Genetic diversity and phylogeographic structure of Bactrian camels shown by mitochondrial sequence variations

The Bactrian camel includes various domestic (Camelus bactrianus) and wild (Camelus ferus) breeds that are important for transportation and for their nutritional value. However, there is a lack of extensive information on their genetic diversity and phylogeographic structure. Here, we studied these parameters by examining an 809‐bp mtDNA fragment from 113 individuals, representing 11 domestic breeds, one wild breed and two hybrid individuals. We found 15 different haplotypes, and the phylogenetic analysis suggests that domestic and wild Bactrian camels have two distinct lineages. The analysis of molecular variance placed most of the genetic variance (90.14%, P < 0.01) between wild and domestic camel lineages, suggesting that domestic and wild Bactrian camel do not have the same maternal origin. The analysis of domestic Bactrian camels from different geographical locations found there was no significant genetic divergence in China, Russia and Mongolia. This suggests a strong gene flow due to wide movement of domestic Bactrian camels.     

Population genetic analysis of the domestic Bactrian camel in China by RAD‐seq

Restriction site‐associated DNA sequencing (RAD‐seq) is one of the most effective high‐throughput sequencing technologies for SNP development and utilization and has been applied to studying the origin and evolution of various species. The domestic Bactrian camels play an important role in economic trade and cultural construction. They are precious species resources and indispensable animals in China's agricultural production. Recently, the rapid development of modern transportation and agriculture, and the deterioration of the environment have led to a sharp decline in the number of camels. Although there have been some reports on the evolution history of the domestic Bactrian camel in China, the origin, evolutionary relationship, and genetic diversity of the camels are unclear due to the limitations of sample size and sequencing technology. Therefore, 47 samples of seven domestic Bactrian camel species from four regions (Inner Mongolia, Gansu, Qinghai, and Xinjiang) were prepared for RAD‐seq analysis to study the evolutionary relationship and genetic diversity. In addition, seven domestic Bactrian camel species are located in different ecological zones, forming different characteristics and having potential development value. A total of 6,487,849 SNPs were genotyped. On the one hand, the filtered SNP information was used to conduct polymorphism mapping construction, LD attenuation analysis, and nucleotide diversity analysis. The results showed that the number of SNPs in Dongjiang camel was the highest, the LD coefficient decayed the fastest, and the nucleotide diversity was the highest. It indicates that Dongjiang camel has the highest genetic diversity. On the other hand, the filtered SNPs information was used to construct the phylogenetic tree, and F_ST analysis, inbreeding coefficient analysis, principal component analysis, and population structure analysis were carried out. The results showed that Nanjiang camel and Beijiang camels grouped together, and the other five Bactrian camel populations gathered into another branch. It may be because the mountains in the northern part of Xinjiang and the desert in the middle isolate the two groups from the other five groups.     

SNP discovery and population structure analysis in Lassi and Marecha camel breeds using a genotyping by sequencing method

Pakistani camels have been classified socio-geographically into 20 breeds, but they have not yet been subjected to substantial selective pressures and the genetic basis for these breeds is not understood. However, it should be possible to distinguish them by use of molecular data. This study investigated the genetic diversity and population structure within and between two major Pakistani camel breeds, Marecha and Lassi. As no SNP array is currently available, we first identified 63 619 SNPs using a genotyping by sequencing approach. After quality control, a panel of 36 926 SNPs was used in the analysis. Population structure was investigated with a principal coordinate analysis as well as a cluster analysis using NetView , and multilocus heterozygosity analysis to explore between- and within-breed genetic variation. In addition, between-breed variation was explored using the fixation index, F_ST. We also compared relationship matrices computed using the VanRaden SNP-based method and a method developed specifically for genotyping by sequencing data. Among the two camel breeds, Lassi showed a lower level of genetic diversity whereas Marecha showed a higher level. As a genotyping platform has not yet been developed for the camel, the SNPs discovered in this study will be useful in future genetic studies in camels.     

采用微卫星标记分析10个双峰驼群体的遗传变异和群体间的遗传关系

为从分子水平上对我国双峰驼(Camelus bactrianus)群体的遗传多样性、群体间遗传关系、群体遗传分化及近交情况进行全面、系统地研究,为双峰驼种质资源保护和新品种培育提供基础数据,本文利用18对微卫星引物,分析了我国9个双峰驼群体和1个蒙古双峰驼群体的遗传多样性和遗传关系。结果显示:10个群体均具有较高的遗传多样性,共检测到242个等位基因,平均等位基因数为13.44,平均有效等位基因数为4.18,平均观察杂合度(Ho)为0.5528。10个群体间存在显著的遗传分化,有9.6%的遗传变异来自群体间,90.4%的遗传变异来自群体内部的个体间。聚类分析、主成分分析和群体遗传结构分析结果都表明10个群体被分成2个明显的分支,新疆4个群体单独聚为一类,剩下的6个群体聚为一类。这一结果可能与它们的地理分布和群体间的地理屏障有关。     

Genetic diversity and population genetic structure of six dromedary camel (camelus dromedarius) populations in Saudi Arabia

Camels are an integral and essential component of the Saudi Arabian heritage. The genetic diversity and population genetic structure of dromedary camels are poorly documented in Saudi Arabia so this study was carried out to investigate the genetic diversity of both local and exotic camel breeds. The genetic diversity was evaluated within and among camel populations using 21 microsatellite loci. Hair and blood samples were collected from 296 unrelated animals representing 4 different local breeds, namely Majaheem (MG), Maghateer (MJ), Sofr (SO), and Shaul (SH), and two exotic breeds namely Sawahli (SL) and Somali (SU). Nineteen out of 21 microsatellite loci generated multi-locus fingerprints for the studied camel individuals, with an average of 13.3 alleles per locus. Based on the genetic analyses, the camels were divided into two groups: one contained the Saudi indigenous populations (MG, MJ, SH and SO) and the other contained the non-Saudi ones (SU and SL). There was very little gene flow occurring between the two groups. The African origin of SU and SL breeds may explain their close genetic relationship. It is anticipated that the genetic diversity assessment is important to preserve local camel genetic resources and develop future breeding programs to improve camel productivity.     

Microscopic and molecular detection of Deraiophoronema evansi (Lewis, 1882) in domestic Bactrian camels (Camelus bactrianus) of Mongolia

Cameline filarosis is an important parasitic disease having an economic impact on the camel industry around the world. However, there has been no study on filarosis in Bactrian camels of Mongolia. Therefore, the aim of the present study was to detect and identify microfilariae of Deraiophoronema evansi (D. evansi) in Bactrian camels from three provinces, located in southern and southwestern Mongolia. Blood samples were obtained from 400 healthy two-humped camels of different ages and both sexes. All blood samples were analysed using a variety of diagnostic techniques. Microfilariae were detected in 30 Bactrian camels (7.5%) by the Knott technique, while 13 Bactrian camels (3.3%) tested positive in a direct smear test. D. evansi was detected in 18 Bactrian camels (4.5%) by PCR assay. Prevalence was shown to be high among Bactrian camels in the age group up to 5 years, while the lowest positive results were obtained for Bactrian camels in the 5–10-year age group and the over 10-year age group. To confirm the morphological identification, D. evansi–COI gene sequences were subjected to phylogenetic analyses. The D. evansi–COI gene sequences from Mongolian two-humped camels were identical to sequences from Iranian one-humped camels and were clustered together with these sequences in the phylogeny. This is the first report of molecular detection and identification of microfilariae of D. evansi in Bactrian camels of Mongolia.     

Results of <Emphasis Type="Italic">Camelus dromedarius</Emphasis> and <Emphasis Type="Italic">Camelus bactrianus</Emphasis> Genotyping by Alpha-S<Subscript>1</Subscript>-Casein,Kappa-Casein Loci,and DNA Fingerprinting

Genotyping of Kazakh camels Camelus dromedarius (milk breed) (n = 18) and Camelus bactrianus (meat breed) (n = 18) by alpha-S₁-casein (αs₁-CN) and kappa-casein (κ-CN) loci was conducted using the PCR–RFLP analysis method. A new pair of primers was suggested for the amplification of the CSN3 gene fragment with subsequent cleavage of the reaction products by AluI restriction endonuclease in order to identify the gene genetic variants. DNA polymorphism was detected only for the kappa-casein locus; no genetic polymorphism for alpha-S₁-casein gene was found in the studied populations. Analysis of the results of DNA fingerprinting demonstrated that the band sharing (BS) coefficient between the groups was low enough (0.13), and the genetic distance (D) between Dromedary and Bactrian breeds was 0.305. The results of genotyping of Bactrian and Dromedary Kazakh camel breeds by alpha-S₁-casein, kappa-casein loci, and DNA fingerprinting indicate that the Dromedary breed female camels are more polymorphic as compared with Bactrian.     

Diversity and distribution of CYP gene family in Bactrian camel

Cytochrome P450 (CYP) enzymes belong to a superfamily of monooxygenases which are phase I enzymes responsible for the first pass metabolism of about 90% of drugs in animals. However, these enzymes are often polymorphic and metabolism of the same drug in different species or different individuals is influenced by genetic and non-genetic factors. Bactrian camels are capable of survival in harsh living environments, being able to consume diets that are often toxic to other mammals and can tolerate extreme water and food deprivation. The aim of this study was to investigate whether the Bactrian camel’s special metabolic pathways and unique detoxification capabilities are attributable to particularities of the CYP gene family. The Bactrian camel’s whole genome sequencing data were systemically analyzed and annotated, and then, CYP gene family was searched from the whole protein database and compared with CYP gene families of cattle, horse, chicken, and human. The total of 63 CYP gene copies were found in Bactrian camel’s whole genome and were classified into 17 families and 38 subfamilies. Among them, 9 multi-gene families were found, and CYP2, CYP3, and CPY4 have 27, 6, and 7 subfamilies, accounting for 43, 10, and 11% in camel CYP gene, respectively. In comparison with cattle, chicken, horse, and human, the distribution of CYP gene subfamilies in camel is different, with more CYP2J and CYP3A copies in the Bactrian camel, which may contribute to the Bactrian camel’s specific biological characteristics and metabolic pathways. Comparing to the cow, horse, chicken, and human CYP genes, the distribution of CYP gene subfamilies is distinct in the Bactrian camel. The higher copy number of CYP2J gene and CYP3A gene in Bactrian camel may be the important factors contributing to the distinct biological characteristics and metabolic pathways of Bactrian camels for adaptation to the harsh environments.     

High mitochondrial differentiation levels between wild and domestic Bactrian camels: a basis for rapid detection of maternal hybridization

Hybridization between wild species and their domestic congeners often threatens the gene pool of the wild species. The last wild Bactrian camel (Camelus ferus) populations in Mongolia and China are examples of populations facing such a hybridization threat. To address this key issue in the conservation of wild camels, we analysed wild, hybrid and domestic Bactrian camels (Camelus bactrianus) originating from Mongolia, China and Austria. Through screening of an 804‐base‐pair mitochondrial fragment, we identified eight mitochondrial haplotypes and found high sequence divergence (1.9%) between C. ferus and C. bactrianus. On the basis of a mitochondrial DNA sequence fixed difference, we developed a diagnostic PCR restriction fragment length polymorphism (PCR‐RFLP) assay to differentiate between wild and domestic camel samples. We applied the assay to 81 individuals and confirmed the origin of all samples including five hybrids with known maternal ancestry. The PCR‐RFLP system was effective for both traditional (blood, skin) and non‐invasive samples (faeces, hair), as well as for museum specimens. Our results demonstrate high levels of mitochondrial differentiation between wild and domestic Bactrian camels and that maternal hybridization can be detected by a rapid and reliable PCR‐RFLP system.     

Identification of a new Indian camel germplasm by microsatellite markers based genetic diversity and population structure of three camel populations

Camel invokes fascinating chapter of Indian desert history and is integral component of its ecosystem. Camel population has reached a crisis point after three decades of decline (75%) causing major concern to the policy makers. >28% of Indian camel is not yet characterized. It is imperative to describe country’s camel germplasm and its existing diversity for designing conservation plan. One such population is Sindhi, distributed along border with Pakistan. Twenty five microsatellite markers being valuable tool for estimating genetic diversity were selected to elucidate genetic variability and relationship of Sindhi with two registered camel breeds of India- Marwari and Kharai. The standard metrics of genomic diversity detected moderate variability in all the three populations. A total of 303 alleles with a mean of 8.116 ± 0.587 alleles per locus were found in total of 143 animals. Sindhi population had intermediate allelic diversity with 8.522 ± 1.063 alleles per locus. Corresponding values in Marwari and Kharai were 8.783 ± 0.962 and 7.043 ± 1.030, respectively. Genetic variability within the breeds was moderate as evidenced by the mean observed heterozygosity of 0.556 ± 0.025. Sindhi camel population harbors higher genetic variability (Ho = 0.594) as compared to the two registered camel breeds (Marwari, 0.543 and Kharai, 0.531). Mean expected heterozygosity under Hardy-Weinberg equilibrium was higher than the observed values across the three camel groups, indicating deviations from assumptions of this model. In fact, average positive F value of 0.084 to 0.206 reflected heterozygote deficiency in these populations. These Indian camel populations have not experienced serious demographic bottlenecks in the recent past. Differences among populations were medium and accounted for 7.3% of total genetic variability. Distinctness of three camel populations was supported by all the approaches utilized to study genetic relationships such as genetic distances, phylogenetic relationship, correspondence analysis, clustering method based on Bayesian approach and individual assignment. Sindhi camel population was clearly separated from two registered breeds of Indian camel. Results conclude Sindhi to be a separate genepool. Moderate genetic diversity provides an optimistic viewpoint for the survival of severely declining indigenous camel populations with appropriate planning strategies for conserving the existing genetic variation and to avoid any escalation of inbreeding.     

Use of assisted reproduction for the improvement of milk production in dairy camels (Camelus dromedarius)

Despite their production potential and ability to survive on marginal resources in extreme conditions, dromedaries have not been exploited as an important food source. Camels have not been specifically selected for milk production, and genetic improvement has been negligible. High individual variation in milk production both within the population and within breeds provides a good base for selection and genetic progress. In this paper, we discuss the possibilities and constraints of selective breeding for milk production in camels, and include a summary of the use of embryo transfer at the world's first camel dairy farm. Embryo transfer is an integral part of the breeding strategy at the camel dairy farm because it increases selection intensity and decreases the generation interval. Using high milk-producing camels as donors and low producing camels as recipients, 146 embryos were recovered (6.1 ± 1.0 embryos/donor; range: 0–18). Embryos were transferred non-surgically into 111 recipients (83 single and 28 twin embryo transfers). Pregnancy rate at 21 days and 5 months was 55% (61/111) and 45% (50/111), respectively. Finally, a total of 46 recipients delivered a live calf. These results document the utility of embryo transfer using high milk producing dromedaries as donors.     

Old World camels in a modern world – a balancing act between conservation and genetic improvement

Old World camels have served humans in cross‐continental caravans, transporting people and goods, connecting different cultures and providing milk, meat, wool and draught since their domestication around 3000–6000 years ago. In a world of modern transport and fast connectivity, these beasts of burden seem to be out‐dated. However, a growing demand for sustainable milk and meat production, especially in countries affected by climate change and increasing desertification, brings dromedaries (Camelus dromedarius) and Bactrian camels (Camelus bactrianus) back onstage and into the focus of animal breeders and scientists. In this review on the molecular genetics of these economically important species we give an overview about the evolutionary history, domestication and dispersal of Old World camels, whereas highlighting the need for conservation of wild two‐humped camels (Camelus ferus) as an evolutionarily unique and highly endangered species. We provide cutting‐edge information on the current molecular resources and on‐going sequencing projects. We cannot emphasise enough the importance of balancing the need for improving camel production traits with maintaining the genetic diversity in two domestic species with specific physiological adaptation to a desert environment.     

Identification of Bactrian camel cell lines using genetic markers

Iranian Bactrian camel population is less than 100 animals. Iranian biological resource center produced more than 50 Bactrian camel fibroblast cell lines as a somatic cell bank for conservation animal genetic resources. We compared two type markers performance, including 14 random amplified polymorphic DNA (RAPDs) (dominant) and eight microsatellite (co-dominant) for cell line identification, individual identification and investigation genetic structure of these samples. Based on clarity, polymorphism, and repeatability, four RAPD primers were selected for future analysis. Four RAPD primers and eight microsatellite markers have generated a total of 21 fragments and 45 alleles, respectively. RAPD primers revealed fragment size between 150 to 2000 bp and gene diversity since 0.27 (IBRD) to 0.46 (GC10), with an average of 0.37. Microsatellite markers generated number of alleles per locus ranged from 3 to 11, with an average of 5.62 alleles. The observed heterozygosity ranged from 0.359 (IBRC02) to 0.978 (YWLL08), and expected heterozygosity ranged from 0.449 (IBRC02) to 0.879 (YWLL08). Bottleneck analysis and curve showed that Bactrian camel population did not experience a low diversity. RAPD profiles were especially suitable for investigation population genetics. All primers generated novel and polymorphic fragments. Briefly, our results show that a multiplex PCR based on these markers can still be valuable and suitable for authentication of cell lines, investigating gene diversity and conservation genetic resources in Bactrian camel, while new technologies are continuously developed.     

Cloning and Molecular Characterization of HSL and Its Expression Pattern in HPG Axis and Testis during Different Stages in Bactrian Camel

Hormone-sensitive lipase (HSL) is a key enzyme in animal fat metabolism and is involved in the rate-limiting step of catalyzing the decomposition of fat and cholesterol. It also plays an important regulatory role in maintaining seminiferous epithelial structure, androgen synthesis and primordial germ cell differentiation. We previously reported that HSL is involved the synthesis of steroids in Bactrian camels, although it is unclear what role it plays in testicular development. The present study was conducted to characterize the biological function and expression pattern of the HSL gene in the hypothalamic pituitary gonadal (HPG) axis and the development of testis in Bactrian camels. We analyzed cloning of the cDNA sequence of the HSL gene of Bactrian camels by RT-PCR, as well as the structural features of HSL proteins, using bioinformatics software, such as ProtParam, TMHMM, Signal P 4.1, SOPMA and MEGA 7.0. We used qRT-PCR, Western blotting and immunofluorescence staining to clarify the expression pattern of HSL in the HPG axis and testis of two-week-old (2W), two-year-old (2Y), four-year-old (4Y) and six-year-old (6Y) Bactrian camels. According to sequence analysis, the coding sequence (CDS) region of the HSL gene is 648 bp in length and encodes 204 amino acids. According to bioinformatics analysis, the nucleotide and amino acid sequence of Bactrian camel HSL are most similar to those of Camelus pacos and Camelus dromedarius, with the lowest sequence similarity with Mus musculus. In adult Bactrian camel HPG axis tissues, both HSL mRNA and protein expression were significantly higher in the testis than in other tissues (hypothalamus, pituitary and pineal tissues) (p < 0.05). The expression of mRNA in the testis increased with age and was the highest in six-year-old testis (p < 0.01). The protein expression levels of HSL in 2Y and 6Y testis were clearly higher than in 2W and 4Y testis tissues (p < 0.01). Immunofluorescence results indicate that the HSL protein was mainly localized in the germ cells, Sertoli cells and Leydig cells from Bactrian camel testis, and strong positive signals were detected in epididymal epithelial cells, basal cells, spermatocytes and smooth muscle cells, with partially expression in hypothalamic glial cells, pituitary suspensory cells and pineal cells. According to the results of gene ontology (GO) analysis enrichment, HSL indirectly regulates the anabolism of steroid hormones through interactions with various targets. Therefore, we conclude that the HSL gene may be associated with the development and reproduction of Bactrian camels in different stages of maturity, and these results will contribute to further understanding of the regulatory mechanisms of HSL in Bactrian camel reproduction.     

Giant cells in the placenta of the bactrian camel in the fetal period of development]

Polyploid giant cells resulting from differentiation of normal cytotrophoblast cells were found in the composition of the placenta cytotrophoblast of the Bactrian camel. In the placenta of the Bactrian camel the transformation of cytotrophoblast cells into multinuclear giant cells is realized via endomitotic polyploidization and distinctly reflects active synthetic processes occurring in these cells related with the trophic function of the placenta. It may be supposed that giant cells of the placenta of female Bactrian camels also participate in the endocrinous function of the placenta. Their histolitical function is completely excluded.     

Genetic diversity and differentiation of dromedarian camel of India

Estimation of genetic variability and relationship among different livestock breeds is important for management of genetic resources for their sustainable utilization and conservation. This is more important when the livestock species, like camel, have shown a sharp decline in head count during the last decade. In the present study we estimated genetic variability and relationship among four camel breeds of India using 23 microsatellite loci. A total of 252 alleles were observed across all the four populations with mean number of alleles per locus as 8.04, 7.30, 6.39, and 7.43 for Bikaneri, Jaisalmeri, Kutchi, and Mewari breeds, respectively. The mean observed heterozygosity of the four breeds were 0.58, 0.57, 0.56, and 0.60 for Bikaneri, Jaisalmeri, Kutchi, and Mewari breeds, respectively and were lower than expected heterozygosity values. The mean estimates of F statistics were 0.227+/-0.044 (F(IT)), 0.157+/-0.038 (F(IS)), and 0.082+/-0.019 (F(ST)). The values were significantly different from zero for all the three measures and point towards the existence of population structure and moderate differentiation in four camel breeds. The exact test also indicated significant population differentiation (P < 0.001). The analysis of molecular variance revealed 12% of the variation attributed to among populations and 88% within populations. Sixty-nine percent of the individuals could be correctly assigned using "leave one out" procedure. All the individuals of Mewari and 42 out of 44 Jaisalmeri were correctly assigned. The existence of strong population structure in Jaisalmeri and Mewari camel was further substantiated by Nei's standard genetic distance as well as interindividual allele sharing distance. Thus these two breeds owing to selection for specific traits are distinct from other camel breeds.     

Variability of Some Milk-Associated Genes and Proteins in Several Breeds of Saudi Arabian Camels

To gain knowledge on the molecular basis of diversity of several clans of Saudi camel (Camelus dromedarius) characterization of these animals was conducted at both genetic and protein levels. To this end, blood and milk samples were collected from several camel breeds at different Saudi Arabia locations (northern Jeddah, Riyadh, and Alwagh governorates). Genomic DNA was extracted from blood of four Saudi camel breeds (Majahem, Safra, Wadha, and Hamara), and DNA fragments of the casein and α-lactalbumin genes were amplified. The retrieved DNA sequences were analyzed for genetic variability. The inter-simple sequence repeat technique was used for confirming the relationships among the analyzed camel breeds, and the PCR–RFLP with two restriction enzymes was utilized for exploring their molecular variations. The number of haplotypes, gene diversity, nucleotide diversity, average number of nucleotide differences, and sequence conservation were calculated for all the analyzed DNA sequences. These analyses revealed the presence of several single nucleotide polymorphisms in the analyzed DNA sequences. A group of neighbor joining trees was built for inferring the evolutionary variations among the studied animals. Protein profiling of milk from different camel clans was also conducted, and differences between and within the Saudi camel clans were easily found based on the isoelectric focusing (IEF) profiles using ampholytes with different IEF range. This study revealed that analyzed camel breeds show low levels of genetic differences. This may be a reflection of the evolutionary history of C. dromedarius that was domesticated based on a highly homogeneous ancestor ecotype.     

New Pliocene remains of Camelus grattardi (Mammalia,Camelidae) from the Shungura Formation,Lower Omo Valley,Ethiopia, and the evolution of African camels

Abstract

Camels are exceptionally rare in the Plio-Pleistocene fossil record of Africa, hindering attempts to understand the evolution of this family on the continent. Here we describe recently collected camel specimens from the Shungura Formation, Lower Omo Valley, Ethiopia, and attribute these remains to Camelus grattardi. The new specimens date to the late Pliocene (~3 to 2.6 Ma) and consist of three upper molars, one upper premolar, and two proximal metatarsals. The dental specimens confirm this species’ small P4 relative to its molars, a trait that differs significantly from all extant and fossil Old World camels. The metatarsals indicate that C. grattardi was similar in size to the living Bactrian camel, C. bactrianus. Phylogenetically, we find no suitable ancestor, sister, or descendant of the eastern African fossil camel, which suggests greater lineage diversity in Plio-Pleistocene Camelus than previously recognised. Microwear analyses suggest that C. grattardi was likely a mixed-feeder preferring browse, which is consistent with carbon isotopes of enamel from the Turkana Basin. A review of the fossil record of African camels suggests no clear paleoenvironmental association, as fossil camels occur in a range of environments from dry savannas with no permanent water bodies to closed woodlands along the paleo-Omo River.     

Genetic characterization of four native Italian shepherd dog breeds and analysis of their relationship to cosmopolitan dog breeds using microsatellite markers

Very little research into genetic diversity of Italian native dog breeds has been carried out so far. In this study we aimed to estimate and compare the genetic diversity of four native Italian shepherd dog breeds: the Maremma, Bergamasco, Lupino del Gigante and Oropa shepherds. Therefore, some cosmopolitan dog breeds, which have been widely raised in Italy for a long time past, have also been considered to check possible influence of these dog populations on the Italian autochthonous breeds considered here. A total of 212 individuals, belonging to 10 different dog breeds, were sampled and genotyped using 18 autosomal microsatellite loci. We analyzed the genetic diversity of these breeds, within breed diversity, breed relationship and population structure. The 10 breeds considered in this study were clearly genetically differentiated from each other, regardless of current population sizes and the onset of separate breeding history. The level of genetic diversity explained 20% of the total genetic variation. The level of H_E found here is in agreement with that found by other studies. The native Italian breeds showed generally higher genetic diversity compared with the long established, well-defined cosmopolitan dog breeds. As the Border Collie seems closer to the Italian breeds than the other cosmopolitan shepherd dogs considered here, a possible utilization of this breed to improve working performance in Italian traditional working shepherd dogs cannot be ignored. The data and information found here can be utilized in the organization of conservation programs planned to reduce inbreeding and to minimize loss of genetic variability.     

双峰驼睾丸和隐睾细胞外基质相关蛋白的组织化学特征及超微结构比较

为探索细胞外基质相关蛋白在隐睾双峰驼的分布情况及其组织化学特征,应用电镜技术和多种组织化学方法比较了隐睾和正常睾丸的超微结构,组织化学特点及层粘连蛋白（LN）、Ⅳ型胶原（Col Ⅳ）和硫酸乙酰肝素糖蛋白（HSPG）的分布特征。结果显示：(1)与正常睾丸间质结构相比,光镜下隐睾生精小管发育不全,间质内胶原纤维稀疏,网状纤维分布明显,间质血管及生精小管固有膜PAS及AB-PAS阳性反应较弱。电镜下,隐睾生精上皮基膜明显增生,外围I型胶原纤维较少,管周肌样细胞不典型;间质毛细血管及Leydig细胞周围纤维细胞多见,而正常睾丸在间质毛细血管及Leydig细胞周围多分布有成纤维细胞。(2) 免疫组织化学染色显示,正常睾丸组织的Col Ⅳ、LN及HSPG在Leydig细胞内均为强阳性表达,Col Ⅳ和LN在毛细血管内皮细胞强阳性表达,后者在Sertoli细胞的表达尤为明显,HSPG在精原细胞无表达;隐睾时Col Ⅳ、LN及HSPG在Leydig细胞内阳性表达均明显减弱,Col Ⅳ、LN在管周肌样细胞及毛细血管内皮细胞阳性表达也减弱明显,HSPG在精原细胞较强阳性表达,且在精子细胞呈强阳性表达。免疫组织化学图像分析结果显示,双峰驼正常睾丸组织中Col Ⅳ和LN的分布显著高于隐睾组织（P<0.05）,HSPG检测结果在正常睾丸与隐睾之间无统计学差异（P>0.01）。该研究表明,双峰驼隐睾生精小管发育异常,间质组织中合成胶原纤维的能力下降,睾丸细胞外基质的重要成分Col Ⅳ,LN与正常组差异显著与生精小管及Leydig细胞异常发育有关,而HSPG在隐睾生精上皮的强阳性表达与精原细胞发育不成熟密切相关。