An SNP molecular marker related to chicken feed conversion rate and its application

Abstract

The present invention relates to the technical field of animal breeding, and particularly relates to an SNP molecular marker related to chicken feed conversion rate and its application. The SNP molecular marker includes one or more of rs15450437, rs314248376 or rs15702068; rs15450437 is located at 143623538 bp on chicken chromosome 1, and the polymorphism is T / C; rs314248376 is located at 52556471 bp on chicken chromosome 3, and the polymorphism is C / T; rs15702068 is located at 12271457 bp on chicken chromosome 13, and the polymorphism is A / G. The present invention provides SNP molecular markers related to chicken feed conversion rate. Based on the polymorphism detection results, the feed conversion rate of chickens can be judged. These SNP molecular markers are further applied to breed chicken varieties with different feed conversion rates, which has important value.

Description

Technical Field

[0001] The present invention relates to the technical field of animal breeding, and particularly relates to an SNP molecular marker related to chicken feed conversion rate and its application. Background Art

[0002] The scarcity of arable land resources is a long-term problem faced by the development of modern agriculture and the guarantee of food security. The key to solving this problem lies in taking the development path of "grain-saving" animal husbandry. Broiler chickens not only contribute to saving food and limited land resources due to their high feed conversion efficiency, but also help reduce carbon emissions, which is of positive significance to both production and environmental protection.

[0003] Feed conversion efficiency is an important economic and technical indicator to measure the feed utilization efficiency of livestock and poultry. In scientific research and production, residual feed intake (RFI) is usually used to measure the level of feed conversion efficiency. At the same time, as a complex quantitative trait, the potential biological regulation process of feed conversion efficiency is affected by various factors. In terms of genetic regulation, with the upgrading of high-throughput sequencing technology and the continuous development of genome mining technology, the method of GWAS (genome-wide association study) has been widely used in the mining of major genes for livestock and poultry economic traits, and multiple candidate genes affecting chicken feed conversion efficiency have also been mapped. However, due to the influence of population specificity, the application of relevant results in broiler chickens is limited. Summary of the Invention

[0004] In order to solve the problems existing in the prior art, the present invention provides an SNP molecular marker related to chicken feed conversion rate and its application.

[0005] The present invention screens 3 key SNP markers (rs15450437, rs314248376 or rs15702068) affecting feed conversion rate through methods such as genome-wide association analysis (GWAS).

[0006] In a first aspect, the present invention provides an SNP molecular marker, including: one or more of rs15450437, rs314248376 or rs15702068;

[0007] Based on the GRCg7b reference genome, the rs15450437 is located at 143623538 bp on chicken chromosome 1, and the polymorphism is T / C; the rs314248376 is located at 52556471 bp on chicken chromosome 3, and the polymorphism is C / T; the rs15702068 is located at 12271457 bp on chicken chromosome 13, and the polymorphism is A / G.

[0008] The present invention further provides an SNP molecular marker, which comprises a nucleotide sequence as shown in any one of SEQ ID NO.1-3 and is located at any one or more of the following sites:

[0009] i) the 97th position of the nucleotide sequence shown in SEQ ID NO.1;

[0010] ii) the 85th position of the nucleotide sequence shown in SEQ ID NO.2;

[0011] iii) the 49th position of the nucleotide sequence shown in SEQ ID NO.3.

[0012] In a second aspect, the present invention further provides a primer pair, which comprises any one or more of the following:

[0013] i) the nucleotide sequences shown in SEQ ID NO.4 and SEQ ID NO.5;

[0014] ii) the nucleotide sequences shown in SEQ ID NO.6 and SEQ ID NO.7;

[0015] iii) the nucleotide sequences shown in SEQ ID NO.8 and SEQ ID NO.9.

[0016] The present invention further provides a kit, which comprises the above-mentioned SNP molecular marker or the above-mentioned primer pair.

[0017] In a third aspect, the present invention provides the use of the above-mentioned SNP molecular marker, or the above-mentioned primer pair, or the above-mentioned kit in the detection of the feed conversion rate of chickens.

[0018] Furthermore, the above-mentioned use includes:

[0019] (1) Extracting the genomic DNA of the chicken to be tested;

[0020] (2) Using the genomic DNA as a template and amplifying with the above-mentioned primer pair;

[0021] (3) Detecting the amplification product and judging the feed conversion rate of the chicken to be tested according to the detection result.

[0022] Furthermore, the judgment of the feed conversion rate of the chicken to be tested according to the detection result includes:

[0023] The feed conversion rate of the chicken to be tested corresponds to the polymorphisms CC, TC and TT of the locus rs15450437 from low to high;

[0024] The polymorphisms CC, CT, and TT of locus rs314248376 correspond to the feed conversion rate of the chickens to be tested from low to high;

[0025] The polymorphisms GG, AG, and AA of locus rs15702068 correspond to the feed conversion rate of the chickens to be tested from low to high.

[0026] The present invention further provides the application of the SNP molecular marker, or the primer pair, or the kit in chicken breeding.

[0027] Furthermore, the application includes: cultivating chicken breeds with low feed conversion rate.

[0028] As a preferred specific embodiment, the present invention provides a method for selecting meat chicken strains with relatively low feed conversion rate, including:

[0029] (1) Obtaining genomic DNA of chicken samples and performing whole-genome SNP genotyping;

[0030] (2) Obtaining the genotyping data of three SNP markers rs15450437, rs314248376, and rs15702068 of all samples;

[0031] (3) Performing whole-genome breeding value estimation on the genotyping data in step (2) and selecting individuals with better breeding values.

[0032] Furthermore, the chicken is a meat chicken.

[0033] Genomic selection (GS) technology is a breeding method widely used in livestock and poultry breeding at present. Its essence is to estimate the effect value of each marker of an individual using genetic markers across the whole genome, and then accumulate the individual marker effect values to obtain the genomic estimated breeding value (GEBV), and finally rank and select individuals. At present, the G matrix calculation method proposed by Vanraden is the most widely used. However, in the actual application process, only a small part of SNP loci have a large effect on the target trait, and most traits are regulated by minor polygenes and multiple loci. Therefore, Zhang et al. established a new method for estimating GEBV by integrating a few significant SNPs to construct a weighted G matrix, namely GA-BLUP, which has a significant improvement effect on the prediction accuracy.

[0034] The SNP markers provided by the present invention and the GEBV estimation method integrating significant SNPs can be used as an important reference for the selection of chicken feed conversion rate traits and have important practical application value in genetic selection. Analysis found that three key SNPs have a significant impact on the feed conversion rate. It is beneficial to improve the breeding progress, shorten the breeding cycle, and accelerate the improvement process of self-bred varieties and the cultivation of new meat chicken strains.

[0035] The present invention has the following beneficial effects:

[0036] Through GWAS analysis and research, the present invention has screened out 3 SNP molecular markers related to chicken feed conversion rate. Based on the polymorphism detection of these 3 SNP molecular markers, the detection of chicken feed conversion rate can be realized, with high accuracy, which is conducive to accelerating the genetic improvement of chicken feed conversion rate traits. The SNP molecular markers provided by the present invention can be applied to chicken breeding to cultivate chicken breeds with different feed conversion rates, which is of great significance in the field of animal breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0038] Figure 1 It is the Meta-association analysis result of the RFI trait of meat-type breeding chickens provided in Example 1 of the present invention.

[0039] Figure 2 It is the forward sequencing map of three genotypes at the rs15450437 locus provided in Example 1 of the present invention.

[0040] Figure 3 It is the forward sequencing map of three genotypes at the rs15450437 locus provided in Example 1 of the present invention.

[0041] Figure 4 It is the forward sequencing map of three genotypes at the rs15450437 locus provided in Example 1 of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0042] To make the objectives, technical solutions, and advantages of the present invention clearer, the following will clearly and completely describe the technical solutions in the present invention with reference to the drawings in the present invention. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art without creative efforts based on the embodiments in the present invention belong to the scope of protection of the present invention.

[0043] Unless otherwise specified, the experimental methods involved in the following embodiments are all conventional methods in the art, which can be referred to the experimental manuals in the art (such as the Molecular Cloning Experiment Manual by Sambrook et al.), or carried out according to the conditions recommended in the manufacturer's instructions.

[0044] In the following examples, the experimental materials and reagents involved can be obtained from commercial sources without special instructions. For example:

[0045] Example 1

[0046] 1. Identification of candidate intervals affecting chicken feed conversion rate traits based on GWAS analysis

[0047] (1) Experimental animals and phenotype determination

[0048] In this example, 939 purebred meat-type chickens were used to measure the feed conversion rate. All experimental individuals were placed in individual cages in the measurement house at about 28 days of age and fed, immunized, and managed according to the conventional feeding method for broilers. The chickens used in the experimental analysis had complete pedigree records and genomic information. To accurately measure the feed conversion rate, the feed conversion rate from 28 days to 40 days was measured in this invention, and the statistical results are shown in the following table.

[0049]

[0050] (2) GWAS analysis of feed conversion rate traits in meat-type breeding chickens

[0051] The 939 individuals of meat-type breeding chickens were genotyped using the 55K SNP genotyping array developed by the Institute of Animal Science, Chinese Academy of Agricultural Sciences (see CN111225986A). First, the PLINK (V1.9) (https: / / www.cog-genomics.org / plink / 1.9 / ) software was used to perform quality control on 54,130 SNPs from 939 individuals. SNPs with a minor allele frequency (MAF) ≥ 5%, a SNP call rate ≥ 90%, and an individual call rate ≥ 90% were retained. Finally, 38,525 SNP loci on chromosomes 1 - 28 and the Z chromosome were retained for subsequent analysis. After removing genotype variations, the Beagle 5.2 software was used to genotype and impute the missing loci.

[0052] The single-trait mixed linear model (MLM) in the GCTA software was used to perform GWAS analysis on the FCR and RFI traits, with the first three principal components used as covariates for analysis. Considering that there are a large number of linkage disequilibrium regions among SNPs, using the Bonferroni correction is too conservative. Therefore, the plink software --indep -pairwise 25 5 0.2 was used to perform independent tests on SNPs, with 0.2 as the r2 threshold. The genome-wide significant P-value threshold was adjusted according to the number of independent SNPs.

[0053] 2. Meta-analysis to determine important candidate loci affecting egg weight traits

[0054] Considering the obvious population stratification in the hybrid offspring, a Meta-analysis was performed on the hybrid offspring. Compared with directly conducting a GWAS analysis on the combined dataset, the efficiency loss of the Meta-analysis is very small, and at the same time, the influence of population stratification on the GWAS analysis results can be avoided. Therefore, Meta-analysis is widely used to combine the summary data of a large number of genetic variations from genome-wide association studies (GWAS). We used the Metal software to perform a Meta-analysis on four hybrid populations.

[0055] Through single-trait GWAS analysis, the present invention calculated the SNP effect values of related traits. In order to further determine the related regions, a meta-analysis was performed on all individuals after reciprocal crosses, and the results Figure 1 are shown as follows. The genomic inflation factor (λ) is 0.997, indicating that population stratification has been effectively controlled. For the RFI trait, multiple SNPs were found to be significantly associated with RFI on chromosomes 1, 3, 4, 13, and 17.

[0056] The results showed that the NALCN gene on GGA1 is related to chicken embryo nervous system development and nerve function. The ESR1 gene on GGA3 is expressed in chicken ovarian and reproductive tract tissues and is closely related to physiological processes such as ovarian development, follicle development, and ovulation. In addition, the ADGRG6 gene, which is also located on chromosome 3, is involved in regulating physiological processes such as chicken nerve development, cell migration, cell adhesion, and signal transduction. Additionally, the located CITED2 gene plays a role in chicken embryo development, organ development, and function maintenance, and has potential functions in aspects such as chicken metabolic regulation, growth and development, and immune response.

[0057] 3. Screening Results

[0058] (1) Finally, 3 SNP loci related to chicken feed conversion rate were screened out, namely rs15450437, rs314248376, and rs15702068. The position information of the three SNP loci (based on the GRCg7b reference genome) is as follows (the sequencing diagrams of the three SNP loci are as Figures 2 - 4 shown):

[0059]

[0060] For the locus rs15450437, the genotype of the polymorphic locus is CC, indicating that the individual has a low feed conversion rate; if the genotype is TC, it indicates that the individual has a medium-level feed conversion rate; if the genotype is TT, it indicates that the individual has a high feed conversion rate;

[0061] For the locus rs314248376, the genotype of the polymorphic locus is CC, indicating that the individual has a low feed conversion rate; if the genotype is CT, it indicates that the individual has a medium feed conversion rate; if the genotype is TT, it indicates that the individual has a high feed conversion rate.

[0062] For the locus rs15702068, the genotype of the polymorphic locus is GG, indicating that the individual has a low feed conversion rate; if the genotype is AG, it indicates that the individual has a medium feed conversion rate; if the genotype is AA, it indicates that the individual has a high feed conversion rate.

[0063] (2) The present invention further provides primer pairs for amplifying the above SNP loci, as shown in the following table:

[0064]

[0065] The PCR reaction system includes:

[0066]

[0067] The PCR reaction conditions include:

[0068] 94°C for 5 min;

[0069] 94°C for 30 s, 60°C for 30 s, 72°C for 1 min, for a total of 35 cycles;

[0070] 72°C for 5 min.

[0071] Example 2

[0072] The present invention further evaluates the genomic selection effect of the feed conversion rate trait of meat - type breeding chickens using 3 key SNPs (rs15450437, rs314248376, and rs15702068), specifically including the following process:

[0073] 1. Experimental population and genotyping

[0074] Using 939 individuals of meat - type breeding chickens, the feed conversion rate was measured. Based on Example 1, whole - genome SNPs were obtained using a 55K SNP chip, and experiments were carried out with the 3 key SNPs (rs15450437, rs314248376, and rs15702068) screened. The SNPs were quality - controlled according to the following criteria: the individual genotype detection rate was less than 90%, the detection rate of a single SNP locus was less than 90%, and the minor allele frequency was less than 5%. The Beagle 5.0 software was used to fill in the genotypes of the missing SNPs.

[0075] 2. Construction of different strategy G - matrices and heritability estimation

[0076] Using the ASReml software, based on the whole-genome SNPs (excluding 3 key SNPs) and the G matrix based on 3 key SNPs, namely G0 and G SNP . Correct G SNP to the G0 level according to the following formula:

[0077]

[0078] where is the corrected G SNP matrix, and G SNP is the matrix constructed from 3 key SNPs. Heritability estimations were performed separately. The calculation formulas for a and b are:

[0079]

[0080]

[0081] Set the relative weight formula for G1 and as:

[0082]

[0083] In the formula, G2 represents the weighted G matrix, and G1 and see the above formula. The weight coefficient c is calculated according to the following formula:

[0084]

[0085] and are the heritabilities of egg weight traits estimated based on the whole-genome SNPs (excluding 3 key SNPs) and 3 key SNPs, respectively. The heritability was estimated by the ASReml v4.1 software.

[0086] 3. Genomic breeding value estimation

[0087] Using the single-trait animal model of the restricted maximum likelihood method in the ASReml v4.1 software to estimate the breeding value of egg weight. The model is as follows:

[0088]

[0089] where y represents the phenotypic vector, b represents the fixed effect vector, including batch and sex, a represents the random additive genetic effect vector, and e represents the random residual effect vector. X and Z represent the incidence matrices of the fixed effect and random additive genetic effect, respectively.

[0090] The (co)variance matrix of the random vectors is as follows:

[0091]

[0092] Among them, and represent the additive genetic variance and the residual environmental variance respectively; G2 is an integration matrix that assigns specific weights to three key SNPs; I represents the identity matrix.

[0093] 4. Prediction accuracy evaluation

[0094] The method of 5-fold cross-validation was used to estimate the prediction accuracy of the RFI trait. Random numbers were generated using the caret package in R v4.1 for random missing, and the prediction accuracy was estimated. The results are shown in the following table.

[0095]

[0096] According to the cross-validation test results, compared with constructing the G matrix based on the whole-genome SNPs, constructing the integrated G matrix by assigning specific weights to three key SNPs can improve the prediction accuracy of the RFI trait by 5.60%, and the matrix ratio, that is, the coefficient c is 0.097.

[0097] Example 3

[0098] The present invention further provides a breeding method for genomic selection of the feed conversion rate trait of meat-type breeding chickens using three key SNPs (rs15450437, rs314248376, and rs15702068),

[0099] 1. Establishment of the reference population, determination of egg weight trait and genotyping

[0100] The individuals that make up the reference population must cover all existing families of this strain. A population of 1000 - 2000 chickens was formed as the reference population. After the reference population entered the measurement house, the feed conversion rate was measured. At the same time, whole blood was collected and stored in an anticoagulant tube for subsequent DNA extraction and genomic variant sequencing. Whole-genome sequencing or "Jingxin No. 1" chicken 55K SNP chip determination was performed on the reference population to obtain whole-genome SNPs for the next step of analysis.

[0101] 2. Establishment of the population to be tested and genotyping

[0102] The population to be tested refers to a candidate breeding chicken population without phenotypic trait records and ready for breeding the next generation. The chickens in the population to be tested are required to have a genetic relationship within 5 generations with the reference population. On the premise of not affecting the survival rate and growth and development of chickens, blood samples of the chickens in the population to be tested were collected as early as possible and sent for chicken whole-genome SNP chip testing. Then, whole-genome SNP detection and quality control were carried out by the methods in the above steps.

[0103] 3. Analysis of individual genomic estimated breeding values (GEBV) of the reference population and the candidate population

[0104] Using the phenotypic values of each individual in the reference population, the genotypes of the whole-genome SNPs of each individual in the reference population, and the whole-genome genotypes of each individual in the population to be tested (including the genotyping of 3 key SNPs, rs15450437, rs314248376, and rs15702068), a total of 3 types of data files, the genomic estimated breeding value (GEBV) can be estimated using this method.

[0105] Example 4

[0106] The present invention further uses the genotyping of 3 key SNPs to directly select the feed conversion rate trait of meat breeding chickens, including the following:

[0107] 1. Population to be selected

[0108] Randomly select 300 test chickens, with a male-female ratio of 1:1. Collect wing vein blood at about 20 days of age and store it in an anticoagulant tube at -20°C.

[0109] 2. DNA extraction

[0110] Extract the genomic DNA of blood samples using the conventional phenol-chloroform method, dissolve it in ultrapure water, and detect the DNA purity and concentration by agarose gel electrophoresis and NanoPhotometer-N50 spectrophotometer. Uniformly dilute the concentration to 50 ng / μL.

[0111] 3. PCR reaction and SNP genotyping

[0112] The primer sequences of the 3 key loci are as shown in Table 3 in Example 1, the PCR reaction system is as shown in Table 4 in Example 1, and the PCR reaction conditions are the same as those in Example 1. The prepared PCR reaction solution is mixed and centrifuged briefly, and the reaction is carried out using an ABI Life ProFlex PCR thermal cycler.

[0113] Use first-generation sequencing to identify the allele genotypes of the amplification products. According to the genotyping results, retain the healthy male and female chickens with the required genotypes of rs15450437, rs314248376, and rs15702068. Keep the breeding stock according to the number of roosters not less than 80 and the male-female ratio not less than 1:3, and establish a new family for breeding at the peak of egg production.

[0114] Finally, it should be noted that the above examples are only used to illustrate the technical solutions of the present invention, and are not intended to limit them; although the present invention has been described in detail with reference to the foregoing examples, those of ordinary skill in the art should understand that they can still modify the technical solutions recorded in the foregoing examples, or perform equivalent replacements for some of the technical features; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Use of a primer pair or kit for detecting SNP molecular markers in the detection of feed conversion rate of meat breeding chickens, The SNP molecular markers include nucleotide sequences shown in any one of SEQ ID NO.1-3, specifically: i) there is a T / C mutation at the 97th position of the nucleotide sequence shown in SEQ ID NO.1; ii) there is a C / T mutation at the 85th position of the nucleotide sequence shown in SEQ ID NO.2; iii) there is a G / A mutation at the 49th position of the nucleotide sequence shown in SEQ ID NO.3; The primer pairs include the following: i) nucleotide sequences shown in SEQ ID NO.4 and SEQ ID NO.5; ii) nucleotide sequences shown in SEQ ID NO.6 and SEQ ID NO.7; iii) nucleotide sequences shown in SEQ ID NO.8 and SEQ ID NO.9; The kit includes the primer pairs described above; The feed conversion rate corresponds to the polymorphisms CC, TC, and TT of locus rs15450437 from low to high; The feed conversion rate corresponds to the polymorphisms CC, CT, and TT of locus rs314248376 from low to high; The feed conversion rate corresponds to the polymorphisms GG, AG, and AA of locus rs15702068 from low to high.

2. The application according to claim 1, wherein The application includes: (1) Extracting genomic DNA of the chicken to be tested; (2) Using the genomic DNA as a template and performing amplification with the primer pairs described in claim 1; (3) Detecting the amplification product and judging the feed conversion rate of the chicken to be tested according to the detection result.

3. The application according to claim 2, characterized in that, The reaction program of the amplification includes: 92~96°C for 5~10 min; 92~96°C for 30~60 s, 60~62°C for 30~60 s, 72~74°C for 1~2 min, for a total of 32~40 cycles; 72~74°C for 5~10 min.

4. Use of a primer pair or kit for detecting SNP molecular markers in the breeding of meat breeding chickens, The SNP molecular markers include nucleotide sequences shown in any one of SEQ ID NO.1-3, specifically: i) there is a T / C mutation at the 97th position of the nucleotide sequence shown in SEQ ID NO.1; ii) there is a C / T mutation at the 85th position of the nucleotide sequence shown in SEQ ID NO.2; iii) there is a G / A mutation at the 49th position of the nucleotide sequence shown in SEQ ID NO.3; The primer pairs include the following: i) nucleotide sequences shown in SEQ ID NO.4 and SEQ ID NO.5; ii) nucleotide sequences shown in SEQ ID NO.6 and SEQ ID NO.7; iii) nucleotide sequences shown in SEQ ID NO.8 and SEQ ID NO.9; The kit includes the primer pairs described above; The application is to breed meat breeding chicken varieties with low feed conversion rate.

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