A SNP site related to feed conversion rate trait of sheldrake and application thereof

Abstract

The application discloses a SNP site related to a feed conversion rate character of a cotton duck and application thereof and belongs to the field of molecular marker-assisted poultry breeding. The SNP site is located at the 6918166th base of a duck chromosome 21, and a G / A mutation exists in the site; the version of the duck genome is GCF_003850225.1_PBH1.5. The application detects the SNP site Chr21:6918166 significantly related to the feed conversion rate, analyzes the correlation between the SNP site and the feed conversion rate character, and the result shows that the Chr21:6918166 site is significantly related to the feed conversion rate, and the feed conversion rate of a population with a GG genotype is lower than that of populations with GA and AA genotypes, thereby filling the research gap of the feed conversion rate of the cotton duck in whole-genome association analysis and providing theoretical support for the breeding of a new high-quality duck variety.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of molecular marker assisted poultry breeding, in particular to a SNP site related to feed conversion ratio trait of sheldrake and application thereof. BACKGROUND

[0002] Poultry development is an integral part of the development of animal husbandry, and its development level is of great significance to the development of animal husbandry and agriculture in China. Feed conversion ratio (FCR) is the most commonly used index to evaluate feed conversion trait, which refers to the ratio of the weight of feed intake to the weight of livestock products obtained by an individual animal. In meat-type livestock, it is referred to as feed-to-meat ratio, and in egg-type poultry, it is referred to as feed-to-egg ratio. FCR can directly reflect the input-output ratio of livestock breeding and has been widely used in animal husbandry production.

[0003] In the current research on sheldrake feed conversion ratio, some key genes and pathways related to FCR have been found, but the mechanism is still not clear. Especially, the relationship between polygenic inheritance and phenotype is complex, and there is a lack of detailed genetic map and GWAS research results. In order to get out of the current predicament, it is necessary to carry out genetic improvement and selection on existing breeds. Poultry breed genetic improvement cannot be separated from the excavation of related key genes. With the development of science and technology, genome-related technologies are increasingly widely and effectively applied in the study of plant and animal quantitative traits. Further through genome selection and other means, the production performance can be significantly improved. GWAS is an analysis method based on the principle of linkage disequilibrium (LD), which detects genetic variation polymorphism in the whole genome of an association population consisting of hundreds of individuals, obtains millions of molecular markers, and then identifies the association between the target traits of the population and the molecular markers to determine the relationship between gene variation and population characteristics, providing ideas for animal breeding and trait improvement. At present, there are few studies on sheldrake feed conversion ratio-related traits using large-scale sequencing methods at home and abroad.

[0004] Genome-wide association analysis method can provide a deep understanding of the genetic mechanism and key genes of sheldrake feed conversion ratio for breeders, provide a scientific basis for sheldrake breed improvement, and accelerate the breeding process. Therefore, there is an urgent need and broad prospects for the application of genome-wide association analysis in sheldrake genetic breeding, and the related genome-wide association analysis method also needs to be developed. SUMMARY

[0005] The application aims to provide a SNP site related to the feed conversion rate trait of muscovy ducks and an application thereof, so as to solve the problems in the prior art.

[0006] To achieve the above-mentioned purpose, the application provides the following solutions.

[0007] The application provides a SNP site related to the feed conversion rate trait of muscovy ducks, wherein the SNP site is located at the 6918166th base of chromosome 21 of a duck genome, and a G / A mutation exists at the site; and the version of the duck genome is GCF_003850225.1_PBH1.5.

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

[0009] The application also provides an application of the SNP site in any one of the following aspects.

[0010] (1) identifying the feed conversion rate trait of muscovy ducks;

[0011] (2) improving the feed conversion rate trait of muscovy ducks through breeding;

[0012] (3) screening new varieties related to the feed conversion rate trait of muscovy ducks.

[0013] Optionally, the muscovy ducks include the Wenshi N111 pure line muscovy ducks.

[0014] Optionally, the feed conversion rate trait of muscovy ducks includes the feed conversion rate of muscovy ducks.

[0015] The application also provides a breeding method for improving the feed conversion rate trait of muscovy ducks, which includes the following steps.

[0016] Genomic DNA of a duck to be tested is extracted, whole genome sequencing is performed or the sequence of 50 kb upstream and downstream of the SNP site is obtained, the feed conversion rate trait of the duck to be tested is determined according to the genotype of the SNP site, and a duck variety with an excellent feed conversion rate trait is selected according to the determination result;

[0017] The muscovy duck with the SNP site genotype of GG has a lower feed conversion rate than the muscovy duck with the SNP site genotype of GA or AA.

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

[0019] Optionally, the muscovy duck comprises Wen's N111 pure line muscovy duck.

[0020] Optionally, the feed conversion rate trait of the muscovy duck comprises a feed conversion rate of the muscovy duck.

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

[0022] The present application selects the whole genome correlation analysis on the phenotype of Wen's N111 pure line muscovy duck, and through the selection of these traits, the single nucleotide polymorphism significantly related to the feed conversion rate is identified, and 14 SNPs significantly associated with the feed conversion rate are detected.

[0023] The present application takes the Chr21:6918166 site as an example to analyze the correlation with the feed conversion rate trait, and the result shows that the Chr21:6918166 site is significantly related to the feed conversion rate, and the feed conversion rate of the GG genotype population is lower than that of the GA and AA populations, the phenotype difference between GG and other genotypes is extremely significant (P<0.01), and the phenotype difference between GA and AA genotypes is not significant (P>0.05).

[0024] The present application fills the gap of the research on the whole genome correlation analysis of the feed conversion rate of muscovy duck, and provides theoretical support for the breeding of new high-quality duck varieties. BRIEF DESCRIPTION OF DRAWINGS

[0025] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed in the embodiments will be briefly introduced below. Obviously, the drawings in the following description only constitute some embodiments of the present application, and those skilled in the art can also obtain other drawings according to these drawings without creative labor.

[0026] Figure 1 It is a genomic DNA stock 1% agarose gel electrophoresis chart (partly shown); wherein, 402-426 represent the number of samples, and M represents a standard DNA molecule;

[0027] Figure 2 It is a Q-Q chart result of the feed conversion rate. DETAILED DESCRIPTION

[0028] Now, various exemplary embodiments of the present application will be described in detail, which should not be considered as limitations of the present application, and should be understood as a more detailed description of some aspects, characteristics and embodiments of the present application.

[0029] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the present application. In addition, where a range of values is provided, it is understood that each intervening value, to the upper and lower limit of the range is also specifically disclosed. Each smaller range between any stated value or intervening value in the stated range and any other stated or intervening value in that stated range is encompassed. The upper and lower limits of these smaller ranges can independently be included or excluded in the range.

[0030] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, the preferred methods and materials are described. All patents, patent applications, publications, and descriptions mentioned herein are incorporated by reference to disclose and describe the methods and / or materials in connection with which the patents, patent applications, publications, and descriptions are cited.

[0031] Many modifications and variations of the present application described in the specification are possible without departing from the scope or spirit of the application. Other embodiments of the application will be apparent to those skilled in the art from consideration of the specification and practice of the application disclosed herein. The specification and examples given are exemplary only. It is to be understood that the application is not limited in scope by the described embodiments.

[0032] As used herein, the terms "comprises", "comprising", "includes", "including", "has", "having", "contains", "containing", or variations thereof, are intended to be open-ended terms that mean inclusion, but not limited to, the listed materials and methods.

[0033] Examples

[0034] 1. Experimental animals

[0035] The experimental animals were purebred Muscovy ducks from N111 of Guangdong Yuncheng Wen Company Limited. The experimental ducks were raised in cages, and the feeding age was 46 days. The duck house was kept well ventilated, and the environmental temperature, humidity, and light were suitable. Routine immunization and epidemic prevention were performed.

[0036] 2. Sample collection and processing

[0037] At 46 days of feeding time, 783 Muscovy ducks (all male) were randomly selected from the cage-feeding population for sub-wing blood collection. 1 mL of blood was extracted using a syringe, mixed with heparin sodium in a blood collection tube to prevent blood clotting, and the individual wing number was marked on the blood collection tube. Then, it was stored in a -80℃ refrigerator.

[0038] 3. Experimental methods

[0039] 3.1 Phenotype data determination

[0040] According to the feeding experiment period record the individual intake of the muscovy duck, the test starting weight and the test ending weight, the feed conversion rate is calculated. The calculation formula of the feed conversion rate is as follows:

[0041]

[0042] Wherein, FI is the intake; BWG is the weight gain of the duck during the determination period.

[0043] 3.2 Extraction and detection of genomic DNA

[0044] The genomic DNA is extracted by the cetyltrimethylammonium bromide method (CTAB), and the genomic DNA is extracted according to the conventional extraction procedure. The extracted genomic DNA needs to be detected for integrity, purity and concentration. The genomic DNA meeting the requirements is reserved, and the genomic DNA not meeting the requirements is eliminated or re-extracted and detected.

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

[0046] The experiment is performed according to the standard protocol provided by the sequencing company. For the qualified genomic DNA sample, the appropriate size fragments are selected by gel electrophoresis, then the library is constructed by PCR amplification, and the constructed library is detected for quality. The qualified library is sequenced by the DNBSEQ-T7 sequencer, and the sequencing is performed by the Baimaikexing Technology Co., Ltd. In order to ensure the quality of information analysis, the base sequencing quality distribution analysis, base type distribution check, insert fragment distribution statistics, depth distribution statistics and filtering of the raw image data (Raw Reads) file obtained by high-throughput sequencing are performed when the DNBSEQ-T7 sequencing system is sequenced.

[0047] The final sequence obtained by sequencing is repositioned to the reference genome for subsequent analysis. The ratio of Clean-Reads that can be positioned to the reference genome to the total Clean-Reads is called alignment efficiency, that is, Mapped (%). The reference gene species is Anas platyrhynchos, the reference genome is GCF_003850225.1_PBH1.5, and the genomic source is the database of the Beijing Baimaikexing Technology 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 arranged through Excel, and the data of each property were removed from abnormal values according to the principle of μ±3σ; the data were subjected to descriptive statistical analysis through R software; and the results of the analysis were sample number, average, standard deviation and coefficient of variation.

[0051] 4.2 Whole genome association analysis

[0052] The samples were sent to Beijing Baimaikesi Biological Science and Technology Co., Ltd. for whole genome association analysis. In order to screen the genes or molecular markers related to the feed conversion rate property in the whole genome range, 783 samples qualified in quality detection were subjected to low-coverage sequencing (6X) by the present application.

[0053] EMMAX software was used to combine the phenotype and genotype data to carry out whole genome association analysis, and the SNPs related to the feed conversion rate property of the muscovy duck were mined. Considering the fixed factor (SNP effect) and random effect (genetic relationship between individuals), the statistical model is as follows:

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

[0055] yi = β0+ β k X ik + η (1)

[0056] Wherein, y is the phenotype, X is the genotype, β0is the fixed effect, β k is the marker effect, η is the error term, and finally each variation site can obtain an association result.

[0057] 4.3 Population stratification

[0058] Population stratification refers to the difference in allele frequency caused by different ancestors, which has been confirmed to be a confounding factor and can cause many false positive results, so when the feed conversion rate of the muscovy duck is subjected to association analysis, the Q-Q plot (Quantile-Quantile Plot) of the feed conversion rate of the muscovy duck is drawn to judge whether there is deviation in the association analysis and the stratification phenomenon of the sample population.

[0059] 4.4 Significant SNP gene annotation

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

[0061] 5, Results and analysis

[0062] 5.1 Genome DNA detection results

[0063] 783 samples of extracted genomic DNA need to pass the quality detection by agarose gel electrophoresis, part of the genomic DNA detection results are shown in Figure 1 The electrophoresis sample hole needs to be clean and pollution-free, the main band needs to be clear and without tailing, and the purity of DNA needs to be 1.6 < OD 260 / OD 280 <2.0, 1.8 < OD 260 / OD 230 <2.1. The detection results of genomic DNA need to meet the above requirements at the same time, and then the library can be constructed. The unqualified samples need to be eliminated or extracted again. All the samples of the present application have been detected, and 783 samples meet the qualified requirements.

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

[0065] The sequencing data quality control results are shown in Table 1. The base type distribution detection is mainly used to check whether there is AT, CG separation phenomenon. This phenomenon may come from sequencing or library construction. If there is obvious separation phenomenon, it will affect the subsequent analysis. The average percentage of G and C bases in total bases (GC (%)) of the sample is 41.0303, the average percentage of bases with quality value greater than or equal to 20 in total bases (Q20 (%)) is 98.8021%, and the average percentage of bases with quality value greater than or equal to 30 in total bases (Q30 (%)) is 96.5225%. The alignment efficiency of sample genomic DNA and reference genomic DNA is more than 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 rate

[0067]

[0068] Note: Clean-Reads: filtered reads; Clean-Base: filtered base, Clean-Reads number multiplied by sequence length.

[0069] 5.3 Statistical results of SNP detection between sample and reference genome

[0070] The mutation of SNP type mainly has two types, which are transition (Transition / Ti, variation of the same type of base) and transversion (Transversion / Tv, variation between different types of bases). Generally, 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 heterozygote ratio (Het-ratio) is 51.24%.

[0071] Table 2 Statistics of detection results of SNPs between reference genomes

[0072]

[0073] Note: Heterozygosity Number is the number of heterozygotes, Homozygosity Number is the number of homozygotes, and Het-ratio is the proportion of heterozygotes.

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

[0075] The descriptive statistical analysis results of the feed conversion rate trait are shown in Table 3. According to the results of the descriptive statistical analysis, a total of 783 ducks (all male ducks) were determined. The average feed conversion rate of the muscovy ducks was 2.12, and the coefficient of variation was 6.18%.

[0076] Table 3 Descriptive statistical analysis results of the feed conversion rate trait

[0077]

[0078] 5.5 Whole genome association analysis results

[0079] The present application uses Fastlmm, Emmax and Gemma three software for association analysis, and performs whole genome association analysis on the feed conversion rate of 783 muscovy ducks (after extracting genomic DNA, the muscovy ducks meeting the quality requirements are 783). In the whole genome range, SNPs significantly related to the feed conversion rate are associated. The duck reference genome of the SNP site is: GCF_003850225.1_PBH1.5, and the genome source is the database of Beijing Baimaikesi Biological Technology Co., Ltd.

[0080] 5.6 Population stratification evaluation results

[0081] The significant SNPs site of the feed conversion rate is shown in the Q-Q plot as Figure 2 , the horizontal coordinate represents the expected value, and the vertical coordinate represents the observed value. The fine line in the figure represents the 45° line, which is the predicted threshold value, and the gray area is the 95% confidence interval of the scatter points on the graph. The farther the distance between the SNP and the solid line, the better the correlation strength. As Figure 2 can be seen, most of the sites in the lower left corner of the graph 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 represent a higher significance with the target trait. The experimental population does not have population stratification phenomenon.

[0082] 5.7 Gene annotation of significant SNPs at the whole genome level

[0083] Through GWAS analysis, 1 SNP reaching a significant correlation level with feed conversion rate was screened, which was located on chromosome Chr 21. The site was preliminarily annotated through NCBI and Ensembl, and the annotation results are shown in Table 4.

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

[0085]

[0086] 5.8 Significant site and feed conversion rate trait association analysis

[0087] Using SPSS software, the genotype and feed conversion rate phenotype trait were analyzed, and the results showed that: the Chr21:6918166 site existed three genotypes (GG, GA and AA) in the mallard population and showed an upward trend, the feed conversion rate of the GG genotype population was lower than that of the GA and AA populations, the phenotype difference between GG and other genotypes was extremely significant (P<0.01), and the phenotype difference between GA and AA genotypes was not significant (P>0.05), as shown in Table 5.

[0088] Table 5 Information of SNPs sites significantly related to feed conversion rate trait

[0089]

[0090] Note: Different letters in the shoulder indicate extremely significant difference (P<0.01).

[0091] The above results also show that the present application provides the SNP marker of Chr21:6918166 site, the mallard corresponding to the GG genotype has a lower feed conversion rate, the mallard corresponding to the GA genotype has a higher feed conversion rate, and the mallard corresponding to the AA genotype has a higher feed conversion rate; the duck feed conversion rate is an important indicator of duck feed conversion rate trait breeding, and a lower feed conversion rate is better, so in the mallard feed conversion rate trait breeding, selecting the feed conversion rate as the GG genotype can improve the breeding efficiency and quickly select duck breeds with excellent feed conversion rate traits.

[0092] By genotyping the feed conversion rate through the SNP site Chr21:6918166 of the molecular marker provided above, the duck breeds with the feed conversion rate of GG genotype are retained, providing a new molecular marker for screening the feed conversion rate trait of ducks.

[0093] The above-described embodiments only describe the preferred modes of the present application, and do not limit the scope of the present application. Without departing from the design spirit of the present application, various modifications and improvements to the technical solutions of the present application made by those skilled in the art shall fall within the protection scope determined by the claims of the present application.

Claims

1. The application of a SNP site associated with feed conversion ratio in Muscovy ducks in any of the following: (1) Identify the feed conversion ratio characteristics of Muscovy ducks; (2) Breeding for improvement of feed conversion ratio in Muscovy ducks; The duck in question is a purebred male Muscovy duck of the Wens N111 strain. The SNP site is located at base 6918166 on chromosome 21 of the duck genome, and a G / A mutation exists at this site; the version of the duck genome is GCF_003850225.1_PBH1.5; The genotypes at the SNP loci are GG, GA, and AA; The feed conversion ratio of Muscovy ducks with the SNP genotype GG is lower than that of Muscovy ducks with the SNP genotypes GA and AA.

2. The application according to claim 1, characterized in that, The feed conversion ratio trait of the Muscovy ducks includes the feed conversion ratio of the Muscovy ducks.

3. A breeding method for improving the feed conversion ratio trait of Muscovy ducks, characterized in that, Includes the following steps: Extract genomic DNA from the duck to be tested, perform whole-genome sequencing or obtain the upstream and downstream 50kb sequences of SNP sites related to feed conversion ratio traits in Muscovy ducks, determine the feed conversion ratio traits of the duck to be tested based on the genotype of the SNP sites, and select duck breeds with excellent feed conversion ratio traits based on the determination results. The SNP site is located at base 6918166 on chromosome 21 of the duck genome, and a G / A mutation exists at this site; the version of the duck genome is GCF_003850225.1_PBH1.5; The genotypes at the SNP loci are GG, GA, and AA; The feed conversion rate of Muscovy ducks with the SNP genotype GG was lower than that of Muscovy ducks with the SNP genotypes GA and AA. The duck in question is a male Muscovy duck of the Wens N111 purebred line.

4. The method according to claim 3, characterized in that, The ducks with the SNP locus genotype GG are ducks with excellent feed conversion ratio.

5. The method according to claim 3, characterized in that, The feed conversion ratio trait of the Muscovy ducks includes the feed conversion ratio of the Muscovy ducks.

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