CN111705145B - SNP marker influencing guanine content in pig individual - Google Patents

SNP marker influencing guanine content in pig individual Download PDF

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CN111705145B
CN111705145B CN202010749748.7A CN202010749748A CN111705145B CN 111705145 B CN111705145 B CN 111705145B CN 202010749748 A CN202010749748 A CN 202010749748A CN 111705145 B CN111705145 B CN 111705145B
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黄路生
麻骏武
黄聪
郑敏
肖石军
杨斌
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Jiangxi Agricultural University
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Abstract

The invention provides an SNP marker influencing the guanine content in pork. The most significant 3SNP markers were: (I) the SNP marker on the nucleic acid I is located at the 55495763 th site from the 5' end on the 12 th chromosome of the 11.1 version international pig genome and is G or A; (II) the SNP marker on the nucleic acid II, which is located at the 55500274 th site from the 5' end on the 12 th chromosome of the 11.1 version of the international pig genome, is C or CT; (III) the SNP marker on the nucleic acid III, which is located at the 54924843 th site from the 5' end on chromosome 12 of the International pig genome version 11.1, is T or C.

Description

SNP marker influencing guanine content in pig individual
Technical Field
The invention provides an SNP marker influencing the guanine content in a pig individual.
Background
For thousands of years, pork plays a very important role in the daily diet of residents in China, serves as a main source for taking animal protein, and can provide various essential amino acids, fatty acids, rich trace elements and carbohydrates for human bodies. With the increase of pork consumption of Chinese residents, the pursuit of pork by people gradually turns from 'quantity' to 'quality', and the main reason is that the meat quality and the taste of commercial pigs represented by 'Du-Dao' in the west in the meat market of China are generally poor and far from that of local pigs in China, so that the pork quality is considered while the 'quantity' of the pork is guaranteed, the problem generally concerned by consumers is the problem, and the research hotspot for improving the meat quality through the genetic improvement of the pigs which are important research components is formed.
The umami taste is one of five basic tastes (the other four tastes are sour, sweet, bitter and salty) of the food to be evaluated, and can obviously enhance the appetite of eaters. The delicate flavor is mainly provided by sodium glutamate (main component of monosodium glutamate), and can be greatly improved under the participation of inosinic acid (inosinic acid, IMP) or guanylic acid (guanylic acid, GMP), so that the increase of the guanylic acid content in food can promote the health of human body and increase the taste of food while reducing the intake of sodium. Pork contains a certain amount of inosinic acid and guanylic acid, and the difference of the two contents is one of important factors causing the difference of the tastes of western commercial pigs and Chinese local pigs. However, studies for improving the taste and quality of pork have mainly focused on the intramuscular fat, tenderness, juiciness and the like of pork, and there are reports for improving the taste and quality of pork from the aspect of improving the content of an umami substance in pork.
Disclosure of Invention
One aspect of the present invention provides an SNP marker for pigs, which includes at least one of the following SNP markers:
(I) the SNP marker on the nucleic acid I, located at the 301 th site from the 5 'end on the SEQ ID No.1, corresponding to the 55495763 th site from the 5' end on chromosome 12 of the 11.1 version of the International pig genome, is G or A;
(II) the SNP marker on the nucleic acid II, located at position 331 from the 5 'end on SEQ ID No.2, corresponding to position 55500274 from the 5' end on chromosome 12 of the version 11.1 International pig genome, is C or CT;
(III) the SNP marker on the nucleic acid III, located at the 301. sup. th site from the 5 'end on SEQ ID No.3, corresponding to the 54924843. sup. th site from the 5' end on chromosome 12 of the International pig genome version 11.1, is T or C;
(IV) the SNP marker on the nucleic acid IV, which is located at the 55496302 th site from the 5' end on the 12 th chromosome of the 11.1 version of the international pig genome, is C or T;
(V) the SNP marker on the nucleic acid V, located at position 55494763 from the 5' end on chromosome 12 of the International pig genome version 11.1, is G or A;
(VI) the SNP marker on nucleic acid VI, at position 55493440 from the 5' end on chromosome 12 of the international pig genome version 11.1, being a or G;
(VII) the SNP marker on nucleic acid VII, located at position 55493401 from the 5' end on chromosome 12 of the International pig genome version 11.1, is G or GGGACC;
(VIII) the SNP marker on nucleic acid VIII, located at position 55369371 from the 5' end on chromosome 12 of the international pig genome version 11.1, being a or T;
(IX) the SNP marker on nucleic acid IX, at position 55494697 from the 5' end on chromosome 12 of the international pig genome version 11.1, being C or T;
(X) the SNP marker on the nucleic acid X, which is located at the 55495244 th site from the 5' end on chromosome 12 of the 11.1 version of the International pig genome, is T or C;
(XI) a SNP marker on nucleic acid XI at position 55493385 from the 5' end on chromosome 12 of the international pig genome version 11.1, as AG or a;
(XII) the SNP marker on nucleic acid XII, located at position 55493473 from the 5' end on chromosome 12 of the international pig genome version 11.1, being T or C;
(XIII) the SNP marker on nucleic acid XIII, located at position 55493963 from the 5' end on chromosome 12 of the international pig genome version 11.1, and being a or G;
(XIV) the SNP marker on nucleic acid XIV, located at position 55377798 from the 5' end on chromosome 12 of the International pig genome version 11.1, is C or A.
In a specific embodiment, the nucleic acids I to XIV are independently selected from at least one of DNA sequences, cDNA sequences and RNA sequences.
In a specific embodiment, the nucleic acids I to XIV independently have a length of 5bp to 26560 bp.
In a specific embodiment, the nucleic acids I to XIV have a length of 5bp to 10000 bp.
In a specific embodiment, the nucleic acids I to XIV independently have a length of 5bp to 5928 bp.
In a specific embodiment, the nucleic acids I to XIV independently have a length of 5bp to 1000 bp.
In a specific embodiment, the nucleic acids I to XIV independently have a length of 5bp to 500 bp.
In a specific embodiment, the nucleic acids I to XIV independently have a length of 5bp to 300 bp.
In one embodiment, the nucleic acid I has the sequence shown in SEQ ID No.1, the nucleic acid II has the sequence shown in SEQ ID No.2, and the nucleic acid III has the sequence shown in SEQ ID No. 3.
The second invention provides the application of the SNP marker in any one of the first invention in determining the content of guanine in pig or pork. Wherein, for (I), the guanine contents of the pork with the A/A, G/A and G/G genotypes at the 55495763 site are reduced in sequence; for (II), the guanine content of pork with CT/CT, C/CT and C/C genotypes at the 55500274 site is reduced in sequence; for (III), the guanine contents of pork with the gene types of C/C, T/C and T/T at the 54924843 th site are reduced in sequence; for (IV), the guanine content of the pork with the T/T, C/T and C/C genotypes at the 55496302 site is reduced in sequence; for (V), the guanine contents of the pork with the A/A, G/A and G/G genotypes at the 55494763 site are reduced in sequence; for (VI), the guanine contents of the pork with the gene types of G/G, A/G and A/A at the 55493440 th site are reduced in sequence; for (VII), the guanine contents of pork with the gene types of GGGACC/GGGACC, G/GGGACC and G/G are reduced in sequence at the 55493401 th site; for (VIII), the guanine contents of pork with T/T, A/T and A/A genotypes at the 55369371 site are reduced in sequence; for (IX), the guanine content of pork of the T/T, C/T and C/C genotypes at the 55494697 site is reduced in sequence; for (X), the guanine content of pork with C/C, T/C and T/T genotypes at the 55495244 site is reduced in sequence; for (XI), the guanine contents of pork with A/A, AG/A and AG/AG genotypes at the 55493385 site are reduced in sequence; for (XII), the guanine content of pork with C/C, T/C and T/T genotypes at the 55493473 site is reduced sequentially; for (XIII), the guanine contents of pork with G/G, A/G and A/A genotypes at the 55493963 site are reduced in sequence; for (XIV), the guanine content of pork of the A/A, C/A and C/C genotypes at the 55377798 position is reduced sequentially.
The third invention provides a method for genetic improvement of pigs, which comprises the following steps: determining the SNP markers according to any one of the invention of the pigs in the core group of the pigs, and making corresponding selection according to the SNP markers:
for (I), selecting breeding pig individuals with G/A and A/A genotypes at the 301 th site from the 5' end on the pig core group, and eliminating the breeding pig individuals with the G/G genotypes at the site to increase the frequency of the allele A at the site by generations; preferably, the breeding pig individual with the 301 th site from the 5' end on the SEQ ID No.1 as the A/A genotype is eliminated, and the breeding pig individual with the G/A and G/G genotypes at the site is eliminated, so that the frequency of the allele A at the site is increased by generations;
for (II), selecting the boar individuals with the 331 st site C/CT and CT/CT genotypes from the 5' end on the SEQ ID No.2 in the boar core group, and eliminating the boar individuals with the C/C genotypes at the site to increase the frequency of the allele CT at the site generation by generation; preferably, the breeding pig individual with the 331 st locus from the 5' end on the SEQ ID No.2 as the CT/CT genotype is eliminated, and the breeding pig individual with the C/CT and C/T genotypes at the locus is eliminated, so that the frequency of the allele CT at the locus is increased generation by generation;
for (III), selecting a boar individual with T/C and C/C genotypes at the 301 th site from the 5' end on the boar core group in the SEQ ID No.3, and eliminating the boar individual with T/T genotype at the site to increase the frequency of allele C at the site by generations; preferably, the breeding pig individuals with the 301 th site from the 5' end on the SEQ ID No.3 as the C/C genotype are eliminated, and the breeding pig individuals with the T/C and T/T genotypes at the site are eliminated, so that the frequency of the allele C at the site is increased generation by generation;
for (IV), selecting boar individuals with C/T and T/T genotypes at 55496302 th site from 5' end on chromosome 12 of the 11.1 version international pig genome in the boar core group, and eliminating boar individuals with C/C genotypes at the site to increase the frequency of allele T at the site by generations; preferably, the boar individuals with T/T genotype at 55496302 th site from the 5' end on the 12 th chromosome of the 11.1 version international pig genome are eliminated, the boar individuals with C/T and C/C genotypes at the site are eliminated, and the frequency of the allele T at the site is increased generation by generation;
for (V), selecting a breeding pig individual having the 55494763 th loci of G/a and a/a genotypes from the 5' end on the chromosome 12 of the 11.1 version international pig genome, and eliminating the breeding pig individual having the G/G genotype at the locus to increase the frequency of the allele a at the locus generation by generation; preferably, the breeding pig individual with the A/A genotype at the 55494763 th site from the 5' end on the 12 th chromosome of the 11.1 version international pig genome is eliminated, and the breeding pig individual with the G/A and G/G genotypes at the site is eliminated, so that the frequency of the allele A at the site is increased by generations;
for (VI), selecting a breeding pig individual with A/G and G/G genotypes at 55493440 th site from the 5' end on the 12 th chromosome of the 11.1 version international pig genome from the pig core group, and eliminating a breeding pig individual with A/A genotype at the site to increase the frequency of allele G at the site by generations; preferably, breeding pig individuals with G/G genotype at 55493440 th site from the 5' end on the 12 th chromosome of the 11.1 version international pig genome are eliminated, and breeding pig individuals with A/G and A/A genotypes at the site are eliminated, so that the frequency of allele G at the site is increased generation by generation;
for (VII), selecting a boar individual having the 55493401 th site of G/GGGACC and GGGACC/GGGACC genotypes from the 5' end on chromosome 12 of the version 11.1 international pig genome, and eliminating a boar individual having a G/G genotype at the site to increase the frequency of the allele GGGACC at the site generation by generation; preferably, the pig individual with the gene type GGGACC/GGGACC at the 55493401 th site from the 5' end on the chromosome 12 of the international pig genome version 11.1 is eliminated, and the pig individual with the gene types G/GGGACC and G/G at the site is eliminated, so that the frequency of the allele GGGACC at the site is increased generation by generation;
for (VIII), selecting a boar individual with A/T and T/T genotypes at 55369371 th site from 5' end on chromosome 12 of the 11.1 version international pig genome, and eliminating the boar individual with A/A genotype at the site to increase the frequency of allele T at the site by generations; preferably, the boar individuals with T/T genotype at 55369371 th site from the 5' end on the 12 th chromosome of the 11.1 version international pig genome are eliminated, and the boar individuals with A/T and A/A genotypes at the site are eliminated, so that the frequency of the allele T at the site is increased generation by generation;
for (IX), selecting a swine individual having C/T and T/T genotypes at the 55494697 th site from the 5' end on the chromosome 12 of the version 11.1 international swine genome from the swine core population, and eliminating a swine individual having a C/C genotype at the site to increase the frequency of the allele T at the site generation by generation; preferably, the boar individuals with T/T genotype at 55494697 th site from the 5' end on the 12 th chromosome of the 11.1 version international pig genome are eliminated, the boar individuals with C/T and C/C genotypes at the site are eliminated, and the frequency of the allele T at the site is increased generation by generation;
for (X), selecting a boar individual with T/C and C/C genotypes at 55495244 th site from 5' end on chromosome 12 of the 11.1 version international pig genome, and eliminating the boar individual with T/T genotype at the site to increase the frequency of allele C at the site by generations; preferably, the boar individuals with the 55495244 th site as C/C genotype from the 5' end on the 12 th chromosome of the 11.1 version international pig genome are eliminated, the boar individuals with the T/C and T/T genotypes at the site are eliminated, and the frequency of the allele C at the site is increased generation by generation;
for (XI), selecting a breeding pig individual with an AG/A and A/A genotype at 55493385 th site from the 5' end on chromosome 12 of the 11.1 version international pig genome from the pig core group, and eliminating a breeding pig individual with an AG/AG genotype at the site to increase the frequency of allele A at the site by generations; preferably, breeding pig individuals with the 55493385 th site from the 5' end on the 12 th chromosome of the 11.1 version international pig genome are of an A/A genotype, and breeding pig individuals with AG/A and AG/AG genotypes at the site are eliminated, so that the frequency of the allele A at the site is increased generation by generation;
for (XII), selecting a swine individual having T/C and C/C genotypes at 55493473 th site from 5' end on chromosome 12 of the international swine genome version 11.1, and eliminating a swine individual having T/T genotype at the site to increase the frequency of allele C at the site generation by generation; preferably, the boar individuals with the 55493473 th site as C/C genotype from the 5' end on the 12 th chromosome of the 11.1 version international pig genome are eliminated, the boar individuals with the T/C and T/T genotypes at the site are eliminated, and the frequency of the allele C at the site is increased generation by generation;
for (XIII), selecting individual breeders of A/G and G/G genotypes at 55493963 th site from 5' end on chromosome 12 of the 11.1 version international pig genome, and eliminating individual breeders of A/A genotypes at the site to increase the frequency of allele G at the site generation by generation; preferably, breeding pig individuals with G/G genotype at 55493963 th site from the 5' end on the 12 th chromosome of the 11.1 version international pig genome are eliminated, and breeding pig individuals with A/G and A/A genotypes at the site are eliminated, so that the frequency of allele G at the site is increased generation by generation;
for (XIV), selecting a breeding pig individual with C/A and A/A genotypes at 55377798 th site from 5' end on chromosome 12 of the 11.1 version international pig genome, and eliminating a breeding pig individual with C/C genotype at the site to increase the frequency of allele A at the site generation by generation; preferably, the breeding pig individual with the 55377798 th site from the 5' end on the 12 th chromosome of the 11.1 version international pig genome is of an A/A genotype, and the breeding pig individuals with the C/A and C/C genotypes at the site are eliminated, so that the frequency of the allele A at the site is increased by generations.
In a specific embodiment, the SNP marker according to any one of the present invention of the breeding pig is determined by analyzing the sequence of the nucleic acid of the breeding pig, wherein the sequence of the nucleic acid is selected from at least one of the group consisting of nucleic acid I, nucleic acid II and nucleic acid III.
The fourth invention provides a method for determining the quality of pork quality traits, which comprises the following steps: determining the SNP marker of any one of the pigs, and determining the pork quality traits according to the SNP marker:
for (I), the pork quality traits are from good to bad, and the genotype sequence of the 301 th site from the 5' end on the SEQ ID No.1 is as follows: A/A genotype, G/A genotype, and G/G genotype;
for (II), the pork quality traits are from good to bad, and the genotype sequence of the 331 st site from the 5' end on the SEQ ID No.2 is as follows: CT/CT genotype, C/CT genotype, and C/C genotype;
for (III), the pork quality traits are from good to bad, and the genotype sequence of the 301 th site from the 5' end on the SEQ ID No.3 is as follows: C/C genotype, T/C genotype and T/T genotype;
for (IV), the pork quality traits are from good to bad, and the genotype ordering of the 55496302 th site from the 5' end on the 12 th chromosome of the 11.1 version international pig genome is as follows: T/T genotype, C/T genotype and C/C genotype;
for (V), the pork quality traits are from good to bad, and the genotype ordering of the 55494763 th site from the 5' end on the 12 th chromosome of the 11.1 version international pig genome is as follows: A/A genotype, G/A genotype, and G/G genotype;
for (VI), the pork quality traits are from good to bad, and the genotype ordering of the 55493440 th site from the 5' end on the 12 th chromosome of the 11.1 version international pig genome is as follows: G/G genotype, A/G genotype and A/A genotype;
for (VII), the pork quality traits are from good to bad, and the genotype ordering of the 55493401 th site from the 5' end on the 12 th chromosome of the 11.1 version international pig genome is as follows: GGGACC/GGGACC genotype, G/GGGACC genotype, and G/G genotype;
for (VIII), the pork quality traits are from good to bad, and the genotype ordering of the 55369371 th site from the 5' end on the 12 th chromosome of the 11.1 version international pig genome is as follows: T/T genotype, A/T genotype, and A/A genotype;
for (IX), the pork quality traits are from good to bad, and the genotype ordering of the 55494697 th site from the 5' end on the 12 th chromosome of the 11.1 version international pig genome is as follows: T/T genotype, C/T genotype and C/C genotype;
for (X), the pork quality traits are from good to bad, and the genotype ordering of the 55495244 th site from the 5' end on the 12 th chromosome of the 11.1 version international pig genome is as follows: C/C genotype, T/C genotype and T/T genotype;
for (XI), the pork quality traits are from good to bad, and the genotype ordering of the 55493385 th site from the 5' end on the 12 th chromosome of the 11.1 version international pig genome is as follows: A/A genotype, AG/A genotype and AG/AG genotype;
for (XII), the pork quality traits are from good to bad, and the genotype ordering of the 55493473 th site from the 5' end on the 12 th chromosome of the 11.1 version international pig genome is as follows: C/C genotype, T/C genotype and T/T genotype;
for (XIII), the pork quality traits were from good to bad, and the genotype ordering of the 55493963 th site from the 5' end on chromosome 12 of the 11.1 version international pig genome was: G/G genotype, A/G genotype and A/A genotype;
for (XIV), the pork quality traits are from good to bad, and the genotype ordering of the 55377798 th site from the 5' end on the 12 th chromosome of the 11.1 version international pig genome is as follows: A/A genotype, C/A genotype and C/C genotype.
The fifth invention provides a method for establishing a new pig strain and/or a new pig variety for improving the quality of pork, which comprises the following steps: the genotype for the SNP marker according to any one of the present invention is
(I) G/A or G/G of the pig is mutated into A/A genotype by site-directed mutagenesis;
(II) C/CT or C/C swine, wherein the C/CT or C/C is mutated into CT/CT genotype by site-directed mutagenesis;
(III) T/C or T/T swine, wherein T/C or T/T is mutated into C/C genotype by site-directed mutagenesis;
(IV) C/T or C/C swine, wherein C/T or C/C is mutated into T/T genotype by site-directed mutagenesis;
(V) G/A or G/G swine, wherein G/A or G/G is mutated to A/A genotype by site-directed mutagenesis;
(VI) A/G or A/A swine, wherein the A/G or A/A is mutated to G/G genotype by site-directed mutagenesis;
(VII) G/GGGACC or G/G swine, wherein G/GGGACC or G/G is mutated into genotype GGGACC/GGGACC by site-directed mutagenesis;
(VIII) A/T or A/A pigs, wherein A/T or A/A is mutated into T/T genotype by site-directed mutagenesis;
(IX) C/T or C/C swine, wherein C/T or C/C is mutated to T/T genotype by site-directed mutagenesis;
(X) T/C or T/T swine, wherein T/C or T/T is mutated into C/C genotype by site-directed mutagenesis;
(XI) AG/A or AG/AG swine, wherein the AG/A or AG/AG is mutated to A/A genotype by site-directed mutagenesis;
(XII) T/C or T/T swine, wherein T/C or T/T is mutated to C/C genotype by site-directed mutagenesis;
(XIII) pigs of a/G or a/a in which a/G or a/a is mutated to the G/G genotype by site-directed mutagenesis;
(XIV) C/A or C/C swine, wherein C/A or C/C is mutated to the A/A genotype by site-directed mutagenesis.
In one embodiment, the mutation is performed by a transgenic method or a gene editing method; more preferably, the mutation is performed using the gene editing method of CRISPR/Cas 9.
The invention has the beneficial effects that:
the 3SNP markers of the invention are closely linked, and particularly the first SNP marker and the second SNP marker are closely linked to a higher degree, and the degree of linkage (r) is higher2) Both reached above 0.96. And the closely linked SNP markers are closely related to the mouthfeel traits, so that the related indexes of the pigs can be detected by at least one of the 3SNP markers, or the genetic improvement is carried out by at least one of the 3SNP markers.
Drawings
Figure 1 shows Manhattan plots (Manhattan Plot) of GWAS analysis and meta-analysis of chimeras F6, F7. Wherein, the X axis is the position of the SNP locus on the chromosome, the Y axis is-log 10(P value) corresponding to the SNP locus, two horizontal lines correspond to the threshold value of screening the significant locus, wherein, the dotted line represents-log 10(1 × 10)-6) The solid line represents-log 10(5 × 10)-8)。
Fig. 2 shows a Box Plot (Box Plot) of the effects of the different genotypes at the three most prominent sites in F6, F7 and the whole population, respectively. Wherein, the X axis represents the genotype of the SNP locus and the number of individuals in a population, the Y axis represents the guanine content of the individuals, the difference of the distribution of the phenotype (guanine content) under each genotype is counted by a multocomp package in an R language, and the letters (a and b) above each box line graph represent whether the phenotype difference of the individuals with the two genotypes is obvious or not (if the same letter exists, the phenotype difference corresponding to the two genotypes is not obvious, and if the letters are different, the phenotype difference is obvious).
FIG. 3 shows the results of analysis of the degree of Linkage Disequilibrium (LD) between significant SNPs and peripheral SNPs.
Detailed Description
The present invention is further illustrated by the following examples, which are intended to be purely exemplary of the invention and are not to be construed as limiting the invention in any way.
The chimeric pedigree F6 and F7 herds used in the present application are offspring generated by crossing 4 western commercial breeding pigs (Duroc, Dabai, Changbai, Pietland) and 4 Chinese local breeding pigs (Erhualian, Laiwu, Bama, Tibetan) through multiple generations, wherein the F7 herd is the offspring generated by matching the F6 herd.
Example 1
1. Obtaining, quality control and typing of pig whole genome re-sequencing data
421 out of the F6 population and 665 out of the F7 population, for a total of 1086, were randomly selected, and one small sample of ear tissue was collected from each individual, and genomic DNA of each individual was extracted by a standard phenol chloroform method, and the extracted genomic DNA was dissolved in TE buffer. The quality of the extracted genome DNA is detected by a Nanodrop-ND1000 spectrophotometer, and the quality standard is reached when the ratio of A260/280 is 1.8-2.0 and the ratio of A260/230 is about 1.7-1.9.
The concentration of the DNA samples meeting the standard was diluted to 50 ng/. mu.l, each DNA sample was subjected to low-depth re-sequencing (average sequencing depth was about 7.8X) using HiSeq X Ten sequencer platform of Illumina, Inc., and the resulting total read-paired ends were mapped to the 11.1 version of International pig genome using BWA software, and then genotype data of each individual was obtained using SAMTools, Platypus, Beagle, and the like software in sequence. Quality control was performed on the obtained genotype data using plink1.9, individuals with Minor Allele Frequency (MAF) <0.03 and pedigree mendelian error rate higher than 0.1 were rejected, and finally 29441528 SNPs in the F6 population and 29198737 SNPs in the F7 population were determined.
Quantitative determination of guanine content in pork by HPLC
Since the present invention is focused on determining the guanylic acid content in pork, accurate quantification of guanine, one of the guanylic acid hydrolysis products, is particularly important. According to the results of studies on biochemical metabolic pathways of guanine, one molecule of guanylic acid is hydrolyzed to one molecule of guanine in an acidic environment, and thus guanine can be accurately quantified using high performance liquid chromatography (hereinafter, referred to as "HPLC").
The detailed preparation of the extract from the longissimus dorsi of 421 pigs in the F6 population and 665 pigs in the F7 population is described in Panhong Zhi, Rongsheng, Zhonglina, Wangkouxu, Yangyoxing. Content of purine in common animal food in China [ J ]. Nutrition journal, 2012, 34 (01): 74-78 deg.
The content of guanine bases (hereinafter referred to as "guanine") in the extract of longisimus dorsi of each individual was measured by HPLC, and the results are shown in Table 1.
TABLE 1
Figure BDA0002609617880000091
3. Whole genome Association analysis (GWAS) analysis and Meta-analysis (Meta-analysis)
Utilizing a Mixed linear Model in GEMMA (Genome-wide Efficient Mixed Model Association algorithm, version number 0.98.1) software to perform GWAS analysis on 29441528 SNP markers in an F6 population and corresponding guanine content data in 421 individuals, and performing GWAS analysis on 29198737 SNP markers in an F7 population and corresponding guanine content data in 665 individuals, wherein the expression is as follows: y is W alpha + x beta + u + epsilon; u-MVNn(0,λτ-1K),∈～MVNn(0,τ-1In). Wherein y represents a phenotypic value vector of all individuals, W represents a covariate matrix, α represents a corresponding coefficient vector containing intercept, x represents a genotype vector of SNP, β represents an influence effect of SNP, u represents a random effect vector, e represents an error vector, λ represents a ratio of two variances, τ -1 represents a variance of residual, K represents a genetic matrix, In represents a unit matrix, and MVNn represents a multivariate normal distribution.
The results of GWAS analysis of the individual populations (F6, F7) are shown in fig. 1, and it can be seen from fig. 1 that there are 2 SNP sites on chromosome 12 of pig that are most significantly associated with guanine content in pork: 12_54924843 at position 301 from the 5' end on SEQ ID No.3 (shown as T in SEQ ID No. 3) corresponding to position 54924843 on chromosome 12 of the international porcine reference sequence (version 11.1); 12_55500274 at position 331 from the 5' end of SEQ ID No.2 (shown as C in SEQ ID No. 2) corresponding to position 55500274 on chromosome 12 of the International pig reference sequence (version 11.1).
The relationship of guanine content (i.e., phenotype) corresponding to individuals with different genotypes at the above two SNP sites is shown in FIG. 2.
The genotypes of each individual at the positions of the 12_54924843SNP and the 12_55500274SNP are extracted from a sequencing file by using PLINK software, after the number of individuals of each genotype is counted, the genotypes of the individuals are in one-to-one correspondence with the corresponding guanine content (namely, the phenotype), then the difference of the genotype distribution under different genotypes is counted by using a mulcomp package in the R language, and the letters (a and b) above each small graph indicate whether the phenotype difference of the individuals with the two genotypes is obvious or not. If the same letter exists, the phenotype difference corresponding to the two genotypes is not obvious; if the letters are different, the two are significantly different. The 12_54924843SNP genotype and the content of guanine in its individuals are shown in table 2 for 421 individuals in the F6 population. The genotype of SNP 12_55500274 among 665 individuals in the F7 population and the content of guanine among their individuals are shown in table 3.
FIG. 2 in combination with Table 2 and Table 3, it can be seen that the change of genotype from T/C to T/T at a single site at the 12_54924843SNP site results in an increase of 1.14mg/100g in guanine content; the change of the genotype of a single site at SNP site 12_55500274 from C/C to C/CT will result in an increase of guanine content by 0.9mg/100 g.
TABLE 2 Effect of SNP 12_54924843 on guanine content of porcine individuals of F6
Figure BDA0002609617880000101
TABLE 3 Effect of SNP 12_55500274 on guanine content of porcine individuals of F7
Figure BDA0002609617880000102
Using the results of the GWAS analysis described above, meta-analysis was performed on the guanine content in the two populations in METAL software, and the correlation analysis results of the different populations were combined by calculating the combined inverse variance-weighted β -coefficient (normalized error), standard error (standard error) and z-score (z-score), as follows:<β>＝(∑i[βi/(SEi)2])/(∑i[1/(SEi)2])；
Figure BDA0002609617880000103
zmeta＝<β>/<SE>wherein beta isiAnd SEiRespectively, the β coefficient and the standard error in study i, and when the number of samples between two or more populations compared is not consistent, the z-score is calculated as follows: z is a radical ofmeta＝∑i(βi/SEi)×wi；
Figure BDA0002609617880000104
Typically, the correlation between the z-score obtained in this formula and the inverse variance weighted z-score is excellent (r)2>0.99)。
The results of meta-analysis showed that there was a SNP site 12_55495763 on chromosome 12 of pig that was significantly associated with guanine content in pork (fig. 1). Located at position 301 from the 5' end of SEQ ID No.1 (shown as G in SEQ ID No. 1), corresponding to position 55495763 on chromosome 12 of the International pig reference sequence (version 11.1).
The relationship of guanine content (i.e., phenotype) corresponding to individuals of different genotypes at the SNP site is shown in FIG. 2.
The genotype of each individual at the 12_55495763SNP site is extracted from a sequencing file by using PLINK software, after the number of individuals of each genotype is counted, the genotypes of the individuals are in one-to-one correspondence with the corresponding guanine content (namely, phenotype), then the difference of the genotype distribution under different genotypes is counted by using a mulcomp package in an R language, and the letters (a and b) above each small graph indicate whether the phenotype difference of the individuals with the two genotypes is obvious or not. If the same letter exists, the phenotype difference corresponding to the two genotypes is not obvious; if the letters are different, the two are significantly different. The 12_55495763SNP genotype and the content of guanine in its individuals were found in table 4 for 421 individuals in the F6 population and 665 individuals in the F7 population.
As shown in FIG. 2 and Table 4, the guanine content in the G/A genotype of the individual at this site is higher than that in the G/G genotype of the individual by about 1 mg/100G.
TABLE 4 Effect of SNP 12_55495763 on guanine content in Whole pig groups F6 and F7
Figure BDA0002609617880000111
Table 5 is directly derived from the analysis results of GWAS. As can be seen from table 5, in the meta-analysis results of both populations F6 and F7, the P values of the 20 most significant sites were decreased by 12-17 orders of magnitude compared to the GWAS analysis results of the single population, and the most significant sites were changed, e.g., the most significant site 12_54924843(4.9E-16) in F6 was the second significant site (3.02E-27) in the meta-analysis; 12_55500274(1.11E-19), which was the most significant site in F7, was the seventh significant site in meta-analysis (4.14E-26); 12_55495763(3.9E-19), the sixth significant site in F7, was the most significant site in meta-analysis (1.68E-27), and its favorable allele A increased the content of guanine in pork by approximately 1mg/100g, the frequency of the A allele in the full population of F6 and F7 being according to the formula: the frequency of the gene was 9.44% (table 4), which was obtained by [ (number of homozygote individuals of the gene × 2+ number of heterozygote individuals) ÷ (total number of individuals × 2) ] × 100%.
TABLE 5 meta-analysis of the most significant 20 SNP site information
Figure BDA0002609617880000112
Figure BDA0002609617880000121
4. Linkage Disequilibrium (LD) analysis
Haploview version4.2 analysis software was used to construct haplotype blocks and the Linkage Disequilibrium (LD) between 3 significant SNPs and 17 SNPs (i.e., SNPs in Table 5) surrounding them was analyzed. The results are shown in FIG. 3. As can be seen from fig. 3, two haplotype blocks (blocks) are formed among the 20 SNPs, in which SNPs numbered 2 to 5 (12_55060526, 12_55096610, 12_55350362 and 12_55355030) are haplotype block 1, and SNPs numbered 7 to 19 are haplotype block 2.SNP loci in the haplotype frame are in a strong linkage state, and the probability of being inherited together to offspring is extremely high. Further analysis revealed that haplotype block 1 contained no 3 significant SNPs affecting guanine content, and haplotype block 2 contained two significant SNPs 12_55495763 and 12_55500274 affecting guanine content, from which it was determined that SNPs numbered 7 to 16 and 19 in haplotype block 2: 12_55369371, 12_55377798, 12_55493385, 12_55493401, 12_55493440, 12_55493473, 12_55493963, 12_55494697, 12_55494763, 12_55495244 and 12_55496302 likewise influence the content of guanine.
While the invention has been described with reference to specific embodiments, those skilled in the art will appreciate that various changes can be made without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, and method to the essential scope and spirit of the present invention. All such modifications are intended to be included within the scope of the present invention as defined in the appended claims.
Sequence listing
<110> university of agriculture in Jiangxi
<120> SNP marker affecting guanine content in pig individual
<130> LHA2060341
<141> 2020-07-29
<160> 3
<170> SIPOSequenceListing 1.0
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ccgcctccta agacacagca ggctactacc cccggttgga attctgggtc cgtttgctcc 60
cctctattgt acgttcctaa tacttggcac gtgtaagaac cacattgtgt gcaaagagcc 120
tttttcctgc cttacctctt tgtgaccatc gcaacaacaa gggcaggtca tgtgcccatt 180
ttacagatag gagtcctgag gctcaaggca tgatgcaccc tgtgcagtgt caccagcgca 240
caagtggtgc aggaccaggt gctcagattt ctcatctctt gcccttcact tgccagagac 300
gaactcggcc tcccctgctg aaacctcttc aaatcgctgc tccgaccctc acttccaagg 360
gacgaatccc ccagaaaggc cgagggtggt tatttctttc ctctttccca ctcggcagcg 420
acctttgctc tcacccgcag ctctctaaag ggagtgcccg gctgcgcctg agtgcaggtc 480
cctgcccagc tgggaggagg ggacgatgac caaacctgcc aggactccaa ctgcggtccc 540
ctggggggag ccggccatct ctaatccagg gtcgtggggg ccagaccagc tctacccttc 600
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ggttactgtg aagattgagt gggtccttaa gtgtaaagtg cttagaacct gcacagtgaa 120
acagacaagc gttactgtgt ggttgccgtg atgtctttta gtacattctt tctgccacag 180
tgtttctgta gagaaaaccc caggtgggaa gggacccatc accaaccctc ctgcaccacc 240
atcctctccc ctttatcagt cttggcttca cctggttgca ggagcctgtc ccccatcccc 300
tggccccact agatccaggc agagccctcc cccatcctag atgactgcag atccaccctg 360
gatggatccc tcggtcacat gagatgcttt ccgggtcccc ggcctctcgt ctctgctgca 420
gctgttcttt atctccatca agttcaagtc tctgcactgc ccctatgctc gtaaccgagt 480
gtgtctccag ccttatgtat atgctctctc cagggttctg ccacagcccc tcgtagtata 540
ctagtctatt tctattattt attatataga atatcatctt aatttgttaa tattacaatg 600
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tcctgtgact aacgccaaac tggaacccag ggcgtggggc caactcgctc atcttgcttc 120
caaaccaacc tcccatgacc acggaggctg gggcgtcctc cgtggatccc gtctgatctg 180
tggatccgtg gatctgtctg tttctgggct tggcctcaga acggtctccc cggggaacat 240
ccaaacccct tggttatttt catctctccg tccagccaga ggcctgggga gtggctggag 300
tcctcggagt ttctcccgac gcagcacaga ggcgctgcgg aaaagaggcc agatttcccc 360
agactggagc aagcaggctt gacacagttc cctctccagg gaagagccaa ggagccacct 420
ctaagatggg cctcttggag ctcctgtggt ggctcagcgg taacaaacgt gactcgtatc 480
ccaaaggact tggttcaacc cctggcctcg ctcggtgggt taaggatccg gcgtggccat 540
gagctatggt gtaggtcgca gatgcagctc tgatcccgcg tggctgtggc tctggcatag 600
g 601

Claims (14)

1. An SNP marker of a pig, wherein the SNP marker is at least one of the following SNP markers:
(I) the nucleotide at position 301 from the 5 'end of SEQ ID number 1, which is G or A, corresponds to the nucleotide at position 55495763 from the 5' end of chromosome 12 of the version 11.1 International pig genome;
(II) the nucleotide at position 331 from the 5 'end of SEQ ID number 2, which is C or CT, corresponding to position 55500274 from the 5' end on chromosome 12 of the version 11.1 international pig genome;
(III) the nucleotide at position 301 from the 5 'terminus of SEQ ID number 3, which is T or C, corresponding to position 54924843 from the 5' terminus on chromosome 12 of the version 11.1 International pig genome.
2. Use of a reagent for detecting a SNP marker according to claim 1 for determining the level of guanine in pork.
3. A method of genetic improvement in a pig, the method comprising: determining the SNP markers of claim 1 for a swine in a swine core group, and making a corresponding selection based on the SNP markers:
(I) selecting breeding pig individuals with G/A and A/A genotypes at the 301 th site from the 5' end on the SEQ ID number 1 in the pig core group, and eliminating the breeding pig individuals with the G/G genotypes at the site to improve the frequency of the allele A at the site by generations;
(II) selecting the swine individuals with the 331 st site from the 5' end of the SEQ ID number 2 as the C/CT and CT/CT genotypes from the swine core group, and eliminating the swine individuals with the C/C genotype at the site to increase the frequency of the allele CT at the site generation by generation;
(III) selecting the boar individuals with T/C and C/C genotypes at the 301 th site from the 5' end on the SEQ ID number 3 in the boar core group, and eliminating the boar individuals with T/T genotypes at the site to increase the frequency of the allele C at the site by generations.
4. The method according to claim 3, wherein for (I), the breeding pig individuals with A/A genotype at the 301. sup. rd site from the 5' end in the SEQ ID number 1 are selected in the core group of the swine, and the breeding pig individuals with G/A and G/G genotypes at the site are eliminated to increase the frequency of allele A at the site generation by generation;
for (II), selecting a swine individual with the 331 st site from the 5' end of the SEQ ID number 2 as the CT/CT genotype from the swine core group, and eliminating swine individuals with the C/CT and C/T genotypes at the site to increase the frequency of the allele CT at the site generation by generation;
for (III), selecting a swine individual with C/C genotype at the 301 th site from the 5' end on the SEQ ID number 3 in the swine core group, and eliminating swine individuals with T/C and T/T genotypes at the site to increase the frequency of allele C at the site by generations.
5. The method according to claim 3, wherein the SNP marker of claim 1 is determined by analyzing the sequence of the nucleic acid of the breeding pig, wherein the sequence of the nucleic acid is selected from at least one of the group consisting of a nucleic acid I comprising the SNP marker in (I), a nucleic acid II comprising the SNP marker in (II) and a nucleic acid III comprising the SNP marker in (III).
6. The method of claim 5, wherein the nucleic acid I, nucleic acid II, and nucleic acid III are independently selected from at least one of a DNA sequence, a cDNA sequence, and an RNA sequence.
7. The method of claim 5, wherein the nucleic acids I to III independently have a length of 5bp to 26560 bp.
8. The method according to claim 5, wherein the nucleic acids I to III independently have a length of 5bp to 10000 bp.
9. The method according to claim 5, characterized in that the nucleic acids I to III independently have a length of 5bp to 5928 bp.
10. The method according to claim 5, wherein the nucleic acids I to III independently have a length of 5bp to 1000 bp.
11. The method according to claim 5, wherein the nucleic acids I to III independently have a length of 5bp to 500 bp.
12. The method according to claim 5, wherein the nucleic acids I to III independently have a length of 5bp to 300 bp.
13. The method according to claim 5, wherein the sequence of nucleic acid I is shown as SEQ ID number 1, the sequence of nucleic acid II is shown as SEQ ID number 2, and the sequence of nucleic acid III is shown as SEQ ID number 3.
14. A method of determining the quality of a pork quality trait, the method comprising: determining the SNP marker of claim 1 of the pig, and determining the pork quality trait according to the SNP marker:
(I) the pork quality traits are from good to bad, and the genotype sequence of the 301 th site from the 5' end on the SEQ ID number 1 is as follows: A/A genotype, G/A genotype, and G/G genotype;
(II) the pork quality traits are from good to bad, and the genotype ordering of the 331 st site from the 5' end on the SEQ ID number 2 is as follows: CT/CT genotype, C/CT genotype, and C/C genotype;
(III) the pork quality traits are from good to bad, and the genotype ordering of the 301 th site from the 5' end on the SEQ ID number 3 sequentially comprises: C/C genotype, T/C genotype, and T/T genotype.
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