CN105543357B - 猪4号染色体上与窝产仔数相关的snp位点 - Google Patents
猪4号染色体上与窝产仔数相关的snp位点 Download PDFInfo
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Abstract
本发明涉及与猪产仔数性状相关的SNP位点，具体涉及猪4号染色体上一个与窝产仔数相关的SNP位点及其引物，属生物技术领域。该SNP位点为基因组版本NCBI Build Sscrofa 10.2的Chr4:113326205；该位点的等位基因为T和C，有C/C、T/C和T/T三种基因型，扩增该位点的引物及延伸引物为：Chr4‑2‑PCRU：5’‑AAA GCA TTA ATG ATA CAG CCT GC‑3’；Chr4‑2‑PCRL：5’‑TTG TGA ACT GGG TAA ATG GAA‑3’；Chr4‑2‑SNPU：5’‑ACT GAC TGA CTG ACT TTG AGT ACT GCT GTA GCT TCC AAT A‑3’。带有T/C基因型的猪产仔数高于T/T基因型，C为提高猪产仔数的有益等位基因并且T/C杂合猪产仔数最高。本发明的有益效果在于：全基因组重测序方法能够快速、批量地筛选与性状相关的SNP位点或基因，基因组版本NCBI Build Sscrofa 10.2的Chr4:37340666这个SNP位点能够作为遗传标记用于猪的遗传育种提高母猪的产仔数。
Description
技术领域：
本发明涉及与猪产仔数性状相关的SNP位点，具体涉及猪4号染色体上一个与窝产仔数相关的SNP位点及其引物，属生物技术领域。
背景技术：
猪的产仔数性状是猪繁殖性状的主要组成部分，提高猪产仔数对提高养猪业的总体经济效益意义重大。我国的太湖猪以高产而闻名，窝产仔数比大多数其它中国地方猪种以及国外猪种的窝产仔数多4-5头(《中国猪品种志》张仲葛主编，上海科技出版社，1986)。太湖猪中与高产仔数相关的多态位点，能够作为遗传标记用于提高母猪的产仔数。
全基因组重测序方法已广泛应用于各个物种的相关研究，它能够快速全面地筛选出与性状、功能相关的基因或突变位点，弥补了QTL定位以及候选基因方法费时费力、单一等不足。
在国内外尚未见本发明中SNP位点与猪窝产仔数相关的公开文献报道。
发明内容：
本发明的目的是提供猪4号染色体上一个与窝产仔数相关的SNP位点及其引物。
本发明选择了二花脸猪(太湖猪的一种)，藏猪，巴马香猪，莱芜猪，及滇南小耳猪5个中国地方猪种以及野猪进行了全基因组重测序。6个猪种的重测序个体数分别为11个，11个，9个，10个，10个和10个。重测序数据跟参考基因组(NCBI Build Sscrofa 10.2)进行比对，利用SAMTools软件进行SNPs位点的提取。发明人比较了二花脸猪与其它5个猪种，在4号染色体上发现一个SNP位点(基因组版本NCBI Build Sscrofa 10.2的Chr4:113326205)与猪的产仔数相关。该位点的等位基因为T和C，有C/C、T/C和T/T三种基因型。
筛选到该位点后，发明人在白色杜洛克×二花脸猪杂交资源群F2代母猪个体中对其进行验证和相关性分析，结果发现该位点与猪产仔数有显著相关性。
本发明的有益效果在于：全基因组重测序方法能够快速、批量地筛选与性状相关的SNP位点或基因，基因组版本NCBI Build Sscrofa 10.2的Chr4:113326205这个SNP位点能够作为遗传标记用于猪的遗传育种提高母猪的产仔数。
附图说明：
图1为白色杜洛克×二花脸猪F2代母猪资源群中Chr4:113326205位点3种基因型的产仔数比较(图中两种基因型产仔数比较时，*表示0.01≤P≤0.05；**表示P＜0.01)。
具体实施方案：
(1)SNP位点的群体分布特征
本发明选择二花脸猪(太湖猪的一种)，藏猪，巴马香猪，莱芜猪，及滇南小耳猪5个中国地方猪种以及野猪进行了全基因组重测序。6个猪种的重测序个体数分别为11个，11个，9个，10个，10个和10个。重测序数据跟参考基因组(NCBI Build Sscrofa 10.2)进行比对，利用SAMTools软件进行SNPs位点的提取。发明人比较了二花脸猪与其它5个猪种，在4号染色体上发现一个SNP位点(基因组版本NCBI Build Sscrofa 10.2的Chr4:113326205)与猪的产仔数相关。
(2)SNP位点的鉴定
在引物设计网站设计SNAPSHOT引物，包括一对PCR扩增引物(Chr4-2-PCRU：5’-AAAGCA TTA ATG ATA CAG CCT GC-3’；Chr4-2-PCRL：5’-TTG TGA ACT GGG TAA ATG GAA-3’)和延伸引物(Chr4-2-SNPU：5’-ACT GAC TGA CTG ACT TTG AGT ACT GCT GTA GCT TCC AATA-3’)。该位点与其它11个位点共24条(12对)引物各取1μl混合为新的多重PCR引物池。首先进行12重PCR反应，反应体系所用试剂为：多重PCR引物池(6.25μM each)8μl、dNTP(10mMeach)8μl、10×PCR Buffer II 116μl、MgCl2(25mM Stock)234μl、Fast Start Taq(5U/μl)24μl和ddH2O 460μl，将以上试剂混合液8.5μl和1.5μl DNA(50ng/μl)混合成10μl体系进行12重PCR扩增反应。扩增反应条件为：94℃4min；94℃30s，55℃30s，72℃1min，共40个循环。下一步进行PCR反应纯化，所需试剂为：Exo I(10U/μl)60μl、SAP(1U/μl)130μl、10×SAPBuffer 35μl和ddH2O 125μl，在每个样品的PCR产物中加入3.5μl上述纯化混合液离心后按以下程序进行反应：37℃40min，96℃10min。然后进行单碱基延伸反应，所需试剂为：SNAPSHOT Multiplex ready reaction Mix 100μl、延伸引物池(6.25μM each，12条延伸引物各取1μl混合)8μl、5×Sequencing Buffer 200μl和ddH2O300μl，将以上试剂混合液6μl和4μl的纯化产物混合成10μl体系进行延伸反应。延伸反应条件为：96℃10s，50℃5s，60℃30s，共25个循环。接着进行延伸反应产物的纯化，所需试剂为：SAP(1U/μl)120μl、10×SAPBuffer 70μl和ddH2O140μl，在每个样品的延伸反应产物中加入上述3.3μl纯化混合液离心后按以下程序进行反应：37℃40min，75℃20min，4℃保存备用。每个样品取5-4μl上述纯化后的SNAPSHOT反应产物和5-6μl的高纯甲酰胺(Hi-Di Formamide)混合成10μl体系离心后上样于ABI 3730XL测序仪进行基因分型，使用GeneMarker软件分析分型结果。
(注：以上4个步骤的反应试剂混合液用量均为100个样品所需试剂量，从单碱基延伸反应开始，需要避光进行。)
(3)白色杜洛克×二花脸猪繁殖性能杂交资源群构建
后续的验证实验所用资源群个体样品及其性状数据由江西农业大学动物生物技术国家重点实验室培育基地提供。
1998年12月，在江西农业大学科研猪场将从江苏省3个二花脸猪保种场购买的二花脸母猪进行场间血缘的交叉配种。2001年1月根据亲本产仔性能，从3头公猪和11头母猪的纯繁后代中选择了17头二花脸母猪作为资源群体的祖代母本。把Sygen PIC公司惠赠的2头优质白色杜洛克公猪作为资源家系的祖代父本。将这2头白色杜洛克和17头二花脸猪杂交产生的F1代个体进行配种，避免全同胞交配，且每头公猪通常与同一头母猪配种，以获得大样本的F2半同胞家系。2003年1月、3月和6月，项目组分别将第1批和第2批F2母猪送往江西省宜春市畜牧良种场(59头)、江西省国营红星种猪场(52头)和江西上犹县四元杂交猪场(118头)用于母猪繁殖性状的测定。
(4)SNP位点在杂交资源群F2代母猪群体的基因分型
按照上述步骤(2)所述的方法，以192个白色杜洛克×二花脸猪杂交资源群F2代母猪个体DNA为模板检测该SNP位点的基因型。192个F2代资源群个体中有171个个体成功分型，3种基因型C/C，T/C和T/T的个体数分别是94，58和19。
(5)SNP位点对产仔数性状的调控效应
在F2代资源群个体中，带有C/C基因型个体的平均产仔数为11.0507，标准差为0.9209；带有T/C基因型个体的平均产仔数为11.4331，标准差为0.9499；带有T/T基因型个体的平均产仔数为9.7304，标准差为1.0823。
本发明采用一般线性模型(GLM)分析了SNP位点对猪总产仔数、产活仔数的影响。全部统计分析采用SAS软件进行。模型如下：
Yijklm＝u+Ai+Gj+Bk+Pl+eijklm
其中Yijklm为猪总产仔数或产活仔数，u为群体均值，Ai为第i个个体的加性效应，Gj为第j个SNP位点基因型的固定效应，Bk为批次效应(k＝1,2,3,4)，Pl为随机效应(l＝1,2,3)。
分析结果发现Chr4:113326205这个SNP位点跟产仔数有显著相关性。带有T/C基因型的F2代母猪的产仔数比带有T/T基因型的母猪平均多1.7027(P＝0.021)，从图1看出，带有T/T基因型的母猪产仔数最低。C为提高猪产仔数的有益等位基因并且T/C杂合猪产仔数最高。
从SNP位点与产仔数相关性分析看出，Chr4:113326205这个SNP位点能够作为分子标记应用于分子标记辅助选择(MAS)来提高母猪的产仔数。
Claims (1)
1.一种检测猪4号染色体上与窝产仔数相关的SNP位点的引物在检测猪产仔数中的应用，其特征在于：所述引物包括PCR扩增引物以及延伸引物，其中PCR扩增引物的核苷酸序列如下所示：Chr4-2-PCRU：5’-AAAGCATTAATGATACAGCCTGC-3’；Chr4-2-PCRL：5’-TTGTGAACTGGGTAAATGGAA-3’；延伸引物的核苷酸序列如下所示：Chr4-2-SNPU：5’-ACTGACTGACTGACTTTGAGTACTGCTGTAGCTTCCAATA-3’，所述猪为白色杜洛克×二花脸猪杂交资源群F2代母猪，所述SNP位点为基因组版本NCBI Sscrofal10.2的Chr4:113326205,该位点的等位基因为T和C，有C/C、T/C和T/T三种基因型，带有T/C基因型的猪产仔数高于带有C/C和T/T基因型的猪，其中基因型T/C为提高猪产仔数的有益基因型。
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| WO2007133075A2 (en) * | 2006-05-12 | 2007-11-22 | Institute For Pig Genetics B.V. | Igf2 marker assisted selection for porcine reproductive traits |
| CN103757003A (zh) * | 2013-12-26 | 2014-04-30 | 中国科学院昆明动物研究所 | 猪13号染色体上与产仔数相关的snp位点及其引物 |
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