广东省江门市捕获鼠类的DNA条形码分析

doi:10.11853/j.issn.1003.8280.2020.03.012

摘要/Abstract

摘要： 目的应用DNA条形码技术对广东省江门市新会区农田捕获的鼠类进行种类鉴定和分析，为研究鼠形动物的多样性及鼠害防治奠定基础。方法以2017年12月在广东省江门市新会区农田捕获的45只鼠为研究对象，分别提取鼠基因组DNA，采用通用引物扩增线粒体细胞色素C氧化酶亚基Ⅰ（COⅠ）基因片段并进行测序，将测序结果在美国国立生物技术信息中心中进行BLAST序列同源性比对，采用邻接法构建分子进化树，基于双参数模型计算遗传距离。结果 45只鼠样本均能通过PCR扩增出特异性基因片段，通过BLAST比对共鉴定出4种鼠（黄毛鼠、黄胸鼠、褐家鼠和黑缘齿鼠），37只鼠样本与形态学鉴定结果一致，为黄毛鼠；8只鼠样本存在形态鉴定错误，经复核，其中5只鼠重新鉴定为黄胸鼠，2只为幼年褐家鼠，另外1只为黑缘齿鼠。对45条序列进行比对分析发现有131个变异位点，单倍型分析结果显示有12个不同的单倍型。黄毛鼠种内遗传距离为0~1.2%，不同鼠种间遗传距离为7.1%~11.6%，平均遗传距离为（10.1±0.7）%，分子进化分析结果显示，同一种鼠类的个体聚为一支，支持率均为100%，能区分不同鼠种。结论 DNA条形码技术能够有效区分形态相似的近缘种，纠正形态学鉴定中的错误。

关键词: 鼠类, DNA条形码, 细胞色素C氧化酶亚基Ⅰ基因

Abstract: Objective To identify and analyze the rodents captured in the farmland in Xinhui district, Jiangmen, Guangdong province, China, using DNA barcoding technology, and to lay a foundation for the diversity study of murine-like animals and rodent prevention and control. Methods Forty-five rodents captured in the farmland in Xinhui district, Jiangmen, Guangdong province, in December 2017, were enrolled as study subjects. Their genetic DNAs were extracted and amplified for mitochondrial cytochrome c oxidase subunit I gene fragments using consensus primers. The gene fragments were sequenced and the results were subjected to sequence homology analysis using BLAST in the National Center for Biotechnology Information. The molecular evolutionary tree was constructed by the neighbor-joining method. Genetic distance was calculated based on a two-parameter model. Results Specific gene fragments of the forty-five samples were obtained by PCR amplification. Four species of rodents (Rattus losea, R. tanezumi, R. norvegicus, and R. andamanensis) were identified through BLAST comparison. Thirty-seven samples had identification results identical to the morphological identification (R. losea), and eight samples had erroneous morphological identification results, among which five were reidentified as R. tanezumi, two were reidentified as young R. norvegicus, and one was reidentified as R. andamanensis, according to the review of the initial results. A total of 131 mutation sites were identified through alignment and analysis of the 45 sequences, and 12 different haplotypes were identified through haplotype analysis. The intraspecific genetic distance of R. losea was 0-1.2%, the interspecific genetic distance was 7.1%-11.6%, and the mean genetic distance was 10.1%±0.7%. The NJ tree showed that individuals of the same rodent species were clustered into one branch with a 100% support rate, which could distinguish among different rodent species. Conclusion The DNA barcoding technology can effectively distinguish between related species with similar morphology and correct the erroneous results of morphological identification.

Key words: Rodent, DNA barcoding, Cytochrome c oxidase subunit Ι gene

中图分类号:

S443

姚丹丹, 姜洪雪, 隋晶晶, 冯志勇. 广东省江门市捕获鼠类的DNA条形码分析[J]. 中国媒介生物学及控制杂志, 2020, 31(3): 305-309.

YAO Dan-dan, JIANG Hong-xue, SUI Jing-jing, FENG Zhi-yong. DNA barcoding analysis of rodents captured in Jiangmen, Guangdong province, China[J]. Chines Journal of Vector Biology and Control, 2020, 31(3): 305-309.

参考文献

[1] 董石,尹玲,杨天天. DNA条形码在动植物检测中的应用[J]. 国土与自然资源研究,2019(1):81-82. DOI:10.16202/j.cnki.tnrs.2019.01.021. Dong S,Yin L,Yang TT. Application of DNA barcode in animal and plant detection[J]. Terr Nat Resour Study,2019(1):81-82. DOI:10.16202/j.cnki.tnrs.2019.01.021.
[2] Hebert PDN,Cywinska A,Ball SL,et al. Biological identifications through DNA barcodes[J]. Proc Roy Soc B,2003,270(1512):313-321. DOI:10.1098/rspb.2002.2218.
[3] Schäffer S,Zachos FE,Koblmüller S. Opening the treasure chest:a DNA-barcoding primer set for most higher taxa of central European birds and mammals from museum collections[J]. PLoS One,2017,12(3):e0174449. DOI:10.1371/journal.pone. 0174449.
[4] Proshek B,Dupuis JR,Engberg A,et al. Genetic evaluation of the evolutionary distinctness of a federally endangered butterfly,Lange's Metalmark[J]. BMC Evol Biol,2015,15:73. DOI:10.1186/s12862-015-0354-9.
[5] Gong SH,Ding YF,Wang Y,et al. Advances in DNA barcoding of toxic marine organisms[J]. Int J Mol Sci,2018,19(10):2931. DOI:10.3390/ijms19102931.
[6] 李争光,丛林,王大伟,等. 黑龙江地区农田害鼠发生特点及DNA条形码鉴定技术的应用[J]. 植物保护,2018,44(6):145-151,167. DOI:10.16688/j.zwbh.2018001. Li ZG,Cong L,Wang DW,et al. Species identification of main pest rodents by DNA barcoding and their occurrence characteristics from croplands in Heilongjiang province[J]. Plant Prot,2018,44(6):145-151,167. DOI:10.16688/j.zwbh.2018001.
[7] Nicolas V,Schaeffer B,Missoup AD,et al. Assessment of three mitochondrial genes (16S,Cytb,COⅠ) for identifying species in the praomyini tribe (Rodentia:Muridae)[J]. PLoS One,2012,7(5):e36586. DOI:10.1371/journal.pone.0036586.
[8] Lu L,Chesters D,Zhang W,et al. Small mammal investigation in spotted fever focus with DNA-barcoding and taxonomic implications on rodents species from Hainan of China[J]. PLoS One,2012,7(8):e43479. DOI:10.1371/journal.pone.0043479.
[9] Zhang B,He K,Wan T,et al. Multi-locus phylogeny using topotype specimens sheds light on the systematics of Niviventer (Rodentia,Muridae) in China[J]. BMC Evol Biol,2016,6(1):261. DOI:10.1186/s12862-016-0832-8.
[10] 郭丽民,席进孝,盖永志,等. 甘肃省鼠疫疫源地啮齿动物DNA条形码分析[J]. 中国媒介生物学及控制杂志,2019,30(2):176-179. DOI:10.11853/j.issn.1003.8280.2019.02.014. Guo LM,Xi JX,Gai YZ,et al. Application of DNA barcoding in the identification of rodent species in plague foci in Gansu province,China[J]. Chin J Vector Biol Control,2019,30(2):176-179. DOI:10.11853/j.issn.1003.8280.2019.02.014.
[11] 陈利伟,赵志亮,邱德义,等. DNA条形码技术在兰州中川机场鼠类鉴定中的应用[J]. 中国国境卫生检疫杂志,2018,41(2):102-105. DOI:10.16408/j.1004-9770.2018.02.008. Chen LW,Zhao ZL,Qiu DY,et al. Application of DNA barcoding in classification of rodents at Lanzhou Zhongchuan airport area[J]. Chin J Front Health Quar,2018,41(2):102-105. DOI:10.16408/j.1004-9770.2018.02.008.
[12] Ndiaye A,Tatard C,Stanley W,et al. Taxonomic hypotheses regarding the genus Gerbillus (Rodentia,Muridae,Gerbillinae) based on molecular analyses of museum specimens[J]. ZooKeys,2016,566:145-155. DOI:10.3897/zookeys.566.7317.
[13] 金圣浩,鲁亮,闫东,等. DNA条形码技术在河北省坝上地区夜行鼠鉴定中的应用[J]. 中国媒介生物学及控制杂志,2015,26(1):55-57. DOI:10.11853/j.issn.1003.4692.2015.01. 014. Jin SH,Lu L,Yan D,et al. Application of DNA barcoding technique in identification of nocturnal rodent species in Bashang area of Hebei province,China[J]. Chin J Vector Biol Control,2015,26(1):55-57. DOI:10.11853/j.issn.1003. 4692.2015.01.014.
[14] Hebert PDN,Ratnasingham S,de Waard JR. Barcoding animal life:cytochrome c oxidase subunit 1 divergences among closely related species[J]. Proc Roy Sci B,2003,270(S1):S96-99. DOI:10.1098/rsbl.2003.0025.
[15] 蒋志刚,马勇,吴毅,等. 中国哺乳动物多样性[J]. 生物多样性,2015,23(3):351-364. DOI:10.17520/biods.2014202. Jiang ZG,Ma Y,Wu Y,et al. China's mammalian diversity[J]. Biodivers Sci,2015,23(3):351-364. DOI:10.17520/biods. 2014202.
[16] Chen Y,Su QQ,Qin J,et al. Call divergence in three sympatric Rattus species[J]. J Acoust Soc Am,2017,142(1):29-34. DOI:10.1121/1.4990022.
[17] Liu SY,He K,Chen SD,et al. How many species of Apodemus and Rattus occur in China? a survey based on mitochondrial cytb and morphological analyses[J]. Zool Res,2018,39(5):309-320. DOI:10.24272/j.issn.2095-8137.2018.053.
[18] 何锴,王文智,李权,等. DNA条形码技术在小型兽类鉴定中的探索:以甘肃莲花山为例[J]. 生物多样性,2013,21(2):197-205. DOI:10.3724/SP.J.1003.2013.09160. He K,Wang WZ,Li Q,et al. DNA barcoding in surveys of small mammal community:a case study in Lianhuashan,Gansu province,China[J]. Biodivers Sci,2013,21(2):197-205. DOI:10.3724/SP.J.1003.2013.09160.
[19] 马英,李海龙,鲁亮,等. DNA条形码技术在青海海东地区小型兽类鉴定中的应用[J]. 生物多样性,2012,20(2):193-198. DOI:10.3724/SP.J.1003.2012.06188. Ma Y,Li HL,Lu L,et al. Application for identification of small mammals by DNA barcoding in Haidong area,Qinghai province,China[J]. Biodivers Sci,2012,20(2):193-198. DOI:10.3724/SP.J.1003.2012.06188.
[20] Ermakov OA,Simonov E,Surin VL,et al. Implications of hybridization,NUMTs,and overlooked diversity for DNA barcoding of Eurasian ground squirrels[J]. PLoS One,2015,10(1):e0117201. DOI:10.1371/journal.pone.0117201.