A molecular marker related to the color of chicken eggshell and application thereof

Abstract

The application relates to the technical field of agricultural breeding, and discloses a molecular marker related to eggshell color and application thereof. The SNP molecular marker corresponds to a site of 18378867 from a 5' end on a chromosome 6 of a chicken reference genome GRCg6a published in an Ensembl genome browser database, the base of the site is C / T, and the site is a missense mutation on an ABCG2 gene. The SNP molecular marker related to the eggshell color is a new molecular marker. By detecting the genotype of the site of a chicken individual to be determined, early selection is carried out on the chicken with an advantageous genotype, the breeding progress of the trait can be accelerated, and the application has great application value and economic benefits.

Description

Technical Field

[0001] This invention relates to the field of agricultural breeding technology, and more specifically, to a molecular marker related to eggshell color and its application. Background Technology

[0002] Chickens, as one of the world's most widely farmed and industrialized agricultural animals, have become a crucial pillar industry for ensuring food security. Providing tens of millions of tons of chicken meat and trillions of eggs annually, chickens are a core source of high-quality, inexpensive animal protein and occupy an irreplaceable position in the global food supply chain. Eggshell color, as a key external indicator of egg quality, directly impacts consumer purchasing decisions and market prices, and is closely related to the commodity attributes and brand premium of eggs. It has become one of the core indicators of focus for egg-laying hen breeding and industry upgrading.

[0003] From the perspective of global consumer market preferences, the market positioning of eggshell color exhibits significant regional characteristics: In major Asian markets such as China and Japan, pink-shelled eggs, with their pleasant appearance and balanced taste, occupy more than 60% of the market share, becoming the mainstream choice for Chinese consumers. In European and American markets, brown-shelled eggs have long been the traditional consumer preference, while white-shelled eggs, due to their low breeding costs and high egg production efficiency, are widely used in large-scale industrial production and circulated as baking ingredients and catering accompaniments. In some Southeast Asian regions and domestic specialty agricultural product markets, green-shelled eggs, due to their perceived "rich in selenium" and "antioxidant" properties, command a market premium of 2-3 times that of ordinary eggs, becoming a representative of high-value-added egg products. This differentiation in consumer preferences is driving an increasingly urgent demand from egg-laying hen farmers for breeding varieties with specific eggshell colors.

[0004] Eggshell color formation is a complex physiological and biochemical process with a heritability of 0.4-0.6, classifying it as a medium-to-high level of heritability, providing a basis for targeted breeding through genetic improvement. After ovulation, the mature oocyte enters the ampulla of the oviduct to complete fertilization (unfertilized eggs proceed directly to the subsequent stages). Subsequently, inner and outer shell membranes form in the isthmus, and finally, calcification and pigment deposition occur in the shell gland (uterus). The main component of the eggshell is calcium carbonate (over 95%), and the core of color formation depends on the types, amounts, and deposition patterns of pigments synthesized by the epithelial cells of the shell gland. Protoporphyrin is the main pigment in brown-shelled eggs, biliverdin and its derivatives are characteristic pigments in green-shelled eggs, while white-shelled eggs, due to blocked pigment synthesis pathways, only exhibit the natural color of calcium carbonate. It is noteworthy that the deposition and calcification processes of eggshell pigments are highly synergistic. Pigment particles embedded in the gaps between calcium carbonate crystals not only determine the depth of color but may also indirectly affect the density and breakage resistance of the eggshell. Therefore, eggshell color is also considered an important indirect indicator of overall egg quality.

[0005] With the full implementation of the "National Egg-Laying Hen Genetic Improvement Program (2021-2035)," the core objective of egg-laying hen breeding in my country has shifted from simply pursuing egg production volume to a new stage of "emphasizing both high production and quality, and coordinating quantity and quality." The program explicitly sets forth the stringent requirement that "high-producing hens should lay more than 500 eggs at 700 days of age, and the coefficient of variation of egg quality indicators in the later laying period (after 50 weeks of age) should be controlled within 10%." This means that while extending the laying cycle and improving production efficiency, it is essential to ensure the stability of key quality traits such as eggshell color, eggshell strength, and albumen height. The problem of late-stage eggshell color decay (in most breeds, eggshell color lightens and uniformity decreases after 60 weeks of age) has become a prominent bottleneck restricting industry upgrading.

[0006] Traditional eggshell color selection methods rely on direct phenotypic measurement, which has three major drawbacks: First, color measurement of 3-5 eggs from each hen must be performed in the mid-to-late laying period (after 43 weeks of age). For large-scale chicken farms (tens of thousands of birds), a single measurement requires the daily workload of 5-10 people, and the measurement needs to be repeated 3-4 times to ensure accuracy, resulting in high breeding costs. Second, phenotypic measurement cannot be carried out in the chick or pullet stage, making early selection impossible. The entire selection cycle takes 2-3 generations (about 3-4 years), severely restricting the speed of genetic progress. Third, eggshell color is a phenotypic specific to hens, and roosters cannot be directly measured. Breeding values ​​can only be estimated indirectly through the phenotypes of siblings or offspring, which reduces accuracy by 30%-40% compared to direct selection and easily leads to the omission of individuals with superior genotypes.

[0007] Genome-wide association analysis (GWAS), a core technology for elucidating the genetic basis of complex quantitative traits, has been widely applied in livestock and poultry breeding. By identifying SNPs (single nucleotide polymorphisms) molecular markers significantly associated with target traits, it provides precise targets for marker-assisted breeding (MAS). Compared to traditional selection methods, SNP-based marker-assisted selection enables "early selection, precise selection, and selection of both sexes"—superior individuals can be selected through genotyping at the chick stage, shortening the breeding cycle by more than 50%, increasing the accuracy of rooster selection to a level comparable to that of hens, and significantly reducing the human and material resources required for phenotyping.

[0008] Although some SNP loci related to eggshell color have been reported in recent years, most have limitations: they are either only validated in a single breed, lacking broad applicability; or have a weak association with eggshell color, resulting in limited selection effects; or do not clearly define the color gradient corresponding to different genotypes, failing to meet the needs of targeted breeding. Therefore, identifying eggshell color-related SNP molecular markers with broad applicability, strong association, and clear phenotypic effects, and establishing standardized detection technologies and breeding systems, can not only effectively solve the pain points of traditional breeding but also meet the core requirements of the National Egg-Laying Hen Genetic Improvement Plan. This will provide technical support for the stable inheritance of eggshell color in the later stages of egg production, and has significant theoretical and practical value in promoting the upgrading of the egg-laying hen industry towards "high quality, high efficiency, and precision." Summary of the Invention

[0009] In view of this, the present invention proposes a molecular marker related to eggshell color and its application, aiming to solve the problems that existing SNP sites related to eggshell color can only be verified in a single variety, have insufficient applicability, and have a weak correlation with eggshell color, resulting in limited selection effect.

[0010] This invention proposes a SNP molecular marker related to eggshell color. The SNP molecular marker corresponds to position 18,378,867 on chromosome 6 of the chicken reference genome GRCg6a published in the Ensembl genome browser database. The base at this position is C / T and is a missense mutation on the ABCG2 gene.

[0011] This invention also provides the application of the above-mentioned eggshell color-related SNP molecular markers in eggshell color selection, and the application method is as follows: 1) Extract genomic DNA from the chickens to be tested; 2) Using specific primer pairs, the extracted genomic DNA was amplified by PCR to obtain an amplification product containing the SNP molecular marker described in claim 1; 3) Perform genotyping analysis on the amplification products to determine whether the genotype of the chicken at the SNP molecular marker site is CC, TC or TT; 4) Select breeding chickens with different eggshell colors based on genotype: CC genotype corresponds to breeding chickens with dark eggshell color, TC genotype corresponds to breeding chickens with medium eggshell color, and TT genotype corresponds to breeding chickens with light eggshell color. The specific primer pair mentioned in step 2) includes ABCG2_SNP18378867_F1 and ABCG2_SNP18378867_R1; The sequence of ABCG2_SNP18378867_F1 is shown in SEQ ID NO.1, and the sequence of ABCG2_SNP18378867_R1 is shown in SEQ ID NO.2.

[0012] Preferably, the method for extracting genomic DNA in step 1) is as follows: Blood was collected from the subwing vein of the chickens to be tested. After adding an anticoagulant, lysis buffer and proteinase K were added for lysis. RNA was degraded by adding RNase A to the lysis buffer. Then, add a phenol-chloroform-isoamyl alcohol mixture, mix well, and allow to stand to separate into layers. Keep the upper aqueous phase. Add a chloroform-isoamyl alcohol mixture to the aqueous phase, mix well, and centrifuge. Keep the aqueous phase. Add sodium acetate solution and anhydrous ethanol to the aqueous phase to obtain DNA precipitate. Wash and redissolve the DNA precipitate to obtain genomic DNA.

[0013] Preferably, the PCR amplification reaction system in step 2) comprises 20 μL of: 8.0 μL of double-distilled water, 10.0 μL of PCR premix, 0.5 μL of 10 μM upstream primer, 0.5 μL of 10 μM downstream primer, and 1.0 μL of genomic DNA.

[0014] Preferably, the reaction conditions for PCR amplification in step 2) are as follows: Pre-denaturation: 95℃ for 10 s Denaturation: 95℃ for 10 seconds Annealing: 60℃ for 5 seconds Extension: 72℃ 5 s Insulation: 4℃ Repeat the denaturation, annealing, and extension process for 35 cycles.

[0015] Preferably, the genotyping analysis of the amplified products described in step 3) specifically involves performing individual whole-genome resequencing using a human whole-genome resequencing platform.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows: The SNP molecular marker of this invention is related to eggshell color and is a novel molecular marker. By detecting the genotype at this locus in the individual chicken being tested, early selection of chickens with the dominant genotype can be carried out, which can accelerate the breeding progress of this trait and has great application value and economic benefits. Attached Figure Description

[0017] Various other advantages and benefits will become apparent to those skilled in the art upon reading the following detailed description of preferred embodiments. The accompanying drawings are for illustrative purposes only and are not intended to limit the invention. Furthermore, the same reference numerals denote the same parts throughout the drawings. In the drawings: Figure 1 This is a graph showing the results of the genome-wide association analysis of the Loch Ness Red chicken in Example 1.

[0018] Figure 2 The eggshell color at different stages for different genotypes at the Chr6:18378867 locus in Example 2.

[0019] Figure 3 This shows the distribution of different genotypes at the Chr6:18378867 locus in 53 varieties / strains in Example 3.

[0020] Figure 4 This study examines the effect of different genotypes at the Chr6:18378867 locus on the shell color of Beijing oil eggs in Example 3.

[0021] Figure 5 The Sanger sequencing results are for the amplification products of different genotypes at the Chr6:18378867 locus in Example 3. Detailed Implementation

[0022] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention. It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the present invention.

[0023] Furthermore, regarding the numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included within this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0024] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0025] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0026] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0027] This invention proposes a SNP molecular marker related to eggshell color. The SNP molecular marker corresponds to position 18,378,867 on chromosome 6 of the chicken reference genome GRCg6a published in the Ensembl genome browser database. The base at this position is C / T and is a missense mutation on the ABCG2 gene.

[0028] This invention also provides the application of the above-mentioned eggshell color-related SNP molecular markers in eggshell color selection, and the application method is as follows: 1) Extract genomic DNA from the chickens to be tested; 2) Using specific primer pairs, the extracted genomic DNA was amplified by PCR to obtain an amplification product containing the SNP molecular marker described in claim 1; 3) Perform genotyping analysis on the amplification products to determine whether the genotype of the chicken at the SNP molecular marker site is CC, TC or TT; 4) Select breeding chickens with different eggshell colors based on genotype: CC genotype corresponds to breeding chickens with dark eggshell color, TC genotype corresponds to breeding chickens with medium eggshell color, and TT genotype corresponds to breeding chickens with light eggshell color. The specific primer pair mentioned in step 2) includes ABCG2_SNP18378867_F1 and ABCG2_SNP18378867_R1; The sequence of ABCG2_SNP18378867_F1 is shown in SEQ ID NO.1, and the sequence of ABCG2_SNP18378867_R1 is shown in SEQ ID NO.2.

[0029] In this invention, the method for extracting genomic DNA in step 1) is as follows: Blood was collected from the subwing vein of the chickens to be tested. After adding an anticoagulant, lysis buffer and proteinase K were added for lysis. RNA was degraded by adding RNase A to the lysis buffer. Then, add a phenol-chloroform-isoamyl alcohol mixture, mix well, and allow to stand to separate into layers. Keep the upper aqueous phase. Add a chloroform-isoamyl alcohol mixture to the aqueous phase, mix well, and centrifuge. Keep the aqueous phase. Add sodium acetate solution and anhydrous ethanol to the aqueous phase to obtain DNA precipitate. Wash and redissolve the DNA precipitate to obtain genomic DNA.

[0030] In this invention, the PCR amplification reaction system described in step 2) is 20 μL and includes: 8.0 μL of double-distilled water, 10.0 μL of PCR premix, 0.5 μL of 10 μM upstream primer, 0.5 μL of 10 μM downstream primer, and 1.0 μL of genomic DNA.

[0031] In this invention, the reaction conditions for PCR amplification in step 2) are as follows: Pre-denaturation: 95℃ for 10 s Denaturation: 95℃ for 10 seconds Annealing: 60℃ for 5 seconds Extension: 72℃ 5 s Insulation: 4℃ Repeat the denaturation, annealing, and extension process for 35 cycles.

[0032] In this invention, the genotyping analysis of the amplified products described in step 3) specifically involves performing individual whole-genome resequencing using a human whole-genome resequencing platform.

[0033] Example 1 (1) Experimental animals: 806 purebred Rhodes Island Red Chickens were raised with free access to feed and water, and the feeding standards met the relevant provisions of the industry standard (NY / T 33-2004).

[0034] (2) Phenotypic determination: The eggshell color of three consecutive eggs from each chicken at 33, 52, 72 and 100 weeks of age was determined.

[0035] (3) Genomic DNA extraction: 0.5 mL of venous blood was collected from the wing vein of all experimental chickens using anticoagulated vacuum blood collection tubes. Whole genomic DNA was extracted using the phenol-chloroform extraction method. DNA samples meeting the required concentration and purity were then subjected to agarose gel electrophoresis to further assess the purity and integrity of the DNA samples. The sample concentration was required to be greater than 50 ng / μL, with a purity OD260 / 280 of 1.8–2.0, and good integrity. DNA samples were stored at -20℃ until use.

[0036] (3) Genome resequencing: DNA samples from all experimental chickens were sent to BGI Genomics, and whole-genome resequencing was performed on the DNBSEQ sequencing platform according to standard operating procedures. FastP (v0.23.4) software was used to clean the data, and BWA-MEM (v0.7.5a-r405) and GATK (v4.6.0.0) software were used for sequence alignment and genotyping with reference genome GRCg6a (v112). After further quality control using the command "--mind 0.05 --geno 0.05 --maf 0.05 --hwe 1e-5", 6,832,796 SNPs and 806 individuals were obtained for subsequent analysis.

[0037] (4) Genome-wide association analysis: After organizing pedigree, phenotypic, and genomic data, to eliminate bias caused by kinship, a single-trait repeatability model was implemented using the DMUAI module of DMU software (v6-R5-2-EM64T) based on pedigree information, and the deregression breeding value (DRP) of each trait was calculated. Subsequently, genome-wide association analysis was performed using GCTA (v1.94.0) software, and the mixed linear model was:

[0038] in y The vector of the corrected phenotype. μ The population mean c Represents covariates (including the first three components of the genotype principal component). x The regression coefficients are the covariates. β This is the effect of SNP. w This is a vector representing the genotypes of SNPs (genotypes AA, Aa, and aa are assigned values ​​of 0, 1, and 2, respectively). g~N(0, Gσ) g 2 ) represents the vector of additive genetic effects, G is the genomic relation matrix, and σ is the vector of additive genetic effects. g 2 It is additive genetic variance. e^(N(0, Iσ)) e 2 ) is the vector of residual effects, σ e 2 It is the residual variance. I It is an identity matrix. Z It is an additive genetic effect. g The corresponding design matrix.

[0039] To ensure the quality of the GWAS data obtained at each time period, EasyQC (v23.8) software was used for quality control. The specific procedures were as follows: First, the summary statistics of individual GWAS data were standardized. Then, using the filelevel_qc.ecf and metalevel_qc.ecf files with default parameter settings, quality control was performed at both the file level and meta-level. After quality control, METAL software (released on March 25, 2011) was used to perform meta-analysis on each eggshell color trait in the four stages. Manhattan plots were drawn using the CMplot (v4.5.1) package in R (v4.3.2). Bonferroni correction was used to set significance thresholds, with the genome-wide significance threshold being (P=0.05 / 6,832,796 (total SNPs) = 7.32 × 10⁻⁶). -9 Meta-GWAS analysis results are as follows: Figure 1 As shown, eggshell color is significantly associated with the Chr6:18378867 locus on chromosome 6.

[0040] Example 2 (1) Experimental animals: Same as above.

[0041] (2) Phenotypic determination: Same as above.

[0042] (3) Extraction of genomic DNA: Same as above.

[0043] (3) Genotyping of Chr6:18378867 locus: Same as above.

[0044] (4) Determination of dominant eggshell color genotypes: At the Chr6:18378867 locus, the eggshell color of the CC genotype was significantly lower than that of the TT genotype at 33, 52, 72, and 100 weeks of age. The CC genotype showed a darker eggshell color. Specific results are as follows: Figure 2 As shown.

[0045] Example 3 Eighty-nine purebred Beijing You chickens were selected as the research subjects. Beijing You chickens primarily lay pink-shelled eggs. Genotyping was performed on these 894 Beijing You chickens, and eggshell color was measured at 33, 43, and 52 weeks of age. Figure 4 As shown, the eggshell color of the CT genotype at the Chr6:18378867 locus in Beijing You chickens was significantly lower than that of the TT genotype. The eggshell color of the CT genotype was darker than that of the TT genotype, which is consistent with the results of Examples 1 and 2. Currently, only one case of the CC genotype has been found in the Beijing You chicken population.

[0046] Example 4 Based on the upstream and downstream DNA sequence information of the Chr6:18378867 locus in the Ensembl genome browser database, specific primers were designed and synthesized for PCR amplification. Primer sequence information is shown in Table 1, PCR amplification system in Table 2, and PCR amplification conditions in Table 3. The genotypes of the Chr6:18378867 locus were analyzed using first-generation sequencing results. Genotype results included CC, CT, and TT. Sanger sequencing was performed on two samples each of the CC, CT, and TT genotypes using the aforementioned primer sequences. Sequencing results for the amplified products of the three genotypes are shown below. Figure 5 As shown.

[0047] Table 1 Primer sequence information

[0048] Table 2 PCR amplification system

[0049] Table 3 PCR amplification conditions

[0050] Example 5 For brown-shelled egg selection breeding, the Loch Ness Red population was selected. The primers of Example 4 were used to detect the Chr6:18378867 locus in Loch Ness Red roosters and hens. Roosters with the CC genotype and hens with the CC and TC genotypes were selected for breeding. Eggshell color phenotype was measured at 33 weeks of age of the offspring. It was found that the eggshell color of the CC genotype was significantly darker than that of the CT genotype, as shown in Table 4.

[0051] Table 4. Eggshell color of Loch Ness Red chicken offspring at 33 weeks of age

[0052] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the specific implementation of the present invention. Any modifications or equivalent substitutions that do not depart from the spirit and scope of the present invention should be covered within the scope of protection of the claims of the present invention.

Claims

1. The application of an SNP molecular marker related to eggshell color in eggshell color selection, characterized in that, The application method is as follows: 1) Extract genomic DNA from the chickens to be tested; 2) Using specific primer pairs, the extracted genomic DNA was amplified by PCR to obtain an amplification product containing the SNP molecular marker; 3) Perform genotyping analysis on the amplification products to determine whether the genotype of the chicken at the SNP molecular marker site is CC, TC or TT; 4) Select breeding chickens with different eggshell colors based on genotype: CC genotype corresponds to breeding chickens with dark eggshell color, TC genotype corresponds to breeding chickens with medium eggshell color, and TT genotype corresponds to breeding chickens with light eggshell color. The specific primer pair mentioned in step 2) includes ABCG2_SNP18378867_F1 and ABCG2_SNP18378867_R1; The sequence of ABCG2_SNP18378867_F1 is shown in SEQ ID NO.1, and the sequence of ABCG2_SNP18378867_R1 is shown in SEQ ID NO.2; The SNP molecular marker corresponds to position 18,378,867 on chromosome 6 of the chicken reference genome GRCg6a published in the Ensembl genome browser database. The base at this position is C / T and is a missense mutation on the ABCG2 gene.

2. The application of the SNP molecular markers related to eggshell color according to claim 1 in eggshell color selection, characterized in that, The method for extracting genomic DNA in step 1) is as follows: Blood was collected from the subwing vein of the chickens to be tested. After adding an anticoagulant, lysis buffer and proteinase K were added for lysis. RNA was degraded by adding RNase A to the lysis buffer. Then, add a phenol-chloroform-isoamyl alcohol mixture, mix well, and allow to stand to separate into layers. Keep the upper aqueous phase. Add a chloroform-isoamyl alcohol mixture to the aqueous phase, mix well, and centrifuge. Keep the aqueous phase. Add sodium acetate solution and anhydrous ethanol to the aqueous phase to obtain DNA precipitate. Wash and redissolve the DNA precipitate to obtain genomic DNA.

3. The application of the SNP molecular markers related to eggshell color according to claim 1 in eggshell color selection, characterized in that, The PCR amplification reaction system described in step 2) is in 20 μL and includes: 8.0 μL of double-distilled water, 10.0 μL of PCR premix, 0.5 μL of 10 μM upstream primer, 0.5 μL of 10 μM downstream primer, and 1.0 μL of genomic DNA.

4. The application of the SNP molecular markers related to eggshell color according to claim 1 in eggshell color selection, characterized in that, The reaction conditions for PCR amplification in step 2) are as follows: Pre-denaturation: 95℃ for 10 s Denaturation: 95℃ for 10 seconds Annealing: 60℃ for 5 seconds Extension: 72℃ 5 s Insulation: 4℃ Repeat the denaturation, annealing, and extension process for 35 cycles.

5. The application of the SNP molecular markers related to eggshell color according to claim 1 in eggshell color selection, characterized in that, Step 3) describes genotyping the amplified products by performing individual whole-genome resequencing using a human whole-genome resequencing platform.

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