A method for constructing a partridge pure line

Abstract

The application belongs to the technical field of genetic breeding, and specifically discloses a method for constructing a pure line of ptilophogus percussor, which mainly utilizes microsatellite amplification, gene sequencing and typing, and family grouping after analyzing the genetic structure of a group; finally, after expansion, subculture, and two generations, the inbreeding coefficient is calculated, and if the inbreeding coefficient is less than 0.01 and the inbreeding increment of generations is less than 0.003, it is indicated that the construction of the pure line is successful. Compared with the prior art, the application can quickly homozygous ptilophogus percussor, keep the purity of the genetic resource variety, guarantee the genetic polymorphism of ptilophogus percussor, lay a foundation for genetic resource protection, effectively avoid the inbreeding depression of ptilophogus percussor, and lay a foundation for the development and utilization of ptilophogus percussor genetic resources and the cultivation of new matching lines.

Description

Technical Field

[0001] This invention relates to the field of genetic breeding technology, and in particular to a method for constructing a purebred strain of the rock partridge. Background Technology

[0002] The partridge, scientifically known as *Symplocos edulis*, also called the quack chicken or red-legged chicken, was initially raised as a pilot project in Jieyang, Guangdong. Subsequently, as a poultry species that can provide meat and eggs, the partridge was gradually promoted to eastern and western Guangdong and even throughout the country, reaching a certain scale in the 1990s.

[0003] The partridge originated from the wild rock partridge. Through artificial domestication and selective breeding focusing on adaptability, meat production, and egg production, the domesticated partridge breed was eventually formed. As a special economic poultry species, the partridge has enriched my country's livestock and poultry breeds, promoted the development of animal husbandry, and increased the economic income of livestock farmers.

[0004] However, partridge farming still faces certain problems. First, there are issues with breed performance. Currently, partridge breed performance is generally low or unstable. This is due to several factors: firstly, long-term self-breeding has led to severe inbreeding within the population, resulting in inbreeding depression and decreased performance; secondly, there is unscientific hybridization. To obtain superior performance and fully utilize interspecific hybrid vigor, other populations are introduced for hybridization. However, existing populations have not been selectively bred, and introduced populations have also not undergone systematic selection. There are even instances of using commercial offspring as parents or grandparents for hybridization, leading to inconsistent performance in the offspring. Secondly, production management is not standardized. There are no unified standards for aspects such as hatching, brooding, egg production, mating, disease control, biosecurity, feed nutrition, and feeding management. This lack of standardized practices results in inconsistent performance and compromised profitability. Thirdly, technology is outdated. While partridges are a special economic animal with a certain scale, their size is relatively small compared to livestock such as pigs, chickens, and cattle. This results in a smaller number of practitioners, lower technical levels, and insufficient investment in research and development, directly impacting the industry's technological development. Ultimately, the lack of standardized partridge breeds and supporting systems hinders the integration of resources across breeding, propagation, promotion, and consumption, preventing the rapid development of this industry. Therefore, establishing standardized partridge breeds or strains has become the primary task for developing the partridge industry. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a method for constructing a purebred partridge strain, enabling the rapid and accurate formation of a purebred partridge strain.

[0006] To achieve the above objectives, the present invention is implemented according to the following technical solution:

[0007] A method for constructing a purebred strain of the rock partridge includes the following steps:

[0008] S1. Based on the existing partridge population, select individuals with full plumage, plumage color that conforms to the characteristics of partridges, full body shape, weight above average, and no disabilities according to the age of the population to form the F0 population; and produce the F1 population by freely pairing males and females from the F0 population.

[0009] S2. Randomly select a number of partridge individuals from the F0 and F1 groups respectively, with half males and half females, and then collect blood samples.

[0010] S3. Extract DNA from blood samples, perform microsatellite amplification, and after agarose gel electrophoresis, perform gene sequencing and genotyping of the amplification results.

[0011] S4. Perform genetic similarity or genetic distance analysis on the genes amplified by microsatellites after genotyping; if the genetic similarity is greater than 95% or the genetic distance is less than 0.05, the two populations are considered to have the same genetic background, and continue to step S5; otherwise, return to step S2.

[0012] S5. Select outstanding male and female partridges from the F0 population whose weight, body length, egg production and other indicators are above the population average for further blood collection, DNA extraction, microsatellite amplification, and gene sequencing and genotyping after agarose gel electrophoresis of the amplification results.

[0013] S6. Perform an individual affiliation probability analysis on the selected male and female partridge individuals with excellent traits, and sort them according to the size of the individual affiliation probability. Select male and female partridge individuals with an individual affiliation probability of more than 50% and perform family pairing to construct the basic population.

[0014] S7. According to the family line, use the natural cross method to produce hatching eggs and breed offspring in pure lineage; hatching eggs are incubated and hatched according to half-sib family lineage, and marked and raised in groups;

[0015] S8. When placing the female partridges into cages after they have grown up, they should be caged according to half-sib family lines. The number of partridges in the cage and the number of eggs laid should be recorded daily. In the second laying cycle, based on the production performance of the female partridges in the first laying cycle, male partridges should be selected, and non-sib male and female partridges should be mated at a ratio of 1:2 to 5 to produce the next generation.

[0016] S9. Record the number of hatching eggs, fertilization rate, hatching rate, and chick survival rate for each half-sib family, as well as the family information and production performance of each chick. After two generations, calculate the inbreeding coefficient. If the inbreeding coefficient is less than 0.01 and the generational inbreeding increment is less than 0.003, it indicates that the purebred partridge strain has been successfully constructed.

[0017] Furthermore, in step S2, at least 100 individual partridges are randomly selected.

[0018] Furthermore, in steps S3 and S5, the number of microsatellite loci is no less than 10, and the number of alleles for each microsatellite is no less than 7; each microsatellite locus is provided with primer F and primer R; each microsatellite locus uses the following amplification system: 7 μL of 2×PCR Mix, 6 μL of sterile deionized water, 0.5 μL of primer F at a concentration of 10 pmol / μL, 0.5 μL of primer R at a concentration of 10 pmol / μL, and 1 μL of DNA template (20 ng / μL).

[0019] Each microsatellite was amplified individually, with the following reaction program: 94℃ for 2 minutes; 94℃ for 30 seconds, annealing at 50-65℃ for 30 seconds, 72℃ for 30 seconds, for 40 cycles; 72℃ for 15 minutes.

[0020] Furthermore, the microsatellite sites are at least 10 of the following microsatellite sites.

[0021]

[0022]

[0023] Furthermore, the family pedigree matching principle in step S6 is as follows:

[0024] According to the male-to-female partridge ratio of 1:2 to 5, the selected male partridges and female partridges with high individual affiliation probability were paired up for family formation. Among them, the semi-sibling family formed with male partridges as the main body should have no less than 60 individuals and female partridges no less than 300 individuals.

[0025] Compared with existing technologies, this invention can rapidly homozygous partridges, maintaining the purity of genetic resources; it can guarantee the genetic polymorphism of partridges, laying the foundation for the protection of genetic resources; and it can effectively avoid the inbreeding depression of partridge performance, laying the foundation for the development and utilization of partridge genetic resources and the breeding of new breeding lines. Attached Figure Description

[0026] Figure 1 The inbreeding coefficient of the second generation population after successful establishment of brood lines in two partridge farms using the brood line establishment method of the present invention. Detailed Implementation

[0027] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention.

[0028] The following embodiments demonstrate the construction of purebred partridge strains in two partridge farms, providing technical support for the protection and development of partridge genetic resources in both farms; the specific construction process is as follows:

[0029] (1) Selecting basic materials: Based on the existing partridge population, select individuals with full feathers, feather color that conforms to the characteristics of partridges, full body shape, weight above average weight, and no disabilities according to the age of the population to form the F0 population; the F1 population is produced by freely combining male and female members of the F0 population.

[0030] (2) Sample collection: 100 partridge individuals, half male and half female, were randomly selected from the F0 and F1 groups respectively for blood sample collection.

[0031] (3) Microsatellite amplification: DNA was extracted from blood samples and microsatellite amplification was performed. At least 10 microsatellite loci were selected, and each microsatellite contained at least 7 alleles. Candidate microsatellite loci information is shown in Table 1.

[0032] Table 1 Microsatellite location information

[0033]

[0034] For each microsatellite locus, the following operating system was used: the amplification system was 15 μL, including 7 μL of 2×PCR Mix, 6 μL of sterile deionized water, 0.5 μL of primer F (10 pmol / μL), 0.5 μL of primer R (10 pmol / μL), and 1 μL of DNA template (20 ng / μL).

[0035] Each microsatellite was amplified individually, with the following reaction program: 94℃ for 2 minutes; 94℃ for 30 seconds, Ta for 30 seconds, 72℃ for 30 seconds, 40 cycles; 72℃ for 15 minutes.

[0036] After amplification, the results were analyzed by agarose gel electrophoresis and then sent to a sequencing company for microsatellite genome scanning.

[0037] (4) Population genetic structure analysis: Genotyping of the amplified microsatellites and analysis of genetic similarity or genetic distance. If the genetic similarity is greater than 95% or the genetic distance is less than 0.05, the two populations are considered to have the same genetic background and can proceed to the next step; otherwise, return to step (2).

[0038] (5) Select outstanding male and female individuals with indicators such as weight, body size and number of eggs produced that are above the population average, based on the performance of the F0 population. Further blood collection, DNA extraction, microsatellite amplification, and microsatellite loci and amplification procedures are performed in the same way as in step (3).

[0039] (6) Perform individual attribution probability analysis on the selected outstanding male and female birds, and sort them according to their individual attribution probability. Select male and female birds with an individual attribution probability of more than 50% to form families and build the basic population. It should be noted that if there are not enough outstanding male and female birds selected, some individuals can be selected from the unselected partridges to supplement the F0 population, and then genetic population analysis and individual attribution probability calculation can be performed to select suitable individuals. Family formation principle: According to the partridge male-to-female ratio of 1:2 to 5, perform family formation on the selected male partridges with high individual attribution probability and female partridges with high individual attribution probability. Among them, the semi-sib families formed with male partridges as the main body should have no less than 60 birds, and the female partridges should have no less than 300 birds.

[0040] (7) Propagation: According to the family line, use the natural cross method to produce hatching eggs and breed offspring. Hatching eggs are incubated and hatched according to half-sib families and marked. Mixed-group rearing.

[0041] (8) Succession: When the female partridges are caged after they have grown up, they are caged according to half-sib family. The number of partridges in the cage and the number of eggs laid are recorded every day. In the second laying cycle, based on the production performance of the female partridges in the first laying cycle, male partridges are selected and non-sib male and female partridges are placed in the cage and mated to produce the next generation at a ratio of 1:2 to 5.

[0042] (9) Data recording: Record the number of hatching eggs, fertilization rate, hatching rate, and chick survival rate of each half-sibling family, as well as the family information and production performance of each chick.

[0043] (10) After two generations, the inbreeding coefficient is calculated using the population phylogenetic tree. If the inbreeding coefficient is less than 0.01 and the generation inbreeding increment is less than 0.003, it indicates that the strain has been successfully established. When assembling families in the future, inbreeding within three generations should be avoided to ensure that the population inbreeding coefficient is within the allowable range and can be bred according to the closed population method.

[0044] Testing revealed that the inbreeding coefficient of the second generation of partridges from the two partridge farms, after successful establishment of purebred strains, was below 0.008, with a generational inbreeding increment of approximately 0.0031. (See [link to relevant documentation]). Figure 1 .

[0045] The technical solutions of the present invention are not limited to the specific embodiments described above. Any technical modifications made in accordance with the technical solutions of the present invention fall within the protection scope of the present invention.

Claims

1. A method for constructing a partridge pure line, characterized by, The method comprises the following steps: S1. Based on an existing partridge population, according to the population age, select individuals with full feathers, feather color consistent with the characteristics of partridge, full body shape, body weight above the average weight, and no disability to form an F0 population; produce an F1 population by free grouping of the F0 population males and females; S2. Randomly select several partridge individuals, half male and half female, from the F0 and F1 populations respectively, and then collect blood samples; S3. Extract DNA from the blood samples, amplify the microsatellite loci, and after agarose gel detection of the amplification results, perform gene sequencing and genotyping; S4. Perform genetic similarity or genetic distance analysis on the genotyped microsatellite locus amplification genes; if the genetic similarity is greater than 95% or the genetic distance is less than 0.05, it is considered that the genetic backgrounds of the two populations are consistent, and step S5 is continued; otherwise, return to step S2; S5. Select excellent male and female partridge individuals with body weight, body length, and egg production number indicators above the population mean from the F0 population, perform blood collection, DNA extraction, microsatellite locus amplification, and after agarose gel detection of the amplification results, perform gene sequencing and genotyping again; S6. Perform individual attribution probability analysis on the selected male and female partridge individuals with excellent traits, and sort them according to the individual attribution probability, select male and female partridge individuals with individual attribution probability greater than 50%, perform family grouping, and construct a foundation population; S7. According to the family, use the method of this cross to produce species eggs and purebred offspring; the species eggs are hatched and marked according to the half-sibling family, and are group-fed; S8. When the partridges are raised, the female partridges are raised according to the half-sibling family, the number of partridges in the cage and the number of eggs produced per day are recorded, and in the second egg production period, the production performance of the female partridges is selected according to the first egg production period, male partridges are selected, and non-sibling male and female partridges are crossbred at a ratio of 1:2-5 to produce the next generation; S9. Record the number of hatching species eggs, fertilization rate, hatching rate, and healthy chick rate of each half-sibling family, the family information and production performance of each chick; after two generations, calculate the inbreeding coefficient, and if the inbreeding coefficient is less than 0.01 and the inbreeding increment is less than 0.003, it is considered that the partridge pure line strain is successfully constructed. The microsatellite loci are selected from at least 10 of the following table, and each microsatellite locus is provided with a primer F and a primer R, as shown in the following table: 。 2. The method of claim 1, wherein the partridge pure line is constructed by crossing a partridge pure line with a chicken pure line, and selecting a partridge pure line from the offspring of the cross. In step S2, at least 100 partridge individuals are randomly selected.

3. The method of claim 1, wherein the partridge pure line is constructed by crossing a partridge pure line with a chicken pure line, and selecting a partridge pure line from the offspring of the cross. The following amplification system is used for each microsatellite locus: 2x PCR Mix 7 μL, sterile deionized water 6 μL, primer F with a concentration of 10 pmol / μL 0.5 μL, primer R with a concentration of 10 pmol / μL 0.5 μL, and DNA template 20 ng / μL 1 μL; Each microsatellite locus is amplified separately, and the reaction program is as follows: 94℃ for 2 minutes; 94℃ for 30 seconds, 50-65℃ for 30 seconds, 72℃ for 30 seconds, 40 cycles; 72℃ for 15 minutes.

4. The method for constructing a purebred partridge strain according to claim 1, characterized in that, The family grouping principle in step S6 is: According to the ratio of 1:2~5 of male and female, the selected individual with high probability of male and female belonging are grouped, wherein the number of half-sibling families established by male is not less than 60, and the total number of female is not less than 300.

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