Mutton sheep breeding method based on molecular marker and genome directional screening

Abstract

The invention belongs to the technical field of mutton sheep genetic breeding, and particularly relates to a mutton sheep breeding method based on molecular markers and genome directional screening. According to the mutton sheep breeding method disclosed by the invention, a'Yu sheep grazing core '10K genome liquid-phase mutton sheep breeding chip is taken as a core, and through molecular marker development, genetic typing screening and multi-generation dynamic breeding, a new mutton sheep variety which is high in reproductive capacity, resistant to damp and heat and capable of efficiently utilizing straws and adapts to the Chinese and original regions is bred. The mutton sheep breeding chip is used for gene detection, individuals with excellent genes can be rapidly and accurately screened out, long-time phenotype selection in a traditional breeding method is avoided, the breeding period is greatly shortened and is shortened to 3-4 generations (5-8 generations are needed traditionally), and the cost is reduced by 20%. The method has the following advantages that the average lambing number of ewes reaches 2.8, and the weaning survival rate is larger than or equal to 96%; the daily weight gain reduction rate in a high-temperature environment is less than or equal to 7.5% (the daily weight gain reduction rate is greater than or equal to 15% The straw conversion rate is increased to 1: 5.0, and the culture benefit is increased by 30%.

Description

Technical Field

[0001] This invention belongs to the field of livestock molecular genetics breeding technology, specifically involving a method for breeding meat sheep based on molecular markers and genome-directed screening. This invention is based on the "Yuyang Muxin" 10K genome liquid phase meat sheep breeding chip, combined with molecular marker screening technology for high fertility-related genes (such as BMPR-IB, FecB, etc.), and through phenotypic-genotypic association analysis and dynamic genetic model optimization, to breed a new breed of prolific meat sheep that is adapted to the Central Plains region, has high fertility, is resistant to humid heat, and makes efficient use of straw. Background Technology

[0002] Against the backdrop of the nation's vigorous promotion of the rural revitalization strategy and the modernization of animal husbandry, the mutton sheep industry, as an important component of animal husbandry, holds strategic significance for increasing agricultural efficiency, farmers' income, and rural economic prosperity through its high-quality development. Simultaneously, with the steady improvement of residents' living standards, the market demand for mutton continues to grow, and the requirements for mutton quality are becoming increasingly stringent, posing greater challenges to the development of the mutton sheep industry. However, for a long time, traditional mutton sheep breeding models have relied primarily on experience and phenotypic selection, which has many drawbacks. Traditional mutton sheep breeding, relying on phenotypic selection, suffers from low efficiency (requiring tracking of the entire growth cycle), poor accuracy (susceptible to environmental interference), and long cycles (requiring multiple generations of selection). Traditional mutton sheep breeding, relying on phenotypic selection, suffers from long cycles (5-8 generations) and low accuracy (error rate 15%-20%). With the development of genomics technology, molecular marker-assisted selection (MAS) and genome selection (GS) have become mainstream, but existing technologies mostly focus on growth rate or meat quality traits, and there are still shortcomings in the systematic screening of genes with high fertility and regional adaptation optimization.

[0003] Henan, a major sheep-raising province, urgently needs breeds adapted to its hot and humid climate and abundant straw resources. While the Small-tailed Han sheep has a high reproductive rate, its meat performance is poor; the White-headed Suffolk sheep grows quickly but lacks heat tolerance. Although crossbreeding can partially optimize traits, its genetic stability is poor, necessitating precision breeding combined with genomics technology.

[0004] The development of the "Yuyang Muxin" 10K genomic liquid phase sheep breeding chip fills the gap in specialized breeding tools for agricultural areas in central China. It covers 6327 loci related to economic traits (including 4049 loci unique to Henan local sheep breeds), significantly improving breeding efficiency (30%) and reducing costs (20%). However, existing patents do not fully integrate targeted screening of high-fertility genes with dynamic genetic model optimization, resulting in the unrealized potential for improving target traits. From an industrial development perspective, the Central Plains region, as an important agricultural production area in my country, possesses abundant crop straw resources. However, the region lacks suitable meat sheep breeds and urgently needs to cultivate breeds that combine high fertility, tolerance to hot / cold climates, and efficient utilization of straw resources. The "multi-parity meat sheep" breeding program is proposed based on this practical need. This breed uses the White-headed Suffolk sheep as the sire and the Small-tailed Han sheep as the dam. White-headed Suffolk sheep have advantages such as rapid early growth and development, outstanding meat performance, high reproductive rate, and tolerance to roughage; however, their adaptability to hot summer environments is relatively weak. Small-tailed Han sheep, on the other hand, excel in local adaptability and reproductive rate, but their meat performance needs further improvement. While Small-tailed Han sheep hybrids possess rapid growth and strong adaptability, their genetic stability is not ideal.

[0005] Therefore, developing a high-fertility meat sheep breed suitable for the Huang-Huai Plain region is of vital importance for promoting the development of the meat sheep industry in the Central Plains and contributing to rural revitalization. This breed must not only possess a medium to large body size and high fertility, adapting well to both hot and cold climates and thriving in both grazing and stall environments, but also be capable of natural mating for efficient reproduction. Ewes can serve as high-quality dams for meat production, while rams can serve as terminal sires to further improve the overall quality of the meat sheep population. More importantly, this breed must be able to fully utilize the abundant straw resources in the Central Plains agricultural area, achieving efficient resource conversion and utilization, reducing breeding costs, and increasing breeding efficiency.

[0006] The emergence of the "Yuyang Muxin" 10K genomic liquid phase sheep breeding chip provides strong technical support for the breeding of new multi-prolific sheep breeds. Through the deep integration of technological innovation and traditional breeding techniques, it breaks through the bottlenecks of traditional sheep breeding, cultivating high-quality new sheep breeds with independent intellectual property rights that are adapted to the environment and industrial needs of the Central Plains region. This will promote the high-quality development of my country's sheep industry and contribute to the comprehensive implementation of the rural revitalization strategy. Summary of the Invention

[0007] This invention aims to address the problems of long breeding cycles and difficulties in the synergistic improvement of multiple traits in traditional breeding techniques. It proposes a method for breeding meat sheep based on molecular markers and genome-directed screening. Using the "Yuyang Muxin" 10K genomic liquid phase meat sheep breeding chip (hereinafter referred to as "Yuyang Muxin" chip) as the core, combined with molecular marker screening technology for high fertility genes (BMPR-IB, FecB), it achieves precision breeding through the following objectives: High fertility: Ewes have an average litter size of ≥2.5 lambs and provide ≥3.7 weaned lambs per year; Resistance to damp heat: Daily weight gain decrease rate ≤8% in summer, survival rate ≥95% in winter; Efficient resource utilization: Straw digestibility increased by 20%, and feed conversion ratio optimized to 1:5.0; Genetic stability: core population genetic diversity index (PIC) ≥ 0.25, heat-resistant genotype frequency ≥ 85%.

[0008] To achieve the above objectives, the technical solution created by this invention is implemented as follows: A method for breeding meat sheep based on molecular markers and genome-directed screening includes the following steps: (1) Basic population construction: Select healthy male and female meat sheep (sires) with typical breed characteristics and clear genetic background, crossbreed them according to a scientific breeding ratio to obtain F1 generation crossbred meat sheep. Use the F1 generation crossbred sheep as the dam, select the sires, and crossbreed them again to form F2 generation crossbred meat sheep. (2) Molecular marker development and chip integration: This includes steps such as primer design, SNP site verification, and microarray function verification for genes related to multiple target traits in meat sheep. The primer design for genes related to multiple target traits in meat sheep includes steps such as conventional primer design, mixed PCR amplification and Sanger sequencing, and specific primer design and FLU-ARMS genotyping. (3) Gene detection and analysis: The process includes steps such as using the "Yuyang Muxin" chip to perform gene testing and genotyping on F1 and / or F2 generation hybrid sheep, phenotype-genotype association analysis, genome selection and dynamic genetic model optimization; (4) Production performance measurement and evaluation The process includes steps such as identifying the body shape and appearance of multiparous meat sheep, measuring their growth and development performance, measuring their reproductive performance, and studying their fattening effects and slaughter performance. (5) Multigenerational selection and phenotypic enhancement This includes steps such as core group construction and updating, and dynamic adjustment of breeding standards; (6) Enhanced adaptability and resource utilization It includes steps such as environmental adaptability assessment and straw resource utilization assessment.

[0009] Specifically, in step (1), the mutton sheep is a prolific mutton sheep; preferably, the male mutton sheep is a white-headed Suffolk sheep; and the female mutton sheep is a small-tailed Han sheep. White-headed Suffolk sheep, as the sire, are characterized by rapid early growth and development, outstanding meat performance, high reproductive rate, and tolerance to roughage, but they are relatively poor adaptable to hot summer environments. Small-tailed Han sheep, as the dam, have the advantages of good local adaptability and high reproductive rate.

[0010] Specifically, in step (1), when crossbreeding again, white-headed Suffolk sheep are selected as the father and F1 generation crossbred sheep are selected as the mother.

[0011] Specifically, in step (2), when designing ordinary primers, based on the sheep reference genome (Oar_v3.1), according to the known genes (e.g., growth hormone gene (GH), multiple birth gene (BMPR-IB), intramuscular fat gene (FABP4)) related to the target traits of prolific meat sheep (e.g., growth rate, reproductive performance, meat quality), ordinary primers are designed with the significant variation sites of the target genes as the center, taking 400bp sequences upstream and downstream; Specifically, in step (2), when performing mixed PCR amplification and Sanger sequencing, the mixed DNA sample is amplified using the common primers shown in sequences 1-14.

[0012] Preferred, the standard PCR amplification program is as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 10 s, 55-65℃ annealing for 30 s, 72℃ extension for 1 min, 35 cycles.

[0013] Specifically, in step (2), when designing specific primers and performing FLU-ARMS typing, specific primers are designed with the new SNP site as the center and the upstream and downstream 200bp sequences are taken. The Touchdown PCR program is used to perform FLU-ARMS typing operation to genotype the core group individuals and perform agarose gel electrophoresis to obtain genotype data.

[0014] Preferably, the Touchdown PCR program is as follows: pre-denaturation at 95℃ for 10 min; annealing at 61℃ → 55℃, with a temperature decrease of 0.6℃ per cycle, for 10 cycles; annealing at 55℃ → 65℃, for 30 cycles.

[0015] Preferably, when designing specific primers, the probe structure used is: 5'-FAM (fluorescent group)-CGCGTCG (specific sequence)-BHQ1 (quencher group)-3', and the 5' end of the reverse primer is labeled with the HEX fluorescent group; The probe design principles are as follows: allele-specific primers (ASP) are designed within 50 bp upstream and downstream of the SNP site; the difference in primer Tm value is ≤1℃ (calculated using Oligo 7.0 software); mismatched bases (such as C→T) are introduced at the third-to-last position of the 3' end to enhance specificity.

[0016] Preferably, the FLU-ARMS classification operation is as follows: PCR amplification system: 2×Taq Master Mix: 10 μL, DNA template (20 ng / μL): 2 μL, FAM / HEX labeled primers (10 μM): 0.5 μL each; sterile ddH2O: add to 20 μL; PCR amplification program: 95℃ pre-denaturation: 5 min Touchdown cycles: 10 cycles: 95℃ 30s → 61℃ (-0.6℃ / cycle) 30s → 72℃ 45s 30 cycles: 95℃ 30s → 55℃ 30s → 72℃ 45s Final extension: 72℃ 5 min.

[0017] Specifically, in step (2), when verifying SNP sites, mixed PCR amplification and Sanger sequencing are used to discover new SNP sites; the genotype is verified by FLU-ARMS typing technology.

[0018] Preferably, the conditions for mixed PCR amplification are: annealing temperature gradient: 55-65℃; the newly discovered SNP sites include g.97048094A; and the PCR program used for FLU-ARMS genotyping is: 61℃→55℃, 10 cycles; 55℃→65℃, 30 cycles.

[0019] Specifically, in step (2), the chip function verification involves comparing the "Yuyang Muxin" chip with the newly discovered SNPs, screening overlapping sites (such as HSP70_rs2023T), and including them in the detection range.

[0020] Specifically, in step (3), before gene testing, blood or tissue samples of F1 generation hybrid sheep are collected. Generally, 5-10 ml of blood is collected from the jugular vein and placed in a blood collection tube containing an anticoagulant, or an appropriate amount of ear tissue sample is collected to ensure that the sample collection process follows the principle of aseptic operation and avoids sample contamination.

[0021] Preferably, DNA is extracted from the collected samples using the conventional phenol-chloroform method or a commercial DNA extraction kit. The extracted DNA samples need to be quality tested to ensure that their purity and concentration meet the requirements of the microarray detection. Generally, the DNA concentration is required to be 50-100 ng / μl, and the A260 / A280 ratio is required to be between 1.8 and 2.0.

[0022] Specifically, in step (3), during gene detection, the extracted DNA sample is processed and loaded onto the "Yuyang Muxin" chip (which covers 6327 loci related to key traits such as sheep reproductive performance, growth rate, feed conversion rate, and muscle quality, including 4049 loci unique to Henan local sheep germplasm resources). Through analysis of these loci, individuals carrying superior gene combinations are screened out.

[0023] Specifically, in step (3), during genotyping, the "Yuyang Muxin" chip is used to perform genotyping on F1 generation hybrid sheep and construct a whole genome SNP database.

[0024] Preferably, when genotyping, the focus is on gene loci related to the desired traits of prolific meat sheep, such as screening for loci related to traits such as high fertility (e.g., prolific genes), rapid growth (e.g., growth hormone genes, myostatin genes), excellent meat quality (e.g., intramuscular fat-related genes) and high feed conversion rate (e.g., nutrient metabolism-related genes). Based on the genotyping results, the F1 generation of hybrid sheep individuals were preliminarily evaluated, and individuals carrying superior gene combinations were screened out. These individuals showed superior genotypes at relevant gene loci of key traits, laying the foundation for subsequent breeding work.

[0025] Specifically, in step (3), during the phenotype-genotype association analysis, a general linear model (SPSS 25.0) is used to perform association analysis between genotype data and phenotype data (including body weight, number of lambs, and intramuscular fat content) to construct a phenotype-genotype model formula.

[0026] Preferably, the formula for constructing the phenotype-genotype model is: Y=Xβ+a+e Where Y is the phenotypic value, X is the genotype fixed effect, a is the sex effect, and e is the residual; Loci that are significantly associated with the target trait (P<0.05) were screened, for example, g.97048094A was significantly associated with body weight (P=0.003), for subsequent core group selection.

[0027] Specifically, in step (3), the genome selection and dynamic genetic model optimization are carried out by a two-dimensional screening model based on chip scoring and FLU-ARMS verification.

[0028] Preferably, the chip score is calculated based on the genome breeding value (GEBV) of the whole genome SNP loci (6327), and individuals with a score ≥90 are selected.

[0029] Preferably, FLU-ARMS verification involves targeted genotyping of key sites such as BMPR-IB_g.746G to ensure homozygous or heterozygous dominant genotypes.

[0030] Preferably, in the two-dimensional screening model process, the dynamic genetic parameters are: Heredity weight: Fertility h 2 =0.40, heat resistance h 2 =0.35, feed conversion ratio h 2 =0.30; Generation interval optimization: Rams should be used for ≤2 generations, and ewes should be culled at a rate of ≤10%.

[0031] Specifically, in step (5), the core group is constructed as follows: based on the F2 generation of the male and female parent hybrids, individuals carrying the BMPR-IB_g.746G allele are selected through open core group breeding technology to form a core group of 1500 individuals.

[0032] Specifically, in step (5), the environmental adaptability assessment includes heat and humidity resistance screening: using the HSP70 gene cluster (e.g., HSP70_rs2023C) in the chip to associate with high temperature phenotype data, individuals with heat resistance are screened.

[0033] Specifically, in step (5), the straw resource utilization assessment includes straw utilization optimization: by cellulase gene (CEL) typing, the straw digestibility is increased to 18%.

[0034] Compared with the prior art, the advantages of the present invention are: 1. Improve breeding efficiency: Using sheep breeding chips for gene testing can quickly and accurately screen individuals with superior genes, avoiding the long phenotypic selection process in traditional breeding methods, greatly shortening the breeding cycle to 3-4 generations (traditionally 5-8 generations), and reducing costs by 20%.

[0035] 2. Reproductive performance: The average number of lambs born to ewes reaches 2.8 (see data in the example), and the weaning survival rate is ≥96%; Heat resistance: The daily weight gain decrease rate under high temperature conditions is ≤7.5% (compared to ≥15% for traditional breeds); Resource utilization: The straw conversion rate is increased to 1:5.0, and the breeding efficiency is increased by 30%.

[0036] Experimental results showed that the reproductive rate of multiparous meat sheep increased by 25%, heat resistance increased by 15%, and the breeding cycle was shortened to 4 years. Attached Figure Description

[0037] Figure 1 A roadmap for breeding multi-parity meat sheep (showing the hybridization, crossbreeding fixation, and herd expansion processes in stages); Figure 2 A timeline chart of the breeding progress of multi-parity meat sheep (key milestones marked on the timeline: 2008 initiation of hybridization, 2013 fixation through crossbreeding, 2023 establishment of the core herd). Figure 3 A flowchart for molecular marker development and chip integration (showing the steps of SNP discovery, verification, and chip integration); Figure 4 A schematic diagram of FLU-ARMS genotyping results (showing the differences in electrophoretic bands for CC, CT, and TT genotypes); Figure 5 A heatmap for phenotype-genotype association analysis (marking significant association sites and P-value distribution). Detailed Implementation

[0038] To better understand the present invention, the present invention will be further described below with reference to the embodiments. However, the content of the present invention is not limited to the following embodiments. All changes or equivalent substitutions that do not depart from the concept of the present invention are included within the protection scope of the present invention.

[0039] The following examples are based on the "Yuyang Muxin" 10K genomic liquid phase meat sheep breeding chip, hereinafter referred to as "Yuyang Muxin" chip; The "Yuyang Muxin" 10K genomic liquid-phase sheep breeding chip was jointly developed by Henan University of Animal Husbandry and Economics and the Beijing Institute of Animal Husbandry and Veterinary Medicine of the Chinese Academy of Agricultural Sciences. This chip fills the gap in specialized breeding tools for agricultural areas in central China. Based on a liquid-phase chip technology platform, the chip employs multiplex PCR and targeted capture strategies, covering 6327 SNP loci related to economic traits, including 4049 loci unique to Henan local sheep germplasm resources. These loci encompass key traits such as reproductive performance, growth rate, heat tolerance, feed conversion ratio, and muscle quality. During the chip design process, genome resequencing data from major meat sheep breeds in Henan (such as the Small-tailed Han sheep and the Huai goat) were used to prioritize the screening of loci significantly associated with regional adaptability, such as the HSP70 gene cluster (heat tolerance), BMPR-IB (multiparity), and CEL (cellulase activity).

[0040] The "Yu Yang Mu Xin" chip technology parameters: It covers 6,327 economic trait loci, including 4,049 loci unique to Henan local sheep. 1,000 breeding sheep have passed the "Yu Yang Mu Xin" test, resulting in a 25% increase in lambing, a heat-resistant genotype frequency of 88%, and a 30% increase in breeding efficiency. The chip covers 6,327 loci related to key traits such as sheep reproductive performance, growth rate, feed conversion rate, and muscle quality, including 4,049 loci unique to local sheep germplasm resources in Henan. Compared to "Lanzhou University-Huayangxin", "Yuyangmuxin" focuses more on regional adaptability and reproductive capacity optimization, filling the technological gap in the central agricultural region.

[0041] Example 1 This embodiment provides a precision breeding method for prolific meat sheep based on the "Yuyang Muxin" 10K genomic liquid phase meat sheep breeding chip and targeted screening of high fertility genes. The final bred meat sheep was named "Zhongyu Meat Sheep". The process is as follows: Figure 1 , 2 As shown, the details are as follows: I. Preparation of Breeding Materials The white-headed Suffolk sheep, a prolific breed of meat sheep, was selected as the sire. It is characterized by rapid early growth and development, outstanding meat performance, high reproductive rate and tolerance to roughage, but it is relatively poor in adapting to hot summer environments. The small-tailed Han sheep, another prolific breed of meat sheep, was selected as the dam. It has the advantages of good local adaptability and high reproductive rate.

[0042] II. Basic Population Construction Healthy Suffolk white-headed rams and Small-tailed Han ewes with typical breed characteristics and clear genetic backgrounds are selected and crossbred according to a scientific breeding ratio to obtain F1 generation hybrid sheep. During the breeding process, the feeding and management of the parent sheep strictly adhere to standardized procedures, including providing sufficient and suitable feed, clean drinking water, and a suitable rearing environment to ensure their physiological state is optimal, thus facilitating reproduction and the healthy development of offspring. Through selection of different breeds, genetic improvement, and mating combinations, the hybrid offspring undergo multiple screenings and identifications.

[0043] Hybridization innovation (2008-2012): The F2 generation was formed by crossing white-headed Suffolk sheep (sire, 75% bloodline) with small-tailed Han sheep (madam, 25% bloodline).

[0044] Cross-linking fixation (2013-2022): Using open core population breeding technology combined with molecular marker-assisted selection (MAS), ideal individuals were selected.

[0045] Breeding and expansion (from 2023 to the end of December 2023): A core breeding group of 1,500 multi-parity meat sheep was successfully established, including about 800 from Henan Yasheng Animal Husbandry Co., Ltd., about 400 from Shenqiu County Agricultural and Animal Husbandry Science and Technology Research and Development Center, and about 300 from Henan Luyuan Meat Sheep Development Co., Ltd.

[0046] III. Development of Molecular Markers for Heat-Resistant Traits This invention targets the heat-resistance-related gene HSP70, designing primers to amplify its promoter and exon regions. Sanger sequencing revealed a novel SNP site, g.HSP70_rs2023T>C. FLU-ARMS genotyping technology was used to genotype 500 sheep in the core flock. Details are as follows: 3.1 Molecular marker development and chip integration (e.g.) Figure 3 (As shown) 3.1.1 Target Gene Primer Design: Based on the sheep reference genome (Oar_v3.1), conventional PCR primers (sequences 1-14) were designed for the growth hormone (GH), multiple birth rate gene (BMPR-IB), and intramuscular fat gene (FABP4) in prolific meat sheep. The specific steps are as follows: Step 1: Target gene primer design Based on known genes related to the target trait in prolific meat sheep, and referring to the sheep reference genome (Oar_v3.1), common primers were designed by taking 400bp upstream and downstream sequences of the target gene as the center (see Sequence 1-14 for details).

[0047] Step 2: Mixed PCR amplification and Sanger sequencing The mixed DNA sample was amplified by conventional PCR using the above-mentioned common primers (sequences 1-14), and new SNP sites (such as g.97048094A>C) were found by comparison with the reference genome.

[0048] The standard PCR amplification program is as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 10 s, 55-65℃ annealing for 30 s, 72℃ extension for 1 min, 35 cycles.

[0049] Step 3: Specific primer design and FLU-ARMS typing Using the newly discovered SNP site (e.g. g.97048094A>C) as the center, allele-specific primers (ASP) were designed by taking 200 bp upstream and downstream sequences (see sequences 15-20 for details). Touchdown PCR program (95℃ pre-denaturation for 10 min; 61℃→55℃ annealing, cooling by 0.6℃ per cycle, 10 cycles; 55℃→65℃ annealing, 30 cycles) was used in conjunction with FLU-ARMS technology to genotype individuals in the core population.

[0050] Probe structure: 5'-FAM (fluorescent group)-CGCGTCG (specific sequence)-BHQ1 (quencher group)-3', with the 5' end of the reverse primer labeled with the HEX fluorescent group. Design principles: Allele-specific primers (ASP) are designed within 50 bp upstream and downstream of the SNP site; the difference in primer Tm value is ≤1℃ (calculated using Oligo 7.0 software); a mismatched base (e.g., C→T) is introduced at the third-to-last position of the 3' end to enhance specificity.

[0051] FLU-ARMS genotyping procedure: PCR amplification: 2×Taq Master Mix: 10 μL, DNA template (20 ng / μL): 2 μL, FAM / HEX labeled primers (10 μM): 0.5 μL each; sterile ddH2O: add to 20 μL.

[0052] 95℃ pre-denaturation: 5 min Touchdown cycle: 10 cycles: 95℃ 30s → 61℃ (-0.6℃ / cycle) 30s → 72℃ 45s 30 cycles: 95℃ 30s → 55℃ 30s → 72℃ 45s Final extension: 72℃ 5 min.

[0053] Electrophoresis detection: Gel preparation: 3% agarose gel (containing 0.5×GelRed); Electrophoresis conditions: 120 V constant voltage electrophoresis for 30 min (0.5×TBE buffer); Imaging analysis: Fluorescence signals were acquired using the Bio-Rad GelDoc XR+ system, and genotypes were interpreted using GeneMarker V2.7.0 software. Genotyping results are shown in Table 1. Figure 4 As shown.

[0054] Table 1 Classification Results

[0055] 3.1.2 SNP site verification: New SNP sites (e.g., g.97048094A>C) were discovered using mixed PCR amplification (annealing temperature gradient: 55-65℃) and Sanger sequencing; the genotype was verified by FLU-ARMS genotyping technology (Touchdown PCR program: 61℃→55℃, 10 cycles; 55℃→65℃, 30 cycles).

[0056] 3.1.3 Chip Function Verification: The sites on the “Yuyang Muxin” chip were compared with the newly discovered SNPs, and overlapping sites (such as HSP70_rs2023T>C) were screened and included in the detection range.

[0057] In this step, the present invention, through searching relevant patents and literature and combining the development requirements of the "Yuyang Muxin" chip, designed primer sequences (sequences 1-20) and their functional descriptions as follows: 1) Primers for genes related to high fertility BMPR-IB gene (FecB mutation site g.746A>G) Sequence 1 (forward primer): 5'-TACGCCTCTGGACGCACAACT-3' (as shown in SEQ ID No. 1) Sequence 2 (reverse primer): 5'-CCTCGCTCGGTCAGGATCTTCA-3' (as shown in SEQ ID No. 2) Function: Used to detect FecB mutation sites in the BMPR-IB gene that are associated with the trait of multiple births, significantly increasing the number of lambs born to ewes.

[0058] GDF9 gene (multiple pregnancy trait) Sequence 3 (forward primer): 5'-AGCCAGGTGCTGAAGGTGAT-3' (as shown in SEQ ID No. 3) Sequence 4 (reverse primer): 5'-GTCCTGGTGCCTCTTGTTCC-3' (as shown in SEQ ID No. 4) Function: To amplify the region of the GDF9 gene that affects ovulation rate and screen for individuals with high fertility.

[0059] 2) Primers for heat resistance-related genes HSP70 gene cluster (rs2023C site) Sequence 5 (forward primer): 5'-GAGCTGGAGCGCGAGAAG-3' Sequence 6 (reverse primer): 5'-CCTCGAGGCCTTCAGCTTC-3' Function: Targeted screening of individuals with a heat- and humidity-tolerant phenotype to reduce the rate of decline in daily weight gain under high-temperature conditions.

[0060] 3) Primers for feed conversion ratio related genes Cellulase gene (CEL) Sequence 7 (forward primer): 5'-TGGACCTGCCTCAACATCCT-3' Sequence 8 (reverse primer): 5'-AGGTGCCATCCACAGTCACA-3' Function: Improves straw digestibility and optimizes feed conversion efficiency.

[0061] 4) Primers for muscle quality-related genes FABP4 gene (intramuscular fat deposition) Sequence 9 (forward primer): 5'-CCAGGAAGACAGCAAGGACA-3' Sequence 10 (reverse primer): 5'-TGCAGGTCCAGGTCTTCATC-3' Function: Select individuals with high intramuscular fat content to improve meat tenderness.

[0062] 5) Primers for growth rate-related genes Growth hormone gene (GH) Sequence 11 (forward primer): 5'-CTGCCTGGAGAGCTACCTGA-3' Sequence 12 (reverse primer): 5'-GCCATCCAGGTCCAGGTAGT-3' Function: Associate daily weight gain traits to screen individuals with superior growth rate.

[0063] 6) Primers for other functional sites GnRHR gene (reproductive performance) Sequence 13 (forward primer): 5'-GCTGCAGCTACCTGACCTCA-3' Sequence 14 (reverse primer): 5'-CAGGTCCAGGTGCTGAAGGT-3' Function: Detect GnRHR gene polymorphism to optimize estrous cycle and litter size.

[0064] Sequence 15, allele T-specific forward primer (5' end marker FAM) for SNP site g.HSP70_rs2023T>C FLU-ARMS typing: 5'- [FAM]GAG CTG GAG CGC GAG AAG TAC G -3'.

[0065] Sequence 16, allele C-specific forward primer (5' end marker FAM) for SNP site g.HSP70_rs2023T>C FLU-ARMS typing: Sequence: 5'- [FAM]GAG CTG GAG CGC GAG AAG CAC G -3'.

[0066] Sequence 17, universal reverse primer (5' end labeled HEX) for SNP site g.HSP70_rs2023T>C FLU-ARMS genotyping: Sequence: 5'- [HEX]CCT CGA GGC CTT CAG CTT C -3'.

[0067] Sequence 18, allele-specific forward primer (5' end marker FAM) for SNP site BMPR-IB_g.746G>A FLU-ARMS typing: Sequence: 5'- [FAM]TAC GCC TCT GGA CGC ACA AGG T -3'.

[0068] Sequence 19, allele A-specific forward primer (5' end marker FAM) for SNP locus BMPR-IB_g.746G>A FLU-ARMS typing: Sequence: 5'- [FAM]TAC GCC TCT GGA CGC ACA AAG T -3'.

[0069] Sequence 20, universal reverse primer (5' end labeled with HEX) for SNP site BMPR-IB_g.746G>A FLU-ARMS genotyping: Sequence: 5'- [HEX]CCT CGC TCG GTC AGG ATC TTC A -3'.

[0070] IV. Gene Detection and Analysis 4.1 Chip Preparation and Sample Collection Genetic testing is performed using the "Yuyang Muxin" chip. Before genetic testing, blood or tissue samples are collected from F1 generation hybrid sheep. Generally, 5-10 ml of blood is collected from the jugular vein and placed in a blood collection tube containing an anticoagulant, or an appropriate amount of ear tissue sample is collected. Ensure that the sample collection process follows aseptic operation principles to avoid sample contamination.

[0071] DNA extraction is performed on the collected samples using either the conventional phenol-chloroform method or a commercial DNA extraction kit. The extracted DNA samples must undergo quality testing to ensure their purity and concentration meet the requirements for microarray detection. Generally, a DNA concentration of 50-100 ng / μl and an A260 / A280 ratio between 1.8 and 2.0 are required.

[0072] 4.2 Gene testing and screening: Genetic testing of hybrid offspring sheep was conducted using the "Yuyang Muxin" microarray. Specifically, extracted DNA samples were processed and loaded onto the "Yuyang Muxin" microarray. This microarray covers 6327 loci related to key traits such as sheep reproductive performance, growth rate, feed conversion ratio, and muscle quality, including 4049 loci unique to Henan local sheep germplasm resources. This provides rich genetic information for the breeding of new prolific meat sheep breeds. Analysis of these loci allows for the selection of individuals carrying superior gene combinations. For example, individuals with genes related to high fertility and rapid growth are selected, laying the foundation for subsequent breeding. The locus composition and functional distribution are shown in Table 2.

[0073] Table 2. Site Composition and Distribution Map

[0074] Key site functional validation data 1) HSP70_rs2023T>C (HEN-SNP001): Functional validation: Dual-luciferase reporter gene assay showed that the C allele increased promoter activity by 42% (P<0.01). Phenotypic association: Sheep carrying the CC genotype had a daily weight gain 18.3% higher than those carrying the TT genotype at 35℃ (n=500, P=0.003). 2) BMPR-IB_g.746G>A (HEN-SNP218): Mutation effect: Leads to Arg→Gln at amino acid position 249 of the BMPR-IB receptor (SIFT score = 0.02); Effect on fertility: GG type ewes had an average litter size of 2.8 lambs, which was significantly higher than AA type (1.9 lambs, P=0.001).

[0075] Association analysis showed that individuals with the g.HSP70_rs2023C genotype had significantly higher daily weight gain than those with the TT genotype under high-temperature conditions (P=0.01). Including this locus in microarray analysis improved the population's heat tolerance by 15%.

[0076] 4.3 Genotyping and Data Interpretation The instruments and software accompanying the chip are used to perform genotyping on the samples, obtaining genotypic information for each sample at the loci covered by the chip. By comparing this information with the chip's reference database, the genotype of each individual can be accurately identified.

[0077] A detailed interpretation of the genotyping data was conducted, with a focus on gene loci associated with the desired traits of prolific meat sheep. This included screening for loci related to traits such as high fertility (e.g., prolific genes), rapid growth (e.g., growth hormone genes, myostatin genes), excellent meat quality (e.g., intramuscular fat-related genes), and high feed conversion rate (e.g., nutrient metabolism-related genes).

[0078] Based on the genotyping results, the F1 generation of crossbred sheep were preliminarily evaluated, and individuals carrying superior gene combinations were selected. These individuals exhibited superior genotypes at relevant gene loci for key traits, laying the foundation for subsequent breeding work. Details are as follows: 4.3.1 Chip typing: The F1 generation of crossbred sheep were genotyped using the "Yuyang Muxin" chip, and a whole-genome SNP database was constructed (PopGen 32 software analysis, PIC>0.25 indicates high polymorphism sites).

[0079] 4.3.2 Phenotype-Gentype Association Analysis: A general linear model (SPSS 25.0) was used to perform association analysis between genotype data and phenotypic data (body weight, number of lambs born, intramuscular fat content). The formula is as follows: Y=Xβ+a+e Where Y is the phenotypic value, X is the genotype fixed effect, a is the sex effect, and e is the residual.

[0080] Loci significantly associated with the target trait (P<0.05) were selected, such as g.97048094A>C, which was significantly associated with body weight (P=0.003), for subsequent core group selection. The analysis results are as follows: Figure 5 As shown.

[0081] 4.3.3 Genome selection and dynamic genetic model optimization: Two-dimensional screening model: Chip scoring: Genomic breeding value (GEBV) is calculated based on whole-genome SNP loci (6327), and individuals with a score ≥90 are selected; FLU-ARMS verification: Targeted genotyping was performed on key loci such as BMPR-IB_g.746G to ensure the presence of homozygous or heterozygous dominant genotypes.

[0082] Dynamic genetic parameters: Heredity weight: Fertility h 2 =0.40, heat resistance h 2 =0.35, feed conversion ratio h 2 =0.30; Generation interval optimization: Rams should be used for ≤2 generations, and ewes should be culled at a rate of ≤10%.

[0083] V. Production Performance Measurement and Evaluation 5.1 Physical Appearance Assessment Detailed observations and records were made on the coat color, skin color, head shape, ear shape, horn shape, neck, body, limbs, hooves, tail, and reproductive organs of both male and female sheep in prolific breeding sheep. The coat color of prolific breeding sheep was entirely white, with large white curls (83.33%) and small, dark white-blue curls (16.67%). The skin color was white. The heads were large, with broad, flat foreheads, large, slightly drooping ears, large, bright eyes with prominent eye sockets. Rams were hornless, while ewes had horns-free or small horns (ratio 86:4). The physical characteristics of each sheep were accurately recorded through visual observation and measurement tools. Image analysis software was used to quantify some characteristics for better comparison and evaluation.

[0084] 5.2 Growth and development performance measurement Weight, height, length, chest circumference, and cannon bone circumference of multiparous meat sheep were measured at different growth stages (birth, weaning, 6 months, 12 months, etc.). For example, the height of 12-month-old rams was 82.78±4.65cm, and the length was 85.40±5.19cm.

[0085] We use a professional electronic scale to measure weight, accurate to 0.1 kg; we use measuring sticks and tape measures to measure height, length, chest circumference, and tube circumference, accurate to 0.1 cm.

[0086] Analyze growth data at each stage, plot growth and development curves, and use statistical analysis software (such as Excel or professional statistical analysis software such as SAS or R) to process and analyze the data. The results show that the cumulative growth curves of weight and body size of male and female sheep are basically consistent. The growth rate is fast in the early stage. The weight and body size of male sheep are greater than those of female sheep at all stages. The growth intensity of male sheep is relatively high from 6 months to 12 months of age, while that of female sheep is relatively slower. The cannon circumference of male and female sheep increases steadily.

[0087] 5.3 Reproductive performance testing Reproductive performance testing experiments were conducted at the Huai Goat Engineering Technology Research Center in Shenqiu County from September 2022 to April 2023. The study collected data on indicators such as estrus rate, estrus cycle, artificial insemination conception rate, number of lambs born, and lamb weaning survival rate in prolific sheep.

[0088] The estrus status of ewes was accurately determined using techniques such as ram estrus detection and ultrasound monitoring. The start and end times of estrus were recorded, and the estrous cycle was calculated. During artificial insemination, standardized procedures were ensured, the number of ewes conceiving was accurately recorded, and the conception rate was calculated. The lambing process was observed and recorded in detail, and the number of lambs born and the lamb weaning survival rate were statistically analyzed. The results showed that prolific ewes had a high estrus rate (52.10%), a short estrus cycle (19.3±1.04 d), an artificial insemination conception rate of 73.26%, an average of 1.68 lambs per litter for primiparous ewes, an average of 2.63 lambs per litter for multiparous ewes, an overall average of 2.58 lambs per litter, a lamb weaning survival rate of 95.83%, and an annual production of 3.75 weaned lambs per ewe.

[0089] The semen of breeding rams was tested for quality. A semen analyzer was used to evaluate the semen volume, semen viability, semen density, and abnormality rate of the breeding rams. The results showed that the semen quality of the breeding rams was excellent, with a semen volume of 1.16 mL, a semen viability of 76.18%, a semen density of 1.728 billion sperm / mL, and an abnormality rate of 12.93%.

[0090] 5.4 Study on fattening effect and slaughter performance Under the same indoor feeding conditions, five male and five female multiparous meat sheep, five male and five female small-tailed Han sheep, and five Du Han F1 sheep were selected at 45 days of weaning to determine the fattening effect over 135 days, and slaughter performance was tested at 6 months of age.

[0091] The measured indicators included body weight, daily weight gain, feed conversion ratio, dressing percentage, lean meat percentage, carcass weight, and lean meat weight. Precise electronic scales and professional slaughtering equipment were used for measurement to ensure data accuracy. Results showed that prolific lambs outperformed both the Small-tailed Han sheep and the Duhan F1 breed in all indicators. At 6 months of age, the average body weights of rams and ewes were 60.37 kg and 53.34 kg, respectively; the average daily weight gain was 319.04 g and 274.07 g / d, respectively; the average dressing percentage was 56.07% and 53.15%, respectively; and the average lean meat percentage was 82.05% and 81.51%, respectively.

[0092] VI. Multigenerational breeding and phenotypic enhancement 6.1 Core Group Selection and Update Combining genetic testing results and production performance measurement data, individuals with excellent overall performance are selected as the core population. Considering individual performance in reproductive performance, growth performance, meat quality, and genotyping results, the core population is ensured to not only exhibit outstanding phenotypic performance but also possess excellent genotypic potential. Individuals that do not meet the standards are removed from the core population, and new superior individuals are introduced to supplement it. The genetic testing and production performance measurement steps are repeated in each generation of breeding to ensure the continuous enrichment and stable inheritance of superior genes within the population.

[0093] Core population construction: Based on the F2 generation of the cross between white-headed Suffolk (paternal) and small-tailed Han sheep (maternal), individuals carrying the BMPR-IB_g.746G allele were selected through open core population breeding technology to form a core population of 1,500 (jointly implemented by Henan Luyuan et al.).

[0094] 6.2 Dynamically adjust breeding standards Based on the characteristics of the growth environment of prolific sheep, such as the hot summer in the local area, the focus is on selecting individuals with strong tolerance to heat. Based on changes in market demand for mutton quality, such as increased requirements for tenderness, the selection of meat quality-related traits is strengthened. For example, if a generation of prolific sheep is found to have reached the expected growth rate, but the tenderness of the meat is not ideal, individuals carrying superior alleles related to tenderness can be selected for subsequent breeding based on microarray analysis results. Furthermore, in subsequent performance testing, greater emphasis is placed on measuring and evaluating meat tenderness.

[0095] VII. Enhancement of Adaptability and Resource Utilization 7.1 Environmental adaptability assessment During the breeding process, the environmental characteristics of the Central Plains region were taken into consideration. The selected sheep flocks were regularly placed in simulated grazing and stall feeding environments to observe their adaptation. Different environmental conditions were set up, such as simulating different seasons' temperatures, humidity, light duration and intensity, as well as different types and methods of forage supply, to observe the sheep's feeding, growth, and health status.

[0096] Individuals that perform poorly under different environmental conditions, such as those that show a significant decrease in food intake, stunted growth, or susceptibility to disease during hot summers, will be marked and recorded. Subsequent genetic testing will be used to analyze their possible genetic factors.

[0097] Screening for heat tolerance: Using HSP70 gene clusters (such as HSP70_rs2023C) in the chip to associate with high-temperature phenotype data (feed intake, body temperature) to screen for heat-tolerant individuals.

[0098] 7.2 Assessment of Straw Resource Utilization Considering the abundant straw resources in the Central Plains agricultural region, this study aimed to evaluate the digestibility and utilization rate of straw feed in prolific sheep. In a feeding trial, different proportions of straw feed were added to the sheep's daily diet, and their feed intake and weight changes were observed. Simultaneously, by combining genetic testing with the feeding trial, gene loci related to digestion and nutrient metabolism were analyzed to select individuals that can efficiently utilize straw resources. For example, gene chip analysis was used to screen for individuals with more efficient cellulase genes or other digestive enzyme genes to improve the flock's ability to utilize straw resources.

[0099] Straw utilization optimization: By typing cellulase gene (CEL), straw digestibility was increased to 18%.

[0100] VIII. Pilot-scale test 8.1 Experimental Objectives To assess the growth and reproductive performance of prolific meat sheep, and to compare their differences with existing breeds in terms of growth rate, weight gain, feed conversion efficiency, and reproductive rate.

[0101] 8.2 Experimental Design Representative meat sheep breeds were selected as the control group, and the subjects were randomly assigned to different experimental groups to ensure sufficient numbers in each group and the reliability of the experimental results. Strict control was exercised over experimental conditions, including feed, water, housing, and husbandry management, ensuring that the experimental and control groups were raised in the same environment to avoid interference from external factors. The nutritional composition of the feed was precisely measured to ensure consistent nutritional levels across different groups. Automated watering and feeding equipment was used to ensure unrestricted but precisely measured water and feed intake for the sheep.

[0102] 8.3 Growth and Development Performance Assessment Regularly measure the weight and height of multiparous sheep and record their growth curves; use electronic tags or other identification systems to mark the sheep for long-term tracking of individual sheep's growth data. Record feed intake and feed conversion ratio; use an intelligent feeding system to accurately record the feed consumption of each sheep and calculate the feed conversion ratio based on weight changes. Measure body conformation indicators such as backfat thickness, chest depth, and shoulder width; use ultrasound equipment to measure backfat thickness to ensure accuracy and repeatability.

[0103] 8.4 Reproductive performance assessment Sexing of multiparous sheep is performed, and the times of first estrus and first mating are recorded. Professional estrus monitoring equipment (such as estrus monitoring collars) and software are used to assist in determining estrus information. Reproductive indicators such as gestational length, farrowing rate, and survival rate are accurately recorded. Ultrasound monitoring of the sheep's condition during pregnancy is used to ensure accurate observation and recording of the farrowing process. Birth weight and weaning weight of lambs are measured using electronic scales to ensure data accuracy.

[0104] 8.5 Data Collection and Analysis Data on multiparous sheep are collected regularly, and a database is established for management. All data is entered into the database for easy storage, retrieval, and analysis. Statistical analysis methods are used to process and compare the data, and statistical software is used to perform difference analysis to evaluate the differences in various performance indicators between the multiparous sheep and the control group.

[0105] 8.6 Test Results and Report A comprehensive analysis of the experimental results should be conducted, and an experimental report should be written, detailing the experimental design, methods, data analysis, and conclusions. The report should include detailed data charts, statistical analysis results, and an in-depth discussion of the experimental results, providing a basis for the promotion and further optimization of the new multi-prolific meat sheep breed.

[0106] Through the above specific implementation methods, the 10K liquid phase meat sheep breeding chip is integrated into the entire process of breeding new prolific meat sheep breeds. From gene-level screening, performance testing, multi-generational breeding optimization to the final pilot-scale test, the environmental adaptability and resource utilization are comprehensively considered. The aim is to breed new prolific meat sheep breeds that are suitable for the Huang-Huai Plain region, with high fertility, good meat performance, strong adaptability, and the ability to make full use of local resources.

[0107] In summary, Suffolk white-headed rams (82.78 kg at 12 months of age) were crossed with Small-tailed Han ewes (2.03 lambs per litter), and the F1 generation was genotyped to select superior individuals (carrying the GH gene g.51238G allele). The ewes had an average litter size of 1.88 lambs and a weaning survival rate of 95.83%. The dressing percentage of 6-month-old rams was 56.07%, and the net meat yield was 82.05%. Feed intake decreased by 7.5% under high-temperature conditions (compared to ≥15% for traditional breeds). Compared with Du Han F1 sheep, the prolific meat sheep showed a 12.3% increase in daily weight gain and an 18% improvement in feed conversion ratio.

[0108] The innovative point of this invention is: Functional chip integration: For the first time, the specific loci of Henan local sheep breed (4049) are combined with high fertility genes (BMPR-IB, FecB) for targeted screening to improve regional adaptability; Dynamic model optimization: Based on heritability weights and generation interval parameters, synergistic improvement of multiple traits is achieved; Efficient utilization of resources: By using CEL genotyping, the ability to digest straw is enhanced, reducing breeding costs.

[0109] References: 1.Meng, Y., Zhang, W., Cheng, Y., Wu, Y., Wu, H., He, M., Chen, S.,Man, C., Gao, H., Du, L., Chen, Q.,&Wang, F. (2024). Development and verification of a 10K liquid chip for Hainan black goat based on genotypingby pinpoint sequencing of liquid captured targets. BMC genomic data , 25(1), 44.https: / / doi.org / 10.1186 / s12863-024-01228-8 2.Zhang, B., Ma, J., Shen, L., Li, Y., Xie, S., Li, H., Li, J., Li,X.,&Wang, Z. (2025). Genomic insights into pigeon breeding: GWAS for economictraits and the development of a high-throughput liquid phase arraychip. Poultry science , 104 (3), 104872. https: / / doi.org / 10.1016 / j.psj.2025.104872 3.Rong, Y., Ao, X., Guo, F., Wang, X., Han, M., Zhang, L., Xia, Q.,Shang, F., Lv, Q., Wang, Z., Su, R., Zhao, Y., Zhang, Y.,&Wang, R. (2025).Genome-Wide Association Analysis Revealed Candidate Genes Related to EarlyGrowth Traits in Inner Mongolia Cashmere Goats. Veterinary sciences , 12 (3),192. https: / / doi.org / 10.3390 / vetsci12030192 4.El-Seedy, A. S., Hashem, N. M., El-Azrak, K. M., Nour El-Din, A.,Ramadan, T. A., Taha, T. A.,&Salem, M. H. (2017). Genetic screening of FecB,FecXG and FecXI mutations and their linkage with litter size in Barki andRahmani sheep breeds. Reproduction in domestic animals = Zuchthygiene , 52 (6),1133–1137. https: / / doi.org / 10.1111 / rda.13002

Claims

1. A method for breeding meat sheep based on molecular markers and genome-directed screening, characterized in that, Includes the following steps: (1) Basic population construction: Select healthy male and female meat sheep with typical breed characteristics and clear genetic background, crossbreed them according to the mating ratio to obtain F1 generation crossbred meat sheep, use the F1 generation crossbred sheep as the female line, select the male line, and crossbreed them again to form F2 generation crossbred meat sheep. (2) Molecular marker development and chip integration: This includes steps such as primer design, SNP site verification, and microarray function verification for genes related to multiple target traits in meat sheep. The primer design for genes related to multiple target traits in meat sheep includes steps such as conventional primer design, mixed PCR amplification and Sanger sequencing, and specific primer design and FLU-ARMS genotyping. (3) Gene detection and analysis: The process includes steps such as using the "Yuyang Muxin" chip to perform gene testing and genotyping on F1 and / or F2 generation hybrid sheep, phenotype-genotype association analysis, genome selection and dynamic genetic model optimization; (4) Production performance measurement and evaluation The process includes steps such as identifying the body shape and appearance of multiparous meat sheep, measuring their growth and development performance, measuring their reproductive performance, and studying their fattening effects and slaughter performance. (5) Multigenerational selection and phenotypic enhancement This includes steps such as core group construction and updating, and dynamic adjustment of breeding standards; (6) Enhanced adaptability and resource utilization It includes steps such as environmental adaptability assessment and straw resource utilization assessment.

2. The method according to claim 1, characterized in that, In step (1), the mutton sheep are prolific mutton sheep; the male mutton sheep are white-headed Suffolk sheep; and the female mutton sheep are small-tailed Han sheep. In step (1), when crossbreeding again, white-headed Suffolk sheep are selected as the father and F1 generation crossbred sheep are selected as the mother.

3. The method according to claim 1, characterized in that, In step (2), when designing ordinary primers, based on the sheep reference genome and the known genes related to the target trait of multiparous meat sheep, ordinary primers are designed with the significant variation sites of the target gene as the center, taking 400bp sequences upstream and downstream.

4. The method according to claim 1, characterized in that, In step (2), when performing mixed PCR amplification and Sanger sequencing, ordinary primers are used to perform ordinary PCR amplification on the mixed DNA sample.

5. The method according to claim 1, characterized in that, In step (2), when designing specific primers and performing FLU-ARMS typing, specific primers are designed with the new SNP site as the center and the upstream and downstream 200bp sequences are taken. The Touchdown PCR program is used to perform FLU-ARMS typing operation to genotype the core group individuals and perform agarose gel electrophoresis to obtain genotype data. When designing specific primers, the probe structure used is: 5'-FAM-CGCGTCG-BHQ1-3', with the 5' end of the reverse primer labeled with the HEX fluorescent group.

6. The method according to claim 1, characterized in that, In step (2), during SNP site verification, mixed PCR amplification and Sanger sequencing were used to discover new SNP sites; the genotype was verified by FLU-ARMS typing technology. The conditions for mixed PCR amplification were: annealing temperature gradient: 55-65℃; the PCR program used for FLU-ARMS genotyping was: 61℃→55℃, 10 cycles; 55℃→65℃, 30 cycles.

7. The method according to claim 1, characterized in that, In step (3), before gene testing, blood or tissue samples are collected from F1 generation hybrid sheep. 5-10 ml of blood is collected from the jugular vein and placed in a blood collection tube containing an anticoagulant, or an appropriate amount of ear tissue sample is collected.

8. The method according to claim 1, characterized in that, In step (3), during gene detection, the extracted DNA sample is processed and loaded onto the "Yuyang Muxin" chip. Through analysis of these sites, individuals carrying superior gene combinations are screened out. In step (3), during genotyping, the "Yuyang Muxin" chip is used to perform genotyping on the F1 generation of hybrid sheep and construct a whole genome SNP database; In step (3), during the phenotype-genotype association analysis, a linear model is used to perform association analysis between genotype data and phenotype data, and to construct the phenotype-genotype model formula.

9. The method according to claim 1, characterized in that, In step (3), the genome selection and dynamic genetic model optimization are carried out by a two-dimensional screening model based on microarray scoring and FLU-ARMS validation.

10. The method according to claim 1, characterized in that, In step (5), the core group is constructed as follows: based on the F2 generation of the male and female parent hybrids, individuals carrying the BMPR-IB_g.746G allele are selected through open core group breeding technology to form a core group of 1500 individuals. In step (5), the environmental adaptability assessment includes heat and humidity resistance screening: using the HSP70 gene cluster in the chip to associate high temperature phenotype data to screen for heat-resistant individuals; In step (5), the straw resource utilization assessment includes straw utilization optimization: by cellulase genotyping, the straw digestibility is increased to 18%.

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