Stone chicken partridge pure line strain construction method

Abstract

The invention belongs to the technical field of genetic breeding, and particularly discloses a stone chicken partridge pure line strain construction method which mainly comprises the following steps: performing gene sequencing and typing after microsatellite amplification, and performing family assembly after analyzing a population genetic structure; and finally, performing propagation and subculture, after two generations, calculating an inbreeding coefficient, and if the inbreeding coefficient is less than 0.01 and the inbreeding increment of the generations is less than 0.003, indicating that the strain is successfully established. Compared with the prior art, the partridge can be rapidly homozygous, and the purity of genetic resource varieties is kept; genetic polymorphism of the partridge can be guaranteed, and a foundation is laid for genetic resource protection; inbreeding decline of partridge performance is effectively avoided, and a foundation is laid for development and utilization of partridge genetic resources and cultivation of a new commercial line.

Description

Technical Field

[0001] The invention relates to the technical field of genetic breeding, in particular to a method for constructing a pure strain of Partridge Partridge. Background Art

[0002] Partridge, also known as Gaga chicken and red-legged chicken, was first bred as a pilot in Jieyang, Guangdong. Later, partridge, as a poultry that can provide meat and eggs, was gradually promoted to the east and west of Guangdong and even the whole country, and formed a certain scale in the 1990s.

[0003] The partridge originated from the wild animal Partridge. After artificial domestication and artificial directional breeding in terms of adaptability, meat production, egg production, etc., the domesticated partridge breed was finally formed. As a special economic poultry, the partridge has enriched the livestock and poultry breeds in my country, promoted the development of animal husbandry, and increased the economic income of animal husbandry practitioners.

[0004] However, there are still some problems in partridge breeding. One is the performance of the breed. At present, the performance of partridge breeds is low or unstable. The reasons are, on the one hand, due to long-term self-breeding and serious inbreeding within the group, which leads to inbreeding depression and performance decline; on the other hand, it is unscientific hybridization. In order to obtain excellent performance and give full play to the hybrid advantages between species, other groups are introduced for hybridization. However, the existing groups have not been selected, and the introduced groups have not been systematically selected. There is even a phenomenon of hybridization with commercial generations as parents and grandparents, which leads to uneven performance of hybrid offspring; second, production management is not standardized. Including incubation, brooding, egg laying, breeding, disease prevention and control, biosafety, feed nutrition, feeding management and other aspects, there is no relatively unified standard, and the production management of each is not orderly, which ultimately leads to unstable performance and the inability to guarantee breeding benefits; third, the technology is backward. As a special economic animal, although partridge has a certain scale, compared with pigs, chickens, cattle and other livestock and poultry, its size is relatively small, the number of practitioners is relatively small, the technical level is low, and the R&D investment is insufficient, which directly affects the development of this industry technology. In the final analysis, it is because the current partridge breed standards are not unified, there is no supporting system, and it is impossible to better integrate the resources of various links such as breeding, propagation, promotion, and consumption to promote the rapid development of this industry. Therefore, establishing a standard partridge breed or strain has become the primary task of developing the partridge industry. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides a method for constructing a pure strain of Partridge, so as to quickly and accurately form a pure strain of Partridge.

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

[0007] A method for constructing a pure strain of Partridge Partridge comprises the following steps:

[0008] S1. Based on the existing quail population, according to the age of the population, select individuals with full feathers, feather colors that meet the characteristics of quails, plump bodies, above average weight, and no disabilities to form an F0 population; the male and female F0 populations are freely mated to produce the F1 population;

[0009] S2. Randomly select a number of partridges from the F0 and F1 groups, half male and half female, and then collect blood samples;

[0010] S3, extracting DNA from blood samples, performing microsatellite amplification, and performing gene sequencing and genotyping after the amplification results are tested on agarose gel;

[0011] S4, performing genetic similarity or genetic distance analysis on the genes amplified by the microsatellite after typing; 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;

[0012] S5. Select excellent male and female partridge individuals whose weight, body length, number of eggs laid and other indicators are above the group average from the F0 group, conduct further blood collection, DNA extraction, microsatellite amplification, and the amplification results are tested by agarose gel, and then gene sequencing is performed again, and genotyping is performed;

[0013] S6. Analyze the individual belonging probability of the selected male and female partridges with excellent traits, sort them according to the individual belonging probability, select male and female partridges with individual belonging probability exceeding 50%, perform family grouping, and construct a basic group;

[0014] S7. According to the family line, adopt the method of inbreeding to produce breeding eggs and pure breeding offspring; the breeding eggs are incubated and hatched according to the half-sib family line, and are marked and raised in groups;

[0015] S8. When the female partridges are put into cages after breeding, they are put into cages according to half-sibling families, and the number of partridges in the cages and the number of eggs laid are recorded every day. In the second egg-laying cycle, male partridges are selected based on the production performance of the female partridges in the first egg-laying cycle, and non-sibling male and female partridges are mated in a ratio of 1:2-5 to produce the next generation;

[0016] S9. Record the number of hatched eggs, fertilization rate, hatching rate, healthy chick rate of each half-sib family, 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 means that the purebred strain of partridge has been successfully constructed.

[0017] Furthermore, in step S2, at least 100 partridge individuals are randomly selected.

[0018] Furthermore, in steps S3 and S5, the number of microsatellite loci is not less than 10, and the number of alleles of each microsatellite is not less than 7; each microsatellite locus is provided with primer F and primer R; each microsatellite locus adopts the following amplification system: 2×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.

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

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

[0021]

[0022]

[0023] Furthermore, the family grouping principle in step S6 is:

[0024] According to the ratio of male to female partridges of 1:2 to 5, the selected male partridges with high individual belonging probability and female partridges with high individual belonging probability are grouped into families, among which the half-sib family coefficient composed mainly of male partridges is not less than 60, and that of female partridges is not less than 300.

[0025] Compared with the prior art, the present invention can quickly produce homozygous partridges and maintain the purity of genetic resource varieties; it can ensure the genetic polymorphism of partridges and lay the foundation for the protection of genetic resources; it can effectively avoid inbreeding depression of partridge performance and lay the foundation for the development and utilization of partridge genetic resources and the breeding of new matching lines. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The invention discloses the inbreeding coefficient of the second generation population after the strain is successfully established in two partridge breeding farms by using the strain establishment method of the invention. DETAILED DESCRIPTION

[0027] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with embodiments. The specific embodiments described herein are only used to explain the present invention and are not used to limit the invention.

[0028] The following example constructs pure strains of quail in two quail breeding farms, providing technical support for the protection and development of quail genetic resources in the two breeding farms. The specific construction process is as follows:

[0029] (1) Selection of basic materials: Based on the existing quail population, select individuals with full feathers, feather colors that meet the characteristics of quails, plump bodies, above-average weights, and no disabilities according to the age of the population to form an F0 population; the F1 population is produced through free mating of male and female quails from the F0 population.

[0030] (2) Sample collection: 100 partridges (half male and half female) were randomly selected from the F0 and F1 populations for blood sample collection.

[0031] (3) Microsatellite amplification: DNA from blood samples was extracted and microsatellite amplification was performed. The number of microsatellite loci was no less than 10, and the number of alleles of each microsatellite was no less than 7. The candidate loci information of microsatellite points is shown in Table 1.

[0032] Table 1 Microsatellite locus information

[0033]

[0034] The following operating system was used for each microsatellite locus: 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, and the reaction program was: 94°C for 2 minutes; 94°C for 30 seconds, Ta for 30 seconds, 72°C for 30 seconds, 40 cycles; 72°C for 15 minutes.

[0036] The amplification results were tested on agarose gel and then sent to a sequencing company for microsatellite gene scanning.

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

[0038] (5) Based on the performance of the F0 group, select excellent male and female individuals whose indicators such as body weight, body size, and number of eggs laid are above the group average, and conduct further blood sampling, DNA extraction, and microsatellite amplification. The microsatellite loci and amplification procedures are the same as step (3).

[0039] (6) Conduct individual attribution probability analysis on the selected excellent males and females, and sort them according to the individual attribution probability. Select males and females with an individual attribution probability of more than 50% to form a family and build a basic group. It should be noted that if the selected excellent male and female individuals are insufficient, some individuals can be selected from the unselected quails and added to the F0 group. Then, genetic population analysis and individual attribution probability calculation are performed to select suitable individuals. Family grouping principle: According to the ratio of male to female quails of 1:2 to 5, the selected male quails with high individual attribution probability and the female quails with high individual attribution probability are family grouped. The coefficient of half-sib families formed mainly with male quails is not less than 60, and the coefficient of female quails is not less than 300.

[0040] (7) Expansion and breeding: According to the family line, adopt the method of inbreeding to produce breeding eggs and pure breeding offspring. Breeding eggs are incubated, hatched and marked according to the half-sib family line. Mixed group breeding.

[0041] (8) Subgeneration: When the female quails are caged after rearing, they are caged according to the half-sibling family, and the number of quails in the cage and the number of eggs laid are recorded every day. In the second egg-laying cycle, based on the production performance of the female quails in the first egg-laying cycle, male quails are selected and non-sibling male quails and female quails are caged in a ratio of 1:2-5 for mating and production of the next generation.

[0042] (9) Data recording: record the number of hatching eggs, fertilization rate, hatching rate, healthy chick rate of each half-sib family, family information of each chick, production performance, etc.

[0043] (10) After two generations, the inbreeding coefficient is calculated using the population pedigree. If the inbreeding coefficient is less than 0.01 and the generational inbreeding increment is less than 0.003, it means that the strain has been successfully established. When subsequently forming families, avoid inbreeding within three generations to ensure that the population inbreeding coefficient is within the allowable range, and breeding can be carried out according to the closed group method.

[0044] After testing, the inbreeding coefficient of the second generation of pure strains of quail in two quail farms was controlled below 0.008, and the generation inbreeding increment was about 0.0031. Figure 1 .

[0045] The technical solution of the present invention is not limited to the above-mentioned specific embodiments. All technical variations made according to the technical solution of the present invention fall within the protection scope of the present invention.

Claims

1. A method for constructing a pure strain of Partridge Partridge, characterized in that: The following steps are involved: S1. Based on the existing quail population, according to the age of the population, select individuals with full feathers, feather colors that meet the characteristics of quails, plump bodies, above average weight, and no disabilities to form an F0 population; the male and female F0 populations are freely mated to produce the F1 population; S2. Randomly select a number of partridges from the F0 and F1 groups, half male and half female, and then collect blood samples; S3, extracting DNA from blood samples, performing microsatellite amplification, and performing gene sequencing and genotyping after the amplification results are tested on agarose gel; S4, performing genetic similarity or genetic distance analysis on the genes amplified by the microsatellite after typing; 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 whose weight, body length and egg number are above the group average from the F0 group, conduct further blood collection, DNA extraction, microsatellite amplification, and the amplification results are tested by agarose gel, and then gene sequencing is performed again, and genotyping is performed; S6. Analyze the individual belonging probability of the selected male and female partridges with excellent traits, sort them according to the individual belonging probability, select male and female partridges with individual belonging probability exceeding 50%, perform family grouping, and construct a basic group; S7. According to the family line, adopt the method of inbreeding to produce breeding eggs and pure breeding offspring; the breeding eggs are incubated and hatched according to the half-sib family line, and are marked and raised in groups; S8. When the female partridges are put into cages after breeding, they are put into cages according to half-sibling families, and the number of partridges in the cages and the number of eggs laid are recorded every day. In the second egg-laying cycle, male partridges are selected based on the production performance of the female partridges in the first egg-laying cycle, and non-sibling male and female partridges are mated in a ratio of 1:2-5 to produce the next generation; S9. Record the number of hatched eggs, fertilization rate, hatching rate, healthy chick rate of each half-sib family, 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 means that the purebred strain of partridge has been successfully constructed.

2. The method for constructing a pure strain of Partridge Partridge according to claim 1, characterized in that: In step S2, at least 100 partridge individuals are randomly selected.

3. The method for constructing a pure strain of Partridge Partridge according to claim 1, characterized in that: In the steps S3 and S5, the number of microsatellite loci is not less than 10, and the number of alleles of each microsatellite is not less than 7; each microsatellite locus is provided with primer F and primer R; each microsatellite locus adopts the following amplification system: 2×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, DNA template (20 ng / μL) 1 μL. Each microsatellite was amplified individually, and the reaction program was: 94°C for 2 minutes; 94°C for 30 seconds, 50-65°C annealing for 30 seconds, 72°C for 30 seconds, 40 cycles; 72°C for 15 minutes.

4. The method for constructing a pure strain of Partridge Partridge according to claim 3, characterized in that: The microsatellite loci are at least 10 of the following microsatellite loci.

5. The method for constructing a pure strain of Partridge Partridge according to claim 1, characterized in that: The family grouping principle in step S6 is: According to the ratio of male to female partridges of 1:2 to 5, the selected male partridges with high individual belonging probability and female partridges with high individual belonging probability are grouped into families, among which the half-sib family coefficient composed mainly of male partridges is not less than 60, and that of female partridges is not less than 300.

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