Simultaneous identification of ryanodine receptor 1 (RYR1) and estrogen receptor (ESR) genotypes with the multiplex PCR-RFLP method in Polish Large White and Polish Landrace pigs

A method allowing simultaneous genotyping of two loci: ryanodine receptor 1 (RYR1) and estrogen receptor (ESR) is presented. In multiplex PCR amplification, two amplicons were simultaneously produced: a 272 bp fragment of RYR1 gene and a 185 bp fragment of ESR gene and were then subjected to "one-tube" restriction enzyme digestion with Hin6 I and Ava I, respectively. A total of 122 Polish Large White and Polish Landrace pigs were genotyped by this method, demonstrating its reliability, convenience and lower costs. This method may be useful in the wide-scale genotyping of both loci in pig breeding programmes.     

Genotyping of the porcine ryanodine receptor 1 (RYR1) and estrogen receptor 1 (ESR1) genes by high resolution melting (HRM) approach

During the last decade, DNA mutations in the porcine ryanodine receptor 1 gene (RYR1, C1843T) and the estrogen receptor 1 gene (ESR1, T1665G), have been widely used in marker-assisted selection (MAS) for the pig industry. These 2 well-characterized SNPs in RYR1 and ESR1 are responsible for porcine stress syndrome (PSS) and litter size, respectively. Here, we describe a reliable, high-efficiency method for the genotyping of these 2 genes using the high-resolution melting (HRM) method. The HRM approach exhibited high-accuracy and repeatability, comparable with the classic PCR-restriction fragment length polymorphism (PCR-RFLP) approach, and is potentially suitable for large-scale genotyping in commercial pig farms.     

Refinement of diagnostic assays for a probable causal mutation for porcine and human malignant hyperthermia.

The substitutions of T for C1843 in the porcine ryanodine receptor (RYR1) gene, which deletes a HinPI restriction endonuclease site and creates a HgiAI site, and of T for C1840 in human RYR1, which deletes a RsaI site, lead to Cys for Arg substitutions in the ryanodine receptors and are probable causal mutations for malignant hyperthermia (MH). To improve the restriction endonuclease assay of these sites, thereby providing an accurate, reliable diagnosis for MH, introns flanking the exon containing the mutation were sequenced, permitting identification and PCR amplification of a 659-bp porcine gene sequence that contains both constant and variant HgiAI sites and a 922-bp human gene sequence that contains both constant and variant RsaI sites. As a result, these PCR-amplified sequences contain constant internal controls for the reliable differentiation by restriction endonuclease digestion of normal, heterozygous, and MH genotypes.     

雌激素受体基因和长白猪繁殖性能相关研究

通过检测612头长白母猪共2 239窝ESR基因的PvuⅡ酶切多态性,分析了不同ESR基因型和长白母猪繁殖性状的相关,以确定ESR基因在猪育种应用的可能性.头胎母猪、第2胎母猪和3胎以上母猪的资料分开统计.群体中B等位基因的频率很低,但似然比检验结果显示群体的基因频率处于哈代-温伯格连锁平衡状态.第1胎BB基因型母猪的总产仔数显著高于AA型母猪(12.05±0.82 vs 10.19±0.24)(P＜0.05),但BB基因型母猪的初生仔猪重显著低于AA型母猪(1.23±0.07vs 1.42±0.02)(P＜0.05).第3胎以上资料合并,BB基因型母猪的总产仔数显著多于AA和AB基因型母猪(11.98±0.63 vs10.90±0.48/10.92±0.51)(P＜0.05),产活仔数显著高于AA型母猪(10.31±0.58 vs 9.43±0.45)(P＜0.05);AB基因型母猪初生仔猪重显著低于AA型母猪(1.44±0.04 vs 1.48±0.04)(P＜0.05).所有资料合并,BB基因型母猪的总产仔数极显著高于AB型母猪(11.63±0.52 VS 10.63±0.42)(P＜0.01),显著高于AA型母猪(11.63±0.52 vs 10.70±0.40)(P＜0.05);BB基因型母猪的产活仔数显著高于AB和AA基因型母猪(10.15±0.50 vs 9.33±0.39/9.41±0.41)(P＜0.05).其余情况下各基因型母猪间繁殖性状间差异不显著(P＞0.05).总之,BB基因型母猪的总产仔数和产活仔数优于其他基因型母猪,但仔猪初生重较低.ESR基因可以作为遗传标记,用于本群长白猪产仔数的选择.     

猪MyoG基因3'端PCR-SSCP遗传多态性及其遗传效应

采用PCR—SSCP方法对长白猪、大白猪、杜洛克猪、山西黑猪和马身猪共636头猪的肌细胞生成素（Myogenin,简称MyoG）基因3’端的遗传多态性进行检测,分析MyoG基因对猪的初生体质量、断奶体质量、6月龄体质量和背膘厚的影响。根据已发表的猪MyoG基因3’端侧翼序列设计3对引物,发现F1／R1引物对扩增的片段有多态性。统计结果发现：长白、大白、杜洛克猪种B基因为优势基因,其基因频率分别为0．8807、0．7256和0．8581：山西黑猪种4基因为优势基因,其基因频率为0．9359;马身猪种只检测到4基因。χ^2独立性检验表明,基因型分布在外来猪种（长白猪、大白猪、杜洛克猪）与地方猪种（山西黑猪、马身猪）间存在极显著差异（P〈0．01）。固定效应模型分析结果表明,初生体质量基因型闻差异显著（P〈0．05）,而断奶体质量、6月龄体质量和背膘厚基因型闻差异不显著（P〉0．05）。最小二乘分析结果表明,BB基因型与其它2种基因型比较有较大的初生质量,同似和AB型比较差异极显著（P〈0．01）。因此,推测MyoG基因对个体的初生体质量存在一足的影响,选择带有B等位基因的个体有望提高个体的初生体质量。     

红鳍东方鲀(Takifugu rubripes)MC4R基因的多态性分析

采用PCR-SSCP(single strand conformation polymorphism)技术和DNA测序方法分析红鳍东方鲀MC4R(Melanocortin-4receptor)基因编码区多态性。在MC4R基因编码区48 nt和264 nt均发生了碱基的转换突变(G→A),两个突变位点分别位于M1和M2引物扩增产物中。引物M1扩增产物SSCP分析得到两种基因型:AA基因型和AB基因型,并且AA基因型和A等位基因频率明显高于AB基因型和B等位基因。引物M2扩增产物也得到两种基因型:CC基因型和CD基因型,CC基因型和C等位基因频率明显高于CD基因型和D等位基因。遗传变异结果分析表明,两个突变位点均属于低度多态性,而且群体遗传杂合度较低,反映了该群体的遗传一致性较高。     

Polymorphism of the estrogen receptor 1 locus in populations of pigs of different genotypes and its association with reproductive traits of large white sows

Populations of Large White, Large Black, Poltava Meaty, Pietrian, and Meishan pigs are characterized by intron PvuII polymorphism in the estrogen receptor 1 gene (ESR1). These breeds differ in ESR1 alleles and genotypes frequencies. In the populations, there are deviations in the genotype distribution from the Hardy-Weinberg equilibrium. The established estrogen receptor 1 locus association with the reproductive traits of Large White sows indicates the possibility of using intron ESR1 PvuII polymorphism as a genetic pregnancies marker in pig selection.     

Simultaneous analysis of bovine K-casein and BLAD alleles by multiplex PCR followed by parallel digestion with two restriction enzymes

An improved and simplified method allowing simultaneous genetic typing of K-casein and CD 18 (bovine leucocyte adhesion deficiency; BLAD) loci has been developed. The method is based on the simultaneous amplification of fragments of the two groups of alleles by multiplex PCR, and on a concurrent, parallel digestion of the products by two restriction enzymes ( Pst I and Hae III) in the same incubation buffer. Digestion with Pst I distinguishes K-casein A and B alleles and does not cut within any of the BLAD alleles, while digestion with Hae III allows the differentiation between normal and mutant allele variants of the CD28 locus. All combinations of the known mutants of the two alleles, characterized to the regions amplified and resulting in phenotypic effect, could be detected by electrophoretic separation performed on the same agarose gel owing to the vast differences in the length of the restriction fragments.     

Genetic polymorphisms of estrogen receptor alpha and catechol-O-methyltransferase genes in Turkish patients with familial prostate carcinoma

OBJECTIVES:

Estrogen is one of the most crucial hormones participating in the proliferation and carcinogenesis of the prostate glands. Genetic polymorphisms in the estrogen metabolism pathway might be involved in the risk of prostate carcinoma development. We evaluated the association between genetic polymorphisms in estrogen receptor alpha (ESR1) and catechol-O-methyltransferase (COMT) genes and the risk of developing familial prostate carcinoma.

MATERIALS AND METHODS:

In this study, 34 cases with prostate carcinoma whose first-degree relatives had prostate carcinoma and 30 healthy age-matched male controls were enrolled. The genotypes of ESR1 and COMT genes were analyzed employing polymerase chain reaction-restriction fragment length polymorphism method. 34 cases with prostate carcinoma, whose first degree relatives had prostate carcinoma and 14 age-matched male controls were enrolled to analyze the genotype of these two genes.

RESULTS:

Among control patients, the ESR1 PvuII genotypes of C/C, C/T and T/T were observed in 37%, 26% and 37%, respectively, whereas the C/C, C/T and T/T genotypes were observed in 18%, 41% and 41% of case patients, respectively. Among controls, the ESR1 PvuII allele frequencies of C and T were equally observed, whereas the C and T allele frequencies were observed in 38% and 62% of patients, respectively. Among ESR1 PvuII genotypes there were not any significant difference in terms of genotype (P = 0.199) and allele (P = 0.181) frequencies. Among controls, the ESR1 XbaI genotypes of G/G, G/A and A/A were observed in 33%, 37% and 33%, respectively, whereas the G/G, G/A and A/A genotypes were observed in 12%, 47% and 41% of patients, respectively. Among controls, the ESR1 XbaI allele frequencies of A and G were observed equally, respectively, whereas the A and G frequencies were observed in 65% and 35% of patients, respectively. Among ESR1 Χ baI, there was not any significant difference in terms of genotype (P = 0.111) and allele (P = 0.093) frequencies. But the C/C genotype of the PvuII site and G/G genotype of the XbaI site in the ESR1 gene were associated significantly with the risk of developing prostate carcinoma. The G/G, G/A and A/A genotypes of the COMT gene were observed in 50%, 29% and 21% of control patients and in 53%, 21% and 26% of case patients, respectively. The A and G allele frequencies of the COMT gene were observed in 36.7%, 63.3% of control patients and in 36.8%, 63.2% of case patients, respectively. In COMT gene, there was not any significant difference in terms of genotype (P = 0.843) and allele (P = 0.991) frequencies. But the G/A genotype of the COMT gene had a weak tendency toward increased risk.

CONCLUSION:

Polymorphisms of ESR1 gene in the estrogen metabolism pathway were associated significantly with familial prostate carcinoma risk. Single nucleotide polymorphisms of low-penetrance genes are targets for understanding the genetic susceptibility of familial prostate carcinoma.     

山羊抑制素α亚基基因HaeⅡ酶切多态性及其与产羔数性状的关联分析

以323 只马头山羊、努比山羊、波尔山羊和海门山羊为试验材料, 利用PCR-SSCP、PCR-RFLP和克隆测序等方法对山羊抑制素α亚基基因(INHA)编码区一个片段进行多态性分析, 发现在该基因第284位(登录号: L28815)存在G→A突变, 该突变引起HaeⅡ酶切位点改变; HaeⅡ-RFLP分型结果显示G等位基因为优势等位基因, INHA不同基因型平均产羔数表现为GG>AG>AA。结果显示: INHA可能是影响山羊产羔数性状的一个主效基因, G等位基因可能与高产性状呈正相关。     

猪HSL基因多态性研究及其部分DNA片段的测序

激素敏感脂肪酶（ＨＳＬ）是动物体脂肪分解的关键酶。采用ＰＣＲ－ＳＳＣＰ法研究猪ＨＣＬ基因的启动子区、部分５’－端转录非翻译区,第７外显子区、第８外显子区和３’－端部分转录非翻译区域ＤＮＡ多态性。结果在对应于ＨＳＬ基因第８外显子区域发现１ＤＮＡ片段的ＳＳＣＰ,表现出３种基因型ＭＭ、ＭＮ和ＮＮ,脂肪型猪种梅山猪和通城猪含有更多的等位基因Ｍ（频率分别为０．６８０和０．７４０）,而瘦肉型猪种大白猪和长白猪     

Relation between Ava I polymorphism within the estrogen receptor gene (ESR) and meatiness in Polish Large White boars

It is currently debated whether identification of ESR (estrogen receptor) genotypes should be introduced into breeding programs of Large White pigs. The aim of this study was to evaluate the possible relations between ESR/Ava I polymorphism and carcass performance traits in Polish Large White boars. We examined 103 boars originating from one herd in NE Poland. ESR/Ava I genotypes were determined by the PCR-RFLP method. By the use of the Duncan test, we found highly significant differences (P < 0.01) between WW and MW genotypes, as well as significant differences (P < 0.05) between WW and MM genotypes for meatiness. No significant differences were found for daily gain and selection index.     

猪Mx1基因第14外显子多态性分析及新突变位点的 发现

采用PCR-RFLP方法对国内外7个猪种Mx1基因第14外显子的多态性进行分析, 共检测到3个等位基因, 6种基因型。其中杜洛克中仅存在AA基因型, 苏太猪中存在全部基因型, 只有在梅山猪和具有梅山猪血统的苏太猪中出现基因型BB。所有猪种中, 只有在地方猪种和培育猪种中出现等位基因B, 所有猪种除松辽黑猪外均以A为优势等位基因。卡方检验结果表明, 不同猪种间基因型分布差异较大, 梅山猪和松辽黑猪与其他所有猪种的基因型频率差异极显著(P＜0.01) , 苏太猪与除皮特兰猪外的所有猪种的基因型频率差异也极显著(P＜0.01) , 淮猪与杜洛克和约克夏这两个国外猪种基因型频率差异不显著(P＞0.05), 而与皮特兰和其他地方猪种的基因型频率均存在极显著差异(P＜0.01) 。通过测序在扩增片段中新发现了3种类型的碱基突变, 前2个分别导致了Thr和Glu向Ala和Arg的替换, 最后一个突变不引起氨基酸的变化, 且后两个突变位点为BB基因型所特有。     

A rapid PCR-based method for the identification of ob mutant mice

With increasing incidence of obesity, there is greater demand for suitable research and therapeutic models. The ob/ob mouse model develops obesity by 5 weeks of age. Previously, a method using DNA purification, PCR, and restriction digestion of products was devised to identify mice bearing the ob allele. Here, we describe a direct PCR method that requires no DNA purification. Wild-type and ob-specific primers are used under the same conditions in two separate and simultaneously run three-primer PCRs. Standard PCR using the wild-type primer mix produces 191 bp and 104 bp bands in +/+ and ob/+ and only the control 191 bp band in ob/ob animals. The ob-specific primer reaction produces 191 bp and 123 bp bands in ob/+ and ob/ob and only the control 191 bp band in +/+ animals. Phenotypic weight gain in offspring of heterozygous intercrosses was used to validate genotypes. This primer-specific PCR method allows simultaneous identification of +/+, ob/+, and ob/ob genotypes prior to breeding age to facilitate breeding and research studies in an important model of clinical obesity.     

Genotyping the bovine kappa-casein locus using polymerase chain reaction]

Polymerase chain reaction (PCR) was used for detecting kappa-casein (kappa-casein) genotype in the synthetic breed (Jersey x Black and White x Holstein-Friesian) dairy cattle. The amplified 228 bp fragment includes a region, where relevant mutations lead to both the appearance of different kappa-casein alleles associated with amino acid substitutions and the appearance of new TaqI and HindIII restriction sites in ae-casein B gene. The specificity of the kappa-casein gene fragment amplification was supported by restriction analysis and Southern blot hybridization. Digestion of amplified fragment with endonucleases PstI, HindIII and/or TaqI allows detection of AA-, AB- and BB genotypes of kappa-casein. A total of 32 animals with known (18 samples) and unknown (14 samples) kappa-casein phenotypes were tested using PCR and blot hybridization. In all known cases the detected genotype confirmed the phenotype. Frequencies of the B allele and of the AB genotype in the breeding population are rather high (53.1 +/- 8.8 and 43.7%, respectively). The possibility of effective use of the PCR analysis for genotyping kappa-casein locus in bulls and their offspring has been shown. The advantages of the PCR method in large breeding programs and linkage analysis have been discussed.     

Rapid detection of the 46C --> T polymorphism in the factor XII gene, a novel genetic risk factor for thrombosis, by melting peak analysis using fluorescence hybridization probes

Factor XII (FXII) level is an important intermediate phenotype associated with thrombotic disease. The 46C --> T transition in the exon 1 of the Factor XII (F12) gene is a significant, prevalent, and independent genetic risk factor for thrombotic disease. It is also associated with interindividual variation of plasma FXII zymogen levels. The aims of this study were to develop a rapid, reproducible, and easy method for 46C --> T genotyping and to compare its reliability with the classical endonuclease digestion methodology. DNA samples from 100 subjects were genotyped for the 46C --> T transition using the classical endonuclease digestion method with Sfna I. The genotypes of three of them (each with a different 46C R T genotype) were confirmed by direct sequencing analysis. We then set out to construct a LightCycler PCR protocol to detect the 46C --> T polymorphism. This protocol was designed to combine a rapid-cycle polymerase chain reaction (PCR) with an allele-specific fluorescent probe melting for mutation detection. In the three sequenced samples, as well as in the remaining 97, the LightCycler PCR procedure unambiguously resulted in the same genotype previously observed by sequencing and endonuclease digestion. Characteristic fluorescent curves were obtained for each genotype; the first derivative of these curves had a maximum at an apparent hybridization temperature (Tm) that was specific for each probe/allele duplex. The whole process took less than 40 min. Thus, if this method is used with a rapid DNA extraction, the genotypes would be obtained within 60 min after receiving a blood sample. In conclusion, the technique presented allows for easy, reliable, and rapid detection of this polymorphism, and is suitable for typing both small and large numbers of DNA samples.     

Estrogen receptor alpha polymorphism and susceptibility to uterine leiomyoma

Uterine leiomyoma is the most frequent pelvic tumor found in female genital tract. Some studies have suggested an association between single nucleotide polymorphisms (SNPs) in estrogen receptors genes with susceptibility in developing uterine leiomyoma. In this work, we estimated the frequency of two SNPs: one located in the intron 1 (rs9322331) and other in the exon 1 (rs17847075) of the estrogen receptor alpha (ESR1) gene in 125 women with uterine leiomyoma and 125 healthy women. To do this we used a PCR-RFLP method with MspI and HaeIII restriction enzymes to respectively detect C/T SNPs in the intron 1 and in the exon 1 of ESR1. To our knowledge this is the first study aimed to investigate the association of ESR1 SNPs with the risk of developing uterine leiomyoma in Brazilian women. Our results showed that the allele frequencies of the exon 1 and the intron 1 of the ESR1 gene did not differ between cases and controls (P = 0.325 and 0.175, respectively). Furthermore, our findings provided little support for the association of these SNPs on ESR1 with leiomyoma. However, we found that the SNP in the intron 1 of the ESR1 gene was underrepresented in the Brazilian female population.     

猪整合素β1基因CRS-PCR多态性与产仔数的关联性分析

文章采用CRS-RFLP技术对长白猪、大白猪和杜洛克猪3个品种的整合素β1基因第5外显子T32207C位点及第7外显子A35230G位点进行单核苷酸多态性分析,并将基因多态性与猪的产仔数进行关联分析。结果表明:32207多态位点的基因型效应对3个品种的总产仔数(TNB)和产活仔数(NBA)影响均不显著;35230多态位点的基因型效应对大白猪和长白猪头胎、二胎及所有胎次的TNB和NBA的影响达到显著(P<0.05)或者极显著水平(P<0.01),基因型GG、AG与AA对产仔数的影响存在差异,其效应为GG,AG>AA。可见整合素β1基因35230位点的G等位基因对大白猪和长白猪的产仔数性状有显著影响。     

SNP exploring in the middle and terminal regions of the IGF-1 gene and association with production and reproduction traits in Holstein cattle

Five primer sets were designed in order to identify single nucleotide polymorphisms (SNPs) in middle and terminal exons (2 to 6) and in some flanking intronic regions of the bovine insulin-like growth factor 1 (IGF-1) gene. Sequencing results of PCR products for 10% of animals showed no variant in exons but a SNP at intron 4 was occurred. Both polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) and high resolution melting (HRM) methods were developed to genotype samples. The PCR–RFLP results showed the presence of three fragments on agarose gel for the C allele due to two cleavage sites while two fragments for the T allele were observed. Melting curves of 123 bp fragments in HRM analysis showed a difference between temperature melting (T_m) of two homozygous genotypes as the CC genotypes had higher T_m than the TT genotypes. Melting curve of the CT genotype was different and crossed two parallel patterns of homozygous genotypes. The frequencies of the CC, CT and TT genotypes were 0.6, 0.37 and 0.03, respectively. Also, the estimated allele frequencies were 0.785 and 0.215 for the C and T alleles, respectively. Results showed higher accuracy of the HRM analysis compared to the PCR–RFLP method. Least square means (LSMs) comparison of the different genotypes in the SNP showed significant association with milk fat yield trait in the first lactation and open days after the second calving. The polymorphism did not have a significant effect on other milk production or reproduction traits. It seems that other variants or QTLs known in this region underlie genetic variation in the production and reproduction of dairy cattle.