A SNP site associated with eggshell color and its application

Abstract

The present invention provides a SNP site related to egg shell color, wherein the site of the SNP marker is a site on chromosomes 1, 3, 4, 5, 7, 8, 9, 12, 17, 20, and 27 of the chicken reference genome GRCg6a. The present invention can predict the egg shell color of breeder chickens in advance by detecting the SNP genotype of the target site. At the same time, breeder chickens with different egg shell colors can be selected according to the breeding goal by optimizing the dominant allele genotype of the SNP site.

Description

[0001] Application technology

[0002] The invention belongs to the field of biotechnology, and in particular relates to a SNP site on a chicken chromosome related to eggshell color and an application thereof. Background Art

[0003] As the most intuitive sensory evaluation index, the eggshell color of poultry is usually more popular with green shells. Jingyuan chicken is an excellent local chicken breed in Ningxia, mainly producing pink and green shell eggs. The increase in the production of green shell eggs, which have a large market demand, can improve the economic benefits of the Jingyuan chicken industry. Eggshell color is a heritable trait with high heritability. Eggshell color is determined by the deposition of eggshell pigments, mainly protoporphyrin-IX, biliverdin-IX, and zinc chelate of biliverdin. Different eggshell colors can be formed by mixing in different proportions. Most studies have shown that both eggshell glands and blood have the function of synthesizing eggshell pigments. Protoporphyrin is mainly synthesized in the eggshell gland, while biliverdin is mainly derived from the degradation of heme in aging red blood cells. Previous studies have shown that the green shell trait of chickens is a quality trait controlled by a dominant single gene (Oocyan, O). In order to locate the O gene, Z. Wang et al. [5] resequenced the genome sequence of the solute carrier organic anion transporter family member 1B3 (SLCO1B3) and found that there was an insertion sequence of the chicken retrovirus EAV-HP in the 5′ flanking region of the gene. This mutation led to abnormal expression of the shell gland of green-shelled laying hens, producing a large amount of OATP1B3 protein, causing biliverdin to deposit on the eggshell. The formation mechanism of the eggshell color of pink-shelled eggs has not yet been fully clarified. Whether brown-shelled eggs are produced is controlled by an incompletely dominant gene, and multiple genes work together to regulate it. H. Rome et al. used a 600K Affymetrix SNP chip to locate QTLs that affect eggshell color. They detected 39 QTLs related to eggshell color traits, of which 22 QTLs directly affected eggshell color. The number of these QTLs was large and the distribution range was wide, which fully proved that eggshell color was controlled by micro-effect polygenes. Zheng et al. [8] used candidate gene association analysis to show that solute carrier organic anion transporter family member 1A2 (SLC01A2) and solute carrier organic anion transporter family member 1C1 (SLCO1C1) were significantly associated with the color depth of brown-shelled eggs. The leucine-rich repeat protein 8 (LRRC8) family genes are involved in a variety of biological processes such as cell adhesion and cell transport, and may be involved in the biological process of the formation of eggshell color in pink-shelled eggs. In addition, the phosphatidylinositol-4-phosphate 3-kinase catalytic subunit 2 type γ (PIK3C2G) gene on chromosome 1, the adenosine triphosphate binding cassette C subfamily 9 member (ABCC9) gene, and the 1,4,5-triphosphate inositol type II receptor (ITPR2

[12] ) gene were also identified as important genes affecting the eggshell color of green-shelled eggs. In recent years, with the development of omics technology and the widespread application of whole-genome association analysis, people have the opportunity to accurately locate QTLs that affect eggshell color. However, there are few reports on the mechanism of eggshell color formation in Shizuoka egg, and the molecular markers related to the eggshell color formation in Shizuoka egg have not yet been explored. Summary of the invention

[0004] In view of the problems existing in the prior art, the present invention provides a method for identifying significant SNPs that affect eggshell color by combining GWAS analysis with transcriptome analysis strategy, which is used in molecular marker-assisted selection and genomic selection to predict the eggshell color of breeder chickens in advance. At the same time, breeder chickens with different eggshell colors can be selected by optimizing the dominant allele genotype of the SNP locus according to the breeding goal, bringing huge economic benefits to the breeding of Jingyuan chickens.

[0005] The primary purpose is to determine the SNP molecular markers associated with egg shell color. The SNP markers include any site on the reference sequence of the chicken genome (Gallus gallus, GRCg6a, Ensembl release-104):

[0006]

[0007]

[0008] In a preferred embodiment, the SNP markers include all the above-mentioned molecular marker sites.

[0009] Another object of the present invention is to provide a method for detecting the above-mentioned SNP molecular marker, characterized in that it comprises the following steps:

[0010] 1) Extracting sample genomic DNA;

[0011] 2) sequencing the above genomic DNA to obtain sequencing reads;

[0012] 3) Aligning the above sequencing reads to the chicken genome;

[0013] 4) Based on the comparison results, determine the genotype of the above SNP sites.

[0014] In a preferred embodiment, the sequencing is first-generation sequencing, second-generation sequencing, or third-generation sequencing.

[0015] Another object of the present invention is to provide an application of the above-mentioned molecular marker in assisting in determining the color of an egg shell, comprising the following steps:

[0016] 1) Extract chicken genomic DNA;

[0017] 2) sequencing the above genomic DNA to obtain sequencing reads;

[0018] 3) Aligning the above sequencing reads to the chicken genome;

[0019] 4) Based on the comparison results, determine the genotype of the SNP site described in claim 1 or claim 3.

[0020] 5) Infer the egg shell color based on the SNP locus genotype determined in step 4).

[0021] In a preferred case, when the 10829854th base of chromosome 20 and / or the 10839766th base of chromosome 20 is CT, it is judged that the eggshell color may be green; when the 10829854th base of chromosome 20 and / or the 10839766th base of chromosome 20 is CC, it is judged that the eggshell color may be pink.

[0022] Another object of the present invention is to provide a molecular assisted breeding method for egg shell color, characterized in that the method comprises the following steps: detecting the genotype of the SNP marker site as described above, and selecting breeder chickens retaining the corresponding genotype according to the egg shell color to be obtained.

[0023] In a preferred embodiment, the method comprises the following steps:

[0024] 1) Extracting genomic DNA from the chicken to be tested;

[0025] 2) sequencing the above genomic DNA to obtain sequencing reads;

[0026] 3) Aligning the above sequencing reads to the chicken genome;

[0027] 4) determining the genotype of the SNP site according to claim 1 or claim 3 based on the comparison results;

[0028] 5) Select breeder chickens with the corresponding genotype based on the desired eggshell color.

[0029] In a preferred embodiment, the aforementioned sequencing is first-generation sequencing, second-generation sequencing, or third-generation sequencing.

[0030] In a preferred case, the aforementioned SNP associated with eggshell color is a C>T mutation at base 10829854 on chromosome 20, and / or a C>T mutation at base 10839766 on chromosome 20.

[0031] In a preferred case, if more green-shell laying hens are to be obtained in the population, the breeder chickens whose base 10829854 on chromosome 20 and / or the base 10839766 on chromosome 20 is CT are retained; if more pink-shell laying hens are to be obtained in the population, the breeder chickens whose base 10829854 on chromosome 20 and / or the base 10839766 on chromosome 20 is CC are retained.

[0032] Compared with the prior art, the present invention has the following advantages and effects:

[0033] First, new SNP molecular markers related to eggshell color were identified, and corresponding detection methods were developed.

[0034] Second, by detecting the SNP locus genotype described in the present invention, the eggshell color of the breeder chickens can be predicted in advance.

[0035] Third, the present invention can select breeder chickens with different eggshell colors by optimizing the dominant allele type of the SNP locus according to the breeding goal. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] The method of the present invention and its beneficial effects are described in detail below in conjunction with the accompanying drawings and specific implementation methods.

[0037] Figure 1 It is the Manhattan plot and QQ plot of GWAS analysis. Figure 1 A and Figure 1 B is the Manhattan plot and QQ plot of GWAS analysis comparing green-shelled eggs and eggs of other colors; Figure 1 A and Figure 1 B are the Manhattan plot and QQ plot of GWAS analysis comparing pink-shelled eggs and eggs of other colors.

[0038] Figure 2 This is a Venn diagram of two GWAS analysis results and transcriptome differentially expressed genes.

[0039] Figure 3 These are the comparison results of the green egg sequencing data of the two CT genotypes at position 10839766 (A, C) and position 10829854 (B, D) on chromosome 20, respectively.

[0040] Figure 4 These are the comparison results of the green egg sequencing data of the other two CT genotypes at position 10839766 (A, C) and position 10829854 (B, D) on chromosome 20, respectively.

[0041] Figure 5 These are the comparison results of the sequencing data of pink eggs of two CC genotypes at positions 10839766 (A and C) and 10829854 (B and D) on chromosome 20, respectively.

[0042] Figure 6 These are the comparison results of the pink egg sequencing data of the other two CC genotypes at the 10839766th site (A, C) and the 10829854th site (B, D) on chromosome 20, respectively. DETAILED DESCRIPTION

[0043] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0044] Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as commonly understood by ordinary technicians in the technical field to which this application belongs.

[0045] Example 1 GWAS analysis

[0046] 1. Experimental Animals

[0047] The samples used in this experiment were all from the Jingyuan chicken breeder farm in Pengyang County, Ningxia. A total of 105 Jingyuan chickens (50 of which laid green-shelled eggs and 50 of which laid pink-shelled eggs, and the remaining 5 were roosters) with obvious breed characteristics, normal development and health under the same feeding conditions were selected for simplified genome sequencing.

[0048] 2. Sample collection

[0049] To simplify the genome sequencing samples, blood was collected from the sub-wing vein into an anticoagulant tube, DNA was extracted for sequencing, and stored in a -20°C refrigerator for future use.

[0050] 3. ddRAD simplifies genome sequencing

[0051] The DNA that has passed the quality test is sent to the sequencing company for simplified genome sequencing. The reference genome sequence is used to simulate the number and distribution of restriction endonuclease sites, and the double enzyme digestion experiment is performed using EcoRI at the end of Reads1 and NlaIII at the end of Read2. Select the appropriate enzyme for digestion and library construction; for details, please refer to the website http: / / www.illumina.com.cn / support / sequencing / kits.aspx.

[0052] The whole chicken genome was sequenced by ddRAD sequencing according to the company's standard process for genotyping. The red junglefowl genome (Gallus gallus, GRCg6a, Ensembl release-104) was used as the reference genome (http: / / ftp.ensembl.org / pub / release-104 / fasta / gallus_gallus). The reads obtained by sequencing were aligned to the reference genome using bwa software (version: 0.7.17-r1188). According to the positioning results of Clean Reads in the reference genome, GATK (version 4.1.4.1) was used for SNP detection to obtain the final SNP site (P < 1.48 × 10-7 ).

[0053] 4. Genome-wide association study (GWAS)

[0054] The general linear model (GLM) was used, and the formula was: y = Xα + Zβ + e, where y represents the phenotypic trait; X represents the indicator matrix of genotype (fixed effect); α represents the estimated parameter of fixed effect; Z represents the indicator matrix of population genetic structure; β represents the SNP effect; e represents the random residual, and e obeys normal distribution. The phenotypes of the test samples were distinguished according to two modes, and GWAS analysis was performed separately. The first mode divided the eggshell color of Jingyuan chicken into 50 green shells and other eggshell colors, and the second mode divided the Jingyuan chicken into 50 pink shells and other eggshell colors.

[0055] The Manhattan plot obtained by genome-wide association analysis of Shizuohara egg shell color ( Figure 1 ) The red line indicates the significant threshold of 0.05 / all SNPs, and the blue line indicates the potential significant threshold of 1 / all SNPs. The green shell trait is associated with 39 significantly associated SNPs, distributed on chromosomes 1 and 20 ( Figure 1 A, Table 1), the threshold of Bonferroni correction was 0.05 / 241008, adjacent to or located on 19 genes including SLCO1C1, SLCO1A2 and PIK3C2G, and the phenotypic variation (R2) explained by a single associated marker locus ranged from 16.67% to 47.45%. Thirty-five significantly associated SNPs were identified for the powder shell trait (P < 1.48 × 10 -7 ), these sites are mainly distributed on 12 chromosomes, including chromosomes 2, 5, 7 and 20 ( Figure 1 C, Table 2), the threshold of Bonferroni correction was significant at 0.05 / 232465. The single associated marker loci explained 16.48% to 46.30% of the phenotypic variation (R2) near or located on 35 genes such as SLC35B3, SLC4A11, and SLC39A10. The QQ plot is an important quality control plot for the association analysis results. λ can determine whether the population is stratified. λ above 1.05 indicates that the population is stratified. The λ values ​​of this study were all less than 1.05, indicating that the individuals were evenly distributed and there was no population stratification phenomenon ( Figure 1 B. Figure 1 D).

[0056] Table 2 SNPs significantly associated with green shell traits

[0057]

[0058] Table 2 SNPs significantly associated with powder shell traits

[0059]

[0060]

[0061] Note: The dash “-” in the 5th column indicates an unnamed gene.

[0062] 5. SNP functional annotation

[0063] The SNP sites and related genes associated at the whole genome level were screened, and the KEGG pathway enrichment and GO analysis were performed on the related genes. A total of 33 pathways were enriched in candidate genes, mainly in metabolic pathways, melanogenesis, calcium signaling pathways, and adrenergic signals in cardiomyocytes; in terms of biological processes, genes were mainly enriched in the regulation of developmental pigmentation and functions related to melanocyte differentiation; in terms of cell components, genes were mainly enriched in the cytoplasm, nucleoplasm, and extracellular regions; in terms of molecular functions, genes were mainly related to catalytic activity, ATP binding, metal ion binding, and endothelin receptor binding.

[0064] Example 2 Analysis of eggshell gland transcriptomes of different eggshell colors

[0065] The samples used in this experiment were all from the Jingyuan chicken breeder farm in Pengyang County, Ningxia. Five green-shelled and five pink-shelled Jingyuan chickens with relatively uniform egg-laying time were selected at 300 days of age under the same feeding conditions for the experiment. The chickens were slaughtered 3 to 5 hours before the estimated egg-laying, and the shell glands were collected after perfusing the tissues with ice-cold phosphate-buffered saline (PBS), immediately frozen in liquid nitrogen, and then stored at -80°C until total RNA was extracted for mRNA sequencing analysis.

[0066] According to the relative expression levels between the two groups of samples, differentially expressed genes can be divided into up-regulated genes and down-regulated genes. The expression level of up-regulated genes in sample B is higher than that in sample A; conversely, they are down-regulated genes. The volcano plot can be used to view the difference in gene expression levels between the two groups of samples, as well as the statistical significance of the difference. The total number of significant DEGs between green shells and pink shells is 277, of which 85 genes are significantly up-regulated and 192 genes are significantly down-regulated.

[0067] Example 3

[0068] Through genome-wide association analysis, 19 genes related to green shell traits and 35 genes related to pink shell traits were screened; while transcriptomic sequencing data screened 277 differentially expressed genes, and joint analysis screened two genes related to eggshell color, namely EDN3 and ZNF831 genes (see Figure 2, Table 3). The corresponding SNP sites are the C>T mutation at base 10829854 of chromosome 20 and the C>T mutation at base 10839766 of chromosome 20. According to the chi-square test results, there are significant differences between the CC genotype and the CT genotype of the above two sites.

[0069] Table 3 Sites identified by joint GWAS and transcriptome analysis

[0070]

[0071] Note: C represents homozygous CC, Y represents heterozygous CT, and N represents not detected. The number before the letter represents the number of the genotype.

[0072] Example 4 Verification of polymorphic sites

[0073] In order to verify the authenticity of the SNP sites obtained by high-pass sequencing and not caused by sequencing errors or alignment errors, 4 pink and 4 green samples were selected respectively, and the complete reads of the target SNP sites were re-aligned using Geneious software to check the SNP base types and quality values ​​of the two sites in Example 3.

[0074] The comparison results are as follows Figure 3-Figure 6 As shown (each figure shows the alignment results of 2 samples at 2 sites respectively), the heterozygous genotype and homozygous genotype of each site are accurate, and the data are quality-filtered reads, and the sequencing and alignment data are accurate and reliable.

[0075] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to the above embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for assisting in determining the color of a Shizuoka egg shell, characterized in that: The steps include: 1) Extract chicken genomic DNA; 2) sequencing the above genomic DNA to obtain sequencing reads; 3) Aligning the above sequencing reads to the chicken genome; 4) Based on the comparison results, the genotype of the SNP site with the C>T mutation at position 10829854 on chromosome 20 of the chicken reference genome version GRCg6a was determined; 5) The eggshell color is determined according to the SNP locus genotype determined in step 4). When the 10829854th base of chromosome 20 is CT, the eggshell color may be green; when the 10829854th base of chromosome 20 is CC, the eggshell color may be pink.

2. The detection method according to claim 1, characterized in that The sequencing is first generation sequencing, second generation sequencing, or third generation sequencing.

3. A breeding method for the color of Shizuohara egg shells, characterized in that: The method comprises the following steps: detecting the genotype of the SNP site of the C>T mutation at the 10829854th base of chromosome 20 of the chicken reference genome GRCg6a version, selecting breeder chickens with the corresponding genotype according to the eggshell color to be obtained, and if more green-shell laying hens are to be obtained in the group, then the breeder chickens with CT at the 10829854th base of chromosome 20 are to be retained, and if more pink-shell laying hens are to be obtained in the group, then the breeder chickens with CC at the 10829854th base of chromosome 20 are to be retained.

4. The method for breeding egg shell color according to claim 3, characterized in that: The method for detecting the genotype of a SNP marker site comprises the following steps: 1) Extracting genomic DNA from the chicken to be tested; 2) sequencing the above genomic DNA to obtain sequencing reads; 3) Aligning the above sequencing reads to the chicken genome; 4) Based on the comparison results, determine the genotype of the SNP site described in claim 3.

5. The method for breeding egg shell color according to claim 3, characterized in that: The sequencing is first generation sequencing, second generation sequencing, or third generation sequencing.

6. Application of a SNP molecular marker in breeding of static egg shell color, wherein the SNP molecular marker is the C>T mutation at base 10829854 of chromosome 20 of the chicken reference genome GRCg6a version; breeding chickens with the corresponding genotype are selected according to the eggshell color to be obtained. If more green-shell laying hens are to be obtained in the group, breeding chickens with CT at base 10829854 of chromosome 20 are to be retained; if more pink-shell laying hens are to be obtained in the group, breeding chickens with CC at base 10829854 of chromosome 20 are to be retained.

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