WO2021207992A1 - 一种与德州驴多胸椎数性状相关的snp位点检测试剂盒及其使用方法 - Google Patents

一种与德州驴多胸椎数性状相关的snp位点检测试剂盒及其使用方法 Download PDF

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WO2021207992A1
WO2021207992A1 PCT/CN2020/085025 CN2020085025W WO2021207992A1 WO 2021207992 A1 WO2021207992 A1 WO 2021207992A1 CN 2020085025 W CN2020085025 W CN 2020085025W WO 2021207992 A1 WO2021207992 A1 WO 2021207992A1
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donkey
vrtn
gene
texas
thoracic
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French (fr)
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王长法
高琦璨
王金鹏
孙艳
李玉华
杨春红
刘桂芹
刘文强
李海静
张新浩
于杰
嵇传良
周祥山
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聊城大学
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms

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  • the invention belongs to the technical field of molecular breeding genetic marker screening, and specifically refers to a SNP site detection kit related to the multi-thoracic vertebral number traits of Texas donkeys and a use method thereof.
  • Donkeys belong to the class Mammalia, Perissodactyla, Equine, Equine, and monogastric herbivore in the taxonomy of vertebrates. Donkeys have strong adaptability, wide eating habits, fast growth, herbivorous, strong disease resistance, high survival rate, easy feeding, and good economic benefits. China has a long history of raising donkeys and rich resources of donkey species. According to their physical size, they can be divided into three types: large, medium and small donkeys. They are mainly concentrated in areas with moderate temperate and warm temperate climates, especially in the middle and lower reaches of the Yellow River and Xinjiang and Gansu. The Hexi Corridor is the most distributed; donkey itself has great development and utilization value.
  • Donkey meat is very rich in nutrients and contains essential amino acids, trace elements and other nutrients that are easily absorbed by the human body. Studies have shown that donkey milk is the closest milk product to breast milk. The content and proportion of 18 amino acids are similar to that of human milk. Its cholesterol content is low, amino acids are complete, and it contains sufficient trace elements. Donkey skin has high medical value and is the main raw material for making donkey-hide gelatin, which has the effects of nourishing blood, nourishing yin, moisturizing dryness, and stopping bleeding.
  • the body length trait is one of the most important economic traits of livestock, and it is also the main measurement index in the conventional breeding of livestock.
  • the length of the body length trait directly affects the carcass length after slaughter, and the carcass length directly affects the economic benefits.
  • the body length trait is mainly affected by the heredity of the number of spine, and has a high heritability, reaching 0.74.
  • the VRTN gene of donkey is located on chromosome 7 with a size of 2141bp and all are exons.
  • QQ homozygous genotype
  • qq Type (qq) individuals have 1.15 more thoracic vertebrae.
  • Garmen Burgos has found that the allele ins of the VRTN gene is a beneficial mutation produced by breeders during the selection and breeding of commercial breeds of pigs.
  • VRTN gene is expressed in high amounts during the critical period of thoracic somite development, indicating that it is a key gene regulating the development of the thoracic spine.
  • the mechanism of VRTN in pigs is to regulate the rate of thoracic vertebral body segmentation by regulating the expression of genes such as Notch2, thereby affecting the number of pig thoracic vertebrae.
  • multi-vertebral traits have high heritability.
  • Borchers and other studies have shown that the heritability of multi-vertebral traits in pigs is 0.62.
  • the present invention provides an indirect detection of multiple thoracic vertebral count traits of Texas donkeys by identifying the single nucleic acid polymorphisms of the Texas donkey VRTN gene. Only a very small amount of Texas donkey genomic DNA can be efficiently identified.
  • the SNP locus of VRTN gene related to multiple thoracic vertebrae count traits and its influence on thoracic vertebrae number are analyzed. According to the analysis results, the g.*1932A>T locus of VRTN gene is used to screen individuals with multiple thoracic vertebrae count traits, which can be widely used in practice.
  • the technical scheme adopted by the present invention is as follows:
  • the present invention is a SNP site detection kit related to the multiple thoracic vertebral number traits of Texas donkey, including a primer pair that amplifies the DNA sequence of g.*1932 at the SNP site of VRTN gene, and PCR reaction System, enzyme digestion reaction system and DNA Marker, the SNP site g.*1932A>T is located at the 47567290 base pair of donkey chromosome 7 and the first base A of the transcription start site of the VRTN gene is + 1. It is significantly related to the traits of multiple thoracic vertebrae in Texas donkey.
  • the amplified primer pair containing the VRTN gene SNP site g.*1932 DNA sequence includes an upstream primer and a downstream primer, and the amplification includes VRTN gene SNP site g. .*
  • the primer pair of DNA sequence at position 1932 is designed and analyzed by Primer 5 software using donkey blood DNA as a template and manually modified.
  • the last 3 base of the upstream primer is a TG mismatch, and the upstream primer is artificially near its 3'end.
  • the Eco47III recognition site AGCGCT is introduced, and the downstream primer is completely matched with the template;
  • the nucleotide sequence of the upstream primer is SEQ ID No. 2: ATTCAGATCTCAGAATCACTAGCG;
  • the nucleotide sequence of the downstream primer is SEQ ID No. 3: GGCGGTGGCGGGAATAATAA.
  • the primer pair for amplifying the DNA sequence of g.*1932 at the SNP site of the VRTN gene contains a nucleotide sequence, the 574th polymorphism of the nucleotide sequence is A/T, and the nuclear The nucleotide sequence is shown below:
  • the PCR reaction system includes 2 ⁇ Taq PCR MasterMix and ddH 2 O; the enzyme digestion reaction system includes FastDigest restriction endonuclease Eco47III, 10 ⁇ FastDigest buffer and ddH 2 O.
  • the present invention also provides a method for using the SNP site detection kit related to the multi-thoracic vertebral number traits of Texas donkey, which includes the following steps:
  • the conditions of the PCR amplification reaction in the step 2) are: 95°C for 5 min pre-denaturation; then at 95°C for 30s, 60°C for 30s, 72°C for 1 min, 35 cycles, and then at Keep at 72°C for 10 min, and finally store at 4°C to obtain PCR amplification products.
  • the PCR reaction system in step 2) is a 25 ⁇ L system including the following components: 2 ⁇ Power Taq PCR Master Mix 12.5 ⁇ l; upstream primers with a concentration of 10 ⁇ mol/l and downstream primers with a concentration of 10 ⁇ mol/l, 1 ⁇ l each ; Template with a concentration of 50ng/ ⁇ l is 1 ⁇ l; ddH 2 O is made up to 25 ⁇ l.
  • the restriction enzyme digestion reaction system in step 3) is calculated as 30 ⁇ l, including the following components: PCR product 10 ⁇ l; restriction enzyme Eco47III 1 ⁇ l with a concentration of 10 U/ ⁇ l; 10 ⁇ FastDigest buffer 2 ⁇ l; ddH 2 O is made up to 30 ⁇ l, The digestion reaction system was digested at 37°C for 1 hour to obtain digestion products, which were detected by 3% and 2.5% agarose gel electrophoresis, respectively.
  • the correlation analysis table between the different genotypes at the 47567290bp position on the chromosome 7 of the experimental donkey and the number of thoracic vertebrae of the Texas donkey was obtained by the following steps:
  • the beneficial effects of the present invention are as follows:
  • the present invention is a SNP site detection kit related to the multiple thoracic vertebral number traits of Texas donkeys and its use method, which indirectly detects Texas donkeys by identifying single nucleic acid polymorphisms in the VRTN gene of Texas donkeys.
  • the method of thoracic vertebral number traits requires only a very small amount of Texas donkey genomic DNA to efficiently identify the SNP locus of the Texas donkey VRTN gene, analyze its influence on the number of thoracic vertebrae, and analyze the SNP locus on the VRTN chromosome 7 of Texas donkey.
  • Genotyping was carried out, and the correlation analysis between different genotypes and the phenotype data of the Texas donkey thoracic vertebrae was performed, and a SNP molecular marker related to the number of multiple thoracic vertebrae characters of the Texas donkey was obtained through screening, and the VRTN gene g.*1932A>T gene was identified.
  • the experimental donkey with type AA and AT is the dominant individual, and this individual has the trait of multiple thoracic vertebrae; and based on this conclusion, the use of VRTN gene g.*1932A>T locus to screen individuals with multiple thoracic vertebrae characters can be widely used in practice.
  • Example 1 is a schematic diagram of the electrophoresis detection result of the PCR product of the donkey VRTN gene used in the experiment in Example 1 of the present invention
  • Figure 2 is the VRTN gene g.*1932A>T(A, AT, T) sequencing result in Example 1 of the present invention
  • Fig. 3 is an agarose gel electrophoresis typing diagram of the SNP locus (g.*1932A>T) of the donkey Texas donkey VRTN gene used in the experiment in Example 3 of the present invention.
  • a and b are AA genotypes
  • c and d are AT genotypes
  • e and f are TT genotypes.
  • the present invention uses the PCR product direct sequencing method to perform SNP identification and typing of VRTN genes;
  • Select 376 donkeys for experiment use disposable vacuum blood collection tube containing EDTA to collect blood from the jugular vein of experimental donkey population to obtain blood samples.
  • the blood volume collected is 7ml/head ⁇ 9ml/head, and the blood samples are kept at a temperature of- Put it in an insulated sampling box under the condition of 20°C ⁇ 0.5°C for long-distance transportation to the laboratory.
  • Store it in the freezer at -20°C ⁇ 0.5°C for later use slaughter the experimental donkey, count the number of thoracic vertebrae and measure the experimental donkey after slaughter The length from the first thoracic vertebra to the last thoracic vertebra.
  • the tested DNA sample is finally diluted to 50ng/ ⁇ l to obtain an intermediate DNA sample solution, which is stored at 4°C for later use.
  • Use Primer5 software to design typing primers based on gene sequences near SNPs Use PrimerPrimer 5 software for design analysis to obtain a primer pair that amplifies the DNA sequence of g.*1932 at the SNP site of VRTN gene, and the upstream primer of the primer pair that amplifies the DNA sequence at g.*1932 of the VRTN gene SNP site is SEQ ID No. 2: ATTCAGATCTCAGAATCACTAGCG and the third base from the bottom of the 3'end is a TG mismatch.
  • This primer artificially introduces the Eco47III recognition site AGCGCT near its 3'end, and the downstream primers are completely paired with the template; the amplification includes VRTN
  • the nucleotide sequence of the primer pair of the gene SNP site g.*1932 DNA sequence is as follows:
  • Upstream primer SEQ ID No. 2 ATTCAGATCTCAGAATCACTAGCG;
  • Downstream primer SEQ ID No. 3 GGCGGTGGCGGGAATAATAA.
  • Configure PCR amplification system 2 ⁇ Power Taq PCR MasterMix 12.5 ⁇ l; upstream primer with a concentration of 10 ⁇ mol/l and downstream primer with a concentration of 10 ⁇ mol/l each 1 ⁇ l; template 1 ⁇ l, 50ng/ ⁇ l; ddH 2 O to make up to 25 ⁇ l.
  • PCR reaction procedure 95°C for 5min pre-denaturation; then 95°C for 30s denaturation, 60°C for 30s annealing, 72°C for 1min extension, after 35 denaturation-annealing-extension cycles, the final 72°C final extension for 10min; Store at 4°C to obtain PCR amplification products.
  • the PCR amplified product was detected by 1% agarose gel electrophoresis, and after it was determined that it was the target band, the PCR amplified product was sequenced.
  • the sequencing result was compared with the reference sequence of the DNAMAN and VRTN gene, combined with the sequencing peak map to confirm The SNP sites in the target fragment are VRTN gene g.*1932A>T.
  • Table 1 shows the statistical results of different genotypes at the 47567290bp locus of the tested Texas donkey on chromosome 7.
  • the length of the thoracic spine is expressed by its mean ⁇ standard deviation
  • VRTN gene g.*1932A>T is closely related to the number of thoracic vertebrae and the length of the thoracic vertebrae of Texas donkeys. Therefore, it is possible to invent a detection reagent suitable for screening of VRTN gene SNP molecular markers with traits of multiple thoracic vertebrae. box.
  • VRTN-F/R primers to amplify the fragment containing VRTN gene g.*1932A>T site (105bp) to obtain PCR product.
  • the fragment containing VRTN gene g.*1932A>T site has restriction enzyme Eco47III The relationship is as follows: when the site is of the TT genotype, it can be cut by the restriction enzyme Eco47III to obtain two fragments of 25bp and 80bp; if the site is of the AA genotype, it cannot be cut by the restriction enzyme Eco47III After cutting, there is only one 105bp fragment; if the site is AT heterozygous, three bands of 25bp, 80bp and 105bp can be detected.
  • PCR product 10 ⁇ l including the following components: PCR product 10 ⁇ l; restriction enzyme Eco47II 1 ⁇ l with a concentration of 10U/ ⁇ l; 10 ⁇ FastDigest buffer 2 ⁇ l; ddH2O fills up to 30 ⁇ l, after detecting that the PCR product is the target fragment, use
  • Table 4 The components of the kit for the detection of multiple thoracic vertebral count traits in two experimental donkeys
  • the PCR reaction system is a 25 ⁇ L reaction system, including the following components: 2 ⁇ Power Taq PCR Master Mix 12.5 ⁇ l; the upstream primer with a concentration of 10 ⁇ mol/l and the downstream primer with a concentration of 10 ⁇ mol/l each 1 ⁇ l; the concentration is 50ng/ ⁇ l 1 ⁇ l of template; ddH 2 O fills up to 25 ⁇ l;
  • the PCR reaction program includes the following steps: 95°C for 5min pre-denaturation; then 95°C for 30s denaturation, 60°C for 30s annealing, 72°C for 1min extension, after 35 denaturation-annealing-extension cycles, the final 72°C final extension 10min; Store at 4°C to obtain PCR amplification product.
  • the digestion reaction system is a 30 ⁇ l reaction system, including the following components: PCR product 10 ⁇ l; restriction enzyme Eco47II with a concentration of 10U/ ⁇ l 1 ⁇ l; 10 ⁇ FastDigest buffer 2 ⁇ l; ddH 2 O fills up to 30 ⁇ l, digestion reaction at 37°C
  • the digested product was obtained after the system for 1 hour; the digested product was detected by 3% and 2.5% agarose gel electrophoresis, respectively.
  • Example 2 the number of thoracic vertebrae of each genotype was analyzed. Individuals carrying allele A have the advantage of longer thoracic vertebral length; this is used to determine whether to reserve seeds.
  • the invention provides a method for indirectly detecting the multiple thoracic vertebral number traits of the Texas donkey by identifying the SNPs of the Texas donkey VRTN gene.
  • This method is suitable for analyzing the correlation between the SNP locus on the donkey VRTN gene and the multiple thoracic vertebral number traits of Texas donkeys, and can be used as a statistical basis for the number of Texas donkey thoracic vertebrae.
  • Theoretical guidance and provides an efficient method for detecting the VRTN gene SNP of Texas donkeys and the number traits of Texas donkey thoracic vertebrae.
  • This method can be used for the development of genetic test kits related to multiple thoracic vertebrae number traits of Texas donkeys.
  • Texas donkey The g.*1932A>T locus of the VRTN gene is significantly related to the number of thoracic vertebrae and the length of the thoracic spine of the donkey. Based on this correlation, a kit for detecting the number of thoracic vertebrae of the Texas donkey is developed by detecting the SNP genotype of the VRTN gene.
  • the SNP site detection kit for screening and detecting Texas donkeys with multiple thoracic vertebral count traits and the SNP site detection kit related to Texas donkeys with multiple thoracic vertebral count traits and the method of use thereof are aimed at accelerating Texas donkeys
  • the process of genetic improvement is not part of the diagnosis and treatment of the disease.

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Abstract

提供了一种与德州驴多胸椎数性状相关的SNP位点检测试剂盒，包括扩增包含VRTN基因SNP位点g.*1932位DNA序列的引物对、PCR反应体系、酶切反应体系和DNA Marker。属于分子育种遗传标记筛选技术领域，具体是提供了一种仅需少量的德州驴基因组DNA就可高效鉴定出与德州驴多胸椎数性状相关的VRTN基因SNP位点并分析其对胸椎数的影响，根据分析结果利用VRTN基因g.*1932A>T位点筛选具有多胸椎数性状的个体，将具有多胸椎数性状的个体选留下来，降低成本的与德州驴多胸椎数性状相关的SNP位点检测试剂盒及其使用方法。

Description

一种与德州驴多胸椎数性状相关的SNP位点检测试剂盒及其使用方法 技术领域
本发明属于分子育种遗传标记筛选技术领域，具体是指一种与德州驴多胸椎数性状相关的SNP位点检测试剂盒及其使用方法。
背景技术
驴在动物分类学上属于脊椎动物门哺乳纲、奇蹄目、马科、马属、单胃草食动物。驴的适应性强、食性广、生长快、喜食草、抗病性强、成活率高、饲养容易、经济效益好。我国养驴历史悠久，驴种资源丰富，根据其体格大小可分为大型、中型和小型驴3种类型，主要集中在属中温带和暖温带气候地区，尤以黄河中下游流域和新疆、甘肃的河西走廊分布最多；驴本身具有很大的开发利用价值，驴肉中的营养物质非常丰富，含有人体必须的氨基酸、微量元素等易于人体吸收的营养物质。研究表明，驴奶成分是最接近母乳的乳品，其中有18种氨基酸含量和比例和人乳比较接近，其胆固醇含量低、氨基酸种类齐全，并含有充足的微量元素。驴皮具有很高的医药价值，是熬制阿胶的主要原材料，阿胶具有补血滋阴、润燥、止血的功效。
体长性状是家畜最重要的经济性状之一，也是家畜常规育种中主要的测定指标，体长性状的长短直接影响屠宰后的胴体长度，而胴体长度直接影响经济效益。体长性状主要受脊椎数遗传影响，并且有很高的遗传力，达到了0.74。经研究发现，德州驴存在多脊椎性状。德州驴胸椎17-19根不等，腰椎5-6根。德州驴经济性状的提高依赖于对其遗传背景的理解，而缺乏遗传信息是了解德州驴发育机制的一个主要障碍，德州驴遗传变异和单核苷酸多态性(SNP)数据的分析对扩展这个物种的遗传信息资源至关重要。因此，很有必要寻找德州驴体长性状的候选基因，对德州驴的遗传变异和起源进化、群体结构等仅分析研究，为我国地方驴品种的育种改良和遗传资源保护提供理论依据。
驴的VRTN基因位于第7号染色体上，大小为2141bp，全部为外显子。近年来发育遗传学和分子遗传学研究结果表明畜禽脊椎数目的变化是由于调控脊椎发育的VRTN基因发生了突变，研究发现具有纯合子基因型(QQ)个体的胸椎数要比具有纯合子基因型(qq)个体的胸椎数多1.15个，Garmen Burgos研究发现VRTN基因的等位基因ins是育种工作者对商业品种猪选育过程中产生的有益突变。研究人员通过小鼠试验发现VRTN基因在胸椎体节发育关键时期高量表达，表明其为调控胸椎发育的关键基因。VRTN在猪上的作用机理是通过调节Notch2等基因的表达来调控胸椎体节分节速率，从而影响猪胸椎数。研究发现多脊椎性状具有高遗传力，Borchers等研究证明，猪的多脊椎性状的遗传力为0.62，中国地方品种棉羊存在4种组合类型，并且这种性状稳定遗传，说明多脊椎性状可稳定遗传，所以可进行定向选择并培育多脊椎数的家畜品种。
发明内容
为解决上述现有难题，本发明提供了一种通过鉴定德州驴VRTN基因单核酸多态性间接检测德州驴多胸椎数性状，仅需极其少量的德州驴基因组DNA就可以高效鉴定出与德州驴多胸椎数性状相关的VRTN基因的SNP位点并分析其对胸椎数的影响，根据分析结果利用VRTN基因g.*1932A>T位点筛选具有多胸椎数性状的个体，可在实践中广泛应用，通过对驴进行早期筛选，将具有多胸椎数性状的个体选留下来，可以减少驴饲养的盲目性，降低成本，增加牧场经济效益的与德州驴多胸椎数性状相关的SNP位点检测试剂盒及其使用方法。
本发明采用的技术方案如下：本发明一种与德州驴多胸椎数性状相关的SNP位点检测试剂盒，包括扩增包含VRTN基因SNP位点g.*1932位DNA序列的引物对、PCR反应体系、酶切反应体系和DNA Marker，所述SNP位点g.*1932A>T位于驴第7号染色体第47567290碱基对并以VRTN基因转录起始位点的第一个碱基A为+1，与德州驴多胸椎数性状显著相关，所述扩增包含VRTN基因SNP位点g.*1932位DNA序列的引物对包括上游引物和下游引物，所述扩增包含VRTN基因SNP位点g.*1932位DNA序列的引物对以驴血液DNA为模板通过Primer 5软件进行设计分析并人工加以修改得到，上游引物倒数第3位碱基为T-G错配，上游引物人为在其3'端附近引入Eco47III识别位点AGCGCT，下游引物完全与模板配对；所述上游引物的核苷酸序列为SEQ ID No.2：ATTCAGATCTCAGAATCACTAGCG；所述下游引物的核苷酸序列为SEQ ID No.3：GGCGGTGGCGGGAATAATAA。
进一步地，所述扩增包含VRTN基因SNP位点g.*1932位DNA序列的引物对含有核苷酸序列，所述核苷酸序列的第574位多态性为A/T，所述核苷酸的序列如下所示：
Figure PCTCN2020085025-appb-000001
进一步地，所述PCR反应体系包括2×Taq PCR MasterMix和ddH 2O；酶切反应体系包括FastDigest 限制性核酸内切酶Eco47III、10×FastDigest buffer和ddH 2O。
本发明还提供了一种与德州驴多胸椎数性状相关的SNP位点检测试剂盒使用方法，包括以下步骤：
1)选取实验用驴抽取静脉血获得血液样本，从血液样本中提取实验用驴的基因中DNA；
2)以提取的实验用驴基因中DNA为模板，通过Primer 5软件进行设计分析并人工加以修改得到核苷酸序列分别为SEQ ID No.2：ATTCAGATCTCAGAATCACTAGCG和SEQ ID No.3：GGCGGTGGCGGGAATAATAA的扩增包含VRTN基因SNP位点g.*1932位DNA序列的引物对，利用扩增包含VRTN基因SNP位点g.*1932位DNA序列的引物对在PCR反应体系内进行PCR扩增反应，得到PCR扩增产物；
3)用酶切反应体系对PCR扩增产物进行消化得到酶切产物，酶切产物分别用3％和2.5％的琼脂糖凝胶电泳检测分析酶切产物片段长度；
4)根据产物片段长度分析结果判定位于驴第7号染色体上的VRTN基因SNP位点基因型，根据实验用驴第7号染色体上第47567290bp位点不同基因型与德州驴胸椎数关联分析表得出：当判定位于驴第7号染色体上的VRTN基因SNP位点基因型为AA、AT时，对应的实验用驴为具有多胸椎性状的个体。
进一步地，所述步骤2)中的PCR扩增反应的条件为：95℃保持5min预变性；然后依次在95℃保持30s、60℃保持30s、72℃保持1min，进行35个循环，再在72℃保持10min，最终4℃保存得到PCR扩增产物。
进一步地，所述步骤2)中的PCR反应体系为25μL体系包括如下组份：2×Power Taq PCR Master Mix12.5μl；浓度为10μmol/l的上游引物和浓度为10μmol/l的下游引物各1μl；浓度为50ng/μl的模板1μl；ddH 2O补足到25μl。
进一步地，步骤3)中酶切反应体系以30μl计，包括如下组份：PCR产物10μl；浓度为10U/μl的限制性内切酶Eco47III1μl；10×FastDigest buffer 2μl；ddH 2O补足到30μl，37℃消化酶切反应体系1h得到酶切产物，酶切产物分别用3％和2.5％的琼脂糖凝胶电泳检测。
进一步地，所述步骤4)中实验用驴第7号染色体上第47567290bp位点不同基因型与德州驴胸椎数关联分析表经以下步骤得到：
1)统计采集血液样本的实验用驴胸椎数目，并测量从第一个胸椎到最后一个胸椎的长度，统计后得到实验用驴的胸椎数及胸椎长度的表型数据；
2)利用PCR产物直接测序法对VRTN基因进行SNP鉴定及分型得到实验用驴第7号染色体第47567290bp位点不同基因型分析统计表，根据实验用驴的胸椎数及胸椎长度的表型数据和实验用驴第7号染色体第47567290bp位点不同基因型分析统计表对实验用驴第7号染色体上第47567290bp位点不同基因型与实验用驴胸椎数进行关联分析得到实验用驴第7号染色体上第47567290bp位点不同基因型与德州驴胸椎数关联分析表。
采用上述方案本发明取得有益效果如下：本发明一种与德州驴多胸椎数性状相关的SNP位点检测试剂盒及其使用方法，通过鉴定德州驴VRTN基因单核酸多态性间接检测德州驴多胸椎数性状的方式，只需要极其少量的德州驴基因组DNA就可以高效鉴定出德州驴VRTN基因的SNP位点，分析其对胸椎数的影响，对德州驴第7号染色体VRTN上的SNP位点进行基因分型，将不同基因型与德州驴胸椎表型数据进行关联分析，通过筛选获得一种与德州驴多胸椎数性状相关的SNP分子标记，并鉴定当VRTN基因g.*1932A>T基因型为AA、AT的实验用驴是优势个体，该个体具有多胸椎数性状；并基于该结论利用VRTN基因g.*1932A>T位点筛选具有多胸椎数性状的个体，可在实践中广泛应用，通过对驴进行早期筛选，将具有多胸椎数性状的个体选留下来，可以减少驴饲养的盲目性，降低成本，增加牧场经济效益。
附图说明
图1为本发明实施例一中实验用驴VRTN基因PCR产物电泳检测结果示意图；
图2为本发明实施例一中VRTN基因g.*1932A>T(A，AT，T)测序结果；
图3为本发明实施例三中实验用驴德州驴VRTN基因SNP位点(g.*1932A>T)琼脂糖凝胶电泳分型图。
其中，a、b为AA基因型，c、d为AT基因型，e、f为TT基因型。
具体实施方式
下面将对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅是本发明的一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其它实施例，都属于本发明保护的范围。
实施例一实验用驴VRTN基因SNP位点的鉴定
本发明采用PCR产物直接测序法对VRTN基因进行SNP鉴定及分型；
1.1实验材料
选取376头实验用驴，使用一次性含EDTA抗凝的真空采血管对实验用驴群体进行颈静脉采血得到血液样本，采集血量为7ml/头～9ml/头，将血液样本在温度为-20℃±0.5℃的条件下放在保温采样箱长途运输至实验室在-20℃±0.5℃的条件下冷冻保存备用，对实验用驴进行屠宰，在屠宰之后统计胸椎的数目并测量实验用驴从第一个胸椎到最后一个胸椎的长度。
1.2驴全血基因组DNA的提取
准备血液基因组DNA提取试剂盒并使用血液基因组DNA提取试剂盒提取血液样本中的基因组DNA获取DNA样品，准备核算蛋白分析仪并利用核算蛋白分析仪检测DNA样品的纯度和浓度，根据DNA样品纯度和浓度将检测后的DNA样品最终稀释成50ng/μl获得中间状态DNA样品溶液，4℃保存备用。
1.3引物设计
使用Primer5软件根据SNPs附近的基因序列设计分型引物。使用PrimerPrimer 5软件进行设计分析得到扩增包含VRTN基因SNP位点g.*1932位DNA序列的引物对，扩增包含VRTN基因SNP位点g.*1932位DNA序列的引物对的上游引物为SEQ ID No.2：ATTCAGATCTCAGAATCACTAGCG且其3'端倒数第三个碱基为T-G错配，该引物人为地在其3'端附近引入Eco47III识别位点AGCGCT，下游引物完全与模板配对；扩增包含VRTN基因SNP位点g.*1932位DNA序列的引物对的核苷酸序列如下：
上游引物SEQ ID No.2：ATTCAGATCTCAGAATCACTAGCG；
下游引物SEQ ID No.3：GGCGGTGGCGGGAATAATAA。
用该扩增包含VRTN基因SNP位点g.*1932位DNA序列的引物对进行扩增，PCR产物理论长度为105bp，包含多态位点序列AGCGCT，突变型AGCGCT为Eco47III识别位点，野生型AGCGCA不能被Eco47III识别切割。
1.4 PCR扩增反应
调配PCR扩增体系：2×Power Taq PCR MasterMix 12.5μl；浓度为10μmol/l的上游引物和浓度为10μmol/l的下游引物各1μl；模板1μl，50ng/μl；ddH 2O补足到25μl。
PCR反应程序：95℃保持5min预变性；然后依次95℃保持30s变性，60℃保持30s退火，72℃保持1min延伸，经过35个变性-退火-延伸循环后，最终72℃终延伸10min；在4℃保存得到PCR扩增产物。
1.5SNP的检测
用1％琼脂糖凝胶电泳检测PCR扩增产物，确定是目的条带后，对PCR扩增产物进行测序，测序结果用DNAMAN与VRTN基因的参考序列进行序列比对，结合测序峰图，确定目的片段中的SNP位点分别为VRTN基因g.*1932A>T。
1.6统计结果
待测德州驴第7号染色体第47567290bp位点不同基因型分析统计结果见表1。
表1待测德州驴第7号染色体第47567290bp位点不同基因型分析统计
Figure PCTCN2020085025-appb-000002
实施例二实验用驴VRTN基因SNP与多胸椎数性状的关联分析
2.1统计376头实验用驴的胸椎数及胸椎长度的表型数据，胸椎长度以平均数加减标准差的形式表示。结果见表2。
表2 376头实验用驴的胸椎数、胸椎长度表型结果
Figure PCTCN2020085025-appb-000003
注：胸椎长度用其平均值±标准差表示
2.2待测实验用驴第7号染色体上第47567290bp位点不同基因型与实验用驴胸椎数关联分析统计结果见表3。
表3不同基因型与实验用驴胸椎数、胸椎长度的关联分析
Figure PCTCN2020085025-appb-000004
注：表中胸椎数、胸椎长度用其平均值±标准差表示，肩标为不同小写字母时，表示差异显著(大写表示P<0.01)。
2.3根据表3可知，AA基因型与TT基因型个体的胸椎数、胸椎长度之间呈显著差异，这表明该位点与实验用驴胸椎数、胸椎长度之间有极显著的关联。
2.3在育种工作者实际对德州驴进行选育的过程中，如果发现某头驴携带A等位基因的个体，那么此个体具有优势胸椎长度的优良性状；因此，该项技术可以指导种驴的早期筛选，减少驴饲养的盲目性，降低养殖成本，增加驴场的经济效益；该分子标记辅助育种的技术在以后的应用前景极其广泛。
实施例三检测试剂盒
如实施例二所述，VRTN基因g.*1932A>T与德州驴胸椎数、驴胸椎长度性状密切相关，因此，可以发明一种适用于筛选具有多胸椎数性状的VRTN基因SNP分子标记检测试剂盒。
3.1 VRTN基因分型
利用VRTN-F/R引物扩增包含VRTN基因g.*1932A>T位点的片段(105bp)获得PCR产物，包含VRTN基因g.*1932A>T位点的片段与限制性内切酶Eco47III有如下关系：该位点为TT基因型时，可以被限制性内切酶Eco47III切开得到25bp和80bp的两个片段；若该位点为为AA基因型时，无法被限制性内切酶Eco47III切开，只有一条105bp片段；若该位点为为AT杂合型时，则可以检测到25bp、80bp和105bp三条条带。
制作30μl体系的酶切反应体系，包括如下组份：PCR产物10μl；浓度10U/μl的限制性内切酶Eco47II1μl；10×FastDigest buffer 2μl；ddH2O补足到30μl，检测PCR产物为目的片段后，用相应的限制性内切酶Eco47II将PCR产物在37℃消化一个小时得到酶切产物；酶切产物分别用1.5％的琼脂糖凝胶电泳检测；根据琼脂糖凝胶电泳检测得到的条带区分统计不同的基因型，结果见附表4。
表4 2种实验用驴多胸椎数性状检测的试剂盒成分
Figure PCTCN2020085025-appb-000005
首先取实验用驴的静脉血于-20℃±0.5℃保存，使用血液基因组试剂盒从静脉血中提取血液DNA，利用上述试剂盒中的成分，以提取的血液DNA为模板进行PCR扩增，然后将PCR扩增产物在PCR反应体系和酶反应体系内进行反应；
所述PCR反应体系为25μL反应体系，包括如下组份：2×Power Taq PCR Master Mix 12.5μl；浓度为10μmol/l的上游引物和浓度为10μmol/l的下游引物各1μl；浓度为50ng/μl的模板1μl；ddH 2O补足到25μl；
PCR反应程序包括如下步骤：95℃保持5min预变性；然后依次95℃保持30s变性，60℃保持30s退火，72℃保持1min延伸，经过35个变性-退火-延伸循环后，最终72℃终延伸10min；在4℃保存得到PCR扩增产物。
酶切反应体系为30μl反应体系，包括如下组份：PCR产物10μl；浓度为10U/μl的限制性内切酶Eco47II1μl；10×FastDigest buffer 2μl；ddH 2O补足到30μl，37℃消化酶切反应体系1h得到酶切产物；酶切产物分别用3％和2.5％的琼脂糖凝胶电泳检测。
酶切结果分析：选择扩增包含VRTN基因g.*1932A>T位点的片段的PCR扩增产物在酶切反应体系被Eco47III限制性内切酶识别后，得到的片段大小为25bp和80bp的VRTN基因g.*424A>G位点为AA基因型；得到的片段只有一条105bp片段的VRTN基因g.*424A>G位点为TT基因型；若检测到25bp、80bp和105bp三条条带，则该VRTN基因g.*424A>G位点为AT杂合型；
按实施例一和实施例二中所述方法分析各基因型的胸椎数，携带等位基因A的个体，具有较长胸椎长度的优势性状；以此决定留种与否。
在此，虽然直接测序可以检测出驴基因SNP的基因型，但直接测序的方法成本高，且周期长，不适用于大批量样品的检测，该试剂盒则可以很好的弥补直接测序的缺陷，为种驴的早期培育提供技术及理论指导。
本发明提供了一种通过鉴定德州驴VRTN基因SNPs位点，间接检测德州驴多胸椎数性状的方法。该方法适用于分析驴VRTN基因上的SNP位点与德州驴多胸椎数性状的相关性，进一步可以作为德州驴胸椎数的统计依据，为筛选具有多胸椎数性状德州驴的选育提供技术及理论指导；并提供了一种高效检测德州驴VRTN基因SNP和德州驴胸椎数性状的方法，该方法可用于德州驴多胸椎数性状相关的基因检验试剂盒的研发；综上所述，德州驴VRTN基因g.*1932A>T的位点与驴多胸椎数、胸椎长度性状显著相关，依据这种相关性，研发通过检测VRTN基因SNP基因型，来检测德州驴多胸椎数性状的试剂盒。
本发明所述SNP位点的应用、提供的用于筛选和检测具有多胸椎数性状德州驴的与德州驴多胸椎数性状相关的SNP位点检测试剂盒及其使用方法其目的是加速德州驴的遗传改良进程，不属于疾病的诊断和治疗方法。
以上所述仅为本发明的实施例，并非因此限制本发明的专利范围，凡是利用本发明说明书内容所作的等效结构或等效流程变换，或直接或间接运用在其它相关的技术领域，均同理包括在本发明的专利保护范围内。

Claims (8)

  1. 一种与德州驴多胸椎数性状相关的SNP位点检测试剂盒，其特征在于，包括扩增包含VRTN基因SNP位点g.*1932位DNA序列的引物对、PCR反应体系、酶切反应体系和DNA Marker，所述SNP位点g.*1932A>T位于驴第7号染色体第47567290碱基对并以VRTN基因转录起始位点的第一个碱基A为+1，所述扩增包含VRTN基因SNP位点g.*1932位DNA序列的引物对包括上游引物和下游引物，所述扩增包含VRTN基因SNP位点g.*1932位DNA序列的引物对以驴血液DNA为模板通过Primer 5软件进行设计分析并人工加以修改得到，上游引物倒数第3位碱基为T-G错配，上游引物人为在其3'端附近引入Eco47III识别位点AGCGCT，下游引物完全与模板配对；所述上游引物的核苷酸序列为SEQ ID No.2:ATTCAGATCTCAGAATCACTAGCG；所述下游引物的核苷酸序列为SEQ ID No.3:GGCGGTGGCGGGAATAATAA。
  2. 根据权利要求1所述的一种与德州驴多胸椎数性状相关的SNP位点检测试剂盒，其特征在于，所述扩增包含VRTN基因SNP位点g.*1932位DNA序列的引物对含有核苷酸序列，所述核苷酸序列的第574位多态性为A/T，所述核苷酸的序列如下所示：
    ATGTGTTATCAGTGGGGAAGGAGTTCTCTTGGGCCCTTTCTCCTCCTGTCCAGCTTTCCTACAGTGGCTGTCATCCTCATGGTCTTGGGGAGTTATTCTTGGTTCTGCTGTTTTTTCATCCCCATATCCAATCCATCAGAAGTCCTGTTGACTCTGCCTCCAAAGTATGCCCAGAATCTTCCCCATCTTGCTGCTGCCATCATCATCCCACCTGGCCCACATCTCGGCATCCTACCTGTCTCCCTGCTTCCACTCTTGCCCCATACAATCCATTCTCCAGAGAGCAGCCAGAGAGATCTTTAAGCGAAATCTGATTCTATCACTTTCCTGTTTAAAATGTGTCAGTGGCTTCCCTTCCCTCTTAGAATCCCTTACGCTGCCTTACAAAGGCCTTCCTAACCTGGCCCCTGACTGACCCAGCAGACCCCAGCCCACTGGCTGATTCTGATCGGAACGTGCAACACTTATTTCTGCTGTCAGGGCCTTTGCGTTGTTCCTTTGGTCTAGAATGCTTTTCCCTCTCATCTTTACAGGGCAGGATCCTTACCATTCAGATCTCAGAATCACTATCGCATCACCCTTTAAAATTCTCTACGTGGCGTTTATTCCCATCTGATGTTCTTGTTTGTTTGCTTATTATTCCCGCCACCGCCCCACTGCTGCCCGTCTTCCCCACCTCCCCCCTTATGTTAGAACATAAGCTCCATGAAAGGCAGGACTTTTTCCTGACTTGCTTACACCACTGCATTCTCAGCAGGCAAATTAGTTCCTGGCACATAGTAGGTGCTATAATTTGTCAAAAGAAGGAATGAATGAAATGGGGCCACTAGGATTTTCTTGTTGTTATCCTTTGGGATTTAGTTTTATAAGACCTA。
  3. 根据权利要求1所述的一种与德州驴多胸椎数性状相关的SNP位点检测试剂盒，其特征在于，所述PCR反应体系包括2×Taq PCR MasterMix和ddH2O；酶切反应体系包括FastDigest限制性核酸内切酶Eco47III、10×FastDigest buffer和ddH2O。
  4. 一种与德州驴多胸椎数性状相关的SNP位点检测试剂盒使用方法，其特征在于：包括以下步骤：
    1)选取实验用驴抽取静脉血获得血液样本，从血液样本中提取实验用驴的基因中DNA；
    2)以提取的实验用驴基因中DNA为模板，通过Primer 5软件进行设计分析并人工加以修改得到核苷酸序列分别为SEQ ID No.2:ATTCAGATCTCAGAATCACTAGCG和SEQ ID No.3:GGCGGTGGCGGGAATAATAA的扩增包含VRTN基因SNP位点g.*1932位DNA序列的引物对，利用扩增包含VRTN基因SNP位点g.*1932位DNA序列的引物对在PCR反应体系内进行PCR扩增反应，得到PCR扩增产物；
    3)用酶切反应体系对PCR扩增产物进行消化得到酶切产物，酶切产物分别用3％和2.5％的琼脂糖凝胶电泳检测分析酶切产物片段长度；
    4)根据产物片段长度分析结果判定位于驴第7号染色体上的VRTN基因SNP位点基因型，根据实验用驴第7号染色体上第47567290bp位点不同基因型与德州驴胸椎数关联分析表得出：当判定位于驴第7号染色体上的VRTN基因SNP位点基因型为AA、AT时，对应的实验用驴为具有多胸椎性状的个体。
  5. 根据权利要求4所述的一种与德州驴多胸椎数性状相关的SNP位点检测试剂盒使用方法，其特征在于：所述步骤2)中的PCR扩增反应的条件为：95℃保持5min预变性；然后依次在95℃保持30s、60℃保持30s、72℃保持1min，进行35个循环，再在72℃保持10min，最终4℃保存得到PCR扩增产物。
  6. 根据权利要求4所述的一种与德州驴多胸椎数性状相关的SNP位点检测试剂盒使用方法，其特征在于：所述步骤2)中的PCR反应体系为25μL体系包括如下组份：2×Power Taq PCR Master Mix 12.5μl；浓度为10μmol/l的上游引物和浓度为10μmol/l的下游引物各1μl；浓度为50ng/μl的模板1μl；ddH2O补足到25μl。
  7. 根据权利要求4所述的一种与德州驴多胸椎数性状相关的SNP位点检测试剂盒使用方法，其特征在于：步骤3)中酶切反应体系以30μl计为，包括如下组份：PCR产物10μl；浓度为10U/μl的限制性内切酶Eco47III1μl；10×FastDigest buffer 2μl；ddH2O补足到30μl，37℃消化酶切反应体系1h得到酶切产物，酶切产物分别用3％和2.5％的琼脂糖凝胶电泳检测。
  8. 根据权利要求4所述的一种与德州驴多胸椎数性状相关的SNP位点检测试剂盒使用方法，其特征在于：所述步骤4)中实验用驴第7号染色体上第47567290bp位点不同基因型与德州驴胸椎数关联分析表经以下步骤得到：
    1)统计采集血液样本的实验用驴胸椎数目，并测量从第一个胸椎到最后一个胸椎的长度，统计后得到实验用驴的胸椎数及胸椎长度的表型数据；
    2)利用PCR产物直接测序法对VRTN基因进行SNP鉴定及分型得到实验用驴第7号染色体第47567290bp位点不同基因型分析统计表，根据实验用驴的胸椎数及胸椎长度的表型数据和实验用驴第7号染色体第47567290bp位点不同基因型分析统计表对实验用驴第7号染色体上第47567290bp位点不同基因型与实验用驴胸椎数进行关联分析得到实验用驴第7号染色体上第47567290bp位点不同基 因型与德州驴胸椎数关联分析表。
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