A method for recovering a close relative cross of a threatened pig breed boar family

Abstract

The application belongs to the technical field of livestock genetic resource protection and utilization, and particularly relates to a kind of endangered pig breed boar family's close relative hybridization recovery method, which first calculates individual kinship based on whole genome SNP chip and G matrix, selects sows with no kinship with existing boars with kinship coefficient <0.1 as threshold value and divides family; the blood of target pig species is purified by stepwise backcrossing generation by generation, and the blood of new boars is identified with blood purity ≥87.5%; a genetic resource backup library is established synchronously, and key individuals (somatic cells with recovery activity ≥90%) and frozen semen (thawing activity ≥30%) are saved. The method has been successfully applied to the protection of Luopanshan pig, adding one boar family, effectively alleviating its endangered state, ensuring the continuity of the creation process and enhancing the genetic diversity of the population.

Description

Technical Field

[0001] This invention belongs to the field of livestock and poultry genetic resource protection and utilization technology, specifically relating to a method for the crossbreeding and restoration of closely related species in endangered boar breeds. Background Technology

[0002] Inbreeding restoration in pigs is an effective genetic resource creation strategy employed in the protection and pedigree continuation of endangered pig breeds when boar pedigree resources are extremely scarce. When a breed faces the risk of extinction due to factors such as a sharp decline in population size, low market demand, or an incomplete breeding system, this method can gradually restore and reconstruct the boar pedigree of the endangered breed by utilizing closely related pig breeds with similar genetic backgrounds. This enhances the genetic diversity and sustainable reproductive capacity of the population while maintaining its core germplasm characteristics. However, existing technologies still have several shortcomings in practical applications, as detailed below: Traditional kinship analysis methods primarily rely on pedigree information. However, pedigree information for endangered pig breed conservation populations is often severely lacking, leading to significant discrepancies between individual kinship assessments and actual conditions. This makes it difficult to accurately screen sows unrelated to existing boar families. Furthermore, current techniques use only kinship coefficients as a single reference standard for maternal selection, neglecting inbreeding coefficients between individuals. This fails to rigorously mitigate inbreeding risks at the molecular level, potentially reducing genetic diversity in offspring and impacting the quality of newly created boar bloodlines.

[0003] Current progressive backcrossing techniques typically rely on theoretical values ​​to determine the bloodline purity of individuals across generations, assuming that backcrossing four generations (F4 generation) can yield a new boar bloodline with a target breed bloodline content of 93.75%. However, actual results show that the actual bloodline content of F4 generation individuals often falls short of the theoretical value, only slightly exceeding the theoretical value of 87.5% for F3 generation. This indicates a significant bias in judging bloodline purity based solely on theoretical generations, and a lack of precise molecular-level monitoring methods based on whole-genome SNP locus information, leading to inconsistent and unscientific standards for identifying new boar bloodlines.

[0004] The creation of new boar bloodlines is a lengthy process, requiring at least four generations of core parent sows, with each generation's mating only permitted after the hybrid boars have reached reproductive maturity. Given the current prevalence of African swine fever, core parent sows and all generations of hybrid boars are highly susceptible to death from disease or other unforeseen events, disrupting the creation process and causing irreparable loss of genetic resources. Current technology lacks a systematic genetic resource backup system covering the entire restoration process, making it impossible to resume the creation process from its interruption using techniques such as somatic cell cloning and frozen semen, severely impacting the continuity and success rate of new boar bloodline creation.

[0005] Therefore, developing a method for crossbreeding and regenerating closely related boar families of endangered pig breeds that can accurately screen unrelated maternal lines, monitor bloodline purity in real time, and have a complete genetic resource backup mechanism is of great significance for improving the efficiency and success rate of rescue protection of endangered pig breeds. Summary of the Invention

[0006] The purpose of this invention is to provide a method for the crossbreeding and restoration of closely related boar families of endangered pig breeds, which is of vital importance to advancing the rescue and protection of endangered pig breeds.

[0007] The objective of this invention is achieved through the following technical solution: This invention provides a method for the crossbreeding and restoration of closely related boar families of endangered pig breeds, comprising the following steps: (1) Select sow individuals that are not related to existing boar pedigrees from the target endangered pig breed population to be restored, and classify sow pedigrees; (2) Using a progressive backcrossing method, the selected sows are crossbred with the selected closely related boars, and individuals with genetic characteristics close to the target pig breed are selected in each generation to continue backcrossing with the target population, thereby gradually purifying the bloodline of the target pig breed. (3) During the entire restoration process, a genetic resource backup library is established simultaneously to preserve the genetic material of key individuals and ensure the continuity of the creation process.

[0008] Furthermore, the kinship screening described in step (1) specifically includes: systematically collecting ear tissue or blood samples from all individuals within the target population, taking precautions against African swine fever and other biosecurity measures during sampling, extracting genomic DNA, measuring DNA concentration using NanoDrop 2000, and identifying the integrity of DNA samples by 1% agarose gel electrophoresis; genotyping qualified DNA samples using whole-genome SNP chips, and using Plink (V1.90) software for quality control of genotype data, with the quality control standards being: using only sites on autosomes, SNP detection rate ≥90%, individual detection rate ≥90%, minimum allele frequency <0.01, and Hardy Weinberg P value ≥0.000001; constructing a genomic relationship G matrix using Gamatrix (V2) software, calculating the kinship coefficient between individuals, and combining the IBS genetic clustering results, performing population clustering analysis using the neighbor-joining method in Mega X (V10.0) software to complete the molecular pedigree division of the population.

[0009] Furthermore, in step (1), the kinship coefficient between individuals is less than 0.1 as the threshold for determining no kinship; at the same time, when selecting the maternal parent, the inbreeding coefficient between individuals is taken into account, and individuals with shorter ROH length and smaller FRoh value are given priority to avoid inbreeding and maintain the genetic diversity of the offspring population.

[0010] Furthermore, the progressive backcrossing described in step (2) specifically includes: selecting a boar of a closely related pig breed with a similar genetic background to the target pig breed to be restored as the paternal parent for hybridization, and using a sow obtained in step (1) that is not related to the existing boar as the backcrossing mother; selecting the boar that is closest in body shape and appearance to the breed to be restored from the F1 generation individuals produced by the two hybridizations, raising it to sexual maturity and then backcrossing it with the original backcrossing mother to produce the F2 generation, and repeating this progressive backcrossing process.

[0011] Furthermore, in step (2), when the purity of the target pig breed bloodline of the backcross offspring reaches 87.5% or above, it is considered as a newly created boar bloodline; if it is necessary to further increase the proportion of the target pig breed bloodline, the same progressive backcrossing method can be used to create higher generations of boars.

[0012] Furthermore, the bloodline purity identification of each generation of individuals in step (2) specifically includes: collecting ear tissue samples of hybrid offspring, performing SNP typing using the "Shenxin No. 1" SNP chip, extracting and merging the obtained SNP typing data file with the variety identification model file to generate a Tmp.bed file, and using the ADMIXTURE v1.3.0 software to execute the command "AdmixtureTmp.bed 25 -supervised -j12" to perform calculations and obtain the bloodline percentage composition of the hybrid offspring.

[0013] Furthermore, the genetic resource backup library mentioned in step (3) includes a somatic cell preservation section, specifically: all somatic cells of sows that can be used for the creation of new boar bloodlines are preserved, and somatic cells of boars of each generation are also preserved; the quality of the preserved somatic cells is randomly inspected, requiring that the cell viability after cryopreservation and thawing reach more than 90%, and that the cell growth curve trends before cryopreservation and after thawing are basically consistent and both show an "S" shape, and that there is no significant difference in the cell cycle distribution between cryopreserved cells and fresh cells.

[0014] Furthermore, the genetic resource backup library mentioned in step (3) also includes a frozen semen preservation section, specifically: using a programmed freezing method to freeze and preserve the semen of boars of each generation during the restoration process, requiring that the semen motility reach more than 30% after freezing and thawing before it can be stored in the library; at least one frozen semen is preserved for each family and each generation of boars.

[0015] Furthermore, when key pigs die during the creation process, individuals with the same genetic traits can be restored from preserved somatic cells using somatic cell cloning. Somatic cell cloned embryos are cultured to the 2-cell stage for transplantation, and each recipient sow can obtain 1-3 weaned piglets. The cloned offspring can be directly used for subsequent crossbreeding and regeneration.

[0016] Furthermore, the target endangered pig breed to be restored is the Luopanshan pig; the closely related boars are selected from boars in the lake and mountain pig group other than the Luopanshan pig, including the Penzhou Mountain Pig, Quxi Pig, and Hechuan Black Pig.

[0017] The beneficial effects of this invention are as follows: This invention addresses the severe scarcity of boar pedigrees in endangered pig breeds, even facing the risk of extinction. It provides an effective strategy for creating genetic resources, gradually restoring and reconstructing the boar bloodline of the endangered breed by utilizing closely related pig breeds with similar genetic backgrounds. This enhances the genetic diversity and sustainable reproductive capacity of the population while maintaining its core germplasm characteristics. This method has been successfully applied in the rescue conservation of the Luopanshan pig. Through the implementation of a national key research and development program, a mature technical system has been formed, adding a new boar pedigree to the Luopanshan pig, which originally had only one surviving pedigree, effectively alleviating the breed's near extinction crisis.

[0018] This invention employs a genome-wide SNP chip-based kinship analysis method, utilizing the G matrix to calculate the kinship coefficient between individuals. Compared to kinship analysis based solely on pedigree information, the results are more accurate and closer to reality, making it particularly suitable for breeding populations with severely missing pedigree information. During kinship screening, a kinship coefficient of less than 0.1 is used as the threshold for determining no kinship, which is stricter than the traditional standard of no kinship beyond three generations. Simultaneously, inbreeding coefficients are considered during maternal selection, prioritizing individuals with shorter ROH lengths and smaller FRoh values. This strict avoidance of inbreeding at the molecular level effectively maintains greater genetic diversity in offspring populations and improves the success rate of new boar bloodline creation.

[0019] This invention optimizes the pedigree purity assessment criteria during the progressive backcrossing process. Based on actual creation results, it uses a pedigree purity of 87.5% or higher for the target pig breed as the standard for identifying the lineage of newly created boars. Compared to the theoretical standard of 93.75% purity after four generations of backcrossing, this allows for earlier identification of the new boar's lineage, effectively shortening the creation cycle. Furthermore, if it is necessary to further increase the pedigree of the target pig breed, the same progressive backcrossing method can still be used to create higher-generation boars, demonstrating good flexibility and scalability.

[0020] This invention establishes a genetic resource backup library simultaneously throughout the entire restoration process, systematically preserving genetic materials such as somatic cells and semen from key individuals. This effectively solves the problems of long cycles in creating new boar bloodlines and the potential for interruptions in the creation process due to disease or accidental death of core breeding pigs. The preserved somatic cells retain over 90% viability after cryopreservation and thawing. The cell growth curves before and after cryopreservation show a consistent trend, both exhibiting an "S" shape. There is no significant difference in cell cycle distribution between cryopreserved and fresh cells, ensuring stable and reliable quality. When key pigs die during the creation process, individuals with identical genetic traits can be restored from the preserved somatic cells using somatic cell cloning. Somatic cell cloned embryos are cultured to the 2-cell stage for transplantation. Each recipient sow can yield 1-3 weaned piglets, and the cloned offspring can be directly used for subsequent crossbreeding and restoration mating.

[0021] This invention employs a programmed freezing method to cryopreserve semen from boars of each generation during the restoration process. Semen motility must reach at least 30% after thawing before it can be stored in the repository. Furthermore, at least one frozen semen sample must be preserved from each family and each restoration generation, providing additional genetic backup for the restoration work. In the context of the ongoing African swine fever epidemic, this genetic resource backup repository is a key technological contingency plan for addressing disease threats and a core risk management strategy to ensure the smooth continuation of new boar pedigree creation. It is of paramount importance for advancing the rescue and protection of endangered pig breeds. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0023] Figure 1 This is a technical roadmap for the intermediate backcrossing technique in this invention; Figure 2 These are growth curves of different generations of Luopanshan pig cells before cryopreservation and after thawing in this invention; Figure 3 This is a cell cycle distribution diagram of fresh cells and frozen cells in this invention; Figure 4 This is a statistical chart of cell embryo cleavage rate in this invention. Detailed Implementation

[0024] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0025] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0026] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0027] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0028] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0029] Example 1: Reconstruction of closely related breeds from the Luopanshan boar family through hybridization 1. Preparation of reagents, consumables and instruments The instruments, equipment, and reagents required for sample collection and DNA preparation in this embodiment are as follows: 1.1 Main Instruments and Equipment The main equipment required for DNA extraction is shown in Table 1 below.

[0030] Table 1 DNA Extraction Equipment

[0031] 1.2 Main Equipment Micropipettes: 0.5-10μL, 2-20μL, 20-200μL, 100-1000μL, with an accuracy error ≤1%, used for accurately transferring minute amounts of liquid. Graduated cylinders: 100mL and 1000mL, used to measure a specific volume of liquid. Metal scissors and tweezers: used for cutting and picking up tissue samples. 1.3 Main Reagents and Consumables Nuclease-free centrifuge tubes: 1.5 mL / 2.0 mL, for storing DNA or other liquids. Nuclease-free pipettes: 10μL, 200μL, 1000μL, for transferring experimental liquids. Tissue DNA Extraction Kit: Suitable for DNA extraction from 1-30 mg tissue samples. Proteinase K: Working concentration 50-200 μg / mL, used to inactivate protease activity in tissues. RNase A: Working concentration 20-100 μg / mL, used to degrade RNA to obtain pure genomic DNA. All instruments and equipment are in normal operation and are regularly maintained. Measuring instruments, constant temperature chambers, water baths, etc. are calibrated and verified according to the prescribed time. Glassware is soaked in potassium dichromate solution, rinsed with tap water and distilled water, sealed with tin foil and sent to a constant temperature drying oven for drying and sterilization. Metal scissors and tweezers are wiped and disinfected with 75% alcohol and used after the alcohol has evaporated.

[0032] 2. Family structure classification of the core breeding group of Luopanshan pigs 2.1 Sample Collection The system collects ear tissue or blood samples from all individuals within the core breeding population of Luopanshan pigs. Each sample corresponds to an individual ID, and the sampling process strictly adheres to biosecurity control measures such as those against African swine fever.

[0033] Ear tissue collection: Use ear forceps to collect ear tissue, avoiding areas rich in blood vessels. Immediately place the tissue into a sample collection tube containing 75% alcohol, label it, and place it on an ice pack for temporary storage. After all collections are completed, transfer the tissue to a -20°C environment for long-term storage.

[0034] Blood collection: Use a nasal tampon to effectively restrain pigs. Blood should be drawn from the anterior vena cava first. After strict disinfection, perform a smooth puncture and draw blood. Apply sufficient pressure to stop bleeding after collection. Collect blood samples using EDTA anticoagulant tubes (purple) and mix gently immediately. Store at 4°C for several days for short-term storage, and after aliquoting, store at -80°C for several years for long-term storage. Heparin anticoagulation is strictly prohibited to avoid hemolysis and high temperatures.

[0035] 2.2 DNA Preparation DNA was prepared using the phenol-chloroform extraction method or according to the instructions of the tissue DNA extraction kit. DNA concentration was determined using NanoDrop 2000, and DNA sample integrity was assessed by 1% agarose gel electrophoresis. Extracted DNA was stored at -20°C for later use.

[0036] 2.3 Genome-wide SNP genotyping and data quality control Genotyping of qualified DNA samples is performed using a medium-to-high density SNP chip (such as 50K) covering the entire genome. The specific procedure is as follows: DNA is hybridized with probes fixed on the chip, unbound DNA is removed, single base extension or ligation reaction is performed, and fluorescent labeling is added. Finally, the fluorescence signal is read by a scanner to determine the genotype of each SNP locus.

[0037] The quality control of genotype data was performed using Plink (V1.90) software, with the following screening criteria: only autosomal loci were used, SNP detection rate ≥90%, individual detection rate ≥90%, minimum allele frequency <0.01, and Hardy Weinberg P value ≥0.000001.

[0038] 2.4 Kinship Calculation and Molecular Patriarchal Classification Based on the genomic relationship G-matrix construction method proposed by VanRaden, the Gamatrix (V2) software was used to construct the genomic relationship G-matrix and calculate the kinship coefficients among individuals within the preservation core population. The formula for calculating the G-matrix is ​​as follows: \(G=\frac{ZZ'}{2\sum p_i(1-p_i)}\) where G is an n×n genomic relationship symmetric matrix (n is the number of individuals); Z is a centered and normalized n×m genotype matrix (m is the number of SNP markers); Z' is the transpose of Z; and \(p_i\) is the reference allele frequency of the i-th SNP locus.

[0039] Based on the genetic clustering results of state homology (IBS), the Neighbor-Joining (NJ) method in Mega X (V10.0) software was used to perform population clustering analysis and complete the molecular pedigree division of the core group for the preservation of Luopanshan pigs. The results are shown in Table 2 below.

[0040] Table 2. Family pedigree classification results of the core breeding group of Luopanshan pigs.

[0041] 3. Screening of sow families for reconstitution through close hybridization Using a kinship coefficient of less than 0.1 as a threshold (theoretically, animals beyond three generations can be considered unrelated; this embodiment raises the screening threshold to ensure accuracy), further pedigree division is performed on female animals classified as "other" in the pedigree division results. Simultaneously, to strictly avoid the risk of inbreeding and maintain offspring genetic diversity, the maternal selection considers individual inbreeding coefficients, prioritizing individuals with shorter homozygous fragment (ROH) lengths and lower ROH proportions (FRoh).

[0042] In the end, a total of 6 sow families that were not related to the existing Luopanshan boar family were selected, and the results are shown in Table 3 below.

[0043] Table 3 Six sow families unrelated to existing boar families

[0044] 4. Selection of closely related boars and progressive backcrossing 4.1 Selection of male parent for hybridization Boars of pig breeds with a similar genetic background to the breed to be restored were selected as the original sires. In this embodiment, the Luopanshan pig belongs to the Huchuan mountain pig group. Therefore, boars from the six groups of Huchuan mountain pigs, excluding the Luopanshan pig, were selected as the hybrid sires. Based on the specific estrus time of the sows and the actual availability of semen from closely related pig breeds at the time of mating, semen from boars of the Penzhou Mountain Pig, Quxi Pig, and Hechuan Black Pig was finally selected.

[0045] 4.2 Selection of female parent for hybridization Sows that are not related to existing Luopanshan boars, as obtained through the above screening, were selected as backcrossing mothers.

[0046] 4.3 Progressive Backcrossing Technique like Figure 1 As shown, the selected sows are crossed with chosen closely related boars to produce the F1 generation. From the F1 generation, boars that most closely resemble the Luopanshan pig in body shape and appearance are selected and raised to sexual maturity. These boars are then backcrossed with the original sows to produce the F2 generation of boars, and this backcrossing process is repeated. Theoretically, by the F4 generation, the proportion of Luopanshan pig blood in the boars can reach 93.75%.

[0047] like Figure 1 As shown, the technical route for creating new boar bloodlines through progressive backcrossing is as follows: a sow (100% Luopanshan pig bloodline) is crossed with a closely related boar (0% Luopanshan pig bloodline) to produce the F1 generation (50% Luopanshan pig bloodline). The F1 generation boar is backcrossed with the original sow to produce the F2 generation (75% Luopanshan pig bloodline). The F2 generation boar is backcrossed with the original sow to produce the F3 generation (87.5% Luopanshan pig bloodline). The F3 generation boar is backcrossed with the original sow to produce the F4 generation (93.75% Luopanshan pig bloodline).

[0048] During the progressive backcrossing process, strictly follow the requirements of the mating record form and the sow breeding and nursing record form, and simultaneously make mating records and farrowing records, and record in detail the sow's ear tag, breed, parity, estrus time, information of the mating boar, farrowing time, piglet sex, weight, coat color characteristics and other data.

[0049] 5. Identification of the bloodline ratio of boars in each generation Each generation of hybrid offspring must undergo blood purity monitoring, and the specific methods are as follows: (1) Collect ear tissue samples from hybrid offspring and perform SNP typing using the "Shenxin No. 1" chip; (2) Extract and merge the obtained SNP typing data file with the variety identification model file to generate the Tmp.bed file; (3) Use ADMIXTURE v1.3.0 software to perform calculations on the integrated data. The command is: AdmixtureTmp.bed 25 -supervised -j12; (4) Import the results into Excel to obtain the bloodline percentage composition of the hybrid offspring.

[0050] Based on whole-genome SNP locus information, the proportion of Luopanshan pig bloodline in boars of each generation was identified, and the results are shown in Table 4 below.

[0051] Table 4. Proportion of Luopanshan boar bloodline in boars of each generation.

[0052] Actual identification results showed that the Luopanshan pig bloodline content of the F4 generation boars did not reach the theoretical value of 93.75%, but it exceeded the theoretical value of 87.5% for the F3 generation. This indicates that as the number of generations increases, the target bloodline content of newly created boars shows an upward trend, but the rate of increase is slower than theoretically expected. Therefore, this embodiment adjusts the criteria for identifying the bloodline of newly created boars as follows: when the target pig breed bloodline purity of the offspring individuals reaches 87.5% or higher (i.e., the purity after three generations of theoretical backcrossing), it can be identified as a newly created boar bloodline; if it is necessary to further improve the bloodline ratio, the same progressive backcrossing method can be used to create F5 generation boars.

[0053] 6. Construction and validation of genetic resource backup library during restoration process To prevent interruptions in the creation process due to diseases such as African swine fever and accidental deaths, this embodiment constructs a genetic resource backup library covering the entire restoration process, including a somatic cell bank and a frozen semen bank, and verifies the feasibility of somatic cell cloning technology.

[0054] 6.1 Construction and Quality Validation of Somatic Cell Banks Somatic cells from nine selected sows suitable for boar bloodline creation were preserved, and somatic cells from each generation of boars were preserved simultaneously during the creation of each generation. Quality checks of the preserved somatic cells showed that cell viability was over 90%.

[0055] like Figure 2 As shown, the growth curves of Luopanshan pig cells before cryopreservation and after thawing at different generations (P3 and P9 generations) are basically consistent, all showing a typical "S" shape, indicating that the cryopreserved somatic cells have normal proliferative capacity and the cryopreservation effect is good.

[0056] like Figure 3As shown, the cell cycle distribution of fresh and cryopreserved cells was highly consistent: fresh cells accounted for 48.1% of the cell cycle, 20.8% of the cell cycle in S phase, and 20.8% of the cell cycle in G2 phase; while cryopreserved cells accounted for 47.2% of the cell cycle in G1 phase, 22.4% of the cell cycle in S phase, and 22.3% of the cell cycle in G2 phase. The RMSD values ​​for the two groups were 2.18 and 2.45, respectively. There were no significant differences in the mean fluorescence intensity of the cell cycle in G1 phase, the mean fluorescence intensity of the cell cycle in G2 phase, and the coefficient of variation, indicating that cryopreservation has no significant impact on the cell cycle of somatic cells, and the quality of the preserved somatic cells is reliable.

[0057] 6.2 Construction and Quality Assessment of Frozen Semen Banks A programmed freezing method was used to cryopreserve semen from boars of each generation during the rehab process. The standard for acceptance into the storage was that the sperm motility after thawing should be above 30%. At least one semen sample was preserved from each family and each rehab generation. Currently, the frozen semen in this embodiment has not been used in the rehab process, but according to relevant experimental statistics, the conception rate of frozen semen from local pigs is between 50% and 60%.

[0058] 6.3 Validation of somatic cell cloning technology Somatic cell cloning experiments were conducted on parent sows. Embryos were cultured to the 2-cell stage and then transplanted. The cleavage rate was statistically analyzed. Figure 4 As shown.

[0059] The litter size of piglets obtained through somatic cell cloning shows that each recipient sow can produce 1-3 weaned piglets. Currently, one family's cloned sow has been successfully bred with its F1 generation boar, producing four hybrid offspring (F2 generation) (3 males and 1 female). Two of the boars are currently alive, verifying the feasibility of somatic cell cloning technology in the restoration and creation process.

[0060] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A method for the regeneration of closely related male pig families through hybridization, characterized in that, Includes the following steps: (1) Select sow individuals that are not related to existing boar pedigrees from the target endangered pig breed population to be restored, and classify sow pedigrees; (2) Using a progressive backcrossing method, the selected sows are crossbred with the selected closely related boars, and individuals with genetic characteristics close to the target pig breed are selected in each generation to continue backcrossing with the target population, thereby gradually purifying the bloodline of the target pig breed. (3) During the entire restoration process, a genetic resource backup library is established simultaneously to preserve the genetic material of key individuals and ensure the continuity of the creation process.

2. The method according to claim 1, characterized in that, The kinship screening described in step (1) specifically includes: systematically collecting ear tissue or blood samples from all individuals in the target population, taking precautions against African swine fever and other biosecurity measures during sampling, extracting genomic DNA, measuring DNA concentration using NanoDrop 2000, and identifying the integrity of DNA samples by 1% agarose gel electrophoresis; genotyping qualified DNA samples using whole-genome SNP chips, and using Plink (V1.90) software for quality control of genotype data, with the quality control standards being: using only sites on autosomes, SNP detection rate ≥90%, individual detection rate ≥90%, minimum allele frequency <0.01, and Hardy Weinberg P value ≥0.000001; constructing a genomic relationship G matrix using Gamatrix (V2) software, calculating the kinship coefficients between individuals, and combining the IBS genetic clustering results, performing population clustering analysis using the neighbor-joining method in MegaX (V10.0) software to complete the molecular pedigree division of the population.

3. The method according to claim 2, characterized in that, In step (1), the kinship coefficient between individuals is less than 0.1 as the threshold for determining no kinship. At the same time, when selecting the maternal parent, the inbreeding coefficient between individuals is taken into account, and individuals with shorter ROH length and smaller FRoh value are given priority to avoid inbreeding and maintain the genetic diversity of the offspring population.

4. The method according to claim 1, characterized in that, The progressive backcrossing described in step (2) specifically includes: selecting a boar of a closely related breed with a similar genetic background to the target breed of pig to be restored as the paternal parent for crossbreeding, and using a sow that is not related to the existing boar obtained in step (1) as the backcrossing mother; selecting the boar that is closest in body shape and appearance to the breed to be restored from the F1 generation individuals produced by the crossbreeding of the two, raising it to sexual maturity and then backcrossing it with the original backcrossing mother to produce the F2 generation, and repeating this progressive backcrossing process.

5. The method according to claim 4, characterized in that, In step (2), when the purity of the target pig breed bloodline of the offspring reaches 87.5% or above, it is considered a newly created boar bloodline. If it is necessary to further increase the proportion of the target pig breed bloodline, the same progressive backcrossing method can be used to create higher generations of boars.

6. The method according to any one of claims 1 to 5, characterized in that, The bloodline purity identification of each generation of individuals in step (2) specifically includes: collecting ear tissue samples of hybrid offspring, performing SNP typing using the "Shenxin No. 1" SNP chip, extracting and merging the obtained SNP typing data file with the variety identification model file to generate a Tmp.bed file, and using the ADMIXTURE v1.3.0 software to execute the command "Admixture Tmp.bed 25 -supervised -j12" to perform calculations and obtain the bloodline percentage composition of the hybrid offspring.

7. The method according to claim 1, characterized in that, The genetic resource backup library mentioned in step (3) includes a somatic cell preservation section, specifically: all somatic cells of sows that can be used for the creation of new boar bloodlines are preserved, and somatic cells of boars of each generation are also preserved; the quality of the preserved somatic cells is randomly inspected, requiring that the cell viability after cryopreservation and thawing reach more than 90%, and that the cell growth curves before cryopreservation and after thawing are basically consistent and both show an "S" shape, and that there is no significant difference in the cell cycle distribution between cryopreserved cells and fresh cells.

8. The method according to claim 1, characterized in that, The genetic resource backup library mentioned in step (3) also includes a frozen semen preservation section, specifically: using a programmed freezing method to freeze and preserve the semen of boars of each generation during the restoration process, requiring that the semen motility reach more than 30% after freezing and thawing before it can be stored in the library; at least one frozen semen of each family and each generation of boars should be preserved.

9. The method according to claim 7, characterized in that, When key pigs die during the creation process, individuals with the same genetic traits can be restored from the preserved somatic cells using somatic cell cloning. Somatic cell cloned embryos are cultured to the 2-cell stage for transplantation, and each recipient sow can obtain 1-3 weaned piglets. The cloned offspring can be directly used for subsequent crossbreeding and regeneration.

10. The method according to claim 1, characterized in that, The target endangered pig breed to be restored is the Luopanshan pig; the closely related boars are selected from boars in the lake and mountain pig group other than the Luopanshan pig, including the Penzhou Mountain Pig, Quxi Pig, and Hechuan Black Pig.

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