A snp site related to the remaining foraging amount of sheldrake and application thereof

Abstract

The application discloses a SNP site related to a residual feed intake trait of a sheldrake and application thereof and belongs to the field of molecular marker-assisted poultry breeding. The SNP site is located at the 121482995th base of a duck chromosome 1, and a G / A mutation exists in the site; the version of the duck genome is GCF_003850225.1_PBH1.5. The application detects a SNP site Chr1:121482995 significantly related to residual feed intake, analyzes the correlation between the SNP site and the residual feed intake trait, and finds that the Chr1:121482995 site is significantly related to the residual feed intake, and the population with an AA genotype has a lower feed intake than populations with GA and GG genotypes, thereby filling a vacancy in the research on the feed intake of sheldrakes in whole-genome association analysis and providing theoretical support for the breeding of new high-quality duck varieties.

Description

Technical Field

[0001] This invention relates to the field of molecular marker-assisted poultry breeding technology, and in particular to a SNP locus associated with the residual feed intake trait of Muscovy ducks and its application. Background Technology

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

[0003] Current research on residual feed intake (RFI) in Muscovy ducks has identified some key genes and pathways related to RFI, but the mechanisms remain unclear. In particular, the relationship between polygenic inheritance and phenotype is complex, lacking detailed genetic maps and genome-wide association studies (GWAS). To overcome the current predicament, genetic improvement and selection of existing breeds are essential. The discovery of relevant key genes is crucial for the genetic improvement of poultry breeds. With the development of science and technology, genome-related technologies are increasingly widely and effectively applied in the study of quantitative traits in animals and plants, further enhancing their production performance through methods such as genomic selection. GWAS is an analytical method based on the linkage disequilibrium (LD) principle. It involves detecting genetic polymorphism in the whole genome of a linked population of hundreds of individuals, obtaining tens of thousands or even millions of molecular markers, and then identifying the association between the target trait and the molecular markers, determining the link between gene variation and population sample characteristics, providing insights for animal breeding and trait improvement. Currently, there are relatively few studies both domestically and internationally utilizing large-scale sequencing to investigate traits related to residual feed intake in Muscovy ducks.

[0004] Genome-wide association studies (GWAS) can provide breeders with in-depth insights into the genetic mechanisms and key genes involved in Muscovy duck residual feed intake, offering a scientific basis for breed improvement and accelerating the breeding process. Therefore, the application of GWAS in Muscovy duck genetics and breeding is urgently needed and has broad prospects, and related GWAS methods urgently require development. Summary of the Invention

[0005] The purpose of this invention is to provide a SNP locus associated with the residual feed intake trait in Muscovy ducks and its application, thereby addressing the problems existing in the prior art. Taking the screened Chr21:6918166 locus as an example, this invention analyzes its association with the residual feed intake trait. The results show that the Chr21:6918166 locus is significantly associated with residual feed intake, and the residual feed intake of the GG genotype population is lower than that of the GA and AA populations. This fills the gap in genome-wide association analysis of residual feed intake in Muscovy ducks and provides theoretical support for the breeding of high-quality duck breeds.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] This invention provides a SNP locus associated with the residual feed intake trait of Muscovy ducks. The SNP locus is located at base 121482995 on chromosome 1 of the duck genome, and a G / A mutation exists at this locus. 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] This invention also provides the application of the SNP site in any of the following:

[0010] (1) Identify the characteristics of residual feed intake in Muscovy ducks;

[0011] (2) Breeding for improvement of the residual feed intake trait in Muscovy ducks;

[0012] (3) Screening for new breeds related to the residual feed intake trait of Muscovy ducks.

[0013] Optionally, the Muscovy duck includes the Wens N111 purebred Muscovy duck.

[0014] Optionally, the residual feed intake trait of the Muscovy ducks includes the residual feed intake of the Muscovy ducks.

[0015] This invention also provides a breeding method for improving the residual feed intake trait in Muscovy ducks, comprising the following steps:

[0016] Extract genomic DNA from the duck to be tested, perform whole-genome sequencing or obtain the upstream and downstream 50kb sequences of the SNP site, determine the residual feed intake trait of the duck to be tested based on the genotype of the SNP site, and select duck breeds with excellent residual feed intake traits based on the determination results.

[0017] The remaining feed intake of Muscovy ducks with the genotype AA at the SNP locus was lower than that of Muscovy ducks with the genotypes GA and GG at the SNP locus.

[0018] Optionally, the ducks with the AA genotype at the SNP locus are ducks with excellent residual feed intake traits.

[0019] Optionally, the Muscovy duck includes the Wens N111 purebred Muscovy duck.

[0020] Optionally, the residual feed intake trait of the Muscovy ducks includes the residual feed intake of the Muscovy ducks.

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

[0022] This invention selects the purebred Wens N111 Muscovy duck phenotype for genome-wide association analysis. By selecting these traits, single nucleotide polymorphisms that are significantly associated with remaining feed intake are identified, and the SNP site Chr1:121482995, which is significantly associated with remaining feed intake, is detected.

[0023] This invention analyzed the association between the Chr1:121482995 locus and the remaining feed intake trait. The results showed that the Chr1:121482995 locus was significantly associated with remaining feed intake, and the remaining feed intake of the AA genotype population was significantly less than that of the GG genotype (P<0.01) and significantly less than that of the GA genotype (P<0.05). The phenotypic differences between the GG and GA genotypes were significant (P<0.05).

[0024] This invention fills a gap in the study of residual feed intake in Muscovy ducks through genome-wide association analysis, providing theoretical support for the breeding of high-quality new duck breeds. Attached Figure Description

[0025] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0026] Figure 1 The image shows a partial 1% agarose gel electrophoresis of the genomic DNA stock solution; where 051-075 represent sample numbers and M represents standard DNA molecules.

[0027] Figure 2 The result is a QQ graph showing the remaining feed intake. Detailed Implementation

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

[0029] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0030] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0031] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

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

[0033] Example

[0034] 1. Laboratory animals

[0035] The experimental animals used were N111 purebred Muscovy ducks from Yuncheng Wenshi Co., Ltd. in Yunfu City, Guangdong Province. The ducks were cage-raised for 46 days and managed according to the farm's feeding procedures. The duck house was kept well-ventilated, and the ambient temperature, humidity, and lighting were suitable. Routine immunizations and disease prevention measures were implemented.

[0036] 2. Sample collection and processing

[0037] When the rearing period was 46 days, 783 male ducks were randomly selected from the caged and housed group for blood collection under their wings. 1 mL of blood was drawn with a syringe, injected into a blood collection tube containing heparin sodium, and mixed to prevent blood clotting. The individual wing number was marked on the blood collection tube, and then stored in a -80℃ refrigerator.

[0038] 3. Experimental Methods

[0039] 3.1 Phenotypic Data Determination

[0040] The remaining feed intake was calculated based on the individual feed intake, body weight at the start of the experiment, and body weight at the end of the experiment recorded during the feeding trial. The formula for calculating the remaining feed intake is as follows:

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

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

[0043] 3.2 Extraction and Detection of Genomic DNA

[0044] Genomic DNA was extracted using the cetyltrimethylammonium bromide (CTAB) method, following standard extraction procedures. The extracted genomic DNA was then tested for integrity, purity, and concentration. DNA that met the requirements was retained, while DNA that did not meet the requirements was discarded or re-extracted and re-tested.

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

[0046] The experiment was conducted according to the standard protocol provided by the sequencing company. For qualified genomic DNA samples, appropriate fragment sizes were selected using gel electrophoresis, followed by PCR amplification and library construction. The constructed libraries underwent quality testing, and qualified libraries were then sequenced using the DNBSEQ-T7 sequencer, performed by BGI Genomics Co., Ltd. To ensure the quality of information analysis, base sequencing quality distribution analysis, base type distribution checks, insert fragment distribution statistics, depth distribution statistics, and filtering of the raw image data (Raw Reads) files obtained from high-throughput sequencing were performed during sequencing using the DNBSEQ-T7 system.

[0047] The final sequences obtained from sequencing are remapped onto a reference genome for further analysis. The percentage of clean reads that can be mapped onto the reference genome is called the alignment efficiency, or Mapped (%). The reference genome species is Anas platyrhynchos, and the reference genome is GCF_003850225.1_PBH1.5, sourced from the database of Beijing Biomarker Biotechnology Co., Ltd.

[0048] 4. Data processing and statistical analysis

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

[0050] The collected data on remaining feed intake were initially organized using Excel, and outliers were removed from the data of each trait according to the μ±3σ principle. Descriptive statistical analysis was performed on the data using R software. The results of the analysis are sample size, 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 for genes or molecular markers associated with the remaining feed intake trait across the entire genome, this invention performed low-coverage sequencing (6X) on 783 quality-tested samples.

[0053] Genome-wide association analysis (GWA) was performed using a mixed linear model with EMMAX software, combining phenotypic and genotypic data, to identify Special National Product (SNP) trait related to residual feed intake in Muscovy ducks. Considering both fixed factors (SNP effects) and random effects (inter-individual kinship), the statistical model is as follows:

[0054] This invention utilizes EMMAX for association analysis based on developed high-density molecular marker data. The mixed linear model formula is as follows:

[0055] yi=β0+β k X ik +η (1)

[0056] Where y represents the phenotype, X represents the genotype, β0 represents the fixed effect, and β k For the labeling effect, η is the error term, and finally each variant site can obtain an association result.

[0057] 4.3 Group Stratification

[0058] Population stratification refers to the difference in allele frequencies due to different ancestors. It has been proven to be a confounding factor that may lead to many false positive results. Therefore, when conducting association analysis on the remaining feed intake of Muscovy ducks, a Quantile-Quantile Plot is plotted on the remaining feed intake of Muscovy ducks to determine whether there is bias in the association analysis and whether there is stratification in the sample population.

[0059] 4.4 Gene annotation of significant SNPs

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

[0061] 5. Results and Analysis

[0062] 5.1 Genomic DNA Detection Results

[0063] Genomic DNA extracted from 783 samples underwent quality testing via agarose gel electrophoresis. Some genomic DNA test results are shown below. Figure 1 As shown, the electrophoresis wells must be clean and uncontaminated, the main band must be clear, and there must be no tailing. Additionally, DNA purity must be measured at 1.6. <OD 260 / OD 280 <2.0, 1.8 <OD 260 / OD 230 <2.1. Genomic DNA testing results must simultaneously meet the above requirements before library construction can proceed. Unqualified samples must be discarded or DNA extracted again. Of all samples tested in this invention, 783 met the requirements.

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

[0065] The sequencing data quality control results are shown in Table 1. Base type distribution detection was mainly used to check for AT and CG segregation, which may originate from sequencing or library construction. Significant segregation will affect subsequent analysis. The average percentage of G and C bases in the sample (GC(%)) was 41.0303%, the average percentage of bases with a quality value greater than or equal to 20 (Q20(%)) was 98.8021%, and the average percentage of bases with a quality value greater than or equal to 30 (Q30(%)) was 96.5225%. The alignment efficiency between the sample genomic DNA and the reference genomic DNA was above 98%, with an average of 98.2135%, indicating that the library construction and sequencing of this sample were normal.

[0066] Table 1. Evaluation Statistics and Comparison Rate of Sample Sequencing Data

[0067]

[0068] Note: Clean-Reads: Number of filtered reads; Clean-Base: Number of filtered bases, calculated by multiplying the number of Clean-Reads by the sequence length.

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

[0070] There are two main types of SNP mutations: transition (Ti, a variation between bases of the same type) and transversion (Tv, a variation between bases of different types). Generally, the probability of transition is higher than that of transversion, i.e., Ti / Tv is greater than 1. As shown in Table 2, a total of 5,754,001 SNPs were detected in this experiment, with a Ti / Tv value of approximately 2.4257 and a heterozygous ratio of 51.24%.

[0071] Table 2. Statistical analysis of SNP detection results between the genome and the reference genome.

[0072]

[0073] Note: HeterozygosityNumber: number of heterozygotes; HomozygosityNumber: number of homozygotes; Het-ratio: percentage of heterozygotes.

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

[0075] The descriptive statistical analysis results of the remaining feed intake trait are shown in Table 3. The results show that a total of 783 ducks (all males) were measured. The average remaining feed intake of the Muscovy ducks was 0.12.

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

[0077]

[0078] 5.5 Results of Genome-wide Association Analysis

[0079] This invention employed Fastlmm, Emmax, and Gemma software for association analysis, performing genome-wide association analysis on the remaining feed intake of 783 Muscovy ducks (783 ducks met the quality requirements after genomic DNA extraction). Significantly associated SNPs with remaining feed intake were identified across the entire genome. The duck reference genome for these SNP loci is GCF_003850225.1_PBH1.5, sourced from the database of Beijing Biomarker Biotechnology Co., Ltd.

[0080] 5.6 Group Stratification Assessment Results

[0081] The remaining feed intake has significant SNP sites, 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 graph represents the 45° line, which is the prediction threshold. The gray area represents the 95% confidence interval of the scatter plot. The greater the distance between the SNP and the solid line, the stronger the association. Figure 2 As can be seen, most of the sites in the lower left corner of the graph are on the diagonal, indicating that the model selection is reasonable. The sites in the upper right corner that extend beyond the diagonal and confidence interval represent the target trait with high significance. There is no population stratification in the experimental population.

[0082] 5.7 Gene annotation of SNPs that are significantly associated at the whole genome level

[0083] GWAS analysis identified one SNP that was significantly correlated with remaining feed intake, located on chromosome Chr1. Preliminary gene annotation of this site was performed using NCBI and Ensembl, and the annotation results are shown in Table 4.

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

[0085]

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

[0087] Association analysis of genotype and residual feed intake phenotype was performed using SPSS software. The results showed that there were three genotypes (GG, GA and AA) at the Chr1:121482995 locus in the Muscovy duck population. The residual feed intake of the AA genotype population was significantly less than that of the GG genotype (P<0.01) and significantly less than that of the GA genotype (P<0.05). The phenotypic differences between the GG and GA genotypes were significant (P<0.05), as shown in Table 5.

[0088] Table 5. SNPs significantly associated with residual feed intake traits.

[0089]

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

[0091] The above results also show that the SNP marker at the Chr1:121482995 locus provided by this invention indicates that the AA genotype corresponds to Muscovy ducks with lower residual feed intake, the GA genotype corresponds to higher residual feed intake, and the GG genotype corresponds to even higher residual feed intake. Residual feed intake is an important indicator of duck residual feed intake, and lower residual feed intake is better. Therefore, selecting the AA genotype for residual feed intake in Muscovy duck breeding can improve breeding efficiency and quickly select duck breeds with superior residual feed intake traits.

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

[0093] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. The application of a SNP locus associated with the residual feed intake trait in Muscovy ducks in any of the following: (1) Identify the characteristics of residual feed intake in Muscovy ducks; (2) Breeding for improvement of the residual feed intake trait in Muscovy ducks; (3) Screening for new breeds related to the residual feed intake trait of Muscovy ducks; The remaining feed intake trait is the remaining feed intake; The SNP site is located at base 121482995 on chromosome 1 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 site are GG, GA, and AA.

2. The application according to claim 1, characterized in that, The ducks mentioned include Wens N111 purebred Muscovy ducks.

3. A breeding method for improving the residual feed intake trait in Muscovy ducks, characterized in that, Includes the following steps: Extract genomic DNA from the ducks to be tested, perform whole-genome sequencing or obtain the upstream and downstream 50kb sequences of SNP loci related to the residual feed intake trait of Muscovy ducks, determine the residual feed intake trait of the ducks to be tested based on the genotype of the SNP loci, and select duck breeds with excellent residual feed intake trait based on the determination results. The SNP site is located at base 121482995 on chromosome 1 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 locus are GG, GA, and AA; the Muscovy ducks with the genotype AA have a lower remaining feed intake than the Muscovy ducks with the genotypes GA and GG at the SNP locus. The remaining feed intake trait is the remaining feed intake.

4. The method according to claim 3, characterized in that, The ducks with the genotype AA at the SNP locus are ducks with excellent residual feed intake traits.

5. The method according to claim 3, characterized in that, The ducks mentioned include Wens N111 purebred Muscovy ducks.

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