SSCP Patterns Research Articles

Identification of single nucleotide variations in the AGPAT6 gene and its association with milk production traits in Murrah buffalo

The present study investigated the association of genetic variations within the 5′UTR exons 1-1511, 3′UTR exons 1512-1723 of the sn-1-acylglycerol-3-phosphate-O-acyltransferase 6 (AGPAT6) gene of buffalo with milk production traits, using the PCR-SSCP and Sanger sequencing method. The study revealed two unique conformational patterns: ‘1’ and ‘2’ in 100 Murrah buffaloes. Ten primer pairs were used for amplification of the AGPAT6 gene and in total nine single nucleotide polymorphisms (SNPs) were identified: viz., two in Exon 4 and seven in Exon8. The frequency of SSCP pattern 1 was found to be consistently high in the buffalo population under study. The preliminary results showed substantial variations in the distribution of SSCP variant frequencies of pattern 1 and 2 within Murrah buffaloes. However, these variants of exon4 and exon 8 had non-significant association with milk yield, whereas the same variants in terms of SNPs in the coding region were found to have a significant (P≤0.01) association with fat percentage. This suggests that AGPAT6 affects the milk fat percentage, which is an economically important trait, and that further study of this gene is warranted in Murrah buffaloes. The polymorphisms observed can be used as molecular markers to assist selection.

Association of ovine follistatin gene polymorphisms with body measurements, fat-tail traits and morphometric of head in Iranian Mehraban sheep

The mature body size of sheep is a polygenic trait that is used as a main selection criterion for increasing meat production through genetic selection. The purpose of this study was to characterize polymorphisms of the follistatin gene (FST locus), which is known to regulate muscle hypertrophy by inhibiting both myostatin and activin. Using single-strand conformation polymorphism analysis (SSCP) and Sanger sequencing, we examined polymorphisms of the ovine FST gene (OAR_v.3.1; Chr 16, NC_019473.1) in Iranian Mehraban sheep (n = 100, seven-month-old male lambs) with records of body measurements, fat-tails, and head morphometry. Among the nine SSCP patterns A to I, we identified five single nucleotide polymorphisms (SNPs), including g.25632659 G>C, g.25632701 G>A, g.25633069 A>G, g.25633069 A>T, and g.25634085 G>C. The only amino acid-altering variant was g.25632659 G>C in exon 3 (p.216Thr > Ser). According to a subsequent statistical analysis, SSCP patterns A (g.25632659 G/C, g.25632701 G/G), B (g.25632659 C/C, g.25632701 G/A), C (g.25632659 C/C, g.25632701 A/A), D (homozygous reference; g.25633069 A/A), E (heterozygous; g.25633069 A/G) and F (homozygous variant; g.25633069 T/T) were significantly (P < 0.05) connected with body measurements. Furthermore, patterns A and B had significantly (P ≤ 0.05) longer forehead lengths than the lambs with the pattern C. Pattern I (homozygous variant; g.25634085 C/C) was significantly associated with an increased mouth width, and decreased eye length compared to patterns H (heterozygous; g.25634085 G/C) and G (homozygous reference; g.25634085 G/G). The findings of this study provide primary evidence of linking between the polymorphisms of the FST gene and body size in a small panel of Iranian Mehraban sheep population.

Polymorphisms within Intron-4 and Exon-4 regions of SPP1 gene and their association with milk traits in Gaolao cattle

Secreted phosphoprotein 1 (SPP1) is an acidic, phosphorylated glycoprotein found in fluids including milk and is involved in development of mammary gland and exhibits impact on milk composition and yield. The aim of study was to reveal SPP1 gene polymorphisms and association with milk traits in Gaolao cattle. Blood was collected from 280 cows and test day milk was recorded. SPP1G1-BsrI locus in intron-IV was polymorphic and frequency of TT genotype was 0.99 and CT was 0.01, respectively. The association of SPP1G1-BsrI polymorphism with milk traits revealed significant difference in Milk yield and SNF% for CT genotype as compared to TT genotype. Sequencing of SPP1G1-BsrI locus-based TT and CT genotypes revealed G-A SNP at 32nd position. The SPP1G5-SSCP locus at exon-4 region was polymorphic with frequency for A pattern as 0.95 and for B patterns as 0.05. The association study, revealed significant differences for SNF% and Lactose% for SPP1G5-SSCP pattern A as compared to pattern B. Direct sequencing of SSCP pattern A and B of SPP1G5-SSCP revealed SNPs A-G at 71st, A-C at 109th and A-C at 208th position and 02 computational SNPs, viz. C-A at 109th position and C-A at 208th position. Identified significant association needs validation in large data sets with records for development of markers for bovine milk traits.

Mutation and up-regulation of TP53 in ovine pulmonary adenocarcinoma lung cells as a model of human lung cancer.

Ovine pulmonary adenocarcinoma (OPA) is a model of human lung cancer‎ and fatal viral disease that causes neoplasia in sheep respiratory cells. ‎In the current study, 986 lung samples was inspected in the slaughterhouse, and finally twenty OPA ‎ lung organs were clinically diagnosed and five healthy lung organs were assigned as the control sample. Three SSCP patterns were detected for the affected lungs animals in comparison with the healthy lungs. In addition, sequencing results indicated three different single point mutations in exon 4 of TP53 within infected lungs, whereas no mutations were observed in exon 9 of this gene. Real-time PCR results showed up-regulation of the TP53 gene in all the infected lung cells compared to healthy cells. There was significant correlation between the mutations in exon 4 and OPAand can be used as a useful tool in determining the mechanism of lung cancer.

Association of POU1F1 gene polymorphism with pre-pubertal body weight in Attappady Black goats of India

The present study was carried out to identify single nucleotide variations in Pituitary specific Transcription factor – 1 (POU1F1) gene and evaluate their association with body weight at different stages of growth before the onset of puberty in meat-type Attappady Black goats. A total of 100 Attappady goats were screened for polymorphism using PCR-SSCP. Representative samples of each SSCP pattern were sequenced to ascertain the genotypes. Four fragments of POU1F1, 170 bp (exon 1), 234 bp (exon 3), 200 bp (exon 4) and 232 bp (exon 6) were analyzed. The 170 bp and 200 bp fragments were found to be monomorphic. The 234 bp fragment showed two diplotypes (GG and GA) with one synonymous single nucleotide polymorphism (g 0.42 C > T) in exon 3. The GA heterozygotes had significantly higher (p ≤ 0.05) body weight than GG homozygotes. at six months of age. The 232 bp fragment showed two diplotypes (CC and TC) with one novel synonymous single nucleotide polymorphism (g 0.180 C > T) in exon 6. The TC variants had significantly higher (p ≤ 0.05) body weight at six and nine months of age compared to the CC variants. The POU1F1 polymorphisms in the present study significantly contributed to variation in body weights at the onset of puberty at six and nine months of age indicating the role of POU1F1-mediated transcriptional regulation of genes coding for the various hormones of the neuro-endocrine system influencing growth and sexual maturity. The study suggests the possibility of using POU1F1 polymorphisms as markers for the selection of goats for higher pre-pubertal body weights in the genetic improvement programmes.

Association between the Ovine MHC-DRB1 Gene and its Resistance to Gastrointestinal Parasites in Deccani Sheep Raised in Hot Semi-arid Ecosystem of India

Backgroung: Major histocompatibility complex (MHC) is linked with the ability of sheep to resist infection by GIN as measured by feacal egg count. Present study was carried out to genotype Deccani sheep for DRB1 locus and associate it with parasitic resistance. Methods: PCR SSCP analysis for Ovar-DRB1 exon 2 and part of intron-1 was carried out under optimum conditions. Effect of genetic and nongenetic factors along with the genotype was performed using linear model of least square analysis by considering log transferred faecal egg count as a dependent variable. Result: PCR SSCP analysis revealed presence of 14 SSCP patterns. The factors such as age, sex, farm and birth type demonstrated a non significant effect on faecal egg count however, season was observed to be significant source of variation. Further, it was observed that, genotype J and F could be possibly associated with resistance and susceptibility to parasitic infection. Thus, it was inferred that DRB1 gene is may be associated with immune response to parasitic invasion and selection for genotype associated with Ovar-Mhc-DRB1 gene could improve the parasitic resistance in Deccani sheep.

Identification of Polymorphism within Exon 8 of Bovine HSP90AA1 Gene using PCR-SSCP Technique

Heat shock proteins (HSPs) are a type of molecular chaperones that aid in the recovery of stressed cells and serve as a major system for intracellular self-defense. A study was conducted during the year 2018–19 at College of Veterinary Science Rajendranagar, Hyderabad, Telengana State, India to find polymorphisms in exon 8 of the bovine HSP90AA1 gene in Sahiwal (n=50) and crossbred (n=50) cows. Blood samples were collected from the experimental animals and genomic DNA was isolated. Physiological parameters like body temperature and respiration rate for each animal were taken during the experimental period and the heat tolerance coefficient was calculated. The data on production and reproduction traits were obtained from the history sheets of the animals. To detect the polymorphism, a 539 bp fragment of the HSP90AA1 gene covering exon 8 was subjected to the Polymerase Chain Reaction-Single-Strand Conformation Polymorphism (PCR-SSCP) technique.The PCR-SSCP of exon 8 of HSP90AA1 gene yielded two genotypic patterns AA and AB corresponding to two allelic variants with frequencies of 0.85, 0.15, 0.81 and 0.19 in Sahiwal and crossbred cows, respectively. The PCR-SSCP patterns obtained were correlated with the physiological, productive, and reproductive traits in both Sahiwal and crossbred cows. The association analysis of SSCP patterns of the exon 8 of HSP90AA1 gene revealed non-significant effect in Sahiwal cows, although the AB genotype had a significantly longer service period in crossbred cows.

​Genetic Polymorphism within Exon 3 of HSP90AA1 Gene and its Association Studies in Sahiwal and Crossbred Cows

Background: Heat shock proteins (HSPs) are molecular chaperones that play a critical role in recovering cells from stress and form a primary system for intra cellular self defense. They are highly conserved and play a crucial role in cellular thermo tolerance and heat stress response. Though there are many HSP genes, thermo tolerance is mainly correlated with HSP70 and HSP90 genes in Livestock species. Polymorphisms in these genes have shown an association with heat tolerance, milk production, fertility and disease susceptibility in livestock. They can be used as genetic markers for the selection of animals with better climate resilience, immune response and superior performance. Methods: The present study was carried out in Sahiwal (n=50) and Crossbred cows (n=50) with the objective to identify polymorphisms in HSP90AA1 gene. A 450 bp fragment of bovine HSP90AA1 gene covering exon3 was subjected to Polymerase Chain Reaction-Single-Strand Conformation Polymorphism (PCR-SSCP) technique to identify the polymorphism. PCR-SSCP patterns were correlated with the physiological, productive and reproductive traits in Sahiwal and crossbred cows using the univariate GLM model of SPSS 25. Result: The PCR-SSCP of exon 3 of HSP90AA1 gene yielded two conformational patterns AA and AB corresponding to two allelic variants A and B in both Sahiwal and crossbred cows. The allele frequencies of A and B were 0.78 and 0.22 and 0.84 and 0.16 in Sahiwal and crossbred cows, respectively. The association analysis of SSCP patterns revealed that genotype AA had higher lactation length in Sahiwal cows and higher total lactation milk yield and peak yield in crossbred cows.

Identification of polymorphism in ABCG2 gene in Rathi cattle

In present study, Rathi cattle were selected to analyze ABCG2 genes for polymorphism. Total 160 lactating animals of Rathi cattle from livestock research station (LRS), Nohar and Bikaner were selected. Blood samples were collected and DNA isolated by kit method. For Exon 7-partial intronic region, Exon 14 and Intron 15 region of ABCG2 gene primers were constructed based on available sequences of ABCG2 gene in the NCBI GenBank database. Fragments of 393, 207 and 323-bp of exon 7-partial intronic region, exon 14 and intron 15 region of ABCG2 gene, respectively were amplified by a polymerase chain reaction in a final reaction volume of 25μl. The PCR-RFLP and PCR-SSCP method was used according to region of ABCG2 gene. In exon 7-partial intronic region and exon 14 of ABCG2 gene shows monomorphism. In intron 15 region of ABCG2 gene two SSCP patterns were observed and it shows polymorphic nature of this region. In intron 15 region of ABCG2 gene observed two alleles and two genotypes with frequency 0.85, 0.15, 0.70 and 0.30, respectively, were observed. In present study, two regions of ABCG2 gene observed monomorphic nature and one region, intron 15 region of ABCG2 gene revealed polymorphic nature.

Association of inhibin alpha gene polymorphism with litter size and growth in Malabari goats of India

Abstract The present study was carried out to identify single nucleotide variations in inhibin alpha (INHA)geneto evaluate its association with production traits in Malabari goats. A total of 251 Malabari adult goats were screened for polymorphism using PCR-SSCP.Representative samples of each SSCP pattern were sequenced to ascertain the genotypes. Five fragments, 274 bp, 220 bp, 160 bp, 287 bp and 253 bp covering the two exons of INHA were analyzed. The 274 bp fragment showed two diplotypes (AA and AB) and sequencing revealed one synonymous SNP (c.129 G > A) in exon 1. The AA genotype showed significant (p ≤ 0.05) association with body length. Similar monomorphic band pattern was observed for 220 bp fragment of exon 2. The 160 bp fragment exhibited two genotypes DD and DE with a synonymous SNP c.567 G > A in exon 2. Goats carrying DD genotype had significantly (p ≤ 0.05) higher adult body weight and body length compared to DE genotype. Three diplotypes, HH, HJ and JJ with a synonymous SNP, c.651 G > A were observed at 287 bp fragment. Significantly (p C. Genotypes of INHA 253 bp (exon 2) locus had significant (p ≤ 0.05) influence on litter size, body weight and trunk circumference. The PP and PQ showed a significantly higher litter size and trunk circumference (p ≤ 0.05) compared to QQ genotypes.Present study indicated the potential role of INHA gene on reproduction and growth, hence it can be used as a candidate gene in the improvement of Malabari goats.

Polymorphism in inhibin alpha gene and its association with semen quality traits in Murrah bulls

An investigation was conducted to detect single nucleotide polymorphism in Inhibin alpha gene (INHA) and to analyse the association between the observed polymorphisms with semen quality traits in Murrah buffalo bulls. Genomic DNA was isolated from blood samples of 103 bulls, the amplified PCR products were further subjected to polyacrylamide gel electrophoresis. Band patterns were characterised by the number of bands, mobility shifts and scored manually. Representative PCR products giving unique SSCP patterns were custom sequenced using automated ABI DNA Sequencer to confirm the mobility shift in each pattern. Sequence data were analysed using DNA Baser and Bio edit software Clustal omega multiple alignments for detecting single nucleotide polymorphisms in comparison to observed sequence with the Bubalus sbubalis INHA gene reference sequence. INHA gene, exon1 was monomorphic, while exon 2 was found to be polymorphic. For simple interpretation of SSCP patterns, INHA exon 2 was divided into two fragments. The first fragment of INHA exon 2 (INHA exon 2 FR1) revealed a transition at the C267119T position and a transversion at the G267033T position in the coding region in comparison to Bubalus bubalis reference sequence. The second fragment of INHA exon 2 (INHA exon 2 FR2) exhibited one transition at T266510C position in comparison to the Bubalus bubalis reference sequence. The SSCP variants of the first fragment of INHA exon2 had a significant effect on semen volume per ejaculate (P<0.05) and sperm concentration (P<0.01). The SSCP variants of the second fragment of INHA exon 2 had a significant effect on sperm concentration (P<0.01) and functional membrane integrity (P<0.05). The observed association between SSCP variants in INHA gene with semen volume per ejaculate, sperm concentration and functional membrane integrity suggests the possibility of using these genetic variants in INHA gene as prognostic markers for selecting Murrah bulls with high semen quality traits.

Identification of Two Novel Mutations in KDM3A Regulatory Gene in Iranian Infertile Males

Background:KDM3A is a key epigenetic regulator expressed in the testis and is required for packaging and condensation of sperm chromatin. To this point, the association of the KDM3A gene with infertility has not been studied in human. The aim of this study was to screen any new mutation in KDM3A gene to explore more details of human male infertility.Methods:In this work, 150 infertile men (oligozoospermia and azoospermia) and 150 normal healthy fathers were studied. Polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) and sequencing were used to screen any mutation in exons 12, 22, and 24 of KDM3A.Results:The infertile men showed various SSCP patterns for the exons 12 and 24, but not for exon 22. A transversion point mutation in exon 12 and a single nucleotide deletion in exon 24 were detected using sequencing analysis. The transversion mutation was located in the preceding exon of lysine-specific demethylase1 and Jumonji (Jmj)-C domain and the later one (deletion) in the cupin-like motif of KDM3A protein. Neither Y chromosome microdeletions nor partial azoospermia factor deletion was found in these patients.Conclusion:The mutations found in infertile men with otherwise unexplained severe spermatogenic failure could be considered as the origin of their abnormalities.

Polymorphism identification in ovine KISS1R/GPR54 gene among pure and crossbreeds of Iranian sheep

Fertility traits have the greatest financial impact on sheep production. In this study we aimed to characterize polymorphisms of the KISS1 receptor gene (KISS1R), also known as the G-protein-coupled receptor 54 gene (GPR54) that is reported to be involved in the control of puberty and reproductive function. Genomic DNA were obtained from 156 ewes of pure Mehraban and Shal Iranian native sheep and their crossbreeds with Romanov. The exploration of polymorphisms of the KISS1R/GPR54 gene (GenBank No: HM135393.1) was performed by single-strand conformation polymorphism analysis (SSCP) and Sanger sequencing. Seven single-nucleotide polymorphisms (SNPs) including g.396 T > G, g.456 T > C, g.475C > A, g.571 A > C, g.3431C > A, g.4108 G > A and g.4123C > A, were observed in the four breeds. Among these SNPs the g.3431C > A in the exon 4 was the only amino acid altering variant (p.195 Phe > Leu). Subsequent statistical analysis revealed that the minor A allele at this position had a significant (P < 0.01) negative effect on litter size (LS) and birth weight (BW) and could be considered as a causal mutation impairing these traits. No significant (P> 0.05) allelic association with the studied traits was found at the position g.396 T > G, g.456 T > C, g.475C > A and g.571 A > C. In contrast, carrier ewes of the SSCP pattern F (homozygous reference; g.4108 G/G, g.4123 C/C) showed a significantly (P < 0.01) higher LS than ewes carrying the patterns G (heterozygous; g.4108 G/A, 4123 C/A) or E (homozygous variant; g.4108 A/A, g.4123 C/C). The results of the present study provide additional evidences on the potential role of the KISS1R/GPR54 gene in controlling reproductive traits and particularly prolificacy in sheep.

Single Nucleotide Polymorphism in KiSS1 gene and its association with semen quality in Bos taurus and Bos indicus bulls

Selection of high fertile bulls with the help of marker assisted selection has gained importance in recent years. The low heritability of fertility traits hampers improvement of these traits by conventional selection based on phenotypic records. No information is available on the role of SNPs in KiSS1 gene in cattle on semen quality parameters in bovines. KiSS1 genes code for Kisspeptin, which are essential upstream regulators of neurons secreting gonadotropin-releasing hormone and play crucial role in reproduction.The coding regions along with exon-intron boundaries of KiSS1 gene, was characterized using PCR-SSCP method and direct sequencing. Two genotypes were observed which were represented as SSCP pattern 1 and pattern 2 and found to carry one SNPs (T153C) and one insertion of G at 291_292bp. The bulls with pattern 2 were heterozygous with respect to the transition T153C and pattern1 bulls were homozygous with TT genotype. The transition was predicted to cause amino acid change from Valine to Alanine. The frequency of bulls with pattern1 and pattern 2 were 0.67 and 0.33 in 67 Holstein Friesian bulls and 0.73 and 0.27 in 13 Khillari bulls. The association study of genotypes with semen quality parameters revealed significant association of genotypes with acrosome integrity in fresh semen (P less than 0.05) and no association with sperm concentration, volume per ejaculate, percent live sperm and Hypo Osmotic Swelling Test (HOST) with higher acrosome integrity in bulls with pattern2. Upon validation of the results in larger population and identifying the exact role of the novel SNP T153C and insertion of G at 291_292bp, they could be incorporated in selection programme for improving fertility in bulls.as markers for acrosome integrity in cattle.

Identification of NURR1 (Exon 4) and FOXA1 (Exon 3) Haplotypes Associated with mRNA Expression Levels in Peripheral Blood Lymphocytes of Parkinson's Patients in Small Indian Population.

Here, we study the expression of NURR1 and FOXA1 mRNA in peripheral blood lymphocytes and its haplotypes in coding region in a small Chennai population of India. Thirty cases of Parkinson's patients (PD) with anti-PD medications (20 males aged 65.85 ± 1.19 and 10 females aged 65.7 ± 1.202) and 30 age matched healthy people (20 males aged 68.45 ± 1.282 and 10 females aged 65.8 ± 1.133) were included. The expression of NURR1 and FOXA1 in PBL was detected by Q-PCR and haplotypes were identified by PCR-SSCP. In the 30 PD cases examined, NURR1 and FOXA1 expression was significantly reduced in both male and female PD patients. However, NURR1 (57.631% reduced in males; 28.93% in females) and FOXA1 (64.42% in males; 55.76% in females) mRNA expression did differ greatly between male and female PD patients. Polymorphisms were identified at exon 4 of the NURR1 and at exon 3 of the FOXA1, respectively, in both male and female patients. A near significant difference in SSCP patterns between genders of control and PD population was analyzed suggesting that further investigations of more patients, more molecular markers, and coding regions should be performed. Such studies could potentially reveal peripheral molecular marker of early PD and different significance to the respective genders.

Associations of polymorphisms in the prolactin receptor gene with growth trait in Japanese quail (Coturnix coturnix japonica)

Prolactin receptor (PRLR), as an important regulatory gene about growth and differentiation, might be a condition gene for reproductive traits. The prolactin receptor is a specific receptor for prolactin (PRL), which is an anterior pituitary peptide hormone involved in various physiological activities and is essential for reproductive improvement. In chickens, the PRLR gene is on the Z chromosome. In the present study, the polymorphism of exon 2 of PRLR gene in Japanese Quail (Coturnix coturnix japonica) was investigated. Blood samples of 180 Japanese Quail were collected randomly. DNA was extracted from blood samples and amplified. Polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis of the exon 2 of PRLR gene. PCR-SSCP analysis of the exon 2 region revealed two banding patterns. Two different SSCP patterns, representing two different genotypes, were identified. The frequencies of the observed genotypes were 0.939 and 0.061 for AA and BB. Allele frequencies were 0.939 and 0.061 for A and B. Observed heterozygosity (Hobs) value was 0.0. The chi-square test showed significant (P&lt;0.05) deviation from Hardy-Weinberg equilibrium for this locus in studied population. The effect of these genotypes on weight gain was investigated, and the BB genotype was found to be associated with body weight at 46 days. This was the first study on polymorphism of PRLR gene in Japanese quail.

Comparative Study of SSCP Pattern of V3 and V4-V5 Region of 16S rRNA of Metagenomic DNA for the Identification Rumen Microbial Diversity

Comparative Study of SSCP Pattern of V3 and V4-V5 Region of 16S rRNA of Metagenomic DNA for the Identification Rumen Microbial Diversity

Polymorphism in the promoter region of HSP70 gene and its association with performance traits in Deoni cattle

A study was undertaken to analyze associations between HSP70 genetic variants of promoter region with heat tolerance and performance in Deoni (Bos indicus) cattle using PCR-SSCP technique. The analysis revealed three SSCP patterns, viz. AA, AB and BB with frequencies of 0.712, 0.254 and 0.034, respectively. Sequence analysis of representative samples of each pattern showed 7 SNPs, which included four transitions (A859G, A1046G, C1257T and G1294A) and 3 transversions (A897C, A1127C and G1166T). Cows with different SSCP patterns showed similar production and reproduction performances. The studies on physiological parameters and calculation of Heat tolerance coefficient (HTC) of individual cows indicated that Deoni cows maintained at Bengaluru were not under heat stress even after 6 h of exposure to sun during summer (March to May).

Sequence characterization and polymorphism detectionin AQP7 gene of Murrah buffalo

Aquaporin 7 (AQP7) gene is a member of aqua-glyceroporins which transports glycerol and water to spermatids. The present study aimed to investigate the polymorphisms within exons 2, 3, 4, 5 and their flanking intronic regions in AQP7 gene of Murrah bulls. Genomic DNA was extracted from 69 Murrah bulls blood samples and was subjected to polymerase chain reaction- single strand conformation polymorphism (PCR-SSCP) analysis. PCR-SSCP analysis revealed a total of eight different variants for amplicons of exons 4 and 5. Amplicon of exon 4 revealed three different patterns viz. E4P1, E4P2 and E4P3 with the frequency of 0.30, 0.22 and 0.48, respectively. Analysis of exon 5 revealed five unique SSCP patterns viz. E5P1, E5P2, E5P3, E5P4 and E5P5, with the frequency of 0.10, 0.37, 0.20, 0.20 and 0.13, respectively. Sequence analysis showed 16 single nucleotide polymorphisms, 7 of which were observed in coding region. Amplicons of exons 2 and 3 showed monomorphic patterns. However, compared to the reference sequence of taurine cattle one transition (C6878T) in exon 3 and 4 transitions (G2099A, C2116T, A2117G, G6848C) in intron 2 were observed for all the bulls under study. The genetic variability identified in the AQP7 gene may serve as potential genetic marker(s) for semen quality traits in buffalo.

Molecular Characterization and Phylogeny Based Analysis of Intron I Sequence of Myostatin (MSTN) Gene in Iranian Makuei Sheep Breed

Abstract Myostatin (MSTN), a negative regulator of skeletal muscle development, acts as a potential candidate gene used to increase muscle mass. Likewise, sheep MSTN gene has an important role in meat production. MSTN is made up of 376 amino acids, and is synthesized as a precursor protein. To investigate the MSTN in Iranian native Makuei sheep, a polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP) analysis was used. Genomic DNA was isolated from the blood samples. A 417-bp of MSTN intron I segment was amplified using locus-specific primers. Four SSCP patterns were identified and nucleotide sequencing of the Makuei sheep MSTN gene was done and registered in the NCBI GenBank with “KJ526625” number. Three novel single nucleotide polymorphisms (SNPs) were found in the samples. These SNPs are found in 224bp, 226bp and 242bp locations. Accordingly three substitutions (c.224C&gt;T; c.226A&gt;G; c.242G&gt;T) were observed in the intron 1 region of MSTN gene. The effects of the observed SNPs on breeding values of some biometric traits were investigated and the substitution of c.226A&gt;G was found to be associated with heart girth (HG) and leg circumference (LC). Phylogenetic analysis, based on the nucleotide sequences indicated similar evaluation with the GenBank reference sequences. It seems that the observed polymorphisms of the ovine MSTN gene are associated with HG and LC traits.