Microsatellite markers based genetic diversity and bottleneck studies in Zanskari pony

Genetic diversity in Zanskari pony breed was evaluated at 48 microsatellite loci using fifty adult, healthy and unrelated animals. Allele frequency data was used to detect genetic diversity and bottleneck. The estimated average number of alleles (±s.e.) was 8.5208±2.5010 with a total of 409 alleles. A high level of genetic diversity within this breed was observed in terms of number of alleles, observed heterozygosity (0.6763±0.1704), expected Leven's heterozygosity (0.7724±0.795), expected Nei's heterozygosity (0.7644±0.0787) and polymorphism information content (>0.5). In-breeding coefficient (F(is)) was 0.115±0.0209, suggesting moderately high in-breeding in Zanskari breed. Although analysis of bottleneck revealed no bottleneck in recent past but population of Zanskari ponies has decreased drastically and only a few thousand pure-bred animals are left. The information is useful for proposing effective population management strategies for future.     

Genetic diversity and relationships of endangered Spanish cattle breeds

Information on the genetic structure and variability of autochthonous livestock breeds is essential for effective conservation programs. Here we present a molecular characterization on the basis of 30 microsatellite markers of 5 Spanish endangered cattle breeds Betizu (BET), Mallorquina (MAL), Menorquina, Monchina (MON), and Serrana de Teruel (ST) and of 2 fighting bull populations, Casta Navarra (CN) and Casta Vistahermosa. The feral and critically endangered BET is divided into 2 subpopulations, one of which has exceptionally low diversity values. A low number of alleles was also observed in the island population MAL. Although the small population size and genetic drift have caused a considerable divergence between the breeds, phylogenetic analysis is in accordance with historical and geographical data. The 2 northern Spanish feral breeds BET and MON cluster together. The local fighting breed CN is relatively close to the more inbred Casta Vistahermosa, which is the progenitor of most other fighting bulls in Spain. Comparison with nonendangered breeds suggests admixture of Alpine and/or Pyrenean mountain cattle in the ST, which may contribute to the high level of linkage disequilibrium in this population.     

Genetic diversity and population structure of Chinese pony breeds using microsatellite markers

China is one of the principal origins of ponies in the world. We made a comprehensive analysis of genetic diversity and population structure of Chinese ponies based on 174 animals of five indigenous Chinese pony breeds from five provinces using 13 microsatellite markers. One hundred and forty-four alleles were detected; the mean number of effective alleles among the pony breeds ranged from 5.38 (Guizhou) to 6.78 (Sichuan); the expected heterozygosity ranged from 0.82 (Guizhou) to 0.85 (Debao, Sichuan). Although abundant genetic variation was found, the genetic differentiation was low between the ponies, with 6% total genetic variance among the different breeds. All the pairwise F(ST) values were significant; they varied from 0.0424 for the Sichuan-Yunnan pair to 0.0833 for the Guizhou-Sichuan pair. All five pony breeds deviated from Hardy-Weinberg equilibrium, except the Yunnan pony. Phylogenetic trees of the five pony breeds based on genetic distances were constructed using a neighbor-joining method. The Sichuan and Yunnan ponies were grouped into the same branch, with a high bootstrap support value (97%). Guizhou and Ningqiang ponies were clustered into the same branch with a bootstrap value of 56%, whereas the Debao pony was placed in a separate group, with a bootstrap value of 56%. This grouping pattern was supported by genetic structure analysis.     

Genetic diversity and bottleneck studies in the Marwari horse breed

Genetic diversity within the Marwari breed of horses was evaluated using 26 different microsatellite pairs with 48 DNA samples from unrelated horses. This molecular characterisation was undertaken to evaluate the problem of genetic bottlenecks also, if any, in this breed. The estimated mean (± s.e.) allelic diversity was 5.9 (± 2.24), with a total of 133 alleles. A high level of genetic variability within this breed was observed in terms of high values of mean (±s.e.) effective number of alleles (3.3 ± 1.27), observed heterozygosity (0.5306 ± 0.22), expected Levene’s heterozygosity (0.6612 ± 0.15), expected Nei’s heterozygosity (0.6535 ± 0.14), and polymorphism information content (0.6120 ± 0.03). Low values of Wright’s fixation index, F_IS (0.2433 ± 0.05) indicated low levels of inbreeding. This basic study indicated the existence of substantial genetic diversity in the Marwari horse population. No significant genotypic linkage disequilibrium was detected across the population, suggesting no evidence of linkage between loci. A normal ‘L’ shaped distribution of mode-shift test, non-significant heterozygote excess on the basis of different models, as revealed from Sign, Standardized differences and Wilcoxon sign rank tests as well as non-significantM ratio value suggested that there was no recent bottleneck in the existing Marwari breed population, which is important information for equine breeders. This study also revealed that the Marwari breed can be differentiated from some other exotic breeds of horses on the basis of three microsatellite primers.     

Genetic diversity in the Churra tensina and Churra lebrijana endangered Spanish sheep breeds and relationship with other Churra group breeds and Spanish mouflon

The aim of the present study was to estimate the genetic intra-breed variability of Churra tensina and Churra lebrijana endangered breeds and to establish genetic relationships with Churra, Latxa and Merino breeds, as well as Spanish mouflon, by using 28 microsatellite markers, to provide useful information for their conservation. Allele frequencies and heterozygosity revealed high genetic variation in the two endangered breeds despite their small population size. Estimates of inbreeding coefficient (F_IS) were significant for all breeds studied, except for Churra lebrijana breed. The highest inbreeding coefficient (F_IS = 0.143) was found in the Spanish mouflon. Genetic differentiation tests (F_ST = 0.121) and assignment of individuals to populations indicated the existence of defined breed populations, and low genetic flow between these breeds. The highest pairwise Reynolds distance (D_R) values were observed between Mouflon and the domestic sheep breeds. Considering only domestic sheep breeds, the Churra lebrijana breed showed the highest pairwise D_R values_. The lowest values were found between Latxa and the other domestic sheep, except for Churra lebrijana. Results of pairwise D_R values, as well as phylogenetic tree and bottleneck analysis showed an important genetic isolation of the Churra lebrijana breed from the other Churra types, and genetic signatures of a demographic bottleneck. Finally, structure analysis of populations detected a population subdivision in the Latxa sheep breed. In conclusion, this study presents valuable insight into the existing genetic variability of two Spanish endangered breeds, as well as the first study in Spanish mouflon based on microsatellite analysis. The high degree of variability demonstrated in Churra tensina and Churra lebrijana implies that these populations are rich reservoirs of genetic diversity.     

Genetic diversity and variability in Alpine sheep breeds

The aim of this study was to analyze, through the use of microsatellite molecular markers, the genetic diversity and variability of nine Alpine sheep breeds reared in Italy (Bergamasca, Biellese, Schwarzbraunes Bergschaf, Tiroler Bergschaf, and Schnalserschaf), Germany (Brillenschaf and Weisses Bergschaf) and Slovenia (Bovška and Jezerzkǒ-Solčavska), and one Italian crossbreed population. Allelic richness was rather high in each breed highlighting a considerable genetic diversity. However, the study evidenced a significant departure from Hardy–Weinberg equilibrium in all analyzed breeds caused by a heterozygote deficiency. Such lack seems to be caused both to the presence of population substructure within breed and to a rather high level of inbreeding. The genetic differentiation among breed was rather low (F_ST = 0.057) but significant. The clustering analysis performed with STRUCTURE detected the presence of eight clusters; Schwarzbraunes Bergschaf and Tiroler Bergschaf were grouped together and Biellese and Bergamasca as well, evidencing a high similarity of their genetic make up. Reynolds’ genetic distance estimates confirmed the close relationship between these pairs of breeds. Moreover, Bovška, Jezerzkǒ-Solčavska and Brillenschaf resulted rather similar as expected according to their region of origin and to their common ancestors. Molecular coancestry confirmed as well such findings, suggesting that the studied breeds had origin from different ancestral populations. Concluding, microsatellite resulted a useful tool to investigate breed variability and to characterize Alpine sheep breeds. Obtained findings suggest the need to set up a conservation plan aiming to safeguard and increase the genetic variability of the studied breeds compromised by the high level of inbreeding. Microsatellites genotyping could help to monitor breed variability and to organize matings.     

Genetic diversity of eleven European pig breeds

A set of eleven pig breeds originating from six European countries, and including a small sample of wild pigs, was chosen for this study of genetic diversity. Diversity was evaluated on the basis of 18 microsatellite markers typed over a total of 483 DNA samples collected. Average breed heterozygosity varied from 0.35 to 0.60. Genotypic frequencies generally agreed with Hardy-Weinberg expectations, apart from the German Landrace and Schwäbisch-Hällisches breeds, which showed significantly reduced heterozygosity. Breed differentiation was significant as shown by the high among-breed fixation index (overall F_ST = 0.27), and confirmed by the clustering based on the genetic distances between individuals, which grouped essentially all individuals in 11 clusters corresponding to the 11 breeds. The genetic distances between breeds were first used to construct phylogenetic trees. The trees indicated that a genetic drift model might explain the divergence of the two German breeds, but no reliable phylogeny could be inferred among the remaining breeds. The same distances were also used to measure the global diversity of the set of breeds considered, and to evaluate the marginal loss of diversity attached to each breed. In that respect, the French Basque breed appeared to be the most unique in the set considered. This study, which remains to be extended to a larger set of European breeds, indicates that using genetic distances between breeds of farm animals in a classical taxonomic approach may not give clear resolution, but points to their usefulness in a prospective evaluation of diversity.     

Genetic variability and bottleneck studies in Zalawadi, Gohilwadi and Surti goat breeds of Gujarat (India) using microsatellites

Indian goat breeds are recognized as an invaluable component of the world's goat genetic resources. Microsatellite pairs were chosen from the list suggested by International Society for Animal Genetics (ISAG) and amplified in two multiplexes (Set-I: 7 microsatellites and Set-II: 11 microsatellites) for automated fluorescence genotyping to assess bottleneck and analyze genetic variability and genetic distances within and between three goat breeds viz. Zalawadi, Gohilwadi and Surti. The observed number of alleles ranged from 4 (Oar JMP-29) to 15 (ILSTS-030 and -034) with a total of 178 alleles and mean of 9.89 alleles across the three breeds. The overall heterozygosity, PIC and Shannon index values were 0.61, 0.60 and 1.50 indicating high genetic diversity. The maximum observed heterozygosity was found in Gohilwadi and minimum in Surti goat breed. The Nei's standard genetic distance was minimum between Zalawadi and Gohilwadi, and maximum between Gohilwadi and Surti. Non-significant heterozygote excess on the basis of IAM, TPM and SMM models, as revealed from Wilcoxon sign-rank tests, along with a normal ‘L’-shaped distribution of mode-shift test, indicated no bottleneck in Zalawadi and Gohilwadi goat populations, whereas mild bottleneck in the recent past for Surti breed. This research on goat genetic diversity in Gujarat state provides valuable information on Zalawadi, Gohilwadi and Surti goat genetic resources, and will assist in developing a national plan for the conservation and utilization of indigenous goat breeds.     

Genetic diversity and differentiation in Portuguese cattle breeds using microsatellites

Genotype data from 30 microsatellites were used to assess genetic diversity and relationships among 10 native Portuguese cattle breeds, American Charolais and the Brazilian Caracú. Hardy–Weinberg equilibrium was observed for all loci/population combinations except for five loci in Brava de Lide and one locus in Alentejana that exhibited heterozygote deficiency. Estimates of average observed and expected heterozygosities, total number of alleles (TNA) per breed and mean number of alleles (MNA) per locus/population were obtained. A total of 390 alleles were detected. TNA among Iberian cattle ranged from 170 to 237 and MNA ranged from 5.67 to 8.07. The highest observed heterozygosities were found in the Caracú, Maronesa, Garvonesa and Arouquesa and the lowest in Brava de Lide and Mirandesa. Estimation of population subdivision using Wright's F_ST index showed that the average proportion of genetic variation explained by breed differences was 9%. Neighbour‐joining phylogenetic trees based on D_A distances showed that the genetic relationships of present‐day Portuguese native breeds are consistent with historical origins in the Brown Concave (Arouquesa, Mirandesa, Marinhoa) and Red Convex (Mertolenga, Alentejana, Garvonesa, Minhota) evolutionary groups. The Iberian Black Orthoide group, represented by Brava de Lide and Maronesa, and the Barrosã breed appeared to be more closely related to the Brown Concave group but may represent a separate lineage. The Caracú breed was not found to be closely associated with any of the native Portuguese breeds.     

Genetic diversity of three Chinese native sheep breeds

The sheep (Ovis aries L.) has been an important farm animal species since its domestication. A wide array of indigenous sheep breeds with abundant phenotypic diversity exists for domestication and selection. Therefore, assessing the genetic diversity of a local sheep resource using a multi-molecular system is helpful for maintaining and conserving those breeds. This study aimed to investigate the genetic diversity of three native Chinese sheep breeds (Tibetan sheep, Sishui Fur sheep, and Small-tailed Han sheep) using 15 microsatellite markers and the second exon of the DRA gene. In regards to the microsatellites, on average, 19 alleles per loci were observed among all individuals. Across loci, the HO within the population was 0.652 ± 0.022 in Tibetan sheep, 0.603 ± 0.023 in Small-tailed Han sheep and 0.635 ± 0.022 in SFS, and for most populations, the H _E and H _O were inconsistent. In addition, affluent private alleles within the breed indicated that the breeds have different domestication histories or sites. In regards to the 2 exon of the DRA gene, three haplotypes were constructed by seven single-nucleotide polymorphisms (SNPs), which were identified in the second DRA exon and inferred the potential for phenotypic variety in these Chinese native sheep. In summary, the current study reveals the importance of implementing effective conservation strategies for these three native Chinese sheep.     

Genetic variability in the Skyros pony and its relationship with other Greek and foreign horse breeds

In Greece, seven native horse breeds have been identified so far. Among these, the Skyros pony is outstanding through having a distinct phenotype. In the present study, the aim was to assess genetic diversity in this breed, by using different types of genetic loci and available genealogical information. Its relationships with the other Greek, as well as foreign, domestic breeds were also investigated. Through microsatellite and pedigree analysis it appeared that the Skyros presented a similar level of genetic diversity to the other European breeds. Nevertheless, comparisons between DNA-based and pedigree-based results revealed that a loss of genetic diversity had probably already occurred before the beginning of breed registration. Tests indicated the possible existence of a recent bottleneck in two of the three main herds of Skyros pony. Nonetheless, relatively high levels of heterozygosity and Polymorphism Information Content indicated sufficient residual genetic variability, probably useful in planning future strategies for breed conservation. Three other Greek breeds were also analyzed. A comparison of these with domestic breeds elsewhere, revealed the closest relationships to be with the Middle Eastern types, whereas the Skyros itself remained isolated, without any close relationship, whatsoever.     

Genetic diversity and conservation in a small endangered horse population

The Old Kladruber horses arose in the 17th century as a breed used for ceremonial purposes. Currently, grey and black coat colour varieties exist as two sub-populations with different recent breeding history. As the population underwent historical bottlenecks and intensive inbreeding, loss of genetic variation is considered as the major threat. Therefore, genetic diversity in neutral and non-neutral molecular markers was examined in the current nucleus population. Fifty microsatellites, 13 single nucleotide polymorphisms (SNPs) in immunity-related genes, three mutations in coat colour genes and one major histocompatibility (MHC-DRA) gene were studied for assessing genetic diversity after 15 years of conservation. The results were compared to values obtained in a similar study 13 years ago. The extent of genetic diversity of the current population was comparable to other breeds, despite its small size and isolation. The comparison between 1997 and 2010 did not show differences in the extent of genetic diversity and no loss of allele richness and/or heterozygosity was observed. Genetic differences identified between the black and grey sub-populations observed 13 years ago persisted. Deviations from the Hardy–Weinberg equilibrium found in 19 microsatellite loci and in five SNP loci are probably due to selective breeding. No differences between neutral and immunity-related markers were found. No changes in the frequencies of markers associated with two diseases, melanoma and insect bite hypersensitivity, were observed, due probably to the short interval of time between comparisons. It, thus, seems that, despite its small size, previous bottlenecks and inbreeding, the molecular variation of Old Kladruber horses is comparable to other horse breeds and that the current breeding policy does not compromise genetic variation of this endangered population.     

Genetic diversity and fitness in black-footed ferrets before and during a bottleneck

The black-footed ferret (Mustela nigripes) is an endangered North American carnivore that underwent a well-documented population bottleneck in the mid-1980s. To better understand the effects of a bottleneck on a free-ranging carnivore population, we used 24 microsatellite loci to compare genetic diversity before versus during the bottleneck, and compare the last wild population to two historical populations. We also compared genetic diversity in black-footed ferrets to that of two sibling species, the steppe polecat (Mustela eversmanni) and the European polecat (Mustela putorius). Black-footed ferrets during the bottleneck had less genetic diversity than steppe polecats. The three black-footed ferret populations were well differentiated (F(ST) = 0.57 +/- 0.15; mean +/- SE). We attributed the decrease in genetic diversity in black-footed ferrets to localized extinction of these genetically distinct subpopulations and to the bottleneck in the surviving subpopulation. Although genetic diversity decreased, female fecundity and juvenile survival were not affected by the population bottleneck.     

Genetic diversity of dog breeds: within-breed diversity comparing genealogical and molecular data

The genetic diversity of 61 dog breeds raised in France was investigated. Genealogical analyses were performed on the pedigree file of the French kennel club. A total of 1514 dogs were also genotyped using 21 microsatellite markers. For animals born from 2001 to 2005, the average coefficient of inbreeding ranged from 0.2% to 8.8% and the effective number of ancestors ranged from 9 to 209, according to the breed. The mean value of heterozygosity was 0.62 over all breeds (range 0.37–0.77). At the breed level, few correlations were found between genealogical and molecular parameters. Kinship coefficients and individual similarity estimators were, however, significantly correlated, with the best mean correlation being found for the Lynch & Ritland estimator ( r  =   0.43). According to both approaches, it was concluded that special efforts should be made to maintain diversity for three breeds, namely the Berger des Pyrénées, Braque Saint-Germain and Bull Terrier.     

Genetic diversity and population structure of three Indian horse breeds

The genetic relationships of three Indian horse breeds—Marwari, Spiti, and Kathiawari were studied by genotyping 96 individuals with 20 polymorphic microsatellite markers. A total of 157 alleles were detected across 20 polymorphic loci. The Marwari population showed the highest allelic diversity (A = 5.7 and Ar = 5.14), followed by Spiti (A = 4.9 and Ar = 4.74) and Kathiawari (A = 4.1 and Ar = 3.82). The gene diversity was highest in the Spiti population (He = 0.67), followed by Marwari (He = 0.66) and Kathiawari (He = 0.59). Within population inbreeding estimates (f) in Marwari, Spiti and Kathiawari breeds were 0.18, 0.08, and 0.07, respectively, suggesting high level of inbreeding in these breeds. Analysis of bottleneck revealed evidence of recent bottleneck in Spiti and Kathiawari populations. Pair-wise Fst analysis, AMOVA and assignment tests demonstrated high genetic differentiation and low gene flow between populations. The information about genetic diversity and population structure will be useful for the future development of effective breeding management in order to preserve these Indian horse breeds.     

Genetic diversity in Swiss goat breeds based on microsatellite analysis

Genetic diversity in eight Swiss goat breeds was estimated using PCR amplification of 20 bovine microsatellites on 20-40 unrelated animals per breed. In addition, the Creole breed from the Caribbean and samples of Ibex and Bezoar goat were included. A total of 352 animals were tested. The bovine microsatellites chosen amplified well in goat. The average heterozygosity within population was higher in domestic goat (0.51-0.58) than in Ibex (0.17) and Bezoar goat (0.19). Twenty-seven per cent of the genetic diversity in the total population could be attributed to differences between the populations. However, with the exclusion of Ibex from the total population, this proportion dropped to 17%. Principal component analysis showed that all Swiss goat breeds are closely related, whereas the Creole breed, Ibex and Bezoar goat are clearly distinct from all eight Swiss breeds.     

Genetic diversity and subdivision of 57 European and Middle-Eastern sheep breeds

The population structure and genetic diversity of 57 European and Middle Eastern marginal and cosmopolitan sheep breeds from 15 countries were analysed by typing 31 microsatellite markers. Mean unbiased expected heterozygosities ranged from 0.63 in British Exmoor Horn to 0.77 in Albanian Ruda. South-eastern European and Middle-Eastern sheep breeds were significantly more variable than northwestern and western European breeds. An overall heterozygote deficiency (f) across all loci was observed (P < 0.001), while genetic differentiation (theta) was 5.7%. Principal component analysis and Bayesian model-based clustering indicate a south-east to north-west cline, but also revealed distinct groups of Middle-Eastern fat-tailed sheep, south-eastern European sheep and north-western/western European sheep. Within the last group, two less-distinct clusters comprised the Merino-type and Alpine breeds respectively. The incomplete demarcations of most clusters probably reflects cross-breeding and/or upgrading.     

Genetic diversity of dog breeds: between-breed diversity, breed assignation and conservation approaches

Genetic relationships between 61 dog breeds were investigated, using a sampling of 1514 animals and a panel of 21 microsatellite markers. Based on the results from distance-based and Bayesian methods, breed constituted the main genetic structure, while groups including genetically close breeds showed a very weak structure. Depending on the method used, between 85.7% and 98.3% of dogs could be assigned to their breed, with large variations according to the breed. However, breed heterozygosity influenced assignment results differently according to the method used. Within-breed and between-breed diversity variations when breeds were removed were highly negatively correlated ( r  = −0.963, P  <   0.0001), because of the genetic structure of the breed set.