SNP (Single Nucleotide Polymorphism) molecular marker related to pink and green eggshell colors and application of SNP molecular marker
Through GWAS and transcriptome analysis, significant SNP sites related to eggshell color were identified, detection methods were developed, and the problem of localization of molecular markers in the prior art was solved, and the prediction and optimization of eggshell color was achieved, and the economic benefits of static chicken breeding were improved.
Patent Information
- Application Number
- CN202510123577.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2023-08-18
- Publication Date
- 2025-05-27
AI Technical Summary
The prior art is difficult to effectively locate and utilize molecular markers related to egg shell color, resulting in the lack of complete clarity of the color formation mechanism of static raw egg shells and the lack of effective molecular marker-assisted selection and genome selection methods.
GWAS analysis combined with transcriptome analysis strategies were used to identify significant SNP sites that affect the color of eggshells, and corresponding detection methods were developed for molecular marker-assisted selection and genome selection to predict the eggshell color of breeding chickens in advance.
The prediction and optimization of eggshell color has been achieved, and breeding chickens with different eggshell colors can be selected and bred according to the breeding target, bringing economic benefits to the breeding of jingyuan chickens.
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Figure CN120041579A_ABST
Abstract
Description
Technical Field
[0001] This application is a divisional application of a Chinese application with the application number 202311045167.5, the application date of August 18, 2023, and the invention title of "An SNP Locus Related to Eggshell Color and Its Application".
[0002] The present invention belongs to the field of biotechnology, and specifically relates to SNP loci on chicken chromosomes related to eggshell powder and green color, and their applications. Background Art
[0003] As the most intuitive sensory evaluation index, the color of avian eggshells is generally more popular in green. Jingyuan chickens are excellent local chicken breeds in Ningxia, mainly producing eggs with pink and green eggshells. The increase in the production of green-shell eggs with a large market demand can improve the economic benefits of the Jingyuan chicken industry. Eggshell color is an inheritable trait with high heritability. The color of eggshells is determined by the deposition of eggshell pigments, mainly including three pigments: protoporphyrin-IX, biliverdin-IX, and zinc chelate of biliverdin. Different eggshell colors can be formed by mixing these pigments in different proportions. Most studies have shown that both the eggshell gland 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 senescent red blood cells. Existing studies have shown that the green-shell trait in chickens is a qualitative trait controlled by a dominant single gene (Oocyan, O). To map the O gene, Z. Wang et al. [5] re-sequenced the genomic sequence of solute carrier organic anion transporter family member 1B3 (SLCO1B3) and found that there is an insertion sequence of chicken retrovirus EAV-HP in the 5′ flanking region of this gene. This variation leads to abnormal expression in the eggshell gland of green-shell laying hens, producing a large amount of OATP1B3 protein, which deposits biliverdin onto the eggshell. The formation mechanism of the color of pink-shell eggs has not been fully clarified. Whether brown-shell eggs are produced is controlled by an incompletely dominant gene, and multiple genes act together to regulate it. H. Rome et al. used a 600K Affymetrix SNP chip to map QTLs affecting eggshell color and detected 39 QTLs related to eggshell color traits, among which 22 QTLs directly affect eggshell color. The large number and wide distribution range of these QTLs fully prove that eggshell color is controlled by minor 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) are significantly correlated with the color depth of brown-shell eggs. The leucine-rich repeat protein 8 (LRRC8) family genes are involved in various biological processes such as cell adhesion and cell transport, and may be involved in the biological processes of the formation of the color of pink-shell eggs. In addition, genes such as phosphatidylinositol-4-phosphate 3-kinase catalytic subunit type 2 gamma (PIK3C2G), ATP-binding cassette sub-family C member 9 (ABCC9), and inositol 1,4,5-trisphosphate receptor type II (ITPR2
[12] ) on chromosome 1 have also been identified as important genes affecting the color of green-shell eggs. In recent years, with the development of omics technologies and the wide application of genome-wide association analysis, it has provided opportunities for people to accurately map QTLs affecting eggshell color. However, there are few reports on the formation mechanism of Jingyuan chicken eggshell color, and molecular markers related to the formation of Jingyuan chicken eggshell color have not been mined yet. Summary of the Invention
[0004] In view of the problems existing in the prior art, the present invention provides a significant SNP affecting eggshell color identified through a GWAS analysis combined with a transcriptome analysis strategy, which is used in molecular marker-assisted selection and genomic selection to predict the eggshell color of breeding chickens in advance. At the same time, different eggshell-color breeding chickens can be selected according to the breeding goal by optimizing the advantageous allele genotypes of SNP loci, bringing huge economic benefits to the breeding of Jingyuan chickens.
[0005] The primary objective is to determine SNP molecular markers related to eggshell color. The SNP markers include any locus corresponding to the reference sequence of the chicken genome (Gallus gallus, GRCg6a, Ensembl release-104) as follows: In a preferred embodiment, the SNP markers include all of the above molecular marker loci.
[0006] Another objective of the present invention is to provide a method for detecting the above SNP molecular markers, which is characterized by including the following steps: 1) Extract genomic DNA from the sample; 2) Sequence the above genomic DNA to obtain sequencing reads; 3) Align the above sequencing reads to the chicken genome; 4) Based on the alignment result, determine the genotype of the above SNP locus.
[0007] In a preferred embodiment, the sequencing is first-generation sequencing, second-generation sequencing, or third-generation sequencing.
[0008] Another objective of the present invention is to provide the application of the above molecular markers in assisting in judging eggshell color, including the following steps: 1) Extract genomic DNA from the chicken; 2) Sequence the above genomic DNA to obtain sequencing reads; 3) Align the above sequencing reads to the chicken genome; 4) Based on the alignment result, determine the genotype of the SNP locus described in claim 1 or claim 3.
[0009] 5) Infer the eggshell color based on the SNP locus genotype determined in step 4).
[0010] In a preferred case, when the base at position 10,829,854 on chromosome 20 and / or the base at position 10,839,766 on chromosome 20 is CT, it is determined that the eggshell color may be green; when the base at position 10,829,854 on chromosome 20 and / or the base at position 10,839,766 on chromosome 20 is CC, it is determined that the eggshell color may be pink.
[0011] Another object of the present invention is to provide a molecular-assisted breeding method for eggshell color, characterized in that the method comprises the following steps: detecting the genotype of the SNP marker locus as described above, and selecting and retaining the breeding chickens with the corresponding genotype according to the desired eggshell color.
[0012] In a preferred case, it specifically comprises the following steps: 1) Extract the genomic DNA of the chicken to be tested; 2) Sequence the above genomic DNA to obtain sequencing reads; 3) Align the above sequencing reads to the chicken genome; 4) Based on the alignment result, determine the genotype of the SNP locus described in claim 1 or claim 3; 5) Select and retain the breeding chickens with the corresponding genotype according to the desired eggshell color.
[0013] In a preferred embodiment, the aforementioned sequencing is first-generation sequencing, second-generation sequencing, or third-generation sequencing.
[0014] In a preferred case, the SNP related to the eggshell color is the C>T mutation at position 10,829,854 on chromosome 20, and / or the C>T mutation at position 10,839,766 on chromosome 20.
[0015] In a preferred case, if it is desired to obtain more green-shelled laying hens in the population, retain the breeding chickens with CT at position 10,829,854 on chromosome 20 and / or at position 10,839,766 on chromosome 20; if it is desired to obtain more pink-shelled laying hens in the population, retain the breeding chickens with CC at position 10,829,854 on chromosome 20 and / or at position 10,839,766 on chromosome 20.
[0016] The present invention has the following advantages and effects compared with the prior art: First, a new SNP molecular marker related to eggshell color is determined, and a corresponding detection method is developed.
[0017] Second, by detecting the genotype of the SNP locus of the present invention, the eggshell color of the breeding chickens can be predicted in advance.
[0018] Thirdly, according to the breeding goal, the present invention can breed chicken breeds with different eggshell colors by selecting the advantageous allele genotypes of SNP sites. BRIEF DESCRIPTION OF THE DRAWINGS
[0019] The following, in conjunction with the drawings and specific embodiments, details the method of the present invention and its beneficial effects.
[0020] Figure 1 are the Manhattan plot and QQ plot of the GWAS analysis. Among them Figure 1 A and Figure 1 B are respectively the Manhattan plot and QQ plot of the GWAS analysis for the comparison between green-shell eggs and eggs of other colors; Figure 1 C and Figure 1 D are respectively the Manhattan plot and QQ plot of the GWAS analysis for the comparison between pink-shell eggs and eggs of other colors.
[0021] Figure 2 is the Venn diagram of the two GWAS analysis results and the differentially expressed genes in the transcriptome.
[0022] Figure 3 are the alignment results of the sequencing data of two green eggs with CT genotypes at the 10839766th site (A, C) and the 10829854th site (B, D) on chromosome 20 respectively.
[0023] Figure 4 are the alignment results of the sequencing data of another two green eggs with CT genotypes at the 10839766th site (A, C) and the 10829854th site (B, D) on chromosome 20 respectively.
[0024] Figure 5 are the alignment results of the sequencing data of two pink eggs with CC genotypes at the 10839766th site (A, C) and the 10829854th site (B, D) on chromosome 20 respectively.
[0025] Figure 6 are the alignment results of the sequencing data of another two pink eggs with CC genotypes at the 10839766th site (A, C) and the 10829854th site (B, D) on chromosome 20 respectively. DETAILED DESCRIPTION OF THE EMBODIMENTS
[0026] The technical solutions in the embodiments of the present invention are clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.
[0027] Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as commonly understood by those of ordinary skill in the technical field to which this application belongs.
[0028] Example 1 GWAS Analysis 1. Experimental Animals All samples used in this experiment were from Jingyuan Chicken Breeding Farm in Pengyang County, Ningxia. 105 Jingyuan chickens with obvious breed characteristics, normal development and good health under the same feeding conditions (including 50 chickens laying green-shell eggs, 50 chickens laying brown-shell eggs, and 5 roosters) were selected for reduced-representation genome sequencing.
[0029] 2. Sample Collection For reduced-representation genome sequencing samples, blood was collected from the wing vein into anticoagulant tubes, and DNA was extracted for sequencing and stored at -20 °C in a refrigerator for future use.
[0030] 3. ddRAD Reduced-Representation Genome Sequencing Qualified DNA samples were sent to a sequencing company for reduced-representation genome sequencing. The number and distribution of restriction enzyme digestion sites of various restriction endonucleases were simulated using the reference genome sequence, and a double digestion experiment was performed using EcoRI at the 5' end of Reads1 and NlaIII at the 3' end of Read2. Appropriate enzymes were selected for digestion and library construction; for details, see the website http: / / www.illumina.com.cn / support / sequencing / kits.aspx.
[0031] Genotype determination was performed by chicken whole-genome ddRAD sequencing according to the company's standard procedures. 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 obtained reads were aligned to the reference genome using the bwa software (version: 0.7.17-r1188). According to the mapping results of Clean Reads on the reference genome, SNP detection was performed using GATK (version 4.1.4.1) to obtain the final SNP sites (P < 1.48×10 -7 )
[0032] 4. Genome-Wide Association (GWAS) Analysis The general linear model (GLM) is adopted, and the formula is: y = Xα + Zβ + e, where y represents the phenotypic trait; X represents the indicator matrix of the genotype (fixed effect); α represents the estimated parameter of the fixed effect; Z represents the indicator matrix of the population genetic structure; β represents the SNP effect; e represents the random residual, and e follows a normal distribution. The phenotypes of the test samples are distinguished in two modes, and GWAS analyses are performed separately. In the first mode, the eggshell colors of Jingyuan chickens are divided into 50 green-shelled ones and other eggshell colors. In the second mode, Jingyuan chickens are divided into 50 pink-shelled ones and other eggshell colors.
[0033] Through the genome-wide association analysis of the eggshell colors of Jingyuan chickens, in the Manhattan plot ( Figure 1 ), the red line represents the significant threshold of 0.05 / total SNPs, and the blue line represents the potential significant threshold of 1 / total SNPs. The green-shell trait is associated with 39 significantly related SNP loci, which are distributed on chromosomes 1 and 20 (Figure 1A, Table 1). The Bonferroni-corrected threshold is the significant threshold of 0.05 / 241008, and these loci are adjacent to or located on 19 genes such as SLCO1C1, SLCO1A2, and PIK3C2G. The range of the phenotypic variation (R2) explained by a single associated marker locus is 16.67% - 47.45%; 35 significantly related SNP loci (P < 1.48×10 -7 ) are identified for the pink-shell trait. These loci are mainly distributed on 12 chromosomes such as 2, 5, 7, and 20 ( Figure 1 C, Table 2). The Bonferroni-corrected threshold is the significant threshold of 0.05 / 232465. These loci are adjacent to or located on 35 genes such as SLC35B3, SLC4A11, and SLC39A10. The range of the phenotypic variation (R2) explained by a single associated marker locus is 16.48% - 46.30%. The QQ plot is an important quality control plot for the association analysis results. λ can judge whether the population is stratified. If λ is above 1.05, it indicates that the population is stratified. The λ values in this study are all less than 1.05, indicating that the individuals are evenly distributed and there is no population stratification phenomenon (Figure 1B, Figure 1 D).
[0034] Table 2 Significantly related SNP loci for the green-shell trait Table 2 Significantly related SNP loci for the pink-shell trait 5. SNP functional annotation Screen out SNP sites and related genes associated at the whole-genome level, and perform KEGG pathway enrichment and GO analysis on the related genes. A total of 33 pathways were enriched for the candidate genes, mainly enriched in metabolic pathways, melanogenesis, calcium signaling pathways, and adrenergic signaling in cardiomyocytes, etc.; in terms of biological processes, the genes were mainly enriched in the regulation of developmental pigmentation and functions related to melanocyte differentiation; in terms of cellular components, the genes were mainly enriched in the cytoplasm, nucleoplasm, and extracellular region; in terms of molecular functions, the genes were mainly related to catalytic activity, ATP binding, metal ion binding, and endothelin receptor binding.
[0035] Example 2 Transcriptome Analysis of Eggshell Glands with Different Eggshell Colors All samples used in this experiment were from Jingyuan Chicken Breeding Farm in Pengyang County, Ningxia. Under the same feeding conditions, 5 Jingyuan chickens laying green eggs and 5 Jingyuan chickens laying pink eggs with relatively uniform laying times were selected at 300 days old for the experiment. They were slaughtered 3 - 5 h before estimated egg laying. After perfusing the tissues with ice-cold phosphate buffer saline (PBS), the eggshell glands were collected, immediately frozen in liquid nitrogen, and then stored at -80 °C until total RNA needed to be extracted for mRNA sequencing analysis.
[0036] According to the relative levels of expression between the two groups of samples, differentially expressed genes can be divided into up-regulated genes (Up-regulated genes) and down-regulated genes (Down-regulated genes). The expression level of up-regulated genes in sample B is higher than that in sample A; vice versa for down-regulated genes. Through the volcano plot (Volcano Plot), the differences in gene expression levels between the two groups of samples and the statistical significance of the differences can be viewed. The total number of significant DEGs between green eggs and pink eggs was 277, among which 85 genes were significantly up-regulated and 192 genes were significantly down-regulated.
[0037] Example 3 19 genes related to the green eggshell trait and 35 genes related to the pink eggshell trait were screened out through genome-wide association analysis; 277 differentially expressed genes were screened out from the transcriptomic sequencing data, and two genes related to eggshell color, namely EDN3 and ZNF831 genes (see Figure 2 Table 3). The corresponding SNP sites were the mutation of base C to T at position 10829854 on chromosome 20, and the mutation of base C to T at position 10839766 on chromosome 20. According to the chi-square test results, there were significant differences between the CC genotype and CT genotype at the above two sites.
[0038] Table 3 Sites Determined by the Joint Analysis of GWAS and Transcriptome Note: C represents homozygous CC, Y represents heterozygous CT, and N represents not detected. The numbers before the letters represent the quantity of each genotype.
[0039] Example 4 Verification of Polymorphic Loci To verify the authenticity of the SNP loci obtained by high-throughput sequencing rather than caused by sequencing errors or alignment errors, 4 pink samples and 4 green samples were respectively selected, and the Geneious software was used to re-align the complete reads of the target SNP loci to check the SNP base types and quality values of the 2 loci in Example 3.
[0040] The alignment results are as Figures 3 - 6 shown (each figure shows the alignment results of 2 samples at 2 loci respectively). The heterozygous genotypes and homozygous genotypes at each locus are accurate, and the data are reads after quality filtering, and the sequencing and alignment data are accurate and reliable.
[0041] The above description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to the above embodiments will be obvious to those skilled in the art, and the general principles defined herein can 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 these embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.
Claims
1. SNP molecular markers associated with eggshell color, It is characterized in that The SNP marker is located on the chicken chromosome, specifically including any of the following sites: The molecular marker according to claim 1, characterized in that the eggshell color is pink or green.
2. The molecular marker according to claim 1 or 2, It is characterized in that The marker is the C>T mutation at base 10839766 of chromosome 20.
3. A method to assist in determining the color of egg shells. It is 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, determine the genotype of the SNP site described in claim 1 or claim 3.
4. Determine the eggshell color based on the SNP locus genotype determined in step 4).
5. The detection method according to claim 4, It is characterized in that The sequencing is first generation sequencing, second generation sequencing, or third generation sequencing.
6. A breeding method for egg shell color, It is characterized in that The method comprises the following steps: detecting the genotype of the SNP marker site according to claim 1 or claim 3, and selecting breeder chickens retaining the corresponding genotype according to the eggshell color to be obtained.
7. The method for breeding egg shell color according to claim 6, It is characterized in that The method for detecting the genotype of the 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 1 or claim 3.
8. The method for breeding egg shell color according to claim 6, It is characterized in that The sequencing is first generation sequencing, second generation sequencing, or third generation sequencing.
9. The method according to claim 6, It is characterized in that The chicken population includes Shizuohara chicken and its synthetic strains.
10. Use of the SNP molecular marker according to claim 1 or 3 in breeding for egg shell color.