适用于种群遗传管理的小熊猫微卫星亲子鉴定体系的建立

由于标记缺失、生产记录不详、亲权关系不明及部分个体来源不清等历史问题,中国小熊猫圈养种群存在谱系错漏、近亲繁殖等风险。近年来,随着小熊猫种群规模不断扩大,管理者们对谱系的准确性提出了更高的需求,亲子鉴定工作也成为了研究的重点。本文采用26个微卫星标记,对国内3个小熊猫圈养种群进行了亲缘关系运算,完成了相关谱系的查错与整理。26个位点多态性与稳定性良好,联合非亲排除概率达到0.9999以上,可解决国内小熊猫圈养种群的各类亲子鉴定需求。在单亲未知或双亲未知的情况下,8或11个位点组合可实现亲子鉴定。5个位点组合可进行个体识别。在小熊猫圈养管理过程中,应用一套亲子鉴定体系对小熊猫圈养的谱系进行查漏补缺,有利于制定科学的配对计划、避免近亲繁殖,对小熊猫种群保护有着重要意义。     

圈养小熊猫遗传多样性与种群遗传结构分析

小熊猫是亚洲特有的珍稀濒危动物,目前受到栖息地减少、片断化和人类活动干扰等威胁。中国圈养小熊猫已经有60 多年历史,约55 个机构曾经饲养过小熊猫,现今圈养数量有400 多只,评估小熊猫圈养种群的遗传多样性和遗传结构对科学维持圈养种群和保存遗传种质资源意义重大。本研究利用19 个微卫星座位,对中国境内11 个小熊猫圈养种群的116 只个体进行了遗传多样性评估及遗传结构分析。结果显示11 个种群都具有较高的遗传多样性,平均基因丰富度3.505 ± 1.033 (北京)至4.026 ± 1.219 (冕宁),期望杂合度0.631 ± 0.225(黄山)至0.782 ±0.171 (温岭)。其中福州和无锡种群极显著偏离Hardy-Weinberg 平衡。整个圈养群体内各个种群遗传分化系数为0.055,呈显著分化,表明11 个种群遗传分化水平较高。Bayesian 遗传聚类分析将11 个种群聚为三个遗传簇,与野生种群的遗传聚类结果一致。结论:小熊猫圈养种群与野生种群相比,同样具有较高的遗传多样性。因此,圈养小熊猫遗传管理的重点不再是引进野生个体充实圈养种群,应制订科学的繁殖计划,避免近交,从而维持圈养种群的遗传多样性。     

基于微卫星位点的人工圈养豚鹿亲子关系鉴定

豚鹿属我国国家I级重点保护动物,目前国内野生种群已经灭绝,人工圈养数量少,已极度濒危。成都动物园是国内最大的豚鹿饲养单位,对该园内豚鹿进行亲子鉴定及遗传谱系建立是我国豚鹿拯救工程中一重要环节。本文利用7个微卫星标记对成都动物园27只豚鹿个体进行了基因分型,在母本已知情况下成功鉴定了13对父子关系,其中排除法鉴定8对,似然法鉴定5对且置信度达95%。将亲子鉴定的结果辅以动物园豚鹿圈养的历史记录,我们构建了该园豚鹿的遗传谱系图。本文的研究成果将为后续人工繁殖中亲本雌雄配对的个体选择以及种群的遗传管理提供参考。     

微卫星标记对牙鲆有丝分裂雌核发育家系的亲子鉴定

利用18个微卫星标记,对6个家系的26尾有丝分裂雌核发育牙鲆进行亲子鉴定,PCR扩增产物经8％非变性聚丙烯酰胺凝胶电泳检测,结果表明：1个座位在母本中表现为相同的基因型,视为单态座位,其他17个座位为多态;多态座位在亲子鉴定中的累计排除概率和累计个体识别概率分别为0.9985、0.9999;根据被测个体在17个微卫星座位的基因型,最后确认26尾子代的母本,其中7尾子代在某些座位表现出与其母本不完全匹配的基因型。利用微卫星标记可确定雌核发育后代的亲子关系,从而构建牙鲆雌核发育家系系谱,对牙鲆雌核发育的深入研究具有重要意义。     

微卫星标记对牙鲆有丝分裂雌核发育家系的亲子鉴定

利用18个微卫星标记,对6个家系的26尾有丝分裂雌核发育牙鲆进行亲子鉴定,PCR扩增产物经8%非变性聚丙烯酰胺凝胶电泳检测,结果表明:1个座位在母本中表现为相同的基因型,视为单态座位,其他17个座位为多态;多态座位在亲子鉴定中的累计排除概率和累计个体识别概率分别为0.9985、0.9999;根据被测个体在17个微卫星座位的基因型,最后确认26尾子代的母本,其中7尾子代在某些座位表现出与其母本不完全匹配的基因型。利用微卫星标记可确定雌核发育后代的亲子关系,从而构建牙鲆雌核发育家系系谱,对牙鲆雌核发育的深入研究具有重要意义。     

卧龙圈养大熊猫遗传多样性现状及预测,

以中国最大的大熊猫圈养种群—四川卧龙中国大熊猫保护中心的圈养种群为对象,以８个大熊猫微卫星位点为分子标记, 探讨了大熊猫圈养种群的遗传多样性, 并与邛崃野生种群及其他７个濒危物种进行比较。微卫星数据表明, 圈养种群的遗传多样性水平(A＝5.5, He ＝0.620, Ho＝0.574) 低于邛崃野生种群(A＝9.8,He＝0.779,Ho＝0.581),但高于其他7 个濒危物种的种群(He＝0.13~0.46)。在此数据的基础上对未来100个世代内圈养种群遗传多样性的变化情况做出了预测。结果表明假设种群数量比现在扩大一倍, 经历100个世代后也只会使平均等位基因数少减少0.4。因此继续增加野生个体对保持遗传多样性的意义已经不大, 建议该圈养种群的保护策略应将重点放到制定更有效的繁殖计划以避免近交上。     

基于微卫星标记的圆口铜鱼亲子鉴定技术

为快速有效地鉴别不同的圆口铜鱼家系及来源, 研究从已发表的40个微卫星标记中筛选出20个多态性较高且稳定扩增的微卫星位点, 通过对8个圆口铜鱼家系339尾个体进行微卫星基因分型检测, 建立了圆口铜鱼荧光微卫星标记与多重毛细管电泳相结合的亲子鉴定技术。遗传多样性分析结果显示, 圆口铜鱼8个家系群体的平均等位基因数(N_a)为9个, 平均多态信息含量(PIC)为0.616, 平均期望杂合度(H_e)为0.659, 平均观测杂合度(H_o)为0.691, 其中子一代群体的遗传多样性水平明显低于亲本群体。亲子鉴定分析结果显示, 当双亲基因型未知时其单亲累积排除概率(CE-1P)为0.99954473, 当单亲基因型已知时其累积排除概率(CE-2P)为0.99999825, 当双亲基因型未知时其双亲累积排除概率(CE-PP)为1.00000000, 当使用20个微卫星位点进行亲子鉴定时, 297尾子一代均能正确找到其父母本, 亲子鉴定准确率为100%。由此可见, 研究建立的圆口铜鱼亲子鉴定技术是可靠的, 能为圆口铜鱼的家系管理、种群遗传管理和增殖放流效果评估提供科学依据     

华南虎圈养种群的统计分析

对《华南虎谱系》307 只登录个体的统计表明, 其圈养的历史有46 年。在人工繁殖的38 年中产122 胎287仔。现存圈养华南虎分散在全国22 家单位, 有57 只个体, 性比M∶F = 1185 , 能成功繁殖后代的个体仅5 只(只占818 %) , 是一个衰退的种群。通过SPARKS 和GENES 软件分析发现: 从1977 年开始, 圈养种群的基因多样性便逐渐下降而近交系数却不断增加。     

微卫星分型法应用于豢养繁殖长江江豚的父权鉴定

目的:鉴定武汉白鱀豚馆及安徽铜陵淡水豚保护区两个豢养长江江豚繁殖群体中出生的6头幼豚的生物学父亲.方法:选择8对江豚物种特异性微卫星引物对两个待鉴定群体的14个DNA样品进行了荧光标记PCR扩增,将纯化后的扩增产物在ABI3130遗传分析仪上进行基因分型,并根据GeneScan Rox 500内标确定不同等位基因的大小,随后对待鉴定对象进行等位基因分析,并计算主要多态性参数.结果:所采用的8个微卫星座位在待鉴定的两个江豚群体中均表现出不同程度的多态性.在母本已知的条件下,利用其中6个微卫星座位的等位基因数据,通过排除法成功地鉴定出两个繁殖群体中出生的6头幼豚的生物学父亲.结论:本研究首次成功地将6个物种特异性微卫星标记应用于豢养长江江豚的父权鉴定,从而为该物种微卫星亲子鉴定技术体系的建立及迁地保护繁殖群体遗传谱系的构建奠定了技术基础.     

中国动物园白鹤圈养种群统计学与遗传学分析

根据《圈养白鹤中国地区谱系簿2012》提供的数据,应用种群管理软件SPARKSv1.6和PMxv1.0对中国动物园白鹤圈养种群进行统计学和遗传学分析。结果显示该种群面临着许多困难,未知性别比例过高(截止到2012年10月31日,中国动物园白鹤圈养种群数量达到92只,其中雄性24只,雌性29只,未知性别39只)、年龄结构不合理(育龄个体数量充足,但幼龄个体数量仅有14只,约占现存种群的15.22%)、基因多样性丢失快(野外来源个体数量较多,达到58只,约占总数的63.04%,但是实际参与繁殖的奠基者仅有7只;该群体中某些奠基者的后代数量较多,种群平均亲缘关系值MK=0.15,从而导致现有种群只保留了野生个体85.25%的基因多样性)、种群数量增长乏力(2006～2012年种群的平均增长率λ=0.98)。因此,为了建立一个健康的白鹤圈养种群,应尽快开展性别鉴定工作,改善饲养环境条件,减少创伤引发的死亡,同时采用各种技术(如人工授精)增加潜在奠基者的繁殖机会,从而尽快扩大种群数量,并减少后代过多个体奠基者的繁殖机会。     

Molecular paternity determination in captive bonobos and the impact of inbreeding on infant mortality

Inbreeding and the loss of genetic diversity may lower fitness and reduce the potential for a population to adapt to changing environments. In small populations, for example in captive populations or populations of endangered species, this can have considerable consequences for their survival. We investigated the effects of inbreeding on infant mortality in the world captive population of bonobos Pan paniscus . Using a combination of studbook data and high-quality pedigree data from genotyped individuals, inbreeding information was available for 142 captive-born individuals. For the determination of paternities that were unresolved in the studbook, nuclear microsatellite DNA was amplified from hair and blood samples using the Great Ape Kit and PowerPlex^® 16 System. In total, 54 bonobos (17 offspring and their putative parents) were genotyped at eight tetranucleotide repeat microsatellite loci. Inbreeding coefficients were calculated for each individual for whom paternity was confirmed by either studbook data or DNA analysis. We found significantly higher infant mortality in inbred offspring compared with non-inbred offspring, suggesting that inbreeding reduces infant survival in captive bonobos. In addition, we argue that the total magnitude of inbreeding depression is probably underestimated in this captive population. In conclusion, even though the breeding programme of captive bonobos is aimed at avoiding inbreeding, closely related individuals do occasionally produce offspring that do show inbreeding depression. There is, however, no indication that this currently threatens the long-time survival of the captive population of bonobos.     

Integrating microsatellite and pedigree analyses to facilitate the captive management of the endangered Mississippi sandhill crane (Grus canadensis pulla)

The minimization of kinship in captive populations is usually achieved through the use of pedigree information. However, pedigree knowledge alone is not sufficient if pedigree information is missing, questionable, or when the founders of the captive population are related to one another. If this is the case, higher levels of inbreeding and lower levels of genetic diversity may be present in a captive population than those calculated by pedigree analyses alone. In this study, the genetic status of the critically endangered Mississippi sandhill crane (MSC) (Grus canadensis pulla) was analyzed using studbook data from the U.S. Fish and Wildlife Service managed captive breeding program as well as microsatellite DNA data. These analyses provided information on shared founder genotypes, allowing for refined analysis of genetic variation in the population, and the development of a new DNA-based studbook pedigree that will assist in the genetic management of the MSC population.     

A new oligonucleotide probe for the giant panda

Quantifying genetic diversity in populations is one of the fundamental measures for species conservation. This is far more important for critically endangered species like giant pandas, where there are few individuals remaining in the population. However, previous multilocus probes could not identify homozygous loci resulting from inbreeding of giant pandas, and produced few polymorphic loci. As a result, we have prepared a new oligonucleotide probe, which had the highest paternity probability and succeeded in identifying the homozygous loci and in discriminating giant panda individuals.     

Genotyping faeces of red pandas (<Emphasis Type="Italic">Ailurus fulgens</Emphasis>): implications for population estimation

The red panda (Ailurus fulgens) is an endangered species distributed in the Himalaya and Hengduan Mountains and extremely difficult to monitor because it is elusive, wary and nocturnal. However, recent advances in noninvasive genetics are allowing conservationists to indirectly estimate population size of this animal. Here, we present a pilot study of individual identification of wild red pandas using DNA extracted from faeces. A chain of optimal steps in noninvasive studies were used to maximize genotyping success and minimize error rate across sampling, selection of microsatellite loci, DNA extraction and amplification and data checking. As a result, 18 individual red pandas were identified successfully from 33 faecal samples collected in the field using nine red panda-specific microsatellite loci with a low probability of identity of 1.249 × 10⁻³ for full siblings. Multiple methods of tracking genotyping error showed that the faecal genetic profiles possessed very few genotyping errors, with an overall error rate of 1.12 × 10⁻⁵. Our findings demonstrate the feasibility and reliability of using faeces as an effective source of DNA for estimating and monitoring wild red panda populations.     

圈养繁殖在大熊猫保护生物学中的作用

栖息地的日益恶化、片段化和种群孤岛化已成为保护大熊猫的严重障碍。尽管就地保护濒危物种是最有希望的措施,但对于大熊猫而言,研究表明圈养繁殖也是保护这一极危物和中的有效手段之一,对保护遗传多样性起以了重要作用,是延缓绝灭速率的保证。本文阐述了大圈养繁殖的必要性及其在保护生物学中的作用。     

Genome-wide survey and analysis of microsatellites in giant panda (Ailuropoda melanoleuca), with a focus on the applications of a novel microsatellite marker system

Background

The giant panda (Ailuropoda melanoleuca) is a critically endangered species endemic to China. Microsatellites have been preferred as the most popular molecular markers and proven effective in estimating population size, paternity test, genetic diversity for the critically endangered species. The availability of the giant panda complete genome sequences provided the opportunity to carry out genome-wide scans for all types of microsatellites markers, which now opens the way for the analysis and development of microsatellites in giant panda.

Results

By screening the whole genome sequence of giant panda in silico mining, we identified microsatellites in the genome of giant panda and analyzed their frequency and distribution in different genomic regions. Based on our search criteria, a repertoire of 855,058 SSRs was detected, with mono-nucleotides being the most abundant. SSRs were found in all genomic regions and were more abundant in non-coding regions than coding regions. A total of 160 primer pairs were designed to screen for polymorphic microsatellites using the selected tetranucleotide microsatellite sequences. The 51 novel polymorphic tetranucleotide microsatellite loci were discovered based on genotyping blood DNA from 22 captive giant pandas in this study. Finally, a total of 15 markers, which showed good polymorphism, stability, and repetition in faecal samples, were used to establish the novel microsatellite marker system for giant panda. Meanwhile, a genotyping database for Chengdu captive giant pandas (n = 57) were set up using this standardized system. What’s more, a universal individual identification method was established and the genetic diversity were analysed in this study as the applications of this marker system.

Conclusion

The microsatellite abundance and diversity were characterized in giant panda genomes. A total of 154,677 tetranucleotide microsatellites were identified and 15 of them were discovered as the polymorphic and stable loci. The individual identification method and the genetic diversity analysis method in this study provided adequate material for the future study of giant panda.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1268-z) contains supplementary material, which is available to authorized users.     

Polymorphic microsatellite loci in peafowl (Pavo cristatus)

Understanding how the genetic characteristics of parents influence reproductive output is central to predicting the dynamics of small, endangered populations. We conducted a breeding experiment to look at the paternal genetic effects on offspring sex, fertility and growth in the peafowl (Pavo cristatus). Microsatellite loci were developed to allow maternity assignment and thus to allow us to separate maternal from paternal effects. We found 19 polymorphic loci in our inbred, captive population, six of which were only slightly polymorphic (H_E range: 0.04–0.70). The remaining 13 loci were polymorphic enough to determine maternity by exclusion in approximately 85% of offspring.