SNP (Single Nucleotide Polymorphism) molecular marker associated with chicken bile acid character, primer group and application

Abstract

The invention relates to the technical field of molecular markers, in particular to an SNP (Single Nucleotide Polymorphism) molecular marker associated with chicken bile acid characters, a primer group and application. The molecular marker is located at the 641 bp position of a nucleotide sequence as shown in SEQ ID No.1, and a basic group at the position has A / G polymorphism. The molecular marker provided by the invention is closely associated with the bile acid level of the chicken, and compared with the chicken with the genotype of AG / GG at the site, the chicken with the genotype of AA has higher bile acid level in serum or bile, and chicken manure has lower bile acid level; the molecular marker can be applied to auxiliary breeding of high-quality chickens which are high in fat utilization rate, low in protein loss and rich in high protein, so that the economic benefit of chicken feeding is effectively improved.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular markers, and in particular relates to a SNP molecular marker associated with chicken bile acid traits, a primer set and an application thereof. Background Art

[0002] Eggs and chicken are one of the most important food sources in the world today. China is a major producer and consumer of eggs and chicken. In order to improve production efficiency, chicken genetic resources have always been the focus of major breeding companies. In the study of chicken genetic resources, bile acid metabolism is closely related to chicken growth performance, feed conversion rate, meat quality and other traits. Bile acid is an indispensable component in the digestion process, and its level directly affects the digestion and absorption of fat. Bile acid can emulsify fat, break it down into fat particles, activate pancreatic lipase, increase lipase activity, promote the digestion and absorption of lipid substances, improve feed digestion and utilization, and improve the growth performance and slaughter performance of broilers, increase the survival rate of broilers, and at the same time, bile acid also plays a pivotal role in enhancing the immune function of chickens and improving egg quality.

[0003] As an important method in modern chicken breeding, molecular marker-assisted selection can effectively avoid the drawbacks of the traditional phenotype-based selection method, shorten the genetic interval, and improve the accuracy of selection. The premise of molecular marker-assisted selection is to identify candidate genes related to the desired phenotypic traits, and then purposefully test the candidate genes to further determine the direction of chicken breeding.

[0004] The transcription factor HNF1B can regulate the transcription of multiple bile acid transport genes and participate in the reabsorption of bile acids in the small intestine. The chicken HNF1B gene is located on chromosome 19, contains 9 exons and 8 introns, and can be divided into three functional regions: A, B, and C, of ​​which region B is the DNA binding domain.

[0005] Studying molecular markers related to bile acid traits is of great significance for the selection and breeding of chicken breeds. Through molecular marker technology, chicken breeds with excellent bile acid characteristics can be selected more accurately, thereby improving breeding efficiency and product quality. However, in this field, although existing studies have focused on genes related to chicken bile acid metabolism, relatively few molecular markers directly related to chicken bile acid traits have been developed, and there are still many molecular marker sites related to chicken bile acid traits that have not been explored, which limits the application of molecular marker breeding technology in chicken breed improvement. Summary of the invention

[0006] In view of the technical problems existing in the above-mentioned prior art, the purpose of the present invention is to provide SNP molecular markers, primer sets and applications associated with chicken bile acid traits. The molecular markers can be used for screening or auxiliary screening of chicken bile acid traits, which is conducive to more accurately selecting chicken breeds with excellent bile acid characteristics, and has high identification efficiency and accuracy.

[0007] The first aspect of the present invention provides a SNP molecular marker associated with chicken bile acid traits, wherein the molecular marker is located at the 641 bp of the nucleotide sequence shown in SEQ ID No. 1, has an A / G polymorphism, and the A allele frequency is greater than the G allele frequency.

[0008] The second aspect of the present invention provides a primer set for amplifying the SNP molecular marker associated with the chicken bile acid trait, wherein the nucleotide sequences of the upstream primer and the downstream primer of the primer set are shown in SEQ ID NO.2 and SEQ ID NO.3:

[0009] SEQ ID NO.2: 5'-TGGCTCAGCAAAACCATTCCA-3';

[0010] SEQ ID NO. 3: 5'-CCCAACCCCAAAGGATGCAA-3'.

[0011] The third aspect of the present invention provides a kit, which comprises the primer set described in the second aspect of the present invention.

[0012] Furthermore, the storage solution concentration of the forward primer and the reverse primer in the primer set is 100 μM.

[0013] Furthermore, the kit also includes dNTPs, Taq DNA polymerase and PCR reaction buffer.

[0014] A fourth aspect of the present invention provides an application of the above-mentioned molecular marker, the above-mentioned primer set or the above-mentioned kit in chicken breeding.

[0015] Furthermore, the application is to breed chickens with different bile acid levels.

[0016] A fifth aspect of the present invention provides a method for breeding chickens, comprising the following steps:

[0017] Extracting DNA from the sample to be tested;

[0018] Perform PCR amplification using the DNA of the sample to be tested as a template to obtain a PCR product;

[0019] Sequencing the PCR products, and selecting chicken breeds according to the genotypes of the SNP molecular markers;

[0020] When the sample to be tested is serum, the bile acid level of individuals with the AA genotype of the SNP molecular marker is higher than the bile acid level of individuals with the AG or GG genotype;

[0021] When the sample to be tested is liver, the bile acid level of individuals with the AA genotype of the SNP molecular marker is higher than the bile acid level of individuals with the AG or GG genotype;

[0022] When the sample to be tested is feces, the bile acid level of an individual with the SNP molecular marker genotype of AA is lower than the bile acid level of an individual with the genotype of AG or GG.

[0023] Furthermore, the reaction system for PCR amplification is: 2×Taq Master Mix 15 μL, DNA template 1-1.5 μL, upstream primer 1-1.5 μL, downstream primer 1-1.5 μL, ddH2O 10.5-12 μL.

[0024] Preferably, the reaction system for PCR amplification is: 15 μL of 2×Taq MasterMix, 1.5 μL of DNA template, 1.5 μL of upstream primer, 1.5 μL of downstream primer, and 10.5 μL of ddH2O.

[0025] Furthermore, the reaction procedure of PCR amplification was as follows: pre-denaturation at 93-95°C for 1.5 min; denaturation at 93-95°C for 20 s, annealing at 58-60°C for 20 s, extension at 72°C for 30 s, 35 cycles; extension at 72°C for 5 min, and storage at 4°C.

[0026] Preferably, the reaction procedure of PCR amplification is: pre-denaturation at 94°C for 1.5 min; denaturation at 94°C for 20 s, annealing at 58°C for 20 s, extension at 72°C for 30 s, 35 cycles; extension at 72°C for 5 min, and storage at 4°C.

[0027] In summary, compared with the prior art, the present invention has the following advantages and effects:

[0028] (1) The present invention provides a SNP molecular marker associated with bile acid levels by deeply studying the key genes in the bile acid metabolic pathway of chickens, and through verification, it is found that the base at the 641bp of the nucleotide sequence shown in SEQ ID No. 1 has an A / G polymorphism. By designing a primer set for detecting the SNP molecular marker, it is found that compared with chickens with genotypes of AG / GG at this site, chickens with genotypes of AA have higher bile acid levels in serum or bile, and chicken feces have lower bile acid levels.

[0029] (2) The molecular markers and primer sets provided by the present invention can be used for screening or auxiliary screening of bile acid traits in chicken breeds, which is conducive to more accurately selecting chicken breeds with excellent bile acid characteristics, improving the efficiency of chicken breeding, and reducing unnecessary feeding costs and time costs. At the same time, through precision breeding, the elimination rate caused by undesirable traits can be reduced, further reducing production costs, which is also of great significance for understanding the molecular mechanism of bile acid metabolism. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Figure 1 is the correlation analysis between the expression level of HNF1B target genes in the small intestine and the HNF1B binding sites; Figure A is the weighted image of transcription factor binding sites (Gene-set), Figure B is the weighted image of gene expression levels (Gene-list), Figure C is the correlation image of unweighted gene expression levels and unweighted transcription factor binding sites, and Figure D is the correlation image of weighted gene expression levels and weighted transcription factor binding sites; the green dots represent the two-dimensional scatter plot of the binding site scores and gene expression levels of each HNF1B target gene, and the size of the green dots represents the weighted value. The curve in Figure A represents the weighted value of each transcription factor binding site, the curve in Figure B represents the weighted value of each gene expression level, the curve in Figure C is the non-weighted fitting curve of the transcription factor binding site score and gene expression level, and the curve in Figure D is the weighted fitting curve of the transcription factor binding site score and gene expression level.

[0031] Figure 2 Figure 1 is the GSEA functional annotation of HNF1B target genes. Figure A shows the enrichment spectrum of HNF1B target genes; Figure B shows the distribution of HNF1B target genes in the gene list, where the vertical line indicates the position of HNF1B target genes in the gene list, and the gray bar graph indicates the rank value of HNF1B target genes.

[0032] Figure 3 The results of gene sequence comparison of 10 chickens; the red box shows the A641G mutation site.

[0033] Figure 4 Figure 3 is the effect of SNP molecular marker site genotype on serum bile acid traits; Figures A to E are the effect of SNP molecular marker site genotype on serum bile acid traits in the total population, Jianghan chicken, Jingmen green-shell laying hens, Wuhan green-shell laying hens, and White Leghorn chickens, respectively; the ordinate in the figure is the serum bile acid level, and the abscissa is the genotype, where 0 / 0 represents AA genotype, 0 / 1 represents AG genotype, and 1 / 1 represents GG genotype; a, b, and c represent the groups of multiple comparison results, respectively, the same letters indicate that there is no significant difference between the groups, and different letters indicate significant difference between the groups (p<0.05).

[0034] Figure 5Figure 3 is the effect of SNP molecular marker site genotype on liver bile acid traits; Figures A to E are the effect of molecular marker site genotype on liver bile acid traits of the total population, Jianghan chicken, Jingmen green-shell laying hens, Wuhan green-shell laying hens, and White Leghorn chickens, respectively; the ordinate in the figure is the liver bile acid level, and the abscissa is the genotype, where 0 / 0 represents AA genotype, 0 / 1 represents AG genotype, and 1 / 1 represents GG genotype; a, b, and c represent the groups of multiple comparison results, respectively, the same letters indicate that there is no significant difference between the groups, and different letters indicate significant difference between the groups (p<0.05).

[0035] Figure 6 Figure 3 is the effect of SNP molecular marker site genotype on fecal bile acid traits; Figures A to E are the effect of SNP molecular marker site genotype on fecal bile acid traits of the total population, Jianghan chicken, Jingmen green-shell laying hens, Wuhan green-shell laying hens, and White Leghorn chickens, respectively; the ordinate in the figure is the fecal bile acid level, and the abscissa is the genotype, where 0 / 0 represents the AA genotype, 0 / 1 represents the AG genotype, and 1 / 1 represents the GG genotype; a, b, and c represent the groups of multiple comparison results, respectively, the same letters indicate that there is no significant difference between the groups, and different letters indicate that there is a significant difference between the groups (p<0.05). DETAILED DESCRIPTION

[0036] In order to enable those skilled in the art to better understand and implement the technical solution of the present invention, the present invention is further described below in conjunction with specific embodiments and drawings.

[0037] In the description of the present invention, unless otherwise specified, all reagents used are commercially available and all methods used are conventional techniques in the art.

[0038] Bile acid metabolism is closely related to chicken growth performance, feed conversion rate, meat quality and other traits. Molecular marker-assisted selection is an important means in modern domestic chicken breeding projects. However, due to the relatively few molecular markers directly related to chicken bile acid traits currently developed, the application of molecular marker breeding technology in chicken breed improvement is limited.

[0039] The present invention firstly uses Grit / Flaver software and GSEA software to analyze and find that the target gene of HNF1B is related to organic acid transport. By comparing the DNA sequences of green-shell laying hens and white Leghorn chickens, it is found that there is an A>G mutation at the 641bp of the nucleotide sequence shown in SEQ ID No.1. PCR and sequencing are performed by designing a primer set. After sequencing, it is found that the genotype frequency of the SNP site shows a trend of AA>AG>GG. The molecular marker and primer set are used to verify the correlation between the SNP molecular marker genotype and the bile acid level. It is found that the molecular marker genotype provided by the present invention is closely related to the bile acid level of chickens, and compared with chickens with AG / GG genotypes at the site, chickens with AA genotypes have higher bile acid levels in serum or bile, and chicken feces have lower bile acid levels.

[0040] Example 1: Obtaining HNF1B gene fragments and SNP molecular markers

[0041] Blood samples and bile acid measurement records of 218 chickens of different breeds (67 White Leghorn chickens, 104 Jianghan chickens, 10 Jingmen green-shell laying hens, 11 Xiaogan green-shell laying hens, and 26 Wuhan green-shell laying hens) were collected from agricultural and animal husbandry farms in Jingzhou City, Hubei Province, and the whole genome DNA of the 218 chickens was extracted using a peripheral blood DNA extraction kit (purchased from Beijing Solebow Company).

[0042] Transcriptome gene chip analysis was performed on 21 tissues of 4 representative chicken breeds, White Leghorn chickens. The gene expression levels of each tissue were compared based on the gene chip results, and the expression level measurement values ​​of each tissue-specifically expressed gene were calculated. The weighted Kendall correlation statistics of Grit / Flaver software were used to detect the correlation between the expression level of each tissue-specifically expressed gene and the binding sites of the target genes acted by various transcription factors in chickens.

[0043] The results are as follows Figure 1 As shown, the higher the score of the binding site, the higher its expression level. For genes containing HNF1B binding sites, there is a significant positive correlation between their expression levels and transcription factor binding sites (p<0.05).

[0044] GSEA software was used to perform functional annotation on the target genes of HNF1B that were correlated with the gene expression levels in chicken small intestine.

[0045] The results are as follows Figure 2 As shown, genes containing HNF1B binding sites were found to be significantly enriched in the organic acid transport gene set, with a distribution biased to the left and tending to be highly expressed, indicating that genes containing HNF1B binding sites are related to organic acid transport at an extremely significant level (p<0.01).

[0046] The genome of 29 green-shell laying hens (10 Jingmen green-shell laying hens, 10 Wuhan green-shell laying hens, and 9 Xiaogan green-shell laying hens) was resequenced, and the sequencing results were compared with the DNA sequence of white Leghorn chickens.

[0047] The comparison results are as follows Figure 3 As shown, it was found that there was an A>G mutation at the 641bp of the nucleotide sequence shown in SEQ ID No.1, and the red box showed the A641G mutation site.

[0048] SEQ ID NO.1:

[0049]

[0050] Oligo software was used to design specific primer combinations for this region, and PCR amplification reactions were performed using genomic DNA of 218 tested individuals as templates.

[0051] The designed primer combination includes an upstream primer and a downstream primer, and the sequences of the upstream primer and the downstream primer are shown in SEQ ID NO.2 and SEQ ID NO.3:

[0052] SEQ ID NO.2: 5'-TGGCTCAGCAAAACCATTCCA-3';

[0053] SEQ ID NO. 3: 5'-CCCAACCCCAAAGGATGCAA-3'.

[0054] Table 1 shows the specific parameters of the PCR reaction system.

[0055] Table 1 PCR reaction system

[0056] Element Content (μL) 2×TaqMasterMix 15 Upstream primer (10 μM) 1.5 Downstream primer (10 μM) 1.5 DNA template 1.5 <![CDATA[ddH2O]]> 10.5

[0057] The PCR amplification program is shown in Table 2.

[0058] Table 2 PCR amplification program

[0059]

[0060] Note: “-” means the corresponding step will not be cycled.

[0061] 4 μL of each tube of PCR product was taken for agarose gel electrophoresis, and the PCR product containing a single band was sent to Shanghai Bioengineering Company for sequencing after imaging. The sequencing results were compared and analyzed using SeqMan software to find and record the gene type of the SNP molecular marker site in each individual (AA: 0 / 0, AG: 0 / 1, GG: 1 / 1).

[0062] Among the 218 chickens tested, the gene and genotype frequencies of the SNP molecular marker loci in the five breeds are shown in Table 3.

[0063] Table 3 Genotype frequency and allele frequency of SNP loci

[0064]

[0065] As shown in Table 3, the SNP molecular marker loci showed three genotypes in 104 Jianghan chickens, and the genotype frequencies showed a trend of AA>AG>GG, and the frequency of A allele was greater than that of G allele; there were only two genotypes, AA and AG, in 10 Jingmen green-shell laying hens and 11 Xiaogan green-shell laying hens, and the frequency of A allele was greater than that of G allele; there were three genotypes in 26 Wuhan green-shell laying hens, and the genotype frequencies showed a trend of AA>AG>GG, and the frequency of A allele was greater than that of G allele; there was only one genotype in White Leghorn chickens, which was AA type.

[0066] Example 2: Validation of SNP molecular markers associated with bile acid levels

[0067] In order to establish the relationship between SNP molecular markers and chicken bile acid traits, 218 chicken tissues of different strains (67 white Leghorn chickens, 104 Jianghan chickens, 10 Jingmen green-shell laying hens, 11 Xiaogan green-shell laying hens, and 26 Wuhan green-shell laying hens) were selected as experimental materials, and the bile acid levels and genotypes of SNP molecular marker sites of 218 chickens were measured. The ANOVA program in R4.1 software was used to perform association analysis on the genotypes of the SNP molecular marker sites of each strain and the bile acid trait data. The results are as follows: Figure 4 , Figure 5 , Figure 6 shown.

[0068] Depend on Figure 4The results show that the serum bile acid levels of AA genotype in all 218 tested chickens were 4.58±0.51, AG genotype 3.62±0.39, GG genotype 2.32±0.59, and serum bile acid levels of AA>AG>GG showed significant differences (p<0.05); the serum bile acid levels of AA genotype in 104 Jianghan chickens were 4.57±0.47, AG genotype 3.60±0.25, GG genotype 2.07±0.47, and serum bile acid levels of AA>AG>GG showed significant differences (p<0.05); the serum bile acid levels of AA genotype in 10 Jingmen green-shell laying hens were 4.28±0.21, AG genotype 4.22, and no GG genotype Due to genotype, there was no significant difference in serum bile acid levels between AA and AG; the serum bile acid level of 26 Wuhan green-shell laying hens with AA genotype was 4.23±0.45, AG genotype was 3.51±0.27, and GG genotype was 2.95±0.24. The serum bile acid level of AA>AG and GG was significantly different (p<0.05), and there was no significant difference between AG and GG; the serum bile acid level of 11 Xiaogan green-shell laying hens with AA genotype was 3.99±0.43, AG genotype was 3.87±1.53, and there was no GG genotype. There was no significant difference in serum bile acid level between AA and AG; the genotype of 67 White Leghorn chickens was all AA, and the serum bile acid level was 4.81±0.43.

[0069] Depend on Figure 5The results show that the liver bile acid levels of all 218 tested chickens with AA genotype were 2.59±0.33, AG genotype 2.24±0.25, GG genotype 2.02±0.13, and the liver bile acid levels of AA>AG and GG showed significant differences (p<0.05), and there was no significant difference between AG and GG; the liver bile acid levels of AA genotype in 104 Jianghan chickens were 2.51±0.18, AG genotype 2.27±0.16, GG genotype 2.01±0.16, and the liver bile acid levels of AA>AG>GG showed significant differences (p<0.05); the liver bile acid levels of AA genotype in 10 Jingmen green-shell laying hens were 2.22±0.28, A The G genotype was 2.05, and there was no GG genotype. There was no significant difference in the liver bile acid levels of AA and AG. The liver bile acid levels of 26 Wuhan green-shell laying hens with AA genotype were 2.29±0.14, AG genotype was 2.34±0.37, and GG genotype was 2.04±0.07. There was no significant difference in the liver bile acid levels of AA, AG and GG. The liver bile acid levels of 11 Xiaogan green-shell laying hens with AA genotype were 2.0±0.15, AG genotype was 1.73±0.15, and there was no GG genotype. The liver bile acid levels AA>AG showed significant differences (p<0.05). The genotypes of 67 White Leghorn chickens were all AA, and the liver bile acid levels were 2.91±0.19.

[0070] Depend on Figure 6 It can be seen that the fecal bile acid level of all 218 tested chickens with AA genotype was 0.50±0.09, AG genotype was 0.60±0.14, GG genotype was 1.32±0.15, and the fecal bile acid level was GG>AG>AA, showing a significant difference (p<0.05); the fecal bile acid level of AA genotype in 104 Jianghan chickens was 0.49±0.09, AG genotype was 0.59±0.12, GG genotype was 1.37±0.15, and the fecal bile acid level was GG>AG>AA, showing a significant difference (p<0.05); the fecal bile acid level of AA genotype in 10 Jingmen green-shell laying hens was The fecal bile acid level in 26 Wuhan green-shell laying hens was 0.45±0.08, AG genotype was 0.62, no GG genotype, and there was no significant difference in fecal bile acid levels between AA and AG; the fecal bile acid level of AA genotype in 26 Wuhan green-shell laying hens was 0.53±0.12, AG genotype was 0.60±0.08, GG genotype was 1.19±0.02, and the fecal bile acid level of GG>AA genotype (p<0.05) and GG>AG genotype (p<0.05), and there was no significant difference between AA and AG; the fecal bile acid level of AA genotype in 11 Xiaogan green-shell laying hens was 0.45±0.10,

[0071] AG genotype 0.72±0.49, no GG genotype, no significant difference in fecal bile acid levels between AA and AG;

[0072] The genotype of all 67 White Leghorn chickens was AA, and the fecal bile acid level was 0.52±0.08.

Claims

1. A SNP molecular marker associated with chicken bile acid traits, characterized in that: The SNP molecular marker is located at the 641 bp of the nucleotide sequence shown in SEQ ID No. 1, and the base at this position has an A / G polymorphism.

2. A primer set for amplifying the SNP molecular marker associated with chicken bile acid traits according to claim 1, characterized in that: The nucleotide sequences of the upstream primer and the downstream primer of the primer set are shown in SEQ ID NO.2 and SEQ ID NO.3: SEQ ID NO.2: 5'-TGGCTCAGCAAAACCATTCCA-3'; SEQ ID NO. 3: 5'-CCCAACCCCAAAGGATGCAA-3'.

3. A kit, characterized in that: The kit comprises the primer set according to claim 2.

4. The kit according to claim 3, characterized in that The kit also includes dNTPs, Taq DNA polymerase and PCR reaction buffer.

5. Use of the molecular marker according to claim 1, the primer set according to claim 2 or the kit according to claim 3 in chicken breeding.

6. The use according to claim 5, characterized in that: The application is to breed chickens with different bile acid levels.

7. A method for breeding chickens, characterized in that: The following steps are involved: Extracting DNA from the sample to be tested; Perform PCR amplification using the DNA of the sample to be tested as a template to obtain a PCR product; The PCR product is sequenced, and chicken breeds are selected according to the genotype of the SNP molecular marker according to claim 1.

8. The chicken breeding method according to claim 7, characterized in that: When the sample to be tested is serum, the bile acid level of individuals with the AA genotype of the SNP molecular marker is higher than the bile acid level of individuals with the AG or GG genotype; When the sample to be tested is liver, the bile acid level of individuals with the AA genotype of the SNP molecular marker is higher than the bile acid level of individuals with the AG or GG genotype; When the sample to be tested is feces, the bile acid level of an individual with the SNP molecular marker genotype of AA is lower than the bile acid level of an individual with the genotype of AG or GG.

9. The method according to claim 7, characterized in that: The reaction system for PCR amplification is: 2×Taq MasterMix 15μL, DNA template 1~1.5μL, upstream primer 1~1.5μL, downstream primer 1~1.5μL, ddH2O 10.5~12μL.

10. The method according to claim 7, characterized in that The reaction procedure of PCR amplification was as follows: pre-denaturation at 93-95°C for 1.5 min; denaturation at 93-95°C for 20 s, annealing at 58-60°C for 20 s, extension at 72°C for 30 s, 35 cycles; extension at 72°C for 5 min, and storage at 4°C.

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