SNP molecular marker module for egg-laying hen abdominal fat deposition trait assisted breeding and application

Abstract

The application belongs to the technical field of molecular breeding, and provides a SNP molecular marker module for assisted breeding of an egg-laying hen abdominal fat deposition trait and application, wherein the SNP molecular marker module comprises SNPs: rs731034862 and rs736850300; the rs731034862 is located at 6,729,430 bp of chromosome 20, and C / G polymorphism exists; the rs736850300 is located at 7,015,142 bp of chromosome 20, and G / A polymorphism exists. It is found that the rs731034862-GG / rs736850300-AA combination is significantly positively correlated with the abdominal fat weight and abdominal fat rate of an egg-laying hen after the above two sites are combined. Meanwhile, the application provides an efficient molecular tool for early selection and breeding of an egg-laying hen abdominal fat trait, helps to shorten the breeding cycle and reduce the determination cost, and has application prospects in egg-laying hen breeding.

Description

Technical Field

[0001] This invention belongs to the field of molecular breeding technology, specifically relating to an SNP molecular marker module for assisted breeding of abdominal fat deposition traits in laying hens and its application. Background Technology

[0002] Abdominal fat deposition is an important economic trait and health indicator in poultry production. Excessively high abdominal fat percentage not only directly reduces feed conversion ratio and slaughter performance, increasing breeding costs, but is also associated with health problems such as increased metabolic burden and decreased reproductive performance. Therefore, in layer hen breeding, appropriate regulation of abdominal fat deposition to cultivate strains with suitable fat deposition is of great significance for improving production efficiency and ensuring flock health. Traditional selection of abdominal fat traits mainly relies on direct measurement after slaughter, a method that is inefficient, costly, and cannot achieve early selection in vivo, severely hindering breeding progress. With the development of high-throughput sequencing and genotyping technologies, it has become possible to regulate abdominal fat deposition at the genetic level through marker-assisted selection (MAS). Genome-wide association analysis (GWAS) has become a key tool for identifying genetic variations affecting abdominal fat deposition, capable of locating significantly associated single nucleotide polymorphisms (SNPs).

[0003] However, existing research mostly focuses on the effects of single SNP loci, while complex traits are often synergistically regulated by multiple loci, and the accuracy and genetic potential of single marker selection are limited. Therefore, there is an urgent need to provide a stable, efficient, and dedicated molecular marker module for abdominal fat deposition in laying hens, in order to achieve early, in vivo, high-throughput, and high-accuracy molecular marker-assisted selection of abdominal fat traits in laying hens, and to overcome the shortcomings of traditional breeding and existing single marker technologies. Summary of the Invention

[0004] The purpose of this invention is to provide an SNP molecular marker module for assisted breeding of abdominal fat deposition traits in laying hens and its application. The SNP molecular marker module of this invention provides an efficient molecular tool for early selection of abdominal fat deposition traits in laying hens, helping to significantly shorten the breeding cycle, reduce phenotypic testing costs, and accelerate genetic progress. It has significant economic benefits and application prospects in laying hen breeding practice.

[0005] This invention provides the application of reagents for identifying SNP molecular marker modules in assisted breeding and / or identification of abdominal fat deposition traits in laying hens, wherein the SNP molecular marker modules include SNPs: rs731034862 and rs736850300. The rs731034862 is located at 6,729,430 bp on chromosome 20 and exhibits C / G polymorphism; the rs736850300 is located at 7,015,142 bp on chromosome 20 and exhibits G / A polymorphism.

[0006] As a preferred embodiment, the reference chicken genome for the SNP molecular marker module is GRCg6a.

[0007] As a preferred embodiment, the abdominal fat deposition characteristics of laying hens include abdominal fat weight and abdominal fat percentage.

[0008] The present invention also provides a product for identifying abdominal fat deposition traits in laying hens, the product comprising at least one of the following reagents: reagents for nucleic acid extraction and amplification, reagents for detecting nucleic acid amplification products, reagents for constructing sequencing libraries, and reagents for sequencing. The amplified product, nucleic acid amplification product, or sequence used for sequencing contains SNPs: rs731034862 and rs736850300. The rs731034862 is located at 6,729,430 bp on chromosome 20 and exhibits C / G polymorphism; the rs736850300 is located at 7,015,142 bp on chromosome 20 and exhibits G / A polymorphism.

[0009] As a preferred embodiment, the product includes primers for detecting the chicken rs731034862 and rs736850300 loci.

[0010] The present invention also provides a method for identifying abdominal fat deposition traits in laying hens, comprising the following steps: extracting genomic DNA from the laying hen to be tested, and detecting the genotypes at the rs731034862 and rs736850300 loci; Individuals carrying both the GG genotype at the rs731034862 locus and the AA genotype at the rs736850300 locus are laying hens with high abdominal fat deposition. Individuals carrying both the CC genotype at the rs731034862 locus and the GG genotype at the rs736850300 locus are laying hens with low abdominal fat deposition. The rs731034862 is located at 6,729,430 bp on chromosome 20 and exhibits C / G polymorphism; the rs736850300 is located at 7,015,142 bp on chromosome 20 and exhibits G / A polymorphism.

[0011] As a preferred option, the genotype detection method includes any one of whole genome resequencing, PCR-sequencing typing, KASP typing, or time-of-flight mass spectrometry typing.

[0012] This invention also provides a molecular marker-assisted breeding method for laying hens with high abdominal fat deposition, comprising the following steps: Using the above method, the genotypes of rs731034862 and rs736850300 loci were detected. Individuals carrying both the GG genotype at rs731034862 locus and the AA genotype at rs736850300 locus were screened and retained for breeding to obtain laying hens with high abdominal fat deposition. The rs731034862 is located at 6,729,430 bp on chromosome 20 and exhibits C / G polymorphism; the rs736850300 is located at 7,015,142 bp on chromosome 20 and exhibits G / A polymorphism.

[0013] This invention also provides a molecular marker-assisted breeding method for laying hens with low abdominal fat deposition, comprising the following steps: Using the above method, the genotypes of rs731034862 and rs736850300 loci were detected. Individuals carrying both the CC genotype at rs731034862 locus and the GG genotype at rs736850300 locus were screened and retained for breeding to obtain laying hens with low abdominal fat deposition. The rs731034862 is located at 6,729,430 bp on chromosome 20 and exhibits C / G polymorphism; the rs736850300 is located at 7,015,142 bp on chromosome 20 and exhibits G / A polymorphism.

[0014] As a preferred embodiment, the laying hens include the Loch Ness Red.

[0015] Beneficial Effects: This invention provides the application of reagents for identifying SNP molecular marker modules in assisted breeding and / or identification of abdominal fat deposition traits in laying hens. The SNP molecular marker modules include SNPs rs731034862 and rs736850300; wherein rs731034862 is located at 6,729,430 bp on chromosome 20 and exhibits C / G polymorphism; and rs736850300 is located at 7,015,142 bp on chromosome 20 and exhibits G / A polymorphism. This invention reveals for the first time that, after modularly combining the above two loci, a specific genotype combination (rs731034862-GG / rs736850300-AA, i.e., GG&AA type) shows a significant positive correlation with abdominal fat weight and abdominal fat percentage in laying hens, with phenotypic values ​​significantly superior to individuals with other genotypes.

[0016] Compared with traditional marker-assisted selection methods that rely on single molecular markers, the modular strategy employed in this invention more fully reflects the genetic interaction effects between multiple loci, significantly improving the accuracy and stability of predicting abdominal fat deposition traits and overcoming the limitations of single marker effects and insufficient selection potential. This combination of molecular markers is reported for the first time and has clear innovativeness.

[0017] At the application level, this module provides a highly efficient molecular tool for the early selection of abdominal fat traits in laying hens. On one hand, it can be used for the rapid selection of low-abdominal-fat strains to improve feed utilization efficiency and overall health; on the other hand, it can also serve the targeted breeding of high-abdominal-fat strains to meet the needs of specific research or product development. This technology helps to significantly shorten the breeding cycle, reduce the cost of phenotyping, and accelerate genetic progress, demonstrating significant economic benefits and promising application prospects in laying hen breeding practices. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly introduced below.

[0019] Figure 1 The figures show the results of genome-wide association analysis (GWAS) of abdominal fat in laying hens and the effects of different genotype combinations on abdominal fat. Among them, A is the Manhattan plot of significant GWAS sites for abdominal fat weight in laying hens; B is the Manhattan plot of significant GWAS sites for abdominal fat percentage in laying hens; C is the box plot of abdominal fat weight in laying hens under different genotype combinations; and D is the box plot of abdominal fat percentage in laying hens under different genotype combinations. Detailed Implementation

[0020] This invention provides the application of reagents for identifying SNP molecular marker modules in assisted breeding and / or identification of abdominal fat deposition traits in laying hens. The SNP molecular marker modules include SNPs rs731034862 and rs736850300; wherein rs731034862 is located at 6,729,430 bp on chromosome 20 and exhibits C / G polymorphism; and rs736850300 is located at 7,015,142 bp on chromosome 20 and exhibits G / A polymorphism. As a specific embodiment, the reference chicken genome for the SNP molecular marker modules is GRCg6a. As a specific embodiment, the abdominal fat deposition trait in laying hens includes abdominal fat weight and abdominal fat percentage.

[0021] In this embodiment of the invention, genome-wide association analysis was used to locate loci significantly associated with abdominal fat weight and abdominal fat percentage. The intersection of the two results was then analyzed, and two SNP loci, rs731034862 (location: 6,729,430 bp) and rs736850300 (location: 7,015,142 bp), on chromosome 20 were found to be significantly associated with abdominal fat weight and abdominal fat percentage at the genome level at 90 weeks of age.

[0022] The embodiments of the present invention show that laying hens with both the rs731034862 (GG) and rs736850300 (AA) genotypes on chromosome 20 have high abdominal fat weight and abdominal fat percentage, which are 39.19 g and 1.43% higher than the mean, respectively, and 46.85 g and 1.71% higher than individuals with the CC&GG genotypes, respectively.

[0023] Traditional marker-assisted selection (MAG) often relies on a single SNP locus. Single-marker selection fails to fully utilize the interaction effects between loci, limiting the accuracy of breeding selection and the potential for genetic progress. The SNP molecular marker module described in this invention includes two SNP loci for predicting abdominal fat deposition, offering greater accuracy than using a single marker.

[0024] This invention also provides a product for identifying abdominal fat deposition traits in laying hens. The product comprises at least one of the following reagents: reagents for nucleic acid extraction and amplification, reagents for detecting nucleic acid amplification products, reagents for constructing sequencing libraries, and reagents for sequencing. The amplified products, nucleic acid amplification products, or sequences used for sequencing contain SNPs rs731034862 and rs736850300. rs731034862 is located at 6,729,430 bp on chromosome 20 and exhibits C / G polymorphism; rs736850300 is located at 7,015,142 bp on chromosome 20 and exhibits G / A polymorphism. As a specific embodiment, the product includes primers for detecting the chicken rs731034862 and rs736850300 loci.

[0025] This invention also provides a method for identifying abdominal fat deposition traits in laying hens, comprising the following steps: extracting genomic DNA from the laying hen to be tested, and detecting the genotypes at the rs731034862 and rs736850300 loci; wherein rs731034862 is located at 6,729,430 bp on chromosome 20 and exhibits C / G polymorphism; and rs736850300 is located at 7,015,142 bp on chromosome 20 and exhibits G / A polymorphism. Individuals carrying both the GG genotype at the rs731034862 locus and the AA genotype at the rs736850300 locus are laying hens with high abdominal fat deposition. Individuals carrying both the CC genotype at the rs731034862 locus and the GG genotype at the rs736850300 locus are laying hens with low abdominal fat deposition.

[0026] This invention does not specifically limit the method for extracting genomic DNA from laying hens; any method commonly used in the art can be employed. The sample from the laying hen can be venous blood or feather samples with follicles for subsequent genomic DNA extraction. In a specific embodiment of this invention, the sample is obtained by venous blood collection, anticoagulated with EDTA or an anticoagulant, followed by lysis and protease digestion. Genomic DNA is then extracted using the CTAB method or a commercially available kit (such as the Qiagen DNeasy Blood & Tissue Kit). In this embodiment, the Loch Ness Red laying hen is used for illustration.

[0027] The genotype detection method described in this invention includes any one of whole-genome resequencing, PCR-sequencing genotyping, KASP genotyping, or time-of-flight mass spectrometry genotyping. In a specific embodiment of this invention, whole-genome resequencing is used for genotyping. A sequencing library (Illumina TruSeq Nano DNA LT Kit) is constructed, and paired-end sequencing (read length 150 bp) is performed on platforms such as Illumina HiSeq 2500 or NovaSeq. After FastQC quality control, adapter and low-quality sequence removal, the raw data is aligned to the GRCg6a reference genome using software such as BWA. Variance detection and genotyping are performed using workflows such as GATK to obtain genotype information for the target locus. A genotype is the combination of two alleles at the same gene locus. Usually, a gene locus has only two alleles, therefore there are usually three genotypes: "homozygous type I", "homozygous type II", and "heterozygous type".

[0028] The SNP molecular marker module described in this invention provides an efficient molecular tool for the early selection of abdominal fat traits in laying hens. On one hand, it can be used for the rapid selection of low-abdomen fat strains to improve feed utilization efficiency and overall health; on the other hand, it can also serve the targeted breeding of high-abdomen fat strains to meet the needs of specific research or product development. This technology helps to significantly shorten the breeding cycle, reduce the cost of phenotyping, and accelerate genetic progress, demonstrating significant economic benefits and application prospects in laying hen breeding practices.

[0029] This invention also provides a molecular marker-assisted breeding method for laying hens with high abdominal fat deposition, comprising the following steps: using the above method, detecting the genotypes of the rs731034862 and rs736850300 loci, screening and retaining individuals carrying both the GG genotype of the rs731034862 locus and the AA genotype of the rs736850300 locus for breeding to obtain laying hens with high abdominal fat deposition; wherein the rs731034862 is located at 6,729,430 bp on chromosome 20 and exhibits C / G polymorphism; the rs736850300 is located at 7,015,142 bp on chromosome 20 and exhibits G / A polymorphism.

[0030] This invention also provides a molecular marker-assisted breeding method for laying hens with low abdominal fat deposition, comprising the following steps: using the above method, detecting the genotypes of the rs731034862 and rs736850300 loci, screening and retaining individuals carrying both the CC genotype of the rs731034862 locus and the GG genotype of the rs736850300 locus for breeding to obtain laying hens with low abdominal fat deposition; wherein the rs731034862 is located at 6,729,430 bp on chromosome 20 and exhibits C / G polymorphism; the rs736850300 is located at 7,015,142 bp on chromosome 20 and exhibits G / A polymorphism.

[0031] This invention reveals a specific regulatory network of energy allocation in high-yielding laying hens under high-intensity, long-cycle reproductive loads, responding to the industry's urgent need to extend the laying cycle and improve later-stage production performance. It overcomes the limitations of applying conclusions from broiler breeds, and the research results possess clear industry relevance and physiological specificity. It provides a reliable molecular tool for regulating abdominal fat traits in laying hen breeding (whether for breeding high-abdomen fat strains for special purposes or for breeding low-abdomen fat strains to improve feed efficiency and health).

[0032] The method for calculating abdominal fat weight and abdominal fat percentage in this invention is as follows: After slaughtering the chicken, completely remove the fat around the abdomen and internal organs, weigh the abdominal fat using an electronic scale with an accuracy of not less than 0.1 g, and calculate the abdominal fat percentage (abdominal fat weight / pre-slaughter live weight × 100%).

[0033] Unless otherwise specified, the present invention does not have special requirements for the raw materials used in the preparation, and commercially available products well known to those skilled in the art can be used.

[0034] To further illustrate the present invention, the following detailed description, in conjunction with embodiments, of an SNP molecular marker module for assisted breeding of abdominal fat deposition traits in laying hens and its application, should not be construed as limiting the scope of protection of the present invention.

[0035] Example 1: Genome-wide association analysis (GWAS) determined the association between rs731034862 and rs736850300 loci and abdominal fat deposition. I. Measurement of abdominal fat deposition Materials: 686 90-week-old Loch Ness Red chickens.

[0036] Phenotypic determination: The pre-slaughter live weight and abdominal fat weight of each chicken were measured, and the abdominal fat percentage was calculated.

[0037] II. SNP Detection 1. Genomic DNA extraction Blood was collected from the vein of chicken wings, and after anticoagulation with an anticoagulant, genomic DNA was extracted, separated, and purified from the whole blood samples using the Tiangen Genomic DNA Extraction Kit (DP318). The specific operating procedure is as follows: (1) Sample processing: Add 190 μL of buffer GS to a 2 mL centrifuge tube, and then add 10 μL of thawed blood sample.

[0038] (2) Protease digestion: Add 20 μL of proteinase K solution, mix thoroughly, and incubate in a 56 ℃ water bath for 2.5 h with shaking to promote cell lysis and protein degradation.

[0039] (3) Lysis and denaturation: Add 200 μL buffer GB, mix thoroughly, and let stand at 70 °C for 10 min to ensure nucleic acid release.

[0040] (4) DNA precipitation: Add 200 μL of anhydrous ethanol and vortex for 15 s.

[0041] (5) DNA adsorption: Place the adsorption column (CB3) in the collection tube, transfer all lysis buffer and precipitate into the adsorption column, centrifuge at 12,000 rpm for 30 s to remove waste liquid, and put the adsorption column back into the collection tube.

[0042] (6) Column cleaning: Add 500 μL of buffer GD to the adsorption column, centrifuge at 12,000 rpm for 30 s, discard the waste liquid, and put the adsorption column back into the collection tube.

[0043] (7) DNA washing: Add 600 μL of washing buffer PW to the adsorption column, centrifuge at 12,000 rpm for 30 s, discard the waste liquid, and put the adsorption column back into the collection tube.

[0044] (8) Repeat washing: Repeat step (7).

[0045] (9) Remove residual liquid: Centrifuge at 12,000 rpm for 2 min to remove residual liquid from the collection tube, then remove the adsorption column and let it stand at room temperature for 10 min to allow the column membrane to dry completely.

[0046] (10) DNA elution: Transfer the adsorption column CB3 to a new 1.5 mL centrifuge tube, add 80 μL of elution buffer TE to the center of the membrane, let it stand at room temperature for 2 min, centrifuge at 12,000 rpm for 2 min, and collect the elution buffer, which is the extracted genomic DNA.

[0047] (11) DNA quality testing: The concentration and purity of DNA were determined using a NanoDrop-2000 nucleic acid analyzer to ensure that the sample showed a single peak and an OD value of [missing information]. 260 / OD 280 The ratio was between 1.7 and 1.9. DNA integrity was assessed using 1% agarose gel electrophoresis to determine the presence of degradation or protein contamination.

[0048] 2. Genotyping After quality testing of the genomic DNA, 686 qualified samples were retained for subsequent whole-genome resequencing. In the library construction phase, a 500 bp insert fragment was first obtained by PCR amplification, and a sequencing library was constructed using the TruSeq Nano DNA LT Library Preparation Kit (Illumina, CA, USA). Whole-genome resequencing was performed using an Illumina HiSeq 2500 sequencer (Illumina, Inc., San Diego, CA, USA), generating 150 bp paired-end sequencing data. To ensure data quality, FastQC software (http: / / www.bioinformatics.babraham.ac.uk / projects / fastqc / ) was used to remove adapter contamination sequences, low-quality sequences, and sequences with an N base content exceeding 5%. After alignment (using the chicken sixth edition reference genome GRCg6a, https: / / ftp.ensembl.org / pub / release-106 / fasta / gallus_gallus / ), deduplication, variant detection, and quality control, a total of 5,904,820 high-quality SNPs on 32 chromosomes from 686 individuals were obtained for subsequent genetic analysis.

[0049] III. Genome-wide association analysis of abdominal fat deposition traits in hens Statistical analysis was performed using the mixed linear model of the genome-wide efficient mixed-model association (GEMMA) statistical analysis software. For details, please refer to: "Zhou X, Stephens M. Genome-wide efficient mixed-model analysis for association studies[J]. Nature Genetics,2012, 44(7): 821-824".

[0050] The statistical analysis model is as follows: ; y This represents the corrected phenotypic value; W Represents the covariate matrix; α These are the coefficients and intercept vectors corresponding to the fixed effects; x Represents SNP genotype; β SNP effects representing corresponding phenotypes; μ Represents a vector of random multigene effects; the covariance structure follows... μ ~N(0, KVg ), K To construct the kinship matrix, Vg It is the additive variance of multiple genes; ε This represents the residual effect.

[0051] The significance of the association between SNPs and phenotypes was assessed using a likelihood ratio test. (r...) 2 A threshold of 0.2 was used to screen independent loci. The number of valid tests was calculated using the simpleM package in R (https: / / github.com / LTibbs / SimpleM), resulting in 150,802 valid tests. The genome-wide significance and potential significance thresholds were determined to be 3.32 × 10⁻⁶. -7 (0.05 / 150802) and 6.63×10 -6 (1 / 150802).

[0052] Genome-wide association analysis (GWAS) was used to locate loci significantly associated with abdominal fat weight and abdominal fat percentage, and the intersection of the two results was taken. The results showed that 35 SNPs on chromosome 20, from 6,729,430 bp to 7,183,732 bp, were significantly associated with both abdominal fat weight and abdominal fat percentage at 90 weeks of age. These included two loci: rs731034862 (location: 6,729,430 bp) and rs736850300 (location: 7,015,142 bp).

[0053] Example 2: Validation of the association between the C / G genotype at rs731034862 locus and the G / A genotype combination at rs736850300 locus in laying hens and abdominal fat deposition. I. Measurement of abdominal fat deposition Materials: Group 1, 686 90-week-old Loch Ness Red chickens.

[0054] Phenotypic determination: The pre-slaughter live weight and abdominal fat weight of each chicken were measured, and the abdominal fat percentage was calculated.

[0055] II. SNP Detection 1. Genomic DNA extraction Blood was collected from the wing veins of chickens, anticoagulated with an anticoagulant, lysed, and digested with proteases. Genomic DNA was extracted using the CTAB method via the DNeasy Blood & Tissue Kit (Qiagen, MD, USA). The concentration and quality of the DNA were determined using a NanoDrop2000 Spectrophotometer (Thermo Fisher Scientific, MA, USA), and quantification was performed using Qubit 1X dsDNA HS Assay Kits (Invitrogen, MA, USA).

[0056] 2. Genotype determination Genomic DNA was collected from each chicken to be tested, and a 500 bp insert fragment was obtained by PCR amplification. Sequencing libraries were then constructed using the TruSeq NanoDNA LT Library Preparation Kit (Illumina, CA, USA). Sequencing data were generated using an Illumina HiSeq 2500 sequencer (Illumina, Inc., San Diego, CA, USA). Subsequent filtering, alignment, and filling processes were performed to obtain the genotypes for all loci in each individual.

[0057] The results are shown in Tables 1 and 2. Table 1 shows that the CC genotype was the dominant genotype in the experimental population at the rs731034862 locus. Table 2 shows that the GG genotype was the dominant genotype in the experimental population at the rs736850300 locus.

[0058] Table 1. C / G genotype distribution at rs731034862 locus

[0059] Table 2. Distribution of G / A genotypes at the rs736850300 locus in laying hen populations.

[0060] III. Association Analysis between Genotype Combinations and Abdominal Fat Deposition The genotypes of the two molecular markers rs731034862 and rs736850300 were combined, and the abdominal fat weight and abdominal fat percentage under different combinations were statistically analyzed. The results are shown in Table 3. As can be seen from Table 3, laying hens with both the rs731034862 (GG) and rs736850300 (AA) genotypes on chromosome 20 have high abdominal fat weight and abdominal fat percentage, which are 39.19 g and 1.43% higher than the mean, respectively, and 46.85 g and 1.71% higher than individuals with the CC & GG genotypes, respectively.

[0061] Table 3. Abdominal fat deposition in individuals with different genotype combinations.

[0062] The GWAS results for abdominal fat weight and abdominal fat percentage were plotted using the CMplot package in R. Potentially significant sites and rs731034862 and rs736850300 were highlighted. Figure 1 China A and Figure 1 The results showed that both traits had extremely strong significant signals on chromosome 20, corresponding to two core SNPs: rs731034862 and rs736850300, with a -log 10 (P) The significant thresholds for the whole genome (red dashed line) are well above the threshold, indicating that these two loci are major candidate sites for regulating abdominal fat deposition in laying hens. Several weaker association peaks were also observed on other chromosomes, suggesting that abdominal fat traits are regulated by major loci plus multiple minor loci. The significant loci for abdominal fat weight and abdominal fat percentage highly overlap, indicating that they share core genetic regulatory loci.

[0063] To investigate the specific effects of the rs731034862 and rs736850300 genotype combinations on abdominal fat deposition in laying hens, the genotypes at both loci were extracted from all individuals and categorized according to their combinations (a total of 8 combinations are shown in Table 3). Abdominal fat weight (g) and abdominal fat percentage (%) were calculated for each combination, and box plots were generated using the ggplot2 package in R. Figure 1 C and Figure 1 (D).

[0064] The results showed that abdominal fat weight ( Figure 1 (C) Significant differences in abdominal fat weight were observed among different genotype combinations, with the GG&AA combination showing the highest abdominal fat weight and the CC&GG combination showing the lowest, exhibiting a clear gradient effect. Abdominal fat percentage ( Figure 1 (D) The trend is highly consistent with abdominal fat weight, with the GG&AA combination having the highest abdominal fat percentage and the CC&GG combination having the lowest, indicating that genotype combinations have a consistent regulation of abdominal fat deposition. The dispersion of the box plot suggests that there are still phenotypic variations among individuals within the same genotype combination, indicating that environmental factors or other minor genes also participate in the regulation of abdominal fat traits.

[0065] In summary, the major-effect loci are located as follows: rs731034862 and rs736850300 on chromosome 20 are key major-effect SNPs regulating abdominal fat weight and abdominal fat percentage in laying hens, and can serve as core targets for molecular markers. Genotypic combination effects: Different combinations of the two SNPs have significant regulatory effects on abdominal fat traits; CC&GG represents a low-fat combination, and GG&AA represents a high-fat combination, providing clear molecular marker combinations for breeding low-fat laying hen breeds. Consistency of trait regulation: The GWAS signals and genotypic effects of abdominal fat weight and abdominal fat percentage are highly consistent, indicating that they are regulated by the same core genetic network and can be simultaneously bred using the same set of molecular markers.

[0066] Breeding application suggestion: Molecular marker-assisted selection (MAS) can be used to screen individuals carrying the CC&GG genotype to breed laying hen strains with less abdominal fat deposition and higher feed conversion rates. Further, precise mapping of significant regions on chromosome 20 can be performed to clone functional genes regulating abdominal fat deposition and elucidate the molecular mechanisms.

[0067] Example 3: Population validation of abdominal fat deposition in laying hens with the combination of C / G genotype at rs731034862 locus and G / A genotype at rs736850300 locus. I. Measurement of abdominal fat deposition Materials: Group 2, 248 100-week-old Loch Ness Red chickens.

[0068] Phenotypic determination: The pre-slaughter live weight and abdominal fat weight of each chicken were measured, and the abdominal fat percentage was calculated.

[0069] II. SNP Detection 1. Genomic DNA extraction Blood was collected from the wing veins of chickens, anticoagulated with an anticoagulant, lysed, and digested with proteases. Genomic DNA was extracted using the CTAB method via the DNeasy Blood & Tissue Kit (Qiagen, MD, USA). The concentration and quality of the DNA were determined using a NanoDrop2000 Spectrophotometer (Thermo Fisher Scientific, MA, USA), and quantification was performed using Qubit 1X dsDNA HS Assay Kits (Invitrogen, MA, USA).

[0070] 2. Genotype determination Genomic DNA was collected from each chicken to be tested, and a 500 bp insert fragment was obtained by PCR amplification. Sequencing libraries were then constructed using the TruSeq NanoDNA LT Library Preparation Kit (Illumina, CA, USA). Sequencing data were generated using an Illumina HiSeq 2500 sequencer (Illumina, Inc., San Diego, CA, USA). Subsequent filtering, alignment, and filling processes were performed to obtain the genotypes for all loci in each individual.

[0071] The results are shown in Tables 4 and 5. Table 4 shows that the CC genotype was the dominant genotype in the experimental population at the rs731034862 locus. Table 5 shows that the GA genotype was the dominant genotype in the experimental population at the rs736850300 locus.

[0072] Table 4. C / G genotype distribution at rs731034862 locus

[0073] Table 5. Distribution of G / A genotypes at the rs736850300 locus in laying hen populations.

[0074] III. Association Analysis between Genotype Combinations and Abdominal Fat Deposition The genotypes of the two molecular markers rs731034862 and rs736850300 were combined, and the abdominal fat weight and abdominal fat percentage under different combinations were statistically analyzed. The results are shown in Table 6. As can be seen from Table 6, laying hens with both the rs731034862 (GG) and rs736850300 (AA) genotypes on chromosome 20 have high abdominal fat weight and abdominal fat percentage.

[0075] High abdominal fat combination: The average abdominal fat weight of individuals in the GG & AA groups was as high as 122.93 g, with an abdominal fat percentage of 5.80%, significantly higher than the population average (abdominal fat weight 89.90 g, abdominal fat percentage 4.31%). Compared with the low abdominal fat combination CC & GG (abdominal fat weight 83.98 g, abdominal fat percentage 3.96%), the abdominal fat weight increased by 38.95 g (+46.4%), and the abdominal fat percentage increased by 1.84% (+46.5%). Low abdominal fat combination: The CC & GA groups had the largest number of individuals (n=47), with an abdominal fat weight of 79.89 g and an abdominal fat percentage of 3.73%, both the lowest among all combinations.

[0076] Table 6. Abdominal fat deposition in individuals with different genotype combinations.

[0077] Comparative Example (1) Existing genetic and molecular mechanism studies mainly focus on broiler chickens, which are designed for rapid growth. Laying hens, however, endure high-intensity metabolic stress related to egg production, and their energy allocation and fat deposition regulation mechanisms differ, making it difficult to directly apply conclusions from broiler models. (Shen, L., Bai, X., Zhao, L. et al. Integrative 3D genomics with multi-omics analysis and functional validation of genetic regulatory mechanisms of abdominal fat deposition in chickens. Nature Communications, 15, 9274 (2024).) (2) Traditional marker-assisted selection is mostly based on a single SNP locus (Guo J, Qu L, Shao D, Wang Q, Li Y, Dou T, Wang X, Hu Y, Tong H. Genetic Architecture of Abdominal Fat Deposition Revealed by a Genome-Wide Association Study in the Laying Chicken.Genes (Basel). 2023 Dec 20;15(1):10.). If screening is based solely on the GG genotype at rs731034862 or the AA genotype at rs736850300, the selected individuals may show some improvement in abdominal fat deposition, but it does not reach the effect level of the optimal combination of two loci (GG&AA) revealed in this invention. Single marker selection fails to fully utilize the interaction effects between loci, limiting the accuracy of breeding selection and the potential for genetic progress.

[0078] Advantages of this invention: Higher accuracy: This invention proposes for the first time a molecular module consisting of two SNP sites and identifies its optimal genotype combination (the optimal combination is GG&AA) for predicting abdominal fat deposition, which is more accurate than using a single marker.

[0079] Improved breeding efficiency: Based on this module, assisted selection can accurately identify individuals with high or low abdominal fat deposition potential at an early stage (before slaughter), accelerating the breeding process and reducing testing costs.

[0080] Clear application value: Unlike previous studies primarily based on broiler models focused on rapid growth, this invention aims to reveal the specific regulatory network of energy allocation in high-producing laying hens under high-intensity, long-cycle reproductive loads. It addresses the industry's urgent need to extend the laying cycle and improve later-stage production performance, overcoming the limitations of using broiler conclusions. The research results possess clear industry relevance and physiological specificity. It provides a reliable molecular tool for regulating abdominal fat traits in laying hen breeding (whether for selecting high-abdomen fat strains for special purposes or for selecting low-abdomen fat strains to improve feed efficiency and health).

[0081] Therefore, this invention provides the application of reagents for identifying SNP molecular marker modules in assisted breeding and / or identification of abdominal fat deposition traits in laying hens. The SNP molecular marker modules include SNPs rs731034862 and rs736850300; wherein rs731034862 is located at 6,729,430 bp on chromosome 20 and exhibits C / G polymorphism; and rs736850300 is located at 7,015,142 bp on chromosome 20 and exhibits G / A polymorphism. This invention discloses for the first time that, after modularly combining the above two loci, a specific genotype combination (rs731034862-GG / rs736850300-AA, i.e., GG&AA type) shows a significant positive correlation with abdominal fat weight and abdominal fat percentage in laying hens, with phenotypic values ​​significantly superior to individuals with other genotypes. Compared to traditional marker-assisted selection methods that rely on single molecular markers, the modular strategy employed in this invention more fully reflects the genetic interactions between multiple loci, significantly improving the accuracy and stability of predicting abdominal fat deposition traits and overcoming the limitations of single marker effects and insufficient selection potential. This combination of molecular markers is reported for the first time and possesses clear innovation. At the application level, this module provides an efficient molecular tool for early selection of abdominal fat traits in laying hens. On the one hand, it can be used for rapid breeding of low-abdominal-fat strains to improve feed utilization efficiency and overall health; on the other hand, it can also serve the targeted breeding of high-abdominal-fat strains to meet the needs of specific research or product development. This technology helps to significantly shorten the breeding cycle, reduce phenotypic testing costs, and accelerate genetic progress, demonstrating significant economic benefits and application prospects in laying hen breeding practices.

[0082] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. The application of reagents for identifying SNP molecular marker modules in assisted breeding and / or identification of abdominal fat deposition traits in laying hens, characterized in that, The SNP molecular marker module includes SNPs: rs731034862 and rs736850300; The rs731034862 is located at 6,729,430 bp on chromosome 20 and exhibits C / G polymorphism; the rs736850300 is located at 7,015,142 bp on chromosome 20 and exhibits G / A polymorphism.

2. The application according to claim 1, characterized in that, The reference chicken genome for the SNP molecular marker module is GRCg6a.

3. The application according to claim 1, characterized in that, The abdominal fat deposition characteristics of laying hens include abdominal fat weight and abdominal fat percentage.

4. A product for identifying the characteristics of abdominal fat deposition in laying hens, characterized in that, The product includes at least one of the following reagents: reagents for nucleic acid extraction and amplification, reagents for detecting nucleic acid amplification products, reagents for constructing sequencing libraries, and reagents for sequencing. The amplified product, nucleic acid amplification product, or sequence used for sequencing contains SNPs: rs731034862 and rs736850300. The rs731034862 is located at 6,729,430 bp on chromosome 20 and exhibits C / G polymorphism; the rs736850300 is located at 7,015,142 bp on chromosome 20 and exhibits G / A polymorphism.

5. The reagent according to claim 4, characterized in that, The product includes primers for detecting the rs731034862 and rs736850300 loci in chickens.

6. A method for identifying abdominal fat deposition characteristics in laying hens, characterized in that, Includes the following steps: Genomic DNA was extracted from the laying hens to be tested, and the genotypes at the rs731034862 and rs736850300 loci were detected. Individuals carrying both the GG genotype at the rs731034862 locus and the AA genotype at the rs736850300 locus are laying hens with high abdominal fat deposition. Individuals carrying both the CC genotype at the rs731034862 locus and the GG genotype at the rs736850300 locus are laying hens with low abdominal fat deposition. The rs731034862 is located at 6,729,430 bp on chromosome 20 and exhibits C / G polymorphism; the rs736850300 is located at 7,015,142 bp on chromosome 20 and exhibits G / A polymorphism.

7. The method according to claim 6, characterized in that, The genotype detection method includes any one of whole genome resequencing, PCR-sequencing typing, KASP typing, or time-of-flight mass spectrometry typing.

8. A molecular marker-assisted breeding method for laying hens with high abdominal fat deposition, characterized in that, Includes the following steps: Using the method described in claim 6 or 7, the genotypes of the rs731034862 locus and the rs736850300 locus are detected, and individuals carrying both the GG genotype at the rs731034862 locus and the AA genotype at the rs736850300 locus are screened and retained for breeding to obtain laying hens with high abdominal fat deposition. The rs731034862 is located at 6,729,430 bp on chromosome 20 and exhibits C / G polymorphism; the rs736850300 is located at 7,015,142 bp on chromosome 20 and exhibits G / A polymorphism.

9. A molecular marker-assisted breeding method for laying hens with low abdominal fat deposition, characterized in that, Includes the following steps: Using the method described in claim 6 or 7, the genotypes of the rs731034862 locus and the rs736850300 locus are detected, and individuals carrying both the CC genotype of the rs731034862 locus and the GG genotype of the rs736850300 locus are screened and retained for breeding to obtain laying hens with low abdominal fat deposition. The rs731034862 is located at 6,729,430 bp on chromosome 20 and exhibits C / G polymorphism; the rs736850300 is located at 7,015,142 bp on chromosome 20 and exhibits G / A polymorphism.

10. The method according to claim 6 or the molecular marker-assisted breeding method according to claim 8 or 9, characterized in that, The laying hens include the Los Angeles Red.

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