Sequence-based genotyping of the sheep MHC class II DRB1 locus

The immunopolymorphism database (IPD) provides a single nomenclature for alleles at the major histocompatibility complex (MHC) loci for a range of different species. The minimum requirements for inclusion of a sheep class II DRB1 sequence is a submission that includes all polymorphic sites within the second exon from at least two independent polymerase chain reactions (PCR). In order to meet these requirements, we have developed a DNA-based genotyping method for the rapid analysis of allelic diversity at the DRB1 locus in domestic sheep, Ovis aries. Using a series of primers located within introns flanking exon 2 and genomic DNA from a cohort of 214 sheep representing 15 different breeds and crossbreeds, the complete exon 2 sequences of 38 Ovar-DRB1 alleles were obtained. This sequence resource allowed the development of a generic set of locus-specific primers which amplify a fragment that includes all polymorphic sites within the second exon. Bidirectional sequence analysis of the PCR product provides a composite sequence where each polymorphic site is represented by the corresponding International Union of Biochemistry nucleotide code. A Basic Local Alignment Search Tool search of alleles held within the IPD or National Center for Biotechnology Information databases allows individual allele sequences to be identified. Low levels of homozygosity (7.48%) within the cohort and verification of previously genotyped samples confirmed the broad allelic specificity of this method. It improves on currently available methods and is broadly applicable to the analysis of MHC diversity in studies investigating linkages with resistance or susceptibility to disease.     

Restriction fragment length polymorphism of exon 2 Ovar-DRB1 gene in Polish Heath Sheep and Polish Lowland Sheep

Exon 2 of the Ovar-DR gene is known to encode the MHC outer domain (alpha or beta chain) that forms the binding area to antigens presented. The study was aimed at analysing exon 2 Ovar -DRB1 gene polymorphism in Polish Heath Sheep and Polish Lowland Sheep (Zelazna variety). A total of 101 and 99 ewes of the respective breeds were included in this study. We identified 65 different haplotypes in Polish Heath Sheep and 68 in Polish Lowland Sheep. The PCR-RFLP method and PCR products sequencing made it possible to identify two new sequences of exon 2 Ovar-DRB1 gene (AY230000 and AY248695). A distinct polymorphism in the exon 2 sequence presents possibilities for immune response toward a great variety of pathogens.     

Single-strand conformational polymorphism and sequence polymorphism of Mhc-DRB in Latxa and Karrantzar sheep: implications for Caprinae phylogeny

Single-strand conformational polymorphism analysis and DNA sequencing were used to characterize Mhc-DRB second exon variability in the Latxa and Karrantzar breeds of sheep. The presence of more than two sequences in some animals indicates that alleles of two different loci have been amplified. Six new alleles were identified by sequencing. The allele frequency distribution of the DRB1 gene is striking, with two alleles accounting for half of the gene pool in both breeds under study. The most frequent allele in both breeds was the same (named DRB1*0702), with some specific amino acids: Tyr in position 31 and Thr in 51. A species variability analysis was also performed including the entire set of sheep DRB exon 2 sequences. Based on the patchwork patterns of different alleles, interallelic recombination appears to be playing a significant role in the generation of allelic diversity at this locus in sheep. The phylogenetic tree of all known Caprinae DRB sequences shows that certain alleles from one species are more closely related to those from other species than they are to each other. Allele DRB1*0702 merits special attention due to its high similarity to the Mufflon allele. As this is the most frequent in both breeds analyzed, one can hypothesize that in sheep, both Mufflon and Argali have had different influences depending on the sheep breed under study and that the relationship between domestic sheep and Mufflon is greater than previously thought. The data generated in this study can serve as a basis for developing a typing assay for the sheep DRB genes in the Latxa and Karrantzar populations.     

Interdependent MHC-DRB exon-plus-intron evolution in artiodactyls

Exon 2 sequences of an expressed MHC-DRB locus from sheep were examined for polymorphisms in both the antigen-binding regions and the adjacent intronic mixed simple tandem repeat. Twenty-one novel exon 2 Ovar-DRB alleles were identified. Short nucleotide motifs are extensively shared between certain exon 2 regions of Ovar-DRB alleles. The simple repeat variations, the number of different amino acids at usually polymorphic sites, and the number of silent substitutions were reduced in the intraspecies analyses of sheep DRB sequences, compared with those of cattle and goats. It was paradoxical that the abundance of different sheep alleles was similar to that of cattle and goats. This paradox may be explained by postulating a relatively small number of "ancient" alleles, with the present-day Ovar-DRB alleles being generated by reciprocal exchange of nucleotide motifs. At the antigen-binding sites, new combinations of amino acids were maintained in Ovar-DRB alleles by strong positive selection. In sheep--and less pronounced in goats and cattle--the DRB alleles can be divided into two groups. In one group, silent substitutions are increased when compared with the other. This suggests separate evolutionary pathways for certain groups of DRB alleles within a species. The simple repetitive sequences are also discussed with respect to the evolution of DRB alleles.      

MHC-DRB exon 2 allele polymorphism in Indian river buffalo (Bubalus bubalis)

The polymorphism of the major histocompatibility complex (MHC) class II DRB gene of riverine buffalo (Bubalus bubalis) was studied. Second exon sequences from the buffalo DRB locus, homologous to the cattle DRB3 gene, were amplified and characterized. A combination of single strand conformation polymorphism (SSCP) and heteroduplex analysis (HA) in a non-denaturing gel was used to identify new DRB second exon sequences. SSCP, HA and finally sequencing allowed the identification of 22 MHC-DRB exon 2 alleles from 25 unrelated Indian river buffalo. These are the first river buffalo DRB second exon sequences reported. A high degree of polymorphism in the sequences encoding the peptide binding regions was observed and some amino acid substitutions were found unique to the river buffalo.     

Allelic variation of ovine MHC class II DQA1 and DQA2 genes

In the present study we characterize allelic variation of polymorphic OLA-DQA1 and OLA-DQA2 genes in sheep. To achieve this, PCR primers were designed to independently amplify the second exons of OLA-DQA1 and OLA-DQA2 genes. Single strand conformation polymorphism (SSCP) gel analyses reveals that there are at least 12 distinct OLA-DQA2 sequences, 10 of which have been characterized by sequencing. Six distinct OLA-DQA1 alleles have been sequenced in sheep and we can detect at least seven DQA1 alleles, including a null allele, by SSCP analysis. The second exon of the OLA-DQA2 gene is more polymorphic than the equivalent region of the OLA-DQA1 gene. Thirty-two per cent of nucleotide and 49% of amino acid sites showed variation at the DQA2 locus, compared to 20% of nucleotide and 33% of amino acid sites for DQA1 . Phylogenetic analysis of DQA sequences from a number of species show that sheep DQA1 sequences group together and are more similar to bovine DQA1 sequences than to sheep DQA2 alleles. The majority of OLA-DQA2 sequences are on the same main branch of the phylogenetic tree as bovine DQA2 sequences. However, three sheep DQA2 sequences have a tendency to group with putative bovine DQA3 sequences rather than to other ovine DQA2 alleles. A variety of SSCP gel conditions were tried in order to develop a typing system for the OLA-DQA2 gene. We describe a set of PCR and SSCP conditions which distinguish between all known OLA-DQA2 alleles.     

Detection of myostatin gene MSTN in some goat breeds (Capra hircus)

Till now not information about myostatin MSTN gene in Egyptian goat breeds. Here we show more information about MSTN in some Egyptian goat breeds to enrich the database with new sequences for Egyptian goat breeds. Our conducted study focused on detection and identifying the MSTN gene as a candidate gene of the muscles growth trait in three goat breeds (Zaraibi, Baladi and Damascus). We found the similarity between the registered sequences with the accession numbers KY463684 for Zaraibi and KY463685 for Baladi and Chinese goat breeds of the MSTN gene deposited with international gene banks by up to 99% and some other species including sheep, cows and bull breeds with percentages of 95 to 97% and between 95 to 99%, respectively. There is also a correlation between the sequences of the registered pieces of Baladi with KY463686 and Damascus and Chinese breeds with KY441464 of MSTN deposited with international gene banks by up to 99% and some other species including sheep and bull breeds at a ratio of 99% for two pieces. Results demonstrated the deposited sequences of object are part of intron 1, exon 2 is fully sequenced with Zaraibi and Baladi breeds; the intron 1, exon 1 with Baladi breed; and the intron 2, part of exon 3 with Damascus breed. Therefore, the Egyptian goat breeds consider national wealth can be used to develop breeding and improvement programs which helps in more applicable scopes like biotechnology, genetic engineering and molecular biology with the help of bioinformatics tools.     

Detecting novel SNPs and breed-specific haplotypes at calpastatin gene in Iranian fat- and thin-tailed sheep breeds and their effects on protein structure

Calpastatin has been introduced as a potential candidate gene for growth and meat quality traits. In this study, genetic variability was investigated in the exon 6 and its intron boundaries of ovine CAST gene by PCR-SSCP analysis and DNA sequencing. Also a protein sequence and structural analysis were performed to predict the possible impact of amino acid substitutions on physicochemical properties and structure of the CAST protein. A total of 487 animals belonging to four ancient Iranian sheep breeds with different fat metabolisms, Lori-Bakhtiari and Chall (fat-tailed), Zel-Atabay cross-bred (medium fat-tailed) and Zel (thin-tailed), were analyzed. Eight unique SSCP patterns, representing eight different sequences or haplotypes, CAST-1, CAST-2 and CAST-6 to CAST-11, were identified. Haplotypes CAST-1 and CAST-2 were most common with frequency of 0.365 and 0.295. The novel haplotype CAST-8 had considerable frequency in Iranian sheep breeds (0.129). All the consensus sequences showed 98–99%, 94–98%, 92–93% and 82–83% similarity to the published ovine, caprine, bovine and porcine CAST locus sequences, respectively. Sequence analysis revealed four SNPs in intron 5 (C24T, G62A, G65T and T69-) and three SNPs in exon 6 (c.197A > T, c.282G > T and c.296C > G). All three SNPs in exon 6 were missense mutations which would result in p.Gln 66 Leu, p.Glu 94 Asp and p.Pro 99 Arg substitutions, respectively, in CAST protein. All three amino acid substitutions affected the physicochemical properties of ovine CAST protein including hydrophobicity, amphiphilicity and net charge and subsequently might influence its structure and effect on the activity of Ca2 + channels; hence, they might regulate calpain activity and afterwards meat tenderness and growth rate. The Lori-Bakhtiari population showed the highest heterozygosity in the ovine CAST locus (0.802). Frequency difference of haplotypes CAST-10 and CAST-8 between Lori-Bakhtiari (fat-tailed) and Zel (thin-tailed) breeds was highly significant (P < 0.001), indicating that these two haplotypes might be breed-specific haplotypes that distinguish between fat-tailed and thin-tailed sheep breeds.     

The relationship between MHC-DRB1 gene second exon polymorphism and hydatidosis resistance of Chinese Merino (Sinkiang Junken type), Kazakh and Duolang sheep

The present study aimed at detecting the association of ovine major histocompatibility complex class II (Ovar II) DRB1 gene second exon and susceptibility or resistance to hydatidosis in three sheep breeds of Sinkiang. The MHC-DRB1 second exon was amplified by polymerase chain reaction (PCR) from DNA samples of healthy sheep and sheep with hydatidosis. PCR products were characterized by the restriction fragment length polymorphism (RFLP) technique. Five restriction enzymes, Mval, Haelll, Sacl, Sacll, Hin1l, were used, yielding 14 alleles and 31 restriction patterns. Frequencies of patterns Mvalbc, Hin1lab, Sacllab, Haelllde, Haellldf, Haellldd (P < 0.01) in Kazakh sheep, Saclab (P < 0.05) in Duolang sheep, and Haelllab, Haelllce, Haelllde, Haelllee (P < 0.01) in Chinese Merino (Sinkiang Junken type) sheep, were significantly higher in healthy sheep compared with infected sheep. These results indicated a strong association between these patterns and hydatidosis resistance. In contrast, the frequencies of Mvalbb, Saclaa, Hinl lbb, Haelllef (P < 0.01) and Haelllab (P < 0.05) in Kazakh sheep, Saclbb, Haelllae, Hin1lab (P < 0.05), Haelllaa, Haelllbe, Haelllef (P < 0.01) in Duolang sheep, Sacllaa (P < 0.05) and Haelllbd, Hin1lbb, Haelllcf, Haelllef (P < 0.01) in Chinese Merino sheep (Sinkiang Junken type) were significantly lower in healthy sheep compared with infected sheep. This indicated a strong association between these patterns and hydatidosis susceptibility. In addition, sheep with the pattern of Haelllef demonstrated a high hydatidosis susceptibility (P < 0.01) in all three breeds, while sheep with the pattern Haelllde demonstrated significant hydatidosis resistance (P < 0.01) in Kazakh and Chinese Merino sheep (Sinkiang Junken type). These results suggest that the Ovar-DRB1 gene plays a role in resistance to hydatidosis infection in the three sheep breeds.     

Prolificacy genotypes at BMPR 1B,BMP15 and GDF9 genes in North African sheep breeds

The aim of this research was to investigate the genetic structure at BMPR 1B, BMP15 and GDF9 prolificacy genes in five sheep breeds reared in Tunisia: Barbarine, Queue Fine de L’Ouest, Noire de Thibar, Sicilo-Sarde and D’man. Genomic DNA of 204 sheep was investigated for the FecB^B (BMPR 1B), FecX^R, FecX^H, FecX^I, FecX^L, FecX^G, FecX^B (BMP15) and FecG^H (GDF9) mutations. The sequence variability of the different DNA fragments utilised for genotyping was further investigated by Single Stranded Conformation Polymorphism (SSCP) and sequencing. All the above-mentioned mutations were absent in the five sheep breeds examined. SSCP analysis and sequencing allowed the detection of two nucleotide variations. A non-functional mutation (T/C transition at nt 747 of BMP15 cDNA known as B3) was found at the BMP15 gene, in the Noire de Thibar breed; this mutation was first detected in the Belclare sheep. A new nucleotide change G/A at nt 1159 of BMP15 cDNA, causing the amino acid change A119T in the mature peptide, was detected in the Barbarine breed for the first time. The highly prolific D’man ewes were monomorphic for the absence of all the known prolificacy alleles.     

Characterization of the <Emphasis Type="Italic">GHR</Emphasis> gene genetic variation in Chinese indigenous goat breeds

The aim of the present work was to investigate single nucleotide polymorphism (SNP) of growth hormone receptor (GHR) gene exon 10, characterize the genetic variation in three Chinese indigenous goat breeds, and search for its potential association with cashmere traits. In this study, a polymerase chain reaction-single strand conformation polymorphism (PCR–SSCP) protocol has been developed for rapid genotyping of the GHR gene in goats. One hundred seventy-eight goats from Liaoning Cashmere (96), Inner Mongolia White Cashmere (40), and Chengdu Grey (42) breeds in China were genotyped at GHR locus using the protocol developed. In all goat breeds investigated, a SNP in exon 10 of GHR gene has been identified by analyzing genomic DNA. The polymorphism consists of a single nucleotide substitution A → G, resulting in two alleles named, respectively, A and G based on the nucleotide at the position. The allele A was found to be more common in the animals investigated, and seems to be more consistent with cattle and zebu at this polymorphic site found in goats. The Hardy–Weinberg equilibrium of genotype distributions of GHR locus was verified in Liaoning Cashmere, and Inner Mongolia White Cashmere breeds. According to the classification of polymorphism information content (PIC), Chengdu Grey was less polymorphic than Liaoning Cashmere and Inner Mongolia White Cashmere breeds at this locus. The phylogenetic tree of different species based on the nucleotide sequences of GHR gene exon 10 is generally in agreement with the known species relationship. No significant association was found between the polymorphism revealed and the cashmere traits analyzed in present work.     

Heterozygosity excess at the cattle DRB locus revealed by large scale genotyping of two closely linked microsatellites

A method for MHC DRB typing in cattle based on two closely linked and highly polymorphic microsatellites is described. The two microsatellites DRBP1ms and DRB3ms are located in intron 2 of the corresponding DRB gene. The very strong linkage disequilibrium between the two loci made it possible to establish DRB microsatellite haplotypes. The typing results with this method on reference samples followed closely that obtained with RFLP and direct sequence analysis of DRB3 exon 2. The method is well suited for large scale genotyping and was successfully applied for typing more than 600 unrelated animals representing 23 breeds. The data were used to test whether the observed DRB allele frequency distributions were consistent with that expected for selectively neutral alleles in populations at mutation-drift equilibrium. A significant heterozygosity excess was detected and there was an obvious trend across breeds towards a more even allele frequency distribution than expected. The deviation may be due to balancing selection acting on the DRB locus or by recent population bottlenecks.     

Characterization of equine and other vertebrate TLR3, TLR7, and TLR8 genes

Toll-like receptors 3, 7, and 8 (TLR3, TLR7, and TLR8) were studied in the genomes of the domestic horse and several other mammals. The messenger RNA sequences and exon/intron structures of these TLR genes were determined. An equine bacterial artificial chromosome clone containing the TLR3 gene was assigned by fluorescent in situ hybridization to the horse chromosomal location ECA27q16–q17 and this map location was confirmed using an equine radiation hybrid panel. Direct sequencing revealed 13 single-nucleotide polymorphisms in the coding regions of the equine TLR 3, 7, and 8 genes. Of these polymorphisms, 12 were not previously reported. The allelic frequency was estimated for each single-nucleotide polymorphism from genotyping data obtained for 154 animals from five horse breeds. Some of these frequencies varied significantly among different horse breeds. Domain architecture predictions for the three equine TLR protein sequences revealed several conserved regions within the variable leucine-rich repeats between the corresponding horse and cattle TLR proteins. A phylogenetic analysis did not indicate that any significant exchanges had occurred between paralogous TLR7 and TLR8 genes in 20 vertebrate species analyzed. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Extensive Diversity of the ADRB3 Gene in Chinese Sheep Identified by PCR-SSCP

The β₃-adrenergic receptor (ADRB3) is the main mediator of the lipolytic and thermogenic effects of high catecholamine concentrations. Polymorphism in the ovine ADRB3 gene has been reported to be associated with birth weight, growth rate, carcass composition, and cold survival. Eight alleles have been identified in New Zealand sheep, but to date, breeds from other countries have not been studied in detail. In this study, we employed PCR-SSCP to detect ADRB3 polymorphism in the Hu, Tan, small-tailed Han, and Tibet breeds of Chinese sheep. Thirteen SSCP patterns were observed, including the eight previously reported and five new patterns. The newly identified SSCP patterns represent five novel sequences, increasing the reported number of alleles of ADRB3 from 8 to 13. In these Chinese sheep, the most common allele was the previously reported allele C (49.0%); the A allele was the next most common (22.0%). These results suggest that Chinese sheep have more diversity in ADRB3 than New Zealand sheep and therefore have possibly been selected either naturally or via breeding for different traits.     

Trans-Species Polymorphism and Selection in the MHC Class II DRA Genes of Domestic Sheep

Highly polymorphic genes with central roles in lymphocyte mediated immune surveillance are grouped together in the major histocompatibility complex (MHC) in higher vertebrates. Generally, across vertebrate species the class II MHC DRA gene is highly conserved with only limited allelic variation. Here however, we provide evidence of trans-species polymorphism at the DRA locus in domestic sheep (Ovis aries). We describe variation at the Ovar-DRA locus that is far in excess of anything described in other vertebrate species. The divergent DRA allele (Ovar-DRA*0201) differs from the sheep reference sequences by 20 nucleotides, 12 of which appear non-synonymous. Furthermore, DRA*0201 is paired with an equally divergent DRB1 allele (Ovar-DRB1*0901), which is consistent with an independent evolutionary history for the DR sub-region within this MHC haplotype. No recombination was observed between the divergent DRA and B genes in a range of breeds and typical levels of MHC class II DR protein expression were detected at the surface of leukocyte populations obtained from animals homozygous for the DRA*0201, DRB1*0901 haplotype. Bayesian phylogenetic analysis groups Ovar-DRA*0201 with DRA sequences derived from species within the Oryx and Alcelaphus genera rather than clustering with other ovine and caprine DRA alleles. Tests for Darwinian selection identified 10 positively selected sites on the branch leading to Ovar-DRA*0201, three of which are predicted to be associated with the binding of peptide antigen. As the Ovis, Oryx and Alcelaphus genera have not shared a common ancestor for over 30 million years, the DRA*0201 and DRB1*0901 allelic pair is likely to be of ancient origin and present in the founding population from which all contemporary domestic sheep breeds are derived. The conservation of the integrity of this unusual DR allelic pair suggests some selective advantage which is likely to be associated with the presentation of pathogen antigen to T-cells and the induction of protective immunity.     

猪白细胞Ⅱ类抗原基因多态性研究进展

本文对SLAⅡ类基因的染色体图谱定位、分子结构、基因分型及其多态性的研究进展作了介绍。重点介绍了SLAⅡ类基因的RFLP基因分型及其多态性研究。猪的主要组织相容性复合物Ⅱ类抗原基因座位存在不同程度的等位基因多态性。RFLP技术是一种简便而快捷的SLAⅡ类等位基因分型方法。SLAⅡ类基因的多态性和抗原肽结合位点密切相关,这些位点能结合处理过的抗原,然后递呈给T细胞。另外,在编码β1链的SLA-DRB1基因和SLA-DQB基因中有4个富含GC序列区。 Abstract:This article gave a detailed introduction about regional map,molecular structure,genotyping and polymorphism of SLA class Ⅱ genes.The pig major histocompatibility complex (MHC) class Ⅱ antigens have been known to exhibit a different degree of allelic polymorphism.The locus-specific oligonucleotide primers and RFLP analysis provide a simple and rapid method for genotyping expressed SLA class Ⅱ from genomic DNA.SLA class Ⅱ polymorphism was related to the antigenic peptide binding sites.Detailed analysis of sequences showed that there were 4 GC-rich sequences in exon 2 of SLA-DQB and SLA-DRB1 genes.