WO2021207993A1 - 一种与德州驴多腰椎数性状相关的snp位点检测试剂盒及其使用方法 - Google Patents

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

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WO2021207993A1
WO2021207993A1 PCT/CN2020/085027 CN2020085027W WO2021207993A1 WO 2021207993 A1 WO2021207993 A1 WO 2021207993A1 CN 2020085027 W CN2020085027 W CN 2020085027W WO 2021207993 A1 WO2021207993 A1 WO 2021207993A1
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pcr
donkey
texas
snp
donkeys
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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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  • the invention belongs to the technical field of molecular marker detection, and specifically refers to a SNP site detection kit related to the trait of multiple lumbar vertebral counts of Texas donkeys and a method of use thereof.
  • Donkeys are herbivores and belong to the order Perissodactyla, Equidae, and Equus. Donkeys resemble horses and are mostly gray-brown, with a large head and long ears, a slightly narrow chest, and thin limbs. The torso is short but longer than the limbs, so the height and length of the donkey are not equal, and they are small rectangles. The neck has thin skin and thick skin, small and firm hooves, strong physique, and strong resistance. Due to the large differences in natural and socio-economic conditions in various regions with a long history, under the effect of long-term natural and artificial selection, several donkeys with different appearances and production performance have been formed.
  • 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.
  • Germ cell nuclear factor (Nuclear receptor subfamily 6group A member 1, NR6A1) belongs to the nuclear receptor superfamily, which is a family of transcription factors activated by a group of ligands through the establishment of a link between signal molecules and transcriptional responses.
  • NR6A1 is located in the QTL area related to changes in the number of vertebrae. It can regulate the physiological processes of cells, especially the growth and differentiation of embryonic stem cells, affect the development of reproduction and nervous system, and is related to traits such as fat deposition, litter size, and spine number.
  • the donkey NR6A1 gene is located on chromosome 10, with a size of 25042bp; currently the gene is mostly studied in pigs, and there is no report on donkeys. The homology of NR6A1 among different species is very high, so it is also highly conserved in function. sex.
  • the present invention provides a SNP site detection kit related to the number traits of Texas donkey lumbar vertebrae and a method of use thereof, which can indirectly detect the number of donkey lumbar vertebrae by identifying the single nucleic acid polymorphism of the Texas donkey NR6A1 gene
  • a very small amount of Texas donkey genomic DNA can be used to efficiently identify the SNP locus of the donkey NR6A1 gene, and analyze its influence on the number of lumbar vertebrae, which can be widely used in practice.
  • Early selection of Texas donkeys can reduce Texas donkey breeding. Blindness, reduce costs, and increase the economic benefits of pasture.
  • the present invention is a SNP site detection kit related to the multiple lumbar vertebral count traits of Texas donkey, including PCR primer pair for amplifying the SNP molecular marker containing NR6A1 gene, PCR reaction system, and enzyme digestion reaction System and DNA Marker, the PCR primer pair includes an upstream primer and a downstream primer; the SNP molecular marker amplified by the PCR primer pair is located at the 18114688 position of the NR6A1 gene on chromosome 10 of Texas donkey and starts with the transcription start position of the NR6A1 gene
  • the first base A of the dot is +1, the polymorphism of the PCR primer to the amplified SNP molecular marker is A/G;
  • the nucleotide sequence of the upstream primer is SEQ ID No. 2: 5' -TGGAAAGGGGGGGTGTGGCCATACC-3'; the nucleotide sequence of the downstream primer is SEQ ID No. 3: 5'-GCTT
  • PCR primer pair amplified SNP molecular markers contain nucleotides, the 349th polymorphism of the nucleotide sequence is A/G, and the nucleotide sequence is as follows:
  • PCR primer pair is designed and analyzed by Primer 5 software and manually modified.
  • the penultimate 3 base of the upstream primer is a CA mismatch and the Bvel recognition site ACCTGC is artificially introduced near its 3'end.
  • the downstream primer is completely Pair with template.
  • the PCR reaction system includes 2 ⁇ Taq PCR MasterMix and ddH 2 O; the enzyme digestion reaction system includes FastDigest restriction endonuclease Bvel, 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-lumbar vertebral count trait of Texas donkey, which includes the following steps:
  • SEQ ID No. 2 5'-TGGAAAGGGGGGGTGTGGCCATACC-3' and SEQ ID No. 2: 5'-TGGAAAGGGGGGGTGTGGCCATACC-3' and SEQ ID No. 3: PCR primer pair of 5'-GCTTAGTAAACCTCCGAAC-3', using PCR primer pair to perform PCR amplification reaction in PCR reaction system to obtain PCR amplification product;
  • the SNP locus on chromosome NR6A1 of experimental donkey 10 was genotyped and SNP marker detection was performed, and the presence of NR6A1 gene g.*424A>G locus alleles was identified in the experimental donkey individual
  • the individual has the trait of multiple lumbar vertebrae; the correlation analysis between different genotypes and the number of lumbar vertebrae of the Texas donkey is performed to determine the multiple lumbar vertebrae traits of the donkey used in the experiment.
  • the SNP marker is a SNP marker related to the trait of multiple lumbar vertebrae in the donkey. The number of lumbar vertebrae of the AG genotype and GG genotype individuals of the SNP marker is superior to that of the AA genotype individuals.
  • 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: DNA template 1.0 ⁇ L, PCR Mixture 12.5 ⁇ L, upstream primer and downstream primer each 1 ⁇ L, and ddH 2 O is added to a total volume of 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 Bvel with a concentration of 10U/ ⁇ l 1 ⁇ l; 10 ⁇ FastDigest buffer 2 ⁇ l, ddH 2 O makes up to 30 ⁇ l ; Digest the digestion reaction system at 37°C for 1 hour to obtain digestion products, which were detected by 3% and 2.5% agarose gel electrophoresis, respectively.
  • the SNP locus detection kit related to the multiple lumbar vertebral count trait of Texas donkey is used in detecting the NR6A1 genotype of Texas donkey; the SNP locus detection reagent related to the multiple lumbar vertebral count trait of Texas donkey is applied The box is used in molecular assisted breeding of Texas donkey.
  • the beneficial effects of the present invention are as follows:
  • the present invention is a SNP site detection kit related to the multiple lumbar vertebral count traits of Texas donkeys and its use method for detecting polymorphic sites of Texas donkey NR6A1 gene.
  • NR6A1 gene a SNP molecular marker related to the number traits of the lumbar vertebrae of Texas donkey was obtained through screening, and it was identified that when the allele G exists at the g.*424A>G locus of the NR6A1 gene, the individual has the trait of multiple lumbar vertebrae numbers.
  • NR6A1 gene g.*424A>G site detection kit is used to screen individuals with multiple lumbar vertebral count traits. The kit can be widely used in practice.
  • Texas donkey NR6A1 genotype Texas will have multiple lumbar vertebral count traits. The individual selection of donkeys can improve the productivity of Texas donkeys, which has potential application value for large-scale molecular breeding of Texas donkeys.
  • Figure 1 is a schematic diagram of electrophoresis detection results of PCR products of NR6A1 gene in Example 1 of the present invention
  • Figure 2 is the sequencing results of three genotypes in PCR amplified products in Example 1 of the present invention
  • Fig. 3 is an agarose gel electrophoresis typing diagram of the SNP locus (g.*424A>G) of the donkey NR6A1 gene used in the experiment in Example 3 of the present invention.
  • a1 and a1 are AA genotypes
  • a3 and a4 are AG genotypes
  • a5 and a6 are GG genotypes.
  • Select 314 donkeys for the experiment use disposable vacuum blood collection tubes containing EDTA to collect blood samples from the jugular vein of the experimental population.
  • the blood volume collected is 7ml/head ⁇ 9ml/head, and the blood samples are kept at -20°C
  • the donkey After being transported to the laboratory for a long distance under the condition of ⁇ 0.5°C, the donkey will be frozen and stored at -20°C ⁇ 0.5°C.
  • the experimental donkey will be slaughtered and the number of lumbar vertebrae will be counted after the slaughter. The length from the lumbar spine to the last lumbar spine.
  • 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.
  • the nucleotide sequence of the designed upstream primer is SEQ ID No. 2: 5'-TGGAAAGGGGGGGTGTGGCCATACC-3', with the third base from the 3'end The base is a CA mismatch.
  • the primer artificially introduces the Bvel recognition site ACCTGC near its 3'end, and the downstream primer is completely matched to the template.
  • the nucleotide sequence of the PCR primer pair is as follows:
  • Upstream primer SEQ ID No. 2 5'-TGGAAAGGGGGGGTGTGGCCATACC-3';
  • Downstream primer SEQ ID No. 3 5'-ACCTCCGAACACCACTCT-3';
  • the PCR primer pair is used for amplification.
  • the PCR product has a theoretical length of 160 bp and contains the polymorphic site sequence ACCTGC.
  • the mutant ACCTGC is the Bvel recognition site, and the wild-type ACCTAC cannot be recognized and cut by Bvel.
  • 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 with a concentration of 50ng/ ⁇ l 1 ⁇ l; ddH 2 O supplemented 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 products were detected by 1% agarose gel electrophoresis, and after confirming that they were the target bands, the PCR amplified products were sequenced.
  • the sequencing results were compared with the reference sequence of DNAMAN and NR6A1 gene, combined with the sequencing peak diagram, to confirm The SNP sites in the target fragment are NR6A1 gene g.*424A>G.
  • the number of lumbar vertebrae and the length of lumbar vertebrae in the table are expressed by their average value ⁇ standard deviation. When the shoulder is marked with different lowercase letters, it means that the difference is significant (uppercase means P ⁇ 0.01).
  • the NR6A1 gene g.*424A>G is closely related to the multiple lumbar vertebral count traits of Texas donkeys. Therefore, a detection kit suitable for screening SNP molecular markers for individuals with multiple lumbar vertebral traits can be invented.
  • the g.*424A>T site of the NR6A1 gene is related to restriction enzymes as follows: When the locus is GG genotype, it can be cut by restriction enzymes to obtain two fragments of 32bp and 128bp; if it is AA genotype, it cannot be cut, and there is only one 160bp fragment; if it is AG heterozygous In the case of type, three bands of 32bp, 128bp and 160bp can be detected;
  • the enzyme digestion reaction system of 30 ⁇ l system was made, including the following components: PCR product 10 ⁇ l; concentration of 10U/ ⁇ l Bvel restriction enzyme 1 ⁇ l; 10 ⁇ FastDigest buffer 2 ⁇ l; ddH 2 O supplemented to 30 ⁇ l;
  • the PCR product is detected as the target fragment through the restriction enzyme digestion system, the PCR product is digested with the corresponding restriction enzyme at 37°C for one hour to obtain the digestion product; the digestion products are respectively detected by 1.5% agarose gel electrophoresis. ; Different genotypes are distinguished and counted according to the bands obtained by electrophoresis, and the results are shown in Table 4.
  • PCR amplifies the product, and then reacts the PCR amplified product in the PCR reaction system and the enzyme reaction system; the PCR reaction system is a 25 ⁇ L reaction system, including the following components: 2 ⁇ Power Taq PCR Master Mix 12.5 ⁇ l; the concentration is 10 ⁇ mol/l upstream primer and 10 ⁇ mol/l downstream primer each 1 ⁇ l; template with a concentration of 50ng/ ⁇ l 1 ⁇ l; ddH 2 O to make 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 Bvel with a concentration of 10U/ ⁇ l 1 ⁇ l; 10 ⁇ FastDigest buffer 2 ⁇ l; ddH 2 O fills up to 30 ⁇ l, digestion at 37°C
  • the digested product was obtained in the reaction system for 1 hour; the digested product was detected by 3% and 2.5% agarose gel electrophoresis, respectively.
  • NR6A1 gene g.*424A>G locus is GG genotype; the obtained fragment has only one 160bp fragment of NR6A1 gene g.*424A>G locus is AA genotype; if three bands of 32bp, 128bp and 160bp are detected, Then the g.*424A>G locus of the NR6A1 gene is AG heterozygous.
  • the number of lumbar vertebrae of each genotype was analyzed according to the methods described in Example 1 and Example 2, and the experimental donkey individuals with the G allele had the advantage of more lumbar vertebrae number, so as to determine whether to reserve the seed.
  • direct sequencing can detect the genotype of the donkey gene SNP.
  • the direct sequencing method is costly and has a long cycle, and is not suitable for the detection of large quantities of samples.
  • the kit can make up for the shortcomings of direct sequencing and provide technical and theoretical guidance for the early breeding of male donkeys.
  • the present invention provides a method for indirectly detecting the number of lumbar vertebrae of Texas donkey NR6A1 gene by identifying the SNP site of Texas donkey.
  • a statistical basis for the multiple lumbar vertebral count traits of Texas donkeys it provides technical and theoretical guidance for the selection and breeding of Texas donkeys with multiple lumbar vertebral count traits; and provides an efficient method for detecting the SNP of Texas donkey NR6A1 gene and the multiple lumbar vertebral count traits of Texas donkeys
  • This method can be used for the development of a genetic test kit related to the multiple lumbar vertebral count traits of Texas donkeys; in summary, the g.*424A>G locus of the Dezhou donkey NR6A1 gene is significantly related to the multiple lumbar vertebral count traits of donkeys. Correlation, research and development of a kit to detect the multiple lumbar vertebral number traits of Texas donkeys by detecting the SNP genotype of the NR6A1 gene.
  • the application of the SNP site of the present invention the provided SNP site detection kit for screening and/detecting Texas donkeys with multiple lumbar vertebral count traits and the SNP site detection kits related to Texas donkeys with multiple lumbar vertebral count traits and the method of use thereof are aimed at accelerating Texas donkeys
  • the process of genetic improvement does not belong to the diagnosis and/or treatment of the disease.

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Abstract

本发明公开了一种与德州驴多腰椎数性状相关的SNP位点检测试剂盒及其使用方法，包括扩增NR6A1基因的SNP分子标记的PCR引物对、PCR反应体系、酶切反应体系和DNA Marker，所述PCR引物对包括上游引物和下游引物；所述PCR引物对扩增的SNP分子标记位于德州驴第10号染色体NR6A1基因的第18114688处。

Description

一种与德州驴多腰椎数性状相关的SNP位点检测试剂盒及其使用方法 技术领域
本发明属于分子标记检测技术领域，具体是指一种与德州驴多腰椎数性状相关的SNP位点检测试剂盒及其使用方法。
背景技术
驴为食草动物，隶属奇蹄目、马科、马属。驴的形象似马且多为灰褐色，头大且耳朵长，胸部稍窄，四肢瘦弱，躯干虽短但长于四肢，因而体高和身长不相等呈小长方形。颈项皮薄肉厚，蹄小坚实，体质健壮，抵抗能力很强。由于历史悠久各地的自然条件和社会经济条件差异较大，在长期的自然选择和人工选择的作用下形成了若干外貌和生产性能的不同品种的驴。我国驴品种资源丰富，但我国驴遗产改良计划并不系统，各品种均未进行系统化、专门化的培育，产肉、产皮、产奶性能均不突出，因此为保障我国驴产业的健康发展应培育产皮、产肉性能突出的品种，急需运用现代分子生物学技术和繁殖技术加快驴遗传育种的进程。
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。
生殖细胞核因子(Nuclear receptor subfamily 6group A member 1，NR6A1)属于核受体超家族，该家族是一组配体激活的，通过在信号分子和转录应答间建立联系的转录因子家族。NR6A1位于与椎骨数量变化相关的QTL区域，能调节细胞的生理过程，尤其是胚胎干细胞的生长和分化，影响生殖和神经系统的发育，与脂肪沉积、产仔数和脊椎数等性状有关。驴的NR6A1基因位于10号染色体上，大小25042bp；目前该基因在猪上的研究居多，尚没有对驴研究报道，在不同物种间NR6A1的同源性很高，因而在功能上也具高度保守性。
发明内容
为解决上述现有难题，本发明提供了一种与德州驴腰椎数性状相关的SNP位点检测试剂盒及其使用方法，通过鉴定德州驴NR6A1基因单核酸多态性间接检测驴腰椎数目的方式，只需要极其少量的德州驴基因组DNA就可以高效鉴定出驴NR6A1基因的SNP位点，分析其对腰椎数的影响，可在实践中广泛应用，通过对德州驴进行早期筛选，减少德州驴饲养的盲目性，降低成本，增加牧场经济效益。
本发明采用的技术方案如下：本发明一种与德州驴多腰椎数性状相关的SNP位点检测试剂盒，包括扩增包含NR6A1基因的SNP分子标记的PCR引物对、PCR反应体系、酶切反应体系和DNA Marker，所述PCR引物对包括上游引物和下游引物；所述PCR引物对扩增的SNP分子标记位于德州驴第10号染色体的NR6A1基因的第18114688处并以NR6A1基因转录起始位点的第一个碱基A为+1，所述PCR引物对扩增的SNP分子标记的多态性为A/G；所述上游引物的核苷酸序列为SEQ ID No.2：5'-TGGAAAGGGGGGGTGTGGCCATACC-3'；所述下游引物的核苷酸序列为SEQ ID No.3：5'-GCTTAGTAAACCTCCGAAC-3'。
进一步地，所述PCR引物对扩增的SNP分子标记含有核苷酸，所述核苷酸序列的第349位多态性为A/G，所述核苷酸的序列如下所示：
Figure PCTCN2020085027-appb-000001
进一步地，所述PCR引物对通过Primer 5软件进行设计分析并人工加以修改，上游引物倒数第3位碱基为C-A错配并人为在其3'端附近引入Bvel识别位点ACCTGC，下游引物完全与模板配对。
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Bvel、10×FastDigest buffer和ddH 2O。
本发明还提供了一种与德州驴多腰椎数性状相关的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，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.2：5'-TGGAAAGGGGGGGTGTGGCCATACC-3'和SEQ ID No.3：5'-GCTTAGTAAACCTCCGAAC-3'的PCR引物对，利用PCR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，分析产物片段长度。
进一步地，将实验用驴第10号染色体NR6A1上的SNP位点进行基因分型和SNP标记的检测，并鉴定出实验用驴个体中存在NR6A1基因g.*424A>G位点存在等位基因G时，该个体具有多腰椎数性状；将不同基因型与德州驴腰椎数进行关联分析，确定所述实验用驴多腰椎数性状，所述SNP标记为驴多腰椎数性状相关的SNP标记，所述SNP标记的AG基因型和GG基因型个体的腰椎数相较于AA基因型个体有优势。
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：DNA模板1.0μL，PCR Mixture12.5μL，上游引物、下游引物各1μL，加入ddH 2O至总体积为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Bvel 1μl；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。
优选地，所述一种与德州驴多腰椎数性状相关的SNP位点检测试剂盒应用于检测德州驴NR6A1基因型中；所述一种与德州驴多腰椎数性状相关的SNP位点检测试剂盒应用于德州驴分子辅助育种中。
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腰椎数性状相关的SNP位点检测试剂盒及其使用方法对德州驴NR6A1基因进行多态性位点的检测，在德州驴NR6A1基因中，通过筛选获得一种与德州驴腰椎数性状相关的SNP分子标记，并鉴定出某个体NR6A1基因g.*424A>G位点存在等位基因G时，该个体具有多腰椎数性状，并利用NR6A1基因g.*424A>G位点检测试剂盒筛选具有多腰椎数性状的个体，该试剂盒可在实践中广泛应用，通过对德州驴进行早期筛选，可以减少德州驴饲养的盲目性，降低成本，增加牧场经济效益，为了解影响驴体长性状的遗传基础提供参考，为德州驴选育提供辅助方法，通过对德州驴NR6A1基因型的选择，将具有多腰椎数性状的德州驴个体选留下来，从而提高德州驴的生产力，对德州驴大规模分子育种具有潜在的应用价值。
附图说明
图1为本发明实施例一中NR6A1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PCR扩增产物中三种基因型测序结果
图3为本发明实施例三中实验用驴NR6A1基因SNP位点(g.*424A>G)琼脂糖凝胶电泳分型图。
其中，a1、a1为AA基因型，a3、a4为AG基因型，a5、a6为GG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 驴NR6A1基因SNP位点的鉴定
采用PCR产物直接测序法对SNP位点进行鉴定及分型
1.1实验材料准备
选取314头实验用驴，使用含EDTA抗凝的一次性真空采血管对实验群体进行颈静脉采血采集血液样本，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后在-20℃±0.5℃的条件下冷冻保存，对实验用驴进行屠宰并在屠宰之后统计腰椎的数目，并测量从第一个腰椎到最后一个腰椎的长度。
1.2提取实验用驴全血基因组DNA
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析分型PCR引物对，设计的上游引物的核苷酸序列为SEQ ID No.2：5'-TGGAAAGGGGGGGTGTGGCCATACC-3'，其3'端倒数第三个碱基为C-A错配，该引物人为地在其3'端附近引入Bvel识别位点ACCTGC，下游引物完全与模板配对。设计PCR引物对的核苷酸序列如下：
上游引物SEQ ID No.2：5'-TGGAAAGGGGGGGTGTGGCCATACC-3'；
下游引物SEQ ID No.3：5'-ACCTCCGAACACCACTCT-3'；
用该PCR引物对进行扩增，PCR产物理论长度为160bp，包含多态位点序列ACCTGC，突变型ACCTGC为Bvel识别位点，野生型ACCTAC不能被Bvel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；浓度为50ng/μl的模板1μl；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。
1.5 SNP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NR6A1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NR6A1基因g.*424A>G。
1.6统计结果
实验用驴第10号染色体上第18114688bp位点不同基因型分析统计结果见表1。
表1待测德州驴第10号染色体上第18114688bp位点不同基因型分析统计
Figure PCTCN2020085027-appb-000002
实施例二 实验用驴NR6A1基因SNP与腰椎数性状的关联分析
2.1统计314头实验用驴的腰椎数及腰椎长度的表型数据，腰椎长度以平均数加减标准差的形式表示。结果见表2。
表2 314头实验用驴的胸椎表型结果
Figure PCTCN2020085027-appb-000003
2.2实验用驴第10号染色体上第18114688bp位点不同基因型与实验用驴腰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腰椎数关联分析
Figure PCTCN2020085027-appb-000004
Figure PCTCN2020085027-appb-000005
注：表中腰椎数、腰椎长度用其平均值±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GA、GG基因型的实验用驴个体的腰椎数、腰椎长度之间呈显著差异，这表明该位点与实验用驴的腰椎数、腰椎长度之间有极显著的关联。
2.4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G等位基因的个体，那么此个体具有多腰椎数的优良性状；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，这种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
如实施例一所述，NR6A1基因g.*424A>G与德州驴多腰椎数性状密切相关，因此，可以发明一种适用于筛选具有多腰椎性状个体SNP分子标记的检测试剂盒。
3.1 NR6A1基因分型
利用NR6A1-F/R引物扩增包含NR6A1基因g.*424A>T位点的片段(160bp)获得PCR产物，包含NR6A1基因g.*424A>T位点与限制性内切酶有如下关系：该位点为GG基因型时，可以被限制性内切酶切开得到32bp和128bp的两个片段；若为AA基因型时，则无法被切开，只有一条160bp片段；若为AG杂合型时，则可以检测到32bp、128bp和160bp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Bvel限制性内切酶1μl；10×FastDigest buffer 2μl；ddH 2O补足到30μl；
通过酶切反应体系检测PCR产物为目的片段后，用相应的限制性内切酶将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。；根据电泳得到的条带区分统计不同的基因型，结果见附表4。
表4 2种实验用驴腰椎长度性状检测的试剂盒成分
Figure PCTCN2020085027-appb-000006
Figure PCTCN2020085027-appb-000007
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扩增得到PCR扩增产物，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Bvel 1μl；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NR6A1基因g.*424A>G位点的片段的PCR扩增产物在酶切反应体系被Bvel限制性内切酶识别后，得到的片段大小为32bp和128bp的NR6A1基因g.*424A>G位点为GG基因型；得到的片段只有一条160bp片段的NR6A1基因g.*424A>G位点为AA基因型；若检测到32bp、128bp和160bp三条条带，则该NR6A1基因g.*424A>G位点为AG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腰椎数，具有G等位基因的的实验用驴个体具有多腰椎数的优势性状，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，为种公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NR6A1基因SNP位点间接检测德州驴腰椎数目的方法，该方法适用于分析驴NR6A1基因上的SNP位点与德州驴多腰椎数性状的相关性，进一步可以作为德州驴多腰椎数性状的统计依据，为筛选具有多腰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NR6A1基因SNP和德州驴多腰椎数性状的方法，该方法可用于德州驴多腰椎数性状相关的基因检验试剂盒的研发；综上所述，德州驴NR6A1基因g.*424A>G的位点与驴多腰椎数性状显著相关，依据这种相关性，研发通过检测NR6A1基因SNP基因型，来检测德州驴多腰椎数性状的试剂盒。
本发明所述SNP位点的应用、提供的用于筛选和/检测具有多腰椎数性状德州驴的与德州驴多腰椎数 性状相关的SNP位点检测试剂盒及其使用方法其目的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 (9)

  1. 一种与德州驴多腰椎数性状相关的SNP位点检测试剂盒，其特征在于，包括扩增包含NR6A1基因的SNP分子标记的PCR引物对、PCR反应体系、酶切反应体系和DNA Marker，所述PCR引物对包括上游引物和下游引物；所述PCR引物对扩增的SNP分子标记位于德州驴第10号染色体的NR6A1基因的第18114688处并以NR6A1基因转录起始位点的第一个碱基A为+1，所述PCR引物对扩增的SNP分子标记的多态性为A/G；所述上游引物的核苷酸序列为SEQ ID No.2：5'-TGGAAAGGGGGGGTGTGGCCATACC-3'；所述下游引物的核苷酸序列为SEQ ID No.3：5'-GCTTAGTAAACCTCCGAAC-3'。
  2. 根据权利要求1所述的一种与德州驴多腰椎数性状相关的SNP位点检测试剂盒，其特征在于，所述PCR引物对扩增的SNP分子标记含有核苷酸，所述核苷酸序列的第349位多态性为A/G，所述核苷酸的序列如下所示：
    GAGAGACCAAGATGGAGGCTGTGGAGCAGTGTTTCCAGTTGCCTCCATAGCAAGAAGAGTTTTTATTTGTTCGTCTGTTTTTTTAACCTCATTTTTCTATATATTTATTTCACGACAGAGTTGAATGTATGGCCTTCAACATGATGCACATGCTTTTGTGTGAATGCAGCCGATGCATTTCCTTGCAGTTTACAGAATGTGAAGATGTTTAATGTTACAGTATTGTCATTGTTTAGAGATAGTTCTTTTGTATTTTGAGGGAGAGGGTGGGATGGGGCTGAGATGACTATTTCCATAATGTTGACAAAGACGACTACCTCAGTGGAAAGGGGGGGTGTGGCCATCCCTACTTTTTCCACGTGTTCTCAGCAGACTCATCCATTTGTCTGTCAGAGAGCAAACTGCCTTTTTCTAGCCACAGAATTGCCAGGTAAAAGAGCACACCATAAAAGGGCAGAGTGGTGTTCGGAGGTTTACTAAGCAAAGTGGGTGAGACATTGGGAACAGGACAGGAGATGAAGATTTAGCATCTCTTTTCTTTGGAAAGCAGGAAAGTTGAGACTGGGGCACAGGCACAGTGACCAAATCTAAGTTGGGTGTCCTCTTTCCAAACACGTCCCCAAGGAGAAGGGTTTTTGTTCTCATGCCATGCTCTGCCCCCCCCCCCCCAACCCCGATCTGAACCACCACTATCAGTTTCTGCCACCATCAACTTTTCATCCAGGCAAAATCCTGGCAGTAAGCTTGAGACTTGCCTACCAGCTCCCTTGGAGCTCACAGCTACCTAGTTTCCTTACCTGGATCTACCAAGGCCTACCTTCCTCAGACTCCTTCCCTCTCCCTTATAAATTTGACTTCTGTTGACCCTTTGACAAAACAACTGAACAAAATGTCACGCCCTGAATCCTGGTGCTCACTACTGATTTATACTCCTTTCTCCTTGCTTTCTTCAAAGGGTAATGGGACAGAGACCAGACTGCTGAAAGAATAAATACTCTTCCTTTGGAGTTCCCTGTTCCTCCCATCACCCCATCCCATCCTACCCCTAGGACTACATCCAAAACGTGCAGTCTCTTTTTCAAACAGTGGGAAGAACATTCACCTGTCTGCTGCCCAGAATTTTTGTCCCTACATTGTGCTTCACATTCTCCTTATCTCTCCA。
  3. 根据权利要求1所述的一种与德州驴多腰椎数性状相关的SNP位点检测试剂盒，其特征在于，所述PCR引物对通过Primer 5软件进行设计分析并人工加以修改，上游引物倒数第3位碱基为C-A错配并人为在其3'端附近引入Bvel识别位点ACCTGC，下游引物完全与模板配对。
  4. 根据权利要求4所述的一种与德州驴多腰椎数性状相关的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ddH 2O；酶切反应体系包括FastDigest限制性核酸内切酶Bvel、10×FastDigest buffer和ddH 2O。
  5. 一种与德州驴多腰椎数性状相关的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，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.2：5'-TGGAAAGGGGGGGTGTGGCCATACC-3'和SEQ ID No.3：5'-GCTTAGTAAACCTCCGAAC-3'的PCR引物对，利用PCR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，分析产物片段长度。
  6. 根据权利要求6所述的一种与德州驴多腰椎数性状相关的SNP位点检测试剂盒使用方法，其特征在于：将实验用驴第10号染色体NR6A1上的SNP位点进行基因分型和SNP标记的检测，并鉴定出实验用驴个体中存在NR6A1基因g.*424A>G位点存在等位基因G时，该个体具有多腰椎数性状；将不同基因型与德州驴腰椎数进行关联分析，确定所述实验用驴多腰椎数性状，所述SNP标记为驴多腰椎数性状相关的SNP标记，所述SNP标记的AG基因型和GG基因型个体的腰椎数相较于AA基因型个体有优势。
  7. 根据权利要求6所述的一种与德州驴多腰椎数性状相关的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。
  8. 根据权利要求6所述的一种与德州驴多腰椎数性状相关的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：DNA模板1.0μL，PCR Mixture 12.5μL，上游引物、下游引物各1μL，加入ddH 2O至总体积为25μL。
  9. 根据权利要求6所述的一种与德州驴多腰椎数性状相关的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，包括如下组份：PCR产物10μl，浓度为10U/μl的限制性内切酶Bvel 1μl；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。
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114134234A (zh) * 2021-10-26 2022-03-04 安徽农业大学 一种基于ovr基因鉴定禽类产蛋性状的分子标记及其鉴定方法和应用

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011118549A1 (ja) * 2010-03-23 2011-09-29 独立行政法人農業生物資源研究所 ブタの椎骨数を支配するVertnin遺伝子、およびその利用
CN109234403A (zh) * 2018-10-09 2019-01-18 石河子大学 一种鉴定绵羊腰椎数相关性状的方法及专用引物
CN110408708A (zh) * 2019-08-06 2019-11-05 山东省农业科学院畜牧兽医研究所 一种多标记辅助选择猪肌内脂肪性状的方法

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011118549A1 (ja) * 2010-03-23 2011-09-29 独立行政法人農業生物資源研究所 ブタの椎骨数を支配するVertnin遺伝子、およびその利用
CN109234403A (zh) * 2018-10-09 2019-01-18 石河子大学 一种鉴定绵羊腰椎数相关性状的方法及专用引物
CN110408708A (zh) * 2019-08-06 2019-11-05 山东省农业科学院畜牧兽医研究所 一种多标记辅助选择猪肌内脂肪性状的方法

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
DATABASE Protein 2 December 2015 (2015-12-02), "PREDICTED: nuclear receptor subfamily 6 group A member 1 isoform X2 [Equus asinus]", XP055859918, retrieved from NCBI Database accession no. XP_014696123 *
HOU HAOBIN, LI HAIJING, ZHANG LI: "Current State and Development Trends of Genetic Breeding in Donkeys", GRASS-FEEDING LIVESTOCK, vol. 190, no. 3, 25 May 2018 (2018-05-25), pages 1 - 8, XP055859908, ISSN: 1003-6377, DOI: 10.16863/j.cnki.1003-6377.2018.03.001 *
YAN GUORONG, QIAO RUIMIN, ZHANG FENG, XIN WENSHUI, XIAO SHIJUN, HUANG TAO, ZHANG ZHIYAN, HUANG LUSHENG: "Imputation-Based Whole-Genome Sequence Association Study Rediscovered the Missing QTL for Lumbar Number in Sutai Pigs", SCIENTIFIC REPORTS, vol. 7, no. 1, 1 December 2017 (2017-12-01), XP055859901, DOI: 10.1038/s41598-017-00729-0 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114134234A (zh) * 2021-10-26 2022-03-04 安徽农业大学 一种基于ovr基因鉴定禽类产蛋性状的分子标记及其鉴定方法和应用

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