A breeding method for a high-breeding, fast-growing, long-legged, slow-feathering Guangxi Ma chicken.

Abstract

This invention relates to the field of poultry breeding technology, specifically to a breeding method for a high-breeding, fast-growing, dwarf, slow-feathering Guangxi Ma chicken. The method includes: constructing a molecular detection system for the sex-linked dwarf gene GHR and the recessive white-feathered RWC gene; hybridizing Guangxi Ma chicken breeding hens with dwarf, recessive white-feathered breeding roosters to obtain a hybrid F1 generation; self-pollinating the F1 generation to produce the F2 generation; using phenotypic selection combined with molecular detection technology to screen out dwarf, slow-feathering Guangxi Ma chicken individuals in the F2 generation; subsequently selecting breeding roosters and hens to form a core group of dwarf, slow-feathering Guangxi Ma chickens; establishing a Generation 0 family; and through successive generations of selection to develop a new high-breeding, fast-growing, dwarf, slow-feathering Guangxi Ma chicken strain. The dwarf, slow-feathering Guangxi Ma chicken strain bred by this invention inherits the dwarf characteristics of dwarf, recessive white-feathered chickens, has a lower feed intake during the laying period compared to Guangxi Ma chickens, and increases egg production, fertilization rate, and egg quality rate, effectively reducing breeding costs; simultaneously, the use of gene detection technology improves the accuracy and efficiency of breeding.

Description

Technical Field

[0001] This invention relates to the field of poultry breeding, and more specifically, to a breeding method for a high-breeding, fast-growing, long-legged, slow-feathering Guangxi Ma chicken. Background Technology

[0002] Guangxi Ma chickens are characterized by their "one mackerel, two slender, and three short" features: moderate weight, tender meat, and slow-growing nature. They are a major slow-growing breed of chicken raised and consumed in Guangxi. Therefore, developing breeding lines for Guangxi Ma chickens with high broiler production, rapid growth, good breast meat quality, good uniformity, and high feed conversion ratio has become an urgent industry need. This invention provides a method for breeding high-breeding, fast-growing, short-legged, slow-feathering Guangxi Ma chickens. This method can quickly cultivate breeding stock for crossbreeding to improve the problems of low meat yield, slow growth rate, low feed conversion ratio, poor uniformity, and high breeding costs associated with Guangxi Ma chickens.

[0003] In yellow-feathered broiler breeding, due to the faster early growth rate and feather growth of fast-feathering strains, purebred lines are often selected towards fast-feathering. In Guangxi Ma chicken production, since purebred lines are often fast-feathering, the sex of newly hatched chicks still requires manual identification. Manual identification is labor-intensive, slow, and causes significant stress and damage to the chicks. This invention develops a slow-feathered, dwarf Ma chicken strain that can be paired with fast-feathered Guangxi Ma chicken purebreds to form a feathering speed self-identification system, simplifying the sexing process and reducing the potential risks of stress, damage, and lateral disease transmission caused by vent examination. Furthermore, it also alleviates the difficulty in recruiting sexing personnel to some extent.

[0004] Due to their low basal metabolic rate, low feed consumption, excellent reproductive performance, strong heat resistance, outstanding stress resistance, and delicious meat, dwarf chickens can effectively increase the stocking density per unit area. Furthermore, the commercial broilers produced by crossbreeding with normal-sized roosters exhibit normal body size and production performance comparable to conventional broilers, resulting in significant economic benefits. Therefore, the introduction of sex-linked dwarf genes has become an important breeding method in the commercial breeding practices of laying hens and broilers both domestically and internationally.

[0005] Traditional methods for introducing sex-linked dwarfism genes rely heavily on breeding experience, selecting offspring based on phenotypic traits rather than directly identifying the genotype of the target gene. This leads to the presence of heterozygous individuals with the sex-linked dwarfism gene in dwarf chicken populations, or the emergence of non-target dwarf individuals due to breeding environmental factors, resulting in low selection accuracy and breeding efficiency. Typically, multiple phenotypic selections are required for gradual purification. Furthermore, dwarf chickens must reach at least 8 weeks of age before homozygous, heterozygous, and homozygous individuals with the sex-linked dwarfism gene can be distinguished based on indicators such as weight and shank length. In contrast, marker-assisted selection technology can effectively overcome the challenges of phenotypic identification, enabling early and precise selection and significantly improving the efficiency of backcross breeding.

[0006] The sex-linked dwarf gene dw (dwarf), as the major gene regulating many quantitative traits such as body weight, feed efficiency, and egg production performance, is the only recessive mutant gene among the eight dwarf genes in chickens that has no adverse effects on the body's health and has positive utilization value. Studies have confirmed that mutations in the chicken growth hormone receptor (GHR) gene are the key factor causing linked dwarfism in chickens. Because dwarf chickens lack growth hormone receptors, the biological function of growth hormone is affected. Currently, four types of GHR gene mutations have been found that lead to sex-linked dwarfism in chickens: (1) a T→C mutation in the last two bases after position 352 of the coding sequence; (2) a 1773bp deletion at position 1744-3516 of the exon 10 untranslated region (UTR); (3) a G to T mutation at position 679 of the coding region; and (4) a single T→C mutation at position 335 of the coding region. In addition, the coexistence of multiple mutations may produce an additive effect, resulting in problems such as large differences in shank length and poor uniformity in dwarf chicken populations.

[0007] Therefore, when using molecular marker-assisted breeding to cultivate sex-linked dwarf chicken breeds, screening for homozygous mutant individuals is crucial for improving the uniformity of the breed and its offspring. Studies by Chen Zhongcan et al. have shown that, compared to normal-sized chickens, dwarf recessive white-feathered chickens induced by GHR gene deletion mutations exhibit significantly enhanced intramuscular fat deposition capacity, a highly significantly reduced muscle fiber diameter, and a highly significantly increased muscle fiber density. This indicates that the muscle histological characteristics and fat deposition capacity of this dwarf chicken are significantly altered, which is beneficial for improving meat quality.

[0008] Recessive white feathering is an important breeding material and is widely used in the breeding of high-quality local chickens in China. Because it is recessive compared to the colored feathers of high-quality local chicken breeds, it has attracted much attention from breeders when using foreign chicken breeds to improve local chickens. There are various types of recessive white feathering genes, among which the tyrosinase (TYR) gene, located on chromosome 1, is the major gene controlling the expression of recessive white feathering and regulates melanin production. Insertion of a retrovirus into either of the two alleles of the TYR gene leads to the expression of the recessive white feathering trait.

[0009] In breeding practice, although there are cases of introducing sex-linked dwarf genes into dwarf yellow-feathered chickens, the offspring of dwarf yellow-feathered chickens and Guangxi Ma chickens have yellow feathers and no speckled features, which cannot meet the specific feather color selection requirements of dwarf slow-feathered Guangxi Ma chickens.

[0010] In the breeding process of introducing desirable traits through hybridization of recessive white-feathered chickens, traditional methods rely on the phenotypic traits of the hybrid offspring for breeding stock, followed by testcross testing, breeding and expansion, etc., resulting in a lengthy breeding cycle. However, molecular marker-assisted identification technology can achieve rapid identification of recessive white-feathered genes.

[0011] Therefore, a breeding method for a high-breeding, fast-growing, short-legged, slow-feathering Guangxi Ma chicken is needed to solve the problems of high breeding cost, slow growth rate, low feed conversion rate, low slaughter rate, and poor uniformity of the existing Guangxi Ma chicken. Summary of the Invention

[0012] In view of this, the present invention addresses the shortcomings of the existing technology by proposing a breeding method for a high-breeding, fast-growing, short-legged, slow-feathering Guangxi Ma chicken, aiming to solve the problems of high breeding cost, slow growth rate, low feed conversion rate, low slaughter rate, and poor uniformity of the existing Guangxi Ma chicken.

[0013] This invention provides a breeding method for the Guangxi Ma Chicken, a high-breeding, fast-growing, long-legged, slow-feathering breed, comprising the following steps:

[0014] Establish molecular detection methods for the sex-linked dwarf gene GHR and the recessive white-feathered RWC gene in chickens;

[0015] Based on body shape and appearance standards, closed-loop breeding was carried out on Guangxi Ma Chicken and Dwarf Recessed White-feathered Chicken to obtain basic populations;

[0016] The dwarf recessive white-feathered roosters and Guangxi Ma chicken hens in the basic population were first screened. The dwarf recessive white-feathered roosters and Guangxi Ma chicken hens selected in the first screening were used as the male and female parents, respectively, and the eggs were collected and incubated to obtain the F1 generation.

[0017] From the F1 generation, select normal-legged, slow-feathered, mottled-feathered roosters that meet the described body shape and appearance standards and contain heterozygotes of the dw gene. Self-cross them with dwarf-legged, slow-feathered, mottled-feathered hens that meet the described body shape and appearance standards. Collect the eggs and incubate them to obtain the F2 generation.

[0018] The F2 generation chicks were raised and then subjected to a second screening. The chicks after the second screening were molecularly tested using the gene molecular detection method. The roosters and hens with mottled feathers that were homozygous for the dw gene and had no recessive white feather gene were selected and raised in individual cages to obtain the basic population of dwarf slow-feathered Guangxi mottled chickens.

[0019] The roosters of the dwarf and slow-feathered Guangxi Ma chickens in the basic population were subjected to a third screening to obtain the final core group of roosters. The roosters of the dwarf and slow-feathered Guangxi Ma chickens in the basic population were subjected to a fourth screening to obtain the final core group of hens.

[0020] The final core group of roosters and hens were randomly assigned to form a 0th generation family, and after successive generations of selective breeding, the high-breeding, fast-growing, short-legged Guangxi Ma Chicken was obtained.

[0021] Furthermore, the specific criteria for body shape and appearance are as follows: the selection criteria for the body shape and appearance of the dwarf recessive white-feathered chicken are recessive white feathers, slow feathers, yellow shanks, and well-developed breast meat; the criteria for the Guangxi Ma chicken are slow feathers, mottled feathers, and yellow shanks.

[0022] Furthermore, the first screening specifically involves: screening for dwarf, recessive white-feathered roosters with shank length between 95-105% of the average, weight between 95% and 110% of the average, ejaculation volume not less than 105% of the average semen volume, semen density greater than 3.5 billion sperm cells / mL, and whose family broodmate hens have a higher family broodmate egg production performance than the family average; and screening for Guangxi Ma chicken hens with an individual egg production of more than 100 eggs at 43 weeks of age and a hatching egg qualification rate not less than 103% of the group average hatching egg qualification rate.

[0023] Furthermore, the second screening specifically involves: when F2 generation chicks are raised to 60-70 days of age, individuals with mottled feathers and a shank length ≤ 6.0 cm are selected.

[0024] Furthermore, the gene molecular detection method specifically includes:

[0025] Blood was collected from the second-selected chicks, and genomic DNA was extracted from the chicken blood.

[0026] Primers for identifying dwarfism genes were designed, as shown in SEQ ID No. 1, SEQ ID No. 2 and SEQ ID No. 3. The genomic DNA of the chicken blood was amplified by PCR and sequenced using the primers for identifying dwarfism genes. Heterozygotes and homozygotes with normal legs were removed, and homozygotes with dwarfism were retained.

[0027] Primers for identifying the recessive white feather gene were designed, as shown in SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6 and SEQ ID No. 7. The recessive white feather gene identification primers were used to perform PCR amplification and sequencing on the chicken blood genomic DNA to eliminate recessive white feather heterozygotes.

[0028] Furthermore, during PCR amplification, the PCR reaction conditions were as follows: 94 ℃ pre-denaturation for 5 min; 94 ℃ denaturation for 30 s, 53 ℃ annealing for 30 s, 72 ℃ extension for 30 s, for 35 cycles; and finally 72 ℃ extension for 5 min.

[0029] Furthermore, the third screening specifically involves: raising dwarf and slow-feathered Guangxi Ma chicken roosters in the basic population of dwarf and slow-feathered Guangxi Ma chickens to 90 days of age, selecting individuals with a weight within the range of average +25% and excellent secondary trait development, and then conducting semen analysis on the selected individuals before they start laying eggs and before they are kept for breeding, selecting roosters with an ejaculation volume greater than or equal to 103% of the average ejaculation volume, a semen viscosity greater than 3.5 billion sperm cells / mL, and a milky white color.

[0030] Furthermore, the fourth screening specifically involves: before the start of egg production in the dwarf and slow-feathered Guangxi Ma chicken basic population, selecting hens with excellent secondary sexual characteristics based on the size and traits of their combs, while collecting the number of eggs laid at 43 weeks, egg weight, and number of eggs laid at 66 weeks, and selecting individuals with more than 100 eggs laid at 43 weeks and a weight greater than the average weight of the hen population.

[0031] Furthermore, when randomly forming a generation 0 family line from the final core group of roosters and hens, the final core group of roosters and hens are randomly formed into a generation 0 family line according to a male-to-female ratio of 1:8.

[0032] Furthermore, in the subsequent generation selection, growth traits were the primary selection trait for roosters, with reproductive traits as a secondary selection trait, while reproductive traits were the primary selection trait for hens, with growth traits as a secondary selection trait. At the same time, slow feathering selection was carried out simultaneously for both males and females.

[0033] Compared with existing technologies, the beneficial effects of this invention are as follows: The dwarf, slow-feathered Guangxi Ma chicken strain bred by this invention not only inherits the dwarf characteristics of the recessive white-feathered chicken, but also reduces feed intake during the laying period by 10%-15% compared to the original Guangxi Ma chicken hens. At 66 weeks of age, daily egg production increases from 157 to 181 eggs, the hatching egg qualification rate increases from 93.5% to 95%, and the hatching egg fertilization rate increases from 93% to 95%, effectively reducing breeding costs. Furthermore, the broiler chickens produced in this strain fully retain the excellent characteristics of the Guangxi Ma chicken breed, such as tender meat and strong disease resistance. They exhibit advantages such as rapid growth, strong disease resistance, high feed conversion rate, good uniformity, excellent meat quality, and protection against breed theft. The market age of the commercial broiler chickens produced from this strain is 5 days earlier than that of the original Guangxi Ma chicken hens. Moreover, because the dwarf mutation type is singular, the uniformity of the commercial broiler chickens produced in this strain is even better. Furthermore, the gene detection technology used in this invention significantly improves the accuracy and efficiency of screening during the breeding process, effectively avoiding genotype segregation in the next generation. Compared to traditional phenotypic selection methods, the breeding cycle is shortened by at least 2-3 generations, greatly accelerating the breeding process. Additionally, this high-breeding, fast-growing, short-legged, slow-feathering Guangxi Ma chicken strain can be paired with a purebred fast-feathering Guangxi Ma chicken to achieve male-feathering / sex identification, simplifying the identification process, reducing stress and injury caused by vent examination, and mitigating the potential for lateral disease transmission. It also alleviates the labor shortage for manual identification to some extent.

[0034] The above general description and the following detailed description are exemplary and explanatory only, and are not intended to limit this disclosure.

[0035] Other features and aspects of this disclosure will become clearer from the following detailed description of exemplary embodiments with reference to the accompanying drawings. Attached Figure Description

[0036] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0037] Figure 1 This is a breeding technology roadmap for the high-breeding-rate, long-legged, slow-feathering Guangxi Ma Chicken provided in this embodiment of the invention;

[0038] Figure 2 This is a graph showing the agarose gel electrophoresis results of the dwarfism gene in an embodiment of the present invention;

[0039] Figure 3 This is a graph showing the agarose gel electrophoresis results of the negative white feather gene in an embodiment of the present invention. Detailed Implementation

[0040] The technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of this application, and not all of the embodiments. Based on the embodiments of this application, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of this application.

[0041] In the description of this application, it should be understood that the terms "center", "upper", "lower", "front", "rear", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer", etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are only for the convenience of describing this application and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on this application.

[0042] The terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. In the description of this application, unless otherwise stated, "a plurality of" means two or more.

[0043] In the description of this application, it should be noted that, unless otherwise expressly specified and limited, the terms "installation," "connection," and "linking" should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral connection; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or an indirect connection through an intermediate medium; and they can refer to the internal connection between two components. Those skilled in the art can understand the specific meaning of the above terms in this application based on the specific circumstances.

[0044] See Figure 1 As shown, a breeding method for a high-breeding, fast-growing, long-legged, slow-feathering Guangxi Ma Chicken according to an embodiment of this application includes the following steps:

[0045] Establish molecular detection methods for the sex-linked dwarf gene GHR and the recessive white-feathered RWC gene in chickens;

[0046] Based on body shape and appearance standards, closed-loop breeding was carried out on Guangxi Ma Chicken and Dwarf Recessed White-feathered Chicken to obtain basic populations;

[0047] The dwarf recessive white-feathered roosters and Guangxi Ma chicken hens in the basic population were first screened. The dwarf recessive white-feathered roosters and Guangxi Ma chicken hens selected in the first screening were used as the male and female parents, respectively, and the eggs were collected and incubated to obtain the F1 generation.

[0048] From the F1 generation, select normal-legged, slow-feathered, mottled-feathered roosters that meet the described body shape and appearance standards and contain heterozygotes of the dw gene. Self-cross them with dwarf-legged, slow-feathered, mottled-feathered hens that meet the described body shape and appearance standards. Collect the eggs and incubate them to obtain the F2 generation.

[0049] The F2 generation chicks were raised and then subjected to a second screening. The chicks after the second screening were molecularly tested using the gene molecular detection method. The roosters and hens with mottled feathers that were homozygous for the dw gene and had no recessive white feather gene were selected and raised in individual cages to obtain the basic population of dwarf slow-feathered Guangxi mottled chickens.

[0050] The roosters of the dwarf and slow-feathered Guangxi Ma chickens in the basic population were subjected to a third screening to obtain the final core group of roosters. The roosters of the dwarf and slow-feathered Guangxi Ma chickens in the basic population were subjected to a fourth screening to obtain the final core group of hens.

[0051] The final core group of roosters and hens were randomly assigned to form a 0th generation family, and after successive generations of selective breeding, the high-breeding, fast-growing, short-legged Guangxi Ma Chicken was obtained.

[0052] Specifically, when randomly forming a generation 0 family line from the final core group roosters and hens, the final core group roosters and hens are randomly formed into a generation 0 family line at a male-to-female ratio of 1:8.

[0053] Understandably, this invention uses dwarf recessive white-feathered chickens and Guangxi Ma chickens as breeding base materials to conduct research on the cultivation of a new high-breeding, fast-growing, dwarf slow-feathering Guangxi Ma chicken strain. The high-breeding, fast-growing, dwarf slow-feathering Guangxi Ma chicken strain prepared by this invention not only possesses the feed-saving characteristics of dwarf chickens, but also reduces feed intake during the laying period by 10%-15% compared to the original Guangxi Ma chicken hens. At 66 weeks of age, the daily egg production reaches over 180 eggs, the qualified rate of hatching eggs increases from 93.5% to 95%, and the fertilization rate of hatching eggs increases from 93% to 95%, effectively reducing breeding costs. The commercial broilers produced by this strain fully retain the excellent characteristics of the Guangxi Ma chicken local breed, such as tender meat and strong disease resistance. They have advantages such as rapid growth, strong disease resistance, high feed conversion rate, good uniformity, excellent meat quality, and anti-theft properties. The market age of the commercial broilers produced by the original Guangxi Ma chicken hens is 3 days earlier. Furthermore, the gene detection technology used in this invention significantly improves the accuracy and efficiency of screening during the breeding process, effectively avoiding genotype segregation in the next generation. Compared to traditional phenotypic selection methods, the breeding cycle is shortened by at least 2-3 generations, greatly accelerating the breeding process. It also serves as a catalyst for the application of molecular marker-assisted selection for other traits in chicken breeding. In addition, this new high-breeding, fast-growing, long-legged, slow-feathering Guangxi Ma chicken strain can be paired with a purebred fast-feathering Guangxi Ma chicken to achieve male / feathering identification, simplifying the identification process, reducing stress and injury caused by vent examination, and mitigating the potential for lateral disease transmission. It also alleviates the difficulty in recruiting identification personnel to some extent.

[0054] In some specific embodiments, the physical appearance criteria are as follows: the physical appearance selection criteria for dwarf reclusive white-feathered chickens are reclusive white feathers, slow feathers, yellow shanks, and well-developed breast meat; the criteria for Guangxi Ma chickens are slow feathers, mottled feathers, and yellow shanks.

[0055] In some specific embodiments, the first screening specifically involves: screening for dwarf, recessive white-feathered roosters with shank length between 95-105% of the average, weight between 95% and 110% of the average, ejaculation volume not less than 105% of the average semen volume, semen density greater than 3.5 billion sperm cells / mL, and whose family lineage of sibling hens has a higher family lineage egg production performance than the average family lineage egg production; and screening for Guangxi Ma chicken hens with an individual egg production of more than 100 eggs at 43 weeks of age and a hatching egg qualification rate not less than 103% of the average hatching egg qualification rate of the group.

[0056] Specifically, during the first screening, the selected dwarf, recessive white-feathered roosters must meet the physical appearance standards and be robust and muscular, while the Guangxi Ma chicken hens must meet the physical appearance standards and be of good shape.

[0057] In some specific embodiments, the second screening specifically involves: when F2 generation chicks are raised to 60-70 days of age, individuals with mottled feathers and a shank length ≤6.0cm are selected.

[0058] In some specific embodiments, the gene molecular detection method is specifically as follows:

[0059] Blood was collected from the second-selected chicks, and genomic DNA was extracted from the chicken blood.

[0060] Primers for identifying dwarfism genes were designed, as shown in SEQ ID No. 1, SEQ ID No. 2 and SEQ ID No. 3. The genomic DNA of the chicken blood was amplified by PCR and sequenced using the primers for identifying dwarfism genes. Heterozygotes and homozygotes with normal legs were removed, and homozygotes with dwarfism were retained.

[0061] Primers for identifying the recessive white feather gene were designed, as shown in SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6 and SEQ ID No. 7. The recessive white feather gene identification primers were used to perform PCR amplification and sequencing on the chicken blood genomic DNA to eliminate recessive white feather heterozygotes.

[0062] In some specific embodiments, the PCR amplification process is performed under the following conditions: 94 °C pre-denaturation for 5 min; 94 °C denaturation for 30 s, 53 °C annealing for 30 s, 72 °C extension for 30 s, for 35 cycles; and finally, 72 °C extension for 5 min.

[0063] Specifically, 0.5 ml of blood was collected from the wing vein of a chicken and placed in an EDTA anticoagulant tube. The sample was inverted and mixed, and stored at -20°C for later use. Genomic DNA was extracted according to the instructions of the blood DNA extraction kit. Based on the previous detection of recessive white-feathered dwarf chickens using PCR amplification and product sequencing, it was found that the sex-linked dwarf gene GHR has four mutation sites that can lead to dwarfism in chickens, namely: (1) a T→C mutation occurring two bases after position 352 of the coding sequence; (2) a 1773bp deletion at position 1744-3516 of exon 10 and UTR; (3) a G→T mutation occurring at position 679 of the coding region; (4) a single-base T→C mutation occurring at position 335 of the coding region. After testing, it was confirmed that only the mutation at the second site (1773bp deletion) exists in the hybrid paternal recessive white-feathered chicken population, and there is only a definite mutation site of 1.7kb in the existing recessive white-feathered dwarf chickens. Based on the DNA sequences of the dwarf gene and the recessive white-feather gene, Primer premier was used to determine the mutation site. Primers were designed using primer design software 5.0 to establish molecular detection methods for chicken dwarfism genes and recessive white feather genes; PCR identification primer information is shown in Table 1.

[0064] Table 1. PCR amplification information for the dwarfism gene and recessive white feather gene.

[0065] ;

[0066] The genomic DNA of the chicken blood was amplified by PCR using primers for identifying dwarfism genes (dw-F1, dw-F2 and dw-R). The PCR reaction system consisted of 25 μL of mixed 12.5 μL of forward and reverse primers, 0.5 μL of template DNA, and 10 μL of ddH2O.

[0067] Reaction conditions: 94 °C pre-denaturation for 5 min; 94 °C denaturation for 30 s, 53 °C annealing for 30 s, 72 °C extension for 30 s, 35 cycles; final extension at 72 °C for 5 min.

[0068] The PCR amplification product was used to detect whether it was the target fragment by 1.5% agarose gel electrophoresis. The results are as follows: Figure 2 As shown, M represents Marker 2000; 1 represents the negative control; 2 represents the dwarf homozygote; 3 represents the normal homozygote; and 4 represents the normal heterozygote. Specifically, when the PCR product size is 400 bp, it indicates no deletion mutation; when the product size is 254 bp, it indicates a deletion mutation homozygote; and when the product shows two bands, it indicates a deletion mutation heterozygote.

[0069] The chicken blood genomic DNA was amplified by PCR using recessive white feather gene identification primers (F1, R1, F2, and R2). The PCR reaction system consisted of 25 μL of mixed 12.5 μL of primers, 0.5 μL of each of the forward and reverse primers, 1 μL of template DNA, and 10 μL of ddH2O.

[0070] Reaction conditions: 94 °C pre-denaturation for 5 min; 94 °C denaturation for 30 s, 53 °C annealing for 30 s, 72 °C extension for 30 s, 35 cycles; final extension at 72 °C for 5 min.

[0071] The PCR amplification product was used to detect whether it was the target fragment by 1.5% agarose gel electrophoresis. The results are as follows: Figure 3 As shown, M represents Marker 2000; 1 represents the negative control; 2 represents the recessive white homozygote; 3 represents the colored feather homozygote; and 4 represents the recessive white heterozygote. Specifically, when the PCR product size is 450 bp, it indicates a colored feather homozygote; when the product size is 226 bp, it indicates a recessive white feather homozygote; and when the product shows two bands, it indicates a recessive white feather heterozygote.

[0072] In some specific embodiments, the third screening specifically involves: when the dwarf slow-feathered Guangxi Ma chicken roosters in the basic population are raised to 90 days of age, individuals with a weight within the range of average +25% and excellent secondary trait development are selected. Subsequently, the selected individuals undergo semen testing before laying eggs and before being used for breeding, and roosters with an ejaculation volume greater than or equal to 103% of the average ejaculation volume, a semen viscosity greater than 3.5 billion sperm cells / mL, and a milky white color are selected.

[0073] Specifically, the third screening also requires screening roosters that meet the aforementioned body shape and appearance standards.

[0074] In some specific embodiments, the fourth screening specifically involves: before the start of egg production in the dwarf slow-feathered Guangxi Ma chicken basic population, selecting hens with excellent secondary sexual characteristics based on the size and traits of their combs, while collecting the number of eggs laid at 43 weeks, egg weight, and number of eggs laid at 66 weeks, and selecting individuals with more than 100 eggs laid at 43 weeks and a weight greater than the average weight of the hen population.

[0075] Specifically, the fourth screening also requires screening hens that meet the aforementioned body shape and appearance standards.

[0076] In some specific embodiments, when the final core group of roosters and hens are randomly assigned to form a generation 0 family, the final core group of roosters and hens are randomly assigned to form a generation 0 family according to a male-to-female ratio of 1:8.

[0077] In some specific embodiments, during the successive breeding process, roosters are selected primarily for growth traits and secondarily for reproductive traits, while hens are selected primarily for reproductive traits and secondarily for growth traits. At the same time, slow feathering selection is carried out simultaneously for both males and females.

[0078] Specifically, in subsequent generations of breeding, roosters were primarily selected for growth traits such as body weight and feed conversion ratio at 90-150 days of age, while reproductive traits such as egg production and semen quality of fraternal hens were also considered. Hens were primarily selected for reproductive traits such as egg production, hatching egg qualification rate, and fertilization rate, while growth traits such as body weight were also considered. Slow-feathering selection was conducted simultaneously for both males and females.

[0079] Chicks are tagged with wing numbers according to their family lineage, and weak or damaged chicks and individuals with green legs are culled.

[0080] Slow-feathering selection: On day 1, spread the wings of each chicken and observe the relative length of the primary wing feathers and the overlying primary wing feathers on the outer wing. Eliminate fast-feathering chickens (primary wing feathers are more than 2mm longer than overlying primary wing feathers). On day 10, select and eliminate individuals with excessively long tail feathers based on the development of the tail feathers.

[0081] Roosters with slow comb development are culled at 42 days of age; hens with underdeveloped combs and yellowish or dull faces are culled at 56 days of age.

[0082] Weigh the entire flock at 70 days old. Before selecting individuals, randomly select 10% of the flock by weight and record the average. For roosters, use -5 to +10% of the average as the breeding range, and for hens, use -8 to +10% as the breeding range, to improve the uniformity of the flock.

[0083] Before converting to egg cages, individuals that do not meet the requirements in terms of body shape and appearance are culled, while individuals with well-developed chest and leg muscles are hand-selected.

[0084] At 90 days old, cull roosters with drooping combs, no combs, excessively high combs, blue legs, or kinked legs are selected. Those with glossy, close-fitting feathers, double wattles, rounded bodies, and medium to high weight are retained. Hens with short combs, no combs, drooping combs, blue legs, or kinked legs are culled. Those with close-fitting feathers, fine down, a few speckled spots, rounded bodies, and medium weight are selected. Chickens testing positive for pullorum disease are culled before being transferred to the laying house.

[0085] From 90 days of age to 150 days of age, roosters were kept in individual cages. Their fasting weight, periodic feed intake, and weight at 150 days of age were recorded. Feed was not controlled during this period, and individual feed conversion ratios were measured. The feed conversion ratio of each rooster was calculated after 150 days of age.

[0086] Semen analysis was performed on 150-day-old roosters, and those with an ejaculation volume greater than or equal to 103% of the average ejaculation volume, a semen viscosity greater than 3.5 billion sperm cells / mL, and a milky white color were selected; roosters and hens were tested for avian leukosis, and positive chickens were culled.

[0087] Before the start of egg production, chickens should be tested for pullorum disease and avian leukosis, and positive and suspected individuals should be culled.

[0088] Before labor begins, the entire group is weighed, and individuals whose weight deviates significantly from the average are eliminated.

[0089] Reproductive performance selection was conducted at 43 weeks of age.

[0090] Hen selection: A combination of family selection and individual selection was used. The egg production at 66 weeks of age of the previous generation was taken into account, and the egg production at 43 weeks of age of the current generation was taken into account. Individuals with egg production higher than the average egg production at 66 weeks of age of the previous generation and a hatching egg qualification rate greater than or equal to 105% of the average hatching egg qualification rate were selected from the offspring of families with egg production higher than the average egg production at 66 weeks of age of the previous generation and from families with egg production higher than the average egg production at 43 weeks of age of the current generation.

[0091] Rooster selection: Based on the performance of siblings, the roosters with the highest egg production of the hens in the current generation at 43 weeks of age are selected from the families with the highest egg production. The roosters must also have a body weight greater than or equal to 110% of the average body weight of roosters, an ejaculation volume greater than or equal to 105% of the average semen volume, a semen consistency greater than 3.5 billion sperm cells / mL, and a low feed conversion ratio between 90 and 150 days of age.

[0092] Selected roosters and hens are paired homogeneously to form families, and pedigree checks are conducted to avoid mating of full and half-siblings. The next generation is bred by repeating the male-to-female ratio of 1:12.

[0093] After 3-4 generations of breeding, a new strain of Guangxi Ma Chicken with high breeding speed, long legs, and slow feathering was finally obtained.

[0094] Example 1

[0095] S1. Collect 0.5 ml of blood sample from the wing vein of chicken and place it in an EDTA anticoagulant tube. Mix by inversion and store at -20℃ for later use. Extract genomic DNA according to the instructions of the blood DNA extraction kit. Based on the DNA sequences of the dwarf gene and recessive white feather gene, design primers using Primer Premier 5.0 primer design software to establish a molecular detection method for the chicken dwarf gene and recessive white feather gene.

[0096] S2. Closed-loop breeding was carried out on Guangxi Ma Chicken and Dwarf Recessed White Feather Chicken according to the body shape and appearance standards to obtain the basic population. The body shape and appearance standards of Guangxi Ma Chicken are recessed white feathers, slow feathers, yellow shanks and well-developed breast meat. The body shape and appearance standards of Dwarf Recessed White Feather Chicken are slow feathers, mottled feathers and yellow shanks.

[0097] S3. Select dwarf, recessive white-feathered roosters with shank length between 95-105% of the average, weight between 95% and 110% of the average, ejaculation volume not less than 105% of the average semen volume, semen density greater than 3.5 billion sperm cells / mL, and whose family sibling hens have higher family egg production performance than the family average, as the sires; select Guangxi Ma chicken hens with an individual egg production of more than 100 eggs at 43 weeks of age and a hatching egg qualification rate not less than 103% of the population average hatching egg qualification rate as the dams. Crossbreed the sires and dams, collect the eggs for incubation, and obtain the F1 generation.

[0098] S4. Select normal-legged, slow-feathered, mottled-feathered roosters that meet the described body shape and appearance standards and contain heterozygotes of the dw gene from the F1 generation, and self-cross them with dwarf-legged, slow-feathered, mottled-feathered hens that meet the described body shape and appearance standards. Collect the eggs and incubate them to obtain the F2 generation.

[0099] S5. The F2 generation chicks are raised until they are 60-70 days old. Individuals with mottled feathers and shank length ≤6.0 cm are selected. The selected chicks are molecularly tested using the gene molecular detection method. Mottled male and female chickens with homozygous dw gene and no recessive white feather gene are selected and raised in individual cages to obtain the basic population of short-legged and slow-feathered Guangxi Ma chicken.

[0100] S6. The roosters in the basic population of the short-legged and slow-feathered Guangxi Ma chickens were screened at 90 days of age. Individuals with a weight within 25% of the average and excellent secondary traits were selected. Semen analysis was then performed on the selected individuals before they started laying eggs and before they were kept for breeding. Roosters with an ejaculation volume greater than or equal to 103% of the average ejaculation volume, a semen consistency greater than 3.5 billion sperm cells / mL, and a milky white color were selected as the final core group of roosters.

[0101] S7. Before the start of egg production in the dwarf and slow-feathered Guangxi Ma chicken basic group, select hens with excellent secondary sexual characteristics based on the size and traits of their combs. At the same time, collect the number of eggs laid at 43 weeks, the egg weight, and the number of eggs laid at 66 weeks. Select individuals with a number of eggs laid at 43 weeks greater than 100 and a weight greater than the average weight of the hen group as the final core group hens.

[0102] S8. Randomly form a generation 0 family line by combining the final core group of roosters and the final core group of hens in a male-to-female ratio of 1:8.

[0103] S9. Conduct successive generational selection and breeding of the aforementioned Generation 0 family lineage;

[0104] S9.1. Hatching chicks should be tagged with wing numbers according to their family lineage, and weak or damaged chicks and individuals with green legs should be culled.

[0105] S9.2 Slow-feathering selection: On day 1, spread the wings of each chicken and observe the relative length of the primary wing feathers and the overlying primary wing feathers on the outer wing. Eliminate fast-feathering type chickens (primary wing feathers are more than 2mm longer than overlying primary wing feathers). On day 10, select and eliminate individuals with excessively long tail feathers based on the development of the tail feathers.

[0106] S9.3. Roosters should be culled at 42 days of age if their combs are underdeveloped; hens should be culled at 56 days of age if their combs are underdeveloped or their faces are yellowish and not rosy.

[0107] S9.4 Weigh the entire flock at 70 days old. Before selecting the weight, randomly select 10% of the individuals in the flock and record the average weight. For roosters, use -5 to +10% of the average weight as the breeding range, and for hens, use -8 to +10% as the breeding range to improve the uniformity of the flock.

[0108] S9.5 Before converting to egg cages, cull individuals that do not meet the requirements in terms of body shape and appearance, and at the same time, hand-select individuals with well-developed chest and leg muscles;

[0109] S9.6. For 90-day-old roosters, culle individuals with drooping combs, no combs, excessively high combs, blue legs, or kinked legs. Select individuals with glossy, close-fitting feathers, double wattles, round body shape, and medium to high weight. For hens, culle individuals with short combs, no combs, drooping combs, blue legs, or kinked legs. Select individuals with close-fitting feathers, fine feathers, a few speckled spots, round body shape, and medium weight. Chickens testing positive for pullorum disease are culled before being transferred to the laying house.

[0110] S9.7 From 90 days of age to 150 days of age, roosters were kept in individual cages. Their fasting weight, periodic feed intake, and weight at 150 days of age were recorded. Feed was not controlled during this period, and individual feed conversion ratios were measured. The feed conversion ratio of each rooster was calculated after 150 days of age.

[0111] S9.8 Semen analysis was performed on 150-day-old male chickens, and those with an ejaculation volume greater than or equal to 103% of the average ejaculation volume, a semen viscosity greater than 3.5 billion sperm cells / mL, and a milky white color were selected; avian leukosis was tested on male and female chickens, and positive chickens were culled.

[0112] S9.9. Before laying eggs, test for pullorum disease and avian leukosis, and cull positive and suspected individuals.

[0113] S9.10. Before labor begins, the entire group is weighed and individuals whose weight deviates significantly from the average are eliminated.

[0114] Reproductive performance selection was conducted at S9.11 and 43 weeks of age.

[0115] S9.12. Selection of Hens: A combination of family selection and individual selection is adopted. The egg production at 66 weeks of age of the previous generation is referenced, and the egg production at 43 weeks of age of the current generation is combined with the egg production at 43 weeks of age of the current generation. From the offspring of families whose egg production at 66 weeks of age of the previous generation was higher than the average egg production, individuals whose egg production at 43 weeks of age of the current generation is higher than the average egg production and whose hatching egg qualification rate is greater than or equal to 105% of the average hatching egg qualification rate are selected.

[0116] S9.13. Selection of roosters: Based on the performance of their siblings, roosters that produce the most eggs are selected from the 43-week-old broods of the current generation, whose egg production is higher than the average. The roosters must also have a body weight greater than or equal to 110% of the average rooster body weight, an ejaculation volume greater than or equal to 105% of the average semen volume, a semen consistency greater than 3.5 billion sperm cells / mL, and a low feed conversion ratio between 90 and 150 days of age. These roosters are selected for breeding.

[0117] S9.14. Selected roosters and hens are homogeneously mated to form families. At the same time, pedigree checks are conducted to avoid mating of full-siblings and half-siblings. After 3 generations, families can be randomly formed.

[0118] S10, after 3-4 generations of breeding, finally yielded a new strain of Guangxi Ma Chicken: high-breeding, fast-growing, long-legged, slow-feathering chicken.

[0119] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A breeding method for a high-breeding, fast-growing, long-legged, slow-feathering Guangxi Ma Chicken, characterized in that, Includes the following steps: Establish molecular detection methods for the sex-linked dwarf gene GHR and the recessive white-feathered RWC gene in chickens; Based on body shape and appearance standards, closed-loop breeding was carried out on Guangxi Ma Chicken and Dwarf Recessed White-feathered Chicken to obtain basic populations; The dwarf recessive white-feathered roosters and Guangxi Ma chicken hens in the basic population were first screened. The dwarf recessive white-feathered roosters and Guangxi Ma chicken hens selected in the first screening were used as the male and female parents, respectively, and the eggs were collected and incubated to obtain the F1 generation. From the F1 generation, select normal-legged, slow-feathered, mottled-feathered roosters that meet the described body shape and appearance standards and contain heterozygotes of the dw gene. Self-cross them with dwarf-legged, slow-feathered, mottled-feathered hens that meet the described body shape and appearance standards. Collect the eggs and incubate them to obtain the F2 generation. The F2 generation chicks were raised and then subjected to a second screening. The chicks after the second screening were molecularly tested using the gene molecular detection method. The roosters and hens with mottled feathers that were homozygous for the dw gene and had no recessive white feather gene were selected and raised in individual cages to obtain the basic population of dwarf slow-feathered Guangxi mottled chickens. The roosters of the dwarf and slow-feathered Guangxi Ma chickens in the basic population were subjected to a third screening to obtain the final core group of roosters, and the hens of the dwarf and slow-feathered Guangxi Ma chickens in the basic population were subjected to a fourth screening to obtain the final core group of hens. The final core group of roosters and hens were randomly formed into a 0th generation family line, and after successive generations of selective breeding, a high-breeding, fast-growing, short-legged Guangxi Ma Chicken was obtained. The specific criteria for body shape and appearance are as follows: the selection criteria for the dwarf recessive white-feathered chicken are recessive white feathers, slow feathers, yellow shanks and well-developed breast meat; the criteria for the Guangxi Ma chicken are slow feathers, mottled feathers and yellow shanks. The first screening specifically involves: screening for dwarf, recessive white-feathered roosters with shank length between 95-105% of the average, weight between 95% and 110% of the average, ejaculation volume not less than 105% of the average semen volume, semen density greater than 3.5 billion sperm cells / mL, and whose family lineage of sibling hens has a higher family lineage egg production performance than the average; and screening for Guangxi Ma chicken hens with an individual egg production of more than 100 eggs at 43 weeks of age and a hatching egg qualification rate not less than 103% of the average hatching egg qualification rate of the group. The second screening process specifically involves: when F2 generation chicks are raised to 60-70 days of age, individuals with mottled feathers and a shank length ≤6.0cm are selected. The specific gene molecular detection method is as follows: Blood was collected from the second-selected chicks, and genomic DNA was extracted from the chicken blood. Primers for identifying dwarfism genes were designed, as shown in SEQ ID No. 1, SEQ ID No. 2 and SEQ ID No.

3. The genomic DNA of the chicken blood was amplified by PCR and sequenced using the primers for identifying dwarfism genes. Heterozygotes and homozygotes with normal legs were removed, and homozygotes with dwarfism were retained. Primers for identifying recessive white feather genes were designed, as shown in SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6 and SEQ ID No.

7. The recessive white feather gene identification primers were used to perform PCR amplification and sequencing on the chicken blood genomic DNA to eliminate recessive white feather heterozygotes. In the selection and breeding of successive generations, growth traits are the primary selection trait for roosters, and reproductive traits are the secondary selection trait for hens. Reproductive traits are the primary selection trait for hens, and growth traits are the secondary selection trait for hens. At the same time, slow feathering selection is carried out simultaneously for both males and females. The specific details of slow-feathering selection and breeding are as follows: Chicks are tagged with wing numbers according to their family lineage, and weak or damaged chicks and individuals with green legs are culled. Slow-feathering selection: On day 1, spread the wings of chickens one by one and observe the relative length of the primary wing feathers and the overlying primary wing feathers on the outer wings. Eliminate fast-feathering chickens. The primary wing feathers of the fast-feathering chickens are more than 2mm longer than the overlying primary wing feathers. On day 10, select and eliminate individuals with excessively long tail feathers based on the development of the tail feathers. Roosters with slow comb development are culled at 42 days of age; hens with underdeveloped combs and yellowish or dull faces are culled at 56 days of age. Weigh the entire flock at 70 days old. Before selecting individuals, randomly select 10% of the flock by weight and record the average. For roosters, use -5 to +10% of the average as the breeding range, and for hens, use -8 to +10% as the breeding range, to improve the uniformity of the flock. Before converting to egg cages, individuals that do not meet the requirements in terms of body shape and appearance are culled, while individuals with well-developed chest and leg muscles are hand-selected. At 90 days old, roosters with drooping combs, no combs, excessively tall combs, blue legs, or angular legs are culled. Individuals with glossy, close-fitting feathers, double wattles, round body shape, and medium to high weight are selected. Hens with short combs, no combs, drooping combs, blue legs, or angular legs are culled. Individuals with close-fitting feathers, fine feathers, a few speckled spots, round body shape, and medium weight are selected. Chickens testing positive for pullorum disease are culled before being transferred to the laying house. From 90 days old to 150 days old, roosters were kept in individual cages. Their fasting weight, periodic feed intake, and weight at 150 days old were recorded. Feed was not controlled during this period. Individual feed conversion ratios were measured. After 150 days old, the feed conversion ratios of each rooster were calculated. Semen analysis was performed on 150-day-old roosters, and those with an ejaculation volume greater than or equal to 103% of the average ejaculation volume, a semen viscosity greater than 3.5 billion sperm cells / mL, and a milky white color were selected; roosters and hens were tested for avian leukosis, and positive chickens were culled. Before the start of egg production, chickens should be tested for pullorum disease and avian leukosis, and positive and suspected individuals should be culled. Before labor begins, the entire group is weighed, and individuals whose weight deviates significantly from the average are eliminated. Reproductive performance selection was conducted at 43 weeks of age. Hen selection: A combination of family selection and individual selection was adopted. The egg production at 66 weeks of age of the previous generation was taken into account, and the egg production at 43 weeks of age of the current generation was taken into account. From the offspring of families whose egg production at 66 weeks of age of the previous generation was higher than the average egg production, individuals whose egg production at 43 weeks of age of the current generation was higher than the average egg production and whose hatching egg qualification rate was greater than or equal to 105% of the average hatching egg qualification rate were selected. Rooster selection: Based on the performance of siblings, the roosters with the highest egg production of the hens in the current generation at 43 weeks of age are selected from the families with the highest egg production. The roosters must also have a body weight greater than or equal to 110% of the average body weight of roosters, an ejaculation volume greater than or equal to 105% of the average semen volume, a semen consistency greater than 3.5 billion sperm cells / mL, and a low feed conversion ratio between 90 and 150 days of age. Selected roosters and hens are paired homogeneously to form families, and pedigree checks are conducted to avoid mating of full and half-siblings. The next generation is bred by passing on the males to females at a ratio of 1:

12. After 3-4 generations of breeding, a new strain of Guangxi Ma Chicken with high breeding speed, long legs, and slow feathering was finally obtained.

2. The breeding method for the high-breeding, fast-growing, long-legged, slow-feathering Guangxi Ma Chicken according to claim 1, characterized in that, When performing PCR amplification, the PCR system reaction conditions are as follows: 94 ℃ pre-denaturation for 5 min; 94 ℃ denaturation for 30 s, 53 ℃ annealing for 30 s, 72 ℃ extension for 30 s, for 35 cycles; and finally 72 ℃ extension for 5 min.

3. The breeding method for the high-breeding, fast-growing, long-legged, slow-feathering Guangxi Ma Chicken according to claim 2, characterized in that, The third screening specifically involves: raising dwarf and slow-feathered Guangxi Ma chicken roosters in the basic population to 90 days of age, selecting individuals with a weight within the average range of +25% and excellent secondary trait development, and then conducting semen analysis on the selected individuals before they start laying eggs and before they are kept for breeding, selecting roosters with an ejaculation volume greater than or equal to 103% of the average ejaculation volume, a semen viscosity greater than 3.5 billion sperm cells / mL, and a milky white color.

4. The breeding method for the high-breeding, fast-growing, long-legged, slow-feathering Guangxi Ma Chicken according to claim 3, characterized in that, The fourth screening specifically involves selecting hens with excellent secondary sexual characteristics from the dwarf and slow-feathered Guangxi Ma chickens in the basic population before they start laying eggs, based on the size and traits of their combs. At the same time, the number of eggs laid at 43 weeks, the egg weight, and the number of eggs laid at 66 weeks are collected. Individuals with a number of eggs laid at 43 weeks greater than 100 and a weight greater than the average weight of the hen population are selected.

5. The breeding method for the high-breeding, fast-growing, long-legged, slow-feathering Guangxi Ma Chicken according to claim 4, characterized in that, When the final core group of roosters and hens are randomly assigned to form a generation 0 family, the final core group of roosters and hens are randomly assigned to form a generation 0 family according to a male-to-female ratio of 1:

8. In the successive breeding process, growth traits are the primary selection trait for roosters, while reproductive traits are the secondary selection trait. For hens, reproductive traits are the primary selection trait, while growth traits are the secondary selection trait. At the same time, slow feathering selection is carried out simultaneously for both males and females.

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