基于粪便DNA的小麂亲权鉴定和婚配制研究

2014年4月至2015年1月,在古田山国家级自然保护区内共收集634份粪便样本,2份肌肉样本。通过严格筛选,最终获得390份可用于PCR扩增的样本。用多态性较高的8个微卫星位点进行基因型分型,共识别出177个小麂个体。SRY基因性别鉴定显示研究样本中雄性84只,雌性93只。所使用的8个微卫星位点在177个样本中,平均等位基因数(A)为11,平均观测杂合度(Ho)在0.960—1.000之间,平均值为0.9685,平均期望杂合度(He)在0.799—0.887之间,平均值为0.8429,多态信息含量(PIC)在0.766—0.872之间,平均多态信息含量为0.8214,基因杂合度水平较高,为遗传多样性丰富的种群。采用Cervus3.0进行亲权分析,当置信度为95%和80%时,8个微卫星位点的鉴定率均达到100%。共鉴定出父-母-子24对,母-子23对,父-子19对,涉及到104只个体。根据亲缘关系分析小麂的婚配制,结果发现小麂的婚配制属于1雄多雌,但并不是目前所知的亚型,而可能是一种被称作"检查策略"的一雄多雌制。     

基于粪便DNA 的九龙山黑麂种群的遗传多样性

近年来,由于人类活动的干扰使黑麂的栖息地明显减少并呈斑块状分布。因此,深入了解该物种各地理分布种群的遗传现状是制定保护及管理策略的重要科学依据。本研究运用非损伤取样技术在浙江省九龙山国家级自然保护区的黑麂分布区共采集61 份粪便样品、2 份肌肉样品和2 份皮张样品,采用8 个特异微卫星位点对61 份粪便样品中提取的DNA 进行检测,肌肉和皮张样品DNA 的检测结果作为对照,分析了九龙山国家级自然保护区黑麂种群的遗传多样性。经多次重复提取和PCR 扩增后,发现61 份粪便样本中有38 份可被稳定扩增,且不同样本间的条带大小有较大差异,因此我们认为38 份粪便样本来源于38 只不同的黑麂个体。8 个微卫星位点共检测到50 个等位基因,平均等位基因数(A)为6 (5 ～ 8);平均有效等位基因数(Ne)为5. 297 (4.031 ～6.353);平均期望杂合度(He) 为0.817 (0.763 ～ 0.855);平均多态信息含量(PIC ) 为0. 775 (0.709 ～0.822)。8 个位点的平均个体识别率(DP)为0.931,累积个体识别率(CDP)为0.999 9。有1 个微卫星位点(BM1706)极显著地偏离了Hardy-Weinberg 平衡(P < 0.01)。以上结果显示该分布区黑麂种群具有较高的遗传多样性。     

Identification of black muntjac (Muntiacus crinifrons) in Tibet, China, by cytochrome b analysis

Full cytochrome b gene sequence of mtDNA was used to identify and analyze four skin samples collected from Tibet, China in this research. By searching for highly similar sequences (megablast) on NCBI, we found all four samples have the highest similarities with the published sequence: AY239042 of the black muntjac (Muntiacus crinifrons). By comparing our sequences to those available on GenBank, all four samples were identified as the black muntjac (Muntiacus crinifrons) by high sequence similarity. We therefore record two new localities for the black muntjac and it is a new distribution in Tibet and hope this study will not only promote more advanced researches on the evolution and phylogeny about this species, but also enhance the conservation work for this species and local biodiversity.     

Isolation and characterization of microsatellite markers in black muntjac (Muntiacus crinifrons)

The black muntjac (Muntiacus crinifrons) is a vulnerable species found in the mountains of eastern China, about which little is known. Here we develop 11 polymorphic microsatellite loci from a (CA)(n) -enrichment library for the animal. In the analyses of 25 individuals sampled, unbiased expected heterozygosity levels varied from 0.686 to 0.876 and the number of alleles per locus varied from five to nine. Results that 11 microsatellite loci were highly polymorphic indicated that these markers are sufficiently powerful to address such questions as parentage, mating system and population genetic structure of M. crinifrons.     

Microsatellite Analysis of Genetic Variation and Population Subdivision for the Black Muntjac, Muntiacus crinifrons

The black muntjac (Muntiacus crinifrons) is a rare deer found only in a restricted region in east China. Recent studies of mitochondrial DNA diversity have shown a markedly low level of nucleotide diversity for the species, and the Suichang population was genetically differentiated from the two other populations, in Huangshan and Tianmushan mountains. In this study, we extended the analysis of genetic diversity and population subdivision for the black muntjac using data from 11 highly polymorphic nuclear DNA microsatellite loci. Contrary to the results based on mtDNA data, the microsatellite loci revealed that the black muntjac retained a rather high nuclear genetic diversity (overall average H (E) = 0.78). Nevertheless, both types of markers supported the idea that the extant black muntjac population is genetically disrupted (overall phi (ST) = 0.16 for mtDNA and overall F (ST) = 0.053 for microsatellite, both P < 0.001). The correlation between genetic differentiation and geographic distance was not significant (Mantel test; P > 0.05), implying that the patterns of genetic differentiation observed in this study might result from recent habitat fragmentation or loss. Based on the results from the mtDNA and nuclear DNA data sets, two management units were defined for the species, Huangshan/Tianmushan and Suichang. We also recommend that a new captive population be established with individuals from the Suichang region as a founder source.     

合肥野生动物园黑麂的繁殖资料

合肥野生动物园自1978年开始进行黑麂(Muntiacus crinifrons)的饲养和繁殖,1989年第一胎圈养条件下繁殖的黑麂出生,到2001年底累计繁殖黑麂51头,繁殖种群正处迅速增长期。13年的繁殖资料统计结果表明,圈养条件下育龄母麂平均每12个月产一胎(多数在11—13个月,少数仅6—9个月),孕期240d左右,哺乳期2—3个月,少数母麂可产后发情,但极少有两年三次产仔现象。值得注意的是圈养条件下黑麂多在9—11月交配,4—7月产仔(80．39％)。圈养条件下黑麂幼年的死亡率较低(7．84％),成年黑麂多死于消化道及呼吸道感染等疾病(56％)。     

Comparative sequence analyses reveal sites of ancestral chromosomal fusions in the Indian muntjac genome

Background

Indian muntjac (Muntiacus muntjak vaginalis) has an extreme mammalian karyotype, with only six and seven chromosomes in the female and male, respectively. Chinese muntjac (Muntiacus reevesi) has a more typical mammalian karyotype, with 46 chromosomes in both sexes. Despite this disparity, the two muntjac species are morphologically similar and can even interbreed to produce viable (albeit sterile) offspring. Previous studies have suggested that a series of telocentric chromosome fusion events involving telomeric and/or satellite repeats led to the extant Indian muntjac karyotype.

Results

We used a comparative mapping and sequencing approach to characterize the sites of ancestral chromosomal fusions in the Indian muntjac genome. Specifically, we screened an Indian muntjac bacterial artificial-chromosome library with a telomere repeat-specific probe. Isolated clones found by fluorescence in situ hybridization to map to interstitial regions on Indian muntjac chromosomes were further characterized, with a subset then subjected to shotgun sequencing. Subsequently, we isolated and sequenced overlapping clones extending from the ends of some of these initial clones; we also generated orthologous sequence from isolated Chinese muntjac clones. The generated Indian muntjac sequence has been analyzed for the juxtaposition of telomeric and satellite repeats and for synteny relationships relative to other mammalian genomes, including the Chinese muntjac.

Conclusions

The generated sequence data and comparative analyses provide a detailed genomic context for seven ancestral chromosome fusion sites in the Indian muntjac genome, which further supports the telocentric fusion model for the events leading to the unusual karyotypic differences among muntjac species.     

非损伤采样提取DNA技术在猫科动物调查中的应用

<正>猫科动物广泛分布于世界各地(Johnson et al.,2006),全球现存37种猫科动物,其中25种已被国际自然保护联盟(IUCN)列为濒危和易危,86%以上的种群数量处于下降或未知状态(IUCN Red List,2011)。我国有13种猫科动物,占世界种类的35%(王应祥,2003),其中属于国家一级保护的有4种,二级保护的有8种,然而,对这些珍稀物种的生态学研究并不充分(高耀亭,     

Camera trap reveals the co‐occurrence patterns of two sympatric muntjac species in southern Anhui Province,China: No spatial segregation

The competitive relationship and coexistence pattern among close related species have long been one of the hot issues in ecological research. Interspecies interactions can exert important influences on the local distribution of rare species. Black muntjac Muntiacus crinifrons is an endemic species to eastern China, currently restricted to limited regions. In contrast, Chinese muntjac Muntiacus reevesi is the most common and widespread deer in southern China. Both species co‐occur in southern Anhui and western Zhejiang Province. Little is known about the interaction of these two sympatric‐related species. In this study, to investigate the site use determinants and co‐occurrence pattern of the two sympatric muntjac species, we conducted a camera trap survey across about 250 km² in mountainous area of southern Anhui Province, China. We adopted a multistep approach to incorporate habitat preferences while modeling occupancy and detection. We found that the two species did not separate along elevation gradient (range from 400 m to 1,400 m) as described in previous studies. Results of single‐species occupancy models indicated that elevation had positive effects on the site use of both species, while slope had an opposite influence on their site use. Positive effects of elevation on the site use implied that both species try to avoid human interference at low elevations. Significant negative effect of slope on the site use of black muntjac suggested that the species prefer habitat with gentle slope and avoided steep. Co‐occurrence models and species interaction factors provided evidence that the two muntjac species had an independent occupancy (ψ ^{BM CM} = ψ ^{BM cm}, SIF = 1) and exhibited a positive species interaction in detection probability (p ^BM < r ^{BM CM}). Combined with the results of previous studies, we suggested that it was fine differentiation in microhabitats and food resources utilization rather spatial or temporal segregation that allowed the two species co‐occurrence. The site use determinants revealed in our study would be useful for the habitat conservation and restoration for the rare black muntjac, and the co‐occurrence pattern of the two sympatric muntjac species could provide useful information for deep understanding of the coexistence mechanism among forest‐dwelling ungulates.     

Gene Expression Analysis with No Sequence Data: Study on Reeves’s Muntjac (Muntiacus reevesi)

Despite scientific progress, the gene sequences for many species not commonly used in research have not yet been analyzed. This makes it difficult to carry out molecular studies on such animals, as the sequence of genes is the basic information used in many techniques. In this study, we attempt to design primers for a real-time PCR analysis, basing on a comparative analysis of selected gene sequences of species related to Reeves’s muntjac (Muntiacus reevesi) and by identifying highly conservative regions. Results of PCR products sequencing and their alignment with the GenBank collection show that all selected primers gave products highly similar (> 90%) to the intended target (among compared species), which led us to the conclusion that our primers may be used for further analyses of gene expression.     

黑麂MHC - DQA2 基因多态性

利用牛特异性扩增DQA2 第2 外显子的嵌套引物,对黑麂基因组DNA 进行PCR 扩增和克隆测序,基于该
序列设计出黑麂DQA2 基因第2 外显子特异性引物。利用该引物,通过PCR - SSCP 以及克隆测序技术,从40 个
黑麂样品中获得4 个不同的DQA2 等位基因。没有一个个体同时具有2 个以上的等位基因,所有序列均不含插入
或缺失突变,不含终止密码子,因此,本研究所扩增的DQA2 基因可能是表达的单基因座位。抗原结合区(Peptide
binding region,PBR)非同义替换率(dn)显著大于同义替换率(ds) (P <0.05),暗示该座位曾经历过明
显的正选择作用;进一步利用CODEML 程序中的相关模型以及贝叶斯法检测出4 个受选择作用的氨基酸位点
(α11、α58、α62、α66),这4 个位点均位于PBR 区。基于NJ 法构建的部分偶蹄类DQA 外显子2 系统发生关系
显示,黑麂4 个DQA2 等位基因与牛、羊以及梅花鹿的DQA2 等位基因构成独立的进化枝,在该进化枝内,黑麂
DQA2 等位基因优先与牛DQA2 等位基因聚类,暗示黑麂DQA2 基因在进化过程中存在跨物种进化现象。上述结
果表明平衡选择是维持黑麂DQA2 基因多态性的主要机制。然而,本研究从40 个样品中仅检测出2 个杂合子,
黑麂DQA2 等位基因之间的频率存在显著差异,推测可能是所检测的样品来源于不同种群,由于华伦德效应
(The Whalund effect)导致杂合度降低,也不排除本文所设计的引物在PCR 扩增过程中存在无效等位基因。     

Molecular evidence for the occurrence of the leaf deer <Emphasis Type="Italic">Muntiacus putaoensis</Emphasis> in Arunachal Pradesh,north-east India

The discovery of the leaf deer Muntiacus putaoensis in northern Myanmar has added to the growing list of large mammals recently discovered in remote, unexplored parts of south and south-east Asia. Its subsequent discovery in eastern Arunachal Pradesh, India, based on morphometric analyses of two skulls collected from local hunters, doubled the size of its known east-west range, which is significant for a newly-discovered and poorly understood species. However, ambiguity remained regarding several other partial skulls and dried skin samples collected during subsequent surveys. The sympatric occurrence of the Indian muntjac Muntiacus muntjak further complicates species identification based primarily on morphometry. In this paper, we develop molecular genetic analyses that can unambiguously identify muntjac species. Further, we test and apply our methods to unknown skin samples to confirm the occurrence of the leaf deer in Arunachal Pradesh. Finally, we use our samples and genetic data from three mitochondrial markers to establish phylogenetic affinities between these samples and other extant members of the Muntiacus genus. Our approach, which combines the use of specific primers and phylogenetic analyses, is generally applicable towards the detection of cryptic biodiversity in unexplored and species-rich areas like north-east India.     

元江鲤种群遗传多样性

选择12对微卫星标记检测了于2011年采集自元江(红河上游中国江段)5个样点192尾鲤的群体遗传多样性.共检测到201个等位基因,每个位点等位基因2-27个.各群体各位点平均等位基因(NA)12.25-14.67个,平均有效等位基因(NE)8.28-9.73个,平均观察杂合度(Ho)o.7765-0.8037,平均期望杂合度(HE)0.7761-0.8080,平均多态信息含量(PIC)0.7534-0.7843.元江鲤种群192个个体各位点NA、NE、Ho、HE、PIC分别为16.50、11.26、0.7927、0.8049、0.7966,种群遗传多样性水平高.元江鲤群体之间遗传分化小,可作为一个种群管理单元进行管理.增殖放流要防止遗传多样性丧失.     

Population structure and gene flow among wild populations of the black muntjac (Muntiacus crinifrons) based on mitochondrial DNA control region sequences

The black muntjac (Muntiacus crinifrons) is a rare species endemic to China and its current distribution is confined to partial mountain ranges in eastern China. To assess the population structure and gene flow among the extant populations, we sequenced a portion of the mitochondrial control region (424 bp) for 47 samples collected from its current three large populations (Huangshan, Tiammushan and Suichang). A total of 18 unique haplotypes were defined based on 22 polymorphic sites. Of these haplotypes, 15 ones were population-specific and only one haplotype was shared among the three populations. Significant genetic differentiation was detected between Suichang and Huangshan populations (?(ST)=0.1677, P<0.001) or between Suichang and Tianmushan populations (?(ST)=0.2002, P<0.001), indicating that the Suichang population may be spatially structured from other two populations along matriline. The Mantel test revealed that this significant differentiation was not driven by geographic distance (P=0.14), implying that genetic divergence of Suichang population might result from recent human disturbances. Phylogenetic analyses suggested the mitochondrial control region haplotypes were split into two well divergent clades (Clade I and Clade II). Interestingly, the two distinct haplotype clades were found to coexist in Suichang area. The nested clade analysis revealed a significant phylogeographic structure among the black muntjac populations (total cladogram: chi2=18.68; P<0.001), which was inferred to result from past fragmentation followed by range expansion. The population expansion was supported by the analysis of mismatch distribution and the tests of neutrality. Therefore, we suggest that the coexistence of distinct haplotypes in Suichang population was induced by historical population expansion after fragmentation and that the current genetic differentiation should be attributed to the reduction of female-mediated gene flow due to recent habitat fragmentation and subsequent loss.     

圈养条件下黑麂雄性标记行为与雌性化学信息存留位置的关系

化学物质被认为是哺乳动物标记行为信息传递的主要载体,而标记在鹿科动物的竞争雌性资源中具有重要的作用,因此本文重点对雄性黑麂的化学标记与雌性化学信息存留点的关系进行了行为学研究。观察发现面腺标记(利用额腺和眶下腺)是雌雄黑麂共有的化学标记行为,而行为性排尿和刨地行为(利用蹄腺)则仅为雄性具有的标记行为;躺卧不具有化学标记作用,而排粪在化学标记上的作用则难以确定。同时分析表明,雄性黑麂更倾向于在靠近雌性化学信息存留区的区域进行化学标记,且可能会因竞争压力的增大而扩大领域范围。     

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的贺兰山岩羊亲权鉴定和婚配制研究

2017年7-8月和11-12月在贺兰山岩画风景区采集到369份岩羊新鲜粪便样本,用多态性较高的10个微卫星位点进行基因分型,共识别出282只岩羊个体。通过复合扩増和牙釉蛋白基因PCR法共鉴定出152只雄性,130只雌性。微卫星分析得到平均等位基因（Na）为10.1,平均观测杂合度（Ho）为0.7737,平均期望杂合度（He）为0.6512,平均多态信息（PIC）为0.6129。运用Cervus3.0软件进行亲权鉴定,并用Kingroup V2计算个体间的亲缘系数（r）,作为亲权鉴定的补充,共鉴定出父-母-子7对,父-子21对,母-子19对。根据亲缘关系对岩羊的婚配制进行推测分析,表明岩羊婚配制应属一雄多雌。     

High-density comparative BAC mapping in the black muntjac (Muntiacus crinifrons): molecular cytogenetic dissection of the origin of MCR 1p+4 in the X1X2Y1Y2Y3 sex chromosome system

The black muntjac (Muntiacus crinifrons, 2n = 8[female symbol]/9[male symbol]) is a critically endangered mammalian species that is confined to a narrow region of southeastern China. Male black muntjacs have an astonishing X1X2Y1Y2Y3 sex chromosome system, unparalleled in eutherian mammals, involving approximately half of the entire genome. A high-resolution comparative map between the black muntjac (M. crinifrons) and the Chinese muntjac (M. reevesi, 2n = 46) has been constructed based on the chromosomal localization of 304 clones from a genomic BAC (bacterial artificial chromosome) library of the Indian muntjac (M. muntjak vaginalis, 2n = 6[female symbol]/7[male symbol]). In addition to validating the chromosomal homologies between M. reevesi and M. crinifrons defined previously by chromosome painting, the comparative BAC map demonstrates that all tandem fusions that have occurred in the karyotypic evolution of M. crinifrons are centromere-telomere fusions. The map also allows for a more detailed reconstruction of the chromosomal rearrangements leading to this unique and complex sex chromosome system. Furthermore, we have identified 46 BAC clones that could be used to study the molecular evolution of the unique sex chromosomes of the male black muntjacs.