 , 刘璐瑶 , 曹玉祥 , 叶长江 , 孙恩涛 . 基于18S和28S rDNA基因的房舍甲螨DNA条形码比较研究[J]. 中国寄生虫学与寄生虫病杂志, 2023 , 41(2) : 163 -169 . DOI: 10.12140/j.issn.1000-7423.2023.02.006

Abstract

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.

参考文献

[1] Lehmitz R, Decker P. The nuclear 28S gene fragment D3 as species marker in oribatid mites (Acari, Oribatida) from German peatlands[J]. Exp Appl Acarol, 2017, 71(3): 259-276.
[2] Koko?ová P, ?urovcová M, ?uptá?ik P, et al. Distinct phylogeographic patterns in populations of two oribatid mite species from the genus Pantelozetes (Acari, Oribatida, Thyrisomidae) in Central Europe[J]. Exp Appl Acarol, 2021, 83(4): 493-511.
[3] Hebert PDN, Cywinska A, Ball SL, et al. Biological identifications through DNA barcodes[J]. Proc Biol Sci, 2003, 270(1512): 313-321.
[4] Liu YL, Guo LX, Qiu JQ, et al. Application of DNA barcoding to identify uncommon flies in Ningbo Port[J]. Chin Port Sci Technol, 2021, 3(9): 15-20. (in Chinese)
  (刘永磊, 郭立新, 邱佳琦, 等. 应用DNA条形码技术鉴定宁波口岸本底非常见蝇类[J]. 中国口岸科学技术, 2021, 3(9): 15-20.)
[5] Yu HJ, Wang Q. Application of DNA barcoding for species identification in aquatic animal[J]. J Anhui Agric Sci, 2021, 49(16): 1-3, 6. (in Chinese)
  (余海军, 王茜. DNA条形码在水生动物物种鉴定中的应用[J]. 安徽农业科学, 2021, 49(16): 1-3, 6.)
[6] Lin CP, Danforth BN. How do insect nuclear and mitochondrial gene substitution patterns differ? Insights from Bayesian analyses of combined datasets[J]. Mol Phylogenetics Evol, 2004, 30(3): 686-702.
[7] Zhao YE, Wu LP, Hu L, et al. Sequencing for complete rDNA sequences (18S, ITS1, 5.8S, ITS2, and 28S rDNA) of Demodex and phylogenetic analysis of Acari based on 18S and 28S rDNA[J]. Parasitol Res, 2012, 111(5): 2109-2114.
[8] Gaonkar CC, Piredda R, Minucci C, et al. Annotated 18S and 28S rDNA reference sequences of taxa in the planktonic diatom family Chaetocerotaceae[J]. PLoS One, 2018, 13(12): e0208929.
[9] Zhao YE, Zhang WY, Wang RL, et al. Divergent domains of 28S ribosomal RNA gene: DNA barcodes for molecular classification and identification of mites[J]. Parasit Vectors, 2020, 13(1): 251.
[10] Wang DY. A molecular systematics research of oribatida on the basis of the RNA gene of ribosome[D]. Jinan: Shandong Normal University, 2007: 23-24, 42. (in Chinese)
  (王德印. 基于核糖体RNA基因的甲螨分子系统学研究[D]. 济南: 山东师范大学, 2007: 23-24, 42.)
[11] Pachl P, Lindl AC, Krause A, et al. The tropics as an ancient cradle of oribatid mite diversity[J]. Acarologia, 2016, 57(2): 309-322.
[12] Yang QQ, Liu SW, Yu XP. Research progress on DNA barcoding analysis methods[J]. Chin J Appl Ecol, 2018, 29(3): 1006-1014. (in Chinese)
  (杨倩倩, 刘苏汶, 俞晓平. DNA条形码分析方法研究进展[J]. 应用生态学报, 2018, 29(3): 1006-1014.)
[13] Puillandre N, Lambert A, Brouillet S, et al. ABGD, automatic barcode gap discovery for primary species delimitation[J]. Mol Ecol, 2012, 21(8): 1864-1877.
[14] Jiang F, Jin Q, Liang L, et al. Existence of species complex largely reduced barcoding success for invasive species of Tephritidae: a case study in Bactrocera spp[J]. Mol Ecol Resour, 2014, 14(6): 1114-1128.
[15] Sch?ffer S, Stabentheiner E, Shimano S, et al. Leaving the tropics: the successful colonization of cold temperate regions by Dolicheremaeus dorni (Acari, Oribatida)[J]. J Zool Syst Evol Res, 2018, 56(4): 505-518.
[16] Pan X. Research on species diversity of soil oribatid mites in agricultural region of Jilin and Liaoning provices[D]. Harbin: Northeast Institute of Geography and Agroecology, Chinese Academy of Sciences, 2021: 25. (in Chinese)
  (潘雪. 吉辽农区土壤甲螨多样性研究[D]. 哈尔滨: 中国科学院大学(中国科学院东北地理与农业生态研究所), 2021: 25.)
[17] Duan ZQ, Cai XP, Wang PY, et al. Study on ecological law of oribatid mites in Huangcheng and Gannan grasslands of Gansu Province[J]. Chin J Vet Sci Technol, 1999, 29(10): 31-32. (in Chinese)
  (段志勤, 才学鹏, 王佩雅, 等. 甘肃皇城和甘南草原甲螨的生态规律研究[J]. 中国兽医科技, 1999, 29(10): 31-32.)
[18] Kreipe V, Corral-Hernández E, Scheu S, et al. Phylogeny and species delineation in European species of the genus Steganacarus (Acari, Oribatida) using mitochondrial and nuclear markers[J]. Exp Appl Acarol, 2015, 66(2): 173-186.
[19] vonSaltzwedel H, Maraun M, Scheu S, et al. Evidence for frozen-niche variation in a cosmopolitan parthenogenetic soil mite species (Acari, Oribatida)[J]. PLoS One, 2014, 9(11): e113268.
[20] Hu L, Zhao YE, Yang YJ, et al. LSU rDNA D5 region: the DNA barcode for molecular classification and identification of Demodex[J]. Genome, 2019, 62(5): 295-304.
[21] Jiang HX, Yao DD, Lin SL, et al. DNA barcoding analysis of small mammals in the agricultural area in Nanxiong of Guangdong Province based on four mitochondrial gene sequences[J]. Chin J Vector Biol Control, 2022, 33(1): 48-53. (in Chinese)
  (姜洪雪, 姚丹丹, 林思亮, 等. 基于4种线粒体基因序列的广东省南雄市农区小型兽类DNA条形码分析[J]. 中国媒介生物学及控制杂志, 2022, 33(1): 48-53.)
文章导航

/

〈 〉