Snps associated with chicken bile acid traits, primer sets and applications

Abstract

The present application relates to the technical field of molecular marker, and particularly relates to a SNP molecular marker associated with chicken bile acid traits, a primer group and application. The molecular marker is located at the 641bp of the nucleotide sequence shown in SEQ ID No. 1, and the base at the position has A / G polymorphism. The molecular marker provided by the present application is closely associated with the bile acid level of chicken. Compared with the chicken with the genotype AG / GG at the site, the chicken with the genotype AA has a higher bile acid level in serum or bile, and the chicken excrement has a lower bile acid level. The molecular marker can be applied to assist in breeding high-quality chicken with high fat utilization rate, low protein loss and high protein content, thereby effectively improving the economic benefits of raising chicken.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of molecular markers, and particularly relates to a SNP molecular marker associated with chicken bile acid traits, a primer set and application. BACKGROUND

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

[0003] Molecular marker-assisted selection, as an important means in modern chicken breeding engineering, can effectively avoid the drawbacks of traditional breeding based on phenotype selection, shorten the genetic interval period, and improve selection accuracy. The premise of molecular marker-assisted selection is to determine the candidate genes related to the desired phenotypic traits, and then detect the candidate genes purposefully, so as to further determine the breeding direction of chickens.

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

[0005] Research on molecular markers related to bile acid traits is of great significance for chicken breeding. Through molecular marker technology, chickens with excellent bile acid characteristics can be more accurately selected, thereby improving breeding efficiency and product quality. However, in this field, although some 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 many molecular marker sites related to chicken bile acid traits have not been explored, which limits the application of molecular marker breeding technology in chicken breeding improvement. SUMMARY

[0006] In view of the technical problems in the prior art, the present application aims to provide a SNP molecular marker associated with chicken bile acid traits, a primer set and application thereof. The molecular marker can be used for screening or auxiliary screening of chicken breed bile acid traits, and is conducive to more accurate selection of chicken breeds with excellent bile acid characteristics, and has high identification efficiency and accuracy.

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

[0008] The second aspect of the present application provides a primer set for amplifying the above-mentioned SNP molecular marker associated with chicken bile acid traits, 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 application provides a kit, which comprises the primer set according to the second aspect of the present application.

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

[0013] Further, the kit further comprises dNTPs, Taq DNA polymerase and PCR reaction buffer.

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

[0015] Further, the application is the selection of chicken breeds with different bile acid levels.

[0016] The fifth aspect of the present application provides a method for chicken breed selection, comprising the following steps:

[0017] extracting DNA of a sample to be tested;

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

[0019] sequencing the PCR product, and selecting chicken breeds according to the genotype of the SNP molecular marker;

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

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

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

[0023] Further, the reaction system of PCR amplification is as follows: 2x Taq Master Mix 15 μL, DNA template 1-1.5 μL, upstream primer 1-1.5 μL, downstream primer 1-1.5 μL, and ddH2O 10.5-12 μL.

[0024] Preferably, the reaction system of PCR amplification is as follows: 2x Taq Master Mix 15 μL, DNA template 1.5 μL, upstream primer 1.5 μL, downstream primer 1.5 μL, and ddH2O 10.5 μL.

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

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

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

[0028] (1) The present application provides a SNP molecular marker associated with the bile acid level by deeply studying the key genes in the bile acid metabolic pathway of chickens, and it is found through verification that the base at the 641bp of the nucleotide sequence shown in SEQ ID No. 1 has A / G polymorphism. It is found through designing primers for detecting the SNP molecular marker that the chicken with the genotype AA has a higher bile acid level in serum or bile than the chicken with the genotype AG / GG, and the chicken with the genotype AA has a lower bile acid level in feces.

[0029] (2) The molecular marker and primer set provided by the application can be used for screening or auxiliary screening of chicken breed bile acid traits, which is beneficial to more accurately select chicken breeds with excellent bile acid characteristics, improve chicken breed selection efficiency, reduce unnecessary feeding costs and time costs. At the same time, through precise breeding, the elimination rate caused by adverse traits can be reduced, further reducing production costs, and also having important significance for understanding the molecular mechanism of bile acid metabolism. BRIEF DESCRIPTION OF DRAWINGS

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

[0031] Figure 2 Functional annotation of GSEA of target genes of HNF1B. A is an enrichment profile of HNF1B target genes; B is a distribution of HNF1B target genes in a gene list, wherein the vertical line marks the position of the HNF1B target gene in the gene list, and the gray columnar chart is the rank value of the HNF1B target gene.

[0032] Figure 3 It is a comparison result of gene sequences of 10 chickens; the red box shows the A641G mutation site.

[0033] Figure 4 It is an influence diagram of the genotype of the SNP molecular marker site on the serum bile acid trait; A-E diagrams are influence diagrams of the genotype of the molecular marker site on the serum bile acid trait in the total population, Jianghan chicken, Jingmen green-shelled egg chicken, Wuhan green-shelled egg chicken and Bailaihang chicken, respectively; the vertical coordinate in the diagram is the serum bile acid level, and the horizontal coordinate is the genotype, wherein 0 / 0 represents the AA genotype, 0 / 1 represents the AG genotype, and 1 / 1 represents the GG genotype; a, b and c respectively represent the groups of multiple comparison results, and the same letter represents that there is no significant difference between groups, and different letters represent that there is a significant difference between groups (p<0.05).

[0034] Figure 5Figure for influence of SNP molecular marker site genotype on liver bile acid traits; A-E figures are respectively for total population, Jianghan chicken, Jingmen green-shelled laying chicken, Wuhan green-shelled laying chicken, Bailaihang chicken; vertical coordinate is liver bile acid level, horizontal coordinate is genotype, wherein 0 / 0 represents AA genotype, 0 / 1 represents AG genotype, and 1 / 1 represents GG genotype; a, b, c respectively represent multiple comparison results of groups, same letter represents no significant difference between groups, and different letter represents significant difference between groups (p<0.05).

[0035] Figure 6 Figure for influence of SNP molecular marker site genotype on fecal bile acid traits; A-E figures are respectively for total population, Jianghan chicken, Jingmen green-shelled laying chicken, Wuhan green-shelled laying chicken, Bailaihang chicken; vertical coordinate is fecal bile acid level, horizontal coordinate is genotype, wherein 0 / 0 represents AA genotype, 0 / 1 represents AG genotype, and 1 / 1 represents GG genotype; a, b, c respectively represent multiple comparison results of groups, same letter represents no significant difference between groups, and different letter represents significant difference between groups (p<0.05). DETAILED DESCRIPTION

[0036] In order to make the skilled in the art better understand the technical solutions of the present application can be implemented, the following specific examples and the present application is further described with reference to the drawings.

[0037] In the description of the present application, if not special, the reagents used are commercially available, and the methods used are conventional techniques in the art.

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

[0039] The application firstly finds that the target gene of HNF1B is related to organic acid transport by using Grit / Flaver software and GSEA software analysis, finds that the base at 641bp of the nucleotide sequence shown in SEQ ID No. 1 exists A>G mutation by comparing the DNA sequences of green shell egg chicken and Bailaihang chicken, and finds that the genotype frequency of the SNP site presents a trend of AA>AG>GG by designing primers for PCR and sequencing. The association between the SNP molecular marker genotype and the bile acid level is verified by using the molecular marker and primer set, and it is found that the molecular marker genotype provided by the application is closely related to the bile acid level of the chicken, and the chicken with genotype AA has higher bile acid level in serum or bile and lower bile acid level in chicken feces compared with the chicken with genotype AG / GG at the site.

[0040] Example 1: Obtaining of HNF1B gene fragment and SNP molecular marker

[0041] 218 blood samples of different strains (67 Bailaihang chickens, 104 Jianghan chickens, 10 Jingmen green shell egg chickens, 11 Xiaogan green shell egg chickens and 26 Wuhan green shell egg chickens) and bile acid determination records were collected from a farm in Jingzhou, Hubei Province, and the whole genome DNA of the 218 chickens was extracted by using a peripheral blood DNA extraction kit (purchased from Beijing Solabio Company).

[0042] Transcriptome gene chip analysis was performed on 21 tissues of 4 representative chicken species Bailaihang chickens, the gene expression levels between tissues were compared according to the gene chip results, the expression levels of the tissue-specific expression genes were calculated, and the correlation between the expression levels of the tissue-specific expression genes and the binding sites of the target genes of the chicken transcription factors was detected by using the weighted Kendall correlation statistics of Grit / Flaver software.

[0043] As shown in the results, Figure 1 the higher the score of the binding site, the higher the expression level, and for the genes containing the HNF1B binding site, the expression level and the transcription factor binding site are significantly positively correlated (p<0.05).

[0044] The target genes of HNF1B related to the gene expression level of chicken small intestine were subjected to functional annotation by using GSEA software.

[0045] As shown in the results, Figure 2 it is found that the genes containing the HNF1B binding site are significantly enriched in the organic acid transport gene set, and the distribution is biased to the left, tending to be highly expressed, indicating that the genes containing the HNF1B binding site are related to organic acid transport, and the correlation is extremely significant (p<0.01).

[0046] 29 Jingmen Green Shell, 10 Wuhan Green Shell, and 9 Xiaogan Green Shell chickens were subjected to whole-genome resequencing, and the sequencing results were compared with the DNA sequence of the White Leghorn chicken.

[0047] The comparison results are shown in Figure 3 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 A641G mutation site is shown in the red box.

[0048] SEQ ID NO. 1:

[0049]

[0050] The specific primer combination was designed for this region fragment using Oligo software, and PCR amplification reaction was carried out using the genomic DNA of 218 individuals as template.

[0051] The designed primer combination contains 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] Ingredients Amount (μL) 2X Taq Master Mix 15 Upstream primer (10 μM) 1.5 Downstream primer (10 μM) 1.5 DNA template 1.5 ddH2O 10.5

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

[0058] Table 2 PCR amplification program

[0059]

[0060] Note: "-" indicates that the corresponding step does not perform the cycle.

[0061] 4 μL of the PCR product in each tube was subjected to agarose gel electrophoresis, and after imaging, the PCR product containing a single band was sent to Shanghai Bioengineering Company for sequencing. The sequencing results were analyzed by SeqMan software, and the SNP molecular marker sites present in each individual were found out and recorded (AA: 0 / 0, AG: 0 / 1, GG: 1 / 1).

[0062] Among the 218 chickens detected, the gene and genotype frequency of the SNP molecular marker site in five breeds is shown in Table 3.

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

[0064]

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

[0066] Example 2: Verification of SNP molecular markers related to bile acid level

[0067] In order to establish the relationship between SNP molecular markers and chicken bile acid traits, 218 chicken tissues of different strains (67 Bailaihang chickens, 104 Jianghan chickens, 10 Jingmen green-shelled laying hens, 11 Xiaogan green-shelled laying hens, and 26 Wuhan green-shelled laying hens) were selected as experimental materials, and the bile acid level and SNP molecular marker site genotype of 218 chickens were determined. The genotype of SNP molecular marker site and the data of bile acid traits of each strain were analyzed for correlation by ANOVA program in R4.1 software, and the results are shown in Table 4. Figure 4 、 Figure 5 、 Figure 6

[0068] As shown in Table 3, the SNP molecular marker site showed three genotypes in 104 Jianghan chickens, the genotype frequency showed a trend of AA > AG > GG, and the A allele frequency was greater than the G allele frequency; in 10 Jingmen green-shelled laying hens and 11 Xiaogan green-shelled laying hens, only AA and AG two genotypes existed, and the A allele frequency was greater than the G allele frequency; in 26 Wuhan green-shelled laying hens, three genotypes were shown, the genotype frequency showed a trend of AA > AG > GG, and the A allele frequency was greater than the G allele frequency; in Bailaihang chickens, only one genotype existed, which was AA type. Figure 4 ​The serum bile acid level of AA genotype in all 218 test chickens was 4.58±0.51, the serum bile acid level of AG genotype was 3.62±0.39, the serum bile acid level of GG genotype was 2.32±0.59, and the serum bile acid level of AA>AG>GG showed significant difference (p<0.05); the serum bile acid level of AA genotype in 104 Jianghan chickens was 4.57±0.47, the serum bile acid level of AG genotype was 3.60±0.25, the serum bile acid level of GG genotype was 2.07±0.47, and the serum bile acid level of AA>AG>GG showed significant difference (p<0.05); the serum bile acid level of AA genotype in 10 Jingmen green-shelled laying hens was 4.28±0.21, the serum bile acid level of AG genotype was 4.22, and there was no GG genotype, and the serum bile acid level of AA and AG showed no significant difference; the serum bile acid level of AA genotype in 26 Wuhan green-shelled laying hens was 4.23±0.45, the serum bile acid level of AG genotype was 3.51±0.27, the serum bile acid level of GG genotype was 2.95±0.24, and the serum bile acid level of AA>AG and GG showed significant difference (p<0.05), and there was no significant difference between AG and GG; the serum bile acid level of AA genotype in 11 Xiaogan green-shelled laying hens was 3.99±0.43, the serum bile acid level of AG genotype was 3.87±1.53, and there was no GG genotype, and the serum bile acid level of AA and AG showed no significant difference; the genotype of 67 white leghorn chickens was all AA type, and the serum bile acid level was 4.81±0.43.

[0069] By Figure 5It can be seen that the liver bile acid level of AA genotype in all 218 test chickens is 2.59±0.33, the AG genotype is 2.24±0.25, and the GG genotype is 2.02±0.13. The liver bile acid level of AA is significantly different from AG and GG (p<0.05), and the difference between AG and GG is not significant. The liver bile acid level of AA genotype in 104 Jianghan chickens is 2.51±0.18, the AG genotype is 2.27±0.16, and the GG genotype is 2.01±0.16. The liver bile acid level of AA is significantly different from AG and GG (p<0.05). The liver bile acid level of AA genotype in 10 Jingmen green shell laying hens is 2.22±0.28, the AG genotype is 2.05, and there is no GG genotype. The liver bile acid level of AA and AG is not significantly different. The liver bile acid level of AA genotype in 26 Wuhan green shell laying hens is 2.29±0.14, the AG genotype is 2.34±0.37, and the GG genotype is 2.04±0.07. The liver bile acid level of AA, AG and GG is not significantly different. The liver bile acid level of AA genotype in 11 Xiaogan green shell laying hens is 2.0±0.15, the AG genotype is 1.73±0.15, and there is no GG genotype. The liver bile acid level of AA is significantly different from AG (p<0.05). The liver bile acid level of 67 White Leghorn chickens is AA, and the liver bile acid level is 2.91±0.19.

[0070] From Figure 6 It can be seen that the fecal bile acid level of AA genotype in all 218 test chickens is 0.50±0.09, the AG genotype is 0.60±0.14, and the GG genotype is 1.32±0.15. The fecal bile acid level of GG is significantly different from AG and AA (p<0.05). The fecal bile acid level of AA genotype in 104 Jianghan chickens is 0.49±0.09, the AG genotype is 0.59±0.12, and the GG genotype is 1.37±0.15. The fecal bile acid level of GG is significantly different from AG and AA (p<0.05). The fecal bile acid level of AA genotype in 10 Jingmen green shell laying hens is 0.45±0.08, the AG genotype is 0.62, and there is no GG genotype. The fecal bile acid level of AA and AG is not significantly different. The fecal bile acid level of AA genotype in 26 Wuhan green shell laying hens is 0.53±0.12, the AG genotype is 0.60±0.08, and the GG genotype is 1.19±0.02. The fecal bile acid level of GG is significantly different from AA genotype (p<0.05) and AG genotype (p<0.05), and AA and AG are not significantly different. The fecal bile acid level of AA genotype in 11 Xiaogan green shell laying hens is 0.45±0.10,

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

[0072] The genotype of 67 white leihang chickens was AA type, and the fecal bile acid level was 0.52 ± 0.08.

Claims

1. The use of a SNP molecular marker associated with chicken bile acid traits in chicken breeding, characterized in that, The application is used for breeding chicken breeds with different levels of bile acids; the SNP molecular marker is located at 641bp of the nucleotide sequence shown in SEQ ID No. 1, and the base at this position has A / G polymorphism; wherein: the bile acid level in serum or bile of an individual with AA genotype at the polymorphism site is higher than that of an individual with AG or GG genotype, and the bile acid level in feces of an individual with AA genotype at the polymorphism site is lower than that of an individual with AG or GG genotype.

2. A method of chicken breeding, characterized by, The method is used for breeding chicken breeds with different levels of bile acids; the method comprises the following steps: extracting DNA of a sample to be tested; performing PCR amplification on the DNA of the sample to be tested as a template to obtain a PCR product; sequencing the PCR product, and breeding chicken breeds according to the genotype of the SNP molecular marker according to claim 1; when the sample to be tested is serum, the bile acid level of an individual with AA genotype at the SNP molecular marker is higher than that of an individual with AG or GG genotype; when the sample to be tested is liver, the bile acid level of an individual with AA genotype at the SNP molecular marker is higher than that of an individual with AG or GG genotype; when the sample to be tested is feces, the bile acid level of an individual with AA genotype at the SNP molecular marker is lower than that of an individual with AG or GG genotype.

3. The method of claim 2, wherein, The reaction system for PCR amplification is: 2x Taq MasterMix 15μL, DNA template 1~1.5μL, upstream primer 1~1.5μL, downstream primer 1~1.5μL, and ddH2O 10.5~12μL.

4. The method of claim 2, wherein, The reaction procedure for PCR amplification is: 93~95℃ pre-denaturation for 1.5min; 93~95℃ denaturation for 20s, 58~60℃ annealing for 20s, 72℃ extension for 30s, 35 cycles; 72℃ extension for 5min, and 4℃ preservation.

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