SNP (Single Nucleotide Polymorphism) molecular marker synergistically related to growth and reproduction traits of chicken and application of SNP molecular marker

Abstract

The invention provides an SNP (Single Nucleotide Polymorphism) molecular marker synergistically related to growth and reproduction traits of chickens and application, and belongs to the technical field of molecular biology. The SNP molecular marker related to reproduction is located at the 28973618th nucleotide of chromosome 6 of a reference sequence of a GRCg6a version of a chicken genome, and has C / G polymorphism. The growth-related SNP molecular marker disclosed by the invention is positioned at the 2897383838th nucleotide of chromosome 6 of a reference sequence of a GRCg6a version of a chicken genome, and has G / A polymorphism. The sperm density and the keel length of a diplotype CCAA individual are the highest, and the growth traits and the reproduction traits are in a synergistic relationship. The SNP molecular marker disclosed by the invention can be used for carrying out marker-assisted selection on characters such as chicken sperm density and keel length, implementing early selection and improving the breeding efficiency. The invention provides a reliable molecular marker for the genetic improvement of chicken growth and reproduction traits, and has important significance for the genetic improvement of chicken.

Description

Technical Field

[0001] The present invention relates to the field of molecular biotechnology, and particularly to an SNP molecular marker synergistically related to chicken growth and reproduction traits and its application. Background Art

[0002] The growth and reproduction traits of chickens are of extremely important significance in the field of poultry breeding and farming. Good growth traits contribute to chickens forming a more ideal meat quality and body shape. Chickens with well-developed muscles have better meat taste and nutritional value, and can better meet consumers' demands for chicken quality. Chickens with excellent reproduction traits can be used as excellent parents in the breeding process, passing on excellent genes to offspring, accelerating the process of variety improvement, and breeding new varieties that are more adaptable to market demands and farming environments. Chicken breeds with excellent growth and reproduction traits can provide support for the efficient development of the entire poultry industry, promote the progress of farming technology and variety improvement, enhance the market competitiveness of the poultry industry, and ensure the stable supply of chicken products.

[0003] Studying the synergistic relationship between chicken growth and reproduction traits is of great significance in both theoretical research and practical applications. Clarifying the synergistic relationship between growth and reproduction traits can select the two traits as a whole, improve the selection efficiency, accelerate the process of breeding excellent chicken breeds with both good growth performance and high reproduction performance, and enhance the comprehensive production performance of poultry breeds. Breeding chicken breeds with synergistic advantages in growth and reproduction traits can enable the chicken flock to have good performance in terms of growth rate, egg production, etc., increase the production of chicken and eggs, reduce farming costs, and improve farming economic benefits. Summary of the Invention

[0004] The purpose of the present invention is to provide an SNP molecular marker synergistically related to chicken growth and reproduction traits and its application, which has potential important value for the genetic improvement of chicken growth and reproduction traits.

[0005] To achieve the above invention purpose, the present invention provides the following technical solutions:

[0006] The present invention provides an SNP molecular marker combination related to chicken growth and reproduction traits, comprising the following two SNP marker sites:

[0007] SNP1: a C / G mutation at position 28973618 on chromosome 6 of the chicken GRCg6a genome;

[0008] SNP2: a G / A mutation at position 28973838 on chromosome 6 of the chicken GRCg6a genome.

[0009] The present invention also provides a primer combination for detecting the above SNP molecular marker combination, and the primer combination includes primers shown in SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.5.

[0010] The present invention also provides a primer combination for detecting the above SNP molecular marker combination, and the primer combination includes primers shown in SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO.10.

[0011] The present invention also provides an SNP molecular marker detection reagent, which is characterized by containing the above primer combination.

[0012] The present invention also provides a molecular breeding method for improving the sperm density trait of chickens, including the following steps:

[0013] a) Detecting the genotype of the SNP1 locus in the genome of the chicken to be tested;

[0014] b) Selecting individuals with the CC genotype at the SNP1 locus as breeding chickens;

[0015] The SNP1 locus is a C / G mutation at position 28973618 on chromosome 6 of the chicken GRCg6a genome.

[0016] The present invention also provides a molecular breeding method for improving the keel length trait of chickens, including the following steps:

[0017] a) Detecting the genotype of the SNP2 locus in the genome of the chicken to be tested;

[0018] b) Selecting individuals with the AA genotype at the SNP2 locus as breeding chickens;

[0019] The SNP2 locus is a G / A mutation at position 28973838 on chromosome 6 of the chicken GRCg6a genome.

[0020] Preferably, detecting the genotype of the SNP1 locus in the genome of the chicken to be tested or detecting the genotype of the SNP2 locus in the genome of the chicken to be tested includes the following steps:

[0021] a) Extracting genomic DNA of the chicken to be tested;

[0022] b) Performing PCR amplification on the SNP1 locus and / or the SNP2 locus using the above SNP molecular marker detection reagent;

[0023] c) Analyzing the amplified product sequence to determine the SNP locus genotype;

[0024] Wherein:

[0025] The amplified product sequence of SNP1 is shown in SEQ ID NO.1, and the 601st base is a C / A polymorphism site;

[0026] The amplified product sequence of SNP2 is shown in SEQ ID NO.6, and the 601st base is a G / A polymorphism site.

[0027] The present invention also provides the application of the above SNP molecular marker combination related to chicken growth and reproduction traits in chicken molecular marker-assisted breeding.

[0028] Preferably, the chicken molecular marker-assisted breeding includes screening breeding chickens with advantages in chicken keel length traits or screening breeding chickens with advantages in chicken sperm density traits.

[0029] The present invention also provides the application of the above primer combination or SNP molecular marker detection reagent in the improvement of chicken growth and / or reproduction traits.

[0030] Beneficial effects of the present invention:

[0031] The SNP molecular markers related to chicken growth and reproduction traits provided by the present invention, the polymorphisms of this locus significantly affect traits such as chicken keel length and sperm density respectively, providing a detection technical means for early selection and improving breeding efficiency. The present invention also provides reliable molecular markers for the genetic improvement of chicken growth and reproduction traits, which is of great significance for the genetic improvement of chickens. Brief Description of the Drawings

[0032] Figure 1 For Figure 1 is the Manhattan plot of the GWAS results of keel length in Example 1 of the present invention;

[0033] Figure 2 is the Manhattan plot of the GWAS results of sperm density in Example 1 of the present invention;

[0034] Figure 3 is the phenotypic values of individuals with different genotypes of two SNPs in Example 1 of the present invention, where, A: sperm density, SNP1; B: keel length, SNP2;

[0035] Figure 4 is the electrophoresis result of PCR amplification of genomic DNA of chickens with three genotypes at the 28973618th nucleotide site on chromosome 6 designed by the primer pair in Example 2 of the present invention;

[0036] Figure 5 is the electrophoresis result of PCR amplification of genomic DNA of chickens with three genotypes at the 28973838th nucleotide site on chromosome 6 designed by the primer pair in Example 2 of the present invention. Detailed Embodiments

[0037] Based on a resource population of 473 adult Wenchang roosters, the present invention measures and records growth and reproductive traits, conducts a GWAS study on growth and reproductive traits using SNP genotyping data obtained by whole-genome sequencing, further analyzes the GWAS results, and identifies SNP molecular markers significantly associated with keel length and sperm density respectively. Using these SNP molecular markers, marker-assisted selection can be carried out for trait indicators such as chicken keel length and sperm density, for early breeding selection, and to improve breeding efficiency. The present invention provides a reliable method for detecting molecular markers for the genetic improvement of chicken growth and reproductive traits.

[0038] The technical solutions provided by the present invention will be described in detail below in conjunction with embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0039] Embodiment

[0040] Obtaining SNP Loci Affecting Chicken Keel Length and Sperm Density

[0041] 1. Test Materials

[0042] Taking the Wen's and Wenchang adult rooster populations as the research objects, through body size measurement and semen quality measurement of 473 individuals, collecting phenotypic measurement data of growth and reproductive traits of all individuals, and collecting whole blood for genomic DNA extraction.

[0043] 2. Test Methods

[0044] 2.1 Semen Quality Measurement

[0045] The roosters are 6 months old, with strong physique and good reproductive performance. Before semen collection, disinfect the semen collection tools such as semen collection tubes and gloves, disinfect and clean the area around the cloaca with a low-concentration potassium permanganate solution, trim the hair around the cloaca at the same time, and use the back massage method for semen collection. The beakers and glass rods used for semen dilution are disinfected in advance, the glass slides and coverslips are placed on the heating stage of the Bei'ang sperm-assisted analyzer in advance for preheating at 37 °C, and the diluent is prepared in advance and placed in a water bath for preheating at 37 °C. After collecting fresh semen, use an electronic thermometer to measure the temperature of the semen and the frozen diluent. When the temperatures are the same, aspirate 0.1 mL of fresh semen and add it to 0.9 mL of preheated diluent for 1:9 dilution. After gently inverting and mixing evenly, aspirate 5 μL and drop it on the glass slide, cover it with a coverslip, and use the above-mentioned Bei'ang sperm analyzer to detect sperm density and motility. This is the sperm density data of fresh chicken semen, and make a good record.

[0046] 2.2 Growth Trait Measurement

[0047] Use a tape measure to measure the distance between the front end and the end of the keel process on the body surface, which is the keel length.

[0048] 2.2, DNA Extraction

[0049] For DNA extraction, the commonly used phenol-chloroform crude extraction method is adopted (for the phenol-chloroform crude extraction method, see Sambrook J, Fritsch EF, Maniatis T. Molecular Cloning: A Laboratory Manual [M]. 2nd ed. Jin Dongyan, Li Mengfeng. Beijing: Science Press, 1999. 465-467), or other recognized extraction methods with the same efficacy. These methods are all commonly reported methods.

[0050] 2.3, Chicken Whole Genome SNP Genotyping Method Based on Whole Genome Sequencing

[0051] By performing whole genome sequencing on 473 individuals with a sequencing depth of 8×, after steps such as read alignment, sorting, marking duplicates, base quality recalibration, and variant detection, 30.1 million SNP sites were initially identified. The Plink software was used to calculate the individual missing rate, SNP site missing rate, and minor allele frequency MAF respectively, and quality control criteria were established. Finally, 18.29 million high-quality SNP markers were obtained.

[0052] 3. Genome-Wide Association Study

[0053] GWAS analysis was performed on keel length and sperm density in growth and reproduction traits based on the mixed linear model of the gemma software. The analysis model is as follows:

[0054] y = Xb + Zu + e

[0055] Among them, y is the vector of trait phenotypic values; b is the fixed effect, and X and Z are the association matrices of b and u respectively; u is the vector that all genetic markers follow the distribution, G is the kinship matrix between individuals; e is the random residual vector.

[0056] The GWAS results are as Figures 1 to 2 shown. Calculate the sperm density ( Figure 1 ) and keel length ( Figure 2)The physical distance between two SNPs with significantly associated traits was retained for loci with a physical distance less than 5000 bp. The difference in allele frequencies (DeltFrequency, DeltF) of these SNP loci was calculated, and loci with DeltF < 0.9 were retained. Finally, 2 (1 pair) SNP loci were obtained, namely SNP1 (6:28973618) (C / G) and SNP2 (6:28973838) (G / A). It is speculated that these two loci produce a synergistic effect by regulating the same gene. Further analysis of the two SNP loci significantly associated with the two traits found that the SNP1 locus, i.e., the nucleotide site C / G at position 28973618 on chromosome 6 of the reference sequence of the chicken reference genome GRCg6a version, was significantly associated with semen volume trait. For a total sample of 473, genotyping was performed using whole-genome sequencing ( Figure 3 A), and the results showed that the number of CC-type individuals was 199, the number of CG-type individuals was 165, the number of GG-type individuals was 83, and 26 individuals had genotype deletions. The specific detection results using the primer set showed that the number of CC-type individuals was 170, the number of CG-type individuals was 215, the number of GG-type individuals was 85, and 3 individuals had genotype deletions. The SNP2 locus, i.e., the nucleotide site G / A at position 28973838 on chromosome 6 of the reference sequence of the chicken genome GRCg6a version, was significantly associated with keel length trait. For a total sample of 473, genotyping was performed using whole-genome sequencing ( Figure 3 B), and the results showed that the number of GG-type individuals was 125, the number of GA-type individuals was 171, the number of AA-type individuals was 151, and 26 individuals had genotype deletions. The specific detection using the primer set showed that the number of GG-type individuals was 107, the number of GA-type individuals was 238, the number of AA-type individuals was 112, and 16 individuals had genotype deletions. The haplotype analysis results are shown in Table 1. The research results showed that the polymorphism of the SNP1 locus was C / G, the polymorphism of the SNP2 locus was G / A, and the sperm density and keel length of the CCAA haplotype individuals were the highest. The CC genotype of the SNP1 locus and the AA genotype of the SNP2 locus can be used as breeding selections. Using these two polymorphic loci, marker-assisted selection can be carried out for the keel length and sperm density trait indicators for early breeding selection to improve breeding efficiency. It provides a reliable detection basis for the genetic improvement of chicken growth and reproductive traits.

[0057] Table 1 Comparison of phenotypic values of keel length and sperm density of different haplotype individuals and their numbers

[0058] Double type Number of individuals Sperm density (number / ml) Number of individuals Keel length CCGG 89 <![CDATA[1.8702E+009±7.40113E+008 AB > 88 <![CDATA[12.3557±0.76817 A > CCGA 68 <![CDATA[1.8634E+009±8.12775E+008 AB > 65 <![CDATA[12.4323±0.80372 A > CCAA 17 <![CDATA[2.2173E+009±1.26591E+009 B > 16 <![CDATA[12.8875±1.09598 B > CGGG 18 <![CDATA[1.8474E+009±1.31869E+009 AB > 18 <![CDATA[12.2778±0.76046 A > CGGA 151 <![CDATA[1.7404E+009±8.80705E+008 A > 147 <![CDATA[12.398±0.80974 A > CGAA 43 <![CDATA[1.7449E+009±8.00290E+008 A > 43 <![CDATA[12.6253±0.85284 AB > GGGA 31 <![CDATA[1.9065E+009±9.72594E+008 AB > 30 <![CDATA[12.58±0.76897 AB > GGAA 53 <![CDATA[1.7480E+009±7.11433E+008 A > 53 <![CDATA[12.5389±0.75528 AB >

[0059] The values in the table are "mean ± standard deviation". Different capital letters in the same row indicate extremely significant differences (P < 0.01), and the same letters indicate no significant differences.

[0060] Example 2

[0061] 1. Detection primer design

[0062] For the nucleotide site C / G at position 28973618 on chromosome 6 obtained in Example 1, a primer combination for detecting this site was designed for PCR detection of this SNP site. The primer combination is shown in SEQ ID NO.2 to SEQ ID NO.5. The upstream and downstream inner primer pairs are shown in SEQ ID NO.2 and SEQ ID NO.3 respectively, and the upstream and downstream outer primer pairs are shown in SEQ ID NO.4 and SEQ ID NO.5 respectively. The amplified product sequence can be used as a molecular marker for detecting sperm density, and its sequence is shown in SEQ ID NO.1. Among them, the base R at position 601 in this sequence is the SNP site, and R represents C or A, resulting in the C / G polymorphism of chicken sperm density at this site. For the nucleotide site G / A at position 28973838 on chromosome 6 obtained in Example 1, a primer combination for detecting this site was designed for PCR detection of this SNP site. The primer combination is shown in SEQ ID NO.7 to SEQ ID NO.10. The upstream and downstream inner primer pairs are shown in SEQ ID NO.7 and SEQ ID NO.8 respectively, and the upstream and downstream outer primer pairs are shown in SEQ ID NO.9 and SEQ ID NO.10 respectively. The amplified product sequence is the molecular marker for detecting keel length, and its sequence is shown in SEQ ID NO.6. Among them, the base R at position 601 in this sequence is the SNP site, and R represents G or A, resulting in the G / A polymorphism of keel length at this site.

[0063] Specifically as follows:

[0064]

[0065] SEQ ID NO.2: 5’-TCAGTGAGATTACTCCTAAAGTCAC-3’

[0066] SEQ ID NO.3: 5’-AAACAGGACACCTAAGTGACTGAC-3’

[0067] SEQ ID NO.4: 5’-ATTGCTATGTCACCAACCGATGTG-3’

[0068] SEQ ID NO.5: 5’-CAGTATTGTCCAGAATGCTGCCA-3

[0069]

[0070] SEQ ID NO.7: 5’-CTAAAATAGCCTGATTTCCGG-3’

[0071] SEQ ID NO.8: 5’-ACATAAGAGATACTTTTACACCGT-3’

[0072] SEQ ID NO.9: 5’-GTCCTGTTTACATTTATGTTCTT-3’

[0073] SEQ ID NO.10: 5’-CATCATTTTTGGGATGTTCTG-3

[0074] 2. DNA template

[0075] According to the whole-genome DNA sequencing results, for the nucleotide site at position 28,973,618 on chromosome 6, genomic DNAs of three individuals with CC, CA, and AA genotypes at the SNP site were respectively selected as DNA templates; for the nucleotide site at position 28,973,838 on chromosome 6, genomic DNAs of three individuals with GG, GA, and AA genotypes at the SNP site were respectively selected as DNA templates.

[0076] 3. PCR amplification of the target fragment

[0077] The PCR reaction system is 10 μL: 5 μL of 2×GS Taq PCR Mix, 0.2 μL of each of the four primers (10 μmol / L), 1.0 μL of DNA template (50 ng / μL), and 3.2 μL of ddH2O. The PCR reaction program is: pre-denaturation at 95°C for 3 min, denaturation at 95°C for 30 s, annealing at the corresponding annealing temperature for 30 s, extension at 72°C for 30 s, for a total of 35 cycles; extension at 72°C for 5 min, and storage at 4°C. The annealing temperature for the nucleotide site at position 28,973,618 on chromosome 6 is 60°C, and the annealing temperature for the nucleotide site at position 28,973,838 on chromosome 6 is 47°C.

[0078] 4. Detection of the PCR amplification products

[0079] The specific operations are as follows: Weigh agarose at a mass concentration of 20 g / L, add it to 1×TAE buffer solution, heat and dissolve it to prepare an agarose solution. Add 5 μL of EB solution to every 100 mL of agarose solution, mix well. After slightly cooling, pour it onto the electrophoresis plate, insert the comb plate, and let it solidify into a gel at room temperature. Then place it in 1×TAE buffer solution and gently pull out the comb plate vertically upward. Take 5 μL of PCR product and add it to the gel sample loading wells. At the same time, add DNA molecular weight standard to one of the sample loading wells, connect the power supply, and perform electrophoresis for 15 - 30 min under the condition of a voltage of 135 V for detection. After the electrophoresis is completed, take out the agarose gel, and image it on a gel imager or an ultraviolet transilluminator. Archive the electrophoresis results as an electronic file or take a photo with a photographic system.

[0080] Judge the size of the amplified band according to the DNA molecular weight standard. For the nucleotide site at position 28973618 on chromosome 6, when there are two bands in the amplified fragment with sizes of 598 bp and 442 bp respectively, the genotype of the sample to be tested is CC genotype; when there are two bands in the amplified fragment with sizes of 598 bp and 204 bp respectively, the genotype of the sample to be tested is AA genotype; when there are three bands in the amplified fragment with sizes of 598 bp, 442 bp and 204 bp respectively, the genotype of the sample to be tested is CA genotype.

[0081] For the nucleotide site at position 28973838 on chromosome 6, when there are two bands in the amplified fragment with sizes of 622 bp and 437 bp respectively, the genotype of the sample to be tested is GG genotype; when there are two bands in the amplified fragment with sizes of 622 bp and 229 bp respectively, the genotype of the sample to be tested is AA genotype; when there are three bands in the amplified fragment with sizes of 622 bp, 437 bp and 229 bp respectively, the genotype of the sample to be tested is GA genotype.

[0082] For each site, DNA was extracted from chicken whole blood samples of three genotypes (three samples were taken for each genotype), and according to the amplification system and PCR reaction program for amplifying the target fragment as described above, 9 DNA samples were detected respectively. The electrophoresis detection results are as Figure 4 、 5 shown. The detection results are consistent with the expectations, indicating that the primer pair provided by the present invention can effectively detect individuals of three different genotypes.

[0083] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. SNP molecular marker combination related to chicken growth and reproductive traits, characterized in that, It contains the following two SNP marker sites: SNP1: a C / G mutation at position 28973618 on chromosome 6 of the chicken GRCg6a genome; SNP2: a G / A mutation at position 28973838 on chromosome 6 of the chicken GRCg6a genome.

2. A primer combination for detecting the SNP molecular marker combination according to claim 1, characterized in that, The primer combination includes the primers shown in SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, and SEQ ID NO.

5.

3. The primer combination for detecting the SNP molecular marker combination according to claim 1, characterized in that, The primer combination includes the primers shown in SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, and SEQ ID NO.

10.

4. A SNP molecular marker detection reagent, characterized in that, It contains the primer combination described in claim 2 or 3.

5. A molecular breeding method for improving the sperm density trait of chickens, characterized in that, It includes the following steps: a) Detect the genotype of the SNP1 site in the genome of the chicken to be tested; b) Select individuals with the CC genotype at the SNP1 site as breeding chickens; The SNP1 site is a C / G mutation at position 28973618 on chromosome 6 of the chicken GRCg6a genome.

6. A molecular breeding method for improving the long-trait of chicken keel bone, characterized in that, It includes the following steps: a) Detect the genotype of the SNP2 site in the genome of the chicken to be tested; b) Select individuals with the AA genotype at the SNP2 site as breeding chickens; The SNP2 site is a G / A mutation at position 28973838 on chromosome 6 of the chicken GRCg6a genome.

7. The method according to claim 5 or 6, characterized in that, The detection of the genotype of the SNP1 site in the genome of the chicken to be tested or the detection of the genotype of the SNP2 site in the genome of the chicken to be tested includes the following steps: a) Extract the genomic DNA of the chicken to be tested; b) Perform PCR amplification on the SNP1 site and / or the SNP2 site using the SNP molecular marker detection reagent described in claim 4; c) Analyze the amplified product sequence to determine the SNP site genotype; Wherein: The amplified product sequence of SNP1 is as shown in SEQ ID NO.1, and the 601st base is a C / A polymorphism site; The amplified product sequence of SNP2 is as shown in SEQ ID NO.6, and the 601st base is a G / A polymorphism site.

8. The application of the SNP molecular marker combination related to chicken growth and reproductive traits described in claim 1 in chicken molecular marker-assisted breeding.

9. The application according to claim 8, wherein The chicken molecular marker-assisted breeding includes screening breeding chickens with advantages in chicken keel length traits or screening breeding chickens with advantages in chicken sperm density traits.

10. The application of the primer combination described in claim 2 or 3 or the SNP molecular marker detection reagent described in claim 4 in the improvement of chicken growth and / or reproductive traits.

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