A dominant SNP molecular marker related to eggshell strength trait of chicken and application thereof

Abstract

This invention discloses a dominant SNP molecular marker associated with eggshell strength and its application, belonging to the field of gene detection technology. The SNP molecular marker is located at position 178474424 on the positive strand of chromosome 1 of the chicken reference genome GRCg7b version published on the Ensembl website. The base here is C or T, denoted as chr1:178474424 T>C or 1_178474424_T_C. The steps for its application are: (1) extracting genomic DNA from the chicken to be tested; (2) detecting the genotype of the SNP locus at position 178474424 on the positive strand of chromosome 1 of the chicken to be tested; (3) determining the eggshell strength level of the chicken to be tested based on the genotype detection results in the steps above. This invention allows for effective selection in the early stages of chicken development, saving production costs and accelerating genetic progress, and has significant application value and potential economic benefits.

Description

Technical Field

[0001] This invention relates to the field of gene detection technology, and in particular to a dominant SNP molecular marker associated with eggshell strength trait and its application. Background Technology

[0002] Eggshell strength is a crucial indicator for evaluating egg quality and laying hen performance. It directly impacts egg breakage rates during collection, transportation, storage, and sales, further affecting the economic benefits of egg farming. Lower eggshell strength leads to a higher proportion of broken, cracked, and soft-shelled eggs, increasing production losses. In breeder chicken production, eggshell quality is also closely related to egg preservation, embryo development, and hatching effectiveness. Therefore, improving eggshell strength is a key breeding objective in laying hen genetic improvement and production management.

[0003] Traditional breeding methods for eggshell strength traits primarily rely on phenotypic testing and pedigree selection. This involves testing eggs using equipment such as eggshell strength meters after the chickens reach a certain laying stage, and then selecting individuals or families based on their phenotypic characteristics. However, eggshell strength is a complex quantitative trait regulated by multiple genes, easily influenced by factors such as breed, age, nutritional level, rearing environment, laying stage, and testing conditions. Furthermore, accurate eggshell strength phenotype testing is typically only possible after the onset of laying, making early screening of candidate individuals difficult. This results in traditional breeding methods suffering from long testing cycles, high costs, and limited selection efficiency.

[0004] With the development of molecular marker technology, whole-genome resequencing technology, and genome-wide association analysis (GWAS), using SNP molecular markers to assist in the selection of eggshell quality-related traits has become an important means to improve the breeding efficiency of laying hens. By screening for genetic variation sites that are significantly associated with eggshell strength, the potential eggshell strength level of an individual can be predicted through genotyping at an early stage. This allows for the early screening of individuals with superior genotypes, shortening the breeding cycle and improving selection accuracy and genetic progress.

[0005] Currently, some candidate genes and SNP loci have been reported that are associated with egg quality traits such as eggshell thickness, eggshell strength, eggshell color, and egg weight. However, genetic backgrounds differ among different breeds and populations, and the discovered molecular markers may not be applicable to all laying hen populations. Therefore, identifying SNP molecular markers associated with stable eggshell strength for specific laying hen breeds and establishing early-assisted selection methods based on these markers is of significant application value for improving eggshell strength, reducing breakage rate, improving egg quality, and enhancing the efficiency of laying hen breeding.

[0006] Therefore, developing a SNP molecular marker related to eggshell strength traits and applying it to the identification, auxiliary identification, and early molecular marker-assisted selection of eggshell strength traits is of great practical significance and promotional value in solving the problems of long selection cycles, high costs, susceptibility to environmental influences, and difficulty in early screening of traditional eggshell strength phenotypic traits. Summary of the Invention

[0007] The purpose of this invention is to provide a dominant SNP molecular marker related to eggshell strength and its application, aiming to solve the technical problems of high cost and low accuracy in the traditional breeding methods for eggshell strength in the prior art.

[0008] To achieve the above objectives, the present invention provides the following solution: One of the technical solutions of the present invention is an SNP molecular marker related to the trait of eggshell strength. The SNP molecular marker is located at the 178474424th base on chromosome 1 of the chicken genome, and the base at this site is T or C. The reference genome for the SNP molecular marker is GRCg7b.

[0009] The second technical solution of the present invention is a method for detecting SNP molecular markers related to eggshell strength traits, comprising the following steps: Genomic DNA was extracted from the chickens to be tested, and the genotype of the chickens at position 178474424 on chromosome 1 was detected.

[0010] The third technical solution of the present invention is the application of the SNP molecular marker in the identification or auxiliary identification of eggshell strength traits, wherein the chicken is a White Leghorn laying hen.

[0011] The fourth technical solution of the present invention is a method for identifying or assisting in the identification of eggshell strength characteristics, wherein the chicken is a White Leghorn laying hen; Includes the following steps: Genomic DNA was extracted from the chickens to be tested, and the genotype of the chickens at position 178474424 on chromosome 1 was detected. The eggshell strength was highest for the TT genotype, followed by the TC genotype, and lowest for the CC genotype.

[0012] The fifth technical solution of the present invention is the application of the SNP molecular marker in the breeding of new breeds with higher eggshell strength, wherein the chicken is a White Leghorn laying hen.

[0013] The sixth technical solution of this invention is a method for breeding a new breed of chicken with higher eggshell strength, wherein the chicken is a White Leghorn laying hen; Includes the following steps: Genomic DNA was extracted from the chickens to be tested, and the genotype of the chickens at position 178474424 on chromosome 1 was detected. Individuals with the TT genotype were selected for breeding.

[0014] Based on the above technical solution, the present invention has the following technical effects: The SNP molecular marker of this invention is associated with eggshell strength and is a novel molecular marker. Compared with molecular markers that select based solely on eggshell strength at a single week of age, the chr1:178474424 C>T locus described in this invention not only shows a significant association in eggshell strength at 32 weeks of age, but also exhibits a consistent genotypic effect in eggshell strength at 43 weeks of age in both the B1 and B3 lines. Individuals with the TT genotype show higher eggshell strength, indicating that this molecular marker has good line applicability and stage stability. By detecting the genotype of this SNP locus, individuals carrying the dominant genotype can be screened at an early stage, improving the efficiency of eggshell strength selection, reducing the risk of egg breakage, and demonstrating high breeding application value. Attached Figure Description

[0015] Figure 1 This is a schematic diagram of the results of the genome-wide association analysis in Example 1 of the present invention.

[0016] Figure 2 This is a graph showing the results of the correlation analysis between different genotypes and eggshell strength in Example 2 of the present invention.

[0017] Figure 3 This is a graph showing the results of the correlation analysis between different genotypes and eggshell strength in Example 3 of the present invention.

[0018] Figure 4 This is a graph showing the results of the correlation analysis between different genotypes and eggshell strength in Example 4 of the present invention.

[0019] Figure 5 This is a graph showing the results of the correlation analysis between different genotypes and eggshell strength in Example 5 of the present invention. Detailed Implementation

[0020] Unless otherwise specified, the technical solutions described in this invention are all conventional solutions in the field, and the reagents or raw materials used are all purchased from commercial channels or are publicly available unless otherwise specified.

[0021] This invention provides an SNP molecular marker related to eggshell strength, wherein the SNP molecular marker is located at base 178474424 on chromosome 1 of the chicken genome, and the base at this site is T or C; The reference genome for the SNP molecular marker is GRCg7b.

[0022] In some specific implementations, the SNP molecular markers include genotypes TT, TC, and CC.

[0023] This invention also provides a method for detecting SNP molecular markers related to eggshell strength traits, comprising the following steps: Genomic DNA was extracted from the chickens to be tested, and the genotype of the chickens at position 178474424 on chromosome 1 was detected.

[0024] This invention also provides the application of the SNP molecular marker in the identification or auxiliary identification of eggshell strength traits, wherein the chicken is a White Leghorn laying hen.

[0025] This invention also provides a method for identifying or assisting in the identification of eggshell strength characteristics, wherein the chicken is a White Leghorn laying hen; Includes the following steps: Genomic DNA was extracted from the chickens to be tested, and the genotype of the chickens at position 178474424 on chromosome 1 was detected. The eggshell strength was highest for the TT genotype, followed by the TC genotype, and lowest for the CC genotype.

[0026] This invention also provides the application of the SNP molecular marker in breeding new breeds with higher eggshell strength, wherein the chicken is a White Leghorn laying hen.

[0027] This invention also provides a method for breeding a new breed of chicken with higher eggshell strength, wherein the chicken is a White Leghorn laying hen; Includes the following steps: Genomic DNA was extracted from the chickens to be tested, and the genotype of the chickens at position 178474424 on chromosome 1 was detected. Individuals with the TT genotype were selected for breeding.

[0028] The SNP molecular marker corresponds to position 178474424 on the positive strand of chromosome 1 in the chicken reference genome GRCg7b version published on the Ensembl website. The base here is T or C, denoted as chr1:178474424 T>C or 1_178474424_T_C.

[0029] In this embodiment of the invention, both the B1 and B3 strains of chickens are White Leghorn laying hens. The B1 strain was collected from Luohe City, Henan Province, and the B3 strain was collected from Nanyang City, Henan Province.

[0030] Example 1 Determination of SNP molecular markers associated with eggshell strength at 32 weeks in B1 breed chickens (1) Experimental animals: 557 individual hens were used as experimental subjects. During the feeding period, they were allowed free access to feed and water, and the feeding conditions strictly followed the relevant provisions of the industry standard (NY / T 33-2004).

[0031] (2) Phenotypic determination: Statistical analysis of eggshell strength phenotypic data over 32 weeks.

[0032] (3) Genomic DNA extraction: 0.5 mL of blood was collected from the wing vein of all experimental chickens using anticoagulated vacuum blood collection tubes. The whole genomic DNA was extracted using the phenol-chloroform extraction method. The qualified DNA samples were subjected to agarose gel electrophoresis to further evaluate the purity and integrity of the DNA samples. The sample concentration should be greater than 50 ng / μL, the purity OD260 / 280 should be between 1.8 and 2.0, and the integrity should be good. The qualified DNA samples were stored at -20 degrees for later use.

[0033] (4) All DNA samples from the experimental chickens were sequenced using the BGISEQ sequencing platform according to standard operating procedures. After raw read filtering, the average coverage depth of each sample was 10×. Sequence alignment and genotype extraction were performed using BWA and GATK software. After SNP call rate and MAF quality control, 6,544,608 SNPs were obtained and used for subsequent analysis.

[0034] (5) Genome-wide association analysis: Pedigree data, phenotypic data, and genomic SNP locus data were compiled, and genome-wide association analysis was performed using GCTA software. The model was as follows: ; in, Represents a vector of phenotypic observations; This represents the fixed effects vector, which includes fixed effects (including group effects and cage effects). This represents the design matrix corresponding to the fixed effects; This indicates the genotype dose of the SNP currently being tested; This indicates the random polygenic background effect caused by genomic relationships between individuals; β This indicates the additive effect of single nucleotide polymorphism sites; This represents random residuals. The significance threshold is... P <1E-05.

[0035] GWAS analysis results are as follows Figure 1 As shown, based on Figure 1 It can be seen that eggshell strength is significantly correlated with the region of chromosome 1, 178 Mb (chr1:178473954-chr1:178484734). P<1E-05), further validation of all loci in the genome-associated region was performed, and the chr1:178474424 locus was identified as a candidate locus (GRCg7b version of the chicken reference genome published on the Ensembl website). Genetic marker data affecting eggshell strength are shown in Table 1.

[0036] Table 1. Genetic marker data affecting eggshell strength

[0037] chr1:178474424 (SEQ ID NO.1): AGGACTCGCGGCGCCAACGG...CGGGGCGGGCCTGTGCTGGAGAGGGCACTGTGGGGTCTTCTGTGAGTACG[C / T]CCCCACGTTGCCTCTCTGGGCGCCCGTGTGGGTGTGCGGATGAGTTTTTA...TGAATAAAGCTTCTGTCTCG.

[0038] Example 2 Correlation between different genotypes at chr1:178474424 locus and eggshell strength of B1 strain at 32 weeks (1) Experimental animals: 557 B1 strain hens were used in the same way as in Example 1, and the feeding method was the same as in Example 1.

[0039] (2) Phenotypic determination: Same as in Example 1.

[0040] (3) Extraction of genomic DNA: Same as in Example 1.

[0041] (4) Genotyping of specific gene loci: Same as in Example 1.

[0042] (5) Identification of phenotypic dominant genotypes: At the chr1:178474424 locus, the average eggshell strength of individuals with the TT genotype was 3.338, the average eggshell strength of individuals with the TC genotype was 3.049, and the average eggshell strength of individuals with the CC genotype was 2.813. Individuals with the TT genotype had the highest eggshell strength, followed by TC, and CC had the lowest. The overall difference in eggshell strength among different genotypes at this locus was significant.

[0043] Table 2 shows the correlation data between different genotypes at the chr1:178474424 locus in B1 breed chickens and eggshell strength at 32 weeks of age.

[0044] Note: Different letters on the abc shoulder labels indicate significant differences in phenotype between groups.

[0045] Example 3: Validation and application of the chr1:178474424 molecular marker in the shell strength of B3 strain eggshells at 32 weeks. (1) Experimental animals: 507 B3 strain hens, fed in the same manner as in Example 1.

[0046] (2) Phenotypic determination: Same as in Example 1.

[0047] (3) Extraction of genomic DNA: Same as in Example 1.

[0048] (4) Genotyping of specific gene loci: Among the obtained genotypes, there were 416 individuals with the TT genotype, 69 individuals with the TC genotype, and 22 individuals with the CC genotype. Based on the dominant genotype for eggshell strength, individuals with the TT genotype were selected for subsequent breeding. After B3 chickens were raised to the egg-laying period, eggshell strength was measured at 32 weeks. The correlation results between different genotypes of individuals with the chr1:178474424 SNP locus in the B3 strain and eggshell strength are shown in Table 3.

[0049] Table 3 shows the correlation data between different genotypes at the chr1:178474424 locus in B3 breed chickens and eggshell strength at 32 weeks of age.

[0050] Note: Different letters on the abc shoulder labels indicate significant differences in phenotype between groups.

[0051] (5) Identification of phenotypic dominant genotypes: At the chr1:178474424 locus, the average eggshell strength of individuals with the TT genotype was 3.621, the average eggshell strength of individuals with the TC genotype was 3.603, and the average eggshell strength of individuals with the CC genotype was 3.116. The phenotypes of the TT and TC genotypes were significantly higher than those of the CC genotype.

[0052] Example 4 Validation and application of the chr1:178474424 molecular marker in the shell strength of B1 strain at 43 weeks of age. (1) Experimental animals: 546 B1 strain hens, fed in the same manner as in Example 1.

[0053] (2) Phenotypic determination: Same as in Example 1.

[0054] (3) Extraction of genomic DNA: Same as in Example 1.

[0055] (4) Genotyping of specific gene loci: Among the obtained genotypes, there were 471 individuals with the TT genotype, 60 individuals with the TC genotype, and 15 individuals with the CC genotype. Based on the dominant genotype for eggshell strength, individuals with the TT genotype were selected for subsequent breeding. After B1 chickens were raised to the egg-laying period, eggshell strength was measured at 43 weeks. The correlation results between different genotypes of the B1 strain chicken chr1:178474424 SNP locus and eggshell strength are shown in Table 4.

[0056] Table 4 shows the correlation data between different genotypes at the chr1:178474424 locus in B1 breed chickens and eggshell strength at 43 weeks of age.

[0057] Note: Different letters on the abc shoulder labels indicate significant differences in phenotype between groups.

[0058] (5) Identification of phenotypic dominant genotypes: At the chr1:178474424 locus, the average eggshell strength of individuals with the TT genotype was 3.042, that of individuals with the TC genotype was 2.847, and that of individuals with the CC genotype was 2.594. Individuals with the TT genotype had the highest eggshell strength, while those with the CC genotype had the lowest. Overall, there was a significant difference in eggshell strength among different genotypes at this locus.

[0059] Example 5 Validation and application of the chr1:178474424 molecular marker in the shell strength of B3 strain at 43 weeks of age. (1) Experimental animals: 501 B3 strain hens, fed in the same manner as in Example 1.

[0060] (2) Phenotypic determination: Same as in Example 1.

[0061] (3) Extraction of genomic DNA: Same as in Example 1.

[0062] (4) Genotyping of specific gene loci: Among the obtained genotypes, there were 409 individuals with the TT genotype, 70 individuals with the TC genotype, and 22 individuals with the CC genotype. Based on the dominant genotype for eggshell strength, individuals with the TT genotype were selected for subsequent breeding. After B3 chickens were raised to the egg-laying period, eggshell strength was measured at 43 weeks. The correlation results between different genotypes of individuals with the chr1:178474424 SNP locus in the B3 strain and eggshell strength are shown in Table 5.

[0063] Table 5 shows the correlation data between different genotypes at the chr1:178474424 locus in B3 breed chickens and eggshell strength at 43 weeks of age.

[0064] Note: Different letters on the abc shoulder labels indicate significant differences in phenotype between groups.

[0065] (5) Identification of phenotypic dominant genotypes: At the chr1:178474424 locus, the average eggshell strength of individuals with the TT genotype was 3.058, that of individuals with the TC genotype was 3.009, and that of individuals with the CC genotype was 2.725. The phenotypes of the TT and TC genotypes were significantly higher than that of the CC genotype. In summary, in both the B1 and B3 strains, individuals with the TT genotype had the highest average eggshell strength at 43 weeks of age, indicating that this locus still has a consistent genotypic effect on eggshell strength across different strains at 43 weeks of age. Combined with the eggshell strength results at 32 weeks of age, this suggests that the TT genotype can be used as a dominant genotype for improving eggshell strength, and can be used for auxiliary identification and early selection of eggshell strength traits.

[0066] In summary, the C>T locus at chr1:178474424 was not only identified as a candidate associated locus in eggshell strength of the B1 strain at 32 weeks of age, but was further validated in eggshell strength of both the B1 and B3 strains at 43 weeks of age. Comparison of different genotypes showed that individuals with the TT genotype exhibited the highest average eggshell strength in both strains at 43 weeks of age, suggesting that this locus has a relatively stable genotypic effect on eggshell strength. Therefore, this SNP locus can serve as an effective marker for auxiliary identification of eggshell strength traits at 32 and 43 weeks of age and for early molecular marker-assisted selection. Prioritizing the selection of individuals with the TT genotype can help improve eggshell strength and reduce the risk of egg breakage during production.

[0067] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively describe all embodiments here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A SNP molecular marker related to eggshell strength trait, characterized in that, The SNP molecular marker is located at base 178474424 on chromosome 1 of the chicken genome, and the base at this site is either T or C. The reference genome for the SNP molecular marker is GRCg7b.

2. The SNP molecular marker according to claim 1, characterized in that, The SNP molecular markers include genotypes TT, TC, and CC.

3. A method for detecting SNP molecular markers related to eggshell strength traits, characterized in that, Includes the following steps: Genomic DNA was extracted from the chickens to be tested, and the genotype of the chickens at position 178474424 on chromosome 1 was detected.

4. The application of the SNP molecular marker as described in claim 1 or 2 in the identification or auxiliary identification of eggshell strength traits, characterized in that, The chickens mentioned are White Leghorn laying hens.

5. A method for identifying or assisting in the identification of eggshell strength characteristics, characterized in that, The chickens mentioned are White Leghorn laying hens; Includes the following steps: Genomic DNA was extracted from the chickens to be tested, and the genotype of the chickens at position 178474424 on chromosome 1 was detected. The eggshell strength was highest for the TT genotype, followed by the TC genotype, and lowest for the CC genotype.

6. The application of the SNP molecular marker as described in claim 1 or 2 in breeding new varieties of eggshells with higher strength, characterized in that, The chickens mentioned are White Leghorn laying hens.

7. A method for cultivating a new variety of eggshell with higher strength, characterized in that, The chickens mentioned are White Leghorn laying hens; Includes the following steps: Genomic DNA was extracted from the chickens to be tested, and the genotype of the chickens at position 178474424 on chromosome 1 was detected. Individuals with the TT genotype were selected for breeding.

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