Molecular marker related to size of top white spot of sex-linked Luhua chick and application of molecular marker

Abstract

The invention discloses a molecular marker related to the size of white spots on the tops of sex-linked Luhua chicken chicks and application of the molecular marker, and belongs to the field of poultry genetic markers. According to the invention, two molecular markers chr2: 6249527 and chr7: 25622689, which are significantly related to the size of the white spot on the top of a chick of the sex-linked Luhua chicken, are screened by taking a new strain (Luhua chicken H strain) of the sex-linked Luhua chicken bred by Jiangsu Institute of Poultry Scientific Research as an experimental subject, and the size of the white spot on the top of the chick of the sex-linked Luhua chicken is determined by determining the genotypes of two sites in the molecular markers. The sex-linked Luhua chickens at any day age stage in matched utilization are selected, chicken individuals with large white spots on the tops are selected more specifically and accurately, a sex-linked Luhua chicken strain with large white spots on the tops is formed, the sex-linked Luhua chicken self-sex distinguishing accuracy of commercial chickens in matched utilization of the sex-linked Luhua chickens is improved, labor is saved, cost is reduced, and the sex-linked Luhua chickens with large white spots on the tops are obtained. And the method has 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] Wenshang Luhua Chicken is a national local chicken breed with sex-linked Luhua feathers as its typical characteristics. Newborn chicks have black fluff with white spots of varying sizes on their heads. Local people have concluded that "black chickens with white tops and white melons will definitely be Luhua when they grow up." The white spots on the heads of chicks gradually disappear with age. After 5 weeks of age, the white spots completely disperse, forming a Luhua feather color with black and white stripes all over the body. Wenshang Luhua Chicken has the characteristics of good egg-laying performance, beautiful carcasses, and excellent meat quality. It is an excellent material for supporting breeding. Luhua Chicken H Series is a new sex-linked Luhua Chicken strain bred by Jiangsu Poultry Science Research Institute with Wenshang Luhua Chicken as the main 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 ), irregular white spots on the head, all female chicks are non-Luhua chickens (Z b W), there is no white spot on the top of the head, so the chicks can be identified by the presence or absence of white spots on their heads. However, due to the different sizes of white spots on the top of the heads of male chicks, under the influence of external factors such as light and the eyesight of the identification personnel, it is difficult to identify the 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 urgently necessary 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 reed chicken chicks and applications thereof, 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 reed chicken chicks are obtained by screening, and the molecular markers are used to assist in chicken genetic breeding, which will help to increase the recognition and consistency of white spots on the heads of commercial generation reed chicken chicks when sex-linked reed 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 the 6249527th position of chicken chromosome 2, and the polymorphic site chr2:6249527 has an A / G mutation; the molecular marker II comprises a polymorphic site chr7:25622689 located at the 25622689th position of chicken chromosome 7, and the polymorphic site chr7:25622689 has an A / G mutation; and the reference genome at the physical positions 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 invention also provides a kit for identifying the size of the white spot on the head of a sex-linked Luhua chicken chick, comprising the primers.

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

[0011] Preferably, it is used to assist in the selection of the size of the white spots on the heads 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 for 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 white spots on the top of 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 with a white spot size trait on the head, comprising the following steps:

[0015] The method comprises the steps of using the genomic DNA of the sex-linked Luhua chicken chicks to be tested as a template, amplifying the molecular markers using primers, and selecting the sex-linked Luhua 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 comprise 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.

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

[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 whole genome 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 breeding and production of sex-linked Luhua chicken hybrids, 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 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 loci, sex-linked reed chickens of any age stage in 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 strain with large white spots on the heads. This is beneficial to improving the accuracy of self-sexing of commercial chicks in 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 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.

[0021] Figure 1 The present 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 top of the heads of individual chicks of the H pure line of Luhua chicken of the present invention;

[0023] Figure 3 The molecular marker genotypes of chr2:6249527 and chr7:25622689 of the present invention and their correlation with the H line of Luhua chicken ♀(Z B W)×yellow-feathered broiler strain♂(Z b Z b )'s results of association analysis of the white spot area 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 should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0026] 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.

[0027] 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.

[0028] 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.

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

[0030] The invention provides a molecular marker related to the size of white spots on the top of the head of a sex-linked Luhua chicken chick and an application thereof. The molecular marker related to the size of white spots on the top of the head of a sex-linked Luhua chicken chick comprises chr2:6249527 and / or chr7:25622689; wherein the chr2:6249527 molecular marker is located at the 6249527th position of the sequence information of chicken chromosome 2 (reference genome: bGalGal1.mat.broiler.GRCg7b), is located at 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 the 25622689th position of the sequence information of chicken chromosome 7 (reference genome: bGalGal1.mat.broiler.GRCg7b), located in the intergenic region, closest to the functional gene TFCP2L1, 28.631Kb, and the ensemble number is rs741063668, where the base is A or G; its genotype includes AA, GA or GG. The above molecular marker screening process and application examples are further described below in an embodiment.

[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 strain) bred by Jiangsu Poultry Science Research Institute was used as the experimental object. The area of ​​white spots 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 light environment, and the tops of their heads were photographed with 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 positions of white spots. ImageJ / Image-Pro Plus 6.0 software was used to measure the areas of irregular white spots and reference objects in the same image, and the area of ​​white spots on the top of the heads of each image 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), so as to obtain accurate phenotypic data of the area of ​​white spots on the top of the heads of chicks.

[0034] When the chickens grow a little bigger and it is convenient to collect blood from the wing vein (7 weeks old 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 a kit (CWE9600 Magbead Blood DNA Kit). The DNA samples were tested, amplified, fragment purified and library constructed in strict accordance with the standard method procedures provided by BGI. After the library construction was completed and the library inspection was qualified, the MGIDNBSEQ-T7 sequencer (BGI Co., Ltd., Shenzhen) was used to select the PE150 sequencing strategy to perform whole genome resequencing on 300 chickens of the H line of Luhua chicken.

[0037] The original reads obtained by sequencing were quality controlled, and the original sequencing reads containing adapters, N and low-quality reads were removed to obtain Clean reads. The clean reads of each individual were aligned to the chicken reference genome (bGalGal1.mat.broiler.GRCg7b) using BWA0.7.15, sorted and indexed using Samtools 1.7, and the Bam file was deduplicated using the module provided by the GATK 4.1.8.0 software. The sequencing depth, genome coverage and other information of each sample were then statistically analyzed based on the Bam file. According to the alignment results of Clean Reads on the reference genome, SNP detection was performed using the software GATK4.1.8.0, and then the SNP was strictly filtered using the Variant Filtration module, and finally 13,906,779 SNP sites were obtained.

[0038] 3. Genotypic and phenotypic quality control

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

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

[0041]

[0042] Phenotypic quality control standard: Delete the individuals with missing phenotypes and check whether the phenotypic data conforms to the normal distribution. After quality control of the phenotypic data, 1 individual with missing phenotype was deleted, and the remaining 299 samples can be used for subsequent analysis. The phenotypic data conforms to the normal distribution as a whole.

[0043] 4. Screening of trait-related loci

[0044] To eliminate false positives caused by population structure, principal component analysis (PCA) was performed on all individuals using the measured genotypes, and the first three principal components were placed in the GWAS model as covariates. Redundant sites were filtered using –indep-pairwise 50100.2 for quality-controlled autosomal and Z chromosome SNPs, resulting in 216,880 independent sites. GWAS used GEMMA software and the Bonferroni multiple test method to determine the association significance threshold. The genome-level 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 P value of each SNPs marker significance test. 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 area of ​​white spots on the heads of experimental chicks, Xβ is the population structure effect, and 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] Through GWAS analysis, two significant SNPs were identified, namely chr2:6249527 and chr7:25622689 (see Table 2, Figure 2 ), NCBI downloaded the chicken reference genome information (bGalGal1.mat.broiler.GRCg7b), and used ANNOVAR software to annotate genes near significant sites. 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 based on the chicken genome (bGalGal1.mat.broiler.GRCg7b); the functional region annotation dist=28631 means 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 above chr2:6249527 and chr7:25622689 genetic marker combination was used to conduct candidate gene association analysis on the F2 generation chicks and roosters of Luhua Chicken H Line (♀) × Yellow Feather Broiler Line (♂). The specific operation 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 top of the head of the F2 generation of 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: According to the physical positions of the chr2:6249527 and chr7:25622689 marker sites in the reference genome (bGalGal1.mat.broiler.GRCg7b), a DNA template sequence containing these two SNP marker sites was downloaded from the NCBI website, and PCR amplification primers were designed using Primer premier 6.0 software. The 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 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 product was sent to a sequencing company for sequence polymorphism detection to obtain the genotype of the individual tested and the sequencing sequence 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] Combination Figure 3 and Figure 4 The results showed that for the molecular marker chr2:6249527, the average area of ​​white spots on the top of the head of individuals with AA genotype was 2.425±0.136cm 2 The average area of ​​white spots on the top of the head of individuals with AG genotype was 2.088±0.103cm 2 The average area of ​​white spots on the top of the head of individuals with GG genotype was 1.496±0.108cm 2 There were significant differences between the genotypes (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 top of the head of individuals with GA genotype was 1.695±0.109cm 2 The average area of ​​white spots on the top of the head of individuals with AA genotype was 1.503±0.149cm 2 There were significant differences between the genotypes (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 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 molecular marker associated with the size of white spots on the head of sex-linked Luhua chicken chicks, characterized in that: It includes molecular marker I and / or molecular marker II, wherein the molecular marker I includes a polymorphic site chr2:6249527 located at the 6249527th position of chicken chromosome 2, and the polymorphic site chr2:6249527 has an A / G mutation; the molecular marker II includes a polymorphic site chr7:25622689 located at the 25622689th position of chicken chromosome 7, and the polymorphic site chr7:25622689 has an A / G mutation; the reference genome at the physical location of the polymorphic site chr2:6249527 and the polymorphic site chr7:25622689 is bGalGal1.mat.broiler.GRCg7b.

2. The molecular marker according to claim 1, characterized in that The genotypes present at the polymorphic site chr2:6249527 and the polymorphic site chr7:25622689 are AA, AG and GG.

3. A primer for identifying the size of white spots on the head of a sex-linked Luhua chicken chick, characterized in that: Comprising a primer pair for amplifying the molecular marker according to claim 1 or 2, the primer pair comprising (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.

4. A kit for identifying the size of white spots on the head of sex-linked Luhua chicken chicks, characterized in that: Comprising the primer described in claim 3.

5. Use of the molecular marker as described in any one of claims 1-2 in assisted chicken genetic breeding.

6. The use according to claim 5, characterized in that It is used to assist in the selection of the size of white spots on the heads of sex-linked Luhua chicken chicks.

7. The use according to claim 6, 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.

8. Use of a reagent for detecting the molecular marker described in any one of claims 1 to 2 in improving the size trait of the white spot on the head of sex-linked Luhua chicken chicks.

9. 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 the genomic DNA of the sex-linked Luhua chicken chicks to be tested as a template, amplifying the molecular markers according to claim 1 or 2 using primers, and selecting the size trait of the white spot on the head of the sex-linked Luhua chicken chicks 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 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.

10. The method according to claim 9, characterized in that The breeding method is: retaining individuals with a genotype of AA at the polymorphic site chr2:6249527 of the molecular marker I, and / or retaining individuals with a genotype of GG at the polymorphic site chr7:25622689 of the molecular marker II.

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