CN116790764A - SNP molecular marker related to boar semen quality character in ATP11A gene, primer pair and application thereof - Google Patents
SNP molecular marker related to boar semen quality character in ATP11A gene, primer pair and application thereof Download PDFInfo
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- 108700028369 Alleles Proteins 0.000 claims description 14
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Abstract
The invention discloses a SNP molecular marker and a primer pair related to boar semen quality traits in an ATP11A gene and application thereof, and relates to the technical field of pig molecular markers. The boar semen quality traits are the characteristics of semen volume, sperm density, sperm motility and the like; the molecular marker is positioned in a promoter region of a pig ATP11A gene, the nucleotide sequence of the molecular marker is shown as SEQ ID NO.1 of a sequence table, and a421 bp position of the sequence has a mutation of A421-G421 bases. The invention also provides a primer pair for amplifying the molecular marker and a method for screening boar semen characters and/or breeding pigs by using the molecular marker. The invention firstly discovers that one SNP molecular marker in the pig ATP11A gene is related to boar semen quality character, so that the invention can be used for pig marker assisted selective breeding, namely provides a new application of one SNP molecular marker in the pig ATP11A gene in marker assisted breeding of boar semen quality character, and realizes early screening of boar reproduction character, and the screening method is simple and rapid.
Description
Technical Field
The invention relates to the technical field of pig molecular markers, in particular to a SNP molecular marker related to boar semen quality traits in an ATP11A gene, a primer pair and application thereof.
Background
The economic benefit of pig farms depends largely on the semen quality of boars. The semen quality-related character not only determines the conception effect of the sow, but also influences the semen storage and utilization efficiency. In recent years, molecular markers affecting reproductive performance of sows are reported successively from the aspect of genetics, however, researches on fertility of boars are still insufficient. Therefore, the key molecular genetic markers for controlling the boar semen quality traits are searched, and the key molecular genetic markers are used for molecular marker assisted breeding, so that the method has important significance for improving the reproductive traits of pigs and increasing economic benefits.
P4 type phosphotransferase 11A (ATP 11A) belongs to the P4 type phosphotransferase family, is an important biological functional protein, and participates in completing functional protein transport. ATP11A maintains the asymmetry of the membrane lipid bilayer and is involved in physiological processes such as apoptosis, secretion of substances, mitosis, host-virus cross-linking, and the like. Meanwhile, the distribution of phosphatidylethanolamine and phosphatidylserine on the cell membrane structure can be maintained, and the phosphatidylethanolamine and the phosphatidylserine are involved in the pathological process of various diseases.
However, no report is made on whether the ATP11A gene can improve the quality performance of boar semen at present, and the molecular mechanism is yet to be reported.
Disclosure of Invention
The invention aims to overcome the defects in the prior art, provides an SNP molecular marker in an ATP11A gene and related to boar semen quality character, a primer pair and application thereof, and explores that one SNP in an ATP11A gene promoter is related to the boar semen quality character, and can be used as a molecular marker for boar reproduction character screening and pig breeding.
In a first aspect, the invention provides an SNP molecular marker related to boar semen quality traits in an ATP11A gene, wherein the semen traits are single semen collection traits; the molecular marker is positioned in a promoter region of a pig ATP11A gene, the nucleotide sequence of the molecular marker is shown as SEQ ID NO.1 of a sequence table, and a421 bp position of the sequence has a mutation of A421-G421 bases.
In a preferred embodiment of the invention, the A or G base polymorphism site at 421bp in the sequence SEQ ID NO.1 is represented as three genotypes of AA, AG or GG, wherein the A allele is the dominant allele.
In a second aspect, the invention provides an application of the SNP molecular marker in boar semen trait screening and/or pig breeding.
In a third aspect, the present invention provides a primer pair for amplifying a molecular marker as described in the first aspect, the primer pair comprising: the nucleotide sequence of the upstream primer is shown as SEQ ID NO.2, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 3.
In a fourth aspect, the invention provides the use of a primer pair as described in the third aspect in boar semen trait screening and/or pig breeding.
In a fifth aspect, the present invention provides a kit for rapid breeding using the SNP molecular marker as set forth in the first aspect, comprising the primer pair as set forth in the third aspect.
In a sixth aspect, the invention provides a method for screening semen traits of a boar and/or breeding a pig, which is characterized by comprising the following steps:
s1, extracting genome DNA of a boar;
s2, carrying out PCR amplification by using the genomic DNA obtained in the step S1 as a template and adopting the primer pair according to the third aspect to obtain the molecular marker according to the first aspect and purifying;
s3, carrying out sequencing analysis on the molecular marker purified in the step S2, reserving an individual carrying the A allele at the 421bp position of the sequence, and eliminating the individual carrying the G allele.
In a preferred embodiment of the present invention, in step S1, genomic DNA is extracted from the ear margin tissue of a boar to be tested.
In a preferred embodiment of the present invention, in step S2, the PCR reaction system is 50. Mu.L, and the components in the system are: 100ng of genomic DNA, 25. Mu.L of PCR mix, 1. Mu.L of each of the upstream primer and the downstream primer, and adding ddH2O to make up to a total volume of 50. Mu.L; the PCR was run as follows: pre-denaturation at 94℃for 3min; denaturation at 94℃for 30s, annealing at 56℃for 30s, elongation at 72℃for 30s,35 cycles; extending at 72 ℃ for 10min; preserving at 4 ℃.
Compared with the prior art, the invention has the beneficial effects that: the invention firstly discovers that one SNP molecular marker in the pig ATP11A gene is related to the reproductive trait of pigs, in particular to the single semen collection trait of boars, so that the molecular marker can be used for screening the semen quality trait of boars and breeding, namely providing a new application of one SNP molecular marker in the pig ATP11A gene in the auxiliary breeding of the marker of the semen quality trait of boars, and realizing the early screening of the semen quality trait of boars, wherein the screening method is simple and rapid.
Drawings
FIG. 1 is a diagram showing agarose gel electrophoresis detection of a PCR product in example 1 of the present invention, wherein lane M is DL2000Marker, lanes 1-5 are amplified fragments in pigs, and the fragment size is 732bp;
FIG. 2 is a sequencing map of the g.421A > G site in example 1 of the present invention.
Detailed Description
The present invention will be described in further detail with reference to the drawings and examples, in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
Example 1 acquisition of pig ATP11A Gene SNP detection fragment and establishment of method for detecting polymorphic site
1. Extraction of boar genomic DNA
Extraction of boar genomic DNA Using animal tissue genomic DNA extraction kit (PureLink) manufactured by Invitrogen company TM Pro 96Genomic DNA Purification Kit,K182104A), and the boar genomic DNA was extracted according to the kit instructions. And (3) detecting the concentration and quality of the extracted DNA, and storing at-20 ℃ for standby.
2. Obtaining of boar ATP11A gene SNP genetic marker detection fragment
(1) PCR amplification
According to the SNP genetic marker detection sequence in the genome sequence of the ATP11A gene of the boar, a pair of primers is designed to amplify fragments of polymorphic sites.
Wherein, the nucleotide sequence of the SNP genetic marker detection sequence is as follows:
CCTGCAAATGCCACTGACTGCGCGGAGGAAGGCCGGCCAAAACCTGGCGCTGGCAGCGGGCTCCTGGCAGAAGGCAGGCAGCGTTGCTGGGAGGAGACGAGGAAAAGTGTGGGACGTCTGCGGCTCGGTCATCGTTCTATGACCCACCAGAGGCGCTGGCCCCAGGGCCCAGCCGCCACCCCTGCCCAGCGGAGGGGGCCTGGGGACAACCGTGGTTCCTGGAGCCTGACATGAGTTGATCGAATGGAAAAGGCAGCTGGACAAGCCCCGTGGCCACTAAGGGAAACAGACACTGCCAGGCGAAGGTGAGGCCAGGGCGTGGTGGAGGGAAGGAAGGCCCCATCTCTCTCCTGGTCTCCAGTTGGCAGCGGGCACCTCTGTTGGTGCAGATCCCCCAGGGGGGCCTTGGGCCTCCTAGACACAAAGCAGGAGAGAGTAGATTTGGAAGGAAAGTGGAGAATCATTGAGTTAGCTGACCCCAAACTGGAGCATTTTCCGCAGAAAAGCACGTCAGCGTCGGGATGGTTTGGGCCTGATTACCCGATTATTCACTAGTGCTCCTTCCTAGTCTCCGCGTCTAACCCCAAGTGCCCGTGCTTCTTCCCGAAAAGCAGCCAGAAGAACACTCAGGTGCGTTCCGGCAAGGCCCGGATCGGATCGGTCGCCAAAGGCCTTGTCAGGCCCCTCGGGTTTCACAAGTGGTGGCCCTTGGCCCCAAGAACGGAAGTTGGA, as shown in SEQ ID NO. 1.
The primers designed were as follows:
an upstream primer: 5'CCTGCAAATGCCACTGACTG 3' is shown in SEQ ID NO. 2.
A downstream primer: 5'TCCAACTTCCGTTCTTGGGG 3' is shown in SEQ ID NO. 3.
By using the primers and taking boar genome DNA as a template, carrying out PCR amplification, wherein a PCR reaction system is 50 mu L, and the components in the system are as follows: 100ng of genomic DNA, 25. Mu.L of PCR mix, 1. Mu.L of each of the above upstream and downstream primers, and ddH 2 O was made up to a total volume of 50. Mu.L.
The PCR was run as follows: pre-denaturation at 94℃for 3min; denaturation at 94℃for 30s, annealing at 56℃for 30s, elongation at 72℃for 30s,35 cycles; extending at 72 ℃ for 10min; preserving at 4 ℃. The PCR products were detected by 1.5% agarose gel electrophoresis, the detection result is shown in FIG. 1, wherein lane M is DL2000Marker, lanes 1-3 are amplified fragments in pigs, and the amplified fragments have the size of 732bp.
(2) PCR product purification
The PCR amplified product was purified using a Gel Extraction Kit kit from Shanghai Biotechnology Co., ltd, and specific steps are shown in the kit specification.
3. Detection of molecular markers by direct sequencing of PCR products
The PCR purified product obtained above is directly sent to Beijing Oreg company for sequencing, and the genotype of the site in the detection population is judged according to the sequencing result. As a result of analysis by using DNA Star software, as shown in FIG. 2, it was found that there was a mutation of the A421-G421 allele, i.e., g.421A > G, at the 421 st bp in the sequence shown in SEQ ID NO.1, which caused polymorphism in the ATP11A gene.
EXAMPLE 2 detection of polymorphism distribution of molecular markers in pigs
In this example, the polymorphism distribution pattern of the g.421A > G site of the ATP11A gene of pigs was detected in 289 pigs, and the detection results are shown in Table 1.
TABLE 1 polymorphism distribution law of ATP11A gene g.421A > G site
From the results in Table 1, it can be seen that: the g.421A > G locus of the ATP11A gene in pigs is represented by three genotypes of AA, AG and GG, with more individuals of the GG genotype and a G allele frequency of 62.6%.
Example 3 correlation analysis of molecular markers and boar semen quality traits
In order to determine whether the g.421A > G locus of the ATP11A gene of a boar is related to the difference of reproductive traits of the boar, polymorphism detection was performed by the method established in example 1, and the correlation of different genotypes of the polymorphic locus with sperm cell volume, sperm density and sperm motility traits was analyzed. The variance analysis of the different SNP genotype combinations was performed using SAS statistical software (SAS Institute Inc, version 9.1) GLM program and the significance test was performed using the following models:
Y ij ＝μ+G i +F j +e ijk ；
Y ij is the character phenotype value, mu is the average value, G i For genotype effects (including gene additive effects and dominant effects; additive effects apply 1,0 and-1 to AA, AG and GG genotypes, respectively, dominant effects apply 1, -1 and 1 to AA, AG and GG genotypes, respectively); f (F) j Is a pig farm comprehensive effect; e, e ijk Is a residual effect.
Correlation analysis between different genotypes and boar semen quality traits is carried out in the boars, and the statistical analysis results are shown in table 2:
TABLE 2 correlation analysis of the quality traits of boar semen at the ATP11A gene g.421A > G locus
Note that: the neutral mean composition of the table is mean ± standard deviation, the difference is significant (P < 0.05) and the effect is significant (P < 0.05) in the capital letters of the shoulder marks
As can be seen from table 2, in boars, the semen volume of AA genotype individuals at g.421a > G locus was significantly higher than AG and GG genotype individuals (P < 0.05), and the additive effect reached an extremely significant level (P < 0.05) with no significant difference in sperm density and sperm motility, so the a allele was the dominant allele.
Example 4 application of SNP molecular marker of ATP11A Gene in boar reproduction trait (boar semen trait) screening and/or pig breeding
The SNP molecular marker g.421A > G site on the ATP11A gene intron is obviously related to boar reproduction traits, especially boar semen traits, and the additive effect of the A allele reaches a significant level. Therefore, in the process of breeding pigs, AA type individuals with good reproductive performance can be selected in an auxiliary way through the SNP molecular marker, individuals carrying the dominant allele should be reserved in breeding, and the semen quantity can be increased under the condition of not changing the sperm density and the sperm activity rate, so that the productivity of the population is improved.
The above-described embodiments of the present invention do not limit the scope of the present invention. Any other corresponding changes and modifications made in accordance with the technical idea of the present invention shall be included in the scope of the claims of the present invention.
Claims (9)
1. An SNP molecular marker related to boar semen quality traits in an ATP11A gene is characterized in that the semen traits are single semen collection traits; the molecular marker is positioned in a promoter region of a pig ATP11A gene, the nucleotide sequence of the molecular marker is shown as SEQ ID NO.1 of a sequence table, and a421 bp position of the sequence has a mutation of A421-G421 bases.
2. The SNP molecular marker associated with boar semen quality trait in the ATP11A gene according to claim 1, wherein the A or G base polymorphism site at 421bp in the sequence SEQ ID NO.1 is represented as three genotypes of AA, AG or GG, wherein the A allele is dominant allele.
3. Use of the SNP molecular marker of any one of claims 1 or 2 in boar semen trait screening and/or pig breeding.
4. A primer pair for amplifying the molecular marker of claim 1, wherein the primer pair comprises: the nucleotide sequence of the upstream primer is shown as SEQ ID NO.2, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 3.
5. The use of the primer pair according to claim 4 in boar semen trait screening and/or pig breeding.
6. A kit for rapid breeding using the SNP molecular marker as set forth in claim 1, characterized by comprising the primer set as set forth in claim 4.
7. A method for screening boar semen characters and/or breeding pigs, which is characterized by comprising the following steps:
s1, extracting genome DNA of a boar;
s2, carrying out PCR amplification by using the genomic DNA obtained in the step S1 as a template and adopting the primer pair as defined in claim 4 to obtain the molecular marker as defined in claim 1 and purifying;
s3, carrying out sequencing analysis on the molecular marker purified in the step S2, reserving an individual carrying the A allele at the 421bp position of the sequence, and eliminating the individual carrying the G allele.
8. The method for boar semen trait selection and/or pig breeding according to claim 7, wherein in step S1 genomic DNA is extracted from the ear margin tissue of the boar to be tested.
9. The method for boar semen trait selection and/or pig breeding according to claim 7, wherein in step S2, the PCR reaction system is 50 μl, and the components in the system are: 100ng of genomic DNA, 25. Mu.L of PCR mix, 1. Mu.L of each of the upstream primer and the downstream primer, and adding ddH2O to make up to a total volume of 50. Mu.L; the PCR was run as follows: pre-denaturation at 94℃for 3min; denaturation at 94℃for 30s, annealing at 56℃for 30s, elongation at 72℃for 30s,35 cycles; extending at 72 ℃ for 10min; preserving at 4 ℃.
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