CN111850136B - Application of MRVI1 gene as marker for screening excellent meat quality traits of beef cattle - Google Patents
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Abstract
Description
技术领域technical field
本发明涉及分子育种技术领域，更具体地，涉及MRVI1基因作为肉牛筛选优良肉质性状的标志物的应用。The present invention relates to the technical field of molecular breeding, more particularly, to the application of MRVI1 gene as a marker for screening excellent meat quality traits in beef cattle.
背景技术Background technique
单核苷酸多态性(Single nucleotide polymorphism,SNP)作为当前应用最广泛的第三代遗传标记技术，在动物遗传育种研究领域具有重要作用及应用价值。SNPs基因分型技术和方法不断涌现，促使分子生物学技术飞跃发展，而基因多态性位点的检测方法主要有DNA直接测序以及限制性片段长度多态性聚合酶链式反应(RFLP-PCR)、单链构象多态性、DNA芯片/阵列分析法等。但针对此多种方法，DNA直接测序法检测SNPs最为简单、准确，并适用于大群体动物样本的检测，对动物个体遗传背景的研究提供了更大的机遇和挑战。Single nucleotide polymorphism (Single nucleotide polymorphism, SNP) is currently the most widely used third-generation genetic marker technology, and has an important role and application value in the field of animal genetics and breeding research. The continuous emergence of SNPs genotyping technologies and methods has prompted the rapid development of molecular biology technology. The detection methods of gene polymorphism sites mainly include direct DNA sequencing and restriction fragment length polymorphism polymerase chain reaction (RFLP-PCR). ), single-strand conformation polymorphism, DNA chip/array analysis, etc. However, in view of these various methods, the direct DNA sequencing method is the most simple and accurate method to detect SNPs, and is suitable for the detection of large groups of animal samples, which provides greater opportunities and challenges for the study of the genetic background of individual animals.
中国西门塔尔牛是我国引进后不断改良优化培育的大型乳肉兼用品种牛，具有其独特的生产性能和良好的肉用性状，表现出了巨大的产业优势和发展潜力。且近年来我国肉牛领域研究重点已经从生长发育性状转移至肉质及胴体性状，而脂肪覆盖率又是重要的胴体及经济性状之一，同时也是衡量牛品质性状的重要标准。脂肪覆盖率是指肉牛胴体表面脂肪覆盖面积占胴体总面积的比率。胴体体表脂肪覆盖率是胴体分级标准的指标之一，特级肉牛的胴体脂肪覆盖率应大于90％；A级肉牛的胴体脂肪覆盖率应在80％～90％；肉牛的胴体脂肪覆盖率小于80％列为B级牛。当用手指压迫牛的背部落在第6～7胸肋至腰椎部位，烧用力便能够碰到脊椎，说明脂肪层很薄，脂肪覆盖率很差；当手指压迫时需要很大力才碰到脊椎，说明脂肪层很厚，脂肪覆盖率很好。眼肌面积是第12到13肋间的肌肉横截面积，是家畜背最长肌的横断面，眼肌面积性状与家畜产肉性能具有密切的相关关系，与牛宰前重、净肉重、屠宰率和净肉率等呈非常显著的正相关关系，所以在动物育种上是作为衡量肉品质的重要指标，占据尤为重要的地位。肾周脂肪是衡量牛屠宰性状的重要指标，肾周脂肪的蓄积直接反映牛体含量。Chinese Simmental is a large-scale dairy-meat dual-purpose breed that has been continuously improved, optimized and cultivated in my country after its introduction. It has its unique production performance and good meat traits, showing huge industrial advantages and development potential. In recent years, the research focus of beef cattle in my country has shifted from growth and development traits to meat quality and carcass traits, and fat coverage is one of the important carcass and economic traits, as well as an important criterion for measuring cattle quality traits. Fat coverage refers to the ratio of the fat coverage area on the surface of beef cattle carcass to the total carcass area. Carcass surface fat coverage is one of the indicators of carcass grading standards. 80% are classified as B grade cattle. When pressing the back of the cow with fingers on the 6th to 7th thoracic ribs to the lumbar vertebrae, the vertebrae can be touched by the burning force, indicating that the fat layer is very thin and the fat coverage rate is poor; when the fingers are pressed, it takes a lot of force to touch the spine , indicating that the fat layer is very thick and the fat coverage is very good. The eye muscle area is the cross-sectional area of the muscle between the 12th and 13th intercostal space, and it is the cross-section of the longissimus dorsi. The eye muscle area is closely related to the meat production performance of livestock, and is closely related to the pre-slaughter weight and net meat weight of cattle. , slaughter rate and net meat rate have a very significant positive correlation, so it is an important indicator to measure meat quality in animal breeding, occupying a particularly important position. Perirenal fat is an important indicator to measure cattle slaughter traits, and the accumulation of perirenal fat directly reflects the body content of cattle.
因此，为提高牛肉品质，从基因角度出发，利用分子的遗传标记作为对研究牛肉品质性状的主要手段，通过改善相关肉质或胴体性状，提高未来牛群的品质，是非常必要的，这将为新品种牛的培育提供重要的基因资源和理论依据。Therefore, in order to improve beef quality, from the perspective of genes, it is very necessary to use molecular genetic markers as the main means to study beef quality traits, and to improve the quality of future cattle herds by improving related meat quality or carcass traits. The breeding of new breeds of cattle provides important genetic resources and theoretical basis.
小鼠逆转录病毒整合位点1同系物(Murine retrovirus integration site 1homolog，MRVI1)基因定位于牛第15位染色体上，编码21个外显子，转录本长度6312bp，开放阅读框2736bp，911个氨基酸。MRVI1基因所编码的蛋白质，能够参与一氧化氮(NO)信号的传导，影响cGMP的合成，能够激活cGMP依赖性蛋白激酶PKG，调控相关的心血管系统的功能，例如心肌、血管平滑肌和血小板等。有相关文献报道MRVI1基因会导致机体的血压改变，主要是通过cGMP依赖性蛋白激酶I型(cGKI)发出信号，使各种平滑肌松弛，血管平滑肌松弛后会导致血管内血流量下降，进而使血压降低，调控血管张力和胃肠动力，主要表现血压异常和严重的胃肠功能障碍。有研究表明(Shaughnessy JD Jr,Largaespada DA,Tian E,etal.Mrvi1,a common MRV integration site in BXH2 myeloid leukemias,encodes aprotein with homology to a lymphoid-restricted membrane proteinJaw1.Oncogene.1999；18(12):2069-2084.doi:10.1038/sj.onc.1202419)，MRVI1基因的MRV整合通过改变对骨髓细胞生长和/或分化关键基因的表达诱导小鼠中的髓样白血病，因此该基因可以作为髓样白血病肿瘤抑制关键基因而发挥重要作用。Murine
目前尚无分子标记能够衡量MRVI1基因与中国西门塔尔牛肉质及胴体性状中的脂肪覆盖率、肾周脂肪和眼肌面积显著相关。There is no molecular marker that can measure the significant association of MRVI1 gene with fat coverage, perirenal fat and eye muscle area in Chinese Simmental beef quality and carcass traits.
发明内容SUMMARY OF THE INVENTION
本发明的目的在于提供MRVI1基因作为在肉牛筛选优良肉质性状的标志物的应用。本发明所提供的利用MRVI1基因的遗传标记检测中国西门塔尔牛肉质及胴体性状脂肪覆盖率、肾周脂肪和眼肌面积的方法，解决当前对牛脂肪覆盖率、肾周脂肪和眼肌面积等标志性性状相关重要分子遗传标记筛查的难题。为遗传工作者的早期选育及改善肉品质奠定基础。The purpose of the present invention is to provide the application of MRVI1 gene as a marker for screening excellent meat quality traits in beef cattle. The method for detecting the fat coverage rate, perirenal fat and eye muscle area of Chinese Simmental beef quality and carcass traits using the genetic marker of the MRVI1 gene provided by the present invention solves the problem of current beef fat coverage rate, perirenal fat and eye muscle area. Difficulties in screening important molecular genetic markers associated with other landmark traits. It lays the foundation for the early selection of genetic workers and the improvement of meat quality.
本发明的第一个目的在于提供MRVI1基因作为肉牛筛选优良肉质性状的标志物的应用。The first object of the present invention is to provide the application of the MRVI1 gene as a marker for screening excellent meat quality traits in beef cattle.
本发明的第二个目的在于提供g.41970426A>G、g.41970520T>C、g.41970609A>G、g.41970714A>G、g.41971686G>A或g.41971818G>A在评价肉牛脂肪覆盖率中的应用。The second object of the present invention is to provide g.41970426A>G, g.41970520T>C, g.41970609A>G, g.41970714A>G, g.41971686G>A or g.41971818G>A in evaluating the fat coverage of beef cattle applications in .
本发明的第三个目的在于提供g.41970426A>G、g.41970520T>C、g.41970609A>G、g.41970626G>A、g.41970714A>G、g.41971818G>A或g.41972009A>C在评价肉牛肾周脂肪性状中的应用。The third object of the present invention is to provide g.41970426A>G, g.41970520T>C, g.41970609A>G, g.41970626G>A, g.41970714A>G, g.41971818G>A or g.41972009A>C Application in the evaluation of perirenal fat traits in beef cattle.
本发明的第四个目的在于提供g.41971686G>A在评价肉牛眼肌面积中的应用。The fourth object of the present invention is to provide the application of g.41971686G>A in evaluating the ocular muscle area of beef cattle.
本发明的第五个目的在于提供g.41970426A>G、g.41970609A>G、g.41970626G>A、g.41970714A>G或g.41971818G>A在评价肉牛胴体长中的应用。The fifth object of the present invention is to provide the application of g.41970426A>G, g.41970609A>G, g.41970626G>A, g.41970714A>G or g.41971818G>A in evaluating the carcass length of beef cattle.
本发明的第六个目的在于提供一种评价肉牛优良肉质性状的方法。The sixth object of the present invention is to provide a method for evaluating the excellent meat quality traits of beef cattle.
本发明的第七个目的在于提供一种评价肉牛脂肪覆盖率的方法。The seventh object of the present invention is to provide a method for evaluating the fat coverage of beef cattle.
本发明的第八个目的在于提供一种评价肉牛肾周脂肪性状的方法。The eighth object of the present invention is to provide a method for evaluating the properties of perirenal fat in beef cattle.
本发明的第九个目的在于提供一种评价肉牛眼肌面积的方法。The ninth object of the present invention is to provide a method for evaluating the area of the ophthalmia of beef cattle.
本发明的第十个目的在于提供一种评价肉牛肾周脂肪性状的方法。The tenth object of the present invention is to provide a method for evaluating the properties of perirenal fat in beef cattle.
本发明的上述目的是通过以下方案予以实现的：Above-mentioned purpose of the present invention is achieved through the following scheme:
发明人经过研究发现MRVI1基因与牛筛选优良肉质性状相关，因此本发明要求保护MRVI1基因作为肉牛筛选优良肉质性状的标志物的应用。Through research, the inventor found that MRVI1 gene is related to the selection of excellent meat quality traits in cattle. Therefore, the present invention claims the application of MRVI1 gene as a marker for selection of excellent meat quality traits in beef cattle.
进一步本发明发现在MRVI1基因上有6个与脂肪覆盖率显著相关的多态性位点，分别为g.41970426A>G、g.41970520T>C、g.41970609A>G、g.41970714A>G、g.41971686G>A和g.41971818G>A；共有7个与肾周脂肪显著相关的多态性位点，分别为g.41970426A>G、g.41970520T>C、g.41970609A>G、g.41970626G>A、g.41970714A>G、g.41971818G>A、g.41972009A>C位点(P<0.01)；有一个位点与眼肌面积显著相关，为g.41971686G>A位点(P<0.05)；g.41970426A>G、g.41970609A>G、g.41970626G>A、g.41970714A>G、g.41971818G>A位点与胴体长显著相关。Further, the present invention finds that there are 6 polymorphic loci in the MRVI1 gene that are significantly related to the fat coverage rate, which are g.41970426A>G, g.41970520T>C, g.41970609A>G, g. g.41971686G>A and g.41971818G>A; there are 7 polymorphic loci significantly associated with perirenal fat, g.41970426A>G, g.41970520T>C, g.41970609A>G, g. 41970626G>A, g.41970714A>G, g.41971818G>A, g.41972009A>C sites (P<0.01); one site was significantly correlated with eye muscle area, which was g.41971686G>A site (P<0.01). <0.05); g.41970426A>G, g.41970609A>G, g.41970626G>A, g.41970714A>G, g.41971818G>A sites were significantly correlated with carcass length.
其中，g.41970426A>G位点：携带AA基因型个体的屠宰率显著高于GA基因型的个体，携带基因型AA个体的脂肪覆盖率显著低于GA基因型的个体，携带基因型AA个体的肾周脂肪重量显著低于GG基因型的个体(P<0.05)；Among them, g.41970426A>G site: the slaughter rate of individuals with AA genotype is significantly higher than that of individuals with GA genotype, the fat coverage rate of individuals with AA genotype is significantly lower than that of individuals with GA genotype, and the individuals with genotype AA The perirenal fat weight was significantly lower in individuals with GG genotype (P<0.05);
g.41970520T>C位点：携带CC基因型个体的肾脏脂肪含量显著高于TT基因型个体(P<0.05)；g.41970520T>C locus: the kidney fat content of individuals with CC genotype was significantly higher than that of individuals with TT genotype (P<0.05);
g.41970609A>G位点：携带AA基因型个体的屠宰率显著高于GA基因型个体，携带基因型AA个体的脂肪覆盖率显著低于GA基因型的个体，携带基因型AA个体的肾周脂肪重量显著低于GG基因型的个体(P<0.05)；g.41970609A>G locus: The slaughter rate of individuals with AA genotype was significantly higher than that of individuals with GA genotype, the fat coverage of individuals with AA genotype was significantly lower than that of individuals with GA genotype, and the peri-renal fat coverage of individuals with genotype AA was significantly lower than that of individuals with GA genotype. The fat weight was significantly lower than that of individuals with GG genotype (P<0.05);
g.41970626G>A位点：携带GG基因型个体的屠宰率显著高于GA基因型的个体，携带GG基因型个体的肾脏脂肪含量和胴体长显著低于AA基因型的个体，(P<0.05)；g.41970626G>A locus: The slaughter rate of individuals with GG genotype was significantly higher than that of individuals with GA genotype, and the kidney fat content and carcass length of individuals with GG genotype were significantly lower than those with AA genotype (P<0.05). );
g.41970714A>G位点：携带AA基因型个体的屠宰率显著高于GA基因型的个体，携带AA基因型的个体的肾脏脂肪含量显著低于GG基因型个体，携带AA基因型的个体的脂肪覆盖率显著低于GA基因型个体(P<0.05)；g.41970714A>G locus: the slaughter rate of individuals with AA genotype was significantly higher than that of individuals with GA genotype, the kidney fat content of individuals with AA genotype was significantly lower than that of individuals with GG genotype, and the The fat coverage rate was significantly lower than that of individuals with GA genotype (P<0.05);
g.41971686G>A位点：携带GG基因型个体的脂肪覆盖率显著低于AG基因型的个体上，携带GG基因型个体的眼肌面积显著高于AA基因型的个体；g.41971686G>A site: the fat coverage rate of individuals with GG genotype is significantly lower than that of individuals with AG genotype, and the eye muscle area of individuals with GG genotype is significantly higher than that of individuals with AA genotype;
g.41971818G>A位点：携带GG基因型个体的肾脏脂肪含量显著低于GA、AA基因型的个体，携带GG基因型个体的胴体脂肪覆盖率显著低于GA基因型的个体，携带GG基因型个体的胴体长显著低于GA、AA基因型的个体，携带GA基因型个体的大腿肌肉厚度显著高于AA基因型的个体；g.41971818G>A locus: the kidney fat content of individuals with GG genotype is significantly lower than that of individuals with GA and AA genotypes, the carcass fat coverage of individuals with GG genotype is significantly lower than that of individuals with GA genotype, and individuals with GG genotype The carcass length of individuals with genotype GA was significantly lower than that of individuals with genotype GA and AA, and the thickness of thigh muscles of individuals with genotype GA was significantly higher than that of individuals with genotype AA;
g.41972009A>C位点：携带AA基因型个体的肾脏脂肪含量显著低于CA基因型个体，携带AA基因型个体的腰部肉厚显著低于CC基因型个体。g.41972009A>C locus: The kidney fat content of individuals with AA genotype was significantly lower than that of CA genotype individuals, and the loin thickness of individuals with AA genotype was significantly lower than that of CC genotype individuals.
因此本发明要求保护以下内容：Therefore the present invention claims the following content:
g.41970426A>G、g.41970520T>C、g.41970609A>G、g.41970714A>G、g.41971686G>A或g.41971818G>A在评价肉牛脂肪覆盖率中的应用；Application of g.41970426A>G, g.41970520T>C, g.41970609A>G, g.41970714A>G, g.41971686G>A or g.41971818G>A in evaluating the fat coverage of beef cattle;
g.41970426A>G、g.41970520T>C、g.41970609A>G、g.41970626G>A、g.41970714A>G、g.41971818G>A或g.41972009A>C在评价肉牛肾周脂肪性状中的应用；G.41970426A>G, g.41970520T>C, g.41970609A>G, g.41970626G>A, g.41970714A>G, g.41971818G>A or g.41972009A>C in the evaluation of beef cattle peri-renal fat traits application;
g.41971686G>A在评价肉牛眼肌面积中的应用；Application of g.41971686G>A in evaluating the area of ophthalmia of beef cattle;
g.41970426A>G、g.41970609A>G、g.41970626G>A、g.41970714A>G或g.41971818G>A在评价肉牛胴体长中的应用。Application of g.41970426A>G, g.41970609A>G, g.41970626G>A, g.41970714A>G or g.41971818G>A in evaluating beef cattle carcass length.
本发明要还有保护：一种评价肉牛优良肉质性状的方法，检测MRVI1基因的基因型。The invention also has protection: a method for evaluating the excellent meat quality traits of beef cattle, detecting the genotype of the MRVI1 gene.
一种评价肉牛脂肪覆盖率的方法，检测g.41970426A>G、g.41970520T>C、g.41970609A>G、g.41970714A>G、g.41971686G>A或g.41971818G>A的基因型。A method for evaluating the fat coverage rate of beef cattle, detecting the genotypes of g.41970426A>G, g.41970520T>C, g.41970609A>G, g.41970714A>G, g.41971686G>A or g.41971818G>A.
一种评价肉牛肾周脂肪性状的方法，检测g.41970426A>G、g.41970520T>C、g.41970609A>G、g.41970626G>A、g.41970714A>G、g.41971818G>A或g.41972009A>C的基因型。A method for evaluating the characteristics of perirenal fat of beef cattle, detecting g. 41972009A>C genotype.
一种评价肉牛眼肌面积的方法，检测g.41971686G>A的基因型。A method for evaluating the ocular muscle area of beef cattle, detecting the genotype of g.41971686G>A.
一种评价肉牛肾周脂肪性状的方法，检测g.41970426A>G、g.41970520T>C、g.41970609A>G、g.41970626G>A、g.41970714A>G、g.41971818G>A或g.41972009A>C的基因型。A method for evaluating the characteristics of perirenal fat of beef cattle, detecting g. 41972009A>C genotype.
与现有技术相比，本发明具有以下有益效果：Compared with the prior art, the present invention has the following beneficial effects:
本发明发明人公开了将MRVI1基因与脂肪覆盖率、肾周脂肪和眼肌面积性状显著相关的遗传标记作为肉牛筛选优良肉质性状的标记辅助选择用途，也应用于早期肉牛的选育。本发明人找到了8个SNP位点，通过对牛群体的多态性位点进行基因分型和基因频率分析以及与牛肉质和胴体性状关联分析，发现MRVI1基因的多态性位点与牛脂肪覆盖率、肾周脂肪、眼肌面积等经济性状相关，解决了当前对牛肉质及胴体性状相关的重要分子遗传标记筛查的难题。也为遗传工作者早期选育优质牛群提高产肉性能及改善肉品质奠定基础。The inventors of the present invention disclose the use of the genetic marker that the MRVI1 gene is significantly correlated with the fat coverage, perirenal fat and eye muscle area traits as a marker-assisted selection for selecting excellent meat quality traits in beef cattle, and it is also used in early-stage beef cattle breeding. The inventors found 8 SNP loci, through genotyping and gene frequency analysis of the polymorphic loci in the cattle population, as well as the correlation analysis with beef quality and carcass traits, and found that the polymorphic loci of the MRVI1 gene are closely related to cattle. Economic traits such as fat coverage, perirenal fat, and eye muscle area are related, which solves the current problem of screening important molecular genetic markers related to beef quality and carcass traits. It also lays a foundation for genetic workers to select high-quality cattle herds early to improve meat production performance and meat quality.
附图说明Description of drawings
图1为MRVI1 SNP1(g.41970426A>G)位点突变位点测序检测图。Figure 1 shows the sequence detection map of the MRVI1 SNP1 (g.41970426A>G) site mutation site.
图2为MRVI1 SNP2(g.41970520T>C)位点突变位点测序检测图。Figure 2 shows the sequence detection map of the MRVI1 SNP2 (g.41970520T>C) site mutation site.
图3为MRVI1 SNP3(g.41970609A>G)位点突变位点测序检测图。Fig. 3 is the sequence detection diagram of the MRVI1 SNP3 (g.41970609A>G) site mutation site.
图4为MRVI1 SNP4(g.41970626G>A)位点突变位点测序检测图。Fig. 4 is the sequence detection map of MRVI1 SNP4 (g.41970626G>A) site mutation site.
图5为MRVI1 SNP5(g.41970714A>G)位点突变位点测序检测图。Fig. 5 is the sequence detection map of the MRVI1 SNP5 (g.41970714A>G) site mutation site.
图6为MRVI1 SNP6(g.41971686G>A)位点突变位点测序检测图。Fig. 6 is the sequence detection map of MRVI1 SNP6 (g.41971686G>A) site mutation site.
图7为MRVI1 SNP7(g.41971818G>A)位点突变位点测序检测图。Fig. 7 is the sequence detection diagram of the MRVI1 SNP7 (g.41971818G>A) site mutation site.
图8为MRVI1 SNP8(g.41972009A>C)位点突变位点测序检测图。Fig. 8 is the sequence detection diagram of the MRVI1 SNP8 (g.41972009A>C) site mutation site.
图9为MRVI1基因的肉牛群体SNPs位点连锁反应。Figure 9 shows the chain reaction of SNPs in the beef cattle population of the MRVI1 gene.
具体实施方式Detailed ways
下面结合具体实施例对本发明做出进一步地详细阐述，所述实施例只用于解释本发明，并非用于限定本发明的范围。下述实施例中所使用的试验方法如无特殊说明，均为常规方法；所使用的材料、试剂等，如无特殊说明，为可从商业途径得到的试剂和材料。The present invention will be further elaborated below with reference to specific embodiments, which are only used to explain the present invention, but not to limit the scope of the present invention. The test methods used in the following examples are conventional methods unless otherwise specified; the materials, reagents, etc. used are commercially available reagents and materials unless otherwise specified.
实施例1中国西门塔尔牛群体全基因组MRVI1基因的扩增Example 1 Amplification of the Whole Genome MRVI1 Gene of the Chinese Simmental Cattle Population
一、实验方法1. Experimental method
1，样本选择1. Sample selection
选用内蒙古乌拉盖地区某大型牛场牛群，采用静脉采血的方式进行样本的采集，采集来自不同个体的50份样本。采用的TIANGEN公司血液基因组DNA提取试剂盒进行全基因组DNA提取，具体操作按照试剂盒说明书。将提取的DNA样品存于-80℃冰箱备用。A large-scale cattle herd in the Wulagai area of Inner Mongolia was selected to collect samples by venous blood collection, and 50 samples from different individuals were collected. The whole genome DNA extraction was performed using the blood genomic DNA extraction kit of TIANGEN company, and the specific operation was in accordance with the kit instructions. The extracted DNA samples were stored in a -80°C refrigerator for later use.
2，引物设计2. Primer Design
设计MRVI1基因引物及进行PCR的扩增。截取取牛的MRVI1基因(ENSBTAT00000046140.4)的两段序列MRVI1-A和MRVI1-B，两段序列的核苷酸序列分别如SEQ ID NO：1～2所示。Design MRVI1 gene primers and carry out PCR amplification. Two sequences of MRVI1-A and MRVI1-B of bovine MRVI1 gene (ENSBTAT00000046140.4) were intercepted, and the nucleotide sequences of the two sequences are shown in SEQ ID NOs: 1-2 respectively.
根据这两个片段进行引物设计，其中扩增片段MRVI1-A(的核苷酸序列分别如SEQID NO：1所示)的扩增引物为：Primer design is carried out according to these two fragments, wherein the amplification primers for amplifying fragment MRVI1-A (the nucleotide sequences of which are shown in SEQ ID NO: 1, respectively) are:
MRVI1-A-F：5’-CTTACTGGCTGTGGAACATCA-3’(SEQ ID NO：3)；MRVI1-A-F: 5'-CTTACTGGCTGTGGAACATCA-3' (SEQ ID NO: 3);
MRVI1-A-R：5’-AATCTGGCAACTCTAGTGGTG-3’(SEQ ID NO：4)。MRVI1-A-R: 5'-AATCTGGCAACTCTAGTGGTG-3' (SEQ ID NO: 4).
扩增片段MRVI1-B(的核苷酸序列分别如SEQ ID NO：2所示)的扩增引物为：The amplification primers for the amplified fragment MRVI1-B (the nucleotide sequences of which are shown in SEQ ID NO: 2 respectively) are:
设计DNA oligos，其具体的引物序列如下：Design DNA oligos, and the specific primer sequences are as follows:
MRVI1-B-F：5’-CCACCACTAGAGTTGCCAGAT-3’(SEQ ID NO：5)；MRVI1-B-F: 5'-CCACCACTAGAGTTGCCAGAT-3' (SEQ ID NO: 5);
MRVI1-B-R：5’-GTCCTTGCTCCTCCACTGAG-3’(SEQ ID NO：6)。MRVI1-B-R: 5'-GTCCTTGCTCCTCCACTGAG-3' (SEQ ID NO: 6).
3，MRVI1基因的扩增3. Amplification of MRVI1 Gene
先将每50个样本的PCR扩增产物进行混合，送到北京金唯智测序，然后对其测序结果进行分析，找出SNP位点。根据MRVI1-A和MRVI1-B的PCR测序结果，找到SNP位点。再将95个样品都进行PCR扩增。测序并进行基因型分析。First, the PCR amplification products of each 50 samples were mixed and sent to Beijing Jinweizhi for sequencing, and then the sequencing results were analyzed to find the SNP site. According to the PCR sequencing results of MRVI1-A and MRVI1-B, SNP sites were found. All 95 samples were PCR amplified. Sequencing and genotyping.
具体操作如下：The specific operations are as follows:
PCR扩增体系如下：2×Power Taq PCR Master Mix 20.0μL；PCR Reverse primer1.0μL；PCR Forward primer 1.0μL；DNA(25mg/L)3.0μL；ddH2O 15μL。The PCR amplification system was as follows: 2×Power Taq PCR Master Mix 20.0 μL; PCR Reverse primer 1.0 μL; PCR Forward primer 1.0 μL; DNA (25 mg/L) 3.0 μL; ddH 2 O 15 μL.
基因PCR扩增反应程序：95℃5min；95℃30s，60℃30s，72℃1min，35个循环；72℃5min；16℃保存。Gene PCR amplification reaction program: 95°C for 5 min; 95°C for 30s, 60°C for 30s, 72°C for 1 min, 35 cycles; 72°C for 5 min; storage at 16°C.
PCR产物按照DNA回收试剂盒说明书步骤回收，并将回收的PCR扩增产物送至生工生物公司测序，利用DNAStar和Chormas软件进行比对和分析。The PCR products were recovered according to the instructions of the DNA recovery kit, and the recovered PCR amplification products were sent to Sangon Bio Inc. for sequencing, and DNAStar and Chormas software were used for comparison and analysis.
二、实验结果2. Experimental results
对于MRVI1基因在中国西门塔尔牛群体共检测到8个SNPs，包括g.41970426A>G、g.41970520T>C、g.41970609A>G、g.41970626G>A、g.41970714A>G、g.41971686G>A、g.41971818G>A、g.41972009A>C，如图1～8所示。选取MRVI1基因(ENSBTAT00000046140.4)启动子区序列，其中，g.41970426A>G、g.41970520T>C、g.41970609A>G、g.41970626G>A和g.41970714A>G位于片段MRVI1-A上，其中，g.41971686G>A、g.41971818G>A和g.41972009A>C位于片段MRVI1-B上。For the MRVI1 gene, 8 SNPs were detected in the Chinese Simmental cattle population, including g.41970426A>G, g.41970520T>C, g.41970609A>G, g.41970626G>A, g.41970714A>G, g. 41971686G>A, g.41971818G>A, g.41972009A>C, as shown in Figures 1 to 8. Select the promoter region sequence of MRVI1 gene (ENSBTAT00000046140.4), wherein g.41970426A>G, g.41970520T>C, g.41970609A>G, g.41970626G>A and g.41970714A>G are located on the fragment MRVI1-A , wherein g.41971686G>A, g.41971818G>A and g.41972009A>C are located on the fragment MRVI1-B.
实施例2Example 2
一、实验方法1. Experimental method
基因型频率是指一个群体中某一性状的某种基因型个体数占总个体数的比率。PAA＝NAA/N，其中PAA代表某一位点的AA基因型频率；NAA表示群体中具有AA基因型的个体数；N为检测群体的总数量。Genotype frequency refers to the ratio of the number of individuals of a certain genotype to the total number of individuals for a certain trait in a population. PAA=NAA/N, where PAA represents the frequency of AA genotype at a certain locus; NAA represents the number of individuals with AA genotype in the population; N is the total number of the detected population.
基因频率是指一个群体中某一基因数对其等位基因总数的相对比率。计算的公式可以写成：PA＝(2NAA+NAa)/2N。公式中，PA表示等位基因A频率，NAA表示群体中具有AA基因型的个体数量，NAa表示群体中具有Aa基因型个体数量。Gene frequency refers to the relative ratio of the number of a gene to the total number of alleles in a population. The calculation formula can be written as: PA=(2NAA+NAa)/2N. In the formula, PA represents the frequency of allele A, NAA represents the number of individuals with the AA genotype in the population, and NAa represents the number of individuals with the Aa genotype in the population.
哈迪—温伯格定律(Hardy-Weinberg equilibrium)也称遗传平衡定律，其定义是：处在最理想的情况下，等位基因的基因型频率和等位基因的频率在遗传中是恒定的，即保持着基因平衡。这种情况需要满足以下几点：①种群足够大；②没有突变发生；③种群中个体间可以随机交配；④没有自然选择；⑤没有新基因加入。此时各基因频率和各基因型频率拥有以下等式：设A＝p，a＝q，则p+q＝1，AA+Aa+aa＝p^2+2pq+q^2＝1。哈代-温伯格平衡定律对于随机交配且群体足够大的族群，基因型频率和基因频率在没有突变、迁移和选择的条件下不会发生改变。The Hardy-Weinberg law, also known as the law of genetic equilibrium, is defined as: in the most ideal case, the genotype frequency of alleles and the frequency of alleles are constant in heredity , that is, to maintain genetic balance. In this case, the following points need to be met: ① the population is large enough; ② no mutation occurs; ③ the individuals in the population can mate randomly; ④ there is no natural selection; ⑤ no new genes are added. At this time, each gene frequency and each genotype frequency have the following equation: set A=p, a=q, then p+q=1, AA+Aa+aa=p^2+2pq+q^2=1. Hardy-Weinberg Equilibrium For populations with random mating and sufficiently large populations, genotype frequencies and gene frequencies do not change in the absence of mutation, migration, and selection.
二、实验结果2. Experimental results
对于MRVI1基因在中国西门塔尔牛群体共检测到8个SNPs，其中SNP1(g.41970426A>G，Ensemble rs208249746)位点，突变纯合型GG个体有8头，GA型有42头，野生型AA有45头。GG的基因型频率为0.084，GA的基因型频率为0.442，AA的基因型频率为0.474，G等位基因频率为0.305，T等位基因频率为0.695；因此A等位基因占优势，以野生型AA为主要的基因型。For the MRVI1 gene, a total of 8 SNPs were detected in the Chinese Simmental cattle population. Among them, SNP1 (g.41970426A>G, Ensemble rs208249746) locus, there were 8 mutant homozygous GG individuals, 42 GA individuals, and 42 wild-type individuals. AA has 45 heads. The genotype frequency of GG is 0.084, the genotype frequency of GA is 0.442, the genotype frequency of AA is 0.474, the frequency of G allele is 0.305, and the frequency of T allele is 0.695; therefore, the A allele is dominant, and the wild Type AA is the predominant genotype.
SNP2(g.41970520T>C，Ensemble rs210035614)位点，CC型个体有7头，CT型有42头，野生TT型有46头。CC的基因型频率为0.074，CT的基因型频率为0.442，TT的基因型频率为0.484，C等位基因频率为0.295，T等位基因频率为0.705；因此T等位基因占优势，以野生型TT为主要的基因型。At the SNP2 (g.41970520T>C, Ensemble rs210035614) locus, there are 7 CC-type individuals, 42 CT-type individuals, and 46 wild-type TT-type individuals. The genotype frequency of CC is 0.074, the genotype frequency of CT is 0.442, the genotype frequency of TT is 0.484, the frequency of C allele is 0.295, and the frequency of T allele is 0.705; therefore, the T allele is dominant, and the wild Type TT was the predominant genotype.
SNP3(g.41970609A>G，Ensemble rs211384477)位点，GG型个体有8头，AG型有42头，AA型有45头。GG的基因型频率为0.084，AG的基因型频率为0.442，AA的基因型频率为0.474，G等位基因频率为0.305，A等位基因频率为0.695；因此A等位基因占优势，以野生型AA为主要的基因型。At the SNP3 (g.41970609A>G, Ensemble rs211384477) locus, there are 8 GG individuals, 42 AG individuals, and 45 AA individuals. The genotype frequency of GG is 0.084, the genotype frequency of AG is 0.442, the genotype frequency of AA is 0.474, the frequency of G allele is 0.305, and the frequency of A allele is 0.695; therefore, the A allele is dominant, and the wild Type AA is the predominant genotype.
SNP4(g.41970626G>A，Ensemble rs208898729)位点，野生型GG个体有46头，AG型有41头，AA型有8头。GG的基因型频率为0.0484，AG的基因型频率为0.432，AA的基因型频率为0.084，G等位基因频率为0.7，A等位基因频率为0.3；因此G等位基因占优势，以野生型GG为主要的基因型。At the SNP4 (g.41970626G>A, Ensemble rs208898729) site, there are 46 wild-type GG individuals, 41 AG-type individuals, and 8 AA-type individuals. The genotype frequency of GG is 0.0484, the genotype frequency of AG is 0.432, the genotype frequency of AA is 0.084, the frequency of G allele is 0.7, and the frequency of A allele is 0.3; Type GG is the predominant genotype.
SNP5(g.41970714A>G，Ensemble rs209751199)位点，突变纯合型GG个体有8头，AG型有42头，野生型AA有45头。GG的基因型频率为0.084，AG的基因型频率为0.442，AA的基因型频率为0.474，G等位基因频率为0.305，A等位基因频率为0.695；因此A等位基因占优势，以野生型AA为主要的基因型。At the SNP5 (g.41970714A>G, Ensemble rs209751199) site, there are 8 mutant homozygous GG individuals, 42 AG individuals, and 45 wild-type AA individuals. The genotype frequency of GG is 0.084, the genotype frequency of AG is 0.442, the genotype frequency of AA is 0.474, the frequency of G allele is 0.305, and the frequency of A allele is 0.695; therefore, the A allele is dominant, and the wild Type AA is the predominant genotype.
SNP6(g.41971686G>A，Ensemble rs207488012)位点，野生型GG型个体有64头，GA型有28头，AA有3头。GG的基因型频率为0.674，GA的基因型频率为0.295，AA的基因型频率为0.031，G等位基因频率为0.821，A等位基因频率为0.179；因此G等位基因占优势，以野生型GG为主要的基因型。At the SNP6 (g.41971686G>A, Ensemble rs207488012) site, there are 64 wild-type GG individuals, 28 GA individuals, and 3 AA individuals. The genotype frequency of GG is 0.674, the genotype frequency of GA is 0.295, the genotype frequency of AA is 0.031, the frequency of G allele is 0.821, and the frequency of A allele is 0.179; therefore, the G allele is dominant, and the wild Type GG is the predominant genotype.
SNP7(g.41971818G>A，Ensemble rs209429084)位点，野生型GG个体有51头，GA型有37头，AA型有7头。GG的基因型频率为0.537，GA的基因型频率为0.389，AA的基因型频率为0.074，G等位基因频率为0.732，A等位基因频率为0.268；因此G等位基因占优势，以野生型GG为主要的基因型。At the SNP7 (g.41971818G>A, Ensemble rs209429084) site, there are 51 wild-type GG individuals, 37 GA-type individuals, and 7 AA-type individuals. The genotype frequency of GG is 0.537, the genotype frequency of GA is 0.389, the genotype frequency of AA is 0.074, the frequency of G allele is 0.732, and the frequency of A allele is 0.268; therefore, the G allele is dominant, and the wild Type GG is the predominant genotype.
SNP8(g.41972009A>C，Ensemble rs110730746)位点，CC个体有11头，CA型有46头，野生型AA型有38头。CC的基因型频率为0.116，CA的基因型频率为0.484，AA的基因型频率为0.400，C等位基因频率为0.358，A等位基因频率为0.642；因此A等位基因占优势，以突变杂合型CA为主要的基因型。MRVI1基因SNP位点基因型频率、等位基因频率及哈代温伯格定律数据如表1所示。At the SNP8 (g.41972009A>C, Ensemble rs110730746) site, there are 11 CC individuals, 46 CA types, and 38 wild-type AA types. The genotype frequency of CC is 0.116, the genotype frequency of CA is 0.484, the genotype frequency of AA is 0.400, the frequency of C allele is 0.358, and the frequency of A allele is 0.642; therefore the A allele is dominant, and the mutation Heterozygous CA was the predominant genotype. The genotype frequency, allele frequency and Hardy-Weinberg's law of MRVI1 gene SNP loci are shown in Table 1.
表1 MRVI1基因SNP位点基因型频率、等位基因频率及哈代温伯格定律数据Table 1 Genotype frequency, allele frequency and Hardy-Weinberg's law of MRVI1 gene SNP loci
实施例3 MRVI1基因8个SNP位点与西门塔尔牛肉质及胴体性状之间的相关性分析Example 3 Correlation analysis between 8 SNP loci of MRVI1 gene and Simmental beef quality and carcass traits
一、实验方法1. Experimental method
中国西门塔尔牛的肉质及胴体性状主要包括：毛重、头重、胴体重、屠宰率、净骨重、前蹄重、后蹄重、皮重、瘤网皱胃、瓣胃、心、肝、肺、肾、肾周脂肪、牛鞭、脾脏、胴体胸深、后腿围、背膘、大理石花纹、眼肌面积等。所有性状的测定根据国家标准GB/T1723821998执行。The meat quality and carcass traits of Chinese Simmental mainly include: gross weight, head weight, carcass weight, slaughter rate, net bone weight, front hoof weight, hind hoof weight, tare weight, tumor net abomasum, ovum, heart, liver , lung, kidney, perirenal fat, bullwhip, spleen, carcass depth, hind leg circumference, back fat, marbling, eye muscle area, etc. The determination of all characters is performed according to the national standard GB/T1723821998.
二、实验结果2. Experimental results
结果显示：与中国西门塔尔肉牛胴体及肉质性状相关性分析的结果表明：g.41970426A>G位点与屠宰率、肾周脂肪、胴体脂肪覆盖率等肉质和胴体性状具有显著的相关性(P<0.05)：携带AA基因型个体的屠宰率显著高于GA基因型的个体，携带基因型AA个体的脂肪覆盖率显著低于GA基因型的个体，携带基因型AA个体的肾周脂肪重量显著低于GG基因型的个体(P<0.05)；The results showed that the results of correlation analysis with Chinese Simmental beef cattle carcass and meat quality traits showed that the g.41970426A>G locus was significantly correlated with slaughter rate, perirenal fat, carcass fat coverage and other meat quality and carcass traits ( P<0.05): The slaughter rate of individuals with AA genotype was significantly higher than that of individuals with GA genotype, the fat coverage rate of individuals with AA genotype was significantly lower than that of individuals with GA genotype, and the perirenal fat weight of individuals with genotype AA significantly lower than individuals with GG genotype (P<0.05);
g.41970520T>C位点与肾周脂肪含量具有显著的相关性(P<0.05)：携带CC基因型个体的肾脏脂肪含量显著高于TT基因型个体(P<0.05)；The g.41970520T>C locus was significantly correlated with the perirenal fat content (P<0.05): the renal fat content of individuals with CC genotype was significantly higher than that of individuals with TT genotype (P<0.05);
g.41970609A>G与屠宰率、肾周脂肪、胴体脂肪覆盖率具有显著的相关性(P<0.05)：携带AA基因型个体的屠宰率显著高于GA基因型个体，携带基因型AA个体的脂肪覆盖率显著低于GA基因型的个体，携带基因型AA个体的肾周脂肪重量显著低于GG基因型的个体(P<0.05)；g.41970609A>G was significantly correlated with slaughter rate, perirenal fat, and carcass fat coverage (P<0.05): the slaughter rate of individuals with AA genotype was significantly higher than that of individuals with GA genotype, and the slaughter rate of individuals with genotype AA was significantly higher The fat coverage rate was significantly lower than that of individuals with GA genotype, and the perirenal fat weight of individuals with AA genotype was significantly lower than that of individuals with GG genotype (P<0.05);
g.41970626G>A位点与屠宰率、肾周脂肪和胴体长具有显著的相关性(P<0.05)：携带GG基因型个体的屠宰率显著高于GA基因型的个体，携带GG基因型个体的肾脏脂肪含量和胴体长显著低于AA基因型的个体，(P<0.05)；The g.41970626G>A locus was significantly correlated with slaughter rate, perirenal fat and carcass length (P<0.05): the slaughter rate of individuals with GG genotype was significantly higher than that of individuals with GA genotype, and individuals with GG genotype Kidney fat content and carcass length were significantly lower in individuals with AA genotype (P<0.05);
g.41970714A>G位点与屠宰率、肾周脂肪和胴体脂肪覆盖率显著相关(P<0.05)：携带AA基因型个体的屠宰率显著高于GA基因型的个体，携带AA基因型的个体的肾脏脂肪含量显著低于GG基因型个体，携带AA基因型的个体的脂肪覆盖率显著低于GA基因型个体(P<0.05)；The g.41970714A>G locus was significantly correlated with slaughter rate, perirenal fat and carcass fat coverage (P<0.05): the slaughter rate of individuals with AA genotype was significantly higher than that of individuals with GA genotype, and individuals with AA genotype The kidney fat content of genotype was significantly lower than that of individuals with GG genotype, and the fat coverage rate of individuals with AA genotype was significantly lower than that of individuals with GA genotype (P<0.05);
g.41971686G>A位点与胴体脂肪覆盖率和眼肌面积显著相关(P<0.05)：携带GG基因型个体的脂肪覆盖率显著低于AG基因型的个体上，携带GG基因型个体的眼肌面积显著高于AA基因型的个体；The g.41971686G>A locus was significantly correlated with carcass fat coverage and eye muscle area (P<0.05): The fat coverage of individuals with GG genotype was significantly lower than that of individuals with AG genotype, and the eyes of individuals with GG genotype were significantly lower The muscle area was significantly higher than that of individuals with AA genotype;
g.41971818G>A位点与肾周脂肪、胴体脂肪覆盖率和胴体长显著相关(P<0.05)：携带GG基因型个体的肾脏脂肪含量显著低于GA、AA基因型的个体，携带GG基因型个体的胴体脂肪覆盖率显著低于GA基因型的个体，携带GG基因型个体的胴体长显著低于GA、AA基因型的个体，携带GA基因型个体的大腿肌肉厚度显著高于AA基因型的个体；The g.41971818G>A locus was significantly correlated with perirenal fat, carcass fat coverage and carcass length (P<0.05): The kidney fat content of individuals with GG genotype was significantly lower than that of individuals with GA and AA genotypes, and individuals with GG gene The carcass fat coverage of individuals with GA genotype was significantly lower than that of individuals with GA genotype, the carcass length of individuals with GG genotype was significantly lower than that of individuals with GA and AA genotypes, and the thickness of thigh muscles of individuals with GA genotype was significantly higher than that of AA genotype individuals the individual;
g.41972009A>C位点肾周脂肪显著相关(P<0.05)：携带AA基因型个体的肾脏脂肪含量显著低于CA基因型个体，携带AA基因型个体的腰部肉厚显著低于CC基因型个体。The g.41972009A>C site was significantly correlated with perirenal fat (P<0.05): the kidney fat content of individuals with AA genotype was significantly lower than that of CA genotype individuals, and the waist thickness of individuals with AA genotype was significantly lower than that of CC genotype individuals individual.
具体数据见表2，3所示。The specific data are shown in Tables 2 and 3.
表2中国西门塔尔牛群胴体及肉质性状与MRVI1基因SNPs的关联性Table 2 Associations between carcass and meat quality traits of Chinese Simmental cattle and MRVI1 gene SNPs
表2注：A，B，C表示极显著差异(P<0.01)，a，b，c表示显著差异(P<0.05)。DW(胴体重量，kg)，DP(屠宰率，％)，KFW(肾周脂肪重量，Kg)，GFW(生殖器脂肪重量，kg)，MBS(大理石花纹得分，评分范围1-9)，FCS(脂肪颜色)得分，得分范围为1-8)，BFT(背部脂肪厚度，cm)，FCR(脂肪覆盖率，％)Note in Table 2: A, B, and C represent extremely significant differences (P<0.01), and a, b, and c represent significant differences (P<0.05). DW (carcass weight, kg), DP (slaughter rate, %), KFW (perirenal fat weight, Kg), GFW (genital fat weight, kg), MBS (marble score, score range 1-9), FCS ( Fat color) score on a scale of 1-8), BFT (back fat thickness, cm), FCR (fat coverage, %)
表3中国西门塔尔牛群胴体及肉质性状与MRVI1基因SNPs的关联性Table 3 Associations between carcass and meat quality traits of Chinese Simmental cattle and MRVI1 gene SNPs
表3注：A，B，C表示极显著差异(P<0.01)，a，b，c表示显著差异(P<0.05)。NWB(骨骼净重，Kg)，CL(胴体长，cm)，CD(胴体深，cm)，CBD(胴体胸深，cm)，TMT(大腿肌肉厚度，cm)，TL(腰部厚度，cm)，REA(眼肌面积，cm2)，MCS(肌肉颜色评分，得分范围1-6)。Note in Table 3: A, B, C represent extremely significant differences (P<0.01), a, b, c represent significant differences (P<0.05). NWB (net bone weight, Kg), CL (carcass length, cm), CD (carcass depth, cm), CBD (carcass depth, cm), TMT (thigh muscle thickness, cm), TL (loin thickness, cm), REA (eye muscle area, cm2), MCS (muscle color score, score range 1-6).
实施例4 MRVI1基因8个SNP位点间连锁反应及单倍型分析Example 4 Chain reaction and haplotype analysis among 8 SNP loci of MRVI1 gene
连锁不平衡(LD)又称为等位基因关联，几个常用于度量LD的符号中，最重要的是D’和r2。当D’和r2的值为零时，连锁完全平衡，D’和r2的值为1时，连锁完全不平衡，而D’<1时，D’的数值表征多大程度的连锁不平衡。Linkage disequilibrium (LD), also known as allelic association, among several symbols commonly used to measure LD, the most important being D' and r 2 . When the value of D' and r2 is zero, the linkage is completely balanced, when the value of D' and r2 is 1, the linkage is completely disequilibrium, and when D'<1, the value of D' indicates how much linkage disequilibrium is. .
一、实验方法1. Experimental method
以此为依据，通过Haploview软件对MRVI1基因的SNPs位点进行连锁分析，在D’值95％可信区间内构建单倍域(haplotype block)。Based on this, the linkage analysis was performed on the SNPs of the MRVI1 gene by Haploview software, and a haplotype block was constructed within the 95% confidence interval of the D' value.
二、实验结果2. Experimental results
结果发现8个SNPs位点间强连锁(D’＝1，r2>0.9)，并且在此位点间构建了单倍结构域，如图9所示。主要有6个单倍型，分别为H1(0.625)、H2(0.151)、H3(0.091)、H4(0.047)、H5(0.042)、H6(0.012)和其他单倍型(0.032)，如表4所示。As a result, 8 SNPs were found to be strongly linked (D'=1, r2>0.9), and a haploid domain was constructed between these loci, as shown in Figure 9. There are mainly 6 haplotypes, namely H1 (0.625), H2 (0.151), H3 (0.091), H4 (0.047), H5 (0.042), H6 (0.012) and other haplotypes (0.032), as shown in the table 4 shown.
表4 MRVI1基因启动子单倍型频率Table 4 MRVI1 gene promoter haplotype frequency
从连续的SNP中形成了6个有研究意义的组合(个体数大于等于3的组合)，进一步分析不同单倍型与肉质和胴体性状的相关关系。结果发现：相关单倍型与胴体重(DW)、屠宰率(DP)、肾周脂肪重量(KFW)、生殖器官脂肪重量(GFW)、胴体脂肪覆盖率(FCR)、胴体长(CL)、胴体胸深(CBD)以及肌肉颜色评分(MCS)有显著相关关系，如表5所示。H1H1和H1H2单倍型与屠宰率(DP)相关(P<0.05)；H1H2，H1H4，H1H5和H2H3单倍型与生殖器官脂肪相关(P<0.05)；H1H1和H1H2与脂肪覆盖率显著相关(P<0.05)。此外，H1H1，H1H3，H1H5以及HH2H3与胴体长显著相关。此外，H1H2与胴体胸深(CBD)和肌肉颜色评分(MCS)显著相关，H1H4和H2H3也与肌肉颜色评分(MCS)显著相关，结果由表5所示。Six interesting combinations (combinations with a number of individuals greater than or equal to 3) were formed from consecutive SNPs, and the correlations between different haplotypes and meat quality and carcass traits were further analyzed. The results showed that the associated haplotypes were associated with carcass weight (DW), slaughter rate (DP), perirenal fat weight (KFW), reproductive organ fat weight (GFW), carcass fat coverage (FCR), carcass length (CL), There was a significant correlation between carcass depth of chest (CBD) and muscle color score (MCS), as shown in Table 5. H1H1 and H1H2 haplotypes were associated with slaughter rate (DP) (P < 0.05); H1H2, H1H4, H1H5 and H2H3 haplotypes were associated with reproductive organ fat (P < 0.05); H1H1 and H1H2 were significantly associated with fat coverage (P < 0.05). P<0.05). In addition, H1H1, H1H3, H1H5 and HH2H3 were significantly associated with carcass length. In addition, H1H2 was significantly associated with carcass depth of chest (CBD) and muscle color score (MCS), and H1H4 and H2H3 were also significantly associated with muscle color score (MCS), the results are shown in Table 5.
表5单倍型组合(个体数≥3)与牛胴体和肉质性状的关联分析结果Table 5 Association analysis results of haplotype combination (number of individuals ≥ 3) and cattle carcass and meat quality traits
表5注：A，B，C表示极显著差异(P<0.01)，a，b，c表示显著差异(P<0.05)。DW(胴体重，kg)，DP(屠宰率，％)，KFW(肾周脂肪重量，Kg)，GFW(生殖脂肪重量，kg)，MBS(大理石花纹得分，评分范围1-9)，FCS(脂肪颜色得分，得分范围为1-8)，BFT(背部脂肪厚度，cm)，FCR(胴体脂肪覆盖率，％)，NWB(骨骼净重，Kg)，CL(胴体长度，cm)，CD(胴体深度，cm)，CBD(胴体胸深，cm)，TMT(大腿肌肉厚度，cm)，TL(腰部厚度，cm)，REA(眼肌面积，cm2)，MCS(肌肉颜色评分，得分范围1-6)。Note in Table 5: A, B, C represent extremely significant differences (P<0.01), a, b, c represent significant differences (P<0.05). DW (carcass weight, kg), DP (slaughter rate, %), KFW (perirenal fat weight, Kg), GFW (reproductive fat weight, kg), MBS (marbling score, score range 1-9), FCS ( Fat color score on a scale of 1-8), BFT (back fat thickness, cm), FCR (carcass fat coverage, %), NWB (net bone weight, Kg), CL (carcass length, cm), CD (carcass length, cm) Depth, cm), CBD (carcass depth, cm), TMT (thigh muscle thickness, cm), TL (loin thickness, cm), REA (eye muscle area, cm2), MCS (muscle color score, score range 1- 6).
综上所述，本发明利用MRVI1基因的遗传标记检测中国西门塔尔牛群体中胴体及肉质性状脂肪覆盖率、肾周脂肪和眼肌面积的方法，将MRVI1基因的遗传标记作为肉牛筛选优良肉质性状的标记辅助选择用途，解决了当前对牛胴体及肉质性状相关的重要分子遗传标记筛查的难题，也为遗传工作者早期选育优质牛群提高产肉性能及改善肉品质奠定基础。MRVI1基因g.41970426A>G、g.41970520T>C、g.41970609A>G、g.41970714A>G、g.41971686G>A和g.41971818G>A位点可作为胴体性状中脂肪覆盖率的分子标记；g.41970426A>G、g.41970520T>C、g.41970609A>G、g.41970626G>A、g.41970714A>G、g.41971818G>A和g.41972009A>C位点可作为屠宰性状中肾周脂肪的分子标记；g.41971686G>A位点可作为肉质性状中眼肌面积的遗传标记，而不同的单倍型组合也与牛胴体和肉质性状显著相关，具有较高应用价值，应用于未来牛的分子育种中，为肉牛脂肪性状的选择和新品种的培育提供基因资源和理论基础。To sum up, the present invention utilizes the genetic marker of MRVI1 gene to detect the fat coverage ratio of carcass and meat quality traits, perirenal fat and eye muscle area in the Chinese Simmental cattle population, and uses the genetic marker of MRVI1 gene as the selection of excellent meat quality in beef cattle. The use of marker-assisted selection of traits solves the current problem of screening important molecular genetic markers related to cattle carcass and meat quality traits, and also lays the foundation for genetic workers to select high-quality cattle herds to improve meat production performance and meat quality early. MRVI1 gene g.41970426A>G, g.41970520T>C, g.41970609A>G, g.41970714A>G, g.41971686G>A and g.41971818G>A can be used as molecular markers of fat coverage in carcass traits ; g.41970426A>G, g.41970520T>C, g.41970609A>G, g.41970626G>A, g.41970714A>G, g.41971818G>A and g.41972009A>C sites can be used as slaughter trait mesonephros Molecular markers of peripheral fat; g.41971686G>A locus can be used as a genetic marker of eye muscle area in meat quality traits, and different haplotype combinations are also significantly correlated with beef carcass and meat quality traits, which have high application value. In the future molecular breeding of cattle, it will provide genetic resources and theoretical basis for the selection of beef cattle fat traits and the cultivation of new breeds.
最后所应当说明的是，以上实施例仅用以说明本发明的技术方案而非对本发明保护范围的限制，对于本领域的普通技术人员来说，在上述说明及思路的基础上还可以做出其它不同形式的变化或变动，这里无需也无法对所有的实施方式予以穷举。凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等，均应包含在本发明权利要求的保护范围之内。Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit the protection scope of the present invention. For those of ordinary skill in the art, on the basis of the above descriptions and ideas, the Variations or changes in other different forms are not required and cannot be exhaustive of all implementations here. Any modifications, equivalent replacements and improvements made within the spirit and principle of the present invention shall be included within the protection scope of the claims of the present invention.
序列表sequence listing
<110> 广东海洋大学<110> Guangdong Ocean University
<120> MRVI1基因作为在肉牛筛选优良肉质性状的标志物的应用Application of <120> MRVI1 Gene as a Marker for Screening Excellent Meat Quality Traits in Beef Cattle
<160> 6<160> 6
<170> SIPOSequenceListing 1.0<170> SIPOSequenceListing 1.0
<210> 1<210> 1
<211> 969<211> 969
<212> DNA<212> DNA
<213> 牛(bos)<213> cattle (bos)
<400> 1<400> 1
cttactggct gtggaacatc acacaggtct ttttgcccct ttaaacctta ttagtttcct 60cttactggct gtggaacatc acacaggtct ttttgcccct ttaaacctta ttagtttcct 60
tttcaaaaga atggtataat agctctattt cacggggcac ataggaaggt acatgctttt 120tttcaaaaga atggtataat agctctattt cacggggcac ataggaaggt acatgctttt 120
atggagagtg tctaatatag tgattagaat atcgtagatg ttcaatataa ggtttatata 180atggagagtg tctaatatag tgattagaat atcgtagatg ttcaatataa ggtttatata 180
acacatacag acacatagac acacagacat acactcacac ctccttcctc acatgcacac 240acacatacag acacatagac acacagacat acactcacac ctccttcctc acatgcacac 240
aaactgaagt ttctagttgt ctttgcagga attgctgcta gattcataaa atcaactttt 300aaactgaagt ttctagttgt ctttgcagga attgctgcta gattcataaa atcaactttt 300
ggagtaccat tccctaaaat ggggtttttg gaccaactgc ataagtgaga atcacccgag 360ggagtaccat tccctaaaat ggggtttttg gaccaactgc ataagtgaga atcacccgag 360
ggccttaaat aaaatatagg tttctgggcc tcactttaga gccacaagat tagaatttct 420ggccttaaat aaaatatagg tttctgggcc tcactttaga gccacaagat tagaatttct 420
agaaaagggg tccagagagt ttcacacttg atgattctat tgcacaatga agttggagaa 480agaaaagggg tccagagagt ttcacacttg atgattctat tgcacaatga agttggagaa 480
acagcagtaa agagtttggt aattcattct aatccctcta cttgcagttc tagtagctat 540acagcagtaa agagtttggt aattcattct aatccctcta cttgcagttc tagtagctat 540
cacgttgtgg ttttgccagt gggagcctgt tcccagtcga tggataggtg taggtgcctg 600cacgttgtgg ttttgccagt gggagcctgt tcccagtcga tggataggtg taggtgcctg 600
agggaaagca tggctagtac ccatgagtct tctctggttc catctacaga aatagttatt 660agggaaagca tggctagtac ccatgagtct tctctggttc catctacaga aatagttatt 660
tatgggtttg acagacagca tgtagtatag tctcatcagg aatctctttc gtaacaagca 720tatgggtttg acagacagca tgtagtatag tctcatcagg aatctctttc gtaacaagca 720
aggagaagag acagtccagg actggatgtc cgtggaaggg gctgggatag aagcacagtg 780aggagaagag acagtccagg actggatgtc cgtggaaggg gctgggatag aagcacagtg 780
ggctaggttt gggggagaaa gcctgccaca gccaggaact gaggataagg actgtatgca 840ggctaggttt gggggagaaa gcctgccaca gccaggaact gaggataagg actgtatgca 840
gccaggcagc tgtcaccagg agggcttgag gccacacgtc tcgaggcacc gacacctaga 900gccaggcagc tgtcaccagg agggcttgag gccacacgtc tcgaggcacc gacacctaga 900
gccctcagca tgactggcga acactttctg aggtggtaag tgatggccca ccactagagt 960gccctcagca tgactggcga acactttctg aggtggtaag tgatggccca ccactagagt 960
tgccagatt 969tgccagatt 969
<210> 2<210> 2
<211> 966<211> 966
<212> DNA<212> DNA
<213> 牛(bos)<213> cattle (bos)
<400> 2<400> 2
ccaccactag agttgccaga tttagcaaat aaaaatacag gatgagccat taaatttcat 60ccaccactag agttgccaga tttagcaaat aaaaatacag gatgagccat taaatttcat 60
taaattagaa tttcagattt tatatatata tatatatacg tacatacaca catatatggg 120taaattagaa tttcagattt tatatatata tatatacg tacatacaca catatatggg 120
gcttccctgg taggtcagct ggtaaagaat tcacctgcaa tgcaggagat gccagtttga 180gcttccctgg taggtcagct ggtaaagaat tcacctgcaa tgcaggagat gccagttttga 180
ttcctgagtt gggaaaatct cctgaagaag gaataggcta cccactccag tattcttggg 240ttcctgagtt gggaaaatct cctgaagaag gaataggcta cccactccag tattcttggg 240
cttctggggt ggcttagatg gtaaagaatc cgcccgcaat gcggcagacc tgggtttgat 300cttctggggt ggcttagatg gtaaagaatc cgcccgcaat gcggcagacc tgggtttgat 300
ccctgggttg ggcagatgcc ctggaagagg gcatggcaac ccactccagt attcttgcct 360ccctgggttg ggcagatgcc ctggaagagg gcatggcaac ccactccagt attcttgcct 360
ggagaatccc catggacaga ggaaccttgt gggttacagt ccatggagtc acaaagagtc 420ggagaatccc catggacaga ggaaccttgt gggttacagt ccatggagtc acaaagagtc 420
agatactact gagcaattaa gcacacagca cagcatatat atgtgtgtgt gtgtatgtaa 480agatactact gagcaattaa gcacacagca cagcatatat atgtgtgtgt gtgtatgtaa 480
gcatgtcctg tgcaatattt gggacatact tatactaaaa tagatatgtg ttatctgaaa 540gcatgtcctg tgcaatattt gggacatact tatactaaaa tagatatgtg ttatctgaaa 540
ttcaagtcta agtaggtgtc ctgcattcta tctggcagtc ctacatagga tttctctgag 600ttcaagtcta agtaggtgtc ctgcattcta tctggcagtc ctacatagga tttctctgag 600
ctgcctctaa ctgctgaaaa gggttggaga ccccatgtga ctggccaggg cccacagcac 660ctgcctctaa ctgctgaaaa gggttggaga ccccatgtga ctggccaggg cccacagcac 660
catgatcttt ctctgggtgg ggggctgcac ccaaggtggt gaggagcgag taagggcaag 720catgatcttt ctctgggtgg ggggctgcac ccaaggtggt gaggagcgag taagggcaag 720
gtgtggactt caggagagag cagctcttcc tcacctgtgt ctgagcaggg gcaggaaact 780gtgtggactt caggagagag cagctcttcc tcacctgtgt ctgagcaggg gcaggaaact 780
cactggtatt gataagcaag tctgagatgc tagcactctg ctaagccttt cagtatcatc 840cactggtatt gataagcaag tctgagatgc tagcactctg ctaagccttt cagtatcatc 840
tcatcaaatc ctcctgcaac cctaggagtt aggcattgtg atcccagtga ttcccacttt 900tcatcaaatc ctcctgcaac cctaggagtt aggcattgtg atcccagtga ttcccacttt 900
agagatgagg agactgaggc tccacgaggt taagaccaca gggctactca gtggaggagc 960agagatgagg agactgaggc tccacgaggt taagaccaca gggctactca gtggaggagc 960
aaggac 966aaggac 966
<210> 3<210> 3
<211> 21<211> 21
<212> DNA<212> DNA
<213> 牛(bos)<213> cattle (bos)
<400> 3<400> 3
cttactggct gtggaacatc a 21cttactggct gtggaacatc a 21
<210> 4<210> 4
<211> 21<211> 21
<212> DNA<212> DNA
<213> 牛(bos)<213> cattle (bos)
<400> 4<400> 4
aatctggcaa ctctagtggt g 21aatctggcaa ctctagtggt g 21
<210> 5<210> 5
<211> 21<211> 21
<212> DNA<212> DNA
<213> 牛(bos)<213> cattle (bos)
<400> 5<400> 5
ccaccactag agttgccaga t 21ccaccactag agttgccaga t 21
<210> 6<210> 6
<211> 20<211> 20
<212> DNA<212> DNA
<213> 牛(bos)<213> cattle (bos)
<400> 6<400> 6
gtccttgctc ctccactgag 20gtccttgctc ctccactgag 20
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