Snps associated with green shell egg shell chlorophyll content and use thereof
Patent Information
- Application Number
- CN202610960757.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-06-30
- Publication Date
- 2026-10-27
AI Technical Summary
本发明检测分析了绿壳蛋鸡32周龄蛋壳的胆绿素含量，利用第二代测序技术进行全基因组重测序和SNP基因分型，通过全基因组关联分析筛选得到与蛋壳胆绿素含量显著相关的SNP标记，为绿壳蛋鸡蛋壳胆绿素含量的选育提供了新的分子标记资源。在育种中，可根据育种目标选择CC基因型个体淘汰CT和TT基因型个体，可提高后代蛋壳的胆绿素含量，对改善绿壳蛋的感官有重要价值。本发明提供的检测方法简单易操作，具有广阔的应用前景。
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of molecular biology technology, specifically, it relates to SNP markers related to the biliverdin content in green-shelled eggs and their applications. Background Technology
[0002] Eggshell color is primarily influenced by the content of two pigments: protoporphyrin IX and biliverdin. Commercially available eggs are mainly brown and pink, both containing protoporphyrin IX and small amounts of biliverdin. As biliverdin concentration increases, the eggshell gradually turns green or blue. If the protoporphyrin IX content is too high in green-shelled eggs, they will appear yellowish-green. The SLCO1B3 gene is the major gene for green eggshells, and the EAV-HP reverse insertion in its promoter region is generally considered the causative mutation for the green eggshell phenotype. This mutation makes it possible for chickens to lay green-shelled eggs; however, not all hens carrying this mutation lay green-shelled eggs. The biliverdin content of eggs laid by individual hens that lay green-shelled eggs varies considerably, ranging from light green to dark green. This indicates that the green color of the eggshell is influenced not only by this major gene but also by other modifying genes. In addition, the varying shades and inconsistencies in the color of green-shelled eggs affect consumers' sensory experience and purchasing desire, making it necessary to improve the color of green-shelled eggs and enhance the uniformity of their color. Summary of the Invention
[0003] The purpose of this invention is to provide SNP markers related to the biliverdin content in green-shelled eggs and their applications.
[0004] To achieve the objective of this invention, in a first aspect, this invention provides an SNP marker related to the biliverdin content of green-shelled eggs, wherein the SNP marker contains a nucleotide sequence with a polymorphism of C / T at position 20261010 on chromosome 6 of the chicken reference genome GRCg7b.
[0005] Furthermore, the genotype of the site with the polymorphism is CC or CT, corresponding to high eggshell biliverdin content, and the genotype of the site with the polymorphism is TT, corresponding to low eggshell biliverdin content.
[0006] Secondly, the present invention provides primer pairs for amplifying the SNP marker.
[0007] Preferably, the primer pair includes primers as shown in SEQ ID NO:1-2.
[0008] Thirdly, the present invention provides reagents or kits containing the said primer pairs.
[0009] Fourthly, the present invention provides a method for identifying genotypes related to the biliverdin content in green-shelled eggs, comprising the following steps: (1) Extract genomic DNA from the chickens to be tested; (2) Detect the bases in the genomic DNA that correspond to the 20261010 site on chromosome 6 in the chicken reference genome GRCg7b, and determine the genotype of the site as CC, CT or TT based on the detection results.
[0010] Further, step (2) includes: using the genomic DNA as a template, performing PCR amplification using the primers shown in SEQ ID NO:1-2 to obtain an amplification product containing the site, and then sequencing the amplification product (such as Sanger sequencing) to determine the bases of the site.
[0011] Furthermore, the annealing temperature used for PCR amplification is 58-62℃.
[0012] Preferably, the PCR amplification reaction program is as follows: 95℃ for 5 min; 95℃ for 30 s, 60℃ for 30 s, 72℃ for 1 min, 35 cycles; 72℃ for 5 min.
[0013] Preferably, the PCR amplification reaction system is as follows: 100-500 ng of chicken DNA to be tested, 10 µL of 2×Taq PCR Mix, 1 µL of 10 µM forward primer, 1 µL of 10 µM reverse primer, and the reaction system is supplemented to 20 µL with ddH2O.
[0014] The 2×Taq PCR Mix is a 2-fold concentrated PCR amplification premix solution containing Taq DNA polymerase, dNTPs, MgCl2, reaction buffer, and stabilizer.
[0015] Preferably, the size of the PCR amplification products is detected by agarose gel electrophoresis.
[0016] Preferably, the concentration of agarose gel in electrophoresis is 1.0-1.5% (preferably 1%).
[0017] Preferably, the electrophoresis conditions are: 120~150V, 20~25min (preferably 120V, 20min).
[0018] Fifthly, the present invention provides a method for breeding green-shelled chickens with high eggshell biliverdin content, characterized in that it includes genotyping candidate chicken flocks using the above method, and selecting individuals with genotype CC or CT as parents for subsequent breeding.
[0019] In a sixth aspect, the present invention provides any of the following applications of the SNP marker, the primer pair, or the reagent or kit: (1) Used to identify the biliverdin content phenotype of green-shelled eggs; (2) Used for the identification or breeding of green-shelled chickens with high eggshell biliverdin content; (3) Used for early prediction of green-shelled chickens with high eggshell biliverdin content; (4) Molecular marker-assisted breeding for green-shelled chickens with high eggshell biliverdin content.
[0020] Preferably, the green-shelled chicken is a Sichuan mountain black-bone chicken.
[0021] By employing the above technical solution, the present invention has at least the following advantages and beneficial effects: This invention detected and analyzed the biliverdin content in eggshells of 32-week-old green-shelled chickens. Using next-generation sequencing (NGS) technology, whole-genome resequencing and SNP genotyping were performed. Genome-wide association analysis (GWAS) was used to screen for SNP markers significantly associated with eggshell biliverdin content, providing a new molecular marker resource for breeding green-shelled eggs with higher biliverdin content. In breeding, selecting individuals with the CC genotype and excluding those with the CT and TT genotypes can increase the biliverdin content of offspring eggshells, which is of significant value in improving the sensory qualities of green-shelled eggs. The detection method provided by this invention is simple and easy to operate, and has broad application prospects. Attached Figure Description
[0022] Figure 1 The Manhattan plot is a genome-wide association study of biliverdin content in eggshells from Sichuan mountain black-boned chickens, representing a preferred embodiment of the present invention.
[0023] Figure 2 The images show the Sanger sequencing results of the PCR amplification products of three genotypes in a preferred embodiment of the present invention. The 424bp indicates that the mutation site is located at the 424th base of the sequences shown in SEQ ID NO:3 and 4. Detailed Implementation
[0024] This invention provides a SNP marker associated with biliverdin content in the eggshells of green-shelled chickens (Sichuan mountain black-boned chickens). The SNP marker is located at base C or T at position 20261010 on chromosome 6 of the chicken reference genome (GRCg7b), with genotypes CC, CT, and TT. C is an enhancing allele, and the average biliverdin content in eggshells of individuals with the CC and CT genotypes is higher than that of individuals with the TT genotype. The primer pair designed in this invention amplifies fragments containing this SNP locus, and the genotype of an individual can be accurately identified by Sanger sequencing of the PCR amplification products. Selecting individuals with the CC genotype for breeding can increase the biliverdin content in the eggshells of offspring, which is of significant value in improving the sensory characteristics of green-shelled eggs.
[0025] The present invention adopts the following technical solution: First, the biliverdin content in the eggshells of 32-week-old Sichuan mountain black-boned chickens was detected. Whole-genome resequencing and SNP genotyping were performed using second-generation sequencing technology. SNP markers significantly associated with eggshell biliverdin content were screened through genome-wide association analysis, providing new molecular marker resources for the breeding of eggshell biliverdin content in Sichuan mountain black-boned chickens.
[0026] This invention provides an SNP marker locus associated with the biliverdin content in green-shelled eggs. By screening for specific genotypes at this locus, the biliverdin content in the mother's eggshell can be increased.
[0027] The present invention also provides primer pairs for identifying the genotype of the chicken SNP site, including forward primer F and reverse primer R, whose nucleotide sequences are shown in SEQ ID NO:1 and 2, respectively.
[0028] SEQ ID NO:1:5'-ATCAGAGGCAGATGTTGGTGG-3' (forward primer F) SEQ ID NO:2: 5'-TGGACAGACAATTCGCTCCAG-3' (reverse primer R) The present invention also provides detection reagents or kits containing the primer pairs described above.
[0029] The present invention also provides the application of the primer pair or detection reagents or kits containing the primer pair.
[0030] This invention also provides a method for identifying the genotype of a chicken at a specific SNP site. The method involves extracting genomic DNA from the chicken to be tested as a template, performing PCR amplification using the primer pair to obtain the amplification product, and then using agarose gel electrophoresis to detect the size of the PCR product fragments. After passing the test, the PCR product is sent to a commercial sequencing company for Sanger sequencing, and the individual genotype of Sichuan mountain black-boned chicken is determined based on the sequencing peak diagram.
[0031] Preferably, the reaction system for PCR amplification is as follows: Chicken DNA to be tested: 100-500 ng 2×Taq PCR Mix 10 µL 10 µM forward primer F 1 µL (10 pm / µL) 10 µM reverse primer R 1 µL (10 pm / µL) Add ddH2O to the reaction mixture to bring the volume to 20 µL. The 2×Taq PCR Mix is a 2-fold concentrated PCR amplification premix solution containing Taq DNA polymerase, dNTPs, MgCl2, and reaction buffer.
[0032] The annealing temperature used for PCR amplification is 58-62℃.
[0033] Preferably, the PCR amplification reaction program is as follows: 95℃ for 5 min; 95℃ for 30 s, 60℃ for 30 s, 72℃ for 1 min, 35 cycles; 72℃ for 5 min; store at 4℃.
[0034] Preferably, the concentration of agarose gel in electrophoresis is 1.0~1.5% (preferably 1%).
[0035] Preferably, the electrophoresis conditions are: 120~150V, 20~25min (preferably 120V, 20min).
[0036] Preferably, genomic DNA is extracted using the Blood / Tissue / Cell Genomic Extraction Kit (DP304) from Tiangen Biotech Co., Ltd., and the specific steps are as follows: (1) Thaw the blood sample, take 20 µL of blood and put it into a 1.5 mL centrifuge tube, and add 180 µL of GA buffer.
[0037] (2) Add 20 µL of Proteinase K solution and repeatedly pipette until the blood and reagent are fully mixed. Place in a 70°C water bath for 10 minutes to digest and then briefly centrifuge to remove water droplets from the inner wall.
[0038] (3) Add 200 µL of GB buffer, mix thoroughly by inverting, place it in a water bath at 56°C, take it out every 5 minutes and mix by inverting, repeat 3 times until the solution becomes clear, and briefly centrifuge to remove water droplets from the inner wall of the tube cap.
[0039] (4) Add 200 µL of anhydrous ethanol and shake thoroughly to mix.
[0040] (5) Add the solution and flocculent precipitate obtained in the previous step to an adsorption column CB3 (place the adsorption column in the collection tube), centrifuge at 12,000 rpm (about 13,400 × g) for 1 min, discard the waste liquid, and put the adsorption column CB3 back into the collection tube.
[0041] (6) Add 500 µL of LGD buffer to the adsorption column CB3 (check that anhydrous ethanol has been added before use), centrifuge at 12,000 rpm (about 13,400 × g) for 1 min, discard the waste liquid, and put the adsorption column CB3 into the collection tube.
[0042] (7) Add 600 µL of washing solution PW to the adsorption column CB3 (check that anhydrous ethanol has been added before use), centrifuge at 12,000 rpm (about 13,400×g) for 1 min, discard the waste liquid, and put the adsorption column CB3 into the collection tube.
[0043] (8) Repeat step (7).
[0044] (9) Place the adsorption column CB3 back into the collection tube, centrifuge at 12,000 rpm (about 13,400×g) for 2 min to remove the residual rinsing liquid in the adsorption column, and place the adsorption column CB3 in a fume hood for 5 min to thoroughly dry the residual rinsing liquid in the adsorption material.
[0045] (10) Transfer the adsorption column CB3 into a clean centrifuge tube, add 30µL of elution buffer TE to the middle of the adsorption membrane, let it stand at room temperature for about 5 min, centrifuge at 12,000 rpm (about 13,400×g) for 2 min, and collect the solution into the centrifuge tube.
[0046] Other possible techniques for extracting DNA from chicken blood or tissues can also be used in this invention.
[0047] When detecting PCR amplified fragments by agarose gel electrophoresis, the target fragment is 511bp. A 1% agarose gel is used, the electrophoresis voltage is 120V, and the electrophoresis time is 20min.
[0048] Different genotypes of green-shelled eggs produce eggs with varying biliverdin content. C is an enhancing allele; the average biliverdin content in the shells of CC genotype individuals is higher than that of CT and TT genotype individuals, with the CT genotype having a higher content than the TT genotype. In breeding, selecting CC genotype individuals and excluding CT and TT genotype individuals according to breeding goals can increase the biliverdin content of offspring eggshells, which is of significant value in improving the sensory qualities of green-shelled eggs.
[0049] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.
[0050] The following examples use Sichuan mountain black-boned chickens that lay green-shelled eggs.
[0051] Example 1: Development of SNP markers associated with biliverdin content in green-shelled eggs This embodiment detected the biliverdin content in the shells of 278 Sichuan mountain black-boned chicken eggs. Genome-wide SNP genotyping was performed using next-generation sequencing technology. Genome-wide association analysis was used to screen for SNP molecular markers significantly associated with eggshell biliverdin content. The results are as follows: Figure 1 As shown.
[0052] This embodiment uses the following experiments to screen and identify molecular markers related to eggshell biliverdin content.
[0053] 1. Phenotypic determination and genotypic testing (1) 278 Sichuan mountain black-bone chickens that lay green-shelled eggs at 32 weeks of age were selected and raised under the same feeding conditions. Eggs laid by each hen for 3 consecutive days were collected, and the biliverdin content of each egg was determined by the methanol-hydrochloric acid method. The average value of the 3 eggs was taken as the biliverdin content of the green-shelled eggs laid by each hen.
[0054] (2) Blood samples were collected from Sichuan mountain black-bone chicken hens via vein and sent to a commercial sequencing company for genome resequencing. The reference genome was GRCg7b. The BWA-bcftools-vcftools workflow was used to detect genomic variations and perform genotyping of each variation site. GEMMA software was used to conduct genome-wide association analysis and estimate the effect of each variation site. Chr6:20261010 was selected as an SNP molecular marker based on the significance and effect size of each marker site.
[0055] 2. Correlation analysis Based on the variation detection results, the genotypes of each sample were confirmed, and correlation analysis was conducted on the biliverdin content in eggshells of different genotypes. Statistical tests were performed using the ANOVA function in R language, and multiple comparisons were conducted using the Tukey HSD test. The results are shown in Table 1. The biliverdin content in eggshells of the CC genotype flock was 49.77 nmol / g, and that of the CT genotype flock was 47.21 nmol / g. The difference between the two was not significant (P=0.36), but both were significantly higher than the 39.00 nmol / g of the TT genotype flock (P<0.01). The results indicate that the Chr6:20261010 SNP molecular marker in Sichuan mountain black-boned chickens is significantly correlated with eggshell biliverdin content. Therefore, according to actual breeding needs, selecting individuals with the CC genotype can increase the biliverdin content in eggshells and improve the sensory characteristics of green-shelled eggs.
[0056] Table 1. Statistical analysis of genotypes and eggshell biliverdin content at Chr6:20261010 SNP marker sites.
[0057] Note: Data with the same subtitle in the same column indicate no significant difference, while data with different subtitles indicate a significant difference (P<0.05); Tukey HSD test was used, CC vs CT, P=0.36; CC vs TT, P=0.0000003; CT vs TT, P=0.0000008.
[0058] Example 2: Verification of the effectiveness of the marker using molecular identification methods. 1. Primer design and preparation Primers were designed based on the SNP molecular marker (Chr6:20261010, reference genome version: GRCg7b) determined in Example 1, and upstream and downstream fragments of the SNP were amplified.
[0059] Primers were synthesized by Sangon Biotech Co., Ltd. (SEQ ID NO:1-2). Forward primer F: 5′-ATCAGAGGCAGATGTTGGTGG-3′, reverse primer R: 5′-TGGACAGACAATTCGCTCCAG-3′.
[0060] 2. Blood samples were collected from 122 Sichuan mountain black-boned chickens that lay green-shelled eggs using a venous blood collection method. At the same time, the biliverdin content of the eggshells of these 122 female chickens was determined using the method in Example 1.
[0061] 3. Extract chicken blood DNA according to the method in Example 1.
[0062] 4. Perform PCR amplification and detect PCR products according to the following PCR amplification system, amplification steps, and agarose gel electrophoresis steps.
[0063] The reaction system for PCR amplification is as follows: Chicken DNA to be tested: 100-500 ng 2×Taq PCR Mix 10 µL 10 µM forward primer F 1 µL (10 pm / µL) 10 µM reverse primer R 1 µL (10 pm / µL) Add ddH2O to the reaction mixture to bring the volume to 20 µL. The 2×Taq PCR Mix is a 2-fold concentrated PCR amplification premix solution containing Taq DNA polymerase, dNTPs, MgCl2, and reaction buffer.
[0064] The PCR amplification reaction program was as follows: 95℃ for 5 min; 95℃ for 30 s, 60℃ for 30 s, 72℃ for 1 min, 35 cycles; 72℃ for 5 min; store at 4℃.
[0065] The agarose gel concentration for electrophoresis was 1%. The electrophoresis conditions were: 120V, 20min.
[0066] PCR product samples with clear electrophoretic bands, approximately 500 bp in length, and unique bands were sent to a commercial sequencing company for Sanger sequencing. The genotype of the individual was determined based on the Sanger sequencing results. The sequences of the amplified products are shown in SEQ ID NO: 3 and 4, respectively, with the 424 bp being the mutation site.
[0067] 5. Correlation analysis was conducted between genotype and phenotype (eggshell biliverdin content), and the results are shown in Table 2.
[0068] Table 2. Statistical analysis of genotypes and eggshell biliverdin content at Chr6:20261010 SNP marker sites.
[0069] Note: Data with the same subheading letter in the same column indicate no significant difference, while data with different subheading letters indicate a significant difference (P<0.05); Tukey HSD test was used, CC vs CT, P=0.42; CC vs TT, P=0.0032; CT vs TT, P=0.042.
[0070] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.
Claims
1. A SNP marker associated with the biliverdin content in green-shelled eggs, characterized in that, The SNP marker contains a nucleotide sequence with a polymorphism of C / T at position 20261010 on chromosome 6 of the chicken reference genome GRCg7b.
2. The SNP marker according to claim 1, characterized in that, The genotype of the site with the polymorphism is CC or CT, which corresponds to high eggshell biliverdin content, and the genotype of the site with the polymorphism is TT, which corresponds to low eggshell biliverdin content.
3. Primer pairs for amplifying the SNP markers of claim 1 or 2.
4. The primer pair according to claim 3, characterized in that, Includes primers as shown in SEQ ID NO:1-2.
5. A reagent or kit containing the primer pair as described in claim 3 or 4.
6. A method for identifying genotypes related to the biliverdin content in green-shelled eggs, characterized in that, Includes the following steps: (1) Extract genomic DNA from the chickens to be tested; (2) Detect the bases in the genomic DNA that correspond to the 20261010 site on chromosome 6 in the chicken reference genome GRCg7b, and determine the genotype of the site as CC, CT or TT based on the detection results.
7. The method according to claim 6, characterized in that, Step (2) includes: using the genomic DNA as a template, performing PCR amplification using the primers shown in SEQ ID NO:1-2 to obtain an amplification product containing the site, and then sequencing the amplification product to determine the bases of the site.
8. The method according to claim 7, characterized in that, The annealing temperature used for PCR amplification is 58-62℃; Preferably, the PCR amplification reaction program is as follows: 95℃ for 5 min; 95℃ for 30 s, 60℃ for 30 s, 72℃ for 1 min, 35 cycles; 72℃ for 5 min.
9. A method for breeding green-shelled chickens with high eggshell biliverdin content, characterized in that, This includes using the method described in any one of claims 6-9 to genotype candidate chicken flocks and selecting individuals with genotypes CC or CT as parents for subsequent breeding.
10. Any of the following applications of the SNP marker of claim 1 or 2, the primer pair of claim 3 or 4, or the reagent or kit of claim 5: (1) Used to identify the biliverdin content phenotype of green-shelled eggs; (2) Used for the identification or breeding of green-shelled chickens with high eggshell biliverdin content; (3) Used for early prediction of green-shelled chickens with high eggshell biliverdin content; (4) Molecular marker-assisted breeding for green-shelled chickens with high eggshell biliverdin content.