Genomic differentiation between swamp and river buffalo using a cattle high-density single nucleotide polymorphisms panel.

Buffalo (Bubalus bubalis) is an important livestock species in many tropical and subtropical regions. In recent decades, the interest in buffalo's milk have expanded and intensive buffalo farms start to emerge. However, breeding programs and population genetics information for this species is scarce or inexistent. The present study aims to test the suitability of the commercial high-density single nucleotide polymorphisms (SNP) genotyping panel, the Illumina BovineHD BeadChip, to estimate population genetics parameters, pedigree control and identification of common variants in major production candidate genes. From a total of 777 962 SNPs included in the panel, 20 479 were polymorphic in water buffalo at a call rate of 86% and an average expected heterozygosity (HE) of 0.306. From these, 357 were mapped within or around the flanking regions of several major candidate genes. A principal components analysis identified three different clusters, each representing pure swamp buffalo type, pure river buffalo type and admixed river buffalo. The hybrids between swamp and river buffalo were clearly identified as an intermediary cluster. The suitability of these SNPs data set for parentage and identity testing demonstrated that the combination of just 30 to 50 SNPs were enough to attain high probabilities of parentage exclusion (0.9999) in both types and identity (2.3×10-5 and 2.0×10-7) for river and swamp buffalo, respectively. Our analysis confirms the suitability of the BovineHD BeadChip to assess population structure, hybridization and identity of the water buffalo populations.

Differences in genetic structure assessed using Y-chromosome and mitochondrial DNA markers do not shape the contributions to diversity in African sires.

Up to 173 African sires belonging to 11 different subpopulations representative of four cattle groups were analysed for six Y-specific microsatellite loci and a mitochondrial DNA fragment. Differences in Y-chromosome and mtDNA haplotype structuring were assessed. In addition, the effect of such structuring on contributions to total genetic diversity was assessed. Thirty-five Y-chromosome and 71 mtDNA haplotypes were identified. Most Y-chromosomes analysed (73.4%) were of zebu origin (11 haplotypes). Twenty-two Y-haplotypes (44 samples) belonged to the African taurine subfamily Y2a. All mtDNA haplotypes belonged to the "African" taurine T1 haplogroup with 16 samples and nine haplotypes belonging to a recently identified subhaplogroup (T1e). Median-joining networks showed that Y-chromosome phylogenies were highly reticulated with clear separation between zebu and taurine clusters. Mitochondrial haplotypes showed a clear star-like shape with small number of mutations separating haplotypes. Mitochondrial-based FST -statistics computed between cattle groups tended to be statistically non-significant (p > .05). Most FST values computed among groups and subpopulations using Y-chromosome markers were statistically significant. AMOVA confirmed that divergence between cattle groups was only significant for Y-chromosome markers (ΦCT  = 0.209). At the mitochondrial level, African sires resembled an undifferentiated population with individuals explaining 94.3% of the total variance. Whatever the markers considered, the highest contributions to total Nei's gene diversity and allelic richness were found in West African cattle. Genetic structuring had no effect on patterns of contributions to diversity.

Lack of haplotype structuring for two candidate genes for trypanotolerance in cattle.

Bovine trypanotolerance is a heritable trait associated to the ability of the individuals to control parasitaemia and anaemia. The INHBA (BTA4) and TICAM1 (BTA7) genes are strong candidates for trypanotolerance-related traits. The coding sequence of both genes (3951 bp in total) were analysed in a panel including 79 Asian, African and European cattle (Bos taurus and B. indicus) to identify naturally occurring polymorphisms on both genes. In general, the genetic diversity was low. Nineteen of the 33 mutations identified were found just one time. Seventeen different haplotypes were defined for the TICAM1 gene, and 9 and 12 were defined for the exon 1 and the exon 2 of the INHBA gene, respectively. There was no clear separation between cattle groups. The most frequent haplotypes identified in West African taurine samples were also identified in other cattle groups including Asian zebu and European cattle. Phylogenetic trees and principal component analysis confirmed that divergence among the cattle groups analysed was poor, particularly for the INHBA sequences. The European cattle subset had the lowest values of haplotype diversity for both the exon1 (monomorphic) and the exon2 (0.077 ± 0.066) of the INHBA gene. Neutrality tests, in general, did not suggest that the analysed genes were under positive selection. The assessed scenario would be consistent with the identification of recent mutations in evolutionary terms.

Lack of mitochondrial DNA structure in Balkan donkey is consistent with a quick spread of the species after domestication.

A total of 132 mtDNA sequences from 10 Balkan donkey populations were analysed to ascertain their regional genetic structure and to contribute to the knowledge of the spreading of the species after domestication. The Balkan donkey sequences were compared with those from 40 Burkina Faso donkeys as an African outgroup to account for possible local Balkan scenarios. The 172 sequences gave 62 different haplotypes (55 in Balkan donkey). Virtually all the analysed populations had haplotypes assigned to either Clade 1 or Clade 2 even though the relative proportion of Clade 1 or 2 haplotypes differed across populations. Geographical maps constructed using factors computed via principal component analysis showed that the Balkan donkey populations are not spatially structured. AMOVA confirmed a lack of genetic structure in Balkan donkey mtDNA. Balkan populations were poorly differentiated (ΦST  = 0.071). Differentiation between the Balkan donkey and the African outgroup also was low. The lack of correspondence between geographical areas and maternal genetic structure is consistent with the hypothesis suggesting a very quick spread of the species after domestication. The current research illustrates the difficulties to trace routes of expansion in donkey, as the species has no geographical structure.

Identification of large selective sweeps associated with major genes in cattle.

Selection for new favorable variants can lead to selective sweeps. However, such sweeps might be rare in the evolution of different species for which polygenic adaptation or selection on standing variation might be more common. Still, strong selective sweeps have been described in domestic species such as chicken lines or dog breeds. The goal of our study was to use a panel of individuals from 12 different cattle breeds genotyped at high density (800K SNPs) to perform a whole-genome scan for selective sweeps defined as unexpectedly long stretches of reduced heterozygosity. To that end, we developed a hidden Markov model in which one of the hidden states corresponds to regions of reduced heterozygosity. Some unexpectedly long regions were identified. Among those, six contained genes known to affect traits with simple genetic architecture such as coat color or horn development. However, there was little evidence for sweeps associated with genes underlying production traits.

Mitochondrial DNA and Y-chromosome diversity in East Adriatic sheep.

Variation in mitochondrial DNA (mtDNA) and Y-chromosome haplotypes was analysed in nine domestic sheep breeds (159 rams) and 21 mouflon (Ovis musimon) sampled in the East Adriatic. Mitochondrial DNA analyses revealed a high frequency of type B haplotypes, predominantly in European breeds, and a very low frequency of type A haplotypes, which are more frequent in some Asian breeds. Mitochondrial haplotype Hmt-3 was the most frequent (26.4%), and 37.1%, 20.8% and 7.6% of rams had haplotypes one, two and three mutations remote from Hmt-3 respectively. In contrast, Y-chromosome analyses revealed extraordinary paternal allelic richness: HY-6, 89.3%; HY-8, 5.0%; HY-18, 3.1%; HY-7, 1.3%; and HY-5, 1.3%. In fact, the number of haplotypes observed is comparable to the number found in Turkish breeds and greater than the number found in European breeds so far. Haplotype HY-18 (A-oY1/135-SRYM18), identified here for the first time, provides a link between the haplotype HY-12 (A-oY1/139-SRYM18) found in a few rams in Turkey and haplotype HY-9 (A-oY1/131-SRYM18) found in one ram in Ethiopia. All mouflons had type B mtDNA haplotypes, including the private haplotype (Hmt-55), and all were paternally monomorphic for haplotype HY-6. Our data support a quite homogeneous maternal origin of East Adriatic sheep, which is a characteristic of European breeds. At the same time, the high number of haplotypes found was surprising and intriguing, and it begs for further analysis. Simultaneous analysis of mtDNA and Y-chromosome information allowed us to detect a large discrepancy between maternal and paternal lineages in some populations. This is most likely the result of breeder efforts to 'upgrade' local populations using rams with different paternal origins.

Usefulness of molecular-based methods for estimating effective population size in livestock assessed using data from the endangered black-coated Asturcón pony.

Empirical evidence of the usefulness of different molecular-based methods to estimate the effective population size (N(e)) for conservation purposes in endangered livestock populations is reported. The black-coated Asturcón pony pedigree (1,981 individuals) was available. Additionally, a total of 267 Asturcón individuals born in 1998, 2002, and 2008 were typed for 15 microsatellites. These yearly cohorts (cohort(1998, 2002, 2008)) included almost all individuals kept for reproduction at the end of the corresponding foaling season. The genealogical realized N(e) was estimated for each cohort by using the individual increase in inbreeding. Molecular N(e) was computed by using 1) linkage disequilibrium [N(e)(()(D)())], 2) a temporal method based on F-statistics [N(e)(()(T)())], 3) an unbiased temporal method [N(e)(()(JR)())], and 4) a Bayesian temporal method [N(e)(()(B)())]. Estimates of increased from cohort(1998) (18.8 ± 5.1) to cohort(2008) (24.9 ± 5.2), illustrating the history of the population and its breeding policy of avoiding matings between close relatives. The estimates of N(e)(()(D)()) were highly biased upward, with the maximum N(e)(()(D)()) value obtained for cohort(2002) (137.0). The estimates of N(e)(()(T)()), N(e)(()(JR)()), and N(e)(()(B)()) showed similar performance. However, N(e)(()(JR)()) estimates were very consistent across cohorts, varying from 14.9 to 15.5 after correcting for the effect of overlapping generations. When the drift signal was not strong (pair cohort(1998)-cohort(2002)), estimates of N(e)(()(T)()) and N(e)(()(B)()) were not realistic. Estimates of N(e)(()(B)()) tended to be biased downward (being 9.0 or below for the sampling pairs including cohort(2008)). Results of N(e)(()(D)()) are more likely to be estimates of the effective number of breeders producing the sample, rather than the effective size for a generation. The temporal methods were strongly affected by a weak drift signal, particularly when samplings were not spaced a sufficient number of generations or a sufficient time apart. The use of molecular-based estimates of N(e) is not straightforward, and their use in livestock conservation programs should be carried out with caution. Sampling strategies (including sampling sizes, sampling periods, and the age structure of the sampled individuals) must be carefully planned to ensure that robust estimates of N(e) are obtained.

Multiple paternal origins of domestic cattle revealed by Y-specific interspersed multilocus microsatellites.

In this study, we show how Y-specific interspersed multilocus microsatellites, which are loci that yield several amplified bands differing in size from the same male individual and PCR reaction, are a powerful source of information for tracing the history of cattle. Our results confirm the existence of three main groups of sires, which are separated by evolutionary time and clearly predate domestication. These three groups are consistent with the haplogroups previously identified by Götherström et al. (2005) using five Y-specific segregating sites: Y1 and Y2 in taurine (Bos taurus) cattle and Y3 in zebu (Bos indicus) cattle. The zebu cattle cluster clearly originates from a domestication process that was geographically and temporally separated from that of taurine clusters. Our analyses further suggest that: (i) introgression of wild sire genetic material into domesticated herds may have a significant role in the formation of modern cattle, including the formation of the Y1 haplogroup; (ii) a putative domestication event in Africa probably included local Y2-like wild sires; (iii) the West African zebu cattle Y-chromosome may have partially originated from an ancient introgression of humped cattle into Africa; and (iv) the high genetic similarity among Asian zebu sires is consistent with a single domestication process.

Y-specific microsatellites reveal an African subfamily in taurine (Bos taurus) cattle.

Five cattle Y-specific microsatellites, totalling six loci, were selected from a set of 44 markers and genotyped on 608 Bos taurus males belonging to 45 cattle populations from Europe and Africa. A total of 38 haplotypes were identified. Haplogroups (Y1 and Y2) previously defined using single nucleotide polymorphisms did not share haplotypes. Nine of the 27 Y2-haplotypes were only present in African cattle. Network and correspondence analyses showed that this African-specific subfamily clustered separately from the main Y2-subfamily and the Y1 haplotypes. Within-breed genetic variability was generally low, with most breeds (78%) showing haplotypes belonging to a single haplogroup. AMOVA analysis showed that partitioning of genetic variation among breeds can be mainly explained by their geographical and haplogroup assignment. Between-breed genetic variability summarized via Principal Component Analysis allowed the identification of three principal components explaining 94.2% of the available information. Projection of principal components on geographical maps illustrated that cattle populations located in mainland Europe, the three European Peninsulas and Mediterranean Africa presented similar genetic variation, whereas those breeds from Atlantic Europe and British Islands (mainly carrying Y1 haplotypes) and those from Sub-Saharan Africa (belonging to Y2-haplogroup) showed genetic variation of a different origin. Our study confirmed the existence of two large Y-chromosome lineages (Y1 and Y2) in taurine cattle. However, Y-specific microsatellites increased analytical resolution and allowed at least two different Y2-haplotypic subfamilies to be distinguished, one of them restricted to the African continent.

Female segregation patterns of the putative Y-chromosome-specific microsatellite markers INRA124 and INRA126 do not support their use for cattle population studies.

Here we tested the segregation and paternal compatibility of markers INRA124 and INRA126 on female DNA in 10 different cattle families, in order to clarify the usefulness of these microsatellites for the study of male-mediated population processes in cattle. Their performance was compared with that of four microsatellites located in the PAR-BTAY (UMN0108, UMN0803, UMN0929 and UMN0905) and another one male-specific microsatellite (INRA189). INRA124 and INRA126 amplified the same sized fragment in both sexes. Same size alleles were sequenced and the high homology found allowed us to rule out non-specific female amplification. INRA124 showed full parental compatibility, whilst the locus INRA126 showed 55% parental incompatibility. Based on these observations, it is recommended that markers INRA124 and INRA126 should not be used in studies to characterize male-mediated genetic events in cattle.

Sry-negative XX true hermaphroditism in a roe deer.

A two-year-old roe deer was brought down in the course of a hunt in the north of Spain (Asturias). On physical examination the individual presented well-developed bared antlers, but surprisingly a female external genitalia. Several anatomical, histological and genetic analyses were performed in order to explain the observed phenotype. Necropsy evidenced ovary-like structures with follicles on the surface; histological analyses of testes evidenced positive immunolabel against testosterone in Leydig cells; genetic analyses showed that the sex of the individual was consistent with a female individual. PCR analysis failed to detect SRY sequences; no PIS deletion, which is responsible for XX sex-reversal in goats, was detected. On the basis of its presumptive normal female sexual karyotype (XX) and the presence of two functional abdominal bilateral testes and ovaries, the roe deer was finally diagnosed as possessing an XX hermaphroditism syndrome. However, as in many other cases, the specific reason for the occurrence of this case of hermaphroditism could not be determined.

Differences in the expression of the ASIP gene are involved in the recessive black coat colour pattern in sheep: evidence from the rare Xalda sheep breed.

Here we have tested the hypothesis of association between different levels of agouti signalling peptide (ASIP) mRNA and the recessive black coat colour in the rare Xalda breed of sheep. To deal with this task, we first tested the possible action of both the dominant black extension allele (E(D)) and a 5-bp deletion (X99692:c.100_104del; A(del)) in the ovine ASIP coding sequence on the black coat colour pattern in 188 Xalda individuals. The E(D) allele was not present in the sample and only 11 individuals were homozygous for the A(del)ASIP allele. All Xalda individuals carrying the A(del)/A(del) genotype were phenotypically black. However, most black-coated individuals (109 out of 120) were not homozygous for the 5-bp deletion, thus rejecting the A(del)/A(del) genotype as the sole cause of recessive black coat colour in sheep. Differences in expression of ASIP mRNA were assessed via RT-PCR in 14 black-coated and 10 white-coated Xalda individuals showing different ASIP genotypes (A(wt)/A(wt), A(wt)/A(del) and A(del)/A(del)). Levels of expression in black animals were significantly (P < 0.0001) lower than those assessed for white-coated individuals. However, the ASIP genotype did not influence the ASIP mRNA level of expression. The consistency of these findings with those recently reported in humans is discussed, and the need to isolate the promoter region of ovine ASIP to obtain further evidence for a role of ASIP in recessive black ovine pigmentation is pointed out.

Technical note: a novel method for routine genotyping of horse coat color gene polymorphisms.

The aim of this note is to describe a reliable, fast, and cost-effective real-time PCR method for routine genotyping of mutations responsible for most coat color variation in horses. The melanocortin-1 receptor, Agouti-signaling peptide, and membrane-associated transporter protein alleles were simultaneously determined using 2 PCR protocols. The assay described here is an alternative method for routine genotyping of a defined number of polymorphisms. Allelic variants are detected in real time and no post-PCR manipulations are required, therefore limiting costs and possible carryover contamination. Data can be copied to a Microsoft Excel spreadsheet for semiautomatic determination of the genotype using a macro freely available at http://www.igijon.com/personales/fgoyache/software_i.htm (last accessed February 26, 2007). The performance of the method is demonstrated on 156 Spanish Purebred horses.