Chicken NCAPG gene SNP molecular marker, detection primer, kit, breeding method and application

Abstract

The present invention relates to a chicken NCAPG gene SNP molecular marker, a detection primer, a kit, a breeding method and an application. The present invention finds through research that a SNP site rs75790569 on the 18th intron of the chicken NCAPG gene has an A>G mutation. After analysis and verification, the SNP molecular marker is significantly correlated with the growth traits, carcass traits and body size traits of chickens, and can be used as a molecular marker for genetic improvement of chicken varieties. The use of the molecular marker to select and breed chickens is conducive to improving the growth traits and / or carcass traits and / or body size traits of a chicken population. The present invention also provides a detection primer, a kit and a breeding method for improving the growth traits and / or carcass traits and / or body size traits of chickens for detecting the SNP molecular marker of the chicken NCAPG gene, which can be used for auxiliary selection and molecular breeding of chickens, and plays an important role in improving the growth traits and / or carcass traits and / or body size traits of chickens.

Description

Technical Field

[0001] The invention relates to a chicken NCAPG gene SNP molecular marker, a detection primer, a kit, a breeding method and an application, and belongs to the technical field of biological breeding. Background Art

[0002] The association analysis between single nucleotide polymorphism (SNP) diversity and economic traits has always been a hot topic in livestock and poultry genetics research. SNP refers to DNA sequence polymorphism caused by the variation of a single nucleotide at the genomic level. Genes are not only involved in the molecular regulatory process of the formation of complex animal phenotypes, but their SNP changes may also lead to abnormal gene function, thereby causing variations in biological phenotypic traits. The formation of gene polymorphism includes insertion, deletion, translocation, amplification and base substitution. Studies have found that polymorphisms occurring in the introns, exons, 3'UTR, and 5'UTR regions of genes can potentially affect the function of genes, leading to changes in the phenotypic traits of animals. The correlation analysis between SNP polymorphism and economic traits can provide auxiliary marker selection for molecular selection and breeding of poultry.

[0003] In the genetic breeding of livestock and poultry, the application of modern breeding technology with molecular markers is an advanced and effective method to accelerate the selection of fine varieties, improve the genetic quality of the population, and increase the production performance of livestock and poultry. The application of molecular marker breeding is first to screen and detect genetic markers closely related to the economic traits of livestock and poultry at the DNA level, followed by the establishment of a rapid detection method for its gene polymorphism, and then to achieve genetic marker-assisted selection and early diagnosis and selection. Chickens are an important economic poultry and an important model organism in scientific research. Growth traits are the main indicators for evaluating chicken production performance and are one of the important economic traits. Domestic high-quality local chickens generally have the problem of slow growth, which affects economic benefits. Chicken growth traits are quantitative traits controlled by micro-effect polygenes and determined by a series of major effect genes or quantitative trait loci. Over the years, chicken breeders have conducted a large number of experimental studies to strive to find candidate genes related to chicken growth traits.

[0004] Non-chromosomal structural maintenance subunit (SMC) condensin I complex subunit G (NCAPG) is a chromosome condensation protein associated with mitosis and widely present in eukaryotic cells. Previous studies have found that the NCAPG gene induces the G0 / G1 cell cycle, thereby promoting cell mitosis. Studies have shown that there are SNPs sites related to the body composition traits of Jinghai yellow chickens at the genomic level of the NCAPG gene, indicating that NCAPG may play an important role in the growth and development of chickens. However, there are few reports on the effects of the NCAPG gene on the growth and development of chickens. Summary of the invention

[0005] In order to solve the above problems, the first object of the present invention is to provide a chicken NCAPG gene SNP molecular marker, which can be applied to marker-assisted selection (MAS) breeding of chicken growth traits and / or carcass traits and / or body size traits, and is conducive to the genetic improvement of chicken populations with excellent traits.

[0006] The second object of the present invention is to provide the application of the SNP molecular marker in the breeding for improving chicken growth traits and / or carcass traits and / or body size traits.

[0007] The third object of the present invention is to provide a detection primer for detecting the genotype of the SNP molecular marker.

[0008] The fourth object of the present invention is to provide a kit for detecting the genotype of the SNP molecular marker.

[0009] A fifth object of the present invention is to provide a detection primer and a kit for use in improving breeding of chicken growth traits and / or carcass traits and / or body size traits.

[0010] A sixth object of the present invention is to provide a breeding method for improving chicken growth traits and / or carcass traits and / or body size traits.

[0011] In order to achieve the above object, the technical solution adopted by the present invention is:

[0012] A chicken NCAPG gene SNP molecular marker, the nucleotide sequence of the SNP molecular marker is shown in SEQ ID NO.1, and the 89th base from the 5' end is A or G.

[0013] The present invention first discovered the above SNP molecular marker, which corresponds to the rs75790569 site in the 18th intron of the chicken NCAPG gene. The SNP site has not been reported before and is a newly discovered molecular marker. The 89th position n of the SEQ ID NO.1 sequence represents base A or G.

[0014] The SNP molecular marker is used in the breeding for improving chicken growth traits and / or carcass traits and / or body size traits.

[0015] Research and analysis found that the SNP molecular marker is significantly correlated with chicken growth traits, carcass traits and body size traits, and can be used in marker-assisted selection breeding of chicken growth traits and / or carcass traits and / or body size traits, which is beneficial to the genetic improvement of chicken populations with excellent traits.

[0016] Specifically, it is used in the breeding of chickens of different ages for the traits of weight, tibia length, tibia circumference, chest angle, sternum length, oblique body length, breast muscle area, breast muscle fiber diameter, full evisceration, half evisceration, abdominal fat, breast muscle weight, leg weight, leg muscle weight, claw weight, head weight, claw rate, heart rate, abdominal fat rate, full evisceration rate and / or half evisceration rate.

[0017] Preferably, the genotype of the SNP molecular marker is detected, and individuals with the SNP molecular marker having a GG genotype are selected.

[0018] Specifically, in practical application, the type of the 89th nucleotide from the 5' end of the sequence shown in SEQ ID NO.1 is detected, and chicken individuals with GG homozygous or AG heterozygous at this site are selected to establish a chicken population with excellent genetic resources.

[0019] A detection primer, which is used to detect the genotype of the above-mentioned SNP molecular marker; the nucleotide sequence of the detection primer is shown in SEQ ID NO.2-3.

[0020] The present invention has found through research that a SNP site rs75790569 on the 18th intron of the chicken NCAPG gene is significantly correlated with the differences in chicken growth traits, carcass traits and body size traits. Specifically, the A>G mutation at the rs75790569 site of the NCAPG gene is significantly correlated with birth weight, 6-week-old weight, 8-week-old weight, 10-week-old weight, 12-week-old weight and slaughter weight (P<0.05), wherein, except for birth weight, the weights of the other different stages are all higher in GG and AG type individuals than in AA type individuals. According to the SNP molecular marker, corresponding detection primers for chicken growth traits and / or carcass traits and / or body size traits are designed, and GG homozygous chicken individuals can be detected and screened out, so the detection primers can be used in auxiliary selection and molecular breeding of chickens, which is helpful to establish fast-growing new chicken varieties or new strains.

[0021] Specifically, the detection primer adopts the design method commonly used in the prior art, and can also be designed according to different detection methods.

[0022] A kit for detecting the genotype of the SNP molecular marker, the kit comprising the above-mentioned detection primer and restriction endonuclease Hpy188I.

[0023] The kit comprises the detection primers and restriction endonuclease Hpy188I. The kit can be used to detect the NCAPG gene SNP molecular marker to determine the genotype according to the number of bands, directly identify the polymorphism, and accurately, quickly, and time-savingly and labor-savingly detect the genotype of the SNP.

[0024] Preferably, the kit further comprises one or more of dNTPs, PCR reaction buffer, and DNA polymerase.

[0025] The application of the above detection primers and detection kit in the improvement and breeding of chicken growth traits and / or carcass traits and / or body size traits.

[0026] The detection primers and detection kit of the present invention have accurate and reliable detection results, strong operability, high cost performance, short detection time, and can be widely promoted.

[0027] A method for improving and breeding chicken growth traits and / or carcass traits and / or body size traits comprises the following steps: using genomic DNA of a chicken to be tested as a template, performing PCR amplification using a primer pair shown in SEQ ID NO.2 and SEQ ID NO.3, performing enzyme digestion reaction on the PCR product using Hpy188I endonuclease, if the enzyme digestion product is a 324 bp band, the mutation site is a homozygous GG type, if the enzyme digestion product is three bands of 324 bp, 234 bp and 87 bp, the site is a heterozygous AG type, if the enzyme digestion product is two bands of 234 bp and 87 bp, the site is a homozygous AA type; and selecting individuals with homozygous GG genotype.

[0028] This method detects the genotypes of the above-mentioned SNP molecular markers and can predict the genotypes of different growth traits, carcass traits and body size traits early and effectively, thereby shortening the breeding time and accelerating the breeding process. It has great economic application value and scientific research value and has broad application prospects in the improvement of chicken germplasm resources.

[0029] Beneficial effects of the present invention:

[0030] 1. The present invention first discovered a SNP site rs75790569 on the 18th intron of the chicken NCAPG gene, which has an A>G mutation. Further detection and analysis showed that the SNP molecular marker was significantly correlated with the growth, body size and carcass traits of chickens, indicating that the SNP molecular marker can be used as an auxiliary selection marker and in chicken breeding, thereby accelerating the breeding speed of improved chickens and improving the quality of chicken populations.

[0031] 2. The present invention designs specific PCR primers for SNP molecular markers of chicken NCAPG gene, performs enzyme digestion reaction on the amplified product, and then performs gel electrophoresis test to determine the genotype according to the number of bands, which can accurately, quickly, save time and effort to detect SNP molecular markers of NCAPG gene.

[0032] 3. The present invention provides a method for improving the breeding of chicken growth traits and / or carcass traits and / or body size traits for the SNP molecular marker of the chicken NCAPG gene. The genotype of the SNP molecular marker can be determined according to the number of bands after PCR and enzyme cutting. The breeding method is simple to operate, low in cost, and short in cycle. It can greatly improve the accuracy of SNP site genotype determination, does not require special instruments, and is easy to promote and popularize. Experiments show that this method can effectively determine the genotype of the SNP site of chicken growth traits, can be used for auxiliary selection and molecular breeding of chickens, and plays an important role in improving the growth traits of chickens. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 This is a position map of the rs75790569 site on the chicken NCAPG gene in Experimental Example 3 of the present invention on the genome;

[0034] Figure 2 This is the agarose gel electrophoresis pattern of the PCR product used for sequencing and detecting polymorphism in Experimental Example 3 of the present invention (M is Marker DNA, and 1-8 are all amplified fragments of the sample);

[0035] Figure 3 The SNP polymorphism sequencing map of the AG mutation at the rs75790569 site of the chicken NCAPG gene screened by PCR cloning in Experimental Example 3 of the present invention;

[0036] Figure 4 The peak diagram of sequencing of individuals with different genotypes of SNP molecular markers in Experimental Example 3 of the present invention (from left to right: AA genotype, AG genotype and GG genotype);

[0037] Figure 5 This is an agarose gel electrophoresis typing diagram of different genotypes at the rs75790569 site of the chicken NCAPG gene in Experimental Example 3 of the present invention. DETAILED DESCRIPTION

[0038] The present invention is further described in detail below in conjunction with specific examples. Unless otherwise specified, the equipment and reagents used in each embodiment, experimental example and comparative example can be obtained from commercial sources.

[0039] Example 1 of a chicken NCAPG gene SNP molecular marker

[0040] The chicken NCAPG gene of this embodiment (located on chromosome 4, sequence information refers to the NCBI database (GRCg6a), and relevant information of the NCAPG gene can be directly queried on this website Genome Data Viewer (https: / / www.ncbi.nlm.nih.gov / genome / gdv / browser / genome / ?id=GCF_000002315.6)) has a nucleotide sequence of the SNP molecular marker as shown in SEQ ID NO.1, and the 89th base from the 5' end is A or G, and the SNP site corresponds to the rs75790569 deoxynucleotide in intron 18 of the chicken NCAPG gene.

[0041] Example 1 of Application of Chicken NCAPG Gene SNP Molecular Marker in Chicken Growth Traits and / or Carcass Traits and / or Body Size Traits Improvement Breeding

[0042] The chicken NCAPG gene SNP molecular marker of this embodiment is used in the improvement and breeding of chicken growth traits and / or carcass traits and / or body size traits. The genotype of the SNP molecular marker is detected, and individuals with the SNP molecular marker GG genotype are selected to establish a chicken population with excellent genetic resources.

[0043] Example 1 of primers for detecting chicken NCAPG gene SNP molecular marker genotype

[0044] The primers for detecting the genotype of the chicken NCAPG gene SNP molecular marker in this embodiment are as follows:

[0045] F Primer: 5'-GTTCCCACTCTAGAAGATAAATAG-3' (shown in SEQ ID NO. 2);

[0046] R Primer: 5'-TTTCCCAGATAGAGTCTATTTTGC-3' (shown in SEQ ID NO. 3).

[0047] Example 1 of a kit for detecting the genotype of the chicken NCAPG gene SNP molecular marker

[0048] In this embodiment, the kit for detecting the genotype of the chicken NCAPG gene SNP molecular marker includes detection primers with nucleotide sequences as shown in SEQ ID NO. 2-3 and restriction endonuclease Hpy188I, as well as dNTPs, PCR reaction buffer, and DNA polymerase.

[0049] Example 1 of the application of detection primers and detection kits in improving chicken growth traits and / or carcass traits and / or body size traits

[0050] The detection primers and the detection kit of this embodiment are used in the improvement and breeding of chicken growth traits and / or carcass traits and / or body size traits, and the genotype of the SNP molecular marker is detected, and individuals with the SNP molecular marker GG genotype are selected to establish a chicken population with excellent genetic resources.

[0051] Example 1 of a method for improving chicken growth traits and / or carcass traits and / or body size traits

[0052] A method for improving chicken growth traits and / or carcass traits and / or body size traits of this embodiment comprises the following steps:

[0053] (1) PCR amplification: using chicken whole genome DNA containing the NCAPG gene as a template, PCR amplification was performed using detection primers with nucleotide sequences as shown in SEQ ID NOs. 2 to 3;

[0054] (2) Enzyme digestion reaction: After PCR amplification, restriction endonuclease Hpy188I was used for digestion;

[0055] (3) Gel electrophoresis: Take the sample treated in step (2) and perform gel electrophoresis to determine the genotype based on the number of bands and perform molecular breeding.

[0056] If the enzyme digestion product is a single band of 324bp, the base at the mutation site is homozygous GG type; if the enzyme digestion product is three bands of 324bp, 234bp and 87bp, the base at the mutation site is heterozygous AG type; if the enzyme digestion product is two bands of 234bp and 87bp, the base at the mutation site is homozygous AA type; select GG homozygous genotype individuals to establish a chicken population with excellent genetic resources.

[0057] Experimental Example 1 Material Preparation

[0058] In this experimental example, biological materials were prepared, and the biological materials used were: Gushi chicken-Anka chicken F2 generation resource group.

[0059] Gushi chicken is a famous local chicken breed in my country. It belongs to the yellow chicken type. It is centered in Gushi County and is naturally formed through long-term closed breeding under special ecological environment and feeding conditions. Gushi chicken has the characteristics of high egg production, large and thick egg shells, and stable genetic performance. It is a dual-purpose chicken for eggs and meat, and one of my country's precious poultry breed resources. It has the following outstanding excellent traits: tolerance to roughage, strong disease resistance, and suitable for grazing in the wild; second, the meat is tender, delicious, the soup is mellow, rich in nutrition, and has a strong tonic effect; third, the hens lay more eggs, large eggs, thick egg whites, dark egg yolk color, thick egg shells, and are resistant to storage and transportation.

[0060] Anka broiler is one of the finest broiler breeds in the world today, and is also the fastest growing red and yellow-feathered broiler in China. It has the characteristics of strong adaptability, stress resistance, fast growth rate, and high feed conversion rate.

[0061] 1.1. Cultivation of biomaterials

[0062] The Gushi-Anka chicken resource group was established according to the F2 distant half-sib design scheme. A total of 7 families were established in the experiment, of which 4 orthogonal lines were Anka chicken ♂×Gushi chicken ♀, and 3 reciprocal lines were Gushi chicken ♂×Anka chicken ♀. The F0 generation was formed by selecting breeders from the pure lines of Gushi chickens for both egg and meat and Anka chickens for meat, and mating them at a ratio of 1:6 between male and female chickens. The selected male and female chickens were required to have the characteristics of this breed, high egg production, medium weight, and pure bloodline to ensure that the F1 generation individuals were heterozygous at each locus. A rooster was selected from the F1 offspring of each family, and mated with hens from other families at a ratio of 1:9 between male and female to produce the F2 generation. It was required that there was no genetic relationship between the mating male and female chickens. The breeding hens of the F1 generation were also scattered in various families as much as possible. When selecting, try to select individuals with rich appearance and heterozygous to ensure that the F2 generation traits have a large separation. The population includes 42 F0 generation chickens, 70 F1 generation chickens, 860 F2 generation chickens, and 772 F2 generation individuals with complete economic traits in the resource population.

[0063] 1.2. Feeding methods

[0064] The chickens were raised in the experimental chicken farm of Henan Agricultural University, and the slaughter experiment was completed in the poultry genetic improvement laboratory of Henan Agricultural University. The nutritional level of the chickens during the feeding period: 0-4 weeks of age, the energy level was 12.40MJ / kg, the protein level was 20.1%; 5-8 weeks of age, the energy level was 12.70MJ / kg, the protein level was 18.2%; 9-12 weeks of age, the energy level was 12.75MJ / kg, the protein level was 16.0%. The families were mixed, caged, free to eat, and had plenty of drinking water.

[0065] Experimental Example 2 Extraction and Detection of Chicken Genomic DNA

[0066] In this experimental example, genomic DNA was extracted from the F2 generation of the Gushi chicken-Anka chicken resource group raised in Experimental Example 1. The specific operation steps are as follows:

[0067] 2.1. Extraction of genomic DNA

[0068] A total of 772 chickens of the F2 generation of Gushi chicken-Anka chicken resource group were used as materials. 5 mL of blood was drawn from the jugular vein of each chicken, anticoagulated with EDTA (10 mmol / L Tris-Cl, 0.2 mmol / L EDTA, PH 8.0), and stored at -80°C. Genomic DNA was extracted from blood samples of the F2 generation resource group using the phenol-chloroform extraction method, dissolved in TE, and stored at 4°C for later use. The specific experimental steps are as follows:

[0069] (1) Take 80 μL of whole blood and place it in a 1.5 mL centrifuge tube. Add 500 μL of TE buffer, 10 μL of proteinase K (10 mg / mL), and 12 μL of 25% SDS lysis buffer. Mix by shaking for 10 min and place in a 55°C water bath overnight.

[0070] (2) Add an equal volume of Tris-saturated phenol, shake and mix for 10 min, and centrifuge at 10,000 rpm for 10 min;

[0071] (3) Take the supernatant, add an equal volume of Tris-saturated phenol, shake and mix for 10 min, and centrifuge at 10,000 rpm for 10 min;

[0072] (4) Take the supernatant, add an equal volume of phenol / chloroform / isoamyl alcohol mixed solution (25:24:1), shake and mix, and centrifuge at 10,000 rpm for 10 min;

[0073] (5) Take the supernatant, add an equal volume of phenol / chloroform mixed solution (24:1), shake and mix, and centrifuge at 10,000 rpm for 10 min;

[0074] (6) Take the supernatant, add 2 volumes of anhydrous ethanol precooled to -20°C, shake slightly, and centrifuge at 10,000 rpm for 10 min;

[0075] (7) Discard the supernatant, add 500 μL of 70% ethanol to wash the precipitate, invert and air-dry at room temperature, add an appropriate amount of TE (pH 8.0) to dissolve the DNA, store at 4°C until the DNA is completely dissolved, detect its quality by 1% agarose gel electrophoresis, and store at -80°C.

[0076] 2.2 Spectrophotometric determination of genomic DNA concentration

[0077] Use a UV photometer to measure the OD value of the DNA sample at 260nm and 280nm. Calculate the DNA content and the ratio of OD260 / OD280. If the OD260 / OD280 ratio is less than 1.6, it means that the sample contains more protein or phenol, and purification should be performed; if the ratio is greater than 1.8, RNA purification should be considered.

[0078] DNA concentration (ng) = 50 × OD260 value × dilution multiple;

[0079] After the DNA test is completed, a certain amount is taken out and diluted to 100 ng / μL, and stored at -20°C for future use, and the rest is stored at -80°C.

[0080] Experimental Example 3 Detection of Chicken NCAPG Gene Polymorphism

[0081] In this experimental example, the chicken NCAPG gene polymorphism was detected, and a SNP site rs75790569 was found in the 18th intron of the chicken NCAPG gene. The SNP molecular marker is shown in SEQ ID NO.1. The 89th base from the 5' end of the sequence is A or G. The position of the SNP molecular marker on the chicken genome is shown in Figure 1 As shown. After testing and analysis, the SNP molecular marker is significantly associated with chicken growth traits, carcass traits and body size traits. The specific operation steps for obtaining the SNP site are as follows:

[0082] 3.1. Detection of mutation sites in chicken NCAPG gene

[0083] (1) Construction of DNA pool

[0084] 100 DNA samples with a concentration of 100 ng / μL in Experimental Example 2 were randomly selected, and 0.5 μL of DNA was taken from each sample to mix and construct a DNA pool.

[0085] (2) Amplification primer design

[0086] The chicken NCAPG gene genome sequence in Genbank was used as a reference (accession number: NC_006091.5), and the sequencing PCR primer pair of chicken NCAPG was designed with oligo. The total length of the amplified fragment was 324 bp. The agarose gel electrophoresis pattern of the PCR product used to detect the polymorphism was shown in the figure. Figure 2 As shown in the figure, it can be seen that the designed primer pair has good specificity and there is no interference from primer dimers and miscellaneous bands.

[0087] The nucleotide sequences of the sequencing PCR primer pairs are:

[0088] F Primer: 5'-GTTCCCACTCTAGAAGATAAATAG-3' (shown in SEQ ID NO. 2);

[0089] R Primer: 5'-TTTCCCAGATAGAGTCTATTTTGC-3' (shown in SEQ ID NO. 3).

[0090] (3) PCR cloning of chicken NCAPG DNA sequence

[0091] Using the DNA pool as a template, sequencing PCR primers were used to perform PCR amplification of P. The reaction system for PCR amplification was 10 μL. The specific reaction system is shown in Table 1.

[0092] Table 1 PCR reaction system

[0093]

[0094] The reaction program of PCR amplification was as follows: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s, annealing at 56°C for 30 s, extension at 72°C for 30 s, 35 cycles; and extension at 72°C for 10 min.

[0095] (4) PCR product sequencing

[0096] After PCR amplification, agarose gel electrophoresis was performed, followed by gel excision, recovery and purification of the PCR product. The gel containing the target fragment was cut from the agarose gel under ultraviolet light and placed in a 2.0 mL centrifuge tube, and then the PCR product was purified using a PCR product recovery and purification kit (Beijing Tiangen Biotechnology Co., Ltd.), and the specific operation was carried out according to the instructions of the kit.

[0097] The PCR purified product was sent to Qingke Company for sequencing. The sequencing peak graph was analyzed and the sequence was compared. The results are as follows Figure 3 , Figure 4 (The shadow in the figure indicates the mutation site). As shown in the figure, there is an A>G polymorphism in the NCAPG gene, which is the single nucleotide polymorphism of the chicken NCAPG gene screened, resulting in the deletion of an Hpy188I restriction endonuclease cleavage site.

[0098] 3.2. Genotyping using restriction enzymes

[0099] Enzyme typing was performed based on the products obtained by PCR amplification. The enzyme digestion reaction system was 15 μL. The specific reaction system is shown in Table 2.

[0100] Table 2 PCR reaction system

[0101]

[0102] The enzyme digestion reaction conditions were: 37°C for 1.5 hours. After the enzyme digestion reaction, 1% agarose gel was used for electrophoresis, 120V for 40 minutes, ethidium bromide (EB) staining was used for detection, and gel imaging system SYNGENE GBOX, Kodak120 (Syngene, USA) was used for imaging. The individual genotype was determined based on the image of the electrophoresis band.

[0103] The cleavage sequence of Hpy188I enzyme is TCNGA (N=A, C, G or T). If the mutation site is A, there is an Hpy188I cleavage sequence TCNGA (N=A, C, G or T); if the mutation site is G, there is no Hpy188I cleavage sequence TCNGA (N=A, C, G or T).

[0104] Therefore, in the gel electrophoresis results, the GG genotype is shown as a band of 324 bp; the AG genotype is shown as three bands of 324 bp, 234 bp and 87 bp, and the AA genotype is shown as two bands of 234 bp and 87 bp. Figure 5 shown.

[0105] 3.3. Frequency statistics of the SNPs (rs75790569) locus of the chicken NCAPG gene and its association analysis with growth, carcass and body size traits

[0106] (1) Genes and gene frequencies

[0107] Allele frequency refers to the relative ratio of a gene to its alleles in a population, with a value between 0 and 1, as shown in formula (1):

[0108] Pi=[2(ii)+(ij1)+(ij2)+………+(ijn)] / 2N (1)

[0109] Where, Pi: frequency of the ith allele;

[0110] i: homozygous multiple allele;

[0111] j1, j2, ……… jn: the 1st to nth alleles co-dominant with i.

[0112] Genotype frequency refers to the relative ratio between various genotypes of a certain trait in a population, as shown in formula (2).

[0113] Genotype frequency = number of genotyped individuals / total number of populations measured (2)

[0114] The genotyping results of the F2 segregating population, two local breeds (Lushi chicken and Gushi chicken), high-yield laying hens (Hai-Line brown laying hens, Luodao red laying hens), dual-purpose broiler chickens (Yao chickens) and fast-growing broilers (Cobb and AA broilers) are shown in Table 3.

[0115] Table 3 Allele and genotype frequencies of SNP loci of NCAPG gene in different chicken populations

[0116]

[0117]

[0118] (2) Related subsystem calculation models

[0119] Association analysis model: SPSS software was used to analyze the correlation between gene loci and growth (weight), carcass and body size traits. To ensure that the data of each trait were normally distributed, the least squares method was used to correct the data; according to the data characteristics, the multivariate linear model was used to analyze the genotype effect and the Benferroni multiple comparison method was used to compare the differences between the genotypes. The results are shown in Tables 4, 5 and 6. Model I is the association analysis model between the polymorphism of the locus and growth traits and body size traits, and Model II is the association analysis model between the polymorphic locus and carcass traits, see formulas (3) and (4) for details.

[0120] Model I:y ijklm =μ + G i + S j + H k + f l + e ijklm (3)

[0121] Model II: y ijklm =μ + G i + S j + H k + f l + b (W ijklm - ) + e ijklm (4)

[0122] Where: yijklm is the individual phenotypic value; μ is the overall mean; Gi is the genotype fixed effect; Sj is the sex effect; Hk is the batch effect; fl is the family effect as a random effect; b is the regression coefficient of slaughter weight; Wijklm is the individual slaughter weight; is the average body weight of the group, and eijklmn is the random error.

[0123] Table 4 Association analysis between chicken NCAPG gene SNP (rs75790569) mutation and chicken growth traits

[0124]

[0125] Note: The same letters in the same column indicate no significant difference (P>0.05), and different letters indicate significant difference (P<0.05).

[0126] As shown in Table 4, the A>G mutation of the SNP (rs75790569) site of the NCAPG gene is significantly associated with birth weight, 6-week-old weight, 8-week-old weight, 10-week-old weight, 12-week-old weight and slaughter weight. In addition to birth weight, the weight of GG and AG type individuals is higher than that of AA type individuals. There is no significant difference between GG and AG types in birth weight, 6-week-old weight and 8-week-old weight; while there is a significant difference between GG and AG types in 10-week-old weight, 12-week-old weight and slaughter weight. The results show that the G allele is conducive to the increase of chicken weight, while the A allele is not conducive to the increase of chicken weight. The GG genotype in the single nucleotide polymorphism of the rs75790569 site of the chicken NCAPG gene is used as a molecular marker for improving chicken weight in marker-assisted selection breeding of chicken weight traits.

[0127] Table 5 Association analysis between chicken NCAPG gene SNP (rs75790569) mutation and chicken carcass traits

[0128]

[0129] Note: The same letters in the same column indicate no significant difference (P>0.05), and different letters indicate significant difference (P<0.05).

[0130] As shown in Table 5, the A>G mutation of the SNP (rs75790569) site of the NCAPG gene is significantly associated with different carcass traits, and almost all of them show that the carcass traits of GG and AG type individuals are higher than those of AA type individuals, and the carcass traits of AG type individuals are lower than those of GG type individuals. This indicates that the G allele is beneficial to the growth of chicken carcasses. The GG genotype in the single nucleotide polymorphism of the rs75790569 site of the chicken NCAPG gene is used as a molecular genetic marker to improve chicken carcasses in marker-assisted selection breeding of chicken carcass traits.

[0131] Table 6 Association analysis between chicken NCAPG gene SNP (rs75790569) mutation and chicken body size traits

[0132]

[0133] Note: The same letters in the same column indicate no significant difference (P>0.05), and different letters indicate significant difference (P<0.05).

[0134] As shown in Table 6, the A>G mutation of the chicken NCAPG gene SNP (rs75790569) site is significantly associated with body size indicators at different ages, and almost all of them show that the body size index of GG and AG type individuals is higher than that of AA type individuals, and the body size index of AG type individuals is lower than that of GG type individuals. This shows that the G allele is conducive to the formation of chicken body shape. The GG and AG genotypes in the single nucleotide polymorphism of the rs75790569 site of the chicken NCAPG gene are used as molecular genetic markers to improve the body shape of chickens in marker-assisted selection breeding of chicken body size indicators.

[0135] In summary, based on the above analysis, the tested chicken individuals are bred to select superior varieties. Specifically, individuals with homozygous GG at the chicken NCAPG gene SNP (rs75790569) site are selected to improve the economic performance of the chickens; AA homozygous individuals are eliminated. If homozygous Chinese local chicken breeds are to be cultivated, individuals with homozygous GG at the chicken NCAPG gene SNP (rs75790569) site are selected.

[0136] The above embodiments are preferred implementations of the present invention. However, those skilled in the art will appreciate that the implementations of the present invention are not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A kind of chicken NCAPG The application of gene SNP molecular markers in the improvement and breeding of chicken growth traits and / or carcass traits and / or body size traits is characterized by: The nucleotide sequence of the SNP molecular marker is shown in SEQ ID NO.1, and the 89th base from the 5' end is A or G; the growth traits are the weight of chickens at 10 weeks, 12 weeks of age and slaughter weight; the carcass traits are weight after molting, head weight, claw weight, double wing weight, gizzard weight, pancreas weight, breast muscle weight, leg weight, leg muscle weight, half-eviscerated weight, full-eviscerated weight, half-eviscerated rate, full-eviscerated rate, and claw rate; the body size traits are tibia length at 12 weeks of age, tibia circumference at 12 weeks of age, sternum length at 8 weeks, sternum length at 12 weeks, and body oblique length at 12 weeks.

2. The chicken according to claim 1 NCAPG The application of gene SNP molecular markers in the improvement and breeding of chicken growth traits and / or carcass traits and / or body size traits is characterized by: The genotype of the SNP molecular marker was detected, and individuals with the SNP molecular marker GG genotype were selected.

3. Use of a detection primer or kit in improving breeding of chicken growth traits and / or carcass traits and / or body size traits, characterized in that: The growth traits are the weight of the chicken at 10 weeks, the weight of the chicken at 12 weeks and the slaughter weight; the carcass traits are the weight after depilation, the head weight, the claw weight, the double wing weight, the gizzard weight, the pancreas weight, the breast muscle weight, the leg weight, the leg muscle weight, the half-eviscerated weight, the full-eviscerated weight, the half-eviscerated rate, the full-eviscerated rate and the claw rate; the body size traits are the shank length at 12 weeks, the shank circumference at 12 weeks, the sternum length at 8 weeks, the sternum length at 12 weeks and the body oblique length at 12 weeks; the primers are used to detect the NCAPG The genotype of the gene SNP molecular marker; the nucleotide sequence of the SNP molecular marker is shown in SEQ ID NO.1, and the 89th base from the 5' end is A or G; the kit includes a kit for detecting chicken NCAPG Primers for genotyping of gene SNP molecular markers.

4. The use of the detection primer or kit according to claim 3 in improving and breeding chicken growth traits and / or carcass traits and / or body size traits, characterized in that: The nucleotide sequence of the detection primer is shown in SEQ ID NO. 2~3.

5. Use of the detection primer or kit according to claim 3 in improving and breeding chicken growth traits and / or carcass traits and / or body size traits, characterized in that: The kit includes restriction endonucleases Hp 188I.

6. Use of the detection primer or kit according to claim 5 in improving and breeding chicken growth traits and / or carcass traits and / or body size traits, characterized in that: The kit also includes one or more of dNTPs, PCR reaction buffer, and DNA polymerase.

7. A method for improving chicken growth traits and / or carcass traits and / or body size traits, characterized in that: The following steps are involved: The genomic DNA of the chicken to be tested was used as a template, and PCR amplification was performed using the primers shown in SEQ ID NO. 2 and SEQ ID NO.

3. The PCR product was amplified by Hp 188I endonuclease is used for enzyme digestion reaction. If the enzyme digestion product is a band of 324 bp, the SNP molecular marker is homozygous GG type, if the enzyme digestion product is three bands of 324 bp, 234 bp and 87 bp, the SNP molecular marker is heterozygous AG type, if the enzyme digestion product is two bands of 234 bp and 87 bp, the SNP molecular marker is homozygous AA type; GG homozygous genotype individuals are selected; the growth traits are chicken weight at 10 weeks, weight at 12 weeks and slaughter weight; the carcass traits are weight after depilation, head weight, claw weight, double wing weight, gizzard weight, pancreas weight, breast muscle weight, leg weight, leg muscle weight, half-eviscerated weight, full-eviscerated weight, half-eviscerated rate, full-eviscerated rate, and claw rate; the body size traits are tibia length at 12 weeks, tibia circumference at 12 weeks, sternum length at 8 weeks, sternum length at 12 weeks, and body oblique length at 12 weeks.

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