Isolation and characterization of 12 novel microsatellite loci for the red panda (Ailurus fulgens)

The information on dispersal patterns and mating systems of red pandas is quite important for the understanding of the genetic diversity and divergence of this species. And microsatellite marker is an ideal tool to analyze dispersal patterns and mating systems. Thus, we describe in this paper the isolation and characterization of 12 microsatellite loci in the red panda from genomic DNA-enriched libraries. These loci were highly polymorphic with numbers of alleles per locus in 24 individuals ranging from 2 to 14, observed heterozygosity from 0.143 to 0.864 and expected heterozygosity from 0.297 to 0.872. All loci except for RP6 locus followed Hardy–Weinberg expectations. No significant linkage association was found among all these loci. The 12 novel polymorphic microsatellite loci will be of use in studying dispersal patterns and mating systems of red pandas.     

Scent-marking in captive red pandas (Ailurus fulgens)

Three male and three female captive red pandas (Ailurus fulgens) were observed during and outside of the breeding season to examine changes in scent-marking behavior. Variation in scent-marking was analyzed with respect to site preferences, sex differences, onset of breeding, behavioral correlates, and environmental factors. Both males and females exhibited significant preferences for particular marking sites, those being prominent points in the enclosures. Males showed significantly higher scent-marking frequencies than females, except in one enclosure where increased marking behavior was associated with the presence of young. Sexual dimorphism in the pattern of marking was also observed, with males showing a bidirectional mark and females a unidirectional mark. Female marking was greater during the breeding season. Sniffing and number of waddles per mark were associated with scent-marking. Temperature change was not related to frequency of scent-marking. These results are compared with scent-marking behaviors in other species of carnivores and functional explanations are suggested.     

小熊猫对同性和异性气味的行为反应

食肉目动物的复杂社会行为在种间和种群间变化较大,这种变化是动物对其生态环境的适应表现特征之一。食肉目的动物与嗅觉通讯相关的气味源有肛周腺、肛囊腺、尾下腺、会阴腺、足腺、尿液、唾液和粪便等十几种,产生的气味物质包含有个体、性别和群体的识别信息,在其繁殖调控中发     

蜂桶寨自然保护区小熊猫巢域初步研究

2002年5~11月,在蜂桶寨自然保护区利用无线电遥测技术对6只小熊猫的巢域利用进行了初步研究。结果表明,6只戴颈圈个体M1、M2、M3、F1、F2、F3的巢域面积分别为330·26hm~2、135·18hm~2、190·67hm~2、98·23hm~2、141·60hm~2、204·80hm~2;雄性个体平均巢域面积为218·70hm~2,雌性个体为148·21hm~2。小熊猫个体间巢域重叠普遍,平均重叠率达25·33%,其中雄性个体之间为26·00%,雌性个体之间为23·67%,两性个体之间为25·67%。可能受人为干扰的影响,M1在6只监测个体中巢域面积、日均移动距离均为最大。     

Genetic structuring and recent demographic history of red pandas (Ailurus fulgens) inferred from microsatellite and mitochondrial DNA

Clarification of the genetic structure and population history of a species can shed light on the impacts of landscapes, historical climate change and contemporary human activities and thus enables evidence‐based conservation decisions for endangered organisms. The red panda (Ailurus fulgens) is an endangered species distributing at the edge of the Qinghai‐Tibetan Plateau and is currently subject to habitat loss, fragmentation and population decline, thus representing a good model to test the influences of the above‐mentioned factors on a plateau edge species. We combined nine microsatellite loci and 551 bp of mitochondrial control region (mtDNA CR) to explore the genetic structure and demographic history of this species. A total of 123 individuals were sampled from 23 locations across five populations. High levels of genetic variation were identified for both mtDNA and microsatellites. Phylogeographic analyses indicated little geographic structure, suggesting historically wide gene flow. However, microsatellite‐based Bayesian clustering clearly identified three groups (Qionglai‐Liangshan, Xiaoxiangling and Gaoligong‐Tibet). A significant isolation‐by‐distance pattern was detected only after removing Xiaoxiangling. For mtDNA data, there was no statistical support for a historical population expansion or contraction for the whole sample or any population except Xiaoxiangling where a signal of contraction was detected. However, Bayesian simulations of population history using microsatellite data did pinpoint population declines for Qionglai, Xiaoxiangling and Gaoligong, demonstrating significant influences of human activity on demography. The unique history of the Xiaoxiangling population plays a critical role in shaping the genetic structure of this species, and large‐scale habitat loss and fragmentation is hampering gene flow among populations. The implications of our findings for the biogeography of the Qinghai‐Tibetan Plateau, subspecies classification and conservation of red pandas are discussed.     

圈养小熊猫育幼行为的初步观察

Nursing behaviors of the captive md pandas were quantitatively studied by focal sampling methods in Chengdu Research Base of Giant Panda Breeding from Jldy to August, 2000. The results indicated that frequencies of activity and rest were low in the first three days after birth, and became higher as time went by. The frequency of licking cub was higher in the first day after birth and deserting cub was only observed in the seventh day. Frequencies of some behaviors, such as rest, licking cub, cherishing cub,sniffing cub, returning to shed and deserting cub, differed sioaificantly in different phases of the first month after birth. Frequencies of licking cub, cherishing cub, sniffing cub and returning to shed were significantly higher in the pre-nursing period than in the midnursing and post-nursing periods. However, frequencies of rest and deserting cub were sioaificantly higher in the post-nursing period than in the per-nursing and mid-nursing periods. The degree of maternal behaviors can be inferred from degree that the mother exposes her babies to the envim~unent and the time that the mother leaves her shed.     

An ARMS-based technique for sex determination of red panda (Ailurus fulgens)

Molecular sexing is a key component in the investigation of wild populations. In this study, we developed a fast, accurate and reliable amplification refractory mutation system (ARMS) technique for sex determination of red panda based on the exon 4 of the ZFX/ZFY gene. The amplicons were distinguished simply by agarose gel electrophoresis, exhibiting one fragment in females (X: 300 bp) and two in males (X: 300 bp, Y: 166 bp). Robustness of this ARMS system was confirmed by testing both 43 captive red pandas using DNA samples with known-sex and 10 wild red pandas using faecal DNA samples with unknown sex.     

小熊猫妊娠期粪便中孕酮、雌二醇的水平变化

2000年3—8月在成都动物园和成都大熊猫繁育研究基地,用放射免疫分析法测定了6只雌性小熊猫粪样中孕酮（P）和雌二醇（E2）的水平变化,同时对其繁殖行为进行了观察。研究结果表明,交配后的雌体粪样中E2浓度下降至基础水平,P浓度在30d后上升至150ng／g以上;怀孕雌体在妊娠期粪样中P水平在200ng／g以上,P峰值可以达500ng／g以上;假妊娠者粪样中P水平一般不超过150ng／g,P峰值在200ng／g以下;雌体产前半个月孕酮迅速下降,E2缓慢上升,产后P保持在基础水平。这意味着可通过测定粪便中P水平进行早孕诊断、产期预测和真假妊娠判断。根据行为观察和粪样激素分析可知,雌兽妊娠期为123—128d（X=124,N=5）,产仔率为1．8／胎（N=5）。     

Isolation and characterization of polymorphic tetranucleotide microsatellite loci in the chestnut-crowned babbler (Pomatostomus ruficeps)

We describe 18 microsatellite markers isolated in the cooperatively breeding chestnut‐crowned babbler (Pomatostomus ruficeps). The number of alleles ranged from seven to 16 per locus (mean N_a = 10.4 ± 0.54 SE) and the expected heterozygosity ranged from 0.732 to 0.889 (mean H_E = 0.836 ± 0.01 SE). Three of the 18 loci exhibited significant heterozygote deficiency, but the remaining 15 will be used to analyse population genetic structure and the mating system of this highly social species.     

小熊猫夏秋季的昼夜活动节律

2002年5~11月,在邛崃山系宝兴蜂桶寨自然保护区采用无线电遥测技术对6只野生小熊猫的昼夜活动节律进行了研究。结果表明,小熊猫具有晨昏活动的习性,5~11月的平均活动率为0.5286。小熊猫以白昼活动为主,兼夜间活动,白昼的活动率(0.5903±0.0538)高于夜晚活动率(0.4468±0.0413)。每昼夜有(52.86±9.8)%的时间处于活动状态,其中64.53%的活动时间在白昼,35.47%的活动时间在夜晚。每昼夜有两个活动高峰,分别出现在08∶00~10∶00和17∶30~18∶30。在休息时间的分配上,小熊猫每昼夜长休息平均2.07次,持续时间平均3.75小时;中等长度休息1.56次,持续1.59小时;短休息1.21次,持续0.84小时;长休息有44.06%在白昼,55.94%在夜晚。     

Characterization of polymorphic tetranucleotide microsatellite loci from the Siberian weasel (Mustela sibirica)

Nine tetrarepeat microsatellite loci for Siberian weasel (Mustela sibirica) were characterized. By using 35 individuals of unknown relationship collected from Taiwan, we evaluated the polymorphism of these loci. The number of alleles for each locus ranged from five to 12 (mean = 8.78 alleles) and the observed heterozygosity from 0.429 to 0.962 (mean = 0.688) and all loci conformed to Hardy–Weinberg expectation.     

大熊猫和小熊猫粪便DNA提取的简易方法

采集了大熊猫和小熊猫的新鲜粪便样品 ,使用 1 0 0 %乙醇保存。通过重复离心富集研究动物的肠道脱落细胞 ,并使用乙醇和双蒸水洗涤以除去抑制物。用 1 %的SDS快速裂解细胞 ,离心除去残渣后 ,向裂解液中加入蛋白酶进行消化。消化结束后使用等体积的酚 /氯仿抽提 ,乙醇沉淀DNA。用双蒸水溶解粪便DNA后 ,使用PCR产物纯化试剂盒对粪便DNA进行纯化。电泳检测结果显示 ,从乙醇保存的大、小熊猫粪便样品中抽提到高质量的粪便DNA。对线粒体控制区、细胞色素b基因、 1 2SrRNA基因的PCR扩增反应以及测序结果也证实了样品保存方法和DNA抽提方法可靠而高效。此方法使用实验室内常用的分子生物学试剂 ,不仅克服了分子粪便学研究中常见的抑制物粪便DNA微量降解严重等障碍 ,与商业化的粪便抽提试剂盒 (QIAampDNAStoolMiniKit,Qiagen)相比还是一种经济的试验方法 (抽提反应成本为试剂盒的 1 / 5 )。文中还对粪便DNA内细菌基因组等背景DNA可能对分子粪便学试验结果的影响进行了探讨。在基于PCR技术的遗传学研究中 ,对于植食性动物而言 ,粪便内的背景DNA对目标动物DNA片断的扩增和序列测定未见影响 ;但对于肉食性动物 ,则必须考虑被捕食者基因组对试验可能产生的影响 ,应谨慎对待     

The current status and future prospects of the Red Panda (Ailurus fulgens) studbook population

Scientifically based captive breeding programs are an essential prerequisite to the long-term preservation of exotic species in zoos. The data provided by studbooks form the foundation on which such a program can be built. In the following paper, information extracted from the International Studbook for the Red Panda (Ailurus fulgens) is used to provide both demographic and genetic analyses of the current captive-born population and simulations of future trends in population structure. From the results of these analyses, conclusions can be drawn which are of importance to future captive management practice.     

Genetic variation in the zoo population of the red panda subspecies Ailurus fulgens fulgens

Genetic variation present in the global and regional zoo populations of the red panda is estimated by computer simulations. In this study it is demonstrated that inbreeding depression could occur on the short term in the regional populations. Migration between regional populations is recommended to ensure the future survival of the red panda in captivity. More genealogical lineages should be represented in the breeding program.     

Isolation and characterization of microsatellite markers in tree lizards (Urosaurus ornatus)

Tree lizards (Urosaurus ornatus) are a highly accessible species useful in testing theories of life-history evolution and behavioural ecology. A polymerase chain reaction-based method was used to isolate nine polymorphic tetranucleotide microsatellite loci from the genome of these lizards. The isolated loci displayed 5 to 9 alleles in the populations screened. Observed heterozygosity ranged from 0.333 to 0.900. Results indicate these loci now provide a basis to study mate choice, parentage, and population genetic structure within this species.     

Isolation and characterization of microsatellite loci from Larix kaempferi

Microsatellites were isolated and characterized for Japanese larch, Larix kaempferi, a conifer species distributed in Japan. A larch genomic DNA library enriched for (AG)_n repeats was screened using the colony polymerase chain reaction method and 145 unique microsatellite containing sequences were obtained. Seventy‐two primer pairs were designed and 30 produced single‐locus products, and 19 of them were polymorphic. The expected heterozygosity ranged from 0.566 to 0.951. These 19 polymorphic microsatellite loci should be valuable markers for genetic studies on Japanese larch.     

Eight novel tetranucleotide and five cross-species dinucleotide microsatellite loci for the ornate chorus frog (Pseudacris ornata)

We describe the cloning and characterization of eight novel tetranucleotide microsatellite loci in the ornate chorus frog (Pseudacris ornata). We also screened 26 loci from GenBank that were isolated from other Pseudacris species and obtained consistent product from five of these dinucleotide loci. All loci are polymorphic. In our sample of 26 frogs from a natural population, polymorphism ranged from 1 to 22 alleles per locus with expected heterozygosities ranging from 0 to 0.958. These loci enable high-resolution studies of P. ornata. Moreover, cross-species amplification success suggests they will also be useful for other chorus frog species.     

Isolation and characterization of microsatellite loci in the ant Myrmica scabrinodis

Five polymorphic microsatellite loci were developed for the ant Myrmica scabrinodis using a magnetic bead hybridization selection protocol. The number of alleles per locus varied between three and six. Cross‐species amplification of four of the loci yielded positive amplification products in four Myrmica species, suggesting their general suitability for microsatellite analysis within this taxonomic group.