涉案偶蹄目动物DNA条形码构建

doi:10.3969/j.issn.1000-2006.202004013

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南京林业大学学报（自然科学版） ›› 2020, Vol. 44 ›› Issue (6): 27-32.doi: 10.3969/j.issn.1000-2006.202004013

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涉案偶蹄目动物DNA条形码构建

刘大伟(), 侯森林^*(), 周用武, 费宜玲, 何建

南京森林警察学院,野生动植物物证技术国家林业和草原局重点实验室,国家林业和草原局森林公安司法鉴定中心, 江苏南京 210023

收稿日期:2020-04-08 修回日期:2020-08-28 出版日期:2020-11-30 发布日期:2020-12-07
通讯作者: 侯森林
基金资助:
中央高校基本科研业务费专项资金项目(LGYB202007);“十三五”江苏省重点建设学科项目(2016-2020)

DNA barcoding of Artiodactyla in cases involved in wildlife for species identification

LIU Dawei(), HOU Senlin^*(), ZHOU Yongwu, FEI Yiling, HE Jian

Key Laboratory of Wildlife Evidence Technology, Forest Police Forensic Identification Center, National Forestry and Grassland Administration,Nanjing Forest Police College, Nanjing 210023, China

Received:2020-04-08 Revised:2020-08-28 Online:2020-11-30 Published:2020-12-07
Contact: HOU Senlin

摘要/Abstract

摘要：

【目的】利用线粒体DNA条形码标记鉴定涉案偶蹄目动物,为解决偶蹄目动物残体、制品的鉴定难题提供技术参考。【方法】对16个偶蹄目动物物种共67份样本,采用试剂盒提取DNA,扩增、测定物种的线粒体DNA的COI基因、Cyt b基因片段;在GenBank上Blast搜索进行同源性分析;利用MEGA 7.0软件分别比较这两个基因片段种内和种间遗传距离,构建系统发育树。【结果】在同源性分析中,西伯利亚狍(Capreolus pygargus)的Cyt b基因片段与GenBank中西伯利亚狍、欧洲狍(Capreolus capreolus)基因序列的同源性均为99.64%~99.82%,该片段不能区分西伯利亚狍与欧洲狍这两个近缘种。其余测序结果与数据库中参考序列一致且相似度较高。对于COI片段,种内遗传距离为0~1.4%,种间遗传距离为1.8%~18.8%。对于Cyt b基因片段种内遗传距离为0~0.5%,种间遗传距离为4.0%~15.8%。两个片段的种内遗传差异均小于种间遗传差异,存在较为明显的条形码间隔。COI和Cyt b基因片段的系统发育树中,同物种的不同个体均能形成具有较高支持度的单分支;不同物种之间界限清晰,每个物种的所有个体均能够形成具有较高支持度的单分支。【结论】基于COI和Cyt b基因片段构建的DNA条形码均可用于对涉案偶蹄目动物的物种鉴定。在物种鉴定中,可采用两个片段联合使用的方式,尤其是对于近缘种的鉴定,以降低错误风险。

关键词: 偶蹄目, DNA条形码, 物种鉴定, COI, Cyt b, 基因片段

Abstract:

【Objective】In this study, we aimed to identify Artiodactyla species in cases of wildlife destruction, using mitochondrial DNA barcoding. 【Method】COI and Cyt b sequences in 67 samples from 16 species were amplified. Animal samples were identified using Blast search on the GenBank sequences. Intra-and inter-specific genetic distances were compared, and phylogenetic trees were constructed using MEGA 7.0. 【Result】In a homology analysis, the Cyt b sequences of Capreolus pygargus and Capreolus capreouls were 99.64%-99.82% homologous; this sequence could not distinguish two closely related species, whereas other COI and Cyt b sequences could. The intra-specific genetic distance was 0-1.4% for COI and 0-0.5% for Cyt b, whereas the inter-specific genetic distance was 1.8%-18.8% for COI and 4.0%-15.8% for Cyt b. An obvious barcoding gap was detected in both markers for all species. The phylogenetic tree showed that samples from the same species formed monophyletic groups with high support values. The gaps between different species were clear, and all samples from each species formed a single branch with a high support value.【Conclusion】COI and Cyt b may serve as molecular markers for Artiodactyla identification in cases involving wildlife resources. To minimize the risk of false identification, the two DNA barcodes could be used simultaneously, especially for the closely related species.

Key words: Artiodactyla, DNA barcoding, species identification, COI, Cyt b, gene fragment

中图分类号:

Q34
S718.6

刘大伟,侯森林,周用武,等. 涉案偶蹄目动物DNA条形码构建[J]. 南京林业大学学报（自然科学版）, 2020, 44(6): 27-32.

LIU Dawei, HOU Senlin, ZHOU Yongwu, FEI Yiling, HE Jian. DNA barcoding of Artiodactyla in cases involved in wildlife for species identification[J].Journal of Nanjing Forestry University (Natural Science Edition), 2020, 44(6): 27-32.DOI: 10.3969/j.issn.1000-2006.202004013.

图/表 5

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参考文献 29

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物种 species	保护级别 protected category	样本数 number of samples	编号 No.	动物体部位 body part
斑羚Naemorhedus goral	II	3	NG1-3	角、肌肉
北山羊Capra ibex	I	5	CI1-5	角、肌肉
藏羚Pantholops hodgsonii	I	3	PH1-3	头骨
鹅喉羚Gazella subgutturosa	II	4	GS1-4	皮张
高鼻羚羊Saiga tatarica	I	6	ST1-6	角
河麂Hydropotes inermis	II	5	HI1-5	肌肉
黒麂 Muntiacus crinifrons	I	2	MC1-2	肌肉
鬣羚Capricornis sumatraensis	II	5	CS1-5	肌肉
羚牛 Budorcas taxicolor	I	3	BT1-3	头骨
马鹿Cervus elaphus	II	5	CE1-5	角
毛冠鹿Elaphodus cephalophus	三有	6	EC1-6	皮张
梅花鹿Cervus nippon	I	5	CN1-5	角
西伯利亚狍Capreolus pygargus	三有	6	CC1-6	肌肉
驼鹿Alces alces	II	3	AA1-3	角
小麂Muntiacus reevesi	三有	4	MR1-4	角、肌肉
马麝Moschus chrysogaster	I	2	MC'1-2	肌肉

物种 species	序列比对结果(相似度/%) sequences alignment result (similarities)
物种 species	相关物种COI基因片段 COI of concerned species	相关物种Cyt b基因片段 Cyt b of concerned species
斑羚	斑羚(99.75~100)	斑羚(98.74~100)
北山羊	北山羊(99.11~99.82)	北山羊(99.58~99.64)
藏羚	藏羚(99.47~99.82)	藏羚(99.47~99.82)
鹅喉羚	鹅喉羚(96.80~100)	鹅喉羚(99.29~99.82)
高鼻羚羊	高鼻羚羊(99.29~100)	高鼻羚羊(98.75~100)
河麂	河麂(98.04~99.64)	河麂(99.29~99.82)
黒麂	黒麂(99.47~100)	黒麂(98.93~99.11)
鬣羚	鬣羚(99.11~100)	鬣羚(98.75~100)
羚牛	羚牛(99.11~99.82)	羚牛(98.58~99.82)
马鹿	马鹿(97.51~99.64)	马鹿(98.76~99.64)
毛冠鹿	毛冠鹿(98.40~100)	毛冠鹿(98.40~100)
梅花鹿	梅花鹿(98.22~99.64)	梅花鹿(98.40~99.64)
西伯利亚狍	西伯利亚狍 (99.11~99.64)	西伯利亚狍、欧洲狍 (99.64~99.82)
驼鹿	驼鹿(98.75~100)	驼鹿(98.74~100)
小麂	小麂(96.98-99.82)	小麂(98.93~99.82)
马麝	马麝(98.93-99.29)	马麝(98.92~99.10)

涉案偶蹄目动物DNA条形码构建

DNA barcoding of Artiodactyla in cases involved in wildlife for species identification

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遗传距离 genetic distance	种内/% intra species		种间/% inter species
遗传距离 genetic distance	COI	Cyt b	COI	Cyt b
最小值maximum	0.0	0.0	1.8	4.0
最大值minimum	1.4	0.5	18.8	15.8
平均值mean	0.1	0.0	12.1	10.9
标准偏差S.D.	0.3	0.1	2.1	1.9

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