SNP (Single Nucleotide Polymorphism) site related to shelduck feed conversion rate character and application

Abstract

The invention discloses an SNP (Single Nucleotide Polymorphism) site related to a shelduck feed conversion rate character and application, and belongs to the field of molecular marker-assisted poultry breeding. The SNP locus is located at the 6918166th basic group of the 21 # 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 locus Chr21: 6918166 obviously related to the feed conversion rate is detected, the relevance between the SNP locus Chr21: 6918166 and the feed conversion rate character is analyzed, the result shows that the Chr21: 6918166 locus is obviously related to the feed conversion rate, and the feed conversion rate of a GG genotype group is lower than that of GA and AA groups, so that the vacancy of research of the feed conversion rate of shelducks on whole genome relevance analysis is filled, and the shelduck feed conversion rate is improved. 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 feed conversion rate of shepherd ducks 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. Feed conversion ratio (FCR) is the most commonly used indicator to evaluate feed conversion traits. It refers to the ratio of the weight of feed ingested by an individual animal to the weight of the livestock products obtained. It is called feed-to-meat ratio in meat-producing livestock and poultry, and feed-to-egg ratio in egg-laying poultry. FCR can directly reflect the input-output ratio of livestock and poultry farming, and has been widely used in livestock production.

[0003] At present, some key genes and pathways related to FCR have been found in the research on feed conversion rate of shepherd ducks, but their 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 varieties. The genetic improvement of poultry varieties 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 traits related to feed conversion rate 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 feed conversion rate of shepherd ducks, provide a scientific basis for the improvement of shepherd duck breeds, and accelerate the breeding process. Therefore, the application of whole genome association analysis in the genetic breeding of shepherd ducks 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 feed conversion rate trait of Mallard duck 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 correlation with the feed conversion rate trait. The results show that the Chr21:6918166 site is significantly correlated with the feed conversion rate, and the feed conversion rate of the GG genotype group is lower than that of the GA and AA groups, which fills the gap in the research of the feed conversion rate of Mallard duck 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 feed conversion rate trait of a shepherd duck, wherein the SNP site is located at the 6918166th base of chromosome 21 of the 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 feed conversion efficiency traits of Mallard ducks;

[0011] (2) Improvement of feed conversion efficiency in Mallard ducks;

[0012] (3) Screening new varieties related to feed conversion efficiency traits of Mallard ducks.

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

[0014] Optionally, the feed conversion rate trait of the shepherd duck includes the feed conversion rate of the shepherd duck.

[0015] The present invention also provides a breeding method for improving the feed conversion rate trait of shepherd ducks, 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 feed conversion rate trait of the duck to be tested according to the genotype of the SNP site, and breeding duck breeds with excellent feed conversion rate traits of the duck according to the judgment result;

[0017] The feed conversion rate of the shepherd duck whose genotype of the SNP site is GG is lower than that of the shepherd duck whose genotype of the SNP site is GA and AA.

[0018] Optionally, the shepherd duck with the SNP site genotype of GG is a shepherd duck with excellent feed conversion rate trait.

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

[0020] Optionally, the feed conversion rate trait of the shepherd duck includes the feed conversion rate of the shepherd duck.

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

[0022] The present invention selects the Wenshi N111 pure line shelduck phenotype for genome-wide association analysis, and through the selection of these traits, identifies single nucleotide polymorphisms significantly associated with feed conversion rate, and as a result, detects 14 SNPs significantly associated with feed conversion rate.

[0023] The present invention takes the Chr21:6918166 locus as an example to analyze its association with the feed conversion rate trait. The results show that the Chr21:6918166 locus is significantly correlated with the feed conversion rate, and the feed conversion rate of the GG genotype group is lower than that of the GA and AA groups. The phenotypic difference between GG and other genotypes is extremely significant (P<0.01), and the phenotypic difference between the GA and AA genotypes is not significant (P>0.05).

[0024] The present invention fills the gap in the research of feed conversion rate 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); wherein 402-426 represent the sample numbers, and M represents the standard DNA molecule;

[0027] Figure 2 This is the QQ plot result of feed conversion rate. 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 feed conversion rate was calculated based on the individual feed intake, body weight at the beginning of the experiment, and body weight at the end of the experiment during the feeding experiment. The calculation formula for the feed conversion rate is as follows:

[0041]

[0042] Among them, FI is feed intake; BWG is the weight gain of ducks during the measurement period.

[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 feed conversion rate-related traits

[0050] The collected feed conversion rate 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 feed conversion rate traits 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 feed conversion rate trait of shepherd ducks. 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 association analysis on the feed conversion rate of Mallard ducks, a QQ plot (Quantile-Quantile Plot) was drawn for the feed conversion rate of Mallard ducks to determine whether there is deviation and stratification of the sample population in the association analysis.

[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: Heterozygosity Number is the number of heterozygotes, Homozygosity Number is the number of homozygotes, and Het-ratio is the ratio of heterozygotes.

[0074] 5.4 Descriptive statistical analysis results of feed conversion rate

[0075] The descriptive statistical analysis results of feed conversion rate are shown in Table 3. From the results of descriptive statistical analysis, it can be seen that the total number of ducks measured was 783 (all male ducks). The average feed conversion rate of Mallard ducks was 2.12, and the coefficient of variation was 6.18%.

[0076] Table 3 Descriptive statistical analysis of feed conversion rate traits

[0077]

[0078] 5.5 Results of genome-wide association analysis

[0079] The present invention uses Fastlmm, Emmax, and Gemma software to perform association analysis, and conducts genome-wide association analysis on the feed conversion rate of 783 ducks (783 ducks that meet quality requirements after genomic DNA extraction). SNPs significantly associated with feed conversion rate 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 sites in feed conversion rate, 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 feed conversion rate was screened and located on chromosome Chr 21. The site was preliminarily annotated using NCBI and Ensembl, and the annotation results are shown in Table 4.

[0084] Table 4 Annotation results of genome-wide association analysis of feed conversion rate

[0085]

[0086] 5.8 Association analysis between significant loci and feed conversion rate traits

[0087] SPSS software was used to perform association analysis between genotype and phenotypic traits of feed conversion rate. The results showed that there were three genotypes (GG, GA and AA) at the Chr21:6918166 locus in the Mallard population and they showed an upward trend. The feed conversion rate of the GG genotype group was higher than that of the GA and AA groups. The phenotypic differences between GG and other genotypes were extremely significant (P<0.01), and the phenotypic differences between the GA and AA genotypes were not significant (P>0.05), as shown in Table 5.

[0088] Table 5 Information on SNPs sites significantly associated with feed conversion rate traits

[0089]

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

[0091] It can also be seen from the above results that the present invention provides a SNP marker for the Chr21:6918166 locus, and the shelduck corresponding to the GG genotype has a lower feed conversion rate, the shelduck corresponding to the GA genotype has a higher feed conversion rate, and the shelduck corresponding to the AA genotype has an even higher feed conversion rate; the duck feed conversion rate is an important indicator for the breeding of duck feed conversion rate traits, and a lower feed conversion rate is better. Therefore, selecting the GG genotype for feed conversion rate in the breeding of shelduck feed conversion rate traits can improve breeding efficiency and quickly select duck breeds with excellent feed conversion rate traits.

[0092] The feed conversion rate was genotyped using the SNP site Chr21:6918166 of the molecular marker provided above, and duck breeds with a feed conversion rate of the GG genotype were retained, providing a new molecular marker for the screening of feed conversion rate traits in 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 site associated with the feed conversion rate trait of shepherd duck, characterized in that: The SNP site is located at base 6918166 of chromosome 21 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 feed conversion efficiency traits of Mallard ducks; (2) Improvement of feed conversion efficiency in Mallard ducks; (3) Screening new varieties related to feed conversion efficiency traits of Mallard ducks.

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 feed conversion rate trait of the shepherd duck includes the feed conversion rate of the shepherd duck.

6. A breeding method for improving the feed conversion rate 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 feed conversion rate trait of the tested shelduck according to the genotype of the SNP site, and select a duck breed with excellent feed conversion rate trait of shelduck according to the determination result; The feed conversion rate of the shepherd duck whose genotype of the SNP site is GG is lower than that of the shepherd duck whose genotype of the SNP site is GA and AA.

7. The method according to claim 6, characterized in that The shepherd duck with the SNP site genotype of GG is a shepherd duck with excellent feed conversion rate 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 feed conversion rate trait of the shepherd duck includes the feed conversion rate of the shepherd duck.

Images