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

doi:10.17520/biods.2022661

Abstract

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

Chao Xing, Yi Lin, Zhiqiang Zhou, Lianjun Zhao, Shiwei Jiang, Zhenzhen Lin, Jiliang Xu, Xiangjiang Zhan. The establishment of terrestrial vertebrate genetic resource bank and species identification based on DNA barcoding in Wanglang National Nature Reserve[J]. Biodiv Sci, 2023, 31(7): 22661.

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URL: https://www.biodiversity-science.net/EN/10.17520/biods.2022661

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

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References 79

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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%)	-