基于DNA条形码技术构建王朗国家级自然保护区陆生脊椎动物遗传资源数据库及物种鉴定

doi:10.17520/biods.2022661

生物多样性 ›› 2023, Vol. 31 ›› Issue (7): 22661. DOI: 10.17520/biods.2022661

• 研究报告: 动物多样性 • 上一篇下一篇

基于DNA条形码技术构建王朗国家级自然保护区陆生脊椎动物遗传资源数据库及物种鉴定

邢超¹^,², 林依²^,³, 周智强⁴, 赵联军⁴, 蒋仕伟⁵, 林蓁蓁²^,^*(), 徐基良¹^,^*(), 詹祥江²

1.北京林业大学生态与自然保护学院, 北京 100083
2.中国科学院动物研究所动物生态与保护生物学重点实验室, 北京 100101
3.中国科学院大学, 北京 100049
4.四川王朗国家级自然保护区管理局, 四川平武 622550
5.平武县林业和草原局, 四川平武 622550

收稿日期:2022-11-25 接受日期:2023-04-14 出版日期:2023-07-20 发布日期:2023-04-25
通讯作者: *E-mail: xujiliang@bjfu.edu.cn; linzz@ioz.ac.cn
作者简介:linzz@ioz.ac.cn
*E-mail: xujiliang@bjfu.edu.cn;
基金资助:
四川王朗国家级自然保护区物种库项目

The establishment of terrestrial vertebrate genetic resource bank and species identification based on DNA barcoding in Wanglang National Nature Reserve

Chao Xing¹^,², Yi Lin²^,³, Zhiqiang Zhou⁴, Lianjun Zhao⁴, Shiwei Jiang⁵, Zhenzhen Lin²^,^*(), Jiliang Xu¹^,^*(), Xiangjiang Zhan²

1. College of Ecology and Nature Conservation, Beijing Forestry University, Beijing 100083
2. Key Laboratory of Animal Ecology and Conservation Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101
3. University of Chinese Academy of Sciences, Beijing 100049
4. Wanglang National Nature Reserve Administration, Pingwu, Sichuan 622550
5. Pingwu Forestry and Grassland Bureau, Pingwu, Sichuan 622550

Received:2022-11-25 Accepted:2023-04-14 Online:2023-07-20 Published:2023-04-25
Contact: *E-mail: xujiliang@bjfu.edu.cn; linzz@ioz.ac.cn

摘要/Abstract

摘要：

DNA条形码(DNA barcode)是基因组中较短的、种内变异相对稳定的基因序列,已经成为生物多样性保护研究中物种鉴定、生物多样性评估的有力手段之一。四川王朗国家级自然保护区地处青藏高原东缘, 属于世界生物多样性热点地区, 具有丰富的生物资源, 在我国珍稀动物保护领域具有重要地位。目前保护区已累积了大量的陆生脊椎动物监测数据, 但缺乏遗传资源本底调查和基础的遗传资源数据库。本研究基于DNA条形码技术, 以四川王朗国家级自然保护区为主要研究区域, 基于样线法和博物馆标本调研, 对所采集的314份样品进行DNA条形码分析, 共鉴定兽类、鸟类、两栖类18目35科74种, 首次获得了王朗齿突蟾(Scutiger wanglangensis)的线粒体基因(COI、12S-16S、16S、Cytb)及核基因(RAG1)的条形码序列信息, 并通过比较不同监测方法说明了DNA条形码技术在动物多样性调查中的应用前景。本研究基于DNA条形码技术最终获得了216份DNA条形码数据, 初步建立了保护区陆生脊椎动物遗传资源数据库, 该数据库将为评估保护区生物多样性提供基础信息, 为动物保护和管理工作提供技术支持。

关键词: DNA条形码, 王朗国家级自然保护区, 陆生脊椎动物, 生物多样性, 条形码数据库

Abstract

Aims: DNA barcoding using the short, highly conserved regions of the genome to make species-level identifications, is widely used in the species identification and diversity assessment of plants and animals in biodiversity conservation research. Wanglang National Nature Reserve is a global biodiversity hot spot located in the eastern part of Qinghai-Tibet Plateau with high level of biodiversity and has a leading role in the wildlife conservation in China. Although a lot of survey data of species diversity have been collected, genetic resource of terrestrial vertebrates in the reserve remains largely unclear. To address this, our study aims to establish a DNA barcoding database for terrestrial vertebrate species for the reserve.

Methods: We collected genetic samples mainly using non-invasive sampling along the line transects in Wanglang National Nature Reserve and from museum specimens. We extracted DNA from the samples to obtain the barcode sequences by PCR. Each DNA barcode sequences were blasted against NCBI database for species identification. For those sequences that we could not directly get the matches in the reference database, we constructed phylogenetic trees using MEGA 11.0 with the aim to identify the species based on the sequences of its relative species from the public database.

Results: The total of 314 samples including tissues from carcasses, feathers, feces and eggshells were collected and 74 species were identified using 16 pairs DNA barcoding primers. The sequences of mitochondrial COI, Cytb, 12S-16S, 16S genes and sequences of nuclear RAG1 gene of Scutiger wanglangensis were firstly reported. Compared with other wildlife monitoring techniques such as camera traps, drift fences and pitfall traps in the same time interval, DNA barcoding is more efficient in all taxon groups and has better performance in bird monitoring. Finally, A terrestrial vertebrate genetic resource bank contains 314 substantial samples and 216 DNA barcodes has been established.

Conclusion: Our study established an elementary DNA barcoding database of 74 terrestrial vertebrate species in the reserve, highlights the utility of DNA barcoding technique in wild animal diversity surveys, and provides a basic catalog for assessing local species diversity to improve conservation research and management in the reserve.

Key words: DNA barcoding, Wanglang National Nature Reserve, terrestrial vertebrates, biodiversity, barcode database

邢超, 林依, 周智强, 赵联军, 蒋仕伟, 林蓁蓁, 徐基良, 詹祥江 (2023) 基于DNA条形码技术构建王朗国家级自然保护区陆生脊椎动物遗传资源数据库及物种鉴定. 生物多样性, 31, 22661. DOI: 10.17520/biods.2022661.

Chao Xing, Yi Lin, Zhiqiang Zhou, Lianjun Zhao, Shiwei Jiang, Zhenzhen Lin, Jiliang Xu, Xiangjiang Zhan (2023) The establishment of terrestrial vertebrate genetic resource bank and species identification based on DNA barcoding in Wanglang National Nature Reserve. Biodiversity Science, 31, 22661. DOI: 10.17520/biods.2022661.

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链接本文: https://www.biodiversity-science.net/CN/10.17520/biods.2022661

https://www.biodiversity-science.net/CN/Y2023/V31/I7/22661

图/表 7

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类别 Category	目标片段所在基因(引物名称) Target gene (primers' names)	上游引物序列5'-3' Forward primer sequence 5'-3'	下游引物序列5'-3' Reverse primer sequence 5'-3'	退火温度 Annealing temperature (℃)	循环次数 Cycles	参考文献 Reference
兽类 Mammals	COI (COIung)	GTACCGCTAATAATTGGTGCTCC	GGGTGGCCAAAGAATCAGAACAAGTG	56	35	Kumar et al, 2017
	16S (16SF/R)	GAGAAGACCCTATGGAGC	ATAGAAACCGACCTGGAT	50	30	Xiong et al, 2016
	COI (BatL/R6036R)	CCTACTCRGCCATTTTACCTATG	ATCTCTGGGTGTCCAAAGAATCA	55	35	Robins et al, 2007
	ATP6 (ATP6-DF3/ATP6-DR2)	AACGAAAATCTATTCGCCTCT	TGGATGGACAGTATTTGTTTTGAT	60/50	10/30	Chaves et al, 2012
	D-loop (BEDL225/H470)	ATGTACATACTGTGCTTGGC	GTCATTAGTCCATCGAGATG	53	38	Zhang et al, 2002
鸟类 Avians	COI (COI-K_Bird_F/R)	CCCCAGACATAGCATTYCC	TTGTGATAGTGGTGGGGTTTTAT	46/53	5/45	Parejo-Farnés et al, 2018
	Cytb (Cytb-L14816/H15173)	CCATCCAACATCTCAGCATGATGAAA	CCCCTCAGAATGATATTTGTCCTCA	50	30	Parson et al, 2000
	COI (L6697Bird_F/H7390Thrush_R)	TCAACYAACCACAAAGAYATCGGYAC	ACGTGGGARATRATTCCAAATCCTG	48/51	5/30	Saitoh et al, 2015
	COI (PasserF/R)	CCAACCACAAAGACATCGGAACC	GTAAACTTCTGGGTGACCAAAGAATC	58	35	Lohman et al, 2009
两栖类 Amphibians	COI (LCO1490/HCO2198)	GGTCAACAAATCATAAAGATATTGG	TAAACTTCAGGGTGACCATCA	50	35	Folmer et al, 1994
	COI (LepF/R)	ATTCAACCAATCATAAAGATATTGG	TAAACTTCTGGATGTCCATCA	46	35	Hebert et al, 2004a
	16S (P7/8)	CGCCTGTTTACCCAT	CCGGTCTGAACTCAGATCACGT	55	36	Simon et al, 1994
	16S (Sar/Sbr)	CGCCTGTTTATCCAT	CCGGTCTGAACTCAGATCACGT	48	40	Palumbi et al, 2002
	12S-16S (1602L/2571H)	GTATACCGGAAGGTGTACTTGGAACAG	TACCTTCGCACGGTCAGAATACCGC	50	35	Fu et al, 2007
	Cytb (FrogGlu-f/Thr-r)	TGATCTGCCACCGTTG	CTCCATTCTTCGRCTTACAAG	46	40	Hillis et al, 1996
	RAG1 (RAG1-F/R)	AGCTGCAGYCARTACCAYAARATGTA	GCAAAGTTTCCGTTCATTCTCAT	50	35	Fu et al, 2007

类别 Category	目标片段所在基因(引物名称) Target gene (primers' names)	上游引物序列5'-3' Forward primer sequence 5'-3'	下游引物序列5'-3' Reverse primer sequence 5'-3'	退火温度 Annealing temperature (℃)	循环次数 Cycles	参考文献 Reference
兽类 Mammals	COI (COIung)	GTACCGCTAATAATTGGTGCTCC	GGGTGGCCAAAGAATCAGAACAAGTG	56	35	Kumar et al, 2017
	16S (16SF/R)	GAGAAGACCCTATGGAGC	ATAGAAACCGACCTGGAT	50	30	Xiong et al, 2016
	COI (BatL/R6036R)	CCTACTCRGCCATTTTACCTATG	ATCTCTGGGTGTCCAAAGAATCA	55	35	Robins et al, 2007
	ATP6 (ATP6-DF3/ATP6-DR2)	AACGAAAATCTATTCGCCTCT	TGGATGGACAGTATTTGTTTTGAT	60/50	10/30	Chaves et al, 2012
	D-loop (BEDL225/H470)	ATGTACATACTGTGCTTGGC	GTCATTAGTCCATCGAGATG	53	38	Zhang et al, 2002
鸟类 Avians	COI (COI-K_Bird_F/R)	CCCCAGACATAGCATTYCC	TTGTGATAGTGGTGGGGTTTTAT	46/53	5/45	Parejo-Farnés et al, 2018
	Cytb (Cytb-L14816/H15173)	CCATCCAACATCTCAGCATGATGAAA	CCCCTCAGAATGATATTTGTCCTCA	50	30	Parson et al, 2000
	COI (L6697Bird_F/H7390Thrush_R)	TCAACYAACCACAAAGAYATCGGYAC	ACGTGGGARATRATTCCAAATCCTG	48/51	5/30	Saitoh et al, 2015
	COI (PasserF/R)	CCAACCACAAAGACATCGGAACC	GTAAACTTCTGGGTGACCAAAGAATC	58	35	Lohman et al, 2009
两栖类 Amphibians	COI (LCO1490/HCO2198)	GGTCAACAAATCATAAAGATATTGG	TAAACTTCAGGGTGACCATCA	50	35	Folmer et al, 1994
	COI (LepF/R)	ATTCAACCAATCATAAAGATATTGG	TAAACTTCTGGATGTCCATCA	46	35	Hebert et al, 2004a
	16S (P7/8)	CGCCTGTTTACCCAT	CCGGTCTGAACTCAGATCACGT	55	36	Simon et al, 1994
	16S (Sar/Sbr)	CGCCTGTTTATCCAT	CCGGTCTGAACTCAGATCACGT	48	40	Palumbi et al, 2002
	12S-16S (1602L/2571H)	GTATACCGGAAGGTGTACTTGGAACAG	TACCTTCGCACGGTCAGAATACCGC	50	35	Fu et al, 2007
	Cytb (FrogGlu-f/Thr-r)	TGATCTGCCACCGTTG	CTCCATTCTTCGRCTTACAAG	46	40	Hillis et al, 1996
	RAG1 (RAG1-F/R)	AGCTGCAGYCARTACCAYAARATGTA	GCAAAGTTTCCGTTCATTCTCAT	50	35	Fu et al, 2007

类别 Category	数量 No. of samples	样品类型 Sample type	数量 No. of samples
鸟类 Avians	156	粪便 Feces	123
兽类 Mammals	137	羽毛(毛发) Feathers (Pelage)	60
两栖类 Amphibians	21	组织 Tissues	37
		标本组织 Tissues from specimens	90
		卵壳 Eggshells	4
总计 Total	314	总计 Total	314

类别 Category	数量 No. of samples	样品类型 Sample type	数量 No. of samples
鸟类 Avians	156	粪便 Feces	123
兽类 Mammals	137	羽毛(毛发) Feathers (Pelage)	60
两栖类 Amphibians	21	组织 Tissues	37
		标本组织 Tissues from specimens	90
		卵壳 Eggshells	4
总计 Total	314	总计 Total	314

物种类型(样品数量/个) Category (No. of samples)	成功扩增样品数(扩增成功率) No. of successfully amplified samples (Success rate)	通用引物鉴定物种数(识别率) No. of species identified by universal primers (Identification rate)	特异性引物鉴定物种数(识别率) No. of species identified by specific primers (Identification rate)
鸟类 Avians (156)	128 (82.1%)	41 (89.1%)	5 (10.9%)
兽类 Mammals (137)	111 (81.0%)	15 (55.6%)	12 (44.4%)
两栖类 Amphibians (21)	21 (100%)	3 (75.0%)	-

基于DNA条形码技术构建王朗国家级自然保护区陆生脊椎动物遗传资源数据库及物种鉴定

The establishment of terrestrial vertebrate genetic resource bank and species identification based on DNA barcoding in Wanglang National Nature Reserve

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物种类别 Category	样品数 No. of samples	物种预判样品数 No. of morphologically identified samples	预判正确样品数 No. of correctly identified samples	预判正确率 Accuracy of morphological identification rate
鸟类 Avians	109	57	30	52.6%
兽类 Mammals	99	38	18	47.4%
两栖类 Amphibians	13	13	9	69.2%
合计 Total	221	108	57	52.8%

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