延边黄牛UCP2、UCP3基因多态性及其与生长性状的关联分析

试验旨在探究延边黄牛解耦联蛋白2（uncoupling protein 2,UCP2）和解耦联蛋白3（uncoupling protein 3,UCP3）基因多态性及其与生长性状的相关性。以120头24月龄的延边黄牛为试验对象,提取血液基因组DNA后构建混池,运用DNA测序法对UCP2、UCP3基因进行测序分析,应用高分辨率熔解曲线分型技术对延边黄牛UCP2、UCP3基因进行分型检测,并结合生长性状数据进行关联分析。结果显示,在24月龄延边黄牛群体中,UCP2基因53 412 568 bp处存在C>G突变（g.53412568 C>G）,产生2个等位基因：C和G,形成3种基因型：CG （0.358）、GG （0.125）和CC （0.516）;UCP3基因53 443 536 bp处存在C>T突变（g.53443536 C>T）,产生2个等位基因：C和T,形成3种基因型：CT （0.383）、CC （0.450）和TT （0.167）。关联分析显示,UCP2基因g.53412568 C>G位点CC基因型体重、腰角宽均显著高于GG基因型,CG基因型个体背高显著高于GG基因型（P<0.05）;UCP3基因g.53443536 C>T位点CC基因型胸围显著高于TT基因型（P<0.05）。以上结果表明,UCP2和UCP3基因在延边黄牛生长发育过程中起到一定作用,可为延边黄牛现代分子育种后续研究提供参考。     

延边黄牛FGF14基因多态性及其对生长性状的影响

本研究旨在探索延边黄牛成纤维细胞生长因子14（fibroblast growth factor 14,FGF14）第3外显子多态性及其对生长性状的影响。应用DNA直接测序法对延边黄牛FGF14基因第3外显子进行测序分析,利用高分辨率熔解曲线HRM分型技术对FGF14基因进行分型检测,探究FGF14基因不同基因型与16和24月龄延边黄牛生长性状的关联性,并进行遗传统计学分析。结果显示,在FGF14基因的631 947 bp处检测到1处A>G突变,有2个等位基因：A和G,存在3种基因型：AA、AG和GG。χ²检验结果表明,FGF14基因的基因型频率和等位基因频率在16和24月龄延边黄牛群体中的分布均符合Hardy-Weinberg平衡定律（P>0.05）。多态信息含量（PIC）分析表明,FGF14基因在这2个群体中均处于中度多态（0.25 < PIC < 0.5）。与生长性状关联分析结果显示,FGF14基因不同基因型对16月龄延边黄牛背高和尻长均具有显著影响（P<0.05）,对24月龄延边黄牛体高、体重、十字部高均具有显著影响（P<0.05）。综上所述,推测FGF14基因对延边黄牛生长发育有一定影响,为后续延边黄牛肉质性状的现代分子育种选育工作提供参考。     

延边黄牛脂蛋白脂肪酶基因多态性及其与肉质性状的关联分析

为探究延边黄牛脂蛋白脂肪酶（lipoprteinlipase,LPL）基因遗传多态性及其对肉质性状的影响,本试验以80头健康的30月龄延边黄牛背最长肌作为试验材料,运用PCR直接测序法和高分辨率熔解曲线技术（HRM）对LPL基因的SNP位点进行检测,分析了试验群体的遗传效应,并与延边黄牛的肉质性状数据进行关联分析。PCR直接测序结果表明,延边黄牛LPL基因存在2个多态性位点（g.6215 A>G和g.18341 C>T）,其中g.6215 A>G位点表现为2种基因型：AA与AG,优势等位基因为A;g.18341 C>T位点表现为2种基因型：CC和CT,C为优势等位基因;对文献中2个位点的检测结果表明,g.355427 T>A位点表现为2种基因型：AA与AT,A为优势等位基因;c.322 G>A位点的HRM分型结果显示并未分型。这些多态性位点全部呈中度多态（0.25<PIC<0.5）,并处于Hardy-Weinberg平衡状态。HRM分型结果显示,这3个SNPs位点与延边黄牛肉质性状存在显著相关：g.6215 A>G位点与延边黄牛胴体重及脂肪、蛋白、水分含量有显著关联,表现为AG基因型个体胴体重、脂肪含量显著高于AA基因型个体（P<0.05）,AA基因型个体蛋白及水分含量显著高于AG基因型个体（P<0.05）;g.18341 C>T位点与延边黄牛宰前活重、胴体重及背膘厚有显著关联,表现为CT基因型个体这些性状均显著高于CC基因型个体（P<0.05）;g.355427 T>A位点AT基因型个体宰前活重、背膘厚和脂肪含量均高于AA基因型个体（P<0.05）。综上,LPL基因g.6215 A>G、g.18341 C>T和g.355427 T>A位点对延边黄牛的肉质有显著影响,LPL基因可以作为延边黄牛品种选育改良工作的优势候选基因和有效分子标记。     

ASB12基因SNPs位点多态性与延边黄牛生长性状关联分析

为研究延边黄牛细胞因子信号转导相关(ASB12)基因多态性与延边黄牛体尺性状的相关性,以203头同等饲养条件下延边黄牛阉牛为研究对象,采用PCR-RFLP技术检测ASB12基因的SNPs位点,并将其与延边黄牛体尺性状进行关联分析。结果:ASB12基因存在2个突变位点,分别为T709C突变,导致氨基酸Leu→Pro的错义突变;C1039T突变,导致氨基酸Pro→Leu的错义突变。χ~2检验显示,2个突变位点在所检测延边黄牛群体中处于Hard-Weinberg平衡状态(P0.05)。关联分析显示,延边黄牛ASB12基因2个突变位点的不同基因型与体高、体斜长差异显著(P0.05)。将2个突变位点合并基因型分析结果表明,组合AACG基因型个体在体高和体斜长显著高于组合BBGG型个体(P0.05)。初步认为ASB12基因对延边黄牛生长性状的有显著影响,其中组合AACG可能是影响延边黄牛生长性状的最佳基因型组合。     

肌联蛋白基因SNPs位点多态性与延边黄牛生长性状间的关联分析

为了研究延边黄牛肌联蛋白(Titin,TTN)基因的多态性对延边黄牛生长性状的影响,选取208头健康的32月龄延边黄牛,利用PCRRFLP和PCR-SSCP方法进行TTN基因遗传变异检测。结果表明:延边黄牛TTN基因存在2个突变位点,分别为A47751G突变,导致氨基酸Ile→Val的错义突变,C73301A同义突变;χ2检验显示,2个突变位点在所检测延边黄牛群体中处于Hard-Weinberg平衡状态(P005);关联分析显示,延边黄牛TTN基因2个突变位点的不同基因型与体高、体斜长、胸围和尻长上差异显著(P005)。将2个突变位点合并基因型分析结果表明,组合AACC基因型个体在体高和体斜长显著高于AATT型个体(P005),初步认为TTN基因对延边黄牛生长性状的有显著影响,其中组合AACC可能是影响延边黄牛生长性状的最佳基因型组合。     

延边黄牛SCD1基因多态性及其与经济性状的关联分析

本研究旨在探索延边黄牛硬脂酰辅酶A去饱和酶1（stearoy-CoA desaturase 1,SCD1）基因多态性及其与经济性状的相关性。以157头36月龄健康无病、体重相近的延边黄牛为研究对象,颈静脉采血提取基因组DNA,根据GenBank中牛SCD1基因序列（登录号：AC_000181.1）,利用Oligo 6.0软件设计特异性引物,应用PCR扩增SCD1全长基因后直接测序,分析其多态位点,并应用生物信息学软件分析其蛋白结构;屠宰前测定活体重、胴体重、屠宰率、背膘厚等胴体性状,屠宰后采集背最长肌,测定眼肌面积、蒸煮损失、滴水损失、嫩度、系水力、pH、脂肪酸含量等肉质性状,并通过一般线性模型分析方法对延边黄牛SCD1基因SNPs与肉质性状的相关性进行了分析。结果发现,延边黄牛SCD1基因846 bp处存在A→G突变（g846 A→G）、1 022 bp处存在C→T突变（g1022 C→T）,其中g1022 C→T突变导致丙氨酸（A）突变为缬氨酸（V）,但未引起三级结构改变。关联分析结果表明,g846 A→G多态位点与延边黄牛宰前活重、胴体重、滴水损失及硬脂酸、亚油酸含量存在显著相关（P<0.05）;g1022 C→T多态位点与延边黄牛宰前活重、胴体重及硬脂酸含量存在显著相关（P<0.05）,说明上述2个位点与延边黄牛相关肉质性状存在显著相关,该突变位点可作为潜在的分子标记。     

延边黄牛PRKAG3基因多态性及其与肉质性状的相关分析

采用PCR-SSCP方法对120头延边黄牛PRKAG3基因的多态性进行检测,共发现C6529T、T6605C、A6608G和G6685A4个SNPs;单倍型分析产生4种单倍型H1(CCGA)、H2(CCGG)、H3(TCGA)、H4(TCGG),其中H1为优势单倍型,其频率为0.590。结果表明:单倍型H3与蒸煮损失存在负相关(P0.05);单倍型H1与pH1、pH24呈正相关(P0.05),与L*24值负相关(P0.05);单倍型H4与油酸存在负相关(P0.05),与谷氨酸、丝氨酸、苏氨酸、精氨酸、丙氨酸和脯氨酸存在正相关(P0.05)。     

延边黄牛甲状腺球蛋白基因的SNPs及其与生长性状的关联分析

The SNPs of Yanbian Yellow cattles thyroglobulin (TG) gene were detected by cloning and sequencing, confirmed by technique of PCR-RFLP,and studied the association of polymorphisms of TG gene with growth traits. The results showed that there were C218T and A430G two mutations in the exon 48 in TG gene of Yanbian Yellow cattle. The association analysis showed that the chest breadth with TT genotype in C218T locus was significantly higher than that of CT genotype（P＜0.05）. A430G locus polymorphism significantly associated with the body weight, rump length and hucklebone width（P＜0.05）. The body weight of GG genotype was higher than AA genotype（P＜0.05）, the rump length of AA and AG genotypes were higher than GG genotype（P＜0.05）,and the hucklebone width of AG genotype was higher than GG genotype within population（P＜0.05）.In a word, C218T and A430G SNPs might be a good DNA marker to be used in MAS for growth traits.     

北京黑猪MYH3和MYH13基因多态性与肉质性状的关联分析

【目的】 试验旨在研究肌球蛋白重链3（myosin heavy chain 3,MYH3）和肌球蛋白重链13（myosin heavy chain 13,MYH13）基因遗传变异对猪肉质性状的影响。【方法】 利用北京黑猪背最长肌样本进行肉质性状（肌内脂肪（intramuscular fat,IMF）、水分、滴水损失、酸碱度（pH）、肉色L^*值、肉色a^*值和肉色b^*值）表型数据的测定。利用PCR和Sanger测序技术对MYH3和MYH13基因启动子区和CDS区进行基因分型。将性别、场、日龄作为协变量,采用协方差分析,确定与猪肉质性状相关的SNPs,利用实时荧光定量PCR检测基因表达水平。【结果】 试验共筛选到9个SNPs,其中MYH3基因CDS区有1个错义突变（c.5782 G>C）与IMF显著相关（P<0.05）,且MYH3基因表达水平与IMF呈正相关;MYH13基因CDS区有4个SNPs,其中2个（c.1923 G>A、c.1308 G>A）与肉色a^*值显著相关,1个（c.963 G>A）与滴水损失显著相关,1个（c.237 G>T）与肉色L^*值显著相关（P<0.05）;MYH13基因启动子区有4个SNPs,其中2个（rs699287502、rs318639161）与肉色L^*值显著相关,2个（rs321315318、rs330770991）与滴水损失显著相关（P<0.05）,且MYH13基因表达水平与滴水损失呈负相关。【结论】 MYH3基因有1个SNP与IMF显著相关,且其基因表达水平与IMF呈正相关。MYH13基因中存在3个SNPs与滴水损失显著相关,且该基因启动子区2个SNPs基因表达水平与滴水损失呈负相关;3个SNPs与肉色L^*值显著相关;2个SNPs与肉色a^*值显著相关。以上SNPs均可作为影响北京黑猪肉质性状的候选基因功能位点。     

延边黄牛α-肌动蛋白1基因的多态性及其与生长性状的相关分析

试验旨在寻找肌内脂肪含量差异极显著的两组个体延边黄牛背最长肌组织差异表达基因,并分析其遗传多态性对生长性状的影响。应用引物退火技术获得了在肌内脂肪含量差异极显著的两组个体背最长肌组织差异表达α-肌动蛋白1(actin alpha 1,ACTA1)基因及其全长cDNA序列,该基因主要在心脏和背最长肌中表达。序列比对结果发现,在5'端非编码区193 bp处存在1个突变。采用PCR-RFLP方法对100头延边黄牛群体进行检测,发现AA、AG、GG 3种基因型,基因型频率分别为0.24、0.50、0.26;A、G等位基因频率分别为0.37、0.63。对该突变位点的多态性与生长性状的关联性进行分析,结果发现,在体长、体重和平均日增重上,GG基因型个体显著高于AA和AG基因型个体(P＜0.05);在胸围上,GG基因型个体显著高于AG基因型个体,但与AA基因型个体差异不显著(P＞0.05);在体高上,不同基因型之间差异不显著(P＞0.05)。初步认为GG基因型是提高延边黄牛体质量和体尺指标性状的有利基因型,提示ACTA1基因有可能作为延边黄牛生长性状的候选基因。     

Polymorphism of FGF14 Gene and Its Effect on Growth Traits in Yanbian Yellow Cattle

The purpose of this study was to explore the polymorphism of exon 3 of fibroblast growth factor 14 (FGF14) gene and its effect on growth traits in Yanbian Yellow cattle.The third exon of FGF14 gene was sequenced and analyzed by DNA direct sequencing method,and the genotyping of FGF14 gene was detected by HRM typing technique with high resolution melting curve,so as to explore the relationship between different genotypes of FGF14 gene and growth traits of 16 and 24 months of age in Yanbian Yellow cattle.The results showed that one A>G mutation was detected at position 631 947 bp of FGF14 gene,two alleles of A and G,and three genotypes of AA,AG,and GG.The results of χ² test showed that the distribution of genotype frequency and allele frequency of FGF14 gene in two Yanbian Yellow cattle populations of 16 and 24 months of age conformed to Hardy-Weinberg equilibrium (P>0.05).Polymorphic information content (PIC) analysis results showed that FGF14 gene was moderately polymorphic in both groups (0.25 < PIC < 0.5).The results of association analysis with growth traits showed that the different genotypes of FGF14 gene had significant influence on the traits of height at back and rump length of Yanbian Yellow cattle at 16 months of age (P<0.05),and had significant influence on the traits of body height,body weight and height at hip cross of Yanbian Yellow cattle at 24 months of age (P<0.05).In conclusion,it was speculated that FGF14 gene had a certain influence on the growth and development of Yanbian Yellow cattle,and provided reference for the subsequent modern molecular breeding and breeding of meat traits in Yanbian Yellow cattle.     

The Correlation Analysis Between the SNPs of MC4R Gene and Meat Quality Traits in Yanbian Yellow Cattle

In order to explore the effects of the key gene of regulating cattle fat deposition on meat quality traits, screening candidate gene which affect beef fattening traits. This study employed 157 cattles (24 months old) with similar weight as research object, analyzed the correlation between SNPs of MC4R gene and meat quality traits in Yanbian Yellow cattle by PCR direct sequencing method. The results indicated that the mutation site of C→G had been found in MC4R gene 1 069 bp, which resulted in L (leucine) translated into V (valine). Protein secondary structure lost α-spiral and β-fold at the 166, 243 and 280 amino acid position, which resulted in the changes of spatial position of β-turns in tertiary structure. This site showed different degrees correlation with backfat thickness, lrightness, yellowness of Yanbian Yellow cattle. The backfat thickness and yellowness (3 d) of GG genotype were significantly higher than CC and CG genotypes (P <0.05). The lrightness of CC and CG genotypes were significantly higher than CC genotype (P <0.05). These results declared that the site had remarkably correlatived with Yanbian Yellow cattle relevant economic traits, and the mutation site could be used as a potential molecular marker.     

延边黄牛黑皮质素受体-4基因多态性及其与肉质性状的相关性分析

试验旨在探索调控肉牛脂肪沉积的关键基因对肉质性状的影响,筛选影响肉牛育肥性状的候选基因。本研究以157头24月龄、体重相近的延边黄牛为研究对象,利用PCR扩增直接测序的方法,分析了延边黄牛黑皮质素受体-4（melanocortin-4 receptor,MC4R）基因的SNPs及其与肉质性状的相关性。结果发现,延边黄牛MC4R基因在1 069 bp处存在C→G突变,导致L （亮氨酸）转变为V （缬氨酸）,引起蛋白质二级结构在166、243、280位氨基酸处出现α螺旋、β折叠丢失,导致三级结构中β转角的空间位置改变。经关联分析表明,该位点与延边黄牛背膘厚、亮度、黄度都存在不同程度的相关,其中GG基因型背膘厚与屠宰后黄度（3 d）显著高于CC和CG基因型（P＜0.05）;GG、CG基因型屠宰后亮度显著高于CC基因型（P＜0.05）。说明该位点与延边黄牛肉质性状存在显著相关,该突变位点可作为潜在的分子标记。     

延边黄牛PLIN基因多态性及其与胴体和肉质性状的相关性分析

为探究延边黄牛脂滴包被蛋白（perilipin,PLIN）基因遗传多态性及其对胴体和肉质性状的影响,试验选取70头30月龄健康延边黄牛公牛,采集颈静脉血液及其12~13肋间背最长肌以提取延边黄牛DNA样本,并测定胴体与肉质性状,采用直接测序方法检测PLIN基因SNP位点,结合延边黄牛胴体、肉质数据与PLIN基因遗传多态性进行关联分析。结果显示,在延边黄牛PLIN基因外显子3 103与156 bp处存在2个突变位点：g.103 C→T和g.156 T→C,均为错义突变。g.103 C→T位点存在CC、CT两种基因型,CC基因型为优势基因型（0.81）,C基因为优势等位基因（0.90）,表现为CT基因型个体脂肪含量、宰前重、胴体重、背膘厚均显著高于CC基因型个体（P<0.05）;g.156 T→C位点存在TC、TT两种基因型,TT基因型为优势基因型（0.83）,T基因为优势等位基因（0.92）,表现为TC基因型个体的宰前重、胴体重及背膘厚均显著高于TT基因型个体（P<0.05）。卡方检验结果表明,延边黄牛在PLIN基因外显子3的2个SNPs位点均达到Hardy-Weinberg平衡（P>0.05）,杂合度较低,属于低度多态（PIC<0.25）。综上,PLIN基因g.103 C→T和g.156 T→C位点对延边黄牛部分胴体与肉质性状存在一定影响,该位点可作为延边黄牛胴体、肉质性状的潜在标记,PLIN基因可以作为延边黄牛品种选育的候选基因和潜在分子标记。     

Polymorphism of PLIN Gene and Its Associaton with Carcass and Meat Quality Traits in Yanbian Yellow Cattle

This experiment was conducted to explore the association between perilipin (PLIN) gene and carcass and meat quality traits in Yanbian Yellow cattle.In this study,70 30-month-old healthy Yanbian Yellow cattle were used as experimental materials,the blood of jugular vein and its 12-13 intercostal longissimus muscle dorsi were collected to extract DNA samples of Yanbian Yellow cattle,and determine the carcass and meat quality characteristics.The SNP of PLIN gene was detected by direct sequencing,and the correlation analysis was conducted based on the carcass and meat quality data of Yanbian Yellow cattle and the genetic polymorphism of PLIN gene.The results showed that there were 2 SNPs of PLIN gene exon 3 in Yanbian Yellow cattle:g.103 C→T and g.156 T→C,both of them were missense mutations.g.103 C→T contained two genotypes:CC and CT,CC genotype was the dominant genotype,the genotype frequency was 0.81,C was the dominant allele,the gene frequency was 0.90.The intramuscular fat,pre slaughter weight,carcass weight and back fat thickness of CT genotype was significantly higher than those of CC genotype(P<0.05).g.156 T→C contained two genotypes:TT and TC,TT genotype was the dominant genotype,the genotype frequency was 0.83,T was the dominant allele,the gene frequency was 0.92.The pre slaughter weight,carcass weight and back fat thickness of TC genotype were significantly higher than those of TT genotype (P<0.05).Chi square test showed that the two SNPs of PLIN gene in Yanbian Yellow cattle reached Hardy-Weinberg equilibrium (P>0.05),and belonged to low polymorphism (PIC<0.25).In conclusion,g.103 C→T and g.156 T→C of PLIN gene had potential effect on carcass and meat quality traits in Yanbian Yellow cattle,and could be used as a candidate gene and potential molecular marker of Yanbian Yellow cattle.