基于粪便DNA的东北马鹿遗传多样性

东北马鹿(Cervus canadensis xanthopygus)为国家二级重点保护野生动物,近些年其种群数量急剧下降、分布区不断退缩、种群基因交流受阻,很多地区更是难觅其踪迹。亟需对其种群的遗传变化,特别是遗传多样性和近交衰退等种群遗传信息开展进一步评价,增强保护与管理的针对性。本研究在大、小兴安岭和长白山脉的6个重点研究区域,共收集409份疑似马鹿粪便样本。首先基于mtDNA Cyt b基因测序技术进行物种鉴定,并对鉴定为马鹿的阳性样本利用微卫星技术进行个体识别。结果共识别出172只东北马鹿个体;Cyt b基因序列共检测出14个变异位点和11个单倍型,单倍型多样性为0.849 (0.105～0.732),核苷酸多样性为0.678%(0.099%～0.775%)。10个微卫星位点检测出种群平均等位基因数为5.7 (5.2～7.2),有效等位基因数为3.3 (2.5～4.1),观测杂合度为0.687 (0.644～0.725),期望杂合度为0.619 (0.564～0.689),近交系数为-0.113 (-0.160～-0.037)。结果表明,东北马鹿种群遗传多样性处于中等水平,其...     

基于粪便DNA的马鹿种群数量和性比

为分析黑龙江省完达山林区马鹿种群生存状态,制定科学有效地保护措施,从分子水平研究了种群数量和性比。2006、2007两年冬季跟踪马鹿足迹链,于五泡林场共搜集210份粪便,以成功提取DNA的167份作为分析样本。通过多态性较高的7个微卫星位点进行了基因分型分析,显示167份粪便DNA分属66只个体。基于非损伤性标志重捕法,统计出林场内马鹿数量2a平均47(39—60)只,密度0.302(0.251—0.386)只/km2,与2002年大样方法调查结果相比有减无增。SRY基因性别鉴定显示,种群雌雄性比1.00∶2.00(22♀,44♂),存在较多雄性个体,分析认为偷猎是导致性比失衡的最主要原因。数量的持续下降和性比失衡提示完达山林区马鹿种群数量的恢复需要更好地保护工作。     

三江源和祁连山国家公园雪豹种群的遗传结构分析

掌握遗传信息对濒危物种的保护和管理具有重要意义。本研究在我国雪豹重要分布区祁连山和三江源国家公园分别采集粪便样品,利用mtDNA的cyt b基因、微卫星多态性位点进行了雪豹的物种鉴定、个体识别和种群遗传结构评估。在采集286份疑似雪豹粪便样品中,成功的对86份雪豹样品进行了扩增鉴定,利用微卫星位点进行个体识别获得41只雪豹个体,其中祁连山国家公园26只,三江源国家公园15只。通过等位基因数、有效等位基因数、观测杂合度、期望杂合度、多态信息含量等指标进行种群遗传多样性评估,认为雪豹种群遗传多样性相对较低,但祁连山国家公园雪豹种群遗传多样性相对较高。STRUCTURE进行群体遗传结构分析表明,4个种群可以划分为3个遗传类群,祁连山国家公园的种群（YCW和QLS）与三江源国家种群(DC和SJ)的遗传差异,可能与种群间的地理隔离存在明显的相似性。     

利用微卫星研究普洱亚洲象种群的遗传多样性

本文采用非损伤性取样法获得普洱地区亚洲象粪便样品113份,试剂盒法提取粪便基因组总DNA,利用9对微卫星引物对DNA进行特异性扩增,CERVUS 3.0软件基因分型得到49个独特的基因型。利用GenAlEx v6.5与Arlequin v3.5进行位点遗传信息检测和种群遗传多样性分析,结果表明:研究采用的所有微卫星位点均未偏离哈迪温伯格平衡,位点的平均等位基因数为3.111,平均香农信息指数为0.804,平均期望杂合度为0.468,平均观测杂合度为0.476。比较相同位点上不同地区亚洲象的遗传杂合度,表明普洱地区亚洲象的种群遗传多样性较低。     

尚勇保护区亚洲象种群数量评估和遗传多样性分析

亚洲象(Elephas maximus)是我国国家一级保护动物,准确地评估其种群大小和遗传多样性对该物种的保护具有十分重要的意义。本文采用非损伤性取样方法,并结合野外观察数据对尚勇保护区当前亚洲象种群数量进行了评估,同时对该种群的遗传多样性进行了分析。采用7对微卫星引物对185份粪便样品进行扩增,通过基因分型,得到59个独特的基因型。文章采用两种方法评估种群数量,即聚集曲线法和分子标记重捕法,估计尚勇保护区的亚洲象种群数量为76±8头(95%置信区间内为67–99头)。遗传多样性方面,平均等位基因数为3.86,平均观察杂合度为0.52,平均期望杂合度为0.42,平均多态信息含量为0.34,表明尚勇保护区亚洲象种群遗传多样性水平为中等偏低,与勐养种群遗传多样性水平相似。     

勐养保护区亚洲象微卫星位点筛选及种群遗传多样性分析

本文以亚洲象肌肉样品提出的DNA为模板,从非洲象31个微卫星位点和5个已知亚洲象微卫星位点中筛选勐养亚洲象的微卫星位点,进而对在西双版纳勐养保护区3年采集到的191 份亚洲象粪便样品中提出的DNA进行特异性PCR扩增、基因分型、检测位点信息和遗传多样性分析.结果表明:36个位点中有14个位点能在亚洲象肌肉样品提出的DNA中成功扩增,且经测序证实为微卫星位点.其中9个多态位点能在185份粪便样品DNA中稳定扩增.勐养种群中,3个位点可能偏离Hardy Weubberg平衡,至少8个位点间无明显连锁存在,平均等位基因数3.78±1.72,平均期望杂和度0.32 ± 0.06,平均观察杂和度0.36±0.02,平均多态信息含量0.28 ,说明这9个位点适用于勐养亚洲象的遗传学研究.根据微卫星位点的杂合度和等位基因频率,相比于其他亚洲象种群,勐养亚洲象种群遗传多样性较低且等位基因频率具有特异性.     

内蒙古赛罕乌拉自然保护区东北马鹿种群遗传多样性与性别结构分析

东北马鹿（Cervus canadensis）种群面临着地理隔绝和生境破碎化等问题,对其种群遗传多样性和性别结构的研究,有助于了解其隔离种群的生存现状,为保护与管理工作提供科学依据。本研究利用8对微卫星分子标记,对内蒙古赛罕乌拉国家级自然保护区的456份马鹿粪便样品进行遗传多样性分析。结果识别出2015年冬季56只个体,2016年秋季41只个体;微卫星位点平均期望杂合度为0.650 1,平均多态信息含量为0.603 5,表明该地区马鹿种群的遗传多样性处于中等偏上水平。种群遗传分化系数为-0.053 99、近交系数为0.086 67、基因流为2.942 11,说明该马鹿种群基因交流普遍,近交水平低。基于SRY基因的方法进行性别鉴定,种群雌雄性比在冬季为1.8：1,秋季为0.71：1。本研究显示,该区域马鹿种群目前遗传多样性较为丰富,但现有种群集中分布于保护区内,处于相对隔离状态,长期发展则存在种群内部近交的风险。因此,加强对种群遗传结构和生存状态的监测,并促进与附近区域马鹿个体的交流,将有助于保持区域性东北马鹿种群长期繁盛。     

可可西里自然保护区藏羚羊的微卫星多态性研究

藏羚羊是我国特有的珍稀濒危动物,对其开展遗传多样性的研究具有非常重要的科学价值。为了获取足够的遗传信息并进一步研究藏羚羊在核基因水平上的遗传多样性,对来自可可西里地区的75个藏羚羊干皮张样本进行了微卫星遗传多样性研究。研究从来自牛和绵羊的25个微卫星基因座中筛选到9个具有高度多态性的微卫星基因座(MCM38,MNS64,IOBT395,MCMAI,TGLA68,BM1329,BMS1341,BM3501和MB066)。用非变性聚丙烯凝胶电泳检测微卫星的PCR扩增产物,计算了这9个微卫星基因座的等位基因频率、多态信息含量、基因杂合度等指标并估算了种群数量。结果在75只藏羚羊中共检测到85个等位基因,9个微卫星基因座的等位基因数为7~12个,平均每个基因座检测到9.4个等位基因,有效等位基因数为处于4.676~9.169之间,平均为6.519;基因频率分布在0.007~0.313之间,多态信息含量在0.753~0.881之间,平均为0.818;观察杂合度为0.791~0.897,平均为0.844,期望杂合度为0.786~0.891之间,平均为0.838±0.0132,各基因座观察杂合度与期望杂合度比较接近。固定指数为-0.269~-0.097,平均为-0.163。Shannon’s指数为1.660~2.315,平均为1.990。种群数量的估算结果显示这75个体均来自同一种群。结果表明该种群在核基因水平仍具有丰富的遗传多样性。     

中国圈养林麝微卫星DNA多样性研究

本文利用13对微卫星标记,对我国3个核心种源地（巴山、秦岭、川西高原）圈养林麝种群进行遗传多样性和遗传结构分析。在167份样品中共检测到142个等位基因（Na）,每个位点等位基因数介于7~16,均值为10.92,平均有效等位基因数（Ne）为6.3730,期望杂合度（He）和观测杂合度（Ho）均值分别为0.8302和0.3897。这些圈养林麝种群遗传多样性水平较高,但较低的观测杂合度表明圈养群体存在近交现象。两两群体间的Fst 值和AMOVA分析结果均表明种群之间分化程度不明显。群体遗传结构分析显示,全部样本聚为3个遗传簇（最佳K值=3）,其主体与3个地理来源相符,但种群间存在基因渗透现象。本研究中的秦岭种群遗传变异最为丰富,可以作为种质改良的基因池。     

喜马拉雅旱獭种群微卫星变异及遗传多样性

为了解不同地理种群喜马拉雅旱獭(Marmota himalayana)的种群数量变化并探讨其内在的遗传学机制,通过构建部分基因组文库的方法筛选出8个高变异微卫星位点,根据微卫星位点的测序结果设计相应引物,PCR扩增检测了青藏高原4个地理种群(德令哈、乌兰、沱沱河、安多)喜马拉雅旱獭的遗传多态性及其种群结构.研究结果显示:8个位点在喜马拉雅旱獭种群中均为高度多态,观察等位基因数、有效等位基因数、多态信息含量分别为4.75、3.033 2、0.610 2;喜马拉雅旱獭种群总的期望杂合度和观察杂合度分别为0.670、0.699;3个喜马拉雅旱獭种群显著(P＜0.05)或极显著(P＜0.01)偏离H-W平衡状态,且这些偏离平衡的位点均由杂合过度导致(FIS＜0);喜马拉雅旱獭种群的部分位点已经偏离了突变-漂移平衡.结论:筛选出的8个微卫星位点适合于喜马拉雅旱獭种群遗传多样性的研究,所研究的喜马拉雅旱獭种群有较高的遗传多样性,安多地理种群在近期可能经历过瓶颈效应,种群数量曾经下降.     

Microsatellite analysis of North American wapiti (Cervus elaphus) populations

Eleven populations of wapiti (Cervus elaphus) were analysed for genetic diversity using 12 microsatellite loci. Samples were taken from Vancouver Island, British Columbia; Burwash and French River herds in Ontario; Ya Ha Tinda Ranch, Alberta; and Banff, Elk Island, Jasper, Kootenay, Riding Mountain, Yellowstone and Yoho National Parks. Overall, wapiti populations have on average three to four alleles per locus and an average expected heterozygosity that ranged from 25.75 to 52.85%. The greatest genetic distances were observed between the Vancouver population and all other populations. Using the assignment test, Roosevelt wapiti (C. e. roosevelti Merriam 1897) assigned only to the Vancouver Island population. The distance and assignment values suggest a divergence of the Roosevelt wapiti from other populations and support the subspecific status for the Vancouver Island population. No evidence was found for the existence of unique Eastern wapiti (C. e. canadensis Erxleben 1777) in the Burwash or French River herds in Ontario. The overlapping distribution of genotypes from indigenous populations from Riding Mountain, Elk Island and Yellowstone National Parks suggests that wapiti were once a continuous population before settlers decimated their numbers. The lack of differentiation between these populations raises questions about the status of Manitoban (C. e.manitobensis Millais 1915) and Rocky Mountain (C. e.nelsoni Bailey 1935) subspecies.     

黑龙江省老爷岭南部穆棱林区马鹿种群分布数量及生境适宜性评价

开展野生动物种群分布数量和生境适宜性评价研究是制定物种科学保护决策的基础和关键。以东北马鹿(Cervus canadensis xanthopygus)为研究对象,于2015—2020年在黑龙江省老爷岭南部穆棱林区,采用大样方调查方法收集马鹿在雪地留下的足迹信息分析马鹿种群数量;通过相机监测、足迹链跟踪和大样方调查3种方法综合收集马鹿活动点信息,利用最大熵(MaxEnt)建模分析马鹿生境适宜性。马鹿种群分布数量研究结果表明,穆棱林区马鹿种群密度为(0.0645±0.009)只/km~2,种群数量为47—61只,主要分布在研究地区南部的和平、龙爪沟和共和林场。生境分析结果表明：人为干扰因子中,居民区对MaxEnt模型的贡献率为44%,马鹿主要在距居民区距离约5 km和10—15 km的区域活动,在偏僻的林间小道和乡村道路马鹿生境适宜性较高,其对模型贡献率分别为16.8%和10.2%;植被因子中,在距常绿针叶林和针阔混交林距离4 km范围内,随距离增加马鹿生境适宜性逐渐降低。生境适宜性分析结果表明,研究地区马鹿适宜生境和次适宜生境面积为87.09 km~2,仅占研究区域的10.39%,主要...     

Characterization of the erythrocyte superoxide dismutase allozymes in the deer Cervus elaphus

The cDNA sequences for Cu,Zn superoxide dismutase from two Cervus elaphus subspecies, North American wapiti and European red deer, were determined. The derived amino acid sequences showed two differences: residue 8 was Leu in wapiti and Met in red deer and residue 25 was His in wapiti and Asn in red deer. The extra positive charge at position 25 in the wapiti isoform accounted for its greater mobility towards the cathode during non-denaturing electrophoresis, a procedure widely used in the genetic analysis of deer. There was no difference in specific activity between the two Cu,Zn superoxide dismutase isoforms, but the wapiti isoform was slightly more susceptible to heat denaturation.     

A phylogenetic comparison of red deer and wapiti using mitochondrial DNA

A phylogeny was constructed for red deer/wapiti (Cervus elaphus) subspecies using sequence data from the control region of mitochondrial DNA (mtDNA). The tree was rooted using Cervus nippon (sika deer), Cervus albirostris (Thorold's white-lipped deer), and several Odocoileinae species. A division between the mtDNA haplotypes of red deer (European) and wapiti (Asian/North American) corresponds to subspecies found on opposite sides of the Himalayan Mountains and Gobi, which suggests wapiti should be reconsidered for the status of C. canadensis. Using parsimony and distance analysis, red deer and wapiti are derived from a single recent common ancestor, which is consistent with current taxonomy that recognizes the subspecies of Cervus elaphus as monophyletic group. However, maximum-likelihood analysis using weighted transitional substitutions caused red deer to form a sister group to sika deer (Cervus nippon) and wapiti. A phenetic comparison revealed wapiti also share more nucleotide similarities with sika deer, although approximately 5% sequence divergence separates wapiti, sika, and red deer. Phylogenetic evidence from the cytochrome b sequences corroborated observations from the control region. Observations from this study suggest that the species status of wapiti should be reinstated.     

Reproductive performance of pubertal red deer (Cervus elaphus) hinds: effects of genetic introgression of wapiti subspecies on pregnancy rates at 18 months of age

Low reproductive productivity of young red deer (Cervus elaphus) hinds on New Zealand deer farms appears to reflect high incidences of puberty failure at 16 months of age. This is despite the general attainment of average liveweights 15–25 kg in excess of the accepted minimum threshold for puberty in subspecies of western European origin (scoticus, elaphus and hippelaphus) that form the basis of the national herd. The present study tests the hypotheses that introgression of the larger North American wapiti subspecies (nelsoni, manitobensis and roosevelti) into breeding herds (1) can be assessed from morphological features of individuals, (2) that there is a relationship between the level of wapiti parentage and non-pregnancy rate at 18 months of age (a proxy for puberty failure) and (3) that minimum liveweight thresholds for puberty increase with increasing levels of wapiti parentage.

A total of 4329 18-month-old hinds across four “red” deer farms in southern New Zealand were scanned for pregnancy status. Each hind was assigned a wapiti score (WS) as a subjective assessment of the obviousness of wapiti features. Various body measurements were additionally recorded for each hind. A hair sample was collected for DNA analysis (14 markers) to objectively assign subspecies pedigree (i.e. “Elkmeter”) on a subset of 1258 individuals. A total of 506 (11.7%) hinds were not pregnant at 18 months of age with rates varying between 4.1 and 37.3% between farms and years. Mean WS differed significantly between farms and reflected the genetic management policy of each farm. WS was positively correlated to Elkmeter for each farm/year (<0.05) although regression slopes varied significantly. WS was able to be adjusted for these differences to assign a corrected WS (CWS) for all 4329 individuals that estimated the proportion wapiti parentage. Discriminant analysis of morphological variables relative to Elkmeter supported the first hypothesis and showed that shoulder height and body length were good indicators of the degree of wapiti parentage within individuals. This enabled the development of an objective estimate of wapiti parentage (EWP). The actual level of such parentage within herds ranged from <5 to >55%. There was a significant negative association between wapiti parentage and pregnancy, which was strongly influenced by liveweight, supporting the second and third hypotheses. This was manifest as marked displacement of pregnancy probability curves in relation to liveweight between genotype groups, particularly for those groups with >20% wapiti parentage. For example, predicted threshold liveweights required to achieve a 90% pregnancy rate for EWP values that represent 0, 10, 20, 30, 40 and 50% wapiti parentage were 81, 81, 85, 106, 127 and 137 kg, respectively. Within the study herds, the majority of hinds of 0–20% wapiti parentage exceeded the predicted 90% threshold liveweights for their genotype cohort. However, hinds with higher levels of wapiti parentage generally fell below the predicted threshold for their genotype group. The data strongly suggest that under liveweight performance levels measured for red deer, hinds with >20% wapiti parentage are at high risk of puberty failure.     

The gestation length of wapiti (Cervus elaphus) revisited

As an ancillary activity to an artificial insemination program in farmed wapiti, the length of gestation of 28 wapiti hinds that delivered single calves of established parentage was calculated. Estrus was synchronized in 47 wapiti using progesterone impregnated devices (controlled internal drug release, CIDR) and an injection of PMSG. All hinds were artificially inseminated between 60 and 63h after CIDR removal. Pregnancy was determined between 45 and 65 days by ultrasound. A verifiable figure for gestation length was obtained based both upon timed-artificial insemination, date of parturition, and confirmation of sire identity through microsatellite DNA technology. The calculated gestational length of 247 +/- 5 days was significantly (P < 0.0001) shorter than the generally quoted figure of 255 +/- 7 days.     

30个祖先信息位点的筛选及应用

摘要：目的 筛选一组祖先信息SNPs位点（AIMs,Ancestry Informative Markers）,构建复合检测体系,用于东亚、欧洲和非洲人群遗传成分描述及个体种族来源推断。方法 以HapMap数据库9个人群的658份样本的分型数据为基础,从30个表型相关基因总共282个SNPs位点中筛选出30个AIMs位点,基于微测序-通用芯片技术构建复合检测体系,并建立人群等位基因频率数据库。使用这组位点分析HapMap数据库中658份人群样本,初步验证位点的区分效能;然后,使用研究构建的体系检验收集的5个人群194份无关个体的DNA样本。最后,通过Structure软件分析获取人群的成分构成以及个体的遗传成分,对个体样本进行种族来源推断。 结果 筛选的30个AIMs位点符合哈迪温伯格平衡（p>0.01）,位点之间没有连锁（r2<0.1）, 658份HapMap数据库样本和194份实验样本的祖先成分分析结果与已知结果完全一致。 结论 本文筛选并建立的30个AIMs位点复合检测体系,能够有效实现东亚、欧洲、非洲人群及混合人群的成分构成和个体遗传成分的分析,有效控制遗传连锁分析中由于人群分层现象带来的误差,也可以用于法医DNA检验中个体祖先来源推断。     

Intraspecific DNA contamination distorts subtle population structure in a marine fish: Decontamination of herring samples before restriction‐site associated sequencing and its effects on population genetic statistics

Wild specimens are often collected in challenging field conditions, where samples may be contaminated with the DNA of conspecific individuals. This contamination can result in false genotype calls, which are difficult to detect, but may also cause inaccurate estimates of heterozygosity, allele frequencies and genetic differentiation. Marine broadcast spawners are especially problematic, because population genetic differentiation is low and samples are often collected in bulk and sometimes from active spawning aggregations. Here, we used contaminated and clean Pacific herring (Clupea pallasi) samples to test (a) the efficacy of bleach decontamination, (b) the effect of decontamination on RAD genotypes and (c) the consequences of contaminated samples on population genetic analyses. We collected fin tissue samples from actively spawning (and thus contaminated) wild herring and nonspawning (uncontaminated) herring. Samples were soaked for 10 min in bleach or left untreated, and extracted DNA was used to prepare DNA libraries using a restriction site‐associated DNA (RAD) approach. Our results demonstrate that intraspecific DNA contamination affects patterns of individual and population variability, causes an excess of heterozygotes and biases estimates of population structure. Bleach decontamination was effective at removing intraspecific DNA contamination and compatible with RAD sequencing, producing high‐quality sequences, reproducible genotypes and low levels of missing data. Although sperm contamination may be specific to broadcast spawners, intraspecific contamination of samples may be common and difficult to detect from high‐throughput sequencing data and can impact downstream analyses.