CN112458188A - Method and primer for identifying farrowing performance of sows - Google Patents
Method and primer for identifying farrowing performance of sows Download PDFInfo
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Abstract
The invention discloses aFSHRA method and a primer pair for analyzing and identifying the farrowing performance of sows by combining two mutation sites. The invention also disclosesFSHR c.532 G>A andFSHR c.1166 C>t amplification primer and a method for detecting, analyzing and identifying the farrowing performance of sows by using the primer. The primer, the method and the scheme can realize the early breeding of the high-yield piglet performance of the sow, shorten the breeding time of excellent pigs, accelerate the breeding process, save the breeding and feeding cost and effectively improve the total number born, the number born alive and the number healthy born of the pigs. The method has high accuracy and simple operation, and has high practical application value in pig breeding.
Description
Technical Field
The invention relates to a molecular breeding technology of two-site combined selection of sow reproductive performance, a primer pair and application of results, belonging to the technical field of biology.
Background
In recent years, due to the influence of African swine fever, the pig raising industry in the world suffers great loss, and the pig raising industry in China is no exception. The number and the quality of the sows can be reduced, and the price of pork is increased. The traditional breeding period of the reproductive performance of the pigs is long. The breeding performance directly influences the production efficiency and economic benefit of the pig farm. The total litter size, the live litter size and the healthy litter size of the pigs are three very important reproductive traits and also very important economic traits. At present, the research on breeding performance molecular breeding focuses on selecting single mutation sites, but the breeding performance is controlled by a micro-effect polygene, so that the molecular breeding by selecting the single mutation sites has certain limitations. The invention relates to a polygene joint breeding method for researching reproductive performance and application thereof.
Follicle-stimulating hormone receptor (FSHR) belongs to the superfamily of G protein-coupled receptors and functions to mediate FSH.FSHRThe gene is an important gene for reproductive performance and plays a key role in the reproduction of animals. Studies have shown that FSHR is expressed only in ovarian granulosa cells, and that FSHR expression levels are closely related to granulosa cell differentiation and follicle maturation (Durlej M, Knappzyk-Stword K, Duda M. The expression of FSH receptor (FSHR) in The neuronal pore space and its regulation by flunomide [ J]. Reprod Domest Anim. 2011 Jun;46(3):377-84. Wu W, Han J, Cao R. Sequence and regulation of the porcine FSHR gene promoter, Anim Reprod Sci, 2015 Mar;154: 95-104.). FSHR and ovarian binding to Follicle Stimulating Hormone (FSH) regulate spermatogenesis and oogenesis (Zhou N, Wang N, Qin X. Expression of follicule-stimulating hormone receptor (FSHR), protein kinase B-2 (AKT2) and adaptor protein with PH domain, PTB domain, and leucosine wrapper (APPL1) in pig ovaries. Pol J Vet Sci.2017 Dec;20(4):661 667.). The research finds thatFSHRThe missense mutation point of the gene is obviously related to the loss of backfat of the first-born sow, and the conclusion is drawnFSHRThe gene is the influence of the sow backfat loss candidate gene (Zhouwei. large white pig lactation backfat loss on reproductive performance and the candidate geneFSHRPolymorphism Association study [ D]Chinese academy of agricultural sciences, 2018). The research shows that the compound has the advantages of high purity,FSHRthe 10 th exon of the gene has 2 polymorphic sites, the 2 sites have obvious influence on litter size of the Meishan pigs, and the polymorphism of the 10 th exon has obvious influence on the litter size characters of southern Anhui black pigs and Bake summer pigs (Zhuang XD, Zhuu HY, Zhou J. Relationship between transgenic genes in exon 10 of FSHR gene and lithium size in swine [ J X D, J D]. Genet Mol Res. 2015 Jul 27, 14(3) 8252-61, Zhu hong Yu, pigFSHRStudy of Gene polymorphism, expression and relationship to litter [ D]University of agriculture, Anhui 2015.).
FSHRThe gene is used as a candidate gene for losing the back fat of the sow and used for regulating the back fat deposition of the pig.FSHRThe gene may also be a candidate for reproductive performance, expressed in ovarian granulosa cells, in combination with FSH to regulate spermatogenesis and oogenesis.FSHRThe polymorphic site of the gene has obvious correlation with the litter traits of the pig, can be used for marker-assisted selection of the pig, and has research and application values and significance.
Disclosure of Invention
The invention discloses an amplification product for early breeding of sow with high farrowing performanceFSHR c.532 G>A andFSHR c.1166 C>a PCR primer pair of T and an amplification primer pair identified by mass spectrometry; wherein the amplification comprisesFSHR c.532 G>A andFSHR c.1166 C>the PCR primer pair of T is shown in SEQ ID NO 1-4; the amplification primer pair identified by mass spectrum is shown in SEQ ID NO: 5-10:
wherein the PCR primer pair is shown in SEQ ID NO: 1-4:
the amplification primer pair identified by mass spectrum is shown as SEQ ID NO: 5-10:
FSHR c.532 G>the position of the GenBank Accession Number XM-021085881.1 mutation site of the A site is Ssc.3: 91750608;FSHR c.1166 C>the position of the GenBank Accession Number XM-021085881.1 mutation site of the T site is Ssc.3: 91773610.
The invention further discloses a kit for identifying the high and low reproductive performance of pigs, which is characterized by comprising the primer pair and an amplification primer identified by mass spectrometry; the identification of the reproductive performance of the pigs comprises the following steps: total litter size, viable litter size, and healthy litter size.
The invention further discloses a single SNP locus identification method, which comprises the steps of carrying out PCR amplification by using the primer pair and taking the DNA of each pig as a template, and carrying out common sequencing on the product obtained by amplification or directly sequencing the DNA of the pig by using a mass spectrum primer; sequencing to obtain a sequencing peak diagram, wherein the sequencing sequence is shown in SEQ ID NO. 11-12 or a plot obtained by mass spectrometry and a sequencing typing result; obtaining the plot graph and sequencing typing result obtained by sequencing peak graph, sequencing sequence or mass spectrum sequencingFSHR c.532 G>A site andFSHR c.1166 C>the type of mutation at the T site; the mutation types of the 2 sites can be used for molecular breeding of the reproductive performance of later-stage sows, and the characteristics and the breeding principle of the sites are as follows:FSHRc.selection and breeding application of total litter size, alive litter size and healthy litter size of GG type individuals at 532 loci,FSHRc, breeding and selecting total litter size, alive litter size and healthy litter size of TT type individuals at the 1166 locus. WhereinFSHRGG type individual at c.532 siteFSHRc.532 G>The bases of the polymorphic site A are G;FSHRTT type individuals at position 1166FSHR c.1166 C>And the basic groups of the T polymorphic sites are all T pigs.
FSHR c.532 G>Sequencing sequence of A related primer amplification product SEQ ID NO. 11
ACTACTTAGTTCTATATGCCCAGGCACAGCCATCAATAAAGATCTGATATACTAAATTAGATATCACTTATTCCCCAAACTTTGAGTTCATCATCCTTTAAAAGAGTAACTATTCCAACATTACCTCAGCTAAAGACCACACCATGCCAAGTAGACTAAGTGTGGGGAGGTGACATGAATGGAGCTGACATAAACATCTAATTTGTCTGTACTTTCTCCTAGAGACATTCAAGATAATATAAACATCCACACTGTTGAAAGAAATTCTTTCGTGGGACTAAGTTTTGAAAGTATGATTCTGTAAGTAAAGAAAAAAAAAATCCAGTGGAGTAACATTGGGCTTTTCATTTAATGACTTCTTTCTCCTAATGGTACACTCCTTTTTTGTTTGGATGACCTTGGCATTTCACCATTCAAATTTCTCTTTGGTTCTATGTCTTTCTCTCATTTCCCATCTTTACATTTCAAAATCACAACATGGAGCCATGTTAATAATTTACGATTCTGTAGTGTGATTCAAGTATTTAAAATTGCTCTTTGAATGACGGTCTCCTGTGATGCAA
FSHR c.1166 C>Sequencing sequence of T related primer amplification product SEQ ID NO 12
AAGCTTCTGGCGAGATGGTGAGTCCAGTTTAGCCAAAGAATTTGACACGATGTACAGTGAATTTGACTATGACTTATGCAATGAAGTGGTTGATGTGATTTGCTCCCCTGAGCCAGATGCCTTCAATCCATGTGAAGATATCATGGGGCATGATATTCTCAGAGTCTTAATATGGTTTATTAGTATCCTTGCCATCACTGGGAACATCATAGTGCTGGTGATCCTGATCACCAGCCAATACAAACTCACAGTCCCTCGGTTCCTTATGTGTAATCTGGCCTTTGCCGATCTCTGCATTGGAATCTACCTCCTGCTCATAGCATCAGTTGATATCCATACCAAAACCCAAATACCACAAA
The invention also disclosesFSHR c.532 G>A site andFSHR c.1166 C>the T site joint identification method adopts the primer pair, takes the DNA of each pig as a template to carry out PCR amplification, and carries out common sequencing on the product obtained by amplification or directly carries out sequencing on the DNA of the pig by utilizing a mass spectrum primer; sequencing to obtain a sequencing sequence, a sequencing peak diagram or a plot obtained by mass spectrometry, and a sequencing typing result; the sequencing peak diagram and the sequencing sequence are shown in SEQ ID NO. 11-12 or the plot obtained by mass spectrometry and the sequencing typing result show that each pigFSHR c.532 G>A site andFSHR c.1166 C>the mutation types of the T locus, namely the mutation types of the 2 loci can be used for molecular breeding of the reproductive performance of later-stage sows, and the characteristics and the breeding principle of the loci are as follows:FSHR c.532 G>a site andFSHR c.1166 C>all results obtained by T site joint analysis; whereinFSHR c.532 G>A site andFSHR c.1166 C>breeding and applying total number born, living number born and healthy number born of the T site combined with GATT individuals;FSHR c.532 G>a site andFSHR c.1166 C>and (3) breeding and application of total number born, living number born and healthy number born of the T site combined GGTT individuals. WhereinFSHR c.532 G>A site andFSHR c.1166 C>the T site is combined with the GATT individualFSHRc.532 in pigs of position G and A, simultaneouslyFSHRc.1166 sites are all T-type individuals;FSHR c.532 G>a site and FSHR c.1166 C>the T site is combined with the GGTT individualFSHRc.532 in all G pigsFSHR c.1166 C>And the basic groups of the T polymorphic sites are all T pigs.
In the process of preparing the matching plan: the filial generation of the two sites, namely the male parent and the female parent (or the female parent and the male parent) of the GATT type individual and the GGTT type individual can be used for reserving seeds, and the hybridization of the male parent and the female parent with the same type is avoided. The primer pair is obtained by amplification by using pig genome DNA as a template. The mutation site or the mutation site combination which is obviously related to the high and low of the reproductive performance of the pig is judged by using the method and is used for selecting corresponding pig individuals.
The experimental results show that:FSHR c.532 G>the total litter size, the viable litter size and the healthy litter size phenotype value of the individual with the type GG at the mutation site of the A gene are higher than those of the individuals with other types.FSHR c.1166 C>The phenotype values of total litter size and healthy litter size, total litter size, viable litter size and healthy litter size of TT type individuals at the T gene mutation sites are higher than those of other types.FSHRc.532G>A site andFSHR c.1166 C>the T locus and GATT combination has better overall indexes of total live litter size, live litter size and healthy litter size of individuals and estimated breeding value. The total litter size in the phenotype index reaches 16, the number of alive litters reaches 13.72 and the healthy litters reaches 13.33; the total litter size in the breeding value index is estimated to reach 0.54, the number of alive litters reaches 0.49 and the number of healthy litters reaches 1.11. As GGTT had 1 individual, dunken analysis of variance was not performed. GGTT type is 1 individual, total number born reaches 16 in phenotype index, number born alive reaches 14.5 and number born healthy reaches 14; the total litter size in the breeding value index is estimated to reach 0.37, the number of alive litters reaches 0.19 and the number of healthy litters reaches 0.96.
The invention further discloses amplification products comprisingFSHR c.532 G>A andFSHR c.1166 C>the PCR primer pair of the T and the amplification primer pair identified by the mass spectrum are applied in the breeding process of the pigs. The application refers to: early breeding of sow with high farrowing performance. The experimental results show that:FSHR c.532 G>the total litter size, the viable litter size and the healthy litter size phenotype value of the individual with the type GG at the mutation site of the A gene are higher than those of the individuals with other types.FSHR c.1166 C>The phenotype values of total litter size and healthy litter size, total litter size, viable litter size and healthy litter size of TT type individuals at the T gene mutation sites are higher than those of other types.FSHR c.532G>A site andFSHR c.1166 C>phenotypic indicators of total viable litter size, viable litter size and healthy litter size and estimated breeding value of T locus combined GATT individualsThe comprehensive index of the method is better. The total litter size in the phenotype index reaches 16, the number of alive litters reaches 13.72 and the healthy litters reaches 13.33; the total litter size in the breeding value index is estimated to reach 0.54, the number of alive litters reaches 0.49 and the number of healthy litters reaches 1.11. As GGTT had 1 individual, dunken analysis of variance was not performed. GGTT type is 1 individual, total number born reaches 16 in phenotype index, number born alive reaches 14.5 and number born healthy reaches 14; the total litter size in the breeding value index is estimated to reach 0.37, the number of alive litters reaches 0.19 and the number of healthy litters reaches 0.96.
The invention is described in more detail below:
the single SNP site identification method disclosed by the invention is to utilize the primer pair to carry out PCR amplification by taking the DNA of each pig as a template, and carry out common sequencing on the product obtained by amplification. Or directly sequencing the DNA of the pig by using a mass spectrum primer. Sequencing to obtain a sequencing sequence, a sequencing peak image or a plot obtained by mass spectrometry sequencing, and sequencing typing results. Obtaining the plot graph and sequencing typing result of each pig through sequencing sequence, sequencing peak graph or mass spectrum sequencingFSHR c.532 G>A site andFSHRc.1166 C>type of mutation at the T site. The mutation types of the 2 sites can be used for molecular breeding of the reproductive performance of later-stage sows, and the characteristics and the breeding principle of the sites are as follows:FSHRc.selection and breeding application of total litter size, alive litter size and healthy litter size of GG type individuals at 532 loci,FSHRc, breeding and applying total litter size, alive litter size and healthy litter size of TT type individuals at the 1166 locus; whereinFSHRGG type individual at c.532 siteFSHR c.532 G>The bases of the polymorphic site A are G;FSHRTT type individuals at position 1166FSHR c.1166 C>And the basic groups of the T polymorphic sites are all T pigs. Note that the method is applicable to Changbai pigs, Dabai pigs and Duroc pig breeds.
The invention disclosesFSHR c.532 G>A site andFSHR c.1166 C>the T site combined identification method is characterized in that the primer pair is utilized, the DNA of each pig is taken as a template for PCR amplification, and the product obtained by amplification is subjected to common sequencing. Or directly sequencing the DNA of the pig by using a mass spectrum primer. Sequencing to obtain a sequencing sequence, and sequencingSequencing peak graph or plot obtained by mass spectrometry sequencing, sequencing typing result. Obtaining the plot graph and sequencing typing result of each pig through sequencing sequence, sequencing peak graph or mass spectrum sequencingFSHR c.532 G>A site andFSHR c.1166 C>type of mutation at the T site.
The mutation types of the 2 sites can be used for molecular breeding of the reproductive performance of later-stage sows, and the characteristics and the breeding principle of the sites are as follows:FSHR c.532 G>a site andFSHR c.1166 C>all results obtained by T site joint analysis; whereinFSHR c.532 G>A site andFSHR c.1166 C>breeding and applying total number born, living number born and healthy number born of the T site combined with GATT individuals;FSHR c.532 G>a site andFSHR c.1166 C>and (3) breeding and application of total number born, living number born and healthy number born of the T site combined GGTT individuals. WhereinFSHR c.532 G>A site andFSHR c.1166 C>the T site is combined with the GATT individualFSHRc.532 pigs with G and A in positionFSHRc.1166 sites are all T-type individuals;FSHRc.532 G>a site andFSHR c.1166 C>the T site is combined with the GGTT individualFSHRc.532 in all G pigsFSHR c.1166 C>And the basic groups of the T polymorphic sites are all T pigs. Note that the method is applicable to Changbai pigs, Dabai pigs and Duroc pig breeds. It includes: FSHR c.532 G>a site andFSHR c.1166 C>the T locus is combined with the overall litter size, the number born alive and the healthy litter size of the GATT individual and the comprehensive index of the estimated breeding value, wherein the overall litter size in the phenotype index reaches 16, the number born alive reaches 13.72 and the healthy litter size reaches 13.33; the total litter size in the breeding value index is estimated to reach 0.54, the number of alive litters reaches 0.49 and the number of healthy litters reaches 1.11.FSHR c.532 G>A site andFSHR c.1166 C>the T site combined GGTT individual has better overall litter size, viable litter size and healthy litter size phenotype indexes and comprehensive indexes of estimated breeding value. The total litter size in the phenotype indexes reaches 16, the number of alive litters reaches 14.5 and the number of healthy litters reaches 14; the total litter size in the breeding value index is estimated to reach 0.37, the number of alive litters reaches 0.19 and the number of healthy litters reaches 0.96. The GATT type individuals and the GGTT type individuals are both advantageousIndividuals, can be used for reproductive performance molecular breeding.
The invention discloses a method for identifying and assisting in selection of a single SNP locus, and belongs to the protection scope of the invention. The method comprisesFSHR Phenotype values for total litter size, litter size alive and healthy size were higher for individuals of GG type at position c.532.FSHR And c.1166 locus TT type individuals have higher total litter size phenotype value, and the estimated breeding values of total litter size, survival litter size and healthy litter size are higher.
As described aboveFSHR GG type individual at c.532 siteFSHR c.532 G>The bases of the polymorphic site A are G; as described aboveFSHR TT type individuals at position 1166FSHR c.1166 C>And the basic groups of the T polymorphic sites are all T pigs.
As described aboveFSHR c.532 G>The GenBank Accession Number XM-021085881.1 mutation site of the A site is Ssc.3: 91750608;FSHR c.1166 C>the GenBank Accession Number XM-021085881.1 mutation site of the T site is Ssc.3: 91773610, and belongs to the scope of the patent claims.
The invention disclosesFSHR c.532 G>A site andFSHR c.1166 C>the T site joint identification and auxiliary selection method belongs to the protection scope of the invention. The method comprises the following steps:FSHR c.532 G>a site andFSHR c.1166 C>all results and applications obtained by T site joint analysis; in particularFSHR c.532 G>A site andFSHR c.1166 C>the T site and GATT combined individual has better overall litter size, viable litter size and healthy litter size phenotype indexes and comprehensive indexes for estimating breeding value. The total litter size in the phenotype index reaches 16, the number of alive litters reaches 13.72 and the healthy litters reaches 13.33; the total litter size in the breeding value index is estimated to reach 0.54, the number of alive litters reaches 0.49 and the number of healthy litters reaches 1.11.FSHR c.532 G>A site andFSHR c.1166 C>the T site combined GGTT individual has better overall litter size, viable litter size and healthy litter size phenotype indexes and comprehensive indexes of estimated breeding value. The total number born in the phenotype indexes reaches 16, the number born alive reaches 14.5 and the number born healthy reaches 14; the total litter size of the breeding value index is estimated to reach 0.37, the number of live litter size is estimated to be 0.19, and the number of healthy litter size is estimated to reach 0.96. GATT type individuals and GGTT individuals are all superior individuals and are used for molecular breeding of reproductive performance. Note that the method is applicable to Changbai pigs, Dabai pigs and Duroc pig breeds.
The above-mentioned joint analysis meansFSHR The GA-type individuals at the c.532 site areFSHR c.532 G>The bases of the polymorphic site A are G and A, and simultaneouslyFSHR c.1166 locus TT type individualsFSHR c.1166 C>The basic groups of the T polymorphic sites are all T pigs;FSHR GG type individual at c.532 siteFSHR c.532 G>The bases of the polymorphic site A are G, and simultaneouslyFSHR c.1166 locus TT type individualsFSHR c.1166 C>And the basic groups of the T polymorphic sites are all T pigs. A kind ofFSHR c.532 G>A site andFSHR c.1166 C>application of T locus joint analysis in breeding practice;FSHR c.532 G>a site andFSHR c.1166 C>and (3) carrying out early seed reserving and breeding on the GATT type individual and the GGTT type individual combined by the T locus. In the process of preparing the matching plan: the offspring of the two sites which are the male parent and the female parent (or the female parent and the male parent) of the GATT type individual and the GGTT type individual can be used for reserving seeds, and the hybridization of the male parent and the female parent with the same type is avoided. Note that the method is applicable to Changbai pigs, Dabai pigs and Duroc pig breeds.
FSHR c.532 G>A site andFSHR c.1166 C>in the T site combined analysis method, a pig DNA sample is used as a template, and the primers are used for PCR amplification. Obtaining PCR products, sequencing and comparing to obtainFSHR c.532 G>A site andFSHR c.1166 C>and (5) breeding the base type of the T site. The primer pair and the method provided by the invention can be used for carrying out early-stage screening on reproductive performance and breeding and matching plan on reproductive performance of the sow to be selected. The primer and the method can realize the early breeding of the high-yield piglet performance of the sow, shorten the breeding time of excellent pigs, save the breeding and feeding cost, and improve the total number born, the number born alive and the number born healthy of the pigs. The method has the advantages of high accuracy, simple operation and low detection cost, and has high practical application value in pig breeding.
The invention mainly solves the molecular breeding problems of total litter size, live litter size and healthy litter size of sows. Key point examinationObserve and makeFSHR c.532 G>A site andFSHR c.1166 C>the method, effect and application of the breeding of single SNP locus and the combined breeding of double SNP loci on the total number born, number born alive and number born healthy of the sows at the T locus. The main difficulty lies in the discovery of mutation sites, the breeding of single SNP sites and the combined breeding and application of double SNP sites.
Compared with the prior art, the method and the primers for identifying the farrowing performance of the sows disclosed by the invention have the positive effects that:
（1）FSHRand the total litter size, the survival litter size and the healthy litter size of the GG type individuals at the position of c.532 are better.FSHRc.1166 locus TT type individual has better overall litter size, alive litter size and healthy litter size.
（2）FSHR c.532 G>A site andFSHR c.1166 C>the T site is better combined with the overall litter size, the number born alive and healthy litter size phenotype indexes and the comprehensive indexes of estimated breeding value of the GATT type individual and the GGTT type individual.
Drawings
FIG. 1 shows a schematic view of aFSHR c.532 G>Sequencing the A site base mutation;
FIG. 2FSHR c.1166 C>Sequencing results of T site base mutation;
FIG. 3FSHR c.532 G>Mass plot of site a; wherein (1) is the mass spectrum result of 82 pigs, and (2) is the mass spectrum result of 289 pigs;
FIG. 4FSHR c.1166 C>Mass plot of T site; wherein (1) is the mass spectrum result of 75 pigs, and (2) is the mass spectrum result of 266 pigs.
Detailed Description
The invention is described below by means of specific embodiments. Unless otherwise specified, the technical means used in the present invention are well known to those skilled in the art. In addition, the embodiments should be considered illustrative, and not restrictive, of the scope of the invention, which is defined solely by the claims. It will be apparent to those skilled in the art that various changes or modifications in the components and amounts of the materials used in these embodiments can be made without departing from the spirit and scope of the invention. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1
The pig sample, reproductive performance data and pedigree data are from a French white pig of Guangxi first-herd pig breeding limited company.
First, pigFSHR c.532 G>A site andFSHR c.1166 C>and (4) determining the mutation site of the T site.
(1) Collecting a pig ear sample, and extracting the DNA of the pig ear tissue by using a DNA extraction kit.
(2) According to the pigFSHRGene GenBank Accession Number XM-021085881.1, and designing and synthesizing primer.FSHRThe primers of (a) were as follows:
(3) taking pig ear DNA as a template, and utilizing an upper primer and a PCR amplification system: 200 ng of genome DNA, 5 muL of 10 XPCR amplification buffer solution, 10 mM of dNTPs final concentration, 50 ng of upstream primer and 50 ng of downstream primer, 0.75 mmol/L of Taq DNA polymerase and the addition of ddH2O to the system to 50 muL.
PCR amplification procedure: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30 s, annealing at 58 ℃ for 30 s, and extension at 72 ℃ for 30 s for 35 cycles; finally, extending for 10 min at 72 ℃; infinity at 4 ℃. After amplification, products 1 and 2 are produced.
(4) The sequencing and sequencing results were as follows:
sequencing the PCR amplification products 1 and 2, and obtaining a sequencing peak image after sequencing as shown in the following figure 1-2. As indicated by the arrow, on chromosome Ssc 3:91750608 and Ssc 3: mutations at positions 91773610 with G > a and C > T are shown in table 2 below.
TABLE 2 mutation site position information and mutation type information
(5) Large population sequencing Using flight Mass Spectrometry sequencing (Small population may not use this part)
Mass spectrometry all primers are shown in table 3 below. All site mutation information was obtained, as shown in FIGS. 3-4 below.
TABLE 3 mutation site location Mass Spectrometry primer information
(6) Correlation analysis is carried out on the phenotype values and estimated breeding values of the pig genotype, the total number born, the number born alive and the number healthy born of the sow by a least square method. The specific method refers to (cattail, Daoyusan, Zhang Bo, Zhang Long super, Guo Liang, Wang Lixian. the correlation analysis of polymorphic locus of HLCS gene of Duroc pigs and growth and development characters [ J ]. Chinese animal husbandry veterinarian, 2020, 47 (06): 1800) 1808. cattail, Zhang Long super, Wang Liang just, Shihui Bi, Wang Lixian. Duroc pig feed utilization efficiency character genetic parameter estimation and correlation analysis [ A ]. Chinese animal husbandry veterinary medical society [ C ]. Chinese animal husbandry veterinary medical society 2015 annual meeting 2015: 1.). Genetic parameter estimation the estimated breeding value is calculated by using the animal model multiple-trait constraint maximum likelihood method (DFREML).
The model used for the correlation analysis was:
Y＝μ＋G＋P＋W＋e
wherein Y is the measured character, mu is the overall mean, and G is the genotype effect; p is the number of births; w the age of the day of initial matching is a covariate; e is the random error.
(7) The unit site association results are as follows:
TABLE 4FSHR c.532 G>Correlation analysis result of A gene mutation site and phenotype character of breeding performance of French-line Changbai pigs
The same superscript different letters indicate significant (< 0.05) differences, and the same letters indicate insignificant differences.
TABLE 5FSHR c.532 G>EBV character correlation analysis result of A gene mutation site and breeding performance of French-breed Changbai pigs
TABLE 6FSHR c.1166 C>Correlation analysis result of T gene mutation site and phenotype character of breeding performance of French-line Changbai pigs
TABLE 7FSHR c.1166 C>T gene mutation site and EBV character correlation analysis result of breeding performance of French-line Changbai pigs
The same upper label and different letter represent significant differences (P <0.05) and the same letter indicates that the difference is not significant.
As is shown by the tables 4-7,FSHR c.532 G>the total litter size, the viable litter size and the healthy litter size phenotype value of the individual with the type GG at the mutation site of the A gene are higher than those of the individuals with other types.FSHR c.1166 C>The phenotype values of total litter size and healthy litter size, total litter size, viable litter size and healthy litter size of TT type individuals at the T gene mutation sites are higher than those of other types.
(8) The results of the combined analysis of the mutation sites are shown below
TABLE 8FSHR c.532 andFSHR c.1166 locus and phenotype character joint analysis result of breeding performance of French-line Changbai pig
The same upper label and different letter represent significant differences (P <0.05) and the same letter indicates that the difference is not significant.
TABLE 9FSHR c.532 andFSHR c.1166 locus and EBV character joint analysis result of breeding performance of French-line Changbai pig
The same upper label and different letter represent significant differences (P <0.05) and the same letter indicates that the difference is not significant.
As can be seen from tables 8 to 9,FSHR c.532G>a site andFSHR c.1166 C>the T locus and GATT combination has better overall indexes of total live litter size, live litter size and healthy litter size of individuals and estimated breeding value. The total litter size in the phenotype index reaches 16, the number of alive litters reaches 13.72 and the healthy litters reaches 13.33; the total litter size in the breeding value index is estimated to reach 0.54, the number of alive litters reaches 0.49 and the number of healthy litters reaches 1.11. As GGTT had 1 individual, dunken analysis of variance was not performed. GGTT type is 1 individual, total number born reaches 16 in phenotype index, number born alive reaches 14.5 and number born healthy reaches 14; the total litter size in the breeding value index is estimated to reach 0.37, the number of alive litters reaches 0.19 and the number of healthy litters reaches 0.96.
GGTT type individuals and GATT type individuals are both more advantageous individuals, and key breeding can be carried out towards the two directions. The offspring produced by the hybridization of the GATT type individuals and the GGTT type individuals are also of the GGTT type and the GATT type, and both the male and the female can be reserved with emphasis on the species.
SEQUENCE LISTING
<110> Tianjin college of agriculture
<120> method and primer for identifying farrowing performance of sows
<160> 12
<170> PatentIn version 3.5
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Claims (5)
1. A composition for early breeding of sow with high farrowing performanceFSHR c.532 G>A andFSHR c.1166 C>a PCR primer pair of T and an amplification primer pair identified by mass spectrometry; wherein the amplification comprisesFSHR c.532 G>A andFSHR c.1166 C>the PCR primer pair of T is shown in SEQ ID NO 1-4; the amplification primer pair identified by mass spectrum is shown in SEQ ID NO. 5-10:
1-4 of SEQ ID NO
The amplification primer pair identified by mass spectrum is shown as SEQ ID NO: 5-10:
FSHR c.532 G>the position of the GenBank Accession Number XM-021085881.1 mutation site of the A site is Ssc.3: 91750608;FSHR c.1166 C>the position of the GenBank Accession Number XM-021085881.1 mutation site of the T site is Ssc.3: 91773610.
2. A kit for identifying the reproductive performance of pigs, which is characterized by comprising the primer pair of claim 1 and amplification primers identified by mass spectrometry; the identification of the reproductive performance of the pigs comprises the following steps: total litter size, viable litter size, and healthy litter size.
3. A single SNP locus identification method, wherein the method comprises the steps of utilizing the primer pair of claim 1, carrying out PCR amplification by taking DNA of each pig as a template, carrying out common sequencing on the product obtained by amplification, and the sequencing sequence is shown in SEQ ID NO. 11-12; or directly sequencing the DNA of the pig by using a mass spectrum primer; sequencing to obtain a sequencing plot and a sequencing typing result;
obtaining the plot graph and sequencing typing result obtained by sequencing peak graph, sequencing sequence or mass spectrum sequencingFSHRc.532 G>A site andFSHR c.1166 C>the type of mutation at the T site; the mutation types of the 2 sites can be used for molecular breeding of the reproductive performance of later-stage sows, and the characteristics and the breeding principle of the sites are as follows:FSHR GG at c.532 siteThe total number born, the number born alive and the number born healthy of the type individual,FSHR c, breeding and selecting total litter size, alive litter size and healthy litter size of TT type individuals at the 1166 locus.
4. A kind ofFSHR c.532 G>A site andFSHR c.1166 C>the T site joint identification method adopts the primer pair of claim 1, takes the DNA of each pig as a template to carry out PCR amplification, and carries out common sequencing on the product obtained by amplification or directly carries out sequencing on the DNA of the pig by using a mass spectrum primer; sequencing to obtain a sequencing peak image, a sequencing sequence or a plot obtained by mass spectrometry sequencing, and a sequencing typing result; the sequencing peak diagram and the sequencing sequence are shown in SEQ ID NO. 11-12 or the plot obtained by mass spectrometry and the sequencing typing result show that each pigFSHR c.532 G>A site andFSHR c.1166 C>the mutation types of the T locus, namely the mutation types of the 2 loci can be used for molecular breeding of the reproductive performance of later-stage sows, and the characteristics and the breeding principle of the loci are as follows:FSHR c.532 G>a site andFSHR c.1166 C>all results obtained by T site joint analysis; whereinFSHR c.532 G>A site andFSHR c.1166 C>breeding and applying total number born, living number born and healthy number born of the T site combined with GATT individuals;FSHR c.532 G>a site andFSHR c.1166 C>and (3) breeding and application of total number born, living number born and healthy number born of the T site combined GGTT individuals.
5. The method of claim 1 comprisingFSHR c.532 G>A andFSHR c.1166 C>the PCR primer pair of the T, the amplification primer pair identified by the mass spectrum and the result are applied in the breeding process of the pigs.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113736890A (en) * | 2021-07-30 | 2021-12-03 | 华南农业大学 | SNP molecular marker related to Jian' er number and survival rate and application thereof |
| CN113736890B (en) * | 2021-07-30 | 2023-07-14 | 华南农业大学 | SNP molecular marker related to healthy number and living number rate and application thereof |
| CN114085914A (en) * | 2021-10-27 | 2022-02-25 | 华南农业大学 | SNP molecular marker located on pig chromosome 9 and related to Jian 'er number and Jian' er rate and application |
| CN114085914B (en) * | 2021-10-27 | 2023-07-14 | 华南农业大学 | SNP molecular marker located on chromosome 9 of pig and related to litter size and application thereof |
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