基于线粒体Cyt b DNA阿拉善马鹿分子系统学研究
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摘要
阿拉善马鹿为国家Ⅱ级重点保护野生动物,仅分布于宁夏和内蒙古交界的贺兰山中段,是我国唯一幸存的该亚种的有效种群。目前关于野生阿拉善马鹿的分子研究尚属空白,本研究于2016年8—9月和2017年2—3月在贺兰山采集新鲜阿拉善马鹿粪便样本396份,使用线粒体Cyt b区DNA引物对提取的DNA进行物种鉴定和9对微卫星引物对其个体识别,共鉴定出278个阿拉善马鹿个体,基于线粒体Cyt b DNA对其全序列进行分析。使用线粒体Cyt b区引物对提取的粪便DNA扩增,获得了1 075 bp的序列,检测到10个变异位点,并定义了10个单倍型。其中10个变异位点占分析长度的0. 93%,均为碱基代换,无碱基插入和缺失。其单倍型多样性为0. 243,核苷酸多样性为0. 000 32。中性检验Tajima's D值为显著负值,Fu和Li's D值为不显著的负值,表示阿拉善马鹿可能经历瓶颈事件并随后出现种群扩张。在系统发育树中,阿拉善马鹿单独聚成一支且与东北马鹿亲缘关系较近,与其他中国马鹿亚种亲缘关系较远。本研究表明野生阿拉善马鹿种群总体遗传多样性较低,建议加强对该亚种的保护力度。
Cervus elaphus alxaicus is a second-class state key protected subspecies in China. It occupies only the middle region of the Helan Mountains between Ningxia and Inner Mongolia. We collected 396 fecal samples at Helan Mountains from August to September 2016 and February to March 2017. We confirmed 278 individuals of C. e. alxaicus by Cyt b sequence analysis and 9 microsatellite loci. We obtained a total of 1 075 bp fragments of Cyt b,detected 10 variable sites,and identified 10 haplotypes. The 10 variable sites accounted for 0. 93% of the analysis length,all of which were base substitutions,without base insertion or deletion. The haplotype diversity was 0. 243,and the nucleotide diversity was 0. 000 32. Estimates of Tajima's D were significantly negative,and the Fu and Li index D was not significantly negative,meaning that the population could experience bottleneck and subsequent population expansion. In the phylogenetic tree, C. e.alxaicus was isolated into one branch and was closely related to C. e. xanthopygus,but was distantly related to other Chinese red deer subspecies. We conclude that the genetic diversity of C.e. alxaicus was low. We recommend that protection for this subspecies be strengthened.
Cervus elaphus alxaicus is a second-class state key protected subspecies in China. It occupies only the middle region of the Helan Mountains between Ningxia and Inner Mongolia. We collected 396 fecal samples at Helan Mountains from August to September 2016 and February to March 2017. We confirmed 278 individuals of C. e. alxaicus by Cyt b sequence analysis and 9 microsatellite loci. We obtained a total of 1 075 bp fragments of Cyt b,detected 10 variable sites,and identified 10 haplotypes. The 10 variable sites accounted for 0. 93% of the analysis length,all of which were base substitutions,without base insertion or deletion. The haplotype diversity was 0. 243,and the nucleotide diversity was 0. 000 32. Estimates of Tajima's D were significantly negative,and the Fu and Li index D was not significantly negative,meaning that the population could experience bottleneck and subsequent population expansion. In the phylogenetic tree, C. e.alxaicus was isolated into one branch and was closely related to C. e. xanthopygus,but was distantly related to other Chinese red deer subspecies. We conclude that the genetic diversity of C.e. alxaicus was low. We recommend that protection for this subspecies be strengthened.
引文
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[25]刘鑫鑫.基于分子粪便学的穆棱地区同域分布梅花鹿(Cervus nippon)和马鹿(Cervus elaphus)遗传多样性比较研究[D].哈尔滨:东北林业大学,2017.
[26]QUINN T W,WILSON A C.Sequence evolution in and around the mitochondrial control region in birds[J].Journal of Molecular Evolution,1993,37(4):417-425.
[27]李楠楠,刘振生,王正寰,等.基于细胞色素b基因的中国岩羊不同地理种群遗传差异分析[J].生态学报,2012,32(8):2381-2389.
[2]李明,王小明,盛和林,等.马鹿四个亚种的起源和遗传分化研究[J].动物学研究,1998,19(3):177-183.
[3]王小明,李明,唐绍祥,等.贺兰山偶蹄类动物资源及保护现状研究[J].动物学杂志,1999,34(5):26-29.
[4]盛和林.中国鹿类动物[M].上海:华东师范大学出版社,1992:1-215.
[5]刘振生,曹丽荣,翟昊,等.贺兰山区马鹿对冬季生境的选择性[J].动物学研究,2004,25(5):403-409.
[6]刘振生,张明明,李志刚,等.贺兰山马鹿冬季取食和卧息生境选择[J].兽类学报,2009,29(2):133-141.
[7]骆颖,张明明,刘振生,等.贺兰山马鹿冬春季生境的选择[J].生态学报,2009,29(5):2757-2763.
[8]高惠,滕丽微,汪洋,等.阿拉善马鹿(Cervus alashanicus)生境适应性评价[J].生态学报,2017,37(11):3926-3931.
[9]MEYER A,KOCHER T D,BASASIBWAKI P,et al.Monophyletic origin of Lake Victoria cichild fishes suggested by mitochondrial DNAsequences[J].Nature,1990,347(6293):550-553.
[10]狄维忠.贺兰山维管植物[M].西安:西北大学出版社,1987.
[11]张丽,滚双宝,雷天云,等.应用mt DNA Cyt b基因全序列分析中国5个马鹿群体的遗传多样性和系统发育[J].华北农学报,2010,25(4):12-16.
[12]KUEHN R,SCHROEDER W,P F,et al.Genetic diversity,gene flow and drift in Bavarian red deer populations(Cervus elaphus)[J].Conservation Genetics,2003,4(2):157-166.
[13]MEREDITH E P,RODZEN J A,LEVINE K F,et al.Characterization of an additional 14 microsatellite loci in California elk(Cervus elaphus)for use in forensic and population applications[J].Conservation Genetics,2005,6(1):151-153.
[14]JONES K C,LEVINE K F,BANKS J D.Characterization of 11polymorphic tetranucleotide microsatellites for forensic applications in California elk(Cervus elaphus canadensis)[J].Molecular Ecology Notes,2002,2(4):425-427.
[15]邢秀梅.中国茸鹿分子遗传多样性研究[D].北京:中国农业科学院,2006.
[16]胡贺娇.西藏马鹿(Cervus wallichii)分子生态学与营养生态学研究[D].哈尔滨:东北林业大学,2016.
[17]HALL T A.BioEdit:a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT[C]//Nucleic acids symposium series.London:Information Retrieval Ltd.,c1979-c2000,1999,41(41):95-98.
[18]ROZAS J,ROZAS R.Dna SP version 3:an integrated program for molecular population genetics and molecular evolution analysis[J].Bioinformatics,1999,15(2):174-175.
[19]KIMURA M.A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences[J].Journal of Molecular Evolution,1980,16(2):111-120.
[20]KUMAR S,STECHER G,TAMURA K.MEGA7:molecular evolutionary genetics analysis version 7.0 for bigger datasets[J].Molecular Biology and Evolution,2016,33(7):1870-1874.
[21]FELSENSTEIN J.Confidence limits on phylogenies:an approach using the tootstrap[J].Evolution,1985,39(4):783-791.
[22]RONQUIST F,HUELSENBECK J P.Mr Bayes 3:Bayesian phylogenetic inference under mixed models[J].Bioinformatics,2003,19(12):1572-1574.
[23]BANDELT H J,FORSTER P,ROHL A.Median-joining networks for inferring intraspecific phylogenies[J].Molecular Biology and E-volution,1999,16(1):37-48.
[24]刘艳华,张明海.基于线粒体Cyt b基因的西藏马鹿种群遗传多样性研究[J].生态学报,2011,31(7):1976-1981.
[25]刘鑫鑫.基于分子粪便学的穆棱地区同域分布梅花鹿(Cervus nippon)和马鹿(Cervus elaphus)遗传多样性比较研究[D].哈尔滨:东北林业大学,2017.
[26]QUINN T W,WILSON A C.Sequence evolution in and around the mitochondrial control region in birds[J].Journal of Molecular Evolution,1993,37(4):417-425.
[27]李楠楠,刘振生,王正寰,等.基于细胞色素b基因的中国岩羊不同地理种群遗传差异分析[J].生态学报,2012,32(8):2381-2389.