CN105755131B - Genetic marker associated with pork quality traits and carcass traits - Google Patents
Genetic marker associated with pork quality traits and carcass traits Download PDFInfo
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Abstract
The invention belongs to the technical field of pig genetic marker preparation, and particularly relates to a genetic marker related to pig meat quality and carcass traits, which is obtained by screening pig BMP-3b gene, wherein the sequence of the genetic marker is shown as SEQ ID NO:1 and figure 4, and A/G base mutation occurs at a base position 425 of the sequence to cause AlwNI-RF L P polymorphism.
Description
Technical Field
The invention belongs to the technical field of molecular marker-assisted selection of pigs, and particularly relates to a genetic marker associated with pork quality and carcass traits. The genetic marker is separated from a CDS region fragment of a BMP-3b gene, and the application of the genetic marker in pig auxiliary selection correlation analysis is also included in the invention.
Background
Traditional breeding mainly depends on phenotype selection, and multiple factors such as environmental conditions, gene interaction, genotype and environment interaction influence phenotype selection efficiency, so that the traditional breeding efficiency is low, and the breeding work progresses slowly. Breeders continually explore and apply genetic markers to improve the selection efficiency and the breeding predictability of breeding in long-term breeding practice. Genetic markers are markers of matter that can be used to distinguish between an individual or population of organisms and their particular genotype and that can be stably inherited, including morphological, cytological, biochemical and molecular markers. At present, molecular markers based on DNA polymorphism are generally regarded by people in molecular marker-assisted selection (MAS) breeding. Molecular marker assisted selection is a new technology generated along with the rapid development of modern molecular biology technology, and can directly select genotypes through molecular markers so as to carry out molecular breeding. The molecular marker assisted selection has high accuracy, can be used for early seed selection, shortens the generation interval, is combined with conventional breeding, and can greatly accelerate the breeding process.
The PCR technology is used for RF L P analysis, namely PCR-RF L P, the technology can use limited DNA as a template, a certain region of a genome is specifically amplified by using a primer, then a PCR product is digested by using a restriction enzyme, and the polymorphism is detected by electrophoresis.
Bone morpholinogenic protein-3b (BMP-3b), also known as GDF10, belongs to a TGF β superfamily member, was first isolated and cloned from rat femur, and was mainly expressed in various Bone tissues, cartilage tissues, Bone tissues, trachea, lung, brain, etc. (1996) BMP-3b gene was mainly associated with the development of cartilage or Bone and embryonic development (1999,2003,2005) Adoligbe C, etc. found that three SNP sites exist in bovine BMP-3b gene exons, forming synonymous or missense mutations, analysis found that these SNP sites are significantly associated with bovine ulnar measurement traits (2011) and further studies showed that BMP-3b gene has a higher expression level in adipose tissues, and the gene was in the form of a non-covalent complex in adipose cells (1995,2011), the expression level in preadipocyte cells was higher than that of mature adipose cells, and is capable of inhibiting adipose cell (2011) and results of this research show that BMP-3b gene plays a role in adipose cells, and further that there is a certain difference in pig adipose tissue differentiation and pig carcass traits, and two pig carcass traits are presumed that there are two pig carcass traits that are two pig traits that are related to pig traits, pig traits that are related to pig meat quality traits.
Disclosure of Invention
The invention aims to screen a genetic marker related to pork quality traits and carcass traits, search SNP sites by cloning CDS sequences of BMP-3b genes of pigs, establish a corresponding SNP detection method, analyze the relation between the SNP sites and the pork quality traits and the carcass traits and provide a useful marker for marker-assisted selection of the pork quality traits and the carcass traits.
The invention is realized by the following technical scheme:
the BMP-3b gene of the big white pig and the Meishan pig obtained by the invention comprises a first intron and a second exon internal fragment, the fragment length is 546bp, the nucleotide sequences are respectively shown in sequence tables SEQID NO:1 (big white pig, shown in figure 2) and SEQ ID NO:2 (Meishan pig, shown in figure 3), 1 nucleotide polymorphism (SNP) site positioned in the amplified fragment is provided by performing blast comparison on the sequences of the 2 pig species, and the SNP site is shown in figure 4. The mutation site is particularly 546bp after the translation initiation site ATG (namely, mutation of one allele occurs at the 425 th base of the sequences shown in SEQ ID NO:1, SEQ ID NO:2 and FIG. 4), and the mutation site is named as G546A in the following.
Local pig breeds of China, namely Meishan pigs and external white pigs are selected as test materials, genomic DNA is extracted from pig blood, and a primer pair is designed according to a pig BMP-3b genomic sequence, wherein the sequence of the primer pair is as follows:
a forward primer F: 5'-TCACTGAAAATAGCAAAGGT-3' (SEQ ID NO: 3),
reverse primer R: 5'-ACTGGGCAGACAGGAGAAGC-3' (SEQ ID NO: 4);
the primer pair is utilized to carry out PCR amplification, purification of PCR products, clone sequencing and sequence comparison analysis.
Screening to obtain a genetic marker related to pork quality trait and carcass trait, wherein the nucleotide sequence of the genetic marker is as follows:
TCACTGAAAATAGCAAAGGTGCCCTCATCAGTTCAATGTGAGGATTACATGAGATAAGGAAAGCCTAGTTGGCCTGGAAGTGCTCCCTCAGTGGGACTTCCTCCTCCACTCCCTGGATGGGAAACCCTATGGCTATGGGGCCTCCCCAGGAAACTGCCTAACAGTCTATGGTCTGTCCTTCCCTCGTAGAAGTGGTCAACCAGAAGGCTGTGTATTTCTTCAACCTGACTTCCATGCAGGACTCAGAAATGATCCTCACAGCCACATTCCATTTTTACTCGGAGCCACGGTTGCCCCGGGCACGTGAGATACCATGCAAGCAGCGGGCCAAGAATGCATCATGCCGCCTGCTGCCCCTGGGTCCACCTGCACGTCAGCACTTGCTCTTCCGCAGCCTCTCCCAGAACACAGCTACACAGGGGCTRCTCCGTGGAGCCATGACCTTGCCGCCCCCACCTCGGGGCCTGTGGCAGGTCAAGGACATCTCCCCCATCGTCAAGGCCGCCCGCCGAGATGGTGAGCTTCTCCTGTCTGCCCAGT
r in the above sequence is A or G, which mutation results in the AlwNI-RF L P polymorphism
The invention provides a method for screening a genetic marker related to pork quality traits and carcass traits, which comprises the following steps:
extracting genome DNA from pig blood, designing a primer pair according to a pig BMP-3b genome sequence, wherein the sequence of the primer pair is a forward primer F: 5'-TCACTGAAAATAGCAAAGGT-3' (shown as SEQ ID NO: 3) and a reverse primer R: 5'-ACTGGGCAGACAGGAGAAGC-3' (shown as SEQ ID NO: 4), carrying out PCR amplification in the pig genome DNA by using the primer pair, carrying out enzyme digestion and typing on the obtained PCR amplified fragment by using AlwNI enzyme to obtain a nucleotide sequence (a large white pig sequence SEQ ID NO:1 and a Meishan pig sequence SEQ ID NO: 2) which causes AlwNI-RF L P polymorphism, wherein a base mutation of A/G exists at the 425 th site of the base positions shown by the SEQ ID NO:1 and the SEQ ID NO:2, and carrying out association analysis on the pork quality and carcass traits by using the mutation sites of the SEQ ID NO:1 and the SEQ ID NO:2 as genetic markers.
The invention also provides an AlwNI-RF L P genotyping method for detecting the sequence G546A variation.
The invention further provides application of the correlation analysis for determining the correlation between different genotype individuals and pork quality traits and carcass traits by using an AlwNI-RF L P method.
The more detailed technical scheme is described in the detailed description.
Drawings
SEQ ID NO. 1 of the sequence Listing is the BMP-3b nucleotide sequence of a white pig of a foreign consanguineous pig variety, i.e., a nucleotide sequence as a genetic marker of the present invention (taking a white pig as an example). Wherein an allelic mutation site occurs at base 425 of the sequence, namely, the allelic mutation site is from 'A' to 'G'.
The sequence table SEQ ID NO 2 is the BMP-3b nucleotide sequence of a Meishan pig, a local pig breed of endemic pig in China; is also a nucleotide sequence that is another genetic marker of the present invention. Wherein an allelic mutation site occurs at base 425 of the sequence, namely the mutation from 'G' to 'A'.
The sequence tables SEQ ID NO 3 and SEQ ID NO 4 are sequences of a primer pair for amplifying BMP-3b gene, and the sequences of the primer pair are also primers for detecting the genetic marker of the present invention.
FIG. 1: the technical flow chart of the invention.
FIG. 2 shows the nucleotide sequence of BMP-3b gene of a white pig of a foreign consanguineous pig breed, wherein the A/G mutation site at base 425 in the sequence is a specific site causing the polymorphism AlwNI-RF L P.
FIG. 3 is the nucleotide sequence of BMP-3b gene of Meishan pig, a endemic pig variety in China, and the G/A mutation site at base 425 in the sequence is the specific site causing the polymorphism AlwNI-RF L P.
FIG. 4 shows that the genetic marker related to pork quality and carcass traits obtained by screening of the present invention has a sequence length of 540bp, and the 'R' at base 425 of the sequence is A or G, which results in the specificity of AlwNI-RF L P polymorphism.
FIG. 5 shows the detection result of BMP-3b gene AlwNI-RF L P of the present invention at 2.5% agarose concentration, and the accompanying drawing marks indicate that in the figure, lane M is D L2000 marker, lane 1 and lane 2 are GG type, 424bp and 116bp, lane 3 and lane 4 are AA genotype and 540bp, lane 5and lane 6 are AG genotype, 540bp, 424bp and 116 bp.
Detailed Description
Example 1: acquisition of pig BMP-3b gene DNA fragment and establishment of SNP detection method
Two types of pig breeds, namely a foreign consanguineous pig breed 'big white pig' and a Chinese local consanguineous pig breed 'Meishan pig' (the biological material is from a competitive pig farm of the university of agriculture in China and is a common breed) are selected as materials in the test, and the following primer pairs are designed according to the genome sequence (the login number Gene ID:100519926) of the BMP-3b Gene of the pig, and the specific sequences are as follows:
a forward primer F: 5'-TCACTGAAAATAGCAAAGGT-3' the flow of the air in the air conditioner,
reverse primer R: 5'-ACTGGGCAGACAGGAGAAGC-3', respectively;
the above primer pairs are used for PCR amplification in genome DNA of 'big white pig' and 'Meishan pig'.
The PCR reaction system is shown in Table 1.
TABLE 1 PCR reaction System
The PCR reaction conditions are shown in Table 2.
TABLE 2 PCR reaction conditions
The obtained PCR products of the two genotype pig species are purified and cloned, and then sequence determination is carried out, and the sequencing work is finished by Shanghai biological engineering technical service company Limited. The analysis of blast alignment revealed the presence of an A/G mutation (interchange) at base 425 in the sequence, which caused the polymorphism of the site for cleavage by AlwNI enzyme.
Mu.l of the PCR product was taken and added to 0.5. mu.l of restriction enzyme AlwNI (5U/. mu.l), 1. mu.l of 10 × buffer and 3.5. mu.l of dH2And O, carrying out enzyme digestion at 37 ℃ for 20min, detecting the enzyme digestion product by 2.5% agarose gel electrophoresis, and observing and recording the enzyme digestion result under an ultraviolet lamp. When the base of the mutation is G, the enzyme cutting site exists, the enzyme cutting result detects that two bands are in GG type (424bp +116bp), when the base of the mutation is A, the site is not identified, the enzyme cutting result detects that one band is in AA type (540bp), and when A and G both exist, the enzyme cutting result detects that 3 bands are in AG type (540bp +424bp +116 bp). The results are shown in FIG. 3.
Example 2: correlation analysis and application of genetic marker in pork quality traits and carcass traits
In order to determine whether SNP in CDS region of BMP-3b gene of pig is related to phenotypic difference of pig, 399-head large white pig × Meishan pig F constructed by pig genetic breeding key laboratory of Ministry of agriculture of Huazhong university of agriculture is selected2A generation resource population (L iu et. Association of MYF5and MYOD1 gene polymorphisms and medium quality traits in L area White × Meiishan F2 pig locations. biochem Genet.2008,46: 720) is used as a test material, a conventional AlwNI-RF L P method is adopted for polymorphism detection, and the correlation between different genotypes of a CDS region of a porcine BMP-3b gene and pork quality traits and carcass traits is analyzed, SAS statistical software G L M program is adopted for single-marker variance analysis, and the model is as follows:
meat quality character analysis model: y isij＝μ+Gi+Sj+Yl+bijkl+eijkl
Carcass trait analysis model: y isij＝μ+Gi+Sj+Yl+βcovijkl+eijkl
In the above model formula: y isijIs a phenotypic value, μ is the mean value, GiThe genotype effect (including gene additive effect and dominant effect; additive effect uses-1, 0 and 1 respectively represent GG, AG and AA genotypes, dominant effect uses 1 represent GG and AA genotypes, and-1 represents AG geneType); sj、YlFor a fixed effect, sex, annual effect, respectively; bijklRegression coefficients for slaughter age, β covijklRegression coefficient for slaughter body weightijklIs the residual effect.
The correlation analysis results are shown in tables 3 and 4. Wherein, table 3 shows the statistical analysis results of different genotypes and pork quality traits. Table 4 shows the statistical analysis results between different genotypes and the pig carcass traits. The following were used:
TABLE 3 Association analysis of polymorphism of porcine BMP-3b gene G546A site and meat quality traits
Description of table 3: the above numerical values are the least square mean value plus or minus standard error; the same row contains the same letter with obvious difference in representation and different lower case letters with obvious difference (P)<0.05), the uppercase alphabet showed significant difference (P)<0.01); gene effect*Represents p<0.05; negative values indicate a reduced phenotype value for the a allele.
TABLE 4 Association analysis of polymorphism of porcine BMP-3b gene G546A site and carcass traits
Description of table 4: the above numerical values are the least square mean value plus or minus standard error; the same row contains the same letter with obvious difference in representation and different lower case letters with obvious difference (P)<0.05), the uppercase alphabet showed significant difference (P)<0.01); gene effect*Represents p<0.05; negative values indicate a reduced phenotype value for the a allele.
Among individuals detected in pigs in 2000, 2003 and 2004, × Meishan pigs in 112 years in 2000 have 17 AA genotypes accounting for 15.17%, AG genotypes accounting for 49 and 43.75%, GG genotypes accounting for 46 and 40%, F2 individuals in 138-year × Meishan pigs in 138-year yellow pig in 2003 have 9 AA genotypes accounting for 7.31%, AG genotypes accounting for 62 and 67, and F2 individuals in 48.43.2004 in × Meishan pigs in F2, 15 and 10.06%, AG genotypes accounting for 61 and 40.93%, and GG genotypes accounting for 73 and 48.99%.
As can be seen from Table 3, for the meat quality traits, BMP-3b gene G546A site has very significant correlation (P <0.01) with longisimus dorsi color value, biceps femoris color value and longisimus dorsi marble score and significant correlation (P <0.05) with intramuscular fat. Wherein the a allele has a significant additive effect on increasing muscle flesh color; while the G allele had a significant additive effect on increasing intramuscular fat content and muscle marbling score.
As can be seen from Table 4, the BMP-3b gene G546A site has significant correlation with average backfat thickness, eye muscle height, leaf fat weight and the like for carcass traits (P < 0.05). Wherein the a allele has a significant additive effect on increasing eye muscle height; the average backfat thickness and the weight average of the suet of AG genotype individuals are obviously higher than those of AA type and GG type, so that the gene is supposed to have obvious dominant effect on the average backfat thickness and the suet weight.
The 9 middle and outer breeds of swine were genotyped, and the frequency distribution of the genotypes of these swine breeds is shown in table 5.
Genotype frequency distribution of pigs of Table 59 middle and outer breeds
Description of table 5: the breeds in Table 5 are native pig breeds in China, except that the white pig is a foreign pig breed.
Primary references
1.Takao M,Hino J,Takeshita N,Konno Y,Nishizawa T,Matsuo H,KangawaK.Identification of rat bone morphogenetic protein-3b(BMP-3b),a new member ofBMP-3.Biochemical&Biophysical Research Communications 1996,219(2):656-662.
2.Hino J,Matsuo H,Kangawa K.Bone morphogenetic protein-3b(BMP-3b)geneexpression is correlated with differentiation in rat calvarialosteoblasts.Biochemical&Biophysical Research Communications 1999,256(2):419-424.
3.Hino J,Nishimatsu S-i,Nagai T,Matsuo H,Kangawa K,NohnoT.Coordination of BMP-3b and cerberus is required for head formation ofXenopus embryos.Developmental Biology 2003,260(1):138-157.
4.Chandar N,Swindle J,Szajkovics A,Kolman K.Relationship of bonemorphogenetic protein expression during osteoblast differentiation to wildtype p53.Journal of Orthopaedic Research 2005,23(6):1345-1353.
5.Hino J,Miyazawa T,Miyazato M,Kangawa K.Bone morphogenetic protein-3b(BMP-3b)is expressed in adipocytes and inhibits adipogenesis as a uniquecomplex.International Journal of Obesity 2011,36(5):725-73。
Claims (5)
1. A genetic marker associated with pork quality traits and carcass traits, having a nucleotide sequence as shown below: TCACTGAAAATAGCAAAGGTGCCCTCATCAGTTCAATGTGAGGATTACATGAGATAAGGAAAGCCTAGTTGGCCTGGAAGTGCTCCCTCAGTGGGACTTCCTCCTCCACTCCCTGGATGGGAAACCCTATGGCTATGGGGCCTCCCCAGGAAACTGCCTAACAGTCTATGGTCTGTCCTTCCCTCGTAGAAGTGGTCAACCAGAAGGCTGTGTATTTCTTCAACCTGACTTCCATGCAGGACTCAGAAATGATCCTCACAGCCACATTCCATTTTTACTCGGAGCCACGGTTGCCCCGGGCACGTGAGATACCATGCAAGCAGCGGGCCAAGAATGCATCATGCCGCCTGCTGCCCCTGGGTCCACCTGCACGTCAGCACTTGCTCTTCCGCAGCCTCTCCCAGAACACAGCTACACAGGGGCT
CTCCGTGGAGCCATGACCTTGCCGCCCCCACCTCGGGGCCTGTGGCAGGTCAAGGACATCTCCCCCATCGTCAAGGCCGCCCGCCGAGATGGTGAGCTTCTCCTGTCTGCCCAGT
R in the above sequence is A or G, resulting in the AlwNI-RF L P polymorphism.
2. Amplifying the pair of genetically labeled primers of claim 1, having the nucleotide sequences set forth below:
a forward primer: TCACTGAAAATAGCAAAGGT the flow of the air in the air conditioner,
reverse primer: ACTGGGCAGACAGGAGAAGC are provided.
3. A method for screening genetic markers associated with pork quality traits and carcass traits, comprising the steps of:
extracting genome DNA from pig blood, designing a primer according to a pig GDF10 gene genome sequence, wherein the sequence of the primer is shown as SEQ ID NO. 3 and SEQ ID NO. 4, carrying out PCR amplification on the pig genome DNA by using the primer, carrying out AlwNI enzyme digestion typing on a fragment amplified by the PCR to obtain a nucleotide sequence which is shown as claim 1 and causes AlwNI-RF L P polymorphism, and carrying out correlation analysis on pork quality traits and carcass traits.
4. The use of the genetic marker of claim 1 in pork quality trait and carcass trait assisted selection.
5. The primer pair of claim 2 is applied to pork quality trait and carcass trait auxiliary selection.
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