CN105567816A - SNP site related with litter size on No.8 chromosome of pig - Google Patents
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Abstract
Description
技术领域：Technical field:
本发明涉及与猪产仔数性状相关的SNP位点，具体涉及猪8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。The invention relates to a SNP site related to piglet size traits, in particular to a SNP site on pig No. 8 chromosome related to litter size and a primer thereof, belonging to the field of biotechnology.
背景技术：Background technique:
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。Pig litter size traits are the main components of pig reproductive traits, and increasing pig litter size is of great significance to improving the overall economic benefits of pig farming. my country's Taihu pigs are famous for their high yield, and the litter size is 4-5 more than that of most other Chinese local pig breeds and foreign pig breeds (Edited by Zhang Zhongge of "China Pig Breeds", Shanghai Science and Technology Publishing Society, 1986). The polymorphic sites associated with high litter size in Taihu pigs can be used as genetic markers to increase the litter size of sows.
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。The whole genome resequencing method has been widely used in the related research of various species. It can quickly and comprehensively screen out genes or mutation sites related to traits and functions, and make up for the time-consuming, laborious and monotonous shortcomings of QTL mapping and candidate gene methods.
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。At home and abroad, there is no public literature report on the correlation between the SNP site and the piglet litter size in the present invention.
发明内容：Invention content:
本发明的目的是提供猪8号染色体上一个与窝产仔数相关的SNP位点及其引物。The purpose of the present invention is to provide a SNP site on the No. 8 chromosome of pigs related to the litter size and the primer thereof.
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(NCBIBuildSscrofa10.2)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8号染色体上发现一个SNP位点(基因组版本NCBIBuildSscrofa10.2的Chr8:109488182)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。In the present invention, five local pig breeds in China, Erhualian pig (a kind of Taihu pig), Tibetan pig, Bamaxiang pig, Laiwu pig, and Diannan small-eared pig, and wild boar were selected for whole-genome resequencing. The resequencing individuals of the 6 pig breeds were 11, 11, 9, 10, 10 and 10, respectively. The resequencing data were compared with the reference genome (NCBIBuildSscrofa10.2), and SNPs were extracted using SAMTools software. The inventor compared the Erhualian pig with five other pig breeds, and found that a SNP site on chromosome 8 (Chr8:109488182 of the genome version NCBIBuildSscrofa10.2) was related to the litter size of pigs. The alleles of this locus are T and C, and there are three genotypes of C/C, T/C and T/T.
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。After screening the locus, the inventor verified and analyzed it in F2 generation sows of the White Duroc×Erhualian pig hybrid resource group, and found that the locus has a significant correlation with piglet size .
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NCBIBuildSscrofa10.2的Chr8:109488182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。The beneficial effect of the present invention is that: the whole genome resequencing method can quickly and batch screen SNP sites or genes related to traits, and the Chr8:109488182 SNP site of the genome version NCBIBuildSscrofa10.2 can be used as a genetic marker for pig heredity Breeding increases litter size in sows.
附图说明：Description of drawings:
图1为白色杜洛克×二花脸猪F2代母猪资源群中Chr8:109488182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)。Figure 1 is the comparison of the litter size of the three genotypes at the Chr8:109488182 site in the F2 generation sow resource group of White Duroc×Erhualian pigs ( when comparing the litter sizes of the two genotypes in the figure, * means 0.01≤P ≤0.05; ** indicates P<0.01).
具体实施方案：Specific implementation plan:
(1)SNP位点的群体分布特征(1) Population distribution characteristics of SNP loci
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(NCBIBuildSscrofa10.2)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8号染色体上发现一个SNP位点(基因组版本NCBIBuildSscrofa10.2的Chr8:109488182)与猪的产仔数相关。The present invention selects Erhualian pig (a kind of Taihu pig), Tibetan pig, Bamaxiang pig, Laiwu pig, and 5 Chinese local pig breeds in southern Yunnan and wild boar for whole genome resequencing. The resequencing individuals of the 6 pig breeds were 11, 11, 9, 10, 10 and 10, respectively. The resequencing data were compared with the reference genome (NCBIBuildSscrofa10.2), and SNPs were extracted using SAMTools software. The inventor compared the Erhualian pig with five other pig breeds, and found that a SNP site on chromosome 8 (Chr8:109488182 of the genome version NCBIBuildSscrofa10.2) was related to the litter size of pigs.
(2)SNP位点的鉴定(2) Identification of SNP sites
在引物设计网站设计SNAPSHOT引物，包括一对PCR扩增引物(Chr8-PCRU：5’-TTGGCTGTCTGTCCACCA-3’；Chr8-PCRL：5’-AATTTCTGACCTGGGTTCATG-3’)和延伸引物(Chr8-SNPU：5’-GACTGACTGACTGACTGACTGAATATATCCGGTTTGCTTCCTTAC-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(6.25μMeach)8μl、dNTP(10mMeach)8μl、10×PCRBufferII116μl、MgCl2(25mMStock)234μl、FastStartTaq(5U/μl)24μl和ddH2O460μl，将以上试剂混合液8.5μl和1.5μlDNA(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：ExoI(10U/μl)60μl、SAP(1U/μl)130μl、10×SAPBuffer35μl和ddH2O125μl，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：SNAPSHOTMultiplexreadyreactionMix100μl、延伸引物池(6.25μMeach，12条延伸引物各取1μl混合)8μl、5×SequencingBuffer200μl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×SAPBuffer70μl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-DiFormamide)混合成10μl体系离心后上样于ABI3730XL测序仪进行基因分型，使用GeneMarker软件分析分型结果。Design SNAPSHOT primers on the primer design website, including a pair of PCR amplification primers (Chr8-PCRU: 5'-TTGGCTGTCTGTCCACCA-3'; Chr8-PCRL: 5'-AATTTCTGACCTGGGTTCATG-3') and extension primers (Chr8-SNPU: 5'-GACTGACTGACTGACTGACTGAATATATCCGGTTTGCTTCCTTAC-3'). This site was mixed with 24 primers (12 pairs) of other 11 sites to form a new multiplex PCR primer pool. First, perform 12-fold PCR reaction, the reagents used in the reaction system are: multiplex PCR primer pool (6.25μMeach) 8μl, dNTP (10mMeach) 8μl, 10×PCRBufferII116μl, MgCl 2 (25mMStock) 234μl, FastStartTaq (5U/μl) 24μl and ddH 2 O460 μl, mix 8.5 μl of the above reagent mixture and 1.5 μl of DNA (50ng/μl) into a 10 μl system for 12-fold PCR amplification reaction. The amplification reaction conditions were: 94°C for 4min; 94°C for 30s, 55°C for 30s, 72°C for 1min, a total of 40 cycles. The next step is to purify the PCR reaction, the required reagents are: ExoI (10U/μl) 60μl, SAP (1U/μl) 130μl, 10×SAPBuffer 35μl and ddH 2 O 125μl, add 3.5μl of the above purification mixture to the PCR product of each sample After the solution was centrifuged, the reaction was carried out according to the following procedure: 37°C for 40 minutes, 96°C for 10 minutes. Then carry out the single base extension reaction, the required reagents are: SNAPSHOTMultiplexreadyreactionMix100μl, the extension primer pool (6.25μMeach, 1μl of each of the 12 extension primers mixed)8μl, 5×SequencingBuffer200μl and ddH 2 O300μl, the above reagent mixture 6μl and 4μl The purified products were mixed into 10 μl system for extension reaction. The extension reaction conditions were: 96°C for 10s, 50°C for 5s, 60°C for 30s, 25 cycles in total. Then purify the extension reaction product, the required reagents are: SAP (1U/μl) 120 μl, 10×SAPBuffer 70 μl and ddH 2 O 140 μl, add the above 3.3 μl purification mixture to the extension reaction product of each sample and centrifuge according to the following procedure Perform the reaction: 37°C for 40 minutes, 75°C for 20 minutes, and store at 4°C for later use. For each sample, take 5-4 μl of the above-mentioned purified SNAPSHOT reaction product and 5-6 μl of high-purity formamide (Hi-DiFormamide) and mix them into a 10 μl system, centrifuge and load the sample on the ABI3730XL sequencer for genotyping, and use GeneMarker software to analyze the typing results .
(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。)(Note: The amount of reaction reagent mixture in the above 4 steps is the amount of reagent required for 100 samples, starting from the single base extension reaction, it needs to be protected from light.)
(3)白色杜洛克×二花脸猪繁殖性能杂交资源群构建(3) Construction of white Duroc × Erhualian pig reproductive performance hybrid resource group
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。The individual samples and trait data of resource groups used in subsequent verification experiments were provided by the breeding base of the State Key Laboratory of Animal Biotechnology, Jiangxi Agricultural University.
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SygenPIC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。In December 1998, the Erhualian sows purchased from 3 Erhualian pig breeding farms in Jiangsu Province were cross-breeded in the scientific research pig farm of Jiangxi Agricultural University. In January 2001, according to the farrowing performance of the parents, 17 Erhualian sows were selected from the purebred offspring of 3 boars and 11 sows as the grandparents of the resource group. Two high-quality white Duroc boars donated by SygenPIC were used as the grandparents of the resource family. The F 1 individuals produced by crossing these 2 white Duroc and 17 Erhualian pigs were bred to avoid full-sib mating, and each boar was usually bred with the same sow to obtain a large sample of F 2 half-sibs family lineage. In January, March and June 2003, the project team sent the first batch and the second batch of F 2 sows to Yichun City Animal Husbandry Breeding Farm (59 heads) in Jiangxi Province and Jiangxi State-owned Hongxing Breeding Pig Farm (52 heads) respectively. ) and the Quaternary Hybrid Pig Farm (118 heads) in Shangyou County, Jiangxi Province for the determination of reproductive traits of sows.
(4)SNP位点在杂交资源群F2代母猪群体的基因分型(4) Genotyping of SNP loci in F 2 generation sow population of hybrid resource group
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185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17，87和81。According to the method described in the above step ( 2 ), the genotype of the SNP locus was detected using the individual DNA of 192 F2 generation sows of the white Duroc×Erhualian pig hybrid resource group as a template. Among the 192 individuals of the F 2 resource group, 185 individuals were successfully typed, and the numbers of individuals of the three genotypes C/C, T/C and T/T were 17, 87 and 81, respectively.
(5)SNP位点对产仔数性状的调控效应(5) Regulatory effects of SNP loci on litter size traits
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10.835，标准差为1.0164；带有T/C基因型个体的平均产仔数为10.3713，标准差为0.8475；带有T/T基因型个体的平均产仔数为11.2106，标准差为0.857。Among individuals in the F 2 resource group, the average litter size of individuals with C/C genotype was 10.835, with a standard deviation of 1.0164; the average litter size of individuals with T/C genotype was 10.3713, with a standard deviation of 0.8475 ; The average litter size of individuals with T/T genotype was 11.2106, with a standard deviation of 0.857.
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：The present invention uses a general linear model (GLM) to analyze the influence of the SNP site on the total litter size and the live piglet size. All statistical analyzes were performed using SAS software. The model is as follows:
Yijklm＝u+Ai+Gj+Bk+Pl+eijklm Y ijklm =u+A i +G j +B k +P l +e ijklm
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)。Among them, Y ijklm is the total litter size or live piglet number, u is the group mean, A i is the additive effect of the i-th individual, G j is the fixed effect of the genotype of the j-th SNP locus, and B k is Batch effect (k=1,2,3,4), P l is random effect (l=1,2,3).
分析结果发现Chr8:109488182这个SNP位点跟产仔数有显著相关性。带有T/T基因型的F2代母猪的产仔数比带有T/C基因型的母猪平均多0.8393(P＝0.0491)，从图1看出，带有T/T基因型的母猪产仔数最高。The analysis results found that the SNP site Chr8:109488182 had a significant correlation with litter size. The litter size of F 2 generation sows with T/T genotype was 0.8393 more than that of sows with T/C genotype on average (P=0.0491), as can be seen from Figure 1, with T/T genotype sows with the highest litter size.
从SNP位点与产仔数相关性分析看出，Chr8:109488182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。From the analysis of the correlation between SNP sites and litter size, it can be seen that the SNP site Chr8:109488182 can be used as a molecular marker in molecular marker-assisted selection (MAS) to increase the litter size of sows.
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| CN108300789A (en) * | 2017-11-14 | 2018-07-20 | 中国农业大学 | With the relevant SNP marker of the multiple important economical traits of pig and its application |
| CN111455071A (en) * | 2020-02-09 | 2020-07-28 | 南京农业大学 | Detection method and application of SNP (single nucleotide polymorphism) marker on chromosome 8 of pig related to total number born of all births of Erhualian pig |
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| CN108300789A (en) * | 2017-11-14 | 2018-07-20 | 中国农业大学 | With the relevant SNP marker of the multiple important economical traits of pig and its application |
| CN111455071A (en) * | 2020-02-09 | 2020-07-28 | 南京农业大学 | Detection method and application of SNP (single nucleotide polymorphism) marker on chromosome 8 of pig related to total number born of all births of Erhualian pig |
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