WO2019153294A1 - 牦牛全基因组snp位点的应用及检测用引物组和试剂盒 - Google Patents

牦牛全基因组snp位点的应用及检测用引物组和试剂盒 Download PDF

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WO2019153294A1
WO2019153294A1 PCT/CN2018/076240 CN2018076240W WO2019153294A1 WO 2019153294 A1 WO2019153294 A1 WO 2019153294A1 CN 2018076240 W CN2018076240 W CN 2018076240W WO 2019153294 A1 WO2019153294 A1 WO 2019153294A1
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seq
nucleotide sequence
yak
snp
primer set
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PCT/CN2018/076240
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姬秋梅
钟金城
信金伟
柴志欣
庞剑会
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西藏自治区农牧科学院畜牧兽医研究所
西南民族大学
成都百因生物科技有限公司
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Priority to US16/321,022 priority Critical patent/US10968488B2/en
Publication of WO2019153294A1 publication Critical patent/WO2019153294A1/zh

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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/16—Primer sets for multiplex assays

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  • the invention belongs to the field of animal husbandry technology, and particularly relates to the application and detection primer set and kit for the whole genome SNP site of yak.
  • the yak is a unique cattle species that is distributed around the Qinghai-Tibet Plateau and its adjacent mountains and sub-alpine regions. It can make full use of the forage resources of alpine grassland and has strong adaptability to the ecological environment conditions of alpine grassland. Living in a harsh environment with thin air, short grass growing period, cold, and long hay period, it will be used for the production and daily necessities of milk, meat, hair, service, fuel, etc. for the local herders. It is a local animal husbandry economy. Indispensable, it can be called "all-round" livestock. Yak is an extremely valuable gene pool in genetic resources. However, there are many types and varieties of yak in China, and the yak type is different for each yak breed. The incomplete information of the genomic variation of Chinese local yak breeds is one of the important reasons for the breeding and related research limitations of yak breeds in China.
  • Single nucleotide polymorphism mainly refers to DNA sequence polymorphism caused by variation of a single nucleotide at the genomic level. It is the most common of the biogenetic variations. SNPs are recognized as third-generation genetic markers, and many phenotypic differences, susceptibility to disease, etc. may be related to SNPs. From the experimental operation, it is easier to find phenotypic-related gene mutations through SNPs; some SNPs do not directly lead to related traits, but because they are adjacent to certain related genes, they can become important markers. There have been no reports of SNP marker loci associated with calf body weight.
  • One of the objects of the present invention is to provide the application of 16 SNPs of the whole genome of yak in yak phenotypic traits or molecular breeding analysis to solve the phenotypic traits or molecules of yak that have not been used in the prior art.
  • the problem of breeding analysis is to provide the application of 16 SNPs of the whole genome of yak in yak phenotypic traits or molecular breeding analysis to solve the phenotypic traits or molecules of yak that have not been used in the prior art.
  • a second object of the invention is a primer set for detecting the 16 SNP sites.
  • a third object of the present invention is to provide a kit comprising the primer set.
  • a fourth object of the present invention is to provide a method for yak phenotypic trait or molecular breeding analysis.
  • the 16 SNP loci of the yak whole genome of the present invention are used in the analysis of yak phenotypic traits or molecular breeding, and the 16 SNP loci are as follows:
  • AX-174702570 the nucleotide sequence is shown in SEQ ID No. 1;
  • AX-174407967 the nucleotide sequence is shown in SEQ ID No. 3;
  • AX-174402854 the nucleotide sequence is shown in SEQ ID No. 4.
  • AX-174929694 the nucleotide sequence is shown in SEQ ID No. 5;
  • AX-174547362 the nucleotide sequence is shown in SEQ ID No. 6;
  • AX-174734142 the nucleotide sequence is shown in SEQ ID No. 7;
  • AX-174706158 the nucleotide sequence is shown in SEQ ID No. 8;
  • AX-174783962 the nucleotide sequence is shown in SEQ ID No. 9;
  • AX-174627015 the nucleotide sequence is shown in SEQ ID No. 10;
  • AX-174928167 the nucleotide sequence is shown in SEQ ID No. 11;
  • AX-174555047 the nucleotide sequence is shown in SEQ ID No. 12;
  • AX-174845027 the nucleotide sequence is shown in SEQ ID No. 13;
  • AX-174891371 the nucleotide sequence is shown in SEQ ID No. 14;
  • AX-174570649 the nucleotide sequence is shown in SEQ ID No. 15;
  • AX-174620133 the nucleotide sequence is shown in SEQ ID No. 16.
  • a primer set of 16 SNP sites according to the present invention comprising an upper primer and a lower primer, the nucleotide sequence of which is shown in Seq ID. No. 17-48.
  • a kit comprising a primer set as described above.
  • the method for analyzing phenotypic traits or molecular breeding of the yak according to the present invention comprises the following steps:
  • Step 1 synthesizing the probe sequences corresponding to the 16 SNP sites as described above;
  • Step 2 Extract the DNA of the yak, perform PCR amplification using the primer set as described above, and judge whether the yak meets the breeding demand of high body weight from the amplified band.
  • the present invention has the following beneficial effects:
  • the 16 SNP loci of the present invention are derived from the unique genetic variation information of 32 Chinese local yak breeds, and can be applied to trait correlation analysis and molecular assisted breeding, etc., and can provide support for the upstream and downstream processes of breeding. It has many advantages such as high measurement accuracy, easy standardization and automatic detection.
  • the idea of the present invention is to first perform genome-wide resequencing data for 96 yak individuals from 32 different yak breeds nationwide. The 32 yak breeds cover all yak species in our country. High-reliability sites were selected from 96 yak SNP mutations to make chips. Then, the sample was expanded, and 268 yak individuals were used to select 16 SNP sites related to meat production using the prepared chips.
  • This example provides a method for selecting the highest site of SNP mutation credibility from the resequencing data of 96 yak.
  • the Illumina X Ten sequencing platform was used to perform genome-wide resequencing data (10X coverage per individual) from 32 different yak populations in China (three individuals in each group) (2XG coverage per individual), resulting in 2592G Sequencing data. On this basis, a quality control is first performed to filter out data with lower sequencing quality. Next, we use the BWA comparison software to compare the total reads to the reference genome, generate the sam format comparison result file, and then use samtools to convert the sam format file into bam format and sort it. Finally, we use the identification. The most common GATK of SNPs identified SNP mutations in 96 calves. Finally, we selected some high-reliability sites from 96 yak SNP mutations to make chips.
  • the detection of DNA samples mainly includes three methods: (1) Agarose gel electrophoresis analysis of the degree of DNA degradation and whether there is contamination of RNA and protein. (2) The purity of DNA (OD260/280 ratio) was measured by Nanodrop method. (3) Qubit method accurately quantifies DNA concentration. According to the above detection results, a DNA sample having an OD value of 1.8 to 2.0 and a content of 1.5 ⁇ g or more was used for the library construction.
  • the above agarose gel electrophoresis, Nanodrop method, and Qubit method are all prior art.
  • the original analysis sequence (Sequenced Reads) reference genome (BosGru_v2.0) for information analysis process which generally includes the following two parts:
  • Sequencing data quality assessment mainly statistics on data volume, base quality, comparison rate, coverage rate, capture rate, homogeneity, etc., to assess whether the sequencing of the database has reached the standard, and the subsequent analysis is carried out in accordance with the standard.
  • Mutation detection The high-quality sequence is aligned to the yak reference genome, the variation information in the sample is detected, and the detected variation is counted and annotated.
  • the SNP information selected by re-sequencing was provided to Affymetrix, and 630209 SNP sites were finally selected for chip design and fabrication through the site screening standard of Affymetrix platform.
  • This example provides a screening method for 268 SNP sites for meat-related traits in yak.
  • the samples were rigorously screened for individual yolk males aged 4-9 years in each group, and each group was screened for healthy individuals with low body weight and high body weight as samples for chip screening.
  • the individual's body weight data were recorded in detail, and GWAS analysis was performed based on the obtained chip screening data, and finally 16 sites most relevant to the meat production of the yak were found.
  • Sample gDNA was quantified using NanoDrop ND-2000 (Thermo Scientific) and detected by gel electrophoresis for DNA integrity. After the DNA quality test is passed, the sample is amplified, fragmented, precipitated, resuspended, chip hybridized, and dyed with reference to the standard process of the chip. After resuspending the gDNA, hybrid MIX was added and then subjected to a quality test. After the quality test was passed, chip hybridization, dyeing and scanning were performed using GeneTitan MC Instrument.
  • This example provides a primer set for detecting 16 SNP sites screened in Example 1.
  • the correspondence between the nucleotide sequence of the primer set and the SNP site is shown in the following table:
  • This embodiment provides a method for applying 16 SNP loci in yak phenotypic traits and molecular assisted breeding, specifically:
  • the probe sequences corresponding to the 16 loci were synthesized, the yak ear samples were collected, and the DNA was extracted. After the quality control, the primer set of the present invention was used for PCR amplification, and the amplified strips were judged to determine whether the yak meets the high-batch breeding requirement.
  • the synthesis of probe sequences, extraction of yak DNA, and PCR amplification are all prior art. If the band mutation site amplified by the primer is marked with "+”, it is proved that the individual is a high-weight individual, and if the band mutation site amplified by the primer is marked with "-”, the individual is proved For individuals with light weight. According to this, the molecular detection of these 16 loci can be carried out on bulls and cows.
  • cows and bulls carry high-weight analytical marker loci, they can be preferentially bred and their offspring can be selected for several generations. Breeding can greatly increase the weight of the calf, increase the ability to produce meat, and bring tangible economic income to the people in the desert area.

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Abstract

提供牦牛全基因组的16个SNP位点在牦牛表型性状或分子育种分析中的应用，所述16个SNP位点的核苷酸序列如SEQ ID No.1-16所示。

Description

牦牛全基因组SNP位点的应用及检测用引物组和试剂盒 技术领域
本发明属于畜牧技术领域，具体地涉及牦牛全基因组SNP位点的应用及检测用引物组和试剂盒。
背景技术
牦牛是分布于以青藏高原为中心，及其毗邻的高山、亚高山地区的特有牛种，能充分利用高寒草地的牧草资源，对高寒草地的生态环境条件具有极强的适应性。在空气稀薄、牧草生长期短、寒冷、枯草期长的恶劣环境条件下生活自如，繁衍后代，为当地牧民提供奶、肉、毛、役力、燃料等生产生活必需品，是当地畜牧业经济中不可或缺的，可称之为“全能”的家畜。牦牛在遗传资源上是一个极为宝贵的基因库。然而，中国牦牛数量和品种类群多，分布广，各牦牛品种产肉产奶，体型特征等表型各不相同。而中国地方牦牛品种基因组变异信息的不完整，是导致我国牦牛品种的育种及相关研究局限的重要原因之一。
单核苷酸多态性(single nucleotide polymorphism，SNP),主要是指在基因组水平上由单个核苷酸的变异所引起的DNA序列多态性。它是生物可遗传的变异中最常见的一种。SNP是公认的第三代遗传标志，许多表型差异、对疾病的易感性等等都可能与SNP有关。且从实验操作来看，通过SNP发现表型相关基因突变要比通过家系来得容易；有些SNP并不直接导致相关性状，但由于它与某些相关基因相邻，可成为重要的标记。目前并未见关于跟牦牛体重相关的SNP标记位点的报道。
因此，提供牦牛表型相关SNP的位点信息，用于在牦牛表型性状或牦牛分子育种分析中，成为了本领域技术人员亟待解决的问题。
发明内容
本发明的目的之一在于，提供牦牛全基因组的16个SNP位点在牦牛表型性状或分子育种分析中的应用，以解决现有技术中尚未有采用SNP位点对牦牛表型性状或分子育种分析的问题。
本发明的目的之二在于用于检测该16个SNP位点的引物组。
本发明的目的之三在于提供包括该引物组的试剂盒。
本发明的目的之四在于提供牦牛表型性状或分子育种分析的方法。
为实现上述目的，本发明采用的技术方案如下：
本发明所述的牦牛全基因组的16个SNP位点在牦牛表型性状或分子育种分析中的应用， 所述16个SNP位点如下：
AX-174702570，核苷酸序列如SEQ ID No.1所示；
AX-174961896，核苷酸序列如SEQ ID No.2所示；
AX-174407967，核苷酸序列如SEQ ID No.3所示；
AX-174402854，核苷酸序列如SEQ ID No.4所示；
AX-174929694，核苷酸序列如SEQ ID No.5所示；
AX-174547362，核苷酸序列如SEQ ID No.6所示；
AX-174734142，核苷酸序列如SEQ ID No.7所示；
AX-174706158，核苷酸序列如SEQ ID No.8所示；
AX-174783962，核苷酸序列如SEQ ID No.9所示；
AX-174627015，核苷酸序列如SEQ ID No.10所示；
AX-174928167，核苷酸序列如SEQ ID No.11所示；
AX-174555047，核苷酸序列如SEQ ID No.12所示；
AX-174845027，核苷酸序列如SEQ ID No.13所示；
AX-174891371，核苷酸序列如SEQ ID No.14所示；
AX-174570649，核苷酸序列如SEQ ID No.15所示；
AX-174620133，核苷酸序列如SEQ ID No.16所示。
本发明所述的16个SNP位点的引物组，该引物组包括上引物和下引物，其核苷酸序列如Seq ID.No.17-48所示。
包括如上所述的引物组的试剂盒。
本发明所述的牦牛表型性状或分子育种分析的方法，包括以下步骤：
步骤1：合成如上所述的16个SNP位点对应的探针序列；
步骤2：提取牦牛的DNA，采用如上所述的引物组进行PCR扩增，从扩增条带判断检测牦牛是否符合高体重的育种需求。
与现有技术相比，本发明具有的有益效果为：
本发明的16个SNP位点来自于32个中国地方牦牛品种特有遗传变异信息，可以应用于性状关联分析和分子辅助育种等方面，可为育种上下游过程提供支持。具有测定准确率高、易实现标准化和自动化检测等诸多优势。
具体实施方式
下面结合实施例对本发明作进一步说明，本发明的方式包括但不仅限于以下实施例。
本发明的思路在于，先对全国范围内32个不同牦牛品种共96个牦牛个体进行全基因组重测序数据。该32个牦牛品种涵盖了我国境内所有的牦牛种类。从96个牦牛的SNP突变中挑选高可信度的位点来制作芯片。然后再扩大样本，用268个牦牛个体，利用制备出的芯片挑选出了16个跟产肉相关的SNP位点。
实施例1
本实施例提供了从96个牦牛的重测序数据中挑选SNP突变可信度最高位点的方法，制备芯片。
首先利用Illumina X Ten测序平台对来自全中国范围内的32个不同地方牦牛群体(每个群体3头个体共计96个牦牛个体)进行全基因组重测序数据(每个个体10X覆盖度)，得到2592G的测序数据。在此基础上，首先进行一个质量控制，过滤掉测序质量较低的数据。接下来我们用BWA比对软件将各个总计的reads比对到参考基因组上，生成sam格式的比对结果文件，然后利用samtools将sam格式的结果文件转换成bam格式并进行排序，最后我们利用鉴定SNP最普遍的GATK来鉴定出96个牦牛中的SNP突变。最后我们从96个牦牛的SNP突变中挑选了一些高可信度的位点来制作芯片。
通过与牦牛现有数据库的比较，鉴定、验证和筛选出与牦牛遗传性状最相关的位点，最终通过Affymetrix公司设计定做出了包含630209个SNP位点的高密度牦牛SNP基因分型芯片。
96个牦牛重测序数据中SNP突变筛选及芯片制备的具体步骤如下：
1.提取DNA。
2.DNA样本检测。
对DNA样品的检测主要包括3种方法：(1)琼脂糖凝胶电泳分析DNA降解程度以及是否有RNA及蛋白等污染。(2)Nanodrop法检测DNA的纯度(OD260/280比值)。(3)Qubit法对DNA浓度进行精确定量。根据上述检测结果，采用OD值在1.8～2.0之间，含量在1.5μg以上的DNA样品用来建库。上述琼脂糖凝胶电泳、Nanodrop法、Qubit法均为现有技术。
3.数据分析
获得原始测序序列(Sequenced Reads)后参考基因组(BosGru_v2.0)进行信息分析流程，大致包括以下两个部分：
1)测序数据质量评估：主要对数据量、碱基质量、比对率、覆盖率、捕获率、均一性等指标进行统计，评估建库测序是否达到了标准，符合标准则进行后续分析。
2)变异检测：将高质量的序列比对到牦牛参考基因组上，检测样本中的变异信息，并对检出的变异进行统计和注释。
4.芯片制备
将重测序挑选出来的SNP信息提供给Affymetrix公司，通过Affymetrix公司平台的位点筛选标准最终选择了630209个SNP位点进行芯片设计及制作。
实施例2
本实施例提供了268个牦牛产肉相关性状SNP位点的筛选方法。
选取了3个国家认证的牦牛群体，包括嘉黎牦牛、帕里牦牛和斯布牦牛总计268个个体，通过实施例1制备的芯片进行SNP位点的筛选。样本严格筛选各群体中4-9岁处于青壮年期的牦牛个体，每个群体都筛选体重偏低和体重偏高的健康个体入组作为芯片筛选的样本。对每个个体的体重数据做详细的记录，根据得到的芯片筛选数据进行GWAS分析，最终找到16个跟牦牛产肉最相关的位点。
具体步骤为：
样品gDNA利用NanoDrop ND-2000(Thermo Scientific)定量并经凝胶电泳检测DNA完整性。DNA质检合格后，样本的扩增、片段化、沉淀、重悬、芯片的杂交以及洗染参照芯片标准流程。gDNA重悬后加入杂交MIX，然后进行质检。质检合格后，利用GeneTitan MC Instrument进行芯片杂交，洗染和扫描。
数据分析部分
将GeneTitan系统扫描得到的原始数据导入到软件Axiom Analysis Suite中，利用该软件对原始数据进行聚类和基因分型。最终通过Axiom AnalysisSuite软件导出PLINK格式数据用于后续的数据分析。
筛选出来的16个SNP位点信息如下表所示：
Figure PCTCN2018076240-appb-000001
Figure PCTCN2018076240-appb-000002
实施例3
本实施例提供了用于检测实施例1筛选出的16个SNP位点的引物组，所述引物组的核苷酸序列与SNP位点的对应关系见下表：
Figure PCTCN2018076240-appb-000003
Figure PCTCN2018076240-appb-000004
实施例4
本实施例提供了16个SNP位点在牦牛表型性状和分子辅助育种中的应用方法，具体为：
合成这16个位点对应的探针序列，采集牦牛耳样，提取DNA，质控后采用本发明的引物组进行PCR扩增，从扩增条带判断检测牦牛是否符合高体重的育种需求。其中探针序列的合成，牦牛DNA的提取，以及PCR扩增均为现有技术。如果根据引物扩增出来的条带突变位点为“+”标记，则证明该个体为体重偏高个体，如果根据引物扩增出来的条带突变位点为“-”标记，则证明该个体为体重偏轻个体。据此，可通过对公牛和母牛进行这16个位点的分子检测，如果母牛和公牛都携带高体重的分析标记位点，则可优先配种，对其后代加以选育，经过几代的繁殖则可极大的提升牦牛的体重，增加产肉能力，给牧区人民带来实实在在的经济收入。
上述实施例仅为本发明的优选实施方式之一，不应当用于限制本发明的保护范围，但凡在本发明的主体设计思想和精神上作出的毫无实质意义的改动或润色，其所解决的技术问题仍然与本发明一致的，均应当包含在本发明的保护范围之内。

Claims (4)

  1. 牦牛全基因组的16个SNP位点在牦牛表型性状或分子育种分析中的应用，所述16个SNP位点如下：
    AX-174702570，核苷酸序列如SEQ ID No.1所示；
    AX-174961896，核苷酸序列如SEQ ID No.2所示；
    AX-174407967，核苷酸序列如SEQ ID No.3所示；
    AX-174402854，核苷酸序列如SEQ ID No.4所示；
    AX-174929694，核苷酸序列如SEQ ID No.5所示；
    AX-174547362，核苷酸序列如SEQ ID No.6所示；
    AX-174734142，核苷酸序列如SEQ ID No.7所示；
    AX-174706158，核苷酸序列如SEQ ID No.8所示；
    AX-174783962，核苷酸序列如SEQ ID No.9所示；
    AX-174627015，核苷酸序列如SEQ ID No.10所示；
    AX-174928167，核苷酸序列如SEQ ID No.11所示；
    AX-174555047，核苷酸序列如SEQ ID No.12所示；
    AX-174845027，核苷酸序列如SEQ ID No.13所示；
    AX-174891371，核苷酸序列如SEQ ID No.14所示；
    AX-174570649，核苷酸序列如SEQ ID No.15所示；
    AX-174620133，核苷酸序列如SEQ ID No.16所示。
  2. 用于检测如权利要求1所述的16个SNP位点的引物组，其特征在于，所述引物组包括上引物和下引物，其核苷酸序列如Seq ID.No.17-48所示。
  3. 包括如权利要求2所述的引物组的试剂盒。
  4. 牦牛表型性状或分子育种分析的方法，其特征在于，包括以下步骤：
    步骤1：合成如权利要求1-3任意一项所述的16个SNP位点对应的探针序列；
    步骤2：提取牦牛的DNA，采用如权利要求2或3所述的引物组进行PCR扩增，从扩增条带判断检测牦牛是否符合高体重的育种需求。
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