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.     

Polymorphism in exon 3 of follicle stimulating hormone beta (FSHB) subunit gene and its association with litter traits and superovulation in the goat

The polymorphism in follicle stimulating hormone beta (FSHB) subunit gene was detected by PCR-SSCP and PCR-RFLP methods in 780 does from four goat breeds including Boer, Matou, Black and Boer-Matou crossbred (BM). The associations of FSHB genotypes with litter size (LS), litter weight at birth (LWB) and gestation length (GL) as well as superovulation performances were analyzed in relatively lower-prolific Boer and higher-prolific Matou breeds. A 1514 bp linear DNA sequence of goat FSHB gene covering the complete 3 exons was cloned by four pairs of primer. A new mutation (A2645G, GenBank Accession no: S64745) locating in exon 3 causing an amino acid change from glutamine (Gln) to arginine (Arg) at residue 115 was identified, which resulted in three genotypes named AA, AB and BB. The three genotypes were detected in the four studied goat breeds. The higher-prolific Matou breed had the highest frequency of genotype AA. Association analysis showed that Boer and Matou does with AA genotype have the largest LS both in average and in parities from first to fourth (P < 0.05). Furthermore, Boer does with AA genotype had the heaviest LWB compared to other two genotypes (P < 0.05). Matou does with AA genotype have more numbers of ova harvested, large follicles and corpus lutea on ovaries after superovulation than those with BB genotype (P < 0.05). Therefore, these results suggest that the FSHB gene is a candidate gene that affects reproduction traits in goats.     

Polymorphism of bone morphogenetic protein 4 gene and its relationship with litter size of Jining Grey goats

Two pairs of primers (P1 and P2) were designed to detect single nucleotide polymorphisms of exon 2 and intron 2 of bone morphogenetic protein 4 (BMP4) gene in both high fecundity breed (Jining Grey goat) and low fecundity breeds (Boer, Angora and Inner Mongolia Cashmere goats) by single strand conformation polymorphism. Results showed that no polymorphism was detected for exon 2 (primer P1) of BMP4 gene in four goat breeds. For intron 2 (primer P2), three genotypes (AA, AB and BB) were detected in Jining Grey and Inner Mongolia Cashmere goats, two genotypes (AB and BB) in Angora goats, and only one genotype (AA) in Boer goats. Sequencing revealed one mutation (2203G>A) of BMP4 gene in the genotype BB in comparison to the genotype AA. The differences of litter size between AA, AB and BB genotypes were not significant (P > 0.05) in Jining Grey goats. A pair of primer (P3) was designed to detect polymorphism in the 3' flanking region of BMP4 gene that contained dinucleotide repeated sequence (CA) in the four goat breeds by microsatellite analysis. For primer P3, three genotypes (CC, CD and DD) were detected in four goat breeds. Sequencing revealed one more CA dinucleotide in genotype DD than in genotype CC. The Jining Grey does with genotype CC had 0.55 (P < 0.05) or 0.72 (P < 0.05) kids more than those with genotype CD or DD. These results preliminarily indicated that allele C of BMP4 gene is a potential DNA marker for improving litter size in goats.     

Polymorphisms in BMP-2 gene and their associations with growth traits in goats

BMPs plays an important role in skeletal development. In this study, PCR-SSCP and DNA sequencing methods were employed to screen the genetic variation of caprine BMP-2 gene in 299 goats from three breeds (Boer goat, Chinese Xuhuai white goat and Chinese Haimen goat). Three fragments of BMP-2 gene were investigated, only 3′ flanking region of BMP-2 gene showed polymorphism. The alignment between nucleotide sequences of AY714781.1 in GenBank and the sequencing results of three PCR products with different patterns revealed that there was one mutation (AY714781.1: g.A172G) in 3′ flanking region of BMP-2 gene, which constructed three genotypes (AA, AB, BB). The frequencies of allele A and genotype AA were dominant in all three breeds. Frequency of allele B in Haimen goat breed was significantly lower than that of the other two breeds. The genotype distributions were in good agreement with Hardy-Weinberg equilibrium (P>0.05) in each breed. The PIC (Polymorphism Information Content) values in three populations were 0.3261, 0.2558, 0.1663 for Boer goat, Xuhuai White goat and Haimen goat respectively. It was indicated that individuals of Boer goats with genotype BB were significantly higher than those of individuals with genotype AA in body trunk index (P<0.01). No polymorphism was detected in the exon3.     

Single nucleotide polymorphisms of myostatin gene in Chinese domestic horses

The myostatin gene (MSTN) is a genetic determinant of skeletal muscle growth. Single nucleotide polymorphisms (SNP) in MSTN are of importance due to their strong associations with horse racing performances. In this study, we screened the SNPs in MSTN gene in 514 horses from 15 Chinese horse breeds. Six SNPs (g.26 T > C, g.156 T > C, g.587A > G, g.598C > T, g.1485C > T, g.2115A > G) in MSTN gene were detected by sequencing and genotyped using PCR-RFLP method. The g.587A > G and g.598C > T residing in the 5′UTR region were novel SNPs identified by this study. The g.2115A > G which have previously been associated with racing performances were present in Chinese horse breeds, providing valuable genetic information for evaluating the potential racing performances in Chinese domestic breeds. The six SNPs together defined thirteen haplotypes, demonstrating abundant haplotype diversities in Chinese horses. Most of the haplotypes were shared among different breeds with no haplotype restricted to a specific region or a single horse breed. AMOVA analysis indicated that most of the genetic variance was attributable to differences among individuals without any significant contribution by the four geographical groups. This study will provide fundamental and instrumental genetic information for evaluating the potential racing performances of Chinese horse breeds.     

Novel SNP of the goatprolactin gene (PRL) associated with cashmere traits

Concentrations of the single-chain polypeptide hormone prolactin (PRL) are associated with wool or cashmere traits, and its seasonal changes may determine patterns of enzymatic activity and may affect cashmere fibre growth and moult. So, thePRL gene is a potential candidate gene for cashmere traits in marker-assisted selection (MAS). In this paper, we report a novel missense single-nucleotide polymorphism (SNP) within the goatPRL gene in 1367 individuals by PCR-SSCP (polymerase chain reaction with single-strand conformation polymorphism) analysis and DNA sequencing. The novel X76049:g.576C>A mutation is confirmed byEco24I PCR-RFLP (restriction fragment length polymorphism) analysis and causes a missense codon (Pro176Thr). The frequencies of allele C varied from 0.79 to 0.93 in 9 analysed goat populations. C allele was correlated with higher fibre length (P = 0.014).     

Polymorphism of Bovine Prolactin Gene: Microsatellites,PCR–RFLP

In the samples of Russian Ayrshire and Gorbatov Red cattle breeds, distribution of frequencies of prolactin (PRL) gene alleles generated due to the presence of polymorphic RsaI site in exon 3 were studied. In the breeds, the frequencies of the Ballele of the PRLgene (with RsaI(+) site) detected by the PCR–RFLP method were 14.1 and 8.6%, respectively. In Black Pied, Ayrshire and Gorbatov Red cattle breeds, variation of the microsatellite dinucleotide repeat in the regulatory region of the gene PRLwas also studied. Gorbatov Red breed was monomorphic at the microsatellite locus with the only allele 164 bp in length. Two alleles (164 bp and 162 bp) were detected in the other breeds studied. The frequencies of 164-bp allele of the microsatellite locus were 93.7 and 90.0% in Black Pied and Ayrshire breeds, respectively. In Gorbatov Red breed of dairy type with good beef qualities and low milk-fat yield, lower level of heterozygosity for PRLgene was demonstrated compared to Ayrshire and Black Pied breeds that have high milk-fat yield. In three cattle breeds, higher mean estimate of polymorphism information content of PCR–RFLP in exon 3 (PIC = 0.21) was revealed compared with the same estimate (PIC = 0.09) for the microsatellite locus variability in the regulatory region of the PRLgene. Characteristics of allele Bdistribution of thePRLgene in the representatives of the Bovidae family are considered.     

Genetic analysis of prolactin gene in Pakistani cattle

Prolactin (PRL) is a polypeptide hormone, secreted mainly by the anterior pituitary gland. It is involved in many endocrine activities. The key functions of PRL are related to reproduction and lactation in mammals. To ascertain the presence of polymorphisms in the bovine PRL gene (bPRL), the bPRL gene was sequenced. Five mutations were identified in exonic region and eleven in associated intronic regions in 100 cattle from four Pakistani cattle breeds. Haplotype of predicted amino acid changes represent a common alteration at codon 222 from R-Arginine into K-Lysine in all four breeds. Significant statistical variations were observed in the distribution of single nucleotide polymorphism (SNP) in various cattle populations. However, on basis of present study, an association of these SNPs with milk performance traits in four Pakistani cow breeds cannot be truly replicated but at least can be effective DNA markers for some of the breeds studied. Linkage analysis between these SNPs on larger populations can be useful for the association with milk production traits. Furthermore, present study may be used for marker-assisted selection and management in cattle breeding program in local cattle breeds.     

Foraging selectivity of three goat breeds in a Mediterranean shrubland

Foraging behaviors of the Damascus, Mamber and Boer goat breeds were compared on shrublands of the South Carmel mountain ridge of Israel. Dietary choice was determined for a group of yearling animals of each breed (n = 11 or 12), housed and grazed separately to prevent social facilitation, during a total of 4 (Mamber) or 5 (Damascus and Boer) sessions of four days each in fall 2004 and spring 2005. The proportions in the diet (including concentrate feed) of three main dietary components – Pistacia lentiscus L. (20% tannins), Phillyrea latifolia L. (3% tannins) and herbaceous vegetation – were determined by application of near-infrared reflectance spectroscopy to faecal samples (n = 147). On average, P. lentiscus constituted 14.9% of the DM ingested by Damascus goats, but only 5.6 and 5.0% of that ingested by Mamber and Boer goats, respectively. In the fall season, Damascus goats ingested diets richer in tannins than did Mamber or Boer goats (P < 0.001). In the spring, the contribution of herbaceous vegetation to DM intake was higher in Mamber goats (38.5%) than in the other breeds (27.7%). Boer goats selected the most nutritious diets in terms of crude protein (CP) content and in vitro dry matter digestibility (IVDMD). Despite their differences in foraging selectivity, the local Damascus and Mamber goats selected similar diets in terms of CP and IVDMD. Our data suggests that, of the three breeds examined, the Damascus should provide the best control of P. lentiscus encroachment and the least competition with cattle for green grass in the spring. These findings may help to develop ecologically sound ways of controlling the spread of the tannin-rich shrub P. lentiscus L., which threatens rangeland biodiversity and amenity values in the Mediterranean climatic region of Israel.     

Combined effects of four SNPs within goat PRLR gene on milk production traits

Xinong Saanen (SN, n = 323) and Guanzhong (GZ, n = 197) goat breeds were used to detect single nucleotide polymorphisms (SNPs) in the coding regions with their intron–exon boundaries of prolactin receptor (PRLR) gene by DNA sequencing, primer-introduced restriction analysis-polymerase chain reaction (PIRA-PCR) and PCR-restriction fragment length polymorphism (PCR-RFLP). Four novel SNPs (g.40452T > C, g.40471G > A, g.61677G > A and g.61865G > A) were identified. The g.61677G > A and g.61865G > A SNPs caused amino acid variations p.Ser485Asn and p.Val548Met, respectively. Both g.40452T>C and g.40471G>A loci were closely linked in SN and GZ goat breeds (r² > 0.33). In addition, there was also a close linkage between g.61677G>A and g.61865G>A loci in both goat breeds. Statistical results indicated that the g.40452T > C, g.61677G > A and g.61865G > A SNPs were significantly associated with milk production traits in SN and GZ breeds. Further analysis revealed that combinative genotype C1 (TTAAGGGG) was better than the others for milk yield in SN and GZ goat breeds. These results are consistent with the regulatory function of PRLR in mammary gland development, milk secretion, and expression of milk protein genes, and extend the spectrum of genetic variation of the caprine PRLR gene, which might contribute to goat genetic resources and breeding.     

SNP detection and haplotype analysis in partial sequence of MSTN gene in sheep

Polymerase chain reaction (PCR) products of the MSTN gene amplified from sixty sheep of nine Chinese indigenous sheep breeds and one imported sheep breed were sequenced to identify the single-nucleotide polymorphisms (SNPs) in a 378-bp fragment including intron 2 and exon 3 of the MSTN gene. A total of fifteen SNPs (A1937C, T1942G, C1956T, A1972C, A1990G, A2008C, A2011G, C2019T, A2025C, A2027C, T2085G, T2173C, C2198T, C2210T and C2213T) were detected among the sixty sequenced individuals and they were all located in intron 2. Twelve haplotypes were identified from these fifteen SNPs, of which haplotype I (CGTCGCGTCCGCTTT) and VIII (ATCAAAACAATTCCC) were the two major and basic ones with frequencies of 12.25% and 77.80%, respectively. Haplotype VIII was distributed in all sheep breeds and all individuals of the meat or meat-wool type sheep breeds were homozygous with respect to this haplotype. This suggests that haplotype VIII might be related to meat production traits in sheep. Haplotype I was only distributed in the fur, lambskin type and fur-meat type sheep breeds. This suggests that haplotype I may have some relationship with fur traits in sheep.     

Single-nucleotide polymorphisms g.151435C>T and g.173057T>C in PRLR gene regulated by bta-miR-302a are associated with litter size in goats

Single-nucleotide polymorphisms (SNPs) located at microRNA-binding sites (miR-SNPs) can affect the expression of genes. This study aimed to identify the miR-SNPs associated with litter size. Guanzhong (n = 321) and Boer (n = 191) goat breeds were used to detect SNPs in the caprine prolactin receptor (PRLR) gene by DNA sequencing, primer-introduced restriction analysis-polymerase chain reaction, and polymerase chain reaction-restriction fragment length polymorphism. Three novel SNPs (g.151435C>T, g.151454A>G, and g.173057T>C) were identified in the caprine PRLR gene. Statistical results indicated that the g.151435C>T and g.173057T>C SNPs were significantly associated with litter size in Guanzhong and Boer goat breeds. Further analysis revealed that combinative genotype C6 (TTAACC) was better than the others for litter size in both goat breeds. Furthermore, the PRLR g.173057T>C polymorphism was predicted to regulate the binding activity of bta-miR-302a. Luciferase reporter gene assay confirmed that 173057C to T substitution disrupted the binding site for bta-miR-302a, resulting in the reduced levels of luciferase. Taken together, these findings suggested that bta-miR-302a can influence the expression of PRLR protein by binding with 3′untranslated region, resulting in that the g.173057T>C SNP had significant effects on litter size.     

Single nucleotide polymorphism (SNP) in alpha-lactalbumin gene of Indian Jamunapari breed of Capra hircus

Alpha-lactalbumin is a major whey protein found in milk. It influences lactose synthesis by modifying the substrate specificity of galactosyl-transferase, is important to milk synthesis since lactose, an impermeable disaccharide, is the major osmole of milk. The present study was undertaken to detect polymorphism in the full coding region of alpha-lactalbumin at the genetic level and to explore allelic variability of this gene. Samples of Jamunapari breed of goat (n = 50) were included under the present study. Jamunapari is the highest milk producer among local Indian goat breeds. PCR-SSCP of all four exons of alpha-lactalbumin (ALA) revealed a total of 9 gel phenotypes of Jamunapari breed of goat. These were sequenced, analyzed and deposited in GenBank, NCBI (accession nos. EU573193–EU573195). Nucleotide and amino acid variations were searched within breeds of Indian goats and homology between caprine, ovine, bovine, bubaline and human. In the present study we describe for the first time two novel gene variants on the goat alpha-lactalbumin gene exon 4.     

Polymorphism and nucleotide sequencing of BMPR1B gene in prolific Assam hill goat

Assam hill goat (Capra hircus) is a prolific local goat in India. bone morphogenetic protein receptor (BMPR1B) gene was studied as a candidate gene for the prolificacy of goats. The objective of the present study was to detect the incidence of mutation in the exonic region of BMPR1B gene of Assam hill goat. Total 90 blood samples were collected randomly from different parts of Assam and genomic DNA were extracted using phenol–chloroform method. The quantity and quality of extracted DNA was examined by spectrophotometry and gel electrophoresis, respectively. PCR amplicon showed a product of 140 bp fragment of BMPR1B gene. The purified product upon digestion with AvaII showed monomorphic banding pattern and revealed wild type alleles with AA genotype. Nucleotide sequencing showed one new mutation 773 (G→C) which is found to be unique in Assam hill goat. Construction of tree at nucleotide level generates from the present experiment lies in common cluster which differs from the other breeds of goat. The analysis of polymorphism for BMPR1B in Assam hill goat indicates that the genetic factor responsible for prolificacy or multiple kidding rates is not related to the reported mutated alleles of BMPR1B gene. Therefore, attempts to be made to detect other SNPs for BMPR1B gene or otherwise effort should be made towards other fecundity gene which might be responsible for the prolificacy of Assam hill goat.     

A promoter polymorphism of <Emphasis Type="Italic">MSTN</Emphasis> g.−371T>A and its associations with carcass traits in Korean cattle

A promoter polymorphism of bovine Myostatin (MSTN) gene g.−371T>A was screened in Holstein and two Korean indigenous cattle breeds, Hanwoo and Jeju Black cattle (JBC). The MSTN g.−371T>A polymorphism was found in all three cattle breeds tested. An allele MSTN g.−371A was the most frequent in the JBC breed among breeds tested. The association of MSTN genotypes for carcass traits was also tested in the Hanwoo population. Significant differences were found between the genotypes and level of meat quality grade index which converted the marbling score levels (P < 0.05), reflecting the metabolic role of MSTN for inhibition of preadipocyte differentiation in intramuscular fat deposition. In addition, significant differences were found for fat color index of backfat according to MSTN genotypes (P < 0.05), suggesting that MSTN may play a role in deposition of white-yellow adipocytes in backfat. However, there was no detection of significant association of genotypes with the live weight, carcass weight, backfat thickness, eye muscle area, marbling score, or meat color index (P > 0.05). Despite the lack of statistical association, wild type g.−371T/-showed association patterns similar to those of A/A homozygotes, such as heavier weights, thinner backfat, larger eye muscle area, and lower marbling score. The results of the present study suggest that MSTN promoter polymorphism g.−371T>A may affect carcass traits, which could be a useful molecular marker for planning improvements in the economic traits of Korean cattle breeds.     

Single nucleotide polymorphisms in the upstream regulatory region alter the expression of myostatin

The expression of the gene encoding myostatin (MSTN), the product of which is a negative regulator of skeletal muscle growth and development in mammals, is regulated by many cis-regulatory elements, including enhancer box (E-box) motifs. While E-box motif mutants of MSTN exhibit altered expression of myostatin in many animal models, the phenotypes of these mutations in chicken are not investigated. In this study, we cloned and sequenced the full encoded DNA sequence of MSTN gene and its upstream promoter region in Wenshang Luhua chicken breed. After analysis of the sequence, 13 E-box motifs were identified in the MSTN promoter region, which were denoted by E1 to E13 according to their positions in the region. Although many single nucleotide polymorphisms (SNPs) were revealed in the MSTN promoter region, only two SNPs were in the E-boxes, i.e., the first nucleotide of the E3 and the fifth nucleotide of E4. The effects of these two polymorphisms on the expression of MSTN gene were explored both with MSTN-GFP reporter constructs in vitro and real-time PCR in vivo. The results suggested that the E-boxes in the chicken MSTN promoter region are involved in the regulation of myostatin expression and the polymorphisms in E3 and E4 altered the expression of myostatin.     

Novel polymorphisms of goat growth hormone and growth hormone receptor genes and their effects on growth traits

In this study, the polymorphisms of growth hormone (GH) gene 5' promoter region and intron 8, exons 4 and 10 of growth hormone receptor (GHR) gene were analyzed in Xinong Saanen goats (SG) and Boer goats (BG). Two alleles (A and B) and three genotypes (AA, AB and BB) were detected at P1 locus of GH gene, and two alleles (G and T) and two genotypes (GG and GT) were detected at P4 locus of GHR gene by PCR-SSCP analysis. In addition, two single nucleotide polymorphisms (SNPs)-A73C (P1 locus) and G114T (P4 locus), were identified by DNA sequencing. The frequencies of alleles A and B in the two goat breeds were 0.61-0.62, and 0.39-0.38, respectively, and the frequencies of alleles G and T in the two goat breeds were 0.82-0.86, and 0.18-0.14, respectively. The SNP loci were in Hardy-Weinberg disequilibrium in both goat breeds (P<0.05). Polymorphisms of GH and GHR genes were shown to be associated with growth traits in BG breed. AA and GG genotypes were associated with superior growth traits in 1-, 2- and 3-month old individuals. Hence, AA and GG genotypes are suggested to be a molecular marker for superior growth traits in BG breed.     

Analysis of STAT5A/AvaI Gene Polymorphism in Four Italian Cattle Breeds

The STAT5A/AvaI polymorphism was investigated with PCR-RFLP in a sample of 339 cattle belonging to four breeds: Italian Friesian, Jersey, Italian Brown, and Podolica reared in south Italy. All three possible genotypes for the C/T polymorphism were identified. In these breeds, PCR-RFLP showed the predominance of the TT genotype in Italian Brown and Jersey cows; in Podolica and Italian Friesian CT is the most frequent genotype. The frequency of the T allele ranged from 0.55 to 0.81 in the analyzed populations. The distribution of genotypic and allelic frequencies at this locus was significantly different among the four populations based on a χ² test (P < 0.001), suggesting that the molecular characteristics of the STAT5A gene could be significantly affected by the breed selection. Gene heterozygosity, gene homozygosity, effective allele number, fixation index, and polymorphism information content (PIC) were calculated. The observed heterozygosity, as well as the N _e and PIC values, indicates high genetic variability in the Podolica breed. Podolica could be considered an interesting reservoir of genetic diversity for a species under high selective pressure elsewhere.