Molecular marker associated with head spot size in sex-linked albino chicken and its application

Abstract

The application discloses a molecular marker related to the size of the head white spot of sex-linked Luzhou chicken chicks and an application thereof, and belongs to the field of poultry genetic markers. The application takes a new strain (Luzhou chicken H strain) of sex-linked Luzhou chicken bred by Jiangsu Poultry Science Institute as an experimental object, and screens two molecular markers chr2:6249527 and chr7:25622689 which are significantly related to the size of the head white spot of sex-linked Luzhou chicken chicks. By determining the genotypes of the two sites in the molecular marker, any sex-linked Luzhou chicken at any age stage can be selected, and the chicken individual with a large head white spot can be accurately selected at the chick stage, so that the sex-linked Luzhou chicken strain with a large head white spot is formed. The application is favorable to improving the accuracy of commodity chick self-alternate sex of the sex-linked Luzhou chicken, saving labor, reducing cost, and having great economic application value.

Description

Technical Field

[0001] The invention relates to the field of poultry genetic markers, in particular to molecular markers related to the size of white spots on the heads of sex-linked Luhua chicken chicks and applications thereof. Background Art

[0002] The Wenshang Luhua chicken is a nationally recognized local chicken breed characterized by its sex-linked reed-feather trait. Newborn chicks have black down with white spots of varying sizes on their heads. Locals have come to believe that "black chickens with white heads and white grosbeaks will grow up to be reed-feathered." The white spots gradually disappear with age, and after five weeks of age, the white spots completely disperse, forming a reed-feather coloration with alternating black and white stripes. The Wenshang Luhua chicken boasts excellent egg-laying performance, an attractive carcass, and superior meat quality, making it an excellent material for breeding complementary lines. The H-line Luhua chicken is a new sex-linked Luhua chicken strain developed by the Jiangsu Poultry Science Research Institute using Wenshang Luhua chickens as primary material.

[0003] Sex-linked reed feather color is controlled by the dominant gene B on the sex chromosome. B W)×yellow-feathered broiler strain ♂ (non-reed-feathered rooster, Z b Z b ), the F2 generation of male chicks are all Luhua chickens (Z B Z b ), with irregular white spots on the head, and all the female chicks are non-Luhua chickens (Z b W), there is no white spot on the head, so the chicks can be identified by the presence or absence of white spots on the head. However, due to the different sizes of white spots on the heads of male chicks, under the influence of external factors such as light and identification personnel's eyesight, it is difficult to identify individuals with smaller spots as roosters at a glance ( Figure 1 ), therefore, in order to increase the recognition and consistency of the white spots on the heads of Luhua chicken chicks, improve the accuracy of self-sexing of chicks, increase the uniformity of chicks, and improve the economic benefits of sales, it is urgent to find molecular markers related to the size of the white spots on the heads of sex-linked Luhua chicken chicks. Summary of the Invention

[0004] The purpose of the present invention is to provide molecular markers related to the size of white spots on the heads of sex-linked Luhua chicken chicks and their applications, so as to solve the problems existing in the above-mentioned prior art. Two molecular markers related to the size of white spots on the heads of sex-linked Luhua chicken chicks are obtained by screening, and the molecular markers are used to assist in chicken genetic breeding, which helps to increase the recognition and consistency of white spots on the heads of commercial generation Luhua chicken chicks when sex-linked Luhua chickens are used in combination, improve the accuracy of self-sexing of chicks, and increase the sales revenue of chicks.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The invention provides a molecular marker related to the size of a white spot on the head of a sex-linked reed chicken chick. The molecular marker comprises a molecular marker I and / or a molecular marker II, wherein the molecular marker I comprises a polymorphic site chr2:6249527 located at position 6249527 of chicken chromosome 2, and an A / G mutation exists in the polymorphic site chr2:6249527; the molecular marker II comprises a polymorphic site chr7:25622689 located at position 25622689 of chicken chromosome 7, and an A / G mutation exists in the polymorphic site chr7:25622689; and the reference genome for the physical locations of the polymorphic site chr2:6249527 and the polymorphic site chr7:25622689 is bGalGal1.mat.broiler.GRCg7b.

[0007] Preferably, the genotypes present at the polymorphic site chr2:6249527 and the polymorphic site chr7:25622689 are AA, AG and GG.

[0008] The present invention also provides a primer for identifying the size of the white spot on the head of a sex-linked Luhua chicken chick, comprising a primer pair for amplifying the molecular marker combination, wherein the primer pair comprises (1) a primer pair for amplifying the molecular marker I, whose nucleotide sequence is shown in SEQ ID NO: 1-2, and / or (2) a primer pair for amplifying the molecular marker II, whose nucleotide sequence is shown in SEQ ID NO: 3-4.

[0009] The present invention also provides a kit for identifying the size of the white spot on the head of sex-linked Luhua chicken chicks, comprising the primers.

[0010] The present invention also provides application of the molecular marker in assisted chicken genetic breeding.

[0011] Preferably, it is used to assist in the selection of the size of the white spot on the head of sex-linked Luhua chicken chicks.

[0012] Preferably, the AA genotype of the molecular marker I or the GG genotype of the molecular marker II is a dominant genotype in sex-linked Luhua chicken chicks with a large area of ​​white spots on the head.

[0013] The present invention also provides application of a reagent for detecting the molecular marker in improving the size trait of the white spot on the head of sex-linked Luhua chicken chicks.

[0014] The present invention also provides a method for auxiliary breeding of sex-linked Luhua chicken chicks for the size trait of white spots on the head, comprising the following steps:

[0015] The method comprises the steps of using the genomic DNA of the sex-linked reed chicken chicks to be tested as a template, amplifying the molecular markers using primers, and selecting the sex-linked reed chicken chicks for the size trait of the white spot on the head according to the genotype of the polymorphic site on the amplified sequence; wherein the primers include a primer pair for amplifying the molecular marker I, whose nucleotide sequence is shown as SEQ ID NO: 1-2, and / or (2) a primer pair for amplifying the molecular marker II, whose nucleotide sequence is shown as SEQ ID NO: 3-4.

[0016] Preferably, the breeding method is: retaining individuals with the genotype of the polymorphic site chr2:6249527 of the molecular marker I being AA, and / or retaining individuals with the genotype of the polymorphic site chr7:25622689 of the molecular marker II being GG.

[0017] The present invention discloses the following technical effects:

[0018] (1) The molecular markers related to the size of the white spots on the heads of sex-linked Luhua chicken chicks of the present invention include chr2:6249527 and / or chr7:25622689, which are significantly correlated with the size of the white spots on the heads of sex-linked Luhua chicken chicks at the genome-wide level and have high reliability. The two molecular markers chr2:6249527 and / or chr7:25622689 are helpful in improving the recognition and consistency of the white spots on the heads of sex-linked Luhua chicken chicks in the hybrid breeding and production of sex-linked Luhua chickens, improving the uniformity of chicks, and increasing the economic benefits of chick sales.

[0019] (2) The present invention screens molecular markers related to the size of the white spots on the heads of sex-linked reed chicken chicks and applies them to the genetic breeding of sex-linked reed chickens. By determining the genotypes of the two sites, sex-linked reed chickens of any age stage in the matching utilization can be selected, and chicken individuals with large white spots on the heads when they are chicks can be more targeted and accurately selected to form a sex-linked reed chicken breed with large white spots on the heads. This is beneficial to improving the accuracy of self-sexing of commercial chicks in the matching utilization of sex-linked reed chickens, saving labor, reducing costs, and having great economic application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. 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 any creative work.

[0021] Figure 1 The invention provides a sex-linked reed chicken chick with white spots on the head of different sizes;

[0022] Figure 2 This is the Manhattan plot of the GWAS analysis of the area of ​​white spots on the head of individual chicks of the H pure line of Luhua chicken of the present invention;

[0023] Figure 3 The present invention is chr2:6249527 and chr7:25622689 molecular marker genotype and its correlation with Luhua chicken H line ♀ (Z B W)×yellow-feathered broiler strain♂(Z b Z b )'s results of correlation analysis of the area of ​​white spots on the head of F2 male chicks;

[0024] Figure 4 For Luhua chicken H series ♀(Z B W)×yellow-feathered broiler strain♂(Z b Z b )'s F2 generation of individuals with different head spot sizes, chr2:6249527 and chr7:25622689 molecular marker genotype detection sequencing peak diagram. DETAILED DESCRIPTION

[0025] 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 rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0026] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0027] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice 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 any conflict with any incorporated document, the contents of this specification shall prevail.

[0028] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be illustrative only.

[0029] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0030] The present invention provides a molecular marker related to the size of the white spot on the head of a sex-linked reed chicken chick and an application thereof. The molecular marker related to the size of the white spot on the head of a sex-linked reed chicken chick comprises chr2:6249527 and / or chr7:25622689; wherein the chr2:6249527 molecular marker is located at position 6249527 of the sequence information of chicken chromosome 2 (reference genome: bGalGal1.mat.broiler.GRCg7b), located in the second intron of gene WDR86, and the ensemble number is rs 794263334, where the base is A or G; its genotype includes AA, AG, or GG; the chr7:25622689 molecular marker is located at position 25622689 of the chicken chromosome 7 sequence (reference genome: bGalGal1.mat.broiler.GRCg7b), in the intergenic region, 28.631 kb closest to the functional gene TFCP2L1. The ensemble number is rs741063668, where the base is A or G; its genotype includes AA, GA, or GG. The following examples further illustrate the molecular marker screening process and its application.

[0031] Example 1 Phenotypic determination of white spot area on the head of sex-linked Luhua chicken chicks and screening of related SNP molecular markers

[0032] 1. Phenotyping and blood sample collection

[0033] A new sex-linked Luhua chicken strain (Luhua chicken H line) bred by the Jiangsu Poultry Science Research Institute was used as the experimental subject. The area of ​​white patches on the top of the heads of 300 chicks was measured on the day of hatching: the chicks were placed in the same position under uniform lighting conditions, and their heads were photographed using a high-definition camera. The chick position and lens position were fixed, and a fixed reference object (such as a one-yuan coin) was set within the shooting range. After image acquisition under uniform conditions, Adobe Photoshop CS6 was used to remove clutter from the images and contour the white patches. ImageJ / Image-Pro Plus 6.0 software was used to measure the area of ​​irregular white patches and the reference object in the same image. The white patch area of ​​each chick's head was standardized and corrected according to the actual area of ​​the reference object (such as the area of ​​a one-yuan coin is 4.90625 square centimeters) to obtain accurate phenotypic data of the white patch area on the top of the chick's head.

[0034] When the chickens grow a little bigger and it is convenient to collect blood from the wing vein (7 weeks of age is selected in the present invention), about 2 mL of blood samples are collected from the wing vein of the above 300 experimental chickens, placed in EDTA anticoagulant tubes, and stored at -70°C.

[0035] 2. Whole-genome resequencing and SNP variation detection

[0036] Genomic DNA was extracted from blood samples using the magnetic bead method using the CWE9600 Magbead Blood DNA Kit. DNA samples were detected, amplified, fragment purified, and library constructed in strict accordance with the standard procedures provided by BGI. After library construction was completed and qualified, whole-genome resequencing was performed on 300 individuals of the H-line of Luhua chicken using the MGIDNBSEQ-T7 sequencer (BGI Genomics Co., Ltd., Shenzhen) using the PE150 sequencing strategy.

[0037] The raw sequencing reads were quality-controlled, and sequences containing adapters, N, and low-quality reads were removed to obtain clean reads. Each individual's clean reads were aligned to the chicken reference genome (bGalGal1.mat.broiler.GRCg7b) using BWA 0.7.15, sorted and indexed using Samtools 1.7, and the Bam file was deduplicated using the module included with GATK 4.1.8.0 software. Sequencing depth, genome coverage, and other information for each sample were then calculated based on the Bam file. Based on the alignment results of the clean reads to the reference genome, SNP detection was performed using GATK 4.1.8.0 software. SNPs were then strictly filtered using the Variant Filtration module, resulting in 13,906,779 SNP sites.

[0038] 3. Genotypic and phenotypic quality control

[0039] Genotype quality control was performed using PLINK v1.90b5.3 to remove sites that did not meet the analysis standards. The specific quality control conditions and site statistics are shown in Table 1 below:

[0040] Table 1 Quality control conditions and site number statistics

[0041]

[0042] Phenotypic quality control criteria: Individuals with missing phenotypes were deleted and the phenotypic data were tested for conformity to a normal distribution. After quality control of the phenotypic data, one individual with missing phenotype was deleted, leaving 299 samples for subsequent analysis. The phenotypic data overall conformed to a normal distribution.

[0043] 4. Screening of trait-related loci

[0044] To eliminate false positives due to population structure, principal component analysis (PCA) was performed on all individuals using the measured genotypes, and the first three principal components were included as covariates in the GWAS model. Redundant loci were filtered using the –indep-pairwise 50100.2 filter for quality-controlled autosomal and Z chromosome SNPs, resulting in 216,880 independent loci. The GWAS used GEMMA software, and the Bonferroni multiple test method was used to determine the association significance threshold. The genome-wide significance threshold was (0.05 / 216,880)2.31×10 -7 The recommended genome threshold (1 / 216880) is 4.61×10 -6 GWAS analysis of the area of ​​white spots on the head of sex-linked Luhua chicken chicks was conducted using a mixed linear model (see Figure 2 ), and obtain the significance test P value of each SNPs marker. The matrix expression of the linear model is:

[0045] y=Xβ+Z k γ k +ξ+е

[0046] Among them, y is the phenotypic value vector of the white spot area on the head of the experimental chicks, Xβ is the group structure effect, Z k γ k is the marker effect to be tested, ξ~N(0,Kφ 2 ) is a polygenic effect, e~N(0,Iσ 2 ) is the residual effect. K in the polygenic effect is the kinship matrix inferred by the markers.

[0047] GWAS analysis identified two significant SNPs, chr2:6249527 and chr7:25622689 (see Table 2, Figure 2 ), the chicken reference genome information (bGalGal1.mat.broiler.GRCg7b) was downloaded from NCBI, and the genes near the significant sites were annotated using ANNOVAR software. chr2:6249527 is located at position 6249527 on chromosome 2, in the second intron of gene WDR86, where the base is A or G; chr7:25622689 is located at position 25622689 on chromosome 7, in the intergenic region, closest to the functional gene TFCP2L1, at 28.631 Kb, where the base is A or G.

[0048] Table 2 Genetic markers associated with the area of ​​white spots on the head of sex-linked Luhua chicken chicks

[0049]

[0050] Note: The physical positions of the marker chromosomes and the annotated candidate genes in the table are all referenced to the chicken genome (bGalGal1.mat.broiler.GRCg7b); the functional region annotation dist=28631 indicates that the distance between the site and the candidate gene is 28631 bp.

[0051] Example 2 A sex-linked molecular marker combination associated with the area of ​​white spots on the head of Luhua chicken chicks and its application

[0052] The candidate gene association analysis was performed using the above-mentioned chr2:6249527 and chr7:25622689 genetic marker combination on the F2 generation of chicks and roosters from the Luhua chicken H line (♀) × yellow-feathered broiler line (♂). The specific steps are as follows:

[0053] 1. Phenotyping and Sample Collection

[0054] The phenotypic determination method in Example 1 was used to obtain the phenotypic data of the white spot area on the head of F2 generation reed chicken male chicks of the reed chicken H line (♀) × yellow-feathered broiler chicken line (♂), a total of 265 data; at the same time, corresponding blood samples were collected, and genomic DNA was extracted and preserved.

[0055] 2. PCR primer design and amplification of relevant marker sites

[0056] Primer Design: Based on the physical locations of the chr2:6249527 and chr7:25622689 SNP markers in the reference genome (bGalGal1.mat.broiler.GRCg7b), DNA template sequences containing these two SNP markers were downloaded from the NCBI website. PCR primers were designed using Primer Premier 6.0 software. Primer information is shown in Table 3. PCR primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0057] Table 3 Amplification primers used to detect sex-linked genetic markers for the size of white spots on the head of Luhua chicken chicks

[0058]

[0059] PCR amplification process: ① Reaction system: 20 μL system includes 1.5 μL DNA template of the material to be identified, 0.5 μL each of forward and reverse primers, 0.5 μL dNTP mixture, 0.3 μL Taq DNA polymerase, 2 μL 10× Buffer, and the remaining volume is made up with ultrapure water.

[0060] ② Reaction procedure: first, denaturation at 95°C for 30 s, annealing temperature as shown in Table 3, annealing time for 30 s, extension at 72°C for 30 s, for a total of 10 cycles; then denaturation at 95°C for 30 s, annealing temperature as shown in Table 3, annealing time for 30 s, extension at 72°C for 30 s, for a total of 32 cycles; extension at 72°C for 5 min, and storage at 10°C.

[0061] Polymorphism detection and association analysis: The amplified products were sent to sequencing companies for sequence polymorphism detection to obtain the genotypes of the tested individuals and the sequencing sequences of samples with different genotypes (see Figure 4 ); IBM SPSS Statistics 20.0 was used to perform one-way analysis of variance on the genotypes of the subjects and the areas of white spots on their heads (see Figure 3 ).

[0062] Combine Figure 3 and Figure 4 The results showed that for the molecular marker chr2:6249527, the average area of ​​white spots on the head of individuals with AA genotype was 2.425±0.136cm 2 The average area of ​​white spots on the head of individuals with AG genotype was 2.088±0.103cm 2 The average area of ​​white spots on the head of individuals with GG genotype was 1.496±0.108cm 2 The differences between the genotypes were significant (P < 0.05). AA was the preferred genotype for the molecular marker chr2:6249527, with the largest and most obvious white spot area and the highest recognition.

[0063] For molecular marker chr7:25622689, the average area of ​​white spots on the top of the head of individuals with GG genotype was 2.297±0.096cm 2 The average area of ​​white spots on the head of individuals with GA genotype was 1.695±0.109cm 2 The average area of ​​white spots on the head of individuals with AA genotype was 1.503±0.149cm 2 The pairwise comparisons between genotypes showed significant differences (P < 0.05). GG was the preferred genotype of genetic marker chr7:25622689, with the largest and most obvious white spot area and the highest recognition.

[0064] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. The use of a reagent for detecting a molecular marker associated with the size of a sex-linked white spot on the head of a Luhua chicken chick in assisting chicken genetic breeding, characterized in that: The molecular markers include molecular marker I and / or molecular marker II, wherein the molecular marker I is a polymorphic site chr2:6249527 located at position 6249527 of chicken chromosome 2, and the polymorphic site chr2:6249527 has an A / G mutation; the molecular marker II is a polymorphic site chr7:25622689 located at position 25622689 of chicken chromosome 7, and the polymorphic site chr7:25622689 has an A / G mutation; the reference genome for the physical locations of the polymorphic site chr2:6249527 and the polymorphic site chr7:25622689 is bGalGal1.mat.broiler.GRCg7b; The application is to assist in selecting the size of the white spots on the heads of sex-linked Luhua chicken chicks; The reed chicken is the Wenshang reed chicken.

2. The use according to claim 1, characterized in that The AA genotype of the molecular marker I or the GG genotype of the molecular marker II is a dominant genotype for sex-linked Luhua chicken chicks with a large area of ​​white spots on the top of their heads.

3. The use of a reagent for detecting molecular markers associated with the size of white spots on the head of sex-linked Luhua chicken chicks in improving the size trait of white spots on the head of sex-linked Luhua chicken chicks, characterized in that: The molecular markers include molecular marker I and / or molecular marker II, wherein the molecular marker I is a polymorphic site chr2:6249527 located at position 6249527 of chicken chromosome 2, and the polymorphic site chr2:6249527 has an A / G mutation; the molecular marker II is a polymorphic site chr7:25622689 located at position 25622689 of chicken chromosome 7, and the polymorphic site chr7:25622689 has an A / G mutation; the reference genome for the physical locations of the polymorphic site chr2:6249527 and the polymorphic site chr7:25622689 is bGalGal1.mat.broiler.GRCg7b; The reed chicken is the Wenshang reed chicken.

4. A method for assisting in the selection of sex-linked white spot size traits on the head of Luhua chicken chicks, characterized in that: The following steps are involved: The method comprises the steps of using genomic DNA of sex-linked Luhua chicken chicks to be tested as a template, amplifying molecular markers using primers, and selecting sex-linked Luhua chicken chicks for the size trait of white spot on the head based on the genotype of the polymorphic site on the amplified sequence; wherein the primers comprise (1) a primer pair for amplifying the molecular marker I, the nucleotide sequence of which is shown in SEQ ID NO: 1-2, and / or (2) a primer pair for amplifying the molecular marker II, the nucleotide sequence of which is shown in SEQ ID NO: 3-4; The molecular markers include molecular marker I and / or molecular marker II, wherein the molecular marker I is a polymorphic site chr2:6249527 located at position 6249527 of chicken chromosome 2, and the polymorphic site chr2:6249527 has an A / G mutation; the molecular marker II is a polymorphic site chr7:25622689 located at position 25622689 of chicken chromosome 7, and the polymorphic site chr7:25622689 has an A / G mutation; the reference genome for the physical locations of the polymorphic site chr2:6249527 and the polymorphic site chr7:25622689 is bGalGal1.mat.broiler.GRCg7b; The reed chicken is the Wenshang reed chicken.

5. The method according to claim 4, wherein The breeding method is: retaining individuals with the genotype of AA at the polymorphic site chr2:6249527 of the molecular marker I, and / or retaining individuals with the genotype of GG at the polymorphic site chr7:25622689 of the molecular marker II.

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