CN111455066B - Genetic molecular marker related to pork quality conductivity character - Google Patents
Genetic molecular marker related to pork quality conductivity character Download PDFInfo
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Abstract
The invention relates to the technical field of molecular markers, in particular to a genetic molecular marker related to pork quality conductivity characteristics, which can be used for breeding pigs with higher pork conductivity, and the breeding method comprises the following steps: sampling, extracting DNA, carrying out quality detection, carrying out genotyping to obtain SNP (Single nucleotide polymorphism) genotyping data, and obtaining a molecular marker related to pork quality conductivity, wherein the nucleotide sequence of the SNP molecular marker is shown as SEQ ID NO.1, and an A/C allele mutation exists at the 51 st base of the sequence, so that the nucleotide of the sequence generates polymorphism, and when the 51 st nucleotide on the nucleotide sequence is C, pork has higher conductivity and better pork quality.
Description
[ field of technology ]
The invention relates to the technical field of molecular markers, in particular to a genetic molecular marker related to pork quality conductivity characteristics.
[ background Art ]
In the past decades, people only pay attention to the growth characters of pigs, such as feed utilization rate, lean meat percentage and the like, but often neglect the meat quality characters, so that the quality of the pork is reduced. Along with the improvement of the living standard of people, the requirements of people on the pork quality reach the conversion stage from quantity to quality, so that the pork quality property becomes an important economic property and is more and more widely paid attention to pig raising enterprises and pig raising improvement companies at home and abroad.
Conductivity is one of important meat quality traits affecting the freshness of pork, sensory inspection and volatile basic total nitrogen (TVB-N) measurement are mainly used as meat freshness indexes in China, sensory inspection is easily affected by personal factors, and the volatile basic total nitrogen (TVB-N) measurement is an important objective index for evaluating the freshness of pork quality, but the method is time-consuming, very complex to operate and unfavorable for rapidly detecting the freshness of pork. Research shows that muscle cells are affected to a certain extent in the process of pork storage, and cell damage is more and more serious, so that matrix in cells flows out, the conductivity of meat quality is increased, and the freshness of meat quality is reduced. Therefore, the freshness of the pork can be accurately evaluated by measuring the conductivity of the pork.
Pork quality traits are low genetic traits and complex traits controlled by multiple genes. It is difficult to find its major genes using conventional genetic means, and to date 30170 Quantitative Trait Loci (QTL) were found in 688 different swine traits (http:// www.Animalgenome.org/cgi-bin/QTLdb/SS/index). Among these QTLs, 419 QTLs associated with the meat quality conductivity trait have been detected, most of which were located using linkage mapping methods. However, genetic improvement of pigs using these QTLs directly is difficult. With the increasing availability of chip data, whole genome association research (GWAS) is widely used by breeding enterprises, and whole genome association analysis based on high-density SNP chips can provide an effective technical means for screening candidate genes for low-genetic traits. Classical GWAS typically perform single-marker regression analysis on all markers one by one based on software such as Plink, followed by setting a significance threshold to screen for significant sites. The method is often faced with the problems of high calculation intensity, overestimation marking effect, unreasonable significance threshold setting and the like. To further increase the efficiency of GWAS, new methods and software are continually being proposed. And how to utilize a genome analysis method to analyze the breeding pigs so as to feed the breeding pigs with higher-quality pork can greatly improve the pork quality and shorten the time of seed selection and breeding.
[ invention ]
In view of the above, it is necessary to provide a genetic molecular marker related to the pork quality conductivity property, which can rapidly screen the breeding pigs with high pork quality conductivity, further select pigs with higher pork quality for feeding and reserving seeds, and can accurately and efficiently select the breeding pigs from genes.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
a genetic molecular marker related to pork quality conductivity property is positioned at a 59537927 nucleic acid site of a 4 th chromosome of a pig, wherein the base of the site is A or C, and the genetic molecular marker is correspondingly positioned at a 101 th nucleic acid site of a nucleic acid sequence table SEQ ID NO. 1.
The invention also includes primers for amplifying the molecular genetic markers or probes for identifying the molecular genetic markers.
The invention also includes kits containing the primers or probes.
The invention also comprises the application of the molecular genetic marker in breeding of high or low pork quality conductivity varieties or strains.
A method for selectively breeding or assisted selective breeding of a pig variety or strain with high/low pork quality and conductivity by using the genetic molecular marker, which is characterized in that the method comprises the following steps: extracting pig DNA, detecting 59537927 deoxyribonucleotide of chromosome 4 of the pig, measuring 59537927 nucleotide sequence as A or C, determining that the genotype of the pig to be detected is AA type, AC type or CC type, and selecting the pig with AA type, AC type or CC type genes for further seed selection and/or breeding according to the requirement; the pork quality conductivity of the CC type gene is higher than that of the AC type and the AA type.
Furthermore, the CC genotype is homozygote of 59537927 th deoxyribonucleotide of chromosome 4 of the pig with C; the AA genotype is the homozygote of which the 59537927 th deoxyribonucleotide of the chromosome 4 of the pig is A; the AC genotype is the heterozygote of A and C of 59537927 th deoxyribonucleotide of pig chromosome 4.
The invention also comprises a method for obtaining the molecular genetic marker related to the pork quality and conductivity characteristics, which comprises the following steps: collecting a pig ear tissue sample or blood sample, extracting pig total DNA, and performing quality control and analysis on the total DNA to obtain genotype data; counting individual conductivity data of pigs as phenotypes, and obtaining SNP obviously related to pork quality conductivity characters by utilizing a whole genome correlation analysis technology; the quality control standard is as follows: the individual detection rate is more than or equal to 90 percent; SNP detection rate is more than or equal to 90%; the minor allele frequency is greater than or equal to 0.01; the Hardy-Winberg equilibrium p value is more than or equal to 10 6 The method comprises the steps of carrying out a first treatment on the surface of the The whole genome association analysis adopts a mixed linear model as follows: y is Y n ＝T ni W i +P nj Q j +e n Wherein Y is n Representing the phenotype vector of the nth individual, T ni Is a fixed effect, comprising sex and genotype of pig farm and i pseudoqtns and controls the first three major components of population genetic background; w (W) i Representing the corresponding effect; p (P) nj A j-th marker representing an nth subject; q (Q) j Representing the jth corresponding effect; e, e n Representing residual vectors, following normal distribution, e.about.N (0,I sigma) e 2 ),σ e 2 Representing the residual variance.
The invention has the following beneficial effects:
1. the method utilizes the FarmCPU model in rMVP software to analyze, utilizes the mixed and alternate use of the fixed effect and the random effect, can rapidly and accurately screen out SNP molecular markers related to the conductivity property, can be applied to the correlation analysis of the genotype or the related property of the gene related to the pork quality conductivity property for non-diagnosis purposes, and provides a new molecular marker resource for the auxiliary selection of the molecular markers related to the pork quality conductivity property. And the genotype of the pig can be detected by adopting a gene chip technology in vitro, so that the pig can be selected for non-diagnosis purposes and the selection of the breeding pig with higher conductivity property can be improved.
The molecular marker can be used for breeding pigs with higher pork conductivity, and the breeding method comprises the following steps: sampling, extracting DNA, carrying out quality detection, carrying out genotyping to obtain SNP (Single nucleotide polymorphism) genotyping data, and obtaining a molecular marker related to pork quality conductivity characteristics, wherein the nucleotide sequence of the SNP molecular marker is shown as SEQ ID NO.1, and an A/C allele mutation exists at the 101 th base of the sequence, so that the nucleotide of the sequence generates polymorphism, and when the 101 st nucleotide on the nucleotide sequence is C, pork has higher conductivity and better pork quality.
[ detailed description ] of the invention
FIG. 1 is a Manhattan diagram of a whole genome correlation analysis of the present invention. Reference numerals illustrate: the pork conductivity, black circle and arrow mark are the molecular mark for screening, and the mark is located on chromosome 4 of pig.
[ detailed description ] of the invention
In order that the above objects, features and advantages of the invention will be readily understood, a more particular description of the invention will be rendered by reference to the appended drawings. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention. The invention may be embodied in many other forms than described herein and similarly modified by those skilled in the art without departing from the spirit or scope of the invention, which is therefore not limited to the specific embodiments disclosed below.
Example 1:
the basic study population of this example was 303 ternary pigs, all from Guangxi Yangxiang farmer, inc., with 131 pigs and 172 sows. The conductivity property measuring part is the longus dorsi muscle between 13 th and 14 th intercostals, and the measurement is completed within 45min after slaughter.
Genotyping and quality control
Pig ear tissue samples or blood samples were collected, total DNA was extracted, and genotyping was performed using GGP 50k SNP (GeneSeek, US) chip to obtain 50679 SNP markers covering the whole genome. NCBI genome alignment program (https:// www.ncb) was used according to the latest edition of porcine reference genome (Srcrofa 11.1)i.nlm.nih.gov /) updates the physical positions of all SNP markers. SNPs with unknown genomic positions are not used for association analysis. For SNP markers on all autosomes, quality control is performed by using Plink software, and the standard is that the individual detection rate is more than or equal to 90%; SNP detection rate is more than or equal to 90%; the minor allele frequency is greater than or equal to 0.01; the Hardy-Winberg equilibrium p value is more than or equal to 10 6 . For the deletion genotypes, the padding was performed using Beagle software (version 4.1). And after filling, controlling the quality again, wherein the quality control conditions are the same as the above.
(II) statistical model
The method of utilizing the multi-marker correlation model uses gender and pig farm as fixed effects, and uses the FarmCPU model of rMVP software package to conduct GWAS analysis under R statistical environment, the research model is as follows:
Y n ＝T ni W i +P nj Q j +e n wherein Y is n Representing the phenotype vector of the nth individual, T ni Is a fixed effect, comprising sex and genotype of pig farm and i pseudoqtns and controls the first three major components of population genetic background; w (W) i Representing the corresponding effect; p (P) nj A j-th marker representing an nth subject; q (Q) j Representing the jth corresponding effect; e, e n Representing residual vectors, following normal distribution, e.about.N (0,I sigma) e 2 ),σ e 2 Representing the residual variance.
(III) marker screening:
the effect values for all markers are plotted in manhattan, displaying and screening SNP markers for large effects. And analyzing the quality conductivity difference of the three-element commercial pig meat of the different genotype group marked by ASGA0019815 by adopting analysis of variance and multiple comparison (R statistical analysis platform).
(IV) analysis of pork mass conductivity of different genotypes:
the conductivity property measuring part is the longus dorsi muscle between 13 th and 14 th intercostals, and the measurement is completed within 45min after slaughter.
The Manhattan diagram is drawn for the effect values of all the markers, and SNP markers with large effects are displayed and screened, and the Manhattan diagram is shown in figure 1; after testing pig conductivity, the pig populations of different genotypes were analyzed for differences in pork quality conductivity using analysis of variance and multiple comparisons (R statistical analysis platform) (table 1).
TABLE 1 genetic markers of pork quality conductivity of different genotypes
As can be seen from Table 1, the pork quality of individuals with genotype CC has higher conductivity; for individuals with genotype AA, they have lower conductivity. In the whole genome association analysis using the mixed linear model, the above marker reached a significant level of association, indicating that the marker was not only significantly associated with the pork quality conductivity trait, but that individuals were more likely to have higher conductivities when the marker was mutated to C.
The sequence table SEQ ID NO.1 is a nucleotide sequence of 100bp upstream and downstream of the molecular marker related to pork quality conductivity characteristics screened by the invention, the sequence length is 201bp, and an A/C allele mutation exists at the 101 th bp of the sequence, and the mutation can cause the nucleotide sequence shown in the SEQ ID NO.1 to generate polymorphism.
Example 2:
according to the gene result obtained by the screening, a molecular genetic marker of the pork quality conductivity character is displayed, wherein the molecular genetic marker is positioned at a 59537927 nucleic acid site of a 4 th chromosome of a pig, the position is a mutation A > C, and the molecular genetic marker is correspondingly positioned at a 101 st nucleic acid site of a nucleic acid sequence table SEQ ID NO. 1.
Example 4:
the person skilled in the art can easily design primers for amplifying the molecular markers or probes for identifying the molecular markers according to the present invention, thereby being used for the detection of the genetic markers, for example, by PCR amplification to obtain the molecular genetic markers, clone sequencing to obtain the corresponding sequences, or Bsm-RFLP polymorphism. Thus, the invention also includes primers for amplifying the molecular genetic markers or probes for identifying the molecular genetic markers, and kits containing the primers or probes.
Example 5:
the molecular genetic marker can be used for assisting pig breeding or assisting breeding work, and the specific method comprises the following steps: extracting pig DNA, detecting 59537927 deoxyribonucleotide of chromosome 4 of the pig, measuring 59537927 nucleotide sequence as A or C, determining that the genotype of the pig to be detected is AA type, AC type or CC type, and selecting the pig with AA type, AC type or CC type genes for further seed selection and/or breeding according to breeding requirements; wherein, the pork quality conductivity of the CC type gene is higher than that of the AC type and the AA type; the CC genotype is homozygote of 59537927 th deoxyribonucleotide of pig chromosome 4 as C; the AA genotype is the homozygote of which the 59537927 th deoxyribonucleotide of the chromosome 4 of the pig is A; the AC genotype is the heterozygote of A and C of 59537927 th deoxyribonucleotide of pig chromosome 4.
In summary, the method can simply, efficiently and accurately obtain the molecular genetic marker related to the pork quality conductivity property, and can design the primer for amplifying the molecular marker and the probe for identifying the molecular marker according to the mutation; pigs with high pork conductivity are rapidly screened, and the method and the device for screening the molecular markers by using the one-step whole genome association analysis method can effectively improve the accuracy and efficiency of screening the molecular markers.
The above examples merely represent a few embodiments of the present invention, which are described in more detail and are not to be construed as limiting the scope of the present invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of the invention should be assessed as that of the appended claims.
Sequence listing
<110> Guangxi Yangxiang Co., ltd
GUANGXI YANGXIANG AGRICULTURE AND ANIMAL HUSBANDRY Co.,Ltd.
<120> a genetic molecular marker related to pork quality and conductivity traits
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 201
<212> DNA
<213> pig of the genus pig (Sus scrofa)
<220>
<221> misc_feature
<222> (101)..(101)
<223> n is a or c
<400> 1
aaggccaggg atccaacccg catcctcatg gatactagtc cggttcattt ccactgagcc 60
atgacggaaa ctcctaaagg agatactttg taagcttaca ntcttccaga ggtgatggcc 120
attaggcttg ctccccaatt tcctccaact gaacaatgaa taacctgagg ccctaaggac 180
ctaggctggg gttcttttca g 201
Claims (3)
1. The application of the molecular genetic marker in breeding of high or low pork quality conductivity varieties or strains is characterized in that the sequence of the molecular genetic marker is shown as a sequence table SEQ ID NO.1, and the base of a 101 st nucleic acid site of the sequence is A or C;
judging whether the genotype of the pig to be detected is AA genotype, AC genotype or CC genotype according to the locus genotype, and selecting the pig with the AA genotype, the AC genotype or the CC genotype according to the requirement for further seed selection and/or breeding; the pork quality conductivity of the CC genotype is higher than that of the AC genotype and the AA genotype.
2. A method for breeding or assisting in breeding a pig variety or strain with high/low pork quality and conductivity by genetic molecular markers, which is characterized in that the method comprises the following steps: extracting pig DNA, detecting 101 th nucleic acid locus of a nucleic acid sequence table SEQ ID NO.1, measuring the sequence of the locus to be A or C, determining that the genotype of the pig to be detected is AA genotype, AC genotype or CC genotype, and selecting the pig with the AA genotype, the AC genotype or the CC genotype according to the requirement for further seed selection and/or breeding; the pork quality conductivity of the CC genotype is higher than that of the AC genotype and the AA genotype.
3. The method of claim 2, wherein the CC genotype is homozygote with the 101 st nucleic acid site of SEQ ID No.1 of the nucleic acid sequence listing being C; the AA genotype is homozygote with 101 th nucleic acid site of a nucleic acid sequence table SEQ ID NO.1 as A; the AC genotype is a hybrid of A and C at the 101 st nucleic acid site of the nucleic acid sequence table SEQ ID NO. 1.
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| CN105624310A (en) * | 2016-02-29 | 2016-06-01 | 华南农业大学 | Molecular marker influencing intramuscular fat character of pig and application |
| CN105755131A (en) * | 2016-04-09 | 2016-07-13 | 华中农业大学 | Genetic marker associated with pig meat quality characters and carcass characters |
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| CN105624310A (en) * | 2016-02-29 | 2016-06-01 | 华南农业大学 | Molecular marker influencing intramuscular fat character of pig and application |
| CN105755131A (en) * | 2016-04-09 | 2016-07-13 | 华中农业大学 | Genetic marker associated with pig meat quality characters and carcass characters |
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| Huynh Thi Phuong Loan等.UBXN1 polymorphism and its expression in porcine M. longissimus dorsi are associated with water holding capacity.Mol Biol Rep.2014,第41卷(第41期),1411-1418. * |
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