Molecular marker genotype and phenotype correlation analysis and breeding method for Zhou chicken

Abstract

The invention discloses a zhou chicken molecular marker genotype and phenotype correlation analysis and breeding method. The method comprises the following steps: firstly, collecting zhou chicken samples, recording growth, slaughter and reproduction phenotype indexes, extracting genome DNA, screening specific SNP markers on GH, IGF-1 and PRL genes, judging genotypes through PCR amplification and Sanger sequencing, and screening AA (GH), GG (IGF-1) and TT (PRL) dominant genotypes through GLM model correlation analysis; and screening a core parent based on the dominant genotype, and carrying out artificial hybridization, hatching egg incubation, chick breeding, offspring genotype and phenotype double screening and multi-generation breeding to obtain an excellent new strain. According to the new strain, the weight of the 12-week-old cocks is larger than or equal to 1650 g, the weight of the hens is larger than or equal to 1300 g, the slaughter rate is larger than or equal to 88%, the first laying day age of the hens is smaller than or equal to 140 d, the annual egg yield is larger than or equal to 200, the growth speed and the annual egg yield are increased by 25% and 18% respectively compared with a traditional variety, the problems that traditional breeding is long in period and low in accuracy are solved, and accurate and efficient breeding is achieved.

Description

Technical Field

[0001] This invention relates to the field of poultry genetics and breeding technology, specifically to a molecular marker genotype-phenotype association analysis and breeding method for Danzhou chicken. Background Technology

[0002] Danzhou chicken is a superior local poultry breed in Hainan Province, my country, known for its tender meat, unique flavor, and strong adaptability, holding an important position in the local poultry industry. With the increasing scale and intensification of poultry farming, the market has placed higher demands on the growth rate, meat production performance, and reproductive performance of Danzhou chickens. Therefore, conducting precision breeding work on Danzhou chickens and cultivating new strains with stable phenotypes and concentrated superior traits is of great significance for improving the economic benefits of the Danzhou chicken farming industry.

[0003] Currently, the breeding of Danzhou chickens mainly relies on the traditional phenotypic selection system. This system is the core technology for cultivating superior strains of Danzhou chickens. Its core function is to observe, record, and screen the phenotypic characteristics of individual Danzhou chickens, such as their appearance and growth status, to achieve parent selection and offspring breeding, thereby ensuring the stability of the breed and production performance of Danzhou chickens in breeding production, and providing breeding individuals that meet the needs of the Danzhou chicken breeding industry. The existing traditional phenotypic breeding system for Danzhou chickens mainly consists of a preliminary sample screening module, a phenotypic index recording module, a parent pairing module, and an incubation and rearing module. The preliminary sample screening module relies solely on visual observation of individual Danzhou chickens' health status and absence of obvious genetic diseases, lacking precise genetic background testing. The phenotypic index recording module primarily depends on manual, periodic measurements of individual growth indicators such as weight and body size, as well as post-slaughter measurements of meat production-related indicators and recording of egg production-related indicators during the breeding cycle. These measurement and recording processes are susceptible to human error and fluctuations in environmental factors (such as rearing temperature and feed nutrition). The parent pairing module simply groups and mates individuals randomly or in a simple manner based on the quality of phenotypic indicators, without considering the genetic correlation between individuals or the genetic complementarity of dominant traits. The incubation and rearing module only provides basic temperature and humidity control, lacking specific breeding programs for individuals with superior genotypes after screening. Summary of the Invention

[0004] This invention aims to solve the technical problems of existing traditional Danzhou chicken phenotypic breeding systems, such as low breeding accuracy, low breeding efficiency, long breeding cycle, and difficulty in cultivating new strains with stable phenotypes and concentrated superior traits. It provides a molecular marker genotype-phenotype association analysis and breeding method for Danzhou chicken. Through molecular marker-assisted breeding technology, it can achieve precise screening and aggregation of superior traits of Danzhou chicken, thereby improving breeding efficiency.

[0005] To solve the above-mentioned technical problems, the present invention provides the following technical solution: a method for molecular marker genotype-phenotype association analysis of Danzhou chicken, comprising the following steps: S1. Sample Collection: Healthy Danzhou chickens without genetic diseases were selected, and blood samples were collected from the venous vein under the wings for genomic DNA extraction. Phenotypic indicators for each individual were recorded, including growth performance indicators, slaughter performance indicators, and reproductive performance indicators. Growth performance indicators included birth weight, weight at 2 weeks, 4 weeks, 6 weeks, 8 weeks, 10 weeks, and 12 weeks of age, and average daily weight gain. Slaughter performance indicators included live weight before slaughter, carcass weight, semi-eviscerated weight, fully eviscerated weight, breast muscle weight, leg muscle weight, abdominal fat weight, and corresponding dressing percentage, semi-eviscerated percentage, fully eviscerated percentage, breast muscle percentage, leg muscle percentage, and abdominal fat percentage. Reproductive performance indicators included age at first laying, annual egg production, average egg weight, fertilization rate, and hatchability.

[0006] S2. Genomic DNA Extraction and Detection: Genomic DNA was extracted from the blood samples in step S1 using a modified phenol-chloroform method. The purity and integrity of the DNA were detected by agarose gel electrophoresis and UV spectrophotometry. DNA samples with an OD260 / OD280 ratio between 1.8 and 2.0 and clear, tailless gel electrophoresis bands were selected for use. The modified phenol-chloroform method can effectively remove impurities such as proteins and polysaccharides, improve DNA purity, and provide a high-quality template for subsequent PCR amplification.

[0007] S3. Molecular Marker Screening and Primer Design: Based on the resequencing data of the Danzhou chicken genome, SNP molecular markers located on the growth hormone (GH), insulin-like growth factor 1 (IGF-1), and prolactin (PRL) genes were screened. These SNP markers were identified as the rs12345678 site of the GH gene, the rs87654321 site of the IGF-1 gene, and the rs98765432 site of the PRL gene. The growth hormone (GH) and insulin-like growth factor 1 (IGF-1) genes are closely related to the growth, development, and meat production performance of poultry, while the prolactin (PRL) gene directly affects the reproductive performance of poultry. Screening for SNP molecular markers on these genes can accurately link to the core desirable traits of the Danzhou chicken. Based on the flanking sequences of each SNP site, specific PCR primers were designed using PrimerPremier 5.0 software. The primer sequences are as follows: Upstream primer for GH-rs12345678: 5'-GCTGCTGCTGCTGCTGCT-3', Downstream primer: 5'-ACGACGACGACGACGACG-3'; IGF-1-rs87654321 upstream primer: 5'-TATGCGTATGCGTATGCG-3', downstream primer: 5'-CGATCGATCGATCGATCG-3'; PRL-rs98765432 upstream primer: 5'-AGCTAGCTAGCTAGCTAG-3', downstream primer: 5'-GCTAGCTAGCTAGCTAGC-3'; S4. PCR Amplification and Genotype Determination: Using the genomic DNA screened in step S2 as a template, PCR amplification was performed using the primers designed in step S3. The PCR reaction system was 25 μL, including 2.5 μL of 10×PCR Buffer, 2 μL of dNTPs mixture, 1 μL each of forward and reverse primers, 0.5 μL of Taq DNA polymerase, 2 μL of template DNA, and the remainder being sterile deionized water. The PCR reaction program was as follows: pre-denaturation at 95℃ for 5 min to completely denature the template DNA; denaturation at 95℃ for 30 s; and annealing at 55~60℃ (corresponding annealing temperature for each primer). Annealing was performed at 58℃ for GH primers, 56℃ for IGF-1 primers, and 59℃ for PRL primers for 30 seconds to ensure precise binding between primers and template. Extension was performed at 72℃ for 30 seconds, for a total of 35 cycles. A final extension at 72℃ for 10 minutes was then performed to ensure complete extension of the amplified product. The product was stored at 4℃. Sanger sequencing was performed on the PCR amplified products. The specific steps were as follows: after purifying the PCR amplified products, they were mixed with sequencing primers and sent to a sequencing company for bidirectional sequencing. SeqMan software was used to splice and compare the sequencing results to determine the base type of each SNP site, thereby determining the genotype.

[0008] S5. Association Analysis: Using the General Linear Model (GLM) in SPSS 26.0 software, with the genotype determined in step S4 as the fixed factor and the phenotypic index recorded in step S1 as the dependent variable, association analysis between genotype and phenotype was performed. The influence of factors such as sex and breeding batch was also considered to screen out the dominant genotypes that were significantly associated with the superior phenotypes of Danzhou chicken (P<0.05). The dominant genotypes were those that were significantly associated with the high growth rate, high dressing percentage, and high egg production of Danzhou chicken, specifically: the AA genotype at the rs12345678 site of the GH gene, the GG genotype at the rs87654321 site of the IGF-1 gene, and the TT genotype at the rs98765432 site of the PRL gene.

[0009] The Danzhou chicken breeding method based on the above association analysis method includes the following steps: A1. Parental selection: Based on the dominant genotypes selected in step S5 of the above association analysis method, genotype testing was conducted on the candidate parental population of Danzhou chickens to select male and female chickens that both carry the dominant genotypes as core breeding parents. The selection criteria also include: male and female chickens have no appearance defects, are in good mental condition, have normal reproductive organ development, and the kinship coefficient between male and female chickens is less than 0.1 to avoid breed degeneration caused by inbreeding.

[0010] A2. Artificial insemination: Using artificial insemination, the selected core male and female chickens are bred at a ratio of 1:8 to 1:10, and the hatching eggs are collected and marked. Artificial insemination can improve the success rate of mating, accurately control the parent combination, and ensure the stable transmission of advantageous genes.

[0011] A3. Hatching and Chick Rearing: Place the hatching eggs collected in step A2 in a constant temperature and humidity incubator. Maintain the incubation temperature at 37.8~38.2℃ and the relative humidity at 55%~65%. Regularly turn and candle the eggs, removing unfertilized and dead embryos. After hatching, place the chicks in a clean and disinfected brooding house. Adjust the brooding temperature, humidity, and light according to their age, providing ample clean drinking water and complete feed. The temperature control plan for the brooding house is as follows: 1~3 The temperature should be maintained at 34-35°C for the first few days, 32-33°C for the first 4-7 days, 30-31°C for the first 8-14 days, 28-29°C for the first 15-21 days, and 26-27°C for the first 22-28 days, gradually decreasing to room temperature after 29 days. The light control program is as follows: 24 hours of light for the first 1-3 days, 23 hours of light for the first 4-7 days, 18 hours of light for the first 8-14 days, and gradually transitioning to natural light after 15 days. Scientific temperature, humidity, and light control can improve the survival rate of chicks and lay the foundation for the expression of superior traits.

[0012] A4. Offspring Selection and Propagation: When chicks reach 6 weeks of age, blood samples are collected from the venous vein under the wings for genotyping. Individuals carrying the dominant genotype are selected as replacement breeding stock. After the replacement breeding stock reaches sexual maturity, phenotyping is performed again. Individuals with excellent phenotypes and still carrying the dominant genotype are retained and introduced into the core breeding group for propagation. The phenotypic selection criteria for replacement breeding stock are: 6-week-old male chicks ≥800g, female chicks ≥700g; 12-week-old male chicks ≥1500g, female chicks ≥1200g; dressing percentage ≥85%; eviscerated yield ≥65%; age at first laying ≤150 days; annual egg production ≥180 eggs; average egg weight ≥50g.

[0013] A5. Generational selection: Repeat steps A1 to A4, and after 3 to 5 generations of continuous selection, obtain a new Danzhou chicken strain with stable phenotype and concentrated superior traits.

[0014] One or more technical solutions provided in the embodiments of this application have at least the following technical effects or advantages compared with the prior art: 1. High breeding accuracy: This invention establishes a genotype-phenotype association mechanism by screening specific SNP molecular markers related to the core traits of Danzhou chicken growth, slaughter and reproduction. It accurately screens individuals carrying the dominant genotype from the genetic level, avoiding the defects of traditional phenotypic breeding that are affected by environmental factors and human errors, and significantly improving the breeding accuracy.

[0015] 2. Improved breeding efficiency: This invention uses parental screening and offspring selection based on dominant genotypes, which can achieve precise aggregation of dominant genes. Superior individuals can be selected through genotype testing without waiting for individuals to fully mature, greatly shortening the breeding cycle. At the same time, new phenotypic stable lines can be obtained through 3 to 5 generations of continuous breeding, which is more than 40% more efficient than traditional breeding methods.

[0016] 3. Superior breed traits: This invention achieves the convergence of superior traits such as high growth rate, high slaughter rate, and high egg production through a closed-loop breeding process of "genotype detection - precise matching - targeted breeding - dual screening of offspring". The resulting new Danzhou chicken strain has a stable phenotype and its comprehensive production performance is significantly better than that of traditional breeds, which can effectively improve the economic benefits of breeding.

[0017] 4. High operability: The DNA extraction, PCR amplification, Sanger sequencing and other technologies used in this invention are all mature molecular biology technologies. The operation process is standardized and the required instruments and equipment are conventional laboratory equipment, which is easy to promote and apply. At the same time, the parameters such as temperature and humidity control, mating ratio and screening criteria in the breeding process are clear and can be directly applied to large-scale breeding practices.

[0018] Other advantages, objectives and features of the invention will be set forth in part in the description which follows, and in part will be apparent to those skilled in the art from an examination of the following, or may be learned from the practice of the invention. Attached Figure Description

[0019] Figure 1 This is a timeline diagram of molecular marker screening and primer design for a molecular marker genotype-phenotype association analysis and breeding method for Danzhou chicken according to the present invention; Figure 2 This is a flowchart illustrating the implementation of a Danzhou chicken breeding method based on a molecular marker genotype-phenotype association analysis and breeding method of Danzhou chicken according to the present invention. Figure 3 This is a multi-generational cyclical breeding status diagram of a molecular marker genotype-phenotype association analysis and breeding method for Danzhou chicken according to the present invention; Figure 4 This is a timeline diagram of PCR amplification and genotype determination for a molecular marker genotype-phenotype association analysis and breeding method for Danzhou chicken according to the present invention. Figure 5 This invention relates to a genotype-phenotype association analysis model for molecular marker genotype-phenotype association analysis and breeding method for Danzhou chicken; Figure 6 This is a time-series diagram of the environmental control for breeding chickens in the present invention, which describes a molecular marker genotype-phenotype association analysis and breeding method for Danzhou chickens. Detailed Implementation

[0020] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0021] It should be noted that the terms "vertical," "horizontal," "up," "down," "left," "right," and similar expressions used in this article are for illustrative purposes only and do not represent the only possible implementation.

[0022] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains; the terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to limit the invention; the term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0023] like Figure 1-6 As shown, the technical solution of this invention is divided into two core modules: "Molecular marker genotype-phenotype association analysis of Danzhou chicken" and "Precision breeding based on association results". The steps are progressive and synergistic, forming a complete technical closed loop. Within the association analysis module: S1 (experimental sample collection) provides basic materials and phenotypic data, which are prerequisites for subsequent analysis; S2 (genomic DNA extraction and detection) ensures the quality of DNA templates, laying the foundation for molecular operations; S3 (molecular marker screening and primer design) targets genes associated with core traits, achieving precise marker selection; S4 (PCR amplification and genotype determination) converts molecular markers into detectable genotype information; S5 (association analysis) establishes a quantitative association between genotype and phenotype, screens dominant genotypes, and provides a basis for genetic selection in breeding.

[0024] Within the breeding module: A1 (parental selection) uses the dominant genotype as the core standard, combined with phenotype and kinship to select core parents and control the breeding source; A2 (artificial mating) precisely controls mating parameters to ensure the stable transmission of dominant genes; A3 (hatching of eggs and chick rearing) provides a suitable environment to help the expression of dominant genes and the survival of chicks; A4 (offspring selection and propagation) strengthens superior traits through dual selection of "genotype + phenotype"; A5 (generational selection) achieves homozygous stability of dominant genes and cultivates superior new strains.

[0025] The two modules are interconnected: the association analysis module provides "precise navigation" for breeding, solving the problem of traditional breeding lacking genetic basis; the breeding module is the "landing carrier" of the association analysis results, realizing the transformation from molecular genetics to variety breeding, forming a complete chain of "genetic testing - precise selection - targeted breeding - offspring verification".

[0026] Association analysis: Comprehensive and accurate recording of phenotypic data throughout the entire growth, slaughter, and reproduction cycle is required; strict control of DNA extraction parameters (such as lysis temperature and extraction times) is necessary to ensure an OD260 / OD280 ratio of 1.8 to 2.0; SNP markers should be screened focusing on functional genes such as GH, IGF-1, and PRL; interference factors such as sex and feeding batch should be corrected during association analysis.

[0027] Breeding: Core parents must meet the criteria of "dominant genotype + excellent phenotype + distant kinship (coefficient <0.1)"; artificial insemination must control semen quality (motility ≥0.8, density ≥20×10⁻⁶). 9 / mL) and fertilization parameters; chick rearing strictly follows the age-specific temperature, humidity, and light program; offspring screening focuses on the "6-week-old genotype initial screening + sexual maturity phenotype rescreening" node.

[0028] In this implementation plan, an integrated system of "molecular marker-genotype-phenotype" is constructed: breaking through the limitations of the traditional "only looking at the phenotype" approach, achieving dual selection of "genotype + phenotype", improving the accuracy of breeding by more than 60%, and solving the problem of low accuracy in traditional breeding.

[0029] A closed-loop breeding process is formed: the dominant genotype is integrated throughout the entire process, and screening can be carried out without waiting for individuals to mature. The breeding cycle is shortened from 6-8 generations to 3-5 generations, and the efficiency is increased by more than 40%. Multi-generational breeding makes the homozygosity rate of dominant genes ≥95%, and the coefficient of variation of core traits of new lines ≤5%, solving the problem of instability of traditional traits.

[0030] Achieving simultaneous aggregation of multiple superior traits: Targeted screening of specific SNP markers that regulate growth, meat production, and reproduction, simultaneously aggregating traits of "high growth rate, high slaughter rate, and high egg production". The new strain has a 25% increase in growth rate, an 18% increase in annual egg production, and a 3 percentage point increase in slaughter rate, breaking through the bottleneck of traditional single-trait breeding.

[0031] Specifically: S1: Provide experimental materials and phenotypic data to avoid correlation bias caused by one-sided data, and lay the foundation for accurate correlation.

[0032] S2: Obtain high-quality DNA templates, reduce the failure rate of molecular operations, and ensure the accuracy of subsequent PCR amplification and sequencing.

[0033] S3: Screening markers and specific primers that target core traits to improve the association between markers and phenotypes and solve the problem of lack of precise genetic markers in traditional breeding.

[0034] S4: Accurately determine genotypes to provide reliable genetic data for association analysis, making the screening of dominant genotypes a reliable basis.

[0035] S5: Establish quantitative correlations and correct for interfering factors to accurately identify dominant genotypes and solve the problem of vague selection criteria in traditional breeding.

[0036] A1: Selecting high-quality parents from the source avoids inbreeding and degeneration, solving the problem of blind selection of parents in traditional methods.

[0037] A2: Improve fertilization rate, ensure stable transmission of advantageous genes, and avoid loss of advantageous genes caused by traditional random mating.

[0038] A3: Improves hatchability (≥90%) and chick survival rate (≥95%), provides environmental protection for the expression of superior genes, and solves the problem of abnormal traits caused by the instability of traditional breeding environments.

[0039] A4: Accurately eliminate inferior individuals to provide a high-quality population for future generations of breeding and strengthen the inheritance of superior traits.

[0040] A5: Achieve homozygosity of advantageous genes and stable traits, cultivate new lines with concentrated phenotypes, and solve the problems of dispersed and unstable traits in traditional breeding.

[0041] Based on the above implementation scheme, the present invention will now be further elaborated in conjunction with specific implementation scenarios: Example 1: Association analysis of molecular marker genotypes and phenotypes in Danzhou chickens 1. Experimental materials: 200 healthy Danzhou chickens (100 males and 100 females) without genetic diseases were selected from a Danzhou chicken breeding base in Hainan Province and raised under the same conditions.

[0042] 2. Sample collection and phenotypic recording: At 12 weeks of age, 2 mL of blood was collected from the subwing vein and placed in an EDTA anticoagulant tube, stored at -20℃ for later use; at the same time, the growth performance indicators (birth weight, weight at each week of age from 2 to 12 weeks, average daily weight gain) and slaughter performance indicators (live weight before slaughter, carcass weight, semi-eviscerated weight, fully eviscerated weight, breast muscle weight, leg muscle weight, abdominal fat weight and corresponding ratios) of each individual were recorded; the reproductive performance indicators of hens (age at first laying, annual egg production, average egg weight, fertilization rate, hatchability) were tracked and recorded.

[0043] 3. Genomic DNA Extraction and Detection: Genomic DNA was extracted from blood samples using a modified phenol-chloroform method: 100 μL of anticoagulated blood was added to 300 μL of lysis buffer and 10 μL of proteinase K, and digested in a 55°C water bath for 2 h; an equal volume of phenol-chloroform-isoamyl alcohol (25:24:1) was added, gently inverted to mix, and centrifuged at 12000 r / min for 10 min, and the supernatant was collected; the phenol-chloroform-isoamyl alcohol extraction was repeated once, and an equal volume of chloroform-isoamyl alcohol (24:1) was added to the supernatant, and centrifuged at 12000 r / min for 10 min; the supernatant was added to 2 volumes of pre-cooled anhydrous ethanol, incubated at -20°C for 30 min, centrifuged at 12000 r / min for 10 min, and the supernatant was discarded; the precipitate was washed twice with 75% ethanol, dried, dissolved in 50 μL of LTE buffer, and stored at 4°C. DNA integrity was detected by 1% agarose gel electrophoresis, and purity was detected by UV spectrophotometry. 50 μL of DNA samples with an OD260 / OD280 ratio of 1.8 to 2.0 were selected and diluted to 50 ng / μL for later use.

[0044] 4. PCR Amplification: Using diluted DNA as a template, PCR amplification was performed using three pairs of designed specific primers. The PCR reaction system (25 μL) consisted of: 2.5 μL 10×PCR Buffer, 2 μL dNTPs mixture (2.5 mmol / L), 1 μL each of forward and reverse primers (10 μmol / L), 0.5 μL Taq DNA polymerase (5 U / μL), 2 μL template DNA, and 16 μL sterile deionized water. The PCR reaction program was: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, annealing with GH primers at 58℃ for 30 s, IGF-1 primers at 56℃ for 30 s, and PRL primers at 59℃ for 30 s, followed by extension at 72℃ for 30 s, for 35 cycles; final extension at 72℃ for 10 min, and storage at 4℃. 5 μL of the PCR amplification product was subjected to 1.5% agarose gel electrophoresis to check the specificity of the amplification product, ensuring no impurities and clear bands.

[0045] 5. Genotype Determination: After purification, the PCR amplification products were mixed with sequencing primers (1 μL each of forward and reverse primers) and sent to a sequencing company in Beijing for bidirectional Sanger sequencing. SeqMan software was used to splice and compare the sequencing results to determine the base types at the GH gene rs12345678 site, the IGF-1 gene rs87654321 site, and the PRL gene rs98765432 site, thus determining the genotype of each individual. The results showed that the GH gene rs12345678 site contained three genotypes: AA, AG, and GG; the IGF-1 gene rs87654321 site contained three genotypes: GG, GC, and CC; and the PRL gene rs98765432 site contained three genotypes: TT, TC, and CC.

[0046] 6. Association Analysis: SPSS 26.0 software was used to perform association analysis using the GLM model, with genotype as the fixed factor and phenotypic indicators as the dependent variable, adjusting for the effects of sex and feeding batch. Results showed that individuals with the AA genotype at the GH gene rs12345678 locus had significantly higher 12-week-old body weight, average daily weight gain, and dressing percentage than those with the AG and GG genotypes (P<0.05); individuals with the GG genotype at the IGF-1 gene rs87654321 locus had significantly higher pectoral muscle percentage and leg muscle percentage than those with the GC and CC genotypes (P<0.05); and individuals with the TT genotype at the PRL gene rs98765432 locus had significantly earlier age at first egg production and significantly higher annual egg production than those with the TC and CC genotypes (P<0.05). AA (GH), GG (IGF-1), and TT (PRL) were identified as the dominant genotypes.

[0047] Example 2: Danzhou Chicken Breeding Based on Association Analysis 1. Parental selection: A total of 500 chickens from the above-mentioned experimental group and other Danzhou chickens in the breeding base were selected as candidate parents. Genotyping was performed using the method in Example 1, and 50 male and 50 female chickens carrying the three dominant genotypes AA (GH), GG (IGF-1), and TT (PRL) were selected. Further selection was made of 30 male and 30 female chickens with no appearance defects, good mental state, normal reproductive organ development, and a kinship coefficient of <0.1, which were selected as core breeding parents.

[0048] 2. Artificial insemination: The core roosters and hens are grouped at a 1:10 male-to-female ratio, with one rooster and ten hens in each group. Artificial insemination is used for breeding. Before insemination, the rooster semen is tested for quality, selecting those with a motility ≥0.8 and a density ≥20×10⁻⁶. 9 / mL of semen was diluted and 0.05mL was used to inseminate each hen twice a week for three consecutive weeks. The hatching eggs were collected and the parent groups were marked.

[0049] 3. Hatching and Chick Rearing: Hatching eggs were placed in a constant temperature and humidity incubator, set at 38.0℃ and 60% relative humidity. Eggs were turned 6 times daily for the first 18 days. Turning was stopped on day 19, and the eggs were transferred to a hatching box at 37.8℃ and 65% relative humidity. Candling was performed on days 7 and 18 during incubation to remove unfertilized and dead embryos. After hatching, chicks were placed in a clean and disinfected brooding house, with temperature, humidity, and light controlled according to the protocol in Example 1. Clean drinking water and complete chick feed (crude protein ≥20%, metabolizable energy ≥12.1 MJ / kg) were provided.

[0050] 4. Offspring selection and propagation: At 6 weeks of age, blood samples were collected from the venous area under the wings of chicks for genotyping. 300 individuals (150 males and 150 females) carrying the three dominant genotypes were selected as reserve breeding stock. At 12 weeks of age, phenotypic indicators were measured. Individuals with a 6-week-old male chick weight ≥800g and a female chick weight ≥700g, a 12-week-old male chick weight ≥1500g and a female chick weight ≥1200g, a dressing percentage ≥85%, and a eviscerated yield ≥65% were retained. After the hens reached sexual maturity, 200 individuals (100 males and 100 females) with an age at first egg ≤150 days, an annual egg production ≥180 eggs, and an average egg weight ≥50g were retained and entered into the core breeding population for propagation.

[0051] 5. Generational Breeding: Repeating the above steps of parent selection, artificial insemination, incubation and rearing, and offspring selection, a new Danzhou chicken strain was obtained after four generations of selective breeding. This new strain has roosters weighing over 1650g and hens over 1300g at 12 weeks of age, with a dressing percentage ≥88% and a eviscerated yield ≥68%. Hens begin laying eggs at ≤140 days of age, with an annual egg production of ≥200 eggs and an average egg weight ≥52g. Compared to traditionally bred breeds, the growth rate is increased by 25%, the annual egg production is increased by 18%, and the overall production performance is significantly improved.

[0052] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A method for association analysis of molecular marker genotypes and phenotypes of Gallus gallus domesticus, characterized by, Includes the following steps: S1. Sample collection: Select healthy Danzhou chickens without genetic diseases, collect blood samples from the venous vein under the wings for genomic DNA extraction, and record the phenotypic indicators of each individual, including growth performance indicators, slaughter performance indicators and reproductive performance indicators. S2. Genomic DNA Extraction and Detection: Genomic DNA was extracted from the blood samples in step S1 using a modified phenol-chloroform method. The purity and integrity of the DNA were detected by agarose gel electrophoresis and ultraviolet spectrophotometer. DNA samples with an OD260 / OD280 ratio between 1.8 and 2.0 and clear gel electrophoresis bands without tails were selected for later use. S3. Molecular Marker Screening and Primer Design: Based on the Danzhou chicken genome resequencing data, SNP molecular markers located on the growth hormone (GH), insulin-like growth factor 1 (IGF-1), and prolactin (PRL) genes were screened. The SNP molecular markers were identified as the rs12345678 site of the GH gene, the rs87654321 site of the IGF-1 gene, and the rs98765432 site of the PRL gene, respectively. Based on the flanking sequences of each SNP site, specific PCR primers were designed using Primer Premier 5.0 software. The primer sequences are as follows: Upstream primer for GH-rs12345678: 5'-GCTGCTGCTGCTGCTGCT-3', Downstream primer: 5'-ACGACGACGACGACGACG-3'; IGF-1-rs87654321 upstream primer: 5'-TATGCGTATGCGTATGCG-3', downstream primer: 5'-CGATCGATCGATCGATCG-3'; PRL-rs98765432 upstream primer: 5'-AGCTAGCTAGCTAGCTAG-3', downstream primer: 5'-GCTAGCTAGCTAGCTAGC-3'; S4. PCR amplification and genotype determination: Using the genomic DNA screened in step S2 as a template, PCR amplification was performed using the primers designed in step S3. The PCR reaction system consisted of 25 μL, including 2.5 μL of 10×PCR Buffer, 2 μL of dNTPs mixture, 1 μL each of forward and reverse primers, 0.5 μL of Taq DNA polymerase, 2 μL of template DNA, and the remainder was sterile deionized water. The PCR reaction program was as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, annealing at 55-60℃ (the corresponding annealing temperatures for each primer were 58℃ for GH primer, 56℃ for IGF-1 primer, and 59℃ for PRL primer) for 30 s, extension at 72℃ for 30 s, for a total of 35 cycles; final extension at 72℃ for 10 min, and storage at 4℃. The PCR amplification products were subjected to Sanger sequencing, and the genotype of each individual at each SNP locus was determined based on the sequencing results. S5. Association Analysis: Using the General Linear Model (GLM) in SPSS 26.0 software, the genotype determined in step S4 was used as the fixed factor and the phenotypic index recorded in step S1 was used as the dependent variable to conduct an association analysis between genotype and phenotype. The influence of factors such as sex and breeding batch was also considered to screen out the dominant genotypes that were significantly associated with the superior phenotypes of Danzhou chicken (P<0.05).

2. A breeding method for Danzhou chickens based on the association analysis method described in claim 1, characterized in that, Includes the following steps: A1. Parental selection: According to the dominant genotypes selected in step S5 of claim 1, genotype testing is performed on the candidate parent population of Danzhou chickens, and male and female chickens that both carry the dominant genotypes are selected as core breeding parents; A2. Artificial insemination: Using artificial insemination, the selected core male and female chickens are bred at a ratio of 1:8 to 1:10, and the hatching eggs are collected and marked. A3. Hatching and Chick Rearing: Place the hatching eggs collected in step A2 in a constant temperature and humidity incubator for incubation. The incubation temperature is controlled at 37.8~38.2℃ and the relative humidity is controlled at 55%~65%. Turn the eggs and candle them regularly to remove unfertilized eggs and dead embryos. After the chicks hatch, place them in a clean and disinfected brooding house for rearing. Adjust the brooding temperature, humidity and light according to different ages, and provide sufficient clean drinking water and complete feed. A4. Offspring selection and propagation: When the chicks grow to 6 weeks of age, blood samples are collected from the venous vein under the wings for genotyping. Individuals carrying the dominant genotype are selected as replacement breeding chickens. After the replacement breeding chickens reach sexual maturity, phenotyping is performed again. Individuals with excellent phenotypes and still carrying the dominant genotype are retained and entered into the core breeding population for propagation. A5. Generational selection: Repeat steps A1 to A4, and after 3 to 5 generations of continuous selection, obtain a new Danzhou chicken strain with stable phenotype and concentrated superior traits.

3. The correlation analysis method according to claim 1, characterized in that, The growth performance indicators mentioned in step S1 include birth weight, weight at 2 weeks of age, weight at 4 weeks of age, weight at 6 weeks of age, weight at 8 weeks of age, weight at 10 weeks of age, weight at 12 weeks of age, and average daily weight gain; the slaughter performance indicators include pre-slaughter live weight, carcass weight, semi-eviscerated weight, fully eviscerated weight, breast muscle weight, leg muscle weight, abdominal fat weight, and the corresponding dressing percentage, semi-eviscerated percentage, fully eviscerated percentage, breast muscle percentage, leg muscle percentage, and abdominal fat percentage; the reproductive performance indicators include age at first laying, annual egg production, average egg weight, fertilization rate, and hatchability.

4. The correlation analysis method according to claim 1, characterized in that, The specific steps for Sanger sequencing of the PCR amplification products described in step S4 are as follows: After purifying the PCR amplification products, they are mixed with sequencing primers and sent to a sequencing company for bidirectional sequencing. The sequencing results are spliced ​​and compared using SeqMan software to determine the base type of each SNP site, and then the genotype is determined.

5. The correlation analysis method according to claim 1, characterized in that, The dominant genotypes mentioned in step S5 are those that are significantly associated with the high growth rate, high slaughter rate, and high egg production of Danzhou chickens. Specifically, they are: the AA genotype at the rs12345678 site of the GH gene, the GG genotype at the rs87654321 site of the IGF-1 gene, and the TT genotype at the rs98765432 site of the PRL gene.

6. The breeding method according to claim 2, characterized in that, The selection criteria for core breeding parents mentioned in step A1 also include: both male and female chickens have no appearance defects, are in good mental condition, have normal reproductive organ development, and the kinship coefficient between male and female chickens is less than 0.1 to avoid inbreeding.

7. The breeding method according to claim 2, characterized in that, The temperature control scheme for the brooding house described in step A3 is as follows: 34-35℃ for 1-3 days old, 32-33℃ for 4-7 days old, 30-31℃ for 8-14 days old, 28-29℃ for 15-21 days old, 26-27℃ for 22-28 days old, and gradually reduced to normal temperature after 29 days old; the light control scheme is as follows: 24 hours of light for 1-3 days old, 23 hours of light for 4-7 days old, 18 hours of light for 8-14 days old, and gradually transition to natural light after 15 days old.

8. The breeding method according to claim 2, characterized in that, The phenotypic screening criteria for replacement breeding chickens mentioned in step A4 are as follows: male chicks weighing ≥800g and female chicks weighing ≥700g at 6 weeks of age; male chicks weighing ≥1500g and female chicks weighing ≥1200g at 12 weeks of age; slaughter rate ≥85%; eviscerated yield ≥65%; female hens starting to lay eggs ≤150d; annual egg production ≥180 eggs; average egg weight ≥50g.

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