Combined use of principal component analysis and random forests identify population‐informative single nucleotide polymorphisms: application in cattle breeds

The genetic identification of the population of origin of individuals, including animals, has several practical applications in forensics, evolution, conservation genetics, breeding and authentication of animal products. Commercial high‐density single nucleotide polymorphism (SNP) genotyping tools that have been recently developed in many species provide information from a large number of polymorphic sites that can be used to identify population‐/breed‐informative markers. In this study, starting from Illumina BovineSNP50 v1 BeadChip array genotyping data available from 3711 cattle of four breeds (2091 Italian Holstein, 738 Italian Brown, 475 Italian Simmental and 407 Marchigiana), principal component analysis (PCA) and random forests (RFs) were combined to identify informative SNP panels useful for cattle breed identification. From a PCA preselected list of 580 SNPs, RFs were computed using ranking methods (Mean Decrease in the Gini Index and Mean Accuracy Decrease) to identify the most informative 48 and 96 SNPs for breed assignment. The out‐of‐bag (OOB) error rate for both ranking methods and SNP densities ranged from 0.0 to 0.1% in the reference population. Application of this approach in a test population (10% of individuals pre‐extracted from the whole data set) achieved 100% of correct assignment with both classifiers. Linkage disequilibrium between selected SNPs was relevant (r² > 0.6) only in few pairs of markers indicating that most of the selected SNPs captured different fractions of variance. Several informative SNPs were in genes/QTL regions that affect or are associated with phenotypes or production traits that might differentiate the investigated breeds. The combination of PCA and RF to perform SNP selection and breed assignment can be easily implemented and is able to identify subsets of informative SNPs useful for population assignment starting from a large number of markers derived by high‐throughput genotyping platforms.     

Genetic characterization of the Burlina cattle breed using microsatellites markers.

The present study was a contribution on the genetic characterization of the Burlina local cattle breed, and an approach to understanding the relationships between Burlina, Holstein Friesian and Brown Swiss which represent the majority of the dairy cattle reared nowadays in North-East Italy. The obtained results helped to clarify the genetic diversity and distinctiveness of Burlina population. In particular, the low genetic distance between Burlina and Holstein Friesian and the assignment of a moderate percentage of Burlina animals to Holstein Friesian suggested that crosses between them took place in the past, while crosses with Brown Swiss seemed to be less frequent. However, analyses of marker genotypes, showed a cluster with only Burlina individuals, which demonstrates the genetic distinctness of this breed. The Burlina breed showed the highest variability among the analysed breeds and its inbreeding coefficient was low. The data contribute to the feasibility of a conservation and selection programme for this breed and the results are useful for the implementation of a conservation strategy that should aim to conserve animals where the contribution from foreign breeds is as small as possible.     

Tuscany autochthonous cattle breeds: an original genetic resource investigated by AFLP markers

The aim of this study was to assess the genetic diversity of four autochthonous cattle breeds of Tuscany and their relationships in comparison with Italian Friesian and Italian Brown, using amplified fragment length polymorphism markers. A total of 212 individuals were genotyped with three primer combinations generating 102 polymorphic markers. Average expected heterozygosity ranged from 0.23 in Mucca Pisana to 0.26 in Chianina, Italian Friesian, Italian Brown and Maremmana. The differences resulted not significant (Kruskall–Wallis test, p = 0.416). Gst‐B index revealed that 86% of the total genetic variance is retained within population and only 14% is accounted by the between populations component. Multivariate analysis at individual and population level indicated that: (i) Calvana and Chianina are quite separate from the other breeds as an effect of the bottleneck experienced or as a signature of different origin; (ii) Podolian, Maremmana and Italian Brown clustered with the double purpose Mucca Pisana, revealing their contribution to its admixed genetic make up; (iii) Italian Friesian behaved always as out group. The ‘analysis of molecular variance’ recovered a significant subdivision clustering the six populations into three groups: Italian Friesian and Italian Brown versus Maremmana and Mucca Pisana versus Chianina and Calvana (6% of the total variance).     

Development of a genomic tool for breed assignment by comparison of different classification models: Application to three local cattle breeds

Assignment of individual cattle to a specific breed can often not rely on pedigree information. This is especially the case for local breeds for which the development of genomic assignment tools is required to allow individuals of unknown origin to be included to their herd books. A breed assignment model can be based on two specific stages: (a) the selection of breed-informative markers and (b) the assignment of individuals to a breed with a classification method. However, the performance of combination of methods used in these two stages has been rarely studied until now. In this study, the combination of 16 different SNP panels with four classification methods was developed on 562 reference genotypes from 12 cattle breeds. Based on their performances, best models were validated on three local breeds of interest. In cross-validation, 14 models had a global cross-validation accuracy higher than 90%, with a maximum of 98.22%. In validation, best models used 7,153 or 2,005 SNPs, based on a partial least squares-discriminant analysis (PLS-DA) and assigned individuals to breeds based on nearest shrunken centroids. The average validation sensitivity of the first two best models for the three local breeds of interest were 98.33% and 97.5%. Moreover, results reported in this study suggest that further studies should consider the PLS-DA method when selecting breed-informative SNPs.     

Genetic variability of three Italian cattle breeds determined by parameters based on probabilities of gene origin

In this study, we have investigated the genetic variability of three Italian cattle breeds, autochthonous of the Tuscany region (Chianina, Maremmana and Mucca Pisana), using the parameters based on probabilities of gene origin: f _e, f _a and N _g. Compared with Chianina (N _g=53.9) and Maremmana (N _g=79.6), Mucca Pisana was found to be the genetically smaller (N _g=10.1) and less diverse population. The genetic variability in Chianina population was lower than in Maremmana probably because of bottlenecks in its pedigree and genetic drift. However, the values found in these breeds were higher than those found in dairy cattle breeds, but lower than those found in beef cattle breeds.     

On the origin of the Slovenian Cika cattle

In 2002, it was discovered that several Cika cattle in the mountain areas of Slovenia had escaped the official policy of cross‐breeding. Here, we report a genetic characterization to assess their status as autochthonous breed. We compared genotypes for 14 microsatellite markers in 150 Cika cattle individuals with data from 16 Central European cattle breeds. We show that Cika cattle are genetically as diverse as other Eastern Alpine breeds, are more diverse than Austrian Simmental but less than the Balkan Busha cattle. STRUCTURE analysis showed Pinzgauer admixture in several individuals but also indicated a unique genetic identity for Cika. This analysis also allowed a selection of the most genetically pure Cika individuals as assessed by the panel of microsatellites. These original Cika cattle form an Eastern Alpine breed cluster together with Pinzgauer and Pustertaler cattle. Cika cattle should be considered as an authentic and valuable genetic resource, which offers clear opportunities for sustainable agriculture and landscape conservation in marginal and mountain areas.     

Genetic diversity among some cattle breeds in the Alpine area

Seven autochthonous Italian cattle breeds bred in the alpine area (Aosta Black Pied, Aosta Red Pied, Aosta Chestnut, Oropa Red Pied, Grey Alpine, Rendena and Burlina) were investigated in order to characterise their genetic structure and to study their phylogenetic origin. Two cattle breeds from Germany (Original German Brown and Holstein) and four from Switzerland (Simmental, Herens, Evolene and Brown Swiss) were included in the study in order to determine the genetic diversity existing among Italian local breeds, similar breeds bred on the other side of the Alps and in the well known Holstein.
Seventeen microsatellites, of the internationally accepted panel for the study of cattle biodiversity, were used for the analysis.
Microsatellites were highly polymorphic with a mean number of 5,5 alleles (ranging from 2 to 12 per locus). For each locus, allelic frequencies, heterozygosity (H) and the Polymorphism Information Content (PIC) were computed. The genetic equilibrium according to Hardy–Weinberg was calculated for each population and for each locus. Allele frequencies were used to estimate genetic distances and to draw a phylogenetic tree. The two closest breeds were Aosta Red Pied and Aosta Black Pied, while the two genetically most different were Holstein and Aosta Chestnut. Aosta valley breeds, Evolene and Herens constituted a tight cluster in the phylogenetic consensus tree. Principal component analysis showed a similar pattern for all the alpine breeds, while Holstein and Original German Brown were far away. The genetic differences among breeds were in accordance with their geographical and historical origins.     

Genetic diversity and assignment tests among seven French cattle breeds based on microsatellite DNA analysis

Genetic variability and relationships among six native French cattle breeds (Abondance, Tarentaise, Villard de Lans, Montbéliarde, Limousin, and Charolais) and one foreign breed (Holstein) were investigated using 23 microsatellite markers. These breeds were also compared with four Swiss breeds genotyped in a previously published study. Interestingly, the French alpine breeds have smaller population sizes but showed higher genetic variability than the larger Holstein breed. Neighbor-joining trees and PCA (principal components analysis) showed that alpine breeds tend to cluster together. Abondance and Tarentaise breeds were closely related, whereas the Holstein was highly differentiated from all breeds analyzed. Two different assignment tests for determining the breed of origin of individuals were compared: "direct" and "exclusion-simulation" approaches. The exclusion-simulation significance test correctly assigns fewer individuals than the direct approach but provides a confidence level (e.g., P < 0.01) for each individual being assigned. Accurate assignment with high statistical confidence is required for animal traceability. Unfortunately, the accuracy of assignment greatly decreases as the threshold level of confidence of assignment increases (e.g., from P < 0.05 to P < 0.001). Assignment accuracy also greatly declines as the level of population differentiation decreases below the level often found between related breeds (e.g., F(ST) < 0.1).     

Genetic differences among native and modern cattle breeds in Lithuania based on milk protein loci polymorphism

The intrabreed and interbreed genetic diversity of Lithuanian cattle breeds - two native, namely Lithuanian Light Grey and Lithuanian White-Backed and two modern, namely Lithuanian Red and Lithuanian Black and White was investigated by determination of genetic markers: 4 milk protein systems, Alpha(s1)-casein, Kappa-casein, Beta-casein and Beta-lactoglobulin, which are comprised of 12 different milk protein types. According to results, the B type of Alpha(s1)-casein was found as predominant in all four studied breeds. The most common A and B types of Kappa-casein were found at high frequency in all investigated cattle breeds. All investigated Lithuanian dairy cattle breeds had high frequency of Beta-lactoglobulin whey protein B types, with the highest frequency in Lithuanian Red breed, and the lowest in Lithuanian Light Grey. After investigation the diversity of alleles and genotypes of milk proteins in Lithuanian dairy cattle breeds was determined that, Lithuanian Red breed was distinguished private C allele and BC genotype of Beta-lactoglobulin and CC genotype of Alpha(s1)-casein. The interbreed genetic diversity was estimated by a principal component analysis (PCA). The first principal component (PC) explains 63.39% and the second principal component (PC) explains 33.67% of the genetics diversity between the breeds. Principal component analysis, suggests the hypothesis that native Lithuanian White Backed and Lithuanian Light Grey breeds still have traits tracing to old native populations.     

Conservation priorities for the different lines of Dutch Red and White Friesian cattle change when relationships with other breeds are taken into account

From a genetic point of view, the selection of breeds and animals within breeds for conservation in a national gene pool can be based on a maximum diversity strategy. This implies that priority is given to conservation of breeds and animals that diverge most and overlap of conserved diversity is minimized. This study investigated the genetic diversity in the Dutch Red and White Friesian (DFR) cattle breed and its contribution to the total genetic diversity in the pool of the Dutch dairy breeds. All Dutch cattle breeds are clearly distinct, except for Dutch Friesian breed (DF) and DFR and have their own specific genetic identity. DFR has a small but unique contribution to the total genetic diversity of Dutch cattle breeds and is closely related to the Dutch Friesian breed. Seven different lines are distinguished within the DFR breed and all contribute to the diversity of the DFR breed. Two lines show the largest contributions to the genetic diversity in DFR. One of these lines comprises unique diversity both within the breed and across all cattle breeds. The other line comprises unique diversity for the DFR but overlaps with the Holstein Friesian breed. There seems to be no necessity to conserve the other five lines separately, because their level of differentiation is very low. This study illustrates that, when taking conservation decisions for a breed, it is worthwhile to take into account the population structure of the breed itself and the relationships with other breeds.     

The Agersoe cattle: the last remnants of the Danish island cattle (Bos taurus)?

A phenotypically interesting strain of cattle existed on the small island of Agersoe, on the west coast of Zealand, Denmark, in the beginning of the last decade. The cattle share a great resemblance to the extinct Danish breed, the Island cattle. The objective of this study was to genetically characterize the Agersoe cattle, using microsatellites, amplified fragment length polymorphism (AFLP) and mtDNA markers, and analyse the genetic variability within the breed and the genetic relationship to 14 European breeds with focus on the Red Danish and Jutland breed. The results show diversity in nuclear markers comparable to that of modern breeds and that the Agersoe cattle are separable from the two native breeds. The absence of inbreeding and the degree of genetic diversity are taken as a sign of recent admixture. The Agersoe cattle did not exhibit a consistent association with any of the European breeds. Several arguments based on this survey have been put forward in favour of characterizing the Agersoe cattle as being the last remnants of the Danish Island Cattle.     

Genetic diversity, structure, and breed relationships in Iberian cattle

In Iberia there are 51 officially recognized cattle breeds of which 15 are found in Portugal and 38 in Spain. We present here a comprehensive analysis of the genetic diversity and structure of Iberian cattle. Forty of these breeds were genotyped with 19 highly polymorphic microsatellite markers. Asturiana de los Valles displayed the greatest allelic diversity and Mallorquina the least. Unbiased heterozygosity values ranged from 0.596 to 0.787. The network based on Reynolds distances was star-shaped with few pairs of interrelated breeds and a clear cluster of 4 breeds (Alistana/Arouquesa/Marinhoa/Mirandesa). The analysis of the genetic structure of Iberian cattle indicated that the most probable number of population clusters included in the study would be 36. Distance results were supported by the STRUCTURE software indicating a relatively recent origin or possible crossbreeding or both between pairs or small groups of breeds. Five clusters included 2 different breeds (Betizu/Pirenaica, Morucha/Avile?a, Parda de Monta?a/Bruna de los Pirineos, Barros?/Cachena, and Toro de Lidia/Brava de Lide), 3 breeds (Berrenda en Negro, Negra Andaluza, and Mertolenga) were divided in 2 independent clusters each, and 2 breeds were considered admixed (Asturiana de los Valles and Berrenda en Colorado). Individual assignation to breeds was not possible in the 2 admixed breeds and the pair Parda de Monta?a/Bruna de los Pirineos. The relationship between Iberian cattle reflects their geographical origin rather than their morphotypes. Exceptions to this geographic clustering are most probably a consequence of crossbreeding with foreign breeds. The relative genetic isolation within their geographical origin, the consequent genetic drift, the adaptation to specific environment and production systems, and the influence of African and European cattle have contributed to the current genetic status of Iberian cattle, which are grouped according to their geographical origin. The greater degree of admixture observed in some breeds should be taken into account before using molecular markers for genetic assignment of individuals to breeds.     

Genetic diversity of Afrikaner cattle in southern Africa

The Afrikaner is an indigenous South African breed of “Sanga” type beef cattle along with breeds such as the Drakensberger and Nguni. Six composite breeds have been developed from crosses with the Afrikaner. Additionally, Afrikaner has been the base from which exotic breeds were established in South Africa through backcrossing. The study examined genetic diversity of Afrikaner cattle by genotyping 1257 animals from 27 herds in different geographic areas of South Africa and Namibia using 11 microsatellite markers. Multiple-locus assignment, performed using the Bayesian clustering algorithm of STRUCTURE, revealed three underlying genotypic groups. These groups were not geographically localized. Across herds and markers, the proportion of unbiased heterozygosity ranged from 0.49 to 0.72 averaging 0.57; mean number of alleles per locus ranged from 3.18 to 7.09, averaging 4.81; and allelic richness ranged from 2.35 to 3.38, averaging 2.67. It is concluded that a low inbreeding level of 2.7% and a moderate to high degree of variation still persists within the Afrikaner cattle breed, despite the recent decline in numbers of animals.     

The effects of dog breed development on genetic diversity and the relative influences of performance and conformation breeding

Genetic diversity was compared among eight dog breeds selected primarily for conformation (Standard Poodle, Italian Greyhound and show English Setter), conformation and performance (Brittany), predominantly performance (German Shorthaired and Wirehaired Pointers) or solely performance (field English Setter and Red Setter). Modern village dogs, which better reflect ancestral genetic diversity, were used as the standard. Four to seven maternal and one to two Y haplotypes were found per breed, with one usually dominant. Diversity of maternal haplotypes was greatest in village dogs, intermediate in performance breeds and lowest in conformation breeds. Maternal haplotype sharing occurred across all breeds, while Y haplotypes were more breed specific. Almost all paternal haplotypes were identified among village dogs, with the exception of the dominant Y haplotype in Brittanys, which has not been identified heretofore. The highest heterozygosity based on 24 autosomal microsatellites was found in village dogs and the lowest in conformation (show) breeds. Principal coordinate analysis indicated that conformation‐type breeds were distinct from breeds heavily used for performance, the latter clustering more closely with village dogs. The Brittany, a well‐established dual show and field breed, was also genetically intermediate between the conformation and performance breeds. The number of DLA‐DRB1 alleles varied from 3 to 10 per breed with extensive sharing. SNPs across the wider DLA region were more frequently homozygous in all pure breeds than in village dogs. Compared with their village dog relatives, all modern breed dogs exhibit reduced genetic diversity. Genetic diversity was even more reduced among breeds under selection for show/conformation.     

Detection of bovine leukocyte adhesion deficiency (BLAD) in Turkish native and Holstein cattle

The purpose of this work was to study whether the bovine leukocyte adhesion deficiency (BLAD) allele is present in native cattle breeds and the Holstein breed in Turkey. Blood samples were obtained from 120 Holstein, 20 Brown Swiss, 20 Anatolian Black, 20 Turkish Grey, 20 South Anatolian Red and 20 East Anatolian Red cattle. The isolated DNA materials were multiplied in PCR using the primer developed by Kriegesmann et al. (1997). In order to determine the area of mutation in PCR products, the PCR products were digested with TaqI endonuclease enzyme. The resulting fragments were analysed on 2% agarose gel for the absence of a TaqI restriction site. It was found that two of the Holstein cattle (a bull and a cow) were heterozygote BLAD carriers. There was no homozygote BLAD animal. The BLAD allele was not found in the other breeds used in the study. The mutant BLAD allele frequency in the 120 Holstein cattle calculations was 0.0084.     

Genetic diversity,population structure,and correlations between locally adapted zebu and taurine breeds in Brazil using SNP markers

Genetic diversity is one of the most important issues in studies on conservation of cattle breeds and endangered species. The objective of this study was to estimate the levels of genetic differentiation between locally adapted taurine (Bos taurus taurus) and zebu (Bos taurus indicus) breeds in Brazil, which were genotyped for more than 777,000 SNPs. The fixation index (F _ST), principal component analysis (PCA), and Bayesian clustering were estimated. The F _ST highlighted genetic differentiation between taurine and zebu breeds. The taurine lines, Caracu and Caracu Caldeano, had significant genetic differentiation (F _ST close to 5%) despite their recent selection for different uses (meat and milk). This genetic variability can be used for conservation of locally adapted animals, as well as for breeding programs on zebu breeds. Introgression of zebu in locally adapted breeds was identified, especially in Curraleiro Pé-Duro breed. The Gyr breed, however, had low breed purity at genomic level due to its very heterogeneous mixing pattern.     

Genetic conservation of five endangered Spanish donkey breeds

The genetic diversity of most livestock species is being reduced, and it is not possible to preserve all of these livestock breeds. In order to preserve as much of the genetic diversity as possible, we must first have a robust method of measuring genetic diversity among breeds. Three different methods of study that graphically represent relationships among breeds are presented; Weitzman's method, principal component analysis (PCA) and a neighbour‐joining tree with allele sharing. Diversity was evaluated on the basis of 15 microsatellite markers typed over a total of 513 DNA samples collected from five Spanish donkey breeds. Breed differentiation was confirmed by the clustering based on the genetic distances between individuals, which essentially grouped all individuals in discrete clusters. The genetic distance among breeds was used to measure the global diversity of the set in breeds considered, and to evaluate the marginal loss of diversity attached to each breed. The Catalana breed appeared to be the most ‘unique’ in the set considered. In addition to this, the usefulness of global evaluations of diversity using molecular markers to choose breeds is worthy of conservation.     

The impact of using old germplasm on genetic merit and diversity—A cattle breed case study

Artificial selection and high genetic gains in livestock breeds led to a loss of genetic diversity. Current genetic diversity conservation actions focus on long‐term maintenance of breeds under selection. Gene banks play a role in such actions by storing genetic materials for future use and the recent development of genomic information is facilitating characterization of gene bank material for better use. Using the Meuse‐Rhine‐Issel Dutch cattle breed as a case study, we inferred the potential role of germplasm of old individuals for genetic diversity conservation of the current population. First, we described the evolution of genetic merit and diversity over time and then we applied the optimal contribution (OC) strategy to select individuals for maximizing genetic diversity, or maximizing genetic merit while constraining loss of genetic diversity. In the past decades, genetic merit increased while genetic diversity decreased. Genetic merit and diversity were both higher in an OC scenario restricting the rate of inbreeding when old individuals were considered for selection, compared to considering only animals from the current population. Thus, our study shows that gene bank material, in the form of old individuals, has the potential to support long‐term maintenance and selection of breeds.     

日本牛的遗传资源

本文扼要回顾了日本牛种资源的发展历史;介绍了日本牛的遗传资源状况和各主要牛种的特征特性;从日本牛的遗传可变性和遗传距离等方面分析了日本牛品种之间的遗传关系;并展望了日本黑毛和牛、日本褐牛、日本短角牛、日本无角牛、见岛牛、口子岛牛等日本牛种资源的发展前景。     

The assessment of genetic diversity within and among the eight subpopulations of Japanese Black cattle using 52 microsatellite markers

Japanese Black cattle are at risk for genetic homogeneity due to intensive use of a few sires. Therefore, assessment of the actual genetic diversity of this breed is important for future breeding plans. In the present study, we investigated the genetic diversity within and among eight subpopulations of Japanese Black cattle using 52 microsatellite markers. The parameters for genetic diversity of Japanese Black cattle were comparable to those of other cattle breeds, suggesting that the relatively high genetic diversity of the breed. However, upon comparison among the eight subpopulations, the Hyogo subpopulation showed markedly low genetic diversity. The results of the pairwise F_ST values, phylogenetic network and structure analysis indicated that the Hyogo population has remarkably high level of genetic differentiation from other populations, while Yamagata, Niigata, Hiroshima and Kagawa populations have low levels of genetic differentiation. Furthermore, multidimensional scaling plots indicated that individuals in some subpopulations were separated from individuals in the other subpopulations. We conclude that while the overall genetic diversity of Japanese Black cattle is still maintained at a relatively high level, that of a particular subpopulation is significantly reduced, and therefore the effective population size of the breed needs to be controlled by correct mating strategies.