SNP (Single Nucleotide Polymorphism) site related to residual feed intake character of sheldrake and application

Abstract

The invention discloses an SNP (Single Nucleotide Polymorphism) site related to residual feed intake traits of sheldrake and application, and belongs to the field of molecular marker-assisted poultry breeding. The SNP locus is located at a 121482995th basic group of a No.1 chromosome of a duck genome, and G / A mutation exists at the locus; the duck genome is characterized in that the version of the duck genome is GCF003850225.1 PBH 1.5. The SNP site Chr1: 121482995 obviously related to the residual feed intake is detected, the relevance between the SNP site Chr1: 121482995 and the residual feed intake character is analyzed, the result shows that the Chr1: 121482995 site is obviously related to the residual feed intake, the feed intake of an AA genotype group is lower than that of GA and GG groups, the vacancy of research on the feed intake of shelducks on whole genome relevance analysis is filled up, and the shelducks can be used for screening the residual feed intake of shelducks. And theoretical support is provided for breeding of new varieties of high-quality ducks.

Description

Technical Field

[0001] The invention relates to the technical field of molecular marker-assisted poultry breeding, and in particular to a SNP site related to a trait of residual feed intake of shelducks and an application thereof. Background Art

[0002] Poultry development is an indispensable part of animal husbandry development, and its development level is of great significance to the development of animal husbandry and agriculture in my country. Residual feed intake is the most commonly used indicator to evaluate the residual feed intake trait. Residual feed intake (RFI) refers to the difference between the expected feed intake required for growth and maintenance and the actual feed intake. It can compare the individual differences of livestock and poultry at different production levels and is considered to be an important indicator that is more suitable for measuring the feed efficiency of livestock and poultry. It has medium heritability. The RFI trait is independent of the production performance and body size of the animal. The body weight gain of livestock and poultry is taken into account during the calculation, and the metabolic weight of livestock and poultry is also corrected, which is conducive to better breeding of high-quality livestock and poultry with high feed efficiency.

[0003] At present, some key genes and pathways related to RFI have been found in the research on the residual feed intake of shepherd ducks, but its mechanism is still not clear enough. In particular, the relationship between polygenic inheritance and phenotype is complex, and there is a lack of detailed genetic maps and genome-wide association analysis (GWAS) research results. In order to get rid of the current predicament, it is necessary to genetically improve and select existing breeds. The genetic improvement of poultry breeds is inseparable from the discovery of relevant key genes. With the development of science and technology, the application of genome-related technologies in the study of quantitative traits of animals and plants has become more and more extensive and effective, and further through means such as genomic selection, its production performance has been significantly improved. GWAS is an analysis method based on the principle of linkage disequilibrium (LD), that is, by detecting the genetic variation polymorphism in the whole genome of an associated population composed of hundreds of individuals, tens of thousands or even millions of molecular markers are obtained, and then the association between the target traits of the population and the molecular markers is identified, and the relationship between genetic variation and population sample characteristics is determined, which provides ideas for animal breeding and trait improvement. At present, there are few studies on the residual feed intake-related traits of shepherd ducks using large-scale sequencing methods at home and abroad.

[0004] The whole genome association analysis method can provide breeders with a deep understanding of the genetic mechanism and key genes of the residual feed intake of shelducks, provide a scientific basis for the improvement of shelduck breeds, and accelerate the breeding process. Therefore, the application of whole genome association analysis in the genetic breeding of shelducks has an urgent need and broad prospects, and the relevant whole genome association analysis methods are also urgently needed to be developed. Summary of the invention

[0005] The purpose of the present invention is to provide a SNP site related to the residual feed intake trait of Mallard ducks and its application to solve the problems existing in the above-mentioned prior art. The present invention takes the screened Chr21:6918166 site as an example to analyze its association with the residual feed intake trait. The results show that the Chr21:6918166 site is significantly correlated with the residual feed intake, and the residual feed intake of the GG genotype group is lower than that of the GA and AA groups, which fills the gap in the study of the residual feed intake of Mallard ducks in the whole genome association analysis and provides theoretical support for the breeding of new high-quality duck varieties.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The invention provides a SNP site related to the residual feed intake trait of a shelduck, wherein the SNP site is located at the 121482995th base of chromosome 1 of a duck genome, and a G / A mutation exists in the site; the version of the duck genome is GCF_003850225.1_PBH1.5.

[0008] Optionally, the genotype at the SNP site is GG, GA and AA.

[0009] The present invention also provides the use of the SNP site in any of the following:

[0010] (1) Identification of residual feed intake traits of Mallard ducks;

[0011] (2) Improvement of residual feed intake traits in Mallard ducks;

[0012] (3) Screening new varieties related to the residual feed intake traits of Mallard.

[0013] Optionally, the shelduck includes the Wenshi N111 pure-line shelduck.

[0014] Optionally, the remaining feed intake trait of the shelduck includes the remaining feed intake of the shelduck.

[0015] The present invention also provides a breeding method for improving the residual feed intake trait of shelduck, comprising the following steps:

[0016] Extracting the genomic DNA of the duck to be tested, performing whole genome sequencing or obtaining the upstream and downstream 50 kb sequence of the SNP site, judging the residual feed intake trait of the duck to be tested according to the genotype of the SNP site, and breeding duck breeds with excellent residual feed intake traits of the duck according to the judgment result;

[0017] The residual feed intake of the shepherd ducks whose genotype of the SNP site is AA is lower than that of the shepherd ducks whose genotype of the SNP site is GA and GG.

[0018] Optionally, the shepherd duck with the genotype of AA at the SNP site is a shepherd duck with excellent residual feed intake trait.

[0019] Optionally, the shelduck includes the Wenshi N111 pure-line shelduck.

[0020] Optionally, the remaining feed intake trait of the shelduck includes the remaining feed intake of the shelduck.

[0021] The present invention discloses the following technical effects:

[0022] The present invention selects the Wen's N111 pure-line shelduck phenotype for whole-genome association analysis, and through the selection of these traits, identifies single nucleotide polymorphisms significantly associated with residual feed intake, and detects the SNP site Chr1:121482995 significantly associated with residual feed intake.

[0023] The present invention analyzes the correlation between the Chr1:121482995 locus and the residual feed intake trait. The results show that the Chr1:121482995 locus is significantly correlated with the residual feed intake, and the residual feed intake of the AA genotype group is extremely significantly lower than that of the GG genotype (P<0.01), and significantly lower than that of the GA genotype (P<0.05). The phenotypic differences between the GG and GA genotypes are significant (P<0.05).

[0024] The present invention fills the gap in the research of residual feed intake of shepherd ducks in whole genome association analysis, and provides theoretical support for the breeding of new high-quality duck varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0026] Figure 1 1% agarose gel electrophoresis of genomic DNA stock solution (partial display); 051-075 represents the sample number, and M represents the standard DNA molecule;

[0027] Figure 2 This is the QQ plot result of residual feed intake. DETAILED DESCRIPTION

[0028] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0029] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0030] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0031] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0032] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0033] Example

[0034] 1. Experimental Animals

[0035] The experimental animals used were purebred N111 ducks from Yuncheng Wenshi Co., Ltd. in Yunfu City, Guangdong Province. The ducks were caged and raised for 46 days, and were managed according to the breeding process of the factory. The duck house was well ventilated, with appropriate environmental temperature, humidity, and light, and routine immunization and epidemic prevention were carried out.

[0036] 2. Sample collection and processing

[0037] When the feeding period was 46 days, 783 Mallards (all male ducks) were randomly selected from the caged group for sub-wing blood sampling. 1 mL of blood was drawn with a syringe, injected into a blood collection tube containing sodium heparin and mixed to prevent blood coagulation. The individual wing number was marked on the blood collection tube and then stored in a -80°C refrigerator.

[0038] 3. Experimental methods

[0039] 3.1 Phenotypic data determination

[0040] The remaining feed intake was calculated based on the individual feed intake of Mallards, their weight at the beginning of the experiment, and their weight at the end of the experiment. The calculation formula for the remaining feed intake is as follows:

[0041] RFI=ADFI-b0+b1×MBW 0.75 +b2×ADG,

[0042] Among them, ADFI = b0 + b1 × MBW 0.75 +b2×ADG+e, ADFI represents the average daily feed intake, b0 represents the regression intercept, ADG is the average daily weight gain, MBW is the mid-term metabolic body weight, MBW=[(measurement starting weight + measurement ending weight) / 2]×0.75, b1 represents the partial regression coefficient of MBW on ADFI, b2 represents the partial regression coefficient of ADG on ADFI, e is the residual value, that is, the RFI value, which is the difference between the actual feed intake and the expected feed intake of the individual.

[0043] 3.2 Extraction and detection of genomic DNA

[0044] The genomic DNA was extracted using the cetyltrimethylammonium ammonium bromide (CTAB) method. The genomic DNA was extracted according to the conventional extraction steps. The extracted genomic DNA was tested for integrity, purity and concentration. Those that met the requirements were retained, and those that did not meet the requirements were eliminated or re-extracted and tested.

[0045] 3.3 Sequencing data quality control and comparison with reference genome

[0046] The experiment was performed according to the standard protocol provided by the sequencing company. For qualified genomic DNA samples, appropriate size fragments were selected by gel electrophoresis, and then amplified by PCR to build a library. The quality of the built library was tested, and the qualified library was sequenced by the sequencer DNBSEQ-T7, which was performed by Biomarker Biotechnology Co., Ltd. To ensure the quality of information analysis, base sequencing quality distribution analysis, base type distribution check, insert fragment distribution statistics, depth distribution statistics, and filtering of the raw image data (RawReads) files obtained by high-throughput sequencing were performed during sequencing on the DNBSEQ-T7 sequencing system.

[0047] The final sequence obtained by sequencing is relocated to the reference genome for subsequent analysis. The ratio of Clean-Reads that can be mapped to the reference genome to the total Clean-Reads is called the alignment efficiency, i.e., Mapped (%). The reference gene species is Anas platyrhynchos, the reference genome is: GCF_003850225.1_PBH1.5, and the genome source is the database of Beijing Biomarker Biotechnology Co., Ltd.

[0048] 4. Data processing and statistical analysis

[0049] 4.1 Descriptive statistical analysis of residual feed intake-related traits

[0050] The collected residual feed intake data were preliminarily sorted out using Excel, and outliers for each trait were removed according to the μ±3σ principle. Descriptive statistical analysis of the data was performed using R software. The results of the analysis included sample number, mean, standard deviation, and coefficient of variation.

[0051] 4.2 Genome-wide association analysis

[0052] The samples were sent to Beijing Biomarker Biotechnology Co., Ltd. for genome-wide association analysis. In order to screen genes or molecular markers related to the residual feed intake trait in the whole genome, the present invention performed low coverage sequencing (6X) on 783 samples that passed the quality inspection.

[0053] Using EMMAX software mixed linear model, combined with phenotypic and genotypic data, genome-wide association analysis was performed to mine SNPs associated with the residual feed intake trait of shelduck. Considering fixed factors (SNP effect) and random effects (kinship between individuals), the statistical model is as follows:

[0054] The present invention is based on the developed high-density molecular marker data and uses EMMAX to perform association analysis. The mixed linear model formula is as follows:

[0055] yi=β0+β k X ik +η (1)

[0056] Among them, y is the phenotype, X is the genotype, β0 is the fixed effect, β k is the marker effect, η is the error term, and ultimately each variant site can get an association result.

[0057] 4.3 Group Stratification

[0058] Population stratification refers to the difference in allele frequencies due to different ancestors, which has been proven to be a confounding factor and may lead to many false positive results. Therefore, when conducting an association analysis on the remaining feed intake of Mallards, a QQ plot (Quantile-Quantile Plot) was drawn for the remaining feed intake of Mallards to determine whether there is bias in the association analysis and stratification of the sample population.

[0059] 4.4 Gene annotation of significant SNPs

[0060] After obtaining the significant SNPs sites of the whole genome association analysis, based on the reference genome, the genes within 50kb upstream and downstream of the site were retrieved for gene annotation.

[0061] 5. Results and Analysis

[0062] 5.1 Genomic DNA test results

[0063] The genomic DNA extracted from 783 samples needs to be tested for quality by agarose gel electrophoresis. The test results of some genomic DNA are as follows: Figure 1 As shown, the electrophoresis sample wells must be clean and free of contamination, the main band must be clear, and there must be no tailing. In addition, the DNA purity test must be 1.6 <OD 260 / OD 280 <2.0, 1.8 <OD 260 / OD 230 <2.1. The test results of genomic DNA must meet the above requirements at the same time before the library can be constructed, and unqualified samples must be eliminated or DNA extracted again. All samples of the present invention were tested and a total of 783 samples met the qualified requirements.

[0064] 5.2 Sequencing data quality control and comparison with reference genome

[0065] The results of sequencing data quality control are shown in Table 1. The base type distribution test is mainly used to check whether there is AT and CG separation, which may come from sequencing or library construction. If there is obvious separation, it will affect the subsequent analysis. The average percentage of G and C bases in the sample is 41.0303, the average percentage of bases with a quality value greater than or equal to 20 in the total number of bases (Q20 (%)) is 98.8021%, and the average percentage of bases with a quality value greater than or equal to 30 in the total number of bases (Q30 (%)) is 96.5225%. The comparison efficiency of the sample genomic DNA and the reference genomic DNA is above 98%, with an average of 98.2135%, indicating that the library construction and sequencing of the sample are normal.

[0066] Table 1 Sample sequencing data evaluation statistics and alignment rates

[0067]

[0068] Note: Clean-Reads: number of reads after filtering; Clean-Base: number of bases after filtering, the number of Clean-Reads multiplied by the sequence length.

[0069] 5.3 Statistics of SNP detection results between samples and reference genome

[0070] There are two main types of SNP mutations, namely transition (Transition / Ti, variation of the same type of base) and transversion (Transversion / Tv, variation between different types of bases). Generally speaking, the probability of transition is higher than that of transversion, that is, Ti / Tv is greater than 1. As shown in Table 2, the total number of SNPs detected in this experiment is 5754001, the Ti / Tv value is about 2.4257, and the heterozygous ratio (Het-ratio) is 51.24%.

[0071] Table 2 Statistics of SNP detection results between the reference genome

[0072]

[0073] Note: HeterozygosityNumber is the number of heterozygotes, HomozygosityNumber is the number of homozygotes, and Het-ratio is the ratio of heterozygotes.

[0074] 5.4 Descriptive statistical analysis results of residual feed intake

[0075] The descriptive statistical analysis results of the residual feed intake traits are shown in Table 3. From the results of the descriptive statistical analysis, it can be seen that the total number of ducks measured was 783 (all male ducks). The average residual feed intake of Mallards was 0.12.

[0076] Table 3 Descriptive statistical analysis of residual feed intake traits

[0077]

[0078] 5.5 Results of genome-wide association analysis

[0079] The present invention uses Fastlmm, Emmax, and Gemma software for association analysis, and conducts genome-wide association analysis on the residual feed intake of 783 ducks (783 ducks that meet quality requirements after genomic DNA extraction). SNPs significantly associated with residual feed intake were found in the whole genome, and the duck reference genome of the SNP site is: GCF_003850225.1_PBH1.5, and the genome source is the database of Beijing Biomarker Biotechnology Co., Ltd.

[0080] 5.6 Group stratification assessment results

[0081] There are significant SNPs loci for residual feed intake, and their QQ plots are as follows Figure 2 As shown, the horizontal axis represents the expected value and the vertical axis represents the observed value. The thin line in the figure represents the 45° line, which is the predicted threshold. The gray area is the 95% confidence interval of the scattered points on the figure. The farther the SNP is from the solid line, the better the association strength. Figure 2 It can be seen that most of the sites in the lower left corner of the figure are on the diagonal line, indicating that the model selection is reasonable, and the sites in the upper right corner that exceed the diagonal line and the confidence interval have a high significance with the target trait. There is no population stratification in the experimental population.

[0082] 5.7 Gene annotation of genome-wide significantly associated SNPs

[0083] Through GWAS analysis, a SNP with a significant correlation with residual feed intake was screened out and located on chromosome Chr1. Preliminary gene annotation of this site was performed through NCBI and Ensembl, and the annotation results are shown in Table 4.

[0084] Table 4 Annotation results of genome-wide association analysis of residual feed intake

[0085]

[0086] 5.8 Association analysis between significant loci and residual feed intake

[0087] SPSS software was used to perform association analysis between genotype and residual feed intake phenotypic traits. The results showed that there were three genotypes (GG, GA and AA) at the Chr1:121482995 locus in the shelduck population, and the residual feed intake of the AA genotype group was extremely significantly lower than that of the GG genotype (P<0.01) and significantly lower than that of the GA genotype (P<0.05). The phenotypes of the GG and GA genotypes were significantly different (P<0.05), as shown in Table 5.

[0088] Table 5 Information on SNPs significantly associated with residual feed intake

[0089]

[0090] Note: Different letters on the shoulders indicate extremely significant differences (P<0.01).

[0091] The above results also show that the present invention provides a SNP marker at the Chr1:121482995 locus, and the shepherd duck corresponding to the AA genotype has a lower residual feed intake, the shepherd duck corresponding to the GA genotype has a higher residual feed intake, and the shepherd duck corresponding to the GG genotype has a higher residual feed intake. The residual feed intake of ducks is an important indicator of the residual feed intake of ducks, and a lower residual feed intake is better. Therefore, in the breeding of the residual feed intake trait of shepherd ducks, selecting the AA genotype for residual feed intake can improve the breeding efficiency and quickly select duck varieties with excellent residual feed intake traits.

[0092] The residual feed intake was genotyped using the SNP site Chr1:121482995 of the molecular marker provided above, and duck breeds with the AA genotype for residual feed intake were retained, providing a new molecular marker for the screening of the residual feed intake trait of ducks.

[0093] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A SNP locus associated with the residual feed intake trait of shelduck, characterized in that: The SNP site is located at base 121482995 of chromosome 1 of the duck genome, and there is a G / A mutation at the site; the version of the duck genome is GCF_003850225.1_PBH1.

5.

2. The SNP site according to claim 1, characterized in that: The genotypes at the SNP site are GG, GA and AA.

3. Use of the SNP site according to claim 1 in any of the following: (1) Identification of residual feed intake traits of Mallard ducks; (2) Improving the residual feed intake trait of Mallard ducks; (3) Screening new varieties related to the residual feed intake traits of Mallard.

4. The use according to claim 3, characterized in that: The shelduck includes the Wenshi N111 pure-line shelduck.

5. The use according to claim 3, characterized in that: The residual feed intake trait of the mallard includes the residual feed intake of the mallard.

6. A breeding method for improving the residual feed intake trait of shepherd ducks, characterized in that: The following steps are involved: Extract the genomic DNA of the duck to be tested, perform whole genome sequencing or obtain the upstream and downstream 50 kb sequence of the SNP site described in claim 1 or 2, determine the residual feed intake trait of the tested shelduck according to the genotype of the SNP site, and select a duck breed with excellent residual feed intake trait of the shelduck according to the determination result; The residual feed intake of the shepherd duck whose genotype of the SNP site is AA is lower than that of the shepherd duck whose genotype of the SNP site is GA and GG.

7. The method according to claim 6, characterized in that The shepherd duck with the AA genotype of the SNP site is a shepherd duck with excellent residual feed intake trait.

8. The method according to claim 6, characterized in that The shelduck includes the Wenshi N111 pure-line shelduck.

9. The method according to claim 6, characterized in that The residual feed intake trait of the mallard includes the residual feed intake of the mallard.

Images