CN109897903B - Molecular marker for identifying breeding traits of white pigs based on FSH beta gene and application - Google Patents
Molecular marker for identifying breeding traits of white pigs based on FSH beta gene and application Download PDFInfo
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- CN109897903B CN109897903B CN201910342801.9A CN201910342801A CN109897903B CN 109897903 B CN109897903 B CN 109897903B CN 201910342801 A CN201910342801 A CN 201910342801A CN 109897903 B CN109897903 B CN 109897903B
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Abstract
The invention discloses a molecular marker for identifying the breeding traits of white pigs based on FSH beta gene and application thereof. The molecular marker is positioned on No.3 exon of porcine FSH beta gene, namely at No.3 exon regions 511, 617, 630, 652, 678, 735, 746 and 921 and is marked as g.511A > G, g.617A > G, g.630C > T, g.652C > T, g.678C > T, g.735C > T, g.746A > G and g.921A > G. The invention also discloses application of the molecular marker in breeding of pig species. The invention provides a new marking method for the molecular marker-assisted selection of the pig and provides a reference for the molecular breeding of the pig.
Description
Technical Field
The invention belongs to the field of animal molecular marker breeding, and particularly relates to a molecular marker for identifying breeding traits of white pigs based on FSH beta gene and application thereof.
Background
Pork is one of the most important meat economic animals in the world at present, China is used as a big pork consuming country, and the number of pigs stored in the market is the first in the world. In order to improve the production benefit, the reproductive performance of the pigs is always one of important indexes concerned by various large breeding enterprises, wherein the total litter size and the live litter size of the pigs are particularly critical.
The molecular marker assisted selection is used as an important means in modern livestock breeding engineering, so that the defects of the traditional phenotype-based breeding means are effectively overcome, the genetic interval is shortened, and the selection accuracy is improved. Molecular marker assisted selection presupposes the identification of candidate genes associated with a desired phenotypic trait, and the purposive detection of these genes determines the direction of breeding.
Porcine Follicle Stimulating Hormone (FSH) is a glycoprotein hormone secreted by anterior pituitary basophils, which binds to gonadal target cells viaSecondary messenger Ca2+And cAMP causes a series of biological reactions, promotes granular cell proliferation, intimal cell differentiation and secretion of the vacuole, induces the generation of receptors of luteinizing hormone and prolactin and the generation of aromatase, and stimulates the synthesis and release of estradiol, thereby coordinating and controlling the development and maturation of gametocytes and playing an important role in the process of animal reproduction. Follicle Stimulating Hormone (FSH) is composed of two subunits, α and β. Wherein the alpha subunit is common to follicle stimulating hormone, luteinizing hormone and thyrotropin, and is highly conserved within the same species and even in all mammals; the beta subunit dominates the biological properties of FSH.
The porcine follicle stimulating hormone beta subunit (FSH beta) gene is located on porcine chromosome 2, has a total length of 10.16kb and consists of 3 exons and 2 introns. The follicle stimulating hormone beta subunit (FSH beta) mainly stimulates the growth and development of follicles, influences the number of growing follicles, and stimulates the final maturation of follicles under the synergistic action of luteinizing hormone to induce ovulation.
Disclosure of Invention
In view of the above, the invention provides a molecular marker for identifying the reproductive traits of white pigs based on FSH beta gene and application thereof.
In order to solve the technical problems, the invention discloses a molecular marker for identifying the reproductive traits of white pigs based on an FSH beta gene, wherein the molecular marker is positioned on the No.3 exon of the FSH beta gene of the pigs, namely at the No.3 exon regions 511, 617, 630, 652, 678, 735, 746 and 921 and is marked as g.511A > G, g.617A > G, g.630C > T, g.652C > T, g.678C > T, g.735C > T, g.746A > G and g.921A > G.
The invention also discloses a primer pair for detecting the molecular marker, which comprises FSH beta-E3-F and FSH beta-E3-R, and the nucleotide sequences of the primer pair are respectively shown as SEQ ID NO.2 and SEQ ID NO. 3.
The invention also discloses a reagent or a kit containing the primer pair.
The invention also discloses a method for detecting the molecular marker, which comprises the following steps: carrying out PCR amplification by using the primer pair by using the genome DNA of the large white pig as a template to obtain a PCR product; and carrying out agarose gel electrophoresis on the PCR product.
Optionally, the PCR amplification reaction system is: 2 XTaq Master Mix 15. mu.L, DNA 1.5. mu.L, 10. mu.M forward primer 1.5. mu.L, 10. mu.M downstream primer 1.5. mu.L, ddH2O 10.5μL。
Alternatively, the PCR amplification reaction procedure is: pre-denaturation at 94 ℃ for 1.5 min; denaturation at 94 ℃ for 20s, annealing at 56.4 ℃ for 20s, and extension at 72 ℃ for 60s, and circulating for 30 times; finally, the extension is carried out for 5min at 72 ℃ and the product is stored at 4 ℃.
The invention also discloses application of the molecular marker in breeding of pig species.
Compared with the prior art, the invention can obtain the following technical effects:
8 mutation sites exist on the No.3 exon of the FSH beta gene, and the 8 mutation sites have correlation with the reproductive traits of pigs, particularly total litter size and live litter size, and are easy to detect. Therefore, the invention provides a molecular marker related to the porcine reproductive traits based on FSH beta gene, and provides a detection method of the molecular marker and application thereof in reproductive trait selection, thereby providing a new marker method for the porcine molecular marker-assisted selection and providing reference for the porcine molecular breeding.
Of course, it is not necessary for any one product in which the invention is practiced to achieve all of the above-described technical effects simultaneously.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention and not to limit the invention. In the drawings:
FIG. 1 is a sequence diagram of the mutation site of exon region A511G of pig FSH beta gene No.3 according to the present invention;
FIG. 2 is a sequence diagram of the mutation site of exon A617G of pig FSH beta gene No.3 according to the present invention;
FIG. 3 is a sequence diagram of the C630T mutation site of the number 3 exon of the porcine FSH beta gene of the present invention;
FIG. 4 is a sequence diagram of the C652T mutation site of the 3 exon of the porcine FSH beta gene of the present invention;
FIG. 5 is a sequence diagram of the mutation site of exon region C678T of pig FSH beta gene No.3 according to the present invention;
FIG. 6 is a sequence diagram of the C735T mutation site of the exon region of the 3-amino acid sequence of the porcine FSH beta gene of the present invention;
FIG. 7 is a sequencing diagram of the mutation site of exon region A746G of porcine FSH beta gene 3 according to the present invention;
FIG. 8 is a sequence diagram of the mutation site of exon region A921G of pig FSH beta gene No.3 according to the present invention;
FIG. 9 is a gel electrophoresis image of the PCR amplification product of exon 3 of porcine FSH beta gene of the present invention; wherein, the 1 st to 7 th holes and the 9 th to 15 th holes are amplification products of the 3 rd exon region of the FSH beta gene of the big white pig, the fragment size is 1022bp, and the 8 th hole is 2000 DNA marker;
FIG. 10 shows the exon nucleotide sequence of the amplified 3-mer of the porcine FSH beta gene of the present invention, the fragment size is 1022bp, the shaded area in the figure is the intron sequence, the base mutation is shown in the box, and the underlined area is the primer sequence.
Detailed Description
The following embodiments are described in detail with reference to the accompanying drawings, so that how to implement the technical features of the present invention to solve the technical problems and achieve the technical effects can be fully understood and implemented.
Example 1: acquisition of pig ESR gene fragments and detection of SNPs
The nucleotide sequence of the FSH beta gene No.3 exon nucleotide sequence related to the pig reproduction traits, which is applied as a molecular marker, is shown as SEQ ID NO.1, wherein gene mutation exists at 511bp, 617bp, 630bp, 652bp, 678bp, 735bp, 746bp and 921bp of the sequence. The SNPs have certain correlation with the reproductive traits (total litter size and live litter size) of the pigs, are easy to detect, and can be used as molecular auxiliary marker selection related to the reproductive traits of the pigs.
Fur samples and breeding production records (total litter size of initial and menstrual products and litter size of live births) of 201 different lines (American line, Dan line and additive line) of the big white pigs are collected from a herding farm of Tianren agriculture and animal husbandry in Leshan City of Sichuan province, and the whole genome DNA of the 201 big white pigs is extracted by using the kit.
Based on the FSH β gene nucleotide sequence (NM — 213875.1) published in GenBank database, No.3 exon regions in FSH β gene were aligned on line, specific primer design was performed on the above three regions using primer5.0 software (see table 1 for details), and PCR amplification reaction was performed using genomic DNA as a template. The reaction system is as follows: 2 XTaq Master Mix 15. mu.L, DNA 1.5. mu.L, upstream primer (10. mu.M) 1.5. mu.L, downstream primer (10. mu.M) 1.5. mu.L, ddH2O10.5. mu.L. Reaction procedure: pre-denaturation at 94 ℃ for 1.5 min; denaturation at 94 ℃ for 20s, annealing at 56.4 ℃ for 20s, and extension at 72 ℃ for 60s, and circulating for 30 times; finally, the extension is carried out for 5min at 72 ℃, and the amplification product is stored at 4 ℃.
TABLE 1 primer information of FSH beta Gene
mu.L of each PCR product was subjected to agarose gel electrophoresis, and the PCR product containing a single band was applied to Kyowas Biotech for sequencing. The sequencing results were analyzed by alignment using BioEdit software, respectively, to find the mutation sites present in the target fragment, and to record the mutation types of the corresponding sites (A511G, A617G, C630T, C652T, C678T, C735T, A746G, A921G) present in each individual (the sequencing maps corresponding to the mutation sites are shown in FIGS. 1-8). The nucleotide sequence of the exon 3 of the FSH beta gene, the corresponding primers and the mutation sites are shown in FIG. 10.
TABLE 2 genotype frequencies and allele frequencies of 8 SNPs sites in the FSH beta Gene
As can be seen from table 2, the 8 mutation sites of the FSH β gene are represented by three genotypes in the 201 big white pigs, respectively. The genotype frequency of the A511G site presents a trend of GG > AG > AA in American white, two genotypes GG > AA in Dan white and one genotype GG in Jia white, and the G allele frequency of the three lines of white pigs is greater than the A allele frequency; the genotype frequency of the A617G site shows a trend of AA > AG > GG in American white line and Dan white line, shows a trend of AA > GG > AG in plus white line, and the A allele frequency is greater than the G allele frequency in the white pigs of the three lines; the genotype frequency of the C630T locus shows the trend of CC > CT > TT in American white line and Dan white line, shows the trend of CC > TT > CT in plus white line, and the C allele frequency is greater than the T allele frequency in three lines of white pigs; the genotype frequency of the C652T locus shows the trend of TT > CT > CC in American white line and Dan white line, shows the trend of TT > CC > CT in plus white line, and the T allele frequency is greater than the C allele frequency in the white pigs of the three lines; the genotype frequency of C678T shows the trend of CC > CT > TT in American line, Dan line and Jia line white, and the C allele frequency is greater than the T allele frequency; the genotype frequency of the C735T locus shows the trend of TT > CT > CC in American white line and Dan white line, shows the trend of TT > CC > CT in plus white line, and the T allele frequency is greater than the C allele frequency in the white pigs of the three lines; the genotype frequency of the A746G locus shows a trend of AA > AG > GG in American white line and Dan white line, and shows a trend of AA > GG > AG in plus white line, and the A allele frequency is greater than the G allele frequency in all three line white pigs; genotype frequencies of A921G showed a trend of AA > AG > GG in American, Dan and Jia lineage large white, and the A allele frequencies were all greater than the G allele frequencies.
Example 2: application of molecular marker in breeding traits of white pigs
In order to establish the relationship between the ESR gene and the breeding traits of the large white pigs, 201 large white pigs in production (the herdship of the Leshan Tian, Sichuan) are selected as experimental materials, the production and breeding records of the 201 large white pigs are collected, and the experimental population is detected by adopting the SNPs detection mode mentioned in the embodiment 1. The GLM model in SAS8.0 software is adopted to carry out relevance analysis on 8 SNPs sites existing in FSH beta gene of each big white pig and the reproduction traits thereof, and the results are all expressed by LSM +/-standard error.
Performing correlation analysis on SNPs existing in FSH beta gene and reproduction traits (total number born and number born alive) by adopting a model as follows: y isij＝μ+Gi+Pj+eijWherein Y isijValues are recorded for reproductive traits, μ is population mean, GiFor genotype effects, PjFor fetal secondary effect, eijIs a random residual effect.
TABLE 3 Effect of different site genotypes of the FSH beta Gene on reproductive traits
As can be seen from table 3, in the 201 large white pig population, the born number of the GG genotype in the american individuals is significantly lower than the AG genotype and the AA genotype at the a617G, a746G, a921G sites in exon 3 of the FSH β gene (p < 0.05); at position C630T, the TT genotype has significantly lower born alive in american individuals than the CT and CC genotypes (p < 0.05); the litter size born alive in american individuals of CC genotype was significantly lower than CT genotype and TT genotype at C652T, C735T site (p < 0.05); at position C678T, the CT genotype has significantly higher born alive in american individuals than the CT genotype (p < 0.05); and the difference between other traits was not significant (p > 0.05).
Example 3: haplotype analysis
Haplotype analysis was performed on 8 SNPs in exon 3 of FSH β gene detected from 201 large white pig individuals, and the following results were obtained:
TABLE 4 haplotype types and frequencies of exon 3 FSH beta Gene
As is clear from Table 4, the 8 mutation sites detected constituted 12 haplotypes, of which the three haplotypes with the highest frequency were H1, H2, and H3, and were 0.642, 0.124, and 0.086, respectively.
While the foregoing description shows and describes several preferred embodiments of the invention, it is to be understood, as noted above, that the invention is not limited to the forms disclosed herein, but is not to be construed as excluding other embodiments and is capable of use in various other combinations, modifications, and environments and is capable of changes within the scope of the inventive concept as expressed herein, commensurate with the above teachings, or the skill or knowledge of the relevant art. And that modifications and variations may be effected by those skilled in the art without departing from the spirit and scope of the invention as defined by the appended claims.
Sequence listing
<110> Sichuan university of agriculture
<120> molecular marker for identifying breeding traits of white pigs based on FSH beta gene and application
<130> 2019
<141> 2019-04-26
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<170> SIPOSequenceListing 1.0
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<213> follicle stimulating hormone beta subunit (FSH beta)
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attcaatgcc tgtctcattt tgattaaata gaaacttctg taatacttta acctaactct 60
ctctctctcc cctgaatccc ttaggacctg gtatacaagg acccagccag gcccaacatc 120
cagaaaacat gtaccttcaa ggagctggtg tacgagaccg tgaaagtacc tggctgtgct 180
caccatgcag actccctgta tacgtatcca gtagccactg aatgtcactg tggcaagtgt 240
gacagtgaca gtactgactg caccgtgaga ggcctggggc ccagctactg ctccttcagt 300
gaaatgaaag aataaagagc agtggacatt tcatgcttcc tacccttgtc tgaaggacca 360
agacgtccaa gaagtttgtg tgtacatgtg cccaggctgc aaaccactat gagagacccc 420
actgatccct gctgtcctgt ggaggaggag ctccaggaat gcagagtgct ggggcctcag 480
tcctatcacc actcaaccct gtattctggg tctggttcca taagttttat tcggtctttt 540
ttttttttaa attactcaat gaattttatt acatttataa ttgtacaatg atcatcacag 600
cccaatttta taggatttcc atcccaaacc cccagcatag acccccatct cccaatctgt 660
ctcatttgga aaccataagt ttttcaaagt ccgtgagtca gtatctactc agtcttatta 720
ccttaaagac atgtgggtgt tttctgttta ataatcttag aaatcctctc aagacaggga 780
tatggaccca gaggaaggaa atgggctaag aatgggtgaa aggactaaat gcagcattct 840
cccactagac acagaagcct acaagagcag ggccagtctc tttgtcatga gtgtggcctc 900
aatacctagc acagtgacta gaattcagta agaaactcaa gaatggcttc cttaaggaaa 960
gtaagattgg aaatgtaggg ggtaggaaaa tactgaaaga agatgttgga ggctatgtga 1020
tg 1022
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catcacatag cctccaa 17
Claims (1)
1. The application of the molecular marker related to the litter size trait of the multiparous live birth of the large white pig in identifying the litter size trait of the multiparous live birth of the individual of the specific strain of the large white pig is characterized in that,
the molecular marker is positioned on the No.3 exon of the pig FSH beta gene and is marked as g.617A > G, g.630C > T, g.652C > T, g.678C > T, g.735C > T, g.746A > G and g.921A > G; particularly at 617, 630, 652, 678, 735, 746 and 921bp of SEQ ID NO.1, wherein,
the born number of the GG genotype in the American line individuals of the big white pig is obviously lower than the AG genotype and the AA genotype at g.617A > G, g.746A > G and g.921A > G;
at the g.630C > T locus, the born number of the TT genotype in the white pig American line individual is obviously lower than that of the CT genotype and the CC genotype;
at the g.652C > T and g.735C > T sites, the born number of the CC genotype in the American line of the big white pig is obviously lower than the CT genotype and the TT genotype;
at the g.678C > T site, the born number of the CT genotype born alive in big white pig Jia-series individuals is obviously higher than that of the CC genotype and the TT genotype.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1357625A (en) * | 2000-12-08 | 2002-07-10 | 李宁 | Partial ESR gene sequence, swine farrowing characteristic related ESR gene and polymorphic FSH-Beta gene determination technology |
| WO2015143691A1 (en) * | 2014-03-28 | 2015-10-01 | Kunming Institute Of Zoology, Chinese Academy Of Sciences | Methods and kits for detecting genetic markers for litter size in pigs |
| CN105567859A (en) * | 2016-03-14 | 2016-05-11 | 江苏农林职业技术学院 | FSHbeta-gene-based molecular marker related to porcine reproduction traits and detection method thereof, and application of molecular marker |
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2019
- 2019-04-26 CN CN201910342801.9A patent/CN109897903B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1357625A (en) * | 2000-12-08 | 2002-07-10 | 李宁 | Partial ESR gene sequence, swine farrowing characteristic related ESR gene and polymorphic FSH-Beta gene determination technology |
| WO2015143691A1 (en) * | 2014-03-28 | 2015-10-01 | Kunming Institute Of Zoology, Chinese Academy Of Sciences | Methods and kits for detecting genetic markers for litter size in pigs |
| CN105567859A (en) * | 2016-03-14 | 2016-05-11 | 江苏农林职业技术学院 | FSHbeta-gene-based molecular marker related to porcine reproduction traits and detection method thereof, and application of molecular marker |
Non-Patent Citations (2)
| Title |
|---|
| Polymorphism of FSHβ Subunit Gene in Six Pig Breeds;ZHANG Dongjie等;《Journal of Northeast Agricultural University (English Edition)》;20100330;第17卷(第1期);摘要，第62页最后一段到63页第一段 * |
| 猪PRLR和FSHβ基因多态性检测及其与繁殖性状的关联分析;范一萍等;《中国畜牧兽医》;20190320;第46卷(第3期);第774-781页 * |
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