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Background:MicroRNA (miRNA) is a short, single-stranded, non-coding sequence of RNA that does not translate into proteins. Because of their ability to control gene expression, recent studies have shown that these short, non-coding sequences play an important role in cancer. So that, detection the polymorphisms or variants in miRNA genes may help to identify their correlation to cancer susceptibility.Objectives:Study the polymorphisms in miRNA genes or variants miRNA genes and their correlation with myeloma disease.Materials and Methods:Twenty samples were collected from people who visited Marjan Hospital in Province of Babylon, Iraq, from February to July 2022. In addition, 20 samples from healthy people were collected as control. Genotyping of miRNA was accomplished using the polymorphism techniques of polymerase chain reaction and single strand conformation polymorphism (SSCP). DNA sequencing was also applied to categorize all the ranges of SSCP models detected using only gel imaging.Results:The genotypes obtained in this experiment verified that several single nucleotide polymorphisms were obtained between 5-band and 6-band miRNA National center for biotechnology information Primer3 plus reference.Conclusion:The results explain that the miRNA gene polymorphisms may have correlation with the susceptibility to myeloma disease.

The experiment was conducted during January, 2022 to January, 2023 at the Department of Animal Genetics & Breeding, College of Veterinary Science and Animal Husbandry, Kamdhenu University, Navsari, Gujarat, India to identify polymorphisms in HSP 70 gene. Heat Shock Proteins (HSPs) known as molecular chaperones are essential for cells' ability to recover from stress and serve as the primary defence mechanism within cells. They are extremely conserved and essential to the response to heat stress and cellular thermotolerance. Even though there are numerous HSP genes, in livestock species, heat tolerance is primarily associated with HSP 70. This gene polymorphisms have been linked to heat tolerance, milk production, fertility and cattle susceptibility to disease. They can be utilised as genetic markers to help choose animals that are more resilient to climate change, have stronger immune systems and perform better overall. A 220 bp fragment of bovine HSP 70 gene was subjected to Polymerase Chain Reaction-Single-Strand Conformation Polymorphism (PCR-SSCP) technique to identify the polymorphism. PCR-SSCP pattern was associated with the thermotolerance traits in Kankrej cattle using the univariate GLM model of SPSS 26. HSP 70 gene (220 bp fragment) was found to be monomorphic documented on SSCP gel which revealed only one genotype (AA) in all Kankrej cattle. It is concluded that genotype and its association with thermotolerance traits were found to be non-significant. However, The HSP 70 polymorphism is expected to strongly predict cattle heat tolerance, aiding in selection for thermotolerance. [ABSTRACT FROM AUTHOR]

The aim of this study was to evaluate the effects of different levels of oak Acorn on the biodiversity of rumen epimural bacterial population using PCR-SSCP molecular technique in Merkhoz goats. A total of 24 Markhoz goats with a mean BW of 16.93±1.25 kg and an average age of 4-5 months were tested in a completely randomized design with 4 treatments and 6 replications for 105 days. Experimental treatments included 1) control diet, 2) diet containing 8% oak acorn, 3) diet containing 17% oak acorn and 4) diet containing 25% oak acorn. The results showed that the effect of diet on biodiversity of rumen bacterial epimural community was significant (P <0.001). There was no significant difference between other experimental treatments (P> 0.05), although treatments containing 8% and 17% oak had more variety than treatments containing 25% oak. Sampling site had no effect on biodiversity of rumen bacterial epimural community (P> 0.05). The highest value of Shannon index was related to ventral ruminal site and the lowest was related to reticulum. The interaction effect of diet and sampling position on biodiversity of rumen bacterial epimural community was significant (P> 0.05). The results showed that the use of oak acorn up to 17% in the diet increased the biodiversity of the of rumen bacterial epimural community, while the use of 25% oak in the diet decreased the biodiversity of the rumen bacterial epimural community. [ABSTRACT FROM AUTHOR]

Cathepsin K (CTSK) is a lysosomal protease existent in the skeletal muscles which is involved in biochemical processes related to obesity. Several studies have reported the effects of CTSK gene on body weight and fat deposition in human, mice and pigs. However, information about its structure and functions in sheep is very limited. Thus, this study was performed to evaluate the association between CTSK gene variants and yearling growth performance in Afshari × Booroola-Merino crossbred sheep. A fragment of 500 bp in exon 6 and partial of intron 5 of CTSK gene was amplified with polymerase chain reaction (PCR). All animals were genotyped by single-stranded conformation polymorphism (SSCP) and further confirmed by sequencing. Association analysis using a fixed linear model indicated that g.106510225G > A SNP was significantly related to average daily weight gain (ADWG) per year, fat-tail weight to carcass weight ratio (FW/CW), muscle thickness (MT) and muscle cross-sectional area (MCSA) of animals (p < 0.05). Due to the low polymorphic information content (PIC <0.25) for targeted locus in studied population, more association studies are needed to confirm the CTSK gene effects on growth traits in sheep. [ABSTRACT FROM AUTHOR]

In recent years, strategic plans for poultry production have emphasized quantitative traits, particularly body weight and carcass traits (meat yield), in response to overpopulation challenges. Candidate genes such as adenylosuccinate lyase (ADSL), melanocortin-4-receptor (MC4R), and calpain 1 (CAPN1) have played vital roles in this context due to their associations with muscle growth and body composition. This study aims to investigate the influence of polymorphisms and gene expressions of the aforementioned genes on body weight (BW), growth rate (GR), breast weight (BrW), and thigh weight (TW) across four distinct chicken breeds: Fayoumi, Matrouh, Mamourah, and Leghorn. The use of PCR-SSCP analysis revealed genetic polymorphisms through the identification of various patterns (genotypes) within the three examined genes. The ADSL, MC4R, and CAPN1 genes exhibited five, three, and two different genotypes, respectively. These polymorphisms displayed promising connections with enhancing economically significant production traits, particularly BW, BrW and TW. Furthermore, gene expression analyses were conducted on breast and thigh tissues obtained from the chicken breeds at 60 days of age, where ADSL and MC4R exhibited a noteworthy up-regulation in Fayoumi and Matrouh breeds, and down-regulation in Mamourah and Leghorn. In contrast, CAPN1 expression decreased across most breeds with a slight increase noted in Fayoumi breed. In conclusion, this investigation underscores the substantial impact of ADSL, MC4R, and CAPN1 genes on economically important production traits within Egyptian domestic chicken breeds. Consequently, these genes emerge as significant molecular markers, holding potential utility in avian selection and breeding programs aimed at enhancing productive performance. [ABSTRACT FROM AUTHOR]

چکیدهسابقه و هدف: ژن میوستاتین به عنوان عامل مهارکننده رشد عضله اسکلتی شناخته شده و اگر جهش در ناحیه کدکننده آن اتفاق افتد، باعث تغییر نقش مهاری آن و افزایش عضله می‏گردد. این پژوهش با هدف شناسایی چندشکلی موجود در اگزون 3 ژن میوستاتین و بررسی صفات بیومتری وزن بدن و لاشه اندازه‏گیری ‌‌‌‌شده با اولتراسونوگرافی در گوسفند کردی انجام شد. مواد و روش: در این پژوهش برای اندازه‌گیری صفات ضخامت چربی پشت و مساحت ماهیچه چشمی از دستگاه اولتراسوند استفاده شد. خون‌گیری به طور تصادفی از تعداد 139 راس گوسفند کردی انجام و پس از استخراج DNA، قطعه 338 جفت بازی اگزون 3 ژن میوستاتین تکثیر شد. جهت تعیین ژنوتیپ‏ ژن میوستاتین، از تکنیک‏هایPCR-RFLP ، PCR-SSCP و تعیین توالی مستقیم استفاده شد. بررسی اثرات ثابت جنس، نوع تولد و سال تولد بر صفات مورد مطالعه با رویه GLM نرم‌افزار SAS و مقایسه میانگین‌های حداقل مربعات سطوح اثرات ثابت با آزمون توکی- کرامر در سطح معنی‌داری 5 درصد انجام شد. برای بررسی ارتباط بین صفات از همبستگی پیرسون استفاده شد.یافته‏ها: در روش‏PCR-RFLP پس از هضم آنزیمی تمام نمونه‏ها، تنها یک ژنوتیپ مشاهده شد و این جایگاه مونومورف (تک شکل) می‏باشد. نتایج به‏دست آمده از روش PCR-SSCP، دو الگوی باندیA و B را نشان داد که شباهت زیادی بین دو الگوی مشاهده شده وجود داشت. لذا برای تعیین ژنوتیپ واقعی، برخی از نمونه‏ها با الگوی متفاوت با روش توالی‌یابی مستقیم مورد ارزیابی قرار گرفتند که نتایج نشان داد که در بین نمونه‌ها چندشکلی وجود ندارد. میانگین ضخامت چربی پشت و مساحت عضله راسته اندازه‏گیری شده با اولتراسونوگرافی در این مطالعه به‏ترتیب 46/0 سانتی‏متر و 78/7 سانتی‏متر مربع با میانگین وزن بدن 42/43 کیلوگرم بود. همبستگی بین دو صفت اولتراسوند (ضخامت چربی پشت و مساحت ماهیچه چشمی)، مثبت و 84/0 برآورد شد (01/0P

The present study was conducted to identify single nucleotide variations in exon 3 region of the bovine Toll-Like Receptor gene (TLR4) associated with somatic cell score (SCS) in crossbred cattle of Kerala using polymerase chain reaction-single stranded conformational polymorphisms (PCR-SSCP) technique. Blood samples were collected from 200 lactating crossbred cattle in different farms of Kerala Veterinary and Animal Sciences University (KVASU) and field centres of ICAR- FPT scheme, KVASU. Genomic DNA was isolated and polymorphisms were identified by SSCP analysis. The 231 bp fragment of exon 3 in TLR4 gene was found to polymorphic and two genotypes viz. CC and CD were obtained with frequencies 77 per cent and 23 per cent respectively. The frequencies of C and D allele in crossbred were found to be 0.88 and 0.12. On sequencing, C to T transition had led to one non-synonymous change at 2021th position of ORF. Cattle with CC genotype were associated with significantly lower SCS (p≤0.05) as compared to CD genotype. The association between TLR4 polymorphism and SCS described in this study suggests the possible role of SNP during mastitis infection. Thus, could be employed as a useful marker for genetic selection to promote mastitis resistance in dairy cattle.

Introduction: Having a fat tail is a characteristic of some Iranian native sheep breeds, whose main role is usually to store energy for using in limited food conditions. However, the amount of energy required to store fat in this tissue directly affects the efficiency of meat production and carcass quality. In Iran, the average weight of each carcass is about 15.3 kg, of which 15% is the fat tail. It requires 1.7 kg more feed per kilogram of fat tail than meat (protein), and customers pay a lower price per unit of weight for sheep with heavier fat tail. Today, researchers in the field of animal breeding have a special focus on reducing the weight of the fat tail and increasing the marketability of sheep carcasses. Bone morphogenetic protein 2 (BMP2) belongs to the β-metamorphic growth factor of the β family and plays an important role in bone and cartilage development and, therefore, seems to be the best candidate for the fat-tailed phenotype. Comparison of the results of genotype obtained from Ovine SNP50K Bead Chip in tow fat-tailed breeds with the results obtained in 13 thin tail breeds showed a missense mutation in BMP2 gene, with the frequency of different alleles in these two different groups.Materials and Methods: In this study, in order to detect the polymorphism in BMP2 gene exon 1 and investigation of its relationship with tail fat trait, blood samples from 150 same age ewes of Lori Bakhtiari breed were randomly taken which are maintained in Gahar Dorud sheep breeding center (Dorud, Lorestan). DNA extraction was performed using a special DNA extraction kit (Pars Toos, Iran) according to the manufacturer's instructions. Determination of quality and quantity of extracted DNA was performed using agarose gel electrophoresis and nanodrop spectrophotometer, respectively. BMP2 gene exon 1 was amplified successfully by a pair of specific primer. The accuracy of this process was confirmed by 1.5% agarose gel. Then, using PCR-SSCP technique, 12% polyacrylamide gel and silver nitrate staining, probable polymorphisms were tracked in this position and finally calculation of Chi-square test (χ2) for deviation from Hardy-Weinberg equilibrium (HWE) has been assessed by Popgen (Ver. 1.32) software. The relationship between genotypes and average fat tail weight (Corrected by body weight (BW)) has been analyzed with the PROC GLM procedures in Statistical Analysis System (SAS) v. 9.1 version.Results and Discussion: Based on the results, amplification of BMP2 gene exon 1 was successfully done and three different patterns of polymorphism have been detected through SSCP analysis. Exon 1 of BMP2 gene in Lori Bakhtiari ewes containing A and B alleles with distributions of 197 and 103 and frequency of 65.7% and 34.33%, respectively, that have generated AA, AB and BB genotypes with distribution of 75, 47 and 28 and frequencies of 50%, 31.33% and 18.67%, respectively. Mean comparison of fat tail weight in each genotype using Duncan procedure showed that the effect of genotype on fat tail weight in Lori Bakhtiari breed was significant (P

The aim of the present study was to detect the FOXP3 gene polymorphisms in Barki sheep at a variable region covering exon 13, intron 13 and the coding sequence in exon 14 and to test the association of these polymorphisms with growth traits. 122 Barki lambs were phenotyped for various growth traits, viz., birth weight (BW), weaning weight (WW), pre-weaning daily gain in weight (ADG1), post-weaning daily gain in weight (ADG2) and marketing bodyweight (MW). The polymerase chain reaction – single-strand conformational polymorphisms (PCR-SSCP) and DNA sequencing methods were used to identify the genetic variants in the FOXP3 gene. The associations between the variation in FOXP3 gene and growth traits were tested using a general linear model. Two variants (F1 and F2 with gene frequencies of 0.64 and 0.36, respectively), and three genotypes (F1F1, F1F2 and F2F2 with frequencies of 0.37, 0.53 and 0.10, respectively) were detected. The association of FOXP3 genotype was significant (p < 0.05) with ADG2 and MW. It is concluded that FOXP3 genotype might be helpful for sheep breeders to produce fast-growing lambs. However, further studies are needed in a large population to confirm the association we found. [ABSTRACT FROM AUTHOR]

For revealing molecular markers related to the traits of multiple lumbar vertebrae in sheep, we analyze the relationship between NR6A1 gene polymorphism and lumbar vertebrae number traits in Xinjiang Kazakh sheep. Lumbar muscle tissues were collected from 6-lumbar spine (L6) Kazak sheep and 7-lumbar spine (L7) Kazak sheep and the intron-8 of NR6A1 gene was amplified by PCR. The SNP locus was detected by the PCR-SSCP method. One-Way ANOVA and an Independent Chi-square Test is adopted to analyze the genotype association with lumbar spine number variation. There were two SNP loci in the intron-8 of the NR6A1 gene: IVS8-188 and IVS8-281. One-Way ANOVA and Independent Chi-square Test indicated a significant association between IVS8-281 and lumbar spine number. The SNP locus of NR6A1 gene intron 8 (IVS8-281G > A) could play a certain role in the variation of lumbar spine number in Xinjiang Kazakh sheep and demonstrates potential to accelerate the sheep breeding of selection process. [ABSTRACT FROM AUTHOR]

ABSTRACT The objective of this study was to evaluate the association of polymorphisms in the diacylglycerol acyltransferase ( DGAT ) and leptin ( LEP ) genes with the performance, carcass characteristics, meat quality, and lipid profile of Nellore cattle. A total of 100 intact male Nelore cattle were used to analyze the performance, carcass, physicochemical and centesimal composition, and fatty acid profile of beef. To identify the polymorphisms, the PCR–single-strand conformation polymorphism (SSCP) technique was applied to genomic DNA extracted from muscle tissue. The SSCP technique revealed the presence of four band patterns for the DGAT gene (AC, AD, AE and BB) with five alleles (A, B, C, D and E). For the LEP gene, five band patterns (AA, AB, AC, BB and BC) with three alleles (A, B and C) were observed. For the LEP gene, the AB genotype was associated with higher backfat thickness and ribs weight, while the BB genotype was associated with lower ribs yield; higher hindquarter yield was associated with AC and BB genotypes. Higher contents of C17:0, C18:0 and lower contents of C18:2ω6C, total polyunsaturated fatty acids, total ω6 and ratio of polyunsaturated and saturated fatty acids (PUFA/SFA) were verified for the AC genotype of the LEP gene. The AC and AA genotypes of the LEP gene were associated with higher means of C15:0 and C18:1ω9t. For the DGAT gene, the highest C24:0 content was associated with the AE genotype and the lowest with the AD and BB genotypes. Polymorphisms in the DGAT and LEP genes influence carcass parameters and the lipid profile of the meat of Nellore cattle.

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The present study was conducted to identify single nucleotide variations in exon 3 region of the bovine Toll-Like Receptor gene (TLR4) associated with somatic cell score (SCS) in crossbred cattle of Kerala using polymerase chain reaction-single stranded conformational polymorphisms (PCR-SSCP) technique. Blood samples were collected from 200 lactating crossbred cattle in different farms of Kerala Veterinary and Animal Sciences University (KVASU) and field centres of ICAR-FPT scheme, KVASU. Genomic DNA was isolated and polymorphisms were identified by SSCP analysis. The 231 bp fragment of exon 3 in TLR4 gene was found to polymorphic and two genotypes viz. CC and CD were obtained with frequencies 77 per cent and 23 per cent respectively. The frequencies of C and D allele in crossbred were found to be 0.88 and 0.12. On sequencing, C to T transition had led to one non-synonymous change at 2021th position of ORF. Cattle with CC genotype were associated with significantly lower SCS (p=0.05) as compared to CD genotype. The association between TLR4 polymorphism and SCS described in this study suggests the possible role of SNP during mastitis infection. Thus, could be employed as a useful marker for genetic selection to promote mastitis resistance in dairy cattle. [ABSTRACT FROM AUTHOR]

The work was oriented to identification of SCD gene polymorphism in population of Slovak pinzgau cattle. The material involved 91 cows of pinzgau breed kept in Slovakia. Bovine genomic DNA was isolated by fenolchlorophorm deproteinization and ethanol precipitation. The polymorphism of SCD gene was detected by PCR-SSCP method. For the checking of correct genotyping was used PCR-RFLP method. The PCR products were digested with SatI restriction enzyme. The mutant allele C (Ala) was detected by 205bp, 70 bp and 40 bp fragments and the wild allele T (Val)was detected by 205 bp and 117 bp fragments on 3 % agarose gel. In the total population included in the study was detected homozygous genotype CC ( 0.31 ), heterozygous genotype CT (0.42) and homozygous genotype TT( 0.27 ). The frequency of occurrence both alleles proved gently of predominance of mutant allele C – 0.52 before wild allele T – 0.48 .

Reproductive traits are regulated by several genes, and genetic polymorphism within these genes which have a role on the reproductive performance could be used in marker assisted selection breeding programs. Several variants of inhibin ßA (INHßA) gene affect the litter size in small ruminant. Therefore, different single nucleotide polymorphisms (SNPs) within this gene could be used as a genetic marker to improve the reproductive efficiency (specially litter size) of sheep and goats. The objective of this study is to find out INHßA subunit-encoding gene polymorphisms by employing PCR-SSCP assay and subsequent analyses of nucleotide sequence, as well as to investigate the potential association of these genetic polymorphisms with twin yield in Egyptian goats and sheep breeds. One hundred and thirty-nine females were chosen, 113 of them were mothers at the 1st., 2nd., 3rd and 4th parity, and the remaining 26 females were young at sexual maturity age. Blood samples were collected for genomic DNA extraction and PCR amplification were performed using five specific primers spanning the entire INHßA CDS partial 5'UTR. Four out of the five PCR amplicons displayed different genetic polymorphisms, and the subsequent nucleotide sequence analyses showed many nucleotide substitutions. In sheep, the amplified fragment 1 showed 3 different genotypes A, C and A/C. The sequence analysis for the 3rd amplicon showed two different genotypes, A and G. Also, the 5th amplicon reveals 3 different genotypes, A, G and A/G. In goats, the sequence analysis of the 4th amplified fragment showed different three genotypes, G, A and A/G. The statistical analysis proposed that the genotype AA (in sheep) could be favorable gene markers for twin production. The present study suggested that the genetic polymorphisms of INHßA gene might have effects on fecundity traits in Egyptian small ruminants. [ABSTRACT FROM AUTHOR]

Introduction1: Having a fat tail is a characteristic of some Iranian native sheep breeds, whose main role is usually to store energy for using in limited food conditions. However, the amount of energy required to store fat in this tissue directly affects the efficiency of meat production and carcass quality. In Iran, the average weight of each carcass is about 15.3 kg, of which 15% is the fat tail. It requires 1.7 kg more feed per kilogram of fat tail than meat (protein), and customers pay a lower price per unit of weight for sheep with heavier fat tail. Today, researchers in the field of animal breeding have a special focus on reducing the weight of the fat tail and increasing the marketability of sheep carcasses. Bone morphogenetic protein 2 (BMP2) belongs to the β-metamorphic growth factor of the β family and plays an important role in bone and cartilage development and, therefore, seems to be the best candidate for the fat-tailed phenotype. Comparison of the results of genotype obtained from Ovine SNP50K Bead Chip in tow fat-tailed breeds with the results obtained in 13 thin tail breeds showed a missense mutation in BMP2 gene, with the frequency of different alleles in these two different groups. Materials and Methods: In this study, in order to detect the polymorphism in BMP2 gene exon 1 and investigation of its relationship with tail fat trait, blood samples from 150 same age ewes of Lori Bakhtiari breed were randomly taken which are maintained in Gahar Dorud sheep breeding center (Dorud, Lorestan). DNA extraction was performed using a special DNA extraction kit (Pars Toos, Iran) according to the manufacturer's instructions. Determination of quality and quantity of extracted DNA was performed using agarose gel electrophoresis and nanodrop spectrophotometer, respectively. BMP2 gene exon 1 was amplified successfully by a pair of specific primer. The accuracy of this process was confirmed by 1.5% agarose gel. Then, using PCRSSCP technique, 12% polyacrylamide gel and silver nitrate staining, probable polymorphisms were tracked in this position and finally calculation of Chi-square test (χ2) for deviation from Hardy-Weinberg equilibrium (HWE) has been assessed by Popgen (Ver. 1.32) software. The relationship between genotypes and average fat tail weight (Corrected by body weight (BW)) has been analyzed with the PROC GLM procedures in Statistical Analysis System (SAS) v. 9.1 version. Results and Discussion: Based on the results, amplification of BMP2 gene exon 1 was successfully done and three different patterns of polymorphism have been detected through SSCP analysis. Exon 1 of BMP2 gene in Lori Bakhtiari ewes containing A and B alleles with distributions of 197 and 103 and frequency of 65.7% and 34.33%, respectively, that have generated AA, AB and BB genotypes with distribution of 75, 47 and 28 and frequencies of 50%, 31.33% and 18.67%, respectively. Mean comparison of fat tail weight in each genotype using Duncan procedure showed that the effect of genotype on fat tail weight in Lori Bakhtiari breed was significant (P <0.05). AA genotype with average fat tail weight of 5.16 showed higher performance than AB genotype with average fat tail weight of 4.29 and BB genotype with average fat tail weight of 3.76. The results of statistical analysis also showed that the presence of allele A causes heavier fat tail weight and the presence of B allele causes lower fat tail weight (P <0.05). Heterozygosity and Homozygosity observed in this study are 0.3154 and 0.6846, respectively. The significance of the calculated chi-square genotypic frequency in each population at the level of 0.05 in comparison with the chi-square table shows that the studied populations are not in Hardy-Weinberg equilibrium. Which can be attributed to the pressure of selecting on reference population for genetic breeding for fat tail weight at Gahar Dorud sheep breeding center. Today, advances in genomic technologies have multiplied, and if information on loci associated with meat quality traits can be obtained and the genes that control these traits are located on chromosomal sites, they can be incorporated into breeding programs. Breeds should be used with MAS and cause genetic growth and development of these traits. Conclusion: Using molecular detection methods and identifying sheep carrying B alleles and selecting them as the parents of the next generation, we can move towards producing herds with lower fat tail weight and more marketability. [ABSTRACT FROM AUTHOR]

Diacylglycerol O-acyltransferase 1 (DGAT1) is a microsomal enzyme that plays a key role in the synthesis of triglycerides. Its gene (DGAT1) is regarded as a candidate gene for variation in milk and meat traits in cattle. The objective of this study was to use a PCR single-strand conformation polymorphism approach to explore sequence variation in two regions of ovine DGAT1 and to assess its effect on meat traits in New Zealand Southdown sheep. Three variant nucleotide sequences were identified in each region, with two single nucleotide polymorphisms (SNPs) and one nucleotide deletion being detected in intron 1 and two SNPs being found in exon 17. The effect of the exon 17 variation was not investigated due to one variant being predominant and the other two variants occurring at low frequencies. In intron 1, one variant (B1) was found to be associated with increase loin meat yield, suggesting that this may have value as a gene marker for improving meat traits. [ABSTRACT FROM AUTHOR]

Despite the key role of casein genes and their effect on quantitative traits and technological assets of milk, few studies manipulated it in camels, unlike other ruminants. Thus, this investigation was performed to lighten up the genetic polymorphism of CSN2 and CSN3 genes, in Maghrebi camels, via PCR-SSCP and DNA sequencing tools, by measuring the impacted milk composition traits. Sixty-eight blood samples were collected from Maghrebi camels in Mersa Matrouh, Egypt. Also, 68 milk samples were collected at different stages of the lactation period. Protein, fat, lactose, total solid, and ash were estimated in all milk samples by biochemical methods. The results of the PCR-SSCP and the sequence analysis showed two genotypes (CC and CA) in CSN2 gene with one SNP (41 T > G) for CC genotype and two SNPs (41 T > G and 462A > C) for CA genotype, as showed three genotypes (AA, AT, and TT) in CSN3 gene with two SNPs (42C > A and 346 T > A) for AA genotype, two SNPs (42C > A and 100 C > T) for TT genotype, and four SNPs (42C > A, 100C > T, 238 T > G, and 346 T > A) for AT genotype. Allele C of CSN2 gene affected milk fat only at the first day and the first seven days of lactation period, while allele T of CSN3 gene affected both the studied milk composition initiated from the first seven days to the end of the lactation period. The genetic discrimination of the understudied genes may be utilized as a valuable marker in the selection of superior animals, through the favorable alleles and genotypes to improve the camel milk composition. [ABSTRACT FROM AUTHOR]

Background: CTCF encodes 11-zinc finger protein which is implicated in multiple tumors including the carcinoma of the breast. The Present study investigates the association of CTCF mutations and their expression in breast cancer cases. Methods: A total of 155 breast cancer and an equal number of adjacent normal tissue samples from 155 breast cancer patients were examined for CTCF mutation(s) by PCR-SSCP and automated DNA sequencing. Immunohistochemistry (IHC) method was used to analyze CTCF expression. Molecular findings were statistically analyzed with various clinicopathological features to identify associations of clinical relevance. Results: Of the total, 16.1% (25/155) cases exhibited mutation in the CTCF gene. Missense mutations Gln > His (G > T) in exon 1 and silent mutations Ser > Ser (C > T) in exon 4 of CTCF gene were analyzed. A significant association was observed between CTCF mutations and some clinicopathological parameters namely menopausal status (p = 0.02) tumor stage (p = 0.03) nodal status (p = 0.03) and ER expression (p = 0.04). Protein expression analysis showed 42.58% samples having low or no expression (+), 38.0% with moderate (++) expression and 19.35% having high (+++) expression for CTCF. A significant association was found between CTCF protein expression and clinicopathological parameters include histological grade (p = 0.04), tumor stage (p = 0.04), nodal status (p = 0.03) and ER status (p = 0.04). Conclusions: The data suggest that CTCF mutations leading to its inactivation significantly contribute to the progression of breast cancer.

پرواپیوملانوکورتین (پومس) یک پروتئین پیش ساز در جوجه­های گوشتی است که از 251 اسیدآمینه تشکیل شده است و در تنظیم غذای مصرفی و تعادل مصرف انرژی نقش دارد. هدف از انجام این پژوهش، تعیین چندشکلی ژن پرواپیوملانوکورتین و بررسی ارتباط آن با صفات رشد (که شامل وزن زنده، وزن لاشه، وزن سینه، وزن ران، وزن پشت وگردن، بال، کبد، قلب، بورس، طحال، پانکراس، پیش معده + سنگدان و چربی محوطه بطنی) در جوجه­های گوشتی سویه راس وکاب بود. بدین منظور از تعداد 100 قطعه جوجه گوشتی سویه راس و 60 قطعه جوجه گوشتی سویه کاب نمونه خون تهیه و استخراج DNA به­وسیله روش نمکی بهینه یافته صورت گرفت. پس از استخراج DNA، قطعه­ای به اندازه bp444 از ناحیه اگزون دوم ژن پرواپیوملانوکورتین با استفاده از تکنیک PCR تکثیر گردید. جهت تعیین ژنوتیپ نمونه­ها، از روش چندشکلی فرم فضایی رشته­های منفرد (SSCP) و الکتروفورز محصولات تک رشته­ای بر روی ژل پلی آکریل آمید و رنگ آمیزی توسط نیترات نقره انجام شد. برای ژن پرواپیوملانوکورتین در جوجه­های گوشتی سویه راس دو الگو بنام ­E و F و در جمعیت جوجه­های گوشتی سویه کاب نیز چهار الگوی A، B،C و D مشاهده شد. ارتباط چندشکلی­ این ژن با صفات رشد به­وسیله رویه­ی GLM نرم افزار SAS آنالیز شد و نتایج نشان داد که در جوجه­های گوشتی سویه راس و کاب، قطعه 444 جفت بازی ژن پرواپیوملانوکورتین در جایگاه اگزون دوم دارای چند شکلی است. و هیچ یک از الگوهای مشاهده شده در ناحیه مورد مطالعه از ژن پرواپیوملانوکورتین، در هیچ کدام از دو جمعیت ­ارتباط معنی­داری با صفات رشد ندارند.

Serum lysozyme gene empowers the immune system due to its bacteriolytic property targeting peptidoglycan layer of cell wall. Polymorphs of serum lysozyme gene are reported to be significantly associated with disease resistance traits. Two different gene fragments viz. fragment I (275 bp) and fragment II (230 bp) of serum lysozyme gene were amplified and screened by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) technique to identify genetic variation in two different goat breeds (Barbari and Marwari) of India. The serum lysozyme activity of different genotypes and mRNA expression across the breed was also estimated. In fragment I, four genotypes (AA, BB, AB, and CC) and three alleles (A, B, and C) were identified. The CC genotype (frequency-0.44) and B allele (frequency-0.46) was found to be prevalent in the population. In fragment II, six genotypes (AA, BB, AC, AD, AE, and FF) and six alleles (A, B, C, D, E, and F) were identified. The AD genotype (frequency-0.44) and A allele (frequency-0.53) were found to be prevalent in the population. A significant difference (p = 0.05) in mean serum lysozyme activity was found among different genotypes of fragment I in Marwari goat only. The genotypes of fragment II did not show any statistically significant effect on serum lysozyme activity. Relative quantification showed that nondescript animals had higher expression levels of serum lysozyme gene as compared to registered goat breeds. A single nucleotide variation was detected at 93rd position (G to A) of full-length coding sequence where guanine in Barbari goat was replaced by adenine in Black Bengal goat. The polymorphic nature of the serum lysozyme gene and its differential expression will help to establish the role of the lysozyme gene in the disease resistance mechanism. [ABSTRACT FROM AUTHOR]

The POU1F1 gene has been identified as one of the major gene for productivity, and its polymorphism is known to be associated with important production traits of various livestock. The present study was designed to investigate POU1F1 (exon-3) gene polymorphism and its association with body weight in Osmanabadi goat (n=217). The PCRSSCP and DNA sequencing revealed single ‘C’ > ‘T’ transition at nucleotide position 42 in the third exonic region (225 bp) POU1F1 gene of Osmanabadi goat. Genotyping revealed ‘TT’ and ‘CC’ genotypes with predominance of ‘C’ allele in the Osmanabadi goat population. Though no association between the said SNP and body weight was observed, these findings can be extended to find the genetic variability of POU1F1 gene associated with growth traits of various Indian goat breeds.

Five keratin-associated protein 6 genes (KRTAP6) have been identified in sheep and variation in some KRTAP6 has been associated with wool fiber diameter-related traits, but none of these homologues have been identified in goats. In this study, we reported the identification of the sheep KRTAP6-5 homologue on goat chromosome 1 and polymerase chain reaction (PCR)-single strand conformation polymorphism analysis in 300 Longdong cashmere goats revealed the existence of 12 variant sequences. Both coding region and 3′UTR of the putative caprine KRTAP6-5 displayed a biggest sequence similarity to ovine KRTAP6-5 gene. This suggested that the gene represents caprine KRTAP6-5 sequences, and these sequences composed 23 genotypes, which was the most polymorphism gene in KRTAPs that have been studied. Among these sequences, 15 nucleotide substitutions and a 24-bp insertion/detection were identified. Variation in goat KRTAP6-5 was associated with variation in mean-fiber diameter, suggesting that KRTAP6-5 is worthy of further study in the context of variation in cashmere traits. [ABSTRACT FROM AUTHOR]

Single-strand conformation polymorphism (SSCP) is a reproducible and sensitive method for the detection of genetic polymorphisms and mutations in a wide range of polymerase chain reaction (PCR) products. However, the applications of this technique are largely confined to a set of cumbersome optimizations. Herein, a non-time-consuming method for PCR-SSCP that can be conducted with minimum efforts and less technical expertise is presented. The main concept of this simplified technique was based on many optimizations that were conducted to ensure the highest possible sensitivity to detect genetic polymorphism without incorporating sophisticated equipment and tedious efforts. The optimum gel concentration, temperature, and time requirements were strictly adjusted so as not to be further modified before applying this method for genotyping purposes. Furthermore, minimized silver staining steps were combined with this method to further minimize time and effort. It was confirmed that the performed adjustments were not reduced the overall sensitivity of the technique. Therefore, the suggested method can be utilized to genotype a wide range of PCR products (mainly from 200 to 600 bp) without the need for further optimizations and modifications. This study proposes a rapid SSCP protocol for genotyping PCR products using simple, low-cost, and friendly to perform recipes. [ABSTRACT FROM AUTHOR]

Hereditary non-polyposis colorectal cancer (HNPCC) is the most frequent autosomal dominant predisposition for development of colorectal cancer (CRC) caused by germline defects in mismatch repair (MMR) genes. Current study was aimed to find genetic variations in MSH2 and MLH1 genes and their correlation with the serum levels of Carcinoembryonic Antigen (CEA) in seven Iranian HNPCC families. Seven unrelated Iranian families including 11 HNPCC patients and 7 affected family members were selected. They were initially screened for mutations in exons 7 of MSH2 and exon 15 of MLH1 gene through polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP). Positive PCR results were further analyzed through exon sequencing. Serum CEA level was determined using the ELISA test. PCR-SSCP was positive in 8 out of 18 patients (44%) for exons 7 of MSH2 gene, whereas two samples (11%) demonstrated to bear a mutation in exon 15 of the MLH1 gene. Sequencing analysis of both amplified exons in positive and negative samples have confirmed no mutation in negative samples while revealed 5 and 7 novel mutations in exons 7 and 15, respectively. The mean serum concentration of CEA had a significant difference between HNPCC patients and their healthy family members. Our results demonstrated that the PCR-SSCP method has high specificity and sensitivity in the first step of mutation screening of HNPCC families. High frequency of novel alterations found in the current assay may revise the mutation screening of MSH2 and MLH1 genes and abet further assessment of their frequency among individual HNPCC patients.

PCR single-strand conformation polymorphism (SSCP) and DNA sequencing techniques were used to analyze the genetic polymorphism of mannose-binding lectin (MBL) gene 3 introns in Hu sheep. The results showed that 3 introns of MBL gene had polymorphism, 3 genotypes were identified in intron1, which were controlled by 2 alleles, respectively; 3 genotypes were identified in intron2, which were controlled by 2 alleles, respectively; 3 genotypes were identified in intron3, which were controlled by 2 alleles, respectively; 6 new single nucleotide polymorphisms(SNPs) mutation sites were found, g.288T>A in intron1, g. 1091 T>C, g.1096A>C, g.1770G>C in intron 2, g.2297C>T, g.2331G>A in intron3. Use ELISA to detect MBL serum level in Hu sheep, then use One-way ANOVA analyze the relationship of different genotypes and MBL serum level. And the result showed that the MBL serum level of AA genotype was higher than BB genotype (P

Flystrike is a major cost and a welfare issue for the New Zealand sheep industry. There are several factors that can predispose sheep to flystrike, such as having fleecerot, a urine-stained breech, and “dags” (an accumulation of fecal matter in the wool of the breech). The FABP4 gene (FABP4) has been associated with variation in ovine fleecerot resistance, with a strong genetic correlation existing between fleecerot and flystrike occurrence. In this study, blood samples were collected from sheep with and without flystrike for DNA typing. PCR-SSCP analyses were used to genotype two regions of ovine FABP4. Sheep with the A1 variant of FABP4 were found to be less likely (odds ratio 0.689, P = 0.014) to have flystrike than those without A1. The likelihood of flystrike occurrence decreased as copy number of A1 increased (odds ratio 0.695, P = 0.006). This suggests that FABP4 might be a candidate gene for flystrike resilience in sheep, although further research is required to verify this association.

Flystrike is a major cost and a welfare issue for the New Zealand sheep industry. There are several factors that can predispose sheep to flystrike, such as having fleecerot, a urine-stained breech, and "dags" (an accumulation of fecal matter in the wool of the breech). The FABP4 gene (FABP4) has been associated with variation in ovine fleecerot resistance, with a strong genetic correlation existing between fleecerot and flystrike occurrence. In this study, blood samples were collected from sheep with and without flystrike for DNA typing. PCR-SSCP analyses were used to genotype two regions of ovine FABP4. Sheep with the A 1 variant of FABP4 were found to be less likely (odds ratio 0.689, P = 0.014) to have flystrike than those without A 1. The likelihood of flystrike occurrence decreased as copy number of A 1 increased (odds ratio 0.695, P = 0.006). This suggests that FABP4 might be a candidate gene for flystrike resilience in sheep, although further research is required to verify this association. [ABSTRACT FROM AUTHOR]

Myeloid differentiation-2 (MD-2), as an essential component of the CD14-TLR4/MD-2 receptor complex, is critical in identifying bacterial Lipopolysaccharide (LPS) and activating innate immune responses. To evaluate the relationship between MD-2 polymorphisms (including 5′ end and exon regions) and clinical mastitis, population genetic analysis was performed via PCR single strand conformation polymorphism (PCR-SSCP) and direct sequencing in Chinese Holstein dairy cows. Eleven pairs of primer PCR products for SSCP analysis: six pairs of primers (P1-P6) for the 5′-end, four (P7–P10) for the exon regions, and one (P11) for 3′-untranslational region. There were six SSCP bands (named: EE, EF, FF, EQ, EM and EN genotype) in the PCR amplification products of primer P1, two bands in P4 (CD and DD) and three bands in P5 (AA, AB, and BB). Total of five (g.-2173 C/G, g.-2148 C/T, g.-2089 G/T, g.-555 G/A and g.-121 C/A) single nucleotides polymorphism sites (SNPs) were identified in 5′-end of the MD-2 gene. Data showed that SNPs g.-555 (G/A) had significant differences (P

Leptin is a protein hormone secreted by white adipose tissue, and variation in leptin gene (LEP) has been described to affect milk production traits in different cattle breeds. The purpose of this study was to investigate whether nucleotide sequence variations identified in exon 3 of LEP were the same as those reported previously, and ascertain whether the variations detected affected milk traits in New Zealand (NZ) Holstein-Friesian × Jersey-cross (HF×J-cross) cows. Polymerase Chain Reaction – Single Strand Conformation Polymorphism (PCR-SSCP) analysis coupled with nucleotide sequencing, enabled 443 HF×J-cross dairy cows to be studied. The PCR-SSCP and nucleotide sequencing revealed three variant sequences (A, B and C), with a total of five single-nucleotide substitutions. There were no associations revealed between LEP genotypes and milk yield and fat percentage. However, cows carrying the AB genotype had a decreased percentage of protein in their milk. This is the first time such findings in NZ HF×J-cross cows have been reported, and they suggest that variation in exon 3 of LEP may be useful for selecting cows for improvement in key milk traits. [ABSTRACT FROM AUTHOR]

The geographical center of species diversity for sheep (Ovis aries) lies around several regions of Iraq and Iran. Therefore, it is necessary to explore the genetic relationships among these domesticated breeds and their maternal ancestors. This study was conducted to assess the mitochondrial D-loop genetic diversity in three breeds of Iraqi/Iranian prolific sheep, exhibiting clear differences in their geographical distributions and phenotypic traits. Five different SSCP-banding patterns were identified in Arabi, while only four distinct patterns were observed in both Karakul and Awassi sheep. One haplotype each in Karakul and Awassi was shared with Arabi. Sequencing reactions confirmed these patterns and validated the presence of 11 haplotypes and 16 variations distributed among studied populations. Arabi exhibited the maximum haplotype diversity, while Awassi exhibited the highest diversity of nucleotide polymorphism. The fixation index among populations showed a high sharing of genetic material among the investigated populations. The observed similar haplotypes showed the highest proportions in Arabi and Karakul and near the highest proportions in Awassi. In conclusion, higher nucleic acid variations were observed in the investigated Arabi and Awassi breeds than in the Karakul breed, signifying a potential role for D-loop-based genotyping in these differences in the three investigated ovine breeds. [ABSTRACT FROM AUTHOR]

The experiment was conducted on Haringhata Black Chicken breed to investigate the polymorphism of exon-2 of Toll-Like Receptor-4 gene. Genomic DNA extracted from 82 birds was used for Polymerase Chain Reaction (PCR). Amplified polymerase chain reaction (PCR) product (248bp) of exon-2 of TLR4 gene revealed two types of SSCP banding pattern arbitrarily assigned as EF and FF genotypes on 10 percent polyacrylamide gel. The genotype frequencies were found to be 0.634 for EF and 0.366 for FF genotype with respect to exon-2 of TLR4 gene. The frequencies of E and F alleles were estimated as 0.317 and 0.683, respectively. The calculated Chi square value revealed that the population was not in Hardy Weinberg Equilibrium with respect to TLR4 gene in Haringhata Black Chicken.

Gene markers are often usefull for selection process of cattle on commercial certain phenotypes. Saanen goat has predominance in milk production traits. Milk fat content is one of the commercial traits of milk production. Diacylglycerol Acyltransferase1 (DGAT1) gene was involved in the final process of triglyceride synthesis and associated with milk fat content. Single nucleotide subtitution or insertion-deletion in certain sequence of DGAT1 gene could be gene markers for milk fat content. The objective of this study was to identify differences variation in milk fat content among individual Saanen goats, to identify exon 14-16 polymorphism of DGAT1 gene and to examine its correlation between polymorphism and milk fat content on Saanen goats. The methodologies was collection blood and milk samples, DNA isolation from blood, amplification of DGAT1 genes, sequencing, detection of polymorphism of exon 14-16 DGAT1 gene with Polymerase Chain Reaction-Single Strand Conformation Polymorphism (PCR-SSCP), and examination milk quality on three lactation months using Lactoscan. The average of daily milk production from 70 Saanen goat was 2340±709 mL, 1880±632 mL, 1432±536 mL and average of milk fat content was 3.07±0.52%, 2.53±0.55%, 3.00±0.55% for the first, second and third months of lactation, respectively. The amplicon size of exon 14-16 of DGAT1 gene was ± 350 bp and showed monomorphic pattern from PCR-SSCP. Variation of milk fat content on Saanen goats not caused by polymorphism of exon 14-16 DGAT1 gene.

Introduction: Congenital heart diseases (CHDs) are structural cardiovascular malformations that arise from abnormal development of the heart during the prenatal life. Mutations in the TBX5 gene, encoding T-box transcription factor, are a major cause of CHD. To evaluate the TBX5 mutations in hotspot exons in sporadic pediatric patients with CHD phenotypes, analytical case/control study performed in an Iranian cohort of unrelated patients with clinical diagnosis of congenital heart malformations. Methods: We investigated TBX5 coding exons 4, 5, 6 and 7 in 95 sporadic patients with CHD phenotypes and compared to 82 healthy controls using PCR-SSCP and DNA sequencing approaches. Results: We report here on a novel and heterozygote Non-sense mutation in exon 5 of TBX5, E128X (G14742T), in two Iranian children. This mutation locates inside the T-box and both of pediatric patients carrying this novel mutation suffer from severe heart malformations. The G14742T mutation leads to the substitution of glutamic acid (E) by stop codon (X) at residue 128, an evolutionarily conserved position in T-box as well as in other species. The non-sense mutation of E128X was predicted to be pathogenic by Mutation Taster and Polyphen software programs. Conclusion: TBX5 E128X mutation results in a translational premature stop. This type of mutation results in a shortened protein that may function improperly and which cannot bind to other transcription factors; therefore, haploinsufficiency of TBX5 protein is presumably causing the severe cardiac malformations in these patients.

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Sheep have been shown to be genetically diverse but this genetic diversity has not been fully described, there are still many sheep populations which have not yet been studied. The purpose of this study were to investigate the polymorphism in the calpain gene (CAPNS1) in Iraqi awassi sheep by using PCR-SSCP method, and their relationship to growth traits. A 192-bp fragment of exons 5 and 6 including intron region of the CAPNS1 gene was amplified and subjected to PCR-SSCP method to screen for polymorphisms. The observed variants had exhibited different distributions in the analyzed samples in terms of the targeted 192 bp amplicons. Were detected five CAPNS1 genetic patterns according to SSCP technology A, B, C, D, and E. Results of sequence analysis revealed five nucleotide substitutions at nt 44, nt 64, nt 94, nt 103 in a part of intron 5, and nt 154 in a part of exon 6 (SNP position). The substitutions at exon 6 nt154 T > C, this SNP is a silent mutation {GGT (Glycine) > GGC (Glycine)}, which creates no substitution effect for amino acid sequence of CAPNS1 protein. Were found Three alleles named C, G and T. Also showing insertion of 'A' nucleotide at nt (72-73) in a part of intron 5. This genetic variation is in noncoding DNA, therefore it is difficult to conclude how this polymorphism may affect the CAPNS1 gene activity. The lambs used in the present study were weaned at three months of age. The average weight gain for weaning weight, six, nine, and twelve months were also recorded the overall average gain 21.62 ± 0.61, 31.32 ± 0.59, 37.51 ± 0.76, 41.42 ± 0.68, and 19.80 ± 0.40 kg, respectively. The results obtained in this study showed the importance of genotypes as sources of variation, where there was significant (P <0.05) effect of genotype in different economic growth traits used in the analysis. Also the study results showed that the breed is unbalanced because the calculated x2 values are greater than the tabular in all positions of the studied CAPNS1 gene, The imbalance, according to Hardy Weinberg rule, may be due to the possibility of out breeding. It may be the result of non-random mating for several years in awassi sheep populations. The results confirmed that the PCRSSCP is an appropriate tool for determining genetic variability in CAPNS1 locus. Since this gene could be considered as a candidate gene of growth and meat quality traits in sheep and may affect lamb quality parameters., further study may be conducted to ascertain the association of these genotypes with the growth and carcass/meat quality traits. [ABSTRACT FROM AUTHOR]