Six microsatellite loci in forest musk deer,Moschus berezovskii

Through an improved enrichment protocol, 20 microsatellite loci were isolated and characterized in an endangered animal, forest musk deer (Moschus berezovskii). In which, six loci showed high polymorphism with five to 13 alleles in 48 sampled individuals. The observed and expected heterozygosities were 0.362–0.955 and 0.784–0.906, respectively. The average polymorphism information content (PIC) was 0.8108 in these six microsatellites and they could be the valuable genetic markers for further investigation of forest musk deer species.     

Genetic quality of the Miyaluo captive forest musk deer (Moschus berezovskii) population as assessed by microsatellite loci

The Miyaluo captive forest musk deer population (Sichuan Province, China) is one of the largest captive breeding populations in the world. In order to evaluate the genetic quality and provide available genetic management strategy, seven polymorphism microsatellite loci were applied to assess the genetic variation of the Miyaluo forest musk deer. The results indicated that a total of 168 alleles were detected from these seven microsatellite loci in 361 individuals, and the number of the alleles per locus ranged from 12 to 41 with a mean of 24. The average observed heterozygosity, expected heterozygosity, and PIC were 0.782, 0.854, and 0.837, respectively. Considering the results of the loci Hardy–Weinberg equilibrium test, the comparison of the common allele frequency as well as the private allele between the adults and juveniles, we concluded that the heterozygosity and the genetic diversity of the Miyaluo captive breeding population are increasing due to the input of new individuals from other populations. However, the frequency of some alleles declined sharply, and some were even lost indicating that there is a risk for diversity loss. Thus, we proposed an improved management and breeding strategy for the captive breeding population of the forest musk deer.     

Genetic diversity of captive forest musk deer (Moschus berezovskii) inferred from the mitochondrial DNA control region

Forest musk deer ( Moschus berezovskii ) were once distributed widely in China. However, wild populations have declined dramatically because of poaching and habitat loss. Captive breeding populations have been established for several decades, but the genetic backgrounds of most captive populations were unclear and the population sizes increased very slowly. To provide useful information for conservation and management of this species, we investigated the genetic diversity and population structure of forest musk deer by analysing a 582-bp fragment of the mitochondrial DNA (mtDNA) control region (CR) in three captive breeding populations in Sichuan Province, China. Ninety-four variable sites and 27 haplotypes were observed in 109 individuals, and the nucleotide and haplotype diversities were relatively high compared with those of other endangered mammals. Of the three investigated populations, the Maerkang population had the highest nucleotide diversity ( π  = 0.0568), haplotype diversity ( h  =   0.836) and average intra-population genetic distance (0.062). The analysis of molecular variance demonstrated that most variation occurred within samples and that there was significant differentiation of the three populations. Estimates of gene flow indicated that there were few genetic exchanges among the three populations. Building pedigree records and increasing gene flow between populations will be helpful for conserving these populations and this species.     

林麝幼体的换毛序和胎毛、夏毛、冬毛的形态

被毛是哺乳动物最主要的特征之一,有关动物换毛序与环境关系的研究,是动物生态学研究的一个重要方面。从1853年Browne开始研究毛发结构以来,人们对毛发的形态学、分类学和种属关系等方面的研究已开展了大量工作,特别是随着电镜技术的应用,这些方面的工作在逐渐深入(曹汉民等,19     

凉山山系林麝夏季利用生境特征

采用样线法对位于四川凉山山系的美姑大风顶自然保护区林麝(Moschus ber-ezovskii)的夏季利用生境特征进行了调查,并对利用生境样地(n=44)和非利用样地(n=209)的生境变量进行了比较.结果表明:大风顶保护区的林麝夏季利用生境的郁闭度(32.4%±1.9%)、灌木盖度(27.1%±2.4%)及竹子盖度...     

圈养林麝的活跃性及与社会亲和度的关系

采用扫描取样和焦点动物取样法对四川马尔康林麝繁育场的45头圈养林麝进行行为取样,以其非静息行为的发生率作为活跃性指标,以社会亲和行为的发生强度作为社会亲和度指标,分析圈养林麝活跃性与其性别、圈群密度及社会亲和度的关系。结果表明:圈群密度和社会亲和度对圈养林麝的活跃性有显著影响(P < 0.05),但活跃性雌雄差异不显著(P > 0.05)。与高密度圈群的林麝(0.25 ± 0.021, n=32)相比,低密度圈群中的个体更活跃(0.40 ± 0.022, n = 4);活跃性与社会亲和度之间显著相关(r = 0.390, P = 0.01),活跃性较高个体倾向于展现高的社会亲和度。建议麝场在进行麝群构建时,充分考虑林麝个体的活跃性差异及与圈群密度的关系,以提高圈群稳定性和驯养生产力。     

圈养林麝社会等级与粪样类固醇激素水平的关系

动物社群的社会等级可对个体的资源分配、社群稳定及个体的行为对策产生重要影响,圈养动物的类固醇激素水平与饲养管理有关,可反映动物的社群紧张水平.深入理解圈养动物的社会等级与其类固醇激素水平的关系是进行濒危动物迁地保育和成功驯养的基础.2018年6月15日—8月15日,用焦点取样法对四川马尔康林麝繁育场的25头林麝进行了冲...     

Molecular polymorphism of MHC-DRB gene and genetic diversity analysis of captive forest musk deer (Moschus berezovskii)

The major histocompatibility complex (MHC) is one of the most important elements in immune system for nearly all vertebrates, and is thought to be essential for an organism to recognize foreign molecules. In this study, we investigated the MHC variation in 51 forest musk deer (Moschus berezovskii) collected from three captive populations in Sichuan Province, China. Seventeen haplotypes were isolated from the 51 samples. A total of 51 mutation sites were identified and yielded a nucleotide diversity of 0.056. These haplotype sequences possessed 83 putative amino acid sites, including 24 PBR sites (peptide binding region) and 59 non-PBR sites. Out of 24 PBR sites, 15 codons showed variation (0.625), while 12 codons showed variation (0.203) in 59 non-PBR sites. Non-synonymous substitutions primarily occurred in PBR, with analyses suggesting that the Mobe-DRB gene had undergone strong positive selection during their evolution. Compared with that of some other endangered species, the forest musk deer had relatively high level of MHC diversity. Our results suggested that the MHC diversity characteristic of captive forest musk deer populations might be not responsible for their high morbidity of abscess disease.     

Major histocompatibility complex class II genetic variation in forest musk deer (Moschus berezovskii) in China

The major histocompatibility complex (MHC) plays an important role in the immune system of vertebrates. We used the second exon of four MHC class II genes (DRA, DQA1, DQA2 and DRB3) to assess the overall MHC variation in forest musk deer (Moschus berezovskii). We also compared the MHC variation in captive and wild populations. We observed 22 alleles at four loci (four at DRA, four at DQA1, four at DQA2 and 10 at DRB3), 15 of which were newly identified alleles. Results suggest that forest musk deer maintain relatively high MHC variation, which may result from balancing selection. Moreover, considerable diversity was observed at the DRA locus. We found a high frequency of Mobe‐DRA*02, Mobe‐DQA1*01 and Mobe‐DQA2*05 alleles, which may be important for pathogen resistance. A Ewens–Watterson test showed that the DRB3 locus in the wild population had experienced recent balancing selection. We detected a small divergence at the DRA locus, suggesting the effect of weak positive selection on the DRA gene. Alternatively, this locus may be young and not yet adapted a wide spectrum of alleles for pathogen resistance. The significant heterozygosity deficit observed at the DQA1 and DRB3 loci in the captive population and at all four loci in the wild population may be the result of a population bottleneck. Additionally, MHC genetic diversity was higher in the wild population than in the captive, suggesting that the wild population may have the ability to respond to a wider range of pathogens.     

圈养林麝春季活动节律及时间分配

2006 年春季,使用红外夜间观察记录仪,对崇明东平麝场12 只成年林麝(8 雌,4 雄)的行为进行了24 h昼夜连续观察。发现春季林麝的行为有37 种,可归并为休息、摄食、运动、社会、维持和排遗6 大行为类型,据此,建构了春季林麝的行为谱。根据各行为持续时间比率和频次分析表明,林麝的主要行为为休息;雌麝的摄食行为所占的比率高于雄麝,这是由于春季雌麝妊娠的能量需求较高所致。活动节律呈典型的晨昏双峰型,清晨(4:00 ～ 6:00)和傍晚(17:00 ～ 19:00)为活动高峰;夜间活动性显著高于白天。     

The reproductive performance of female Forest musk deer (Moschus berezovskii) in captivity

Reproductive performance of 750 adult female Forest musk deer (FMD, Moschus berezovskii) was monitored (from 2005 to 2009) on two Forest musk deer farms in Sichuan province, China. The mean (± SEM) lengths of the estrous cycle, pregnancy, and consecutive fawning intervals were 17.5 ± 0.8 d (n = 64), 182.9 ± 0.8 d (n =190), and 363.2 ± 2.4 d (n = 120), respectively. Copulation occurred from the end of October to early March, with the majority (84.1 ± 1.9%) during November and December (P < 0.05). The fawning rate was 68.7 ± 2.2% in yearling hinds and 73.3 ± 1.3% in adult hinds. Fawning occurred from the end of April to early September, with the majority (88. 9 ± 2.2%) during May and June (P < 0.005), including a peak in May (52.0 ± 1.0%). Yearling hinds fawned later (7.3 ± 1.8 d) than adult hinds. Rates of premature delivery and dystocia were 2.7 ± 0.4 and 2.0 ± 0.3%, respectively. There was a 1.07:1 female-to-male ratio at birth (P > 0.05), with 67.7% twins, but only two cases of triplets and one set of quadruplets among 537 fawns. Birth weight averaged 518.7 ± 10.3 g, ranging from 304 to 775 g (n = 240), with male fawns slightly heavier than females (521.1 ± 14.8 vs 516.9 ± 14.3 g). Postnatal and pre-weaning mortality rates were 25.0 ± 1.2 and 17.9 ± 3.1%, respectively. Postnatal mortality and urinary calculus were much more common in male fawns (P < 0.01). The mortality rate due to suppuration and dyspepsia were different (P < 0.001) between postnatal and pre-weaning periods. Due to higher mortality of male fawns, the adult sex ratio was significantly female-biased. In conclusion, captive FMD had similar seasonal patterns of copulation and fawning as wild FMD and other musk deer species.     

The essential differences in microbial and chemical components of musk of different qualities secreted by captive male forest musk deer (Moschus berezovskii)

Musk is a precious raw material and ingredient in Chinese traditional medicine. The production of unqualified musk has become a puzzling problem in forest musk deer (FMD) breeding. However, what the essential differences between so-called unqualified musk and mature qualified musk have not yet been elucidated. In this study, 12 musk samples were collected and separated into two groups according to their external properties. One group is white or black cream-like secretion with sour or unpleasant odour (MM); the other group is brown or blackish brown solid secretion with pleasant fragrance (DM). Next-generation sequencing and gas chromatography-mass spectrometry were used to explore the essential differences between the DM and MM groups in microbial and chemical compositions. The results indicate that the DM group has more heterogenous microbial structure but simpler relationships among microbial communities. LEfSe analysis showed that 14 taxa at the genus level could be used to distinguish the DM and MM groups and Bacillus, Paracoccus, tenoteophomonas, Mycobacterium and Leuconostoc were more abundant in the DM group (P < 0.05). In addition, six compounds were identified to specifically distinguish the DM and MM groups under the OPLS-DA model. PICRUSt analysis revealed that metabolic pathways such as carbohydrate metabolism, nucleotide metabolism, energy metabolism, transport and catabolism were enriched in the DM group. All these findings of differences in microbiota and chemical compositions would provide potential clues for MM quality improvement and new evidence for the scientific establishment of a quality evaluation standard for musk.     

A rapid sex-identification test for the forest musk deer (Moschus berezovskii) based on the ZFX/ZFY gene

We describe a rapid sex-identification method for the forest musk deer (Moschus berezovskii) using PCR based on zinc-finger protein-encoding genes (ZFX/ZFY) located on the X and Y chromosomes. Fragments of the ZFX and ZFY genes were amplified and sequenced. The ZFX and ZFY fragments were identical in length and 94% similar in nucleotide sequence. Specific primers for forest musk deer sex identification were designed on the basis of sequence differences between ZFX and ZFY. All the primers were multiplexed in single-tube PCR. Both male and female forest musk deer showed amplification bands of 447 bp and 212 bp separated in agarose gels. A sex-specific 278-bp band was amplified only from males. These results show that testing by PCR for the presence of the 278-bp sequence is a rapid and reliable method for sex identification.     

New insights into the karyotypic relationships of Chinese muntjac (Muntiacus reevesi), forest musk deer (Moschus berezovskii) and gayal (Bos frontalis)

To investigate the karyotypic relationships between Chinese muntjac (Muntiacus reevesi), forest musk deer (Moschus berezovskii) and gayal (Bos frontalis), a complete set of Chinese muntjac chromosome-specific painting probes has been assigned to G-banded chromosomes of these three species. Sixteen autosomal probes (i.e. 6-10, 12-22) of the Chinese muntjac each delineated one pair of conserved segments in the forest musk deer and gayal, respectively. The remaining six autosomal probes (1-5, and 11) each delineated two to five pairs of conserved segments. In total, the 22 autosomal painting probes of Chinese muntjac delineated 33 and 34 conserved chromosomal segments in the genomes of forest musk deer and gayal, respectively. The combined analysis of comparative chromosome painting and G-band comparison reveals that most interspecific homologous segments show a high degree of conservation in G-banding patterns. Eleven chromosome fissions and five chromosome fusions differentiate the karyotypes of Chinese muntjac and forest musk deer; twelve chromosome fissions and six fusions are required to convert the Chinese muntjac karyotype to that of gayal; one chromosome fission and one fusion separate the forest musk deer and gayal. The musk deer has retained a highly conserved karyotype that closely resembles the proposed ancestral pecoran karyotype but shares none of the rearrangements characteristic for the Cervidae and Bovidae. Our results substantiate that chromosomes 1-5 and 11 of Chinese muntjac originated through exclusive centromere-to-telomere fusions of ancestral acrocentric chromosomes.     

Development and characterization of 15 novel microsatellite markers from forest musk deer (<Emphasis Type="Italic">Moschus berezovskii</Emphasis>)

A total of 15 polymorphic microsatellite loci were isolated from forest musk deer (Moschus berezovskii). These loci showed high levels of allelic diversity, presenting 6–14 alleles per locus in a group with 31 individuals. The mean observed and expected heterozygosities were 0.82 and 0.68, respectively. The average polymorphic information content value, cumulative discrimination power, overall probability of exclusion with both unknown parents or with only unknown sire for these markers were 0.78, 1.0, 0.9999 and 1.0, respectively, indicating that this suit of microsatellite loci would be a powerful tool for the genetic determination of the forest musk deer.     

Isolation and characterization of microsatellite loci from Asparagus acutifolius (Liliaceae)

The isolation of molecular markers in Asparagus acutifolius, a wild edible plant species, is important to characterize local ecotypes that could be cultivated and preserved. We isolated and characterized polymorphic microsatellite loci from A. acutifolius by constructing and screening an enriched DNA library. Primer pairs were designed for 12 loci. Seven primer pairs worked well during amplification reactions and were tested on a wild population from Pontecagnano (SA), Italy. These loci showed a high level of genetic variability, with the numbers of alleles identified ranging from two to five and observed heterozygosity ranging from 0.20 to 0.73.     

Isolation and characterization of microsatellite loci from Weigela coraeensis (Caprifoliaceae)

Weigela coraeensis var. coraeensis is a deciduous shrub species distributed in Japan on the mainland, Honshu, whereas its variety W. coraeensis var. fragrans is endemic to the Izu Islands located south of Honshu. We isolated eight polymorphic microsatellite loci from the species and characterized these loci for 20 individuals from a population in Honshu. The number of alleles per locus ranged from 7 to 15 and the observed and expected heterozygosities ranged from 0.60 to 0.90 and from 0.65 to 0.90, respectively. These eight polymorphic microsatellites will be useful for examining intraspecific genetic differentiation in W. coraeensis.     

Isolation and characterization of microsatellite loci from Corydalis ambigua (Papaveraceae)

Primers for seven unlinked and highly polymorphic microsatellite loci were developed for Corydalis ambigua to investigate its mating system and population genetics. This species can be used to explore the impact of an alien bumblebee on plant reproduction. Genetic diversity and other population genetic parameters were estimated in two populations with and without the alien bumblebee. The number of alleles per locus ranged from 4 to 20, and observed heterozygosities ranged from 0.355 to 0.969. These markers can be applied to study mating systems and population genetics in C. ambigua.     

Isolation,primary culture,and morphological characterization of gland epithelium from forest musk deer (<Emphasis Type="Italic">Moschus berezovskii</Emphasis>)

Research of epithelial cells in musk gland is lacking. There are no good characterized epithelial cell lines that can provide complementary in vitro models for in vivo research. We successfully cultivated epithelial cells of musk gland for the first time. The protocol described here produces epithelial cell lines from the mature secreting musk gland. Based on morphological observation, epithelial cells of musk gland were isolated and cultured in vitro. After the third passage, the musk gland-derived cells were filled with many lipid droplets and proliferated well. We used gas chromatography and mass spectrometry to explore the chemical composition of lipid droplets in the musk gland-derived cells. The main components of secreted lipid droplet were alkanes, esters, amines, alcohols, ketones, organic acids, and aldehydes. Muscone, which is the main active compound of musk, was not found. This is a new attempt in the field of animal musk to obtain naturally secreted animal musk in vitro by cloning specialized cells. In conclusion, this study provides a reference at the cellular level to further analyze the biology and physiology of the musk gland epithelium and secretion mechanism of musk deer.