阅读说明：本技术 南丹瑶鸡鸡蛋浓蛋白高度和蛋黄颜色关联的snp分子标记的筛选方法和用途 (Screening method and application of SNP molecular marker associated with concentrated egg protein height and yolk color of Nandan Yao chicken ) 是由 杨秀荣 邹乐勤 邓继贤 于 2020-06-11 设计创作，主要内容包括：本发明公开了南丹瑶鸡鸡蛋浓蛋白高度和蛋黄颜色关联的SNP分子标记的筛选方法,直接从与鸡蛋品质相关的基因中筛选得到与鸡蛋浓蛋白高度和蛋黄颜色关联的8个SNP分子标记,这些SNP分子标记所处的序列如SEQ ID NO:1-SEQ ID NO:8所示。本发明提供的SNP分子标记筛选方法简化了全基因组关联分析,直接对性状相关基因进行SNP标记的筛选,比全基因组测序的费用少,适用于高遗传力的数量性状的选育。筛选得到的SNP分子标记可用于分子辅助育种,在早期可以直接在基因组水平上选育个体,不依赖表型信息,对于低遗传力,难以度量的性状可显著提高选择效率,加快育种进程,节省中间饲养费用,提高了育种效率,降低了育种成本,具有广泛的应用前景。(The invention discloses a screening method of SNP molecular markers associated with the height of concentrated egg white and the color of yolk of Nandan Yao chicken eggs, which directly screens 8 SNP molecular markers associated with the height of concentrated egg white and the color of yolk from genes associated with the quality of eggs, wherein the sequences of the SNP molecular markers are shown as SEQ ID NO. 1-SEQ ID NO. 8. The SNP molecular marker screening method provided by the invention simplifies the whole genome association analysis, directly screens the SNP marker of the gene related to the character, has lower cost than whole genome sequencing, and is suitable for the quantitative character breeding with high heritability. The SNP molecular marker obtained by screening can be used for molecular assisted breeding, individuals can be directly selected and bred on the genome level in the early stage, phenotype information is not relied on, the selection efficiency can be obviously improved for the characters with low heritability and difficult measurement, the breeding process is accelerated, the intermediate breeding cost is saved, the breeding efficiency is improved, the breeding cost is reduced, and the application prospect is wide.)

1. A screening method of SNP molecular markers related to the concentrated egg protein height and the yolk color of Nandan Yao chicken eggs is characterized by comprising the following steps:

(1) finding out genes related to the egg quality from known documents, taking Nandan Yao chicken, taking blood, extracting genome DNA, mixing the blood with a pool, sending the mixture to a company for sequencing, and detecting SNP (single nucleotide polymorphism) sites with mutation on the genes related to the egg quality;

(2) taking Nandan Yao hen as a test chicken, measuring the concentrated protein height and yolk color character indexes of the eggs from 24 weeks old, measuring for 1 time every 4 weeks, and continuously measuring for 3 days every time until the test of 60 weeks old is finished;

(3) collecting blood of 60-week-old test chickens, extracting genome DNA, carrying out high-throughput sequencing on the SNP sites detected in the step (1), and collecting genotype information of the test chickens at the SNP sites and the mutation SNP sites around the SNP sites;

(4) and (3) carrying out correlation analysis on the concentrated egg white height and yolk color data detected in the step (2) and the SNP locus detected in the step (3) by adopting SAS software, and screening out the SNP molecular marker which is extremely obvious (Pr > F <0.01) in correlation with the concentrated egg white height and the yolk color.

2. The screening method according to claim 1, wherein: the genes related to the egg quality in the step (1) are ALAS1, CPOX-P6, TF, TSPO, SLC25A5 and SLLCO1A2 genes.

3. The screening method of claim 1, wherein the SNP molecular markers are selected from the Nandan Yao chicken egg concentrated protein height and the yolk color.

4. The SNP molecular marker according to claim 3, wherein the SNP molecular marker is: chr1_68526357, chr12_3349578, chr4_16562030, chr1_68526344, chr4_16562070, chr1_85290991, chr1_65234293 and chr1_ 65258207; among them, chr1_68526357, chr12_3349578, chr4_16562030 and chr1_68526344 are associated with the concentrated egg white height, chr1_85290991, chr1_65234293 and chr1_65258207 are associated with the egg yolk color, and chr4_16562070 are associated with the concentrated egg white height and the egg yolk color at the same time.

5. The SNP molecular marker according to claim 4, wherein:

the chr 1-68526357 site is positioned at the 101 th site of the nucleotide sequence shown in SEQ ID NO. 1, and three genotypes of GG, GA and AA are arranged at the position, wherein the egg concentrated protein height of an AA type individual is the highest;

the chr 12-3349578 site is positioned at the 101 th site of the nucleotide sequence shown in SEQ ID NO. 2, and three genotypes of CC, CT and TT are arranged at the position, wherein the egg concentrated protein height of a CT type individual is the highest;

the chr 4-16562030 site is positioned at the 101 th site of the nucleotide sequence shown in SEQ ID NO. 3, and the position has CC and CT genotypes, wherein the concentrated egg protein height of a CC type individual is the highest;

the chr 1-68526344 site is positioned at the 101 th site of the nucleotide sequence shown in SEQ ID NO. 4, and the position has two genotypes of CC and CA, wherein the concentrated egg protein height of a CC type individual is the highest;

the chr 1-85290991 site is positioned AT the 101 th site of the nucleotide sequence shown in SEQ ID NO. 5, and AA and AT genotypes are positioned AT the position, wherein the egg yolk of an AT individual has the darkest color;

the chr 1-65234293 site is positioned at the 101 th site of the nucleotide sequence shown in SEQ ID NO. 6, and the position has two genotypes of CC and CG, wherein the egg yolk of CG individuals has the darkest color;

the chr 1-65258207 site is positioned AT the 101 th site of the nucleotide sequence shown in SEQ ID NO. 7, and AA and AT genotypes are positioned AT the position, wherein the egg yolk of an AT individual has the darkest color;

the chr4_16562070 site is located at the 101 th site of the nucleotide sequence shown in SEQ ID NO. 8, and two genotypes of GG and GA are arranged at the position, wherein the egg thick albumen height of GG individuals is the highest, and the yolk color is the deepest.

6. Use of the SNP molecular marker related to the egg concentrated protein height and the yolk color of the Nandan Yao chicken as set forth in any one of claims 3 to 5 for breeding of the Nandan Yao chicken.

7. The use of the SNP molecular marker related to the egg white height and the egg yolk color of the Nandan Yao chicken according to any one of claims 3 to 5 in the preparation of a kit or a detection method.

Technical Field

The invention belongs to the technical field of molecular biology, and relates to a screening method and application of SNP molecular markers related to the height of concentrated egg protein and the color of yolk of Nandan Yao chicken eggs.

Background

Eggs are a necessity in the diet of people, are rich in nutrition and low in price, and have a certain health-care function. With the improvement of the living standard quality of people, eggs with high internal quality are favored by more consumers. The body of egg white and the color of egg yolk are important indicators of consumer choice. The consistency index of the egg white is expressed by the height of concentrated protein, which is related to the quality and freshness of eggs, and the higher the height of the concentrated protein is, the better the quality of the eggs is; the most abundant nutrient components in the eggs are egg yolks, the shade of the color of the egg yolk is related to the content of pigment deposited in the egg yolk, and the darker the color of the egg yolk is more popular.

A large number of livestock and poultry varieties have been successfully cultivated at home and abroad by utilizing the traditional breeding method. However, in the traditional breeding, offspring is mainly selected according to phenotype by virtue of breeding experience, and the genotype of a target gene cannot be directly selected, so that the breeding is long in time consumption, the phenotype is complex to measure, and the selection efficiency and accuracy are low. At present, molecular biology technology is applied to breeding, breeding can be carried out on a molecular level, and the selection efficiency of breeding can be greatly improved and the breeding time can be shortened by molecular marker-assisted breeding or genetic modification breeding (transgenic breeding).

The concentrated egg white height and the yolk color belong to quantitative traits, the quantitative traits are influenced by polygene effect together, one or more SNP sites often play a role in genes, and the traditional poultry breeding cannot directly select the SNP sites, so that the traditional breeding selection efficiency and accuracy are low. Therefore, at present, there is an urgent need to screen SNP molecular markers highly correlated with egg yolk color and concentrated egg white, and apply the SNP molecular markers to molecular breeding of poultry.

Disclosure of Invention

The invention aims to provide a screening method of SNP molecular markers related to the egg concentrated protein height and the yolk color of Nandan Yao chicken, aiming at the defects of the prior art.

Another object of the present invention is to provide the SNP molecular markers obtained by the above screening method.

Another object of the present invention is to provide the use of the SNP marker.

The screening method of the SNP molecular marker related to the concentrated egg white height and the yolk color of the Nandan Yao chicken eggs comprises the following steps:

(1) finding out genes related to egg quality from known documents, taking Nandan Yao chicken, taking blood to extract genome DNA, mixing with pool, sending to company for sequencing, and detecting SNP sites with mutation on the genes;

(2) taking Nandan Yao hen as a test chicken, measuring the concentrated protein height and yolk color character indexes of the eggs from 24 weeks old, measuring for 1 time every 4 weeks, and continuously measuring for 3 days every time until the test of 60 weeks old is finished;

(3) collecting blood of 60-week-old test chickens, extracting genome DNA, carrying out high-throughput sequencing on the SNP sites detected in the step (1), and collecting genotype information of the test chickens at the SNP sites and the mutation SNP sites around the SNP sites;

(4) and (3) carrying out correlation analysis on the concentrated egg white height and yolk color data detected in the step (2) and the SNP locus detected in the step (3) by adopting SAS software, and screening out the SNP molecular marker which is extremely obvious (Pr > F <0.01) in correlation with the concentrated egg white height and the yolk color.

Wherein the genes related to the egg quality in the step (1) are ALAS1, CPOX-P6, TF, TSPO, SLC25A5 and SLLCO1A2 genes.

The method can quickly find the SNP molecular markers associated with the concentrated egg protein height and the yolk color of the Nandan Yao chicken from a large amount of data, and the molecular markers obtained by screening according to the method comprise 8 SNP sites, namely chr1_68526357, chr12_3349578, chr4_16562030, chr1_68526344, chr4_16562070, chr1_85290991, chr1_65234293 and chr1_ 65258207; among them, chr1_68526357, chr12_3349578, chr4_16562030 and chr1_68526344 are associated with the concentrated egg white height, chr1_85290991, chr1_65234293 and chr1_65258207 are associated with the egg yolk color, and chr4_16562070 are associated with the concentrated egg white height and the egg yolk color at the same time.

The chr 1-68526357 site is positioned at the 101 th site of the nucleotide sequence shown in SEQ ID NO. 1, and three genotypes of GG, GA and AA are arranged at the position, wherein the egg concentrated protein height of an AA type individual is the highest;

the chr 12-3349578 site is positioned at the 101 th site of the nucleotide sequence shown in SEQ ID NO. 2, and three genotypes of CC, CT and TT are arranged at the position, wherein the egg concentrated protein height of a CT type individual is the highest;

the chr 4-16562030 site is positioned at the 101 th site of the nucleotide sequence shown in SEQ ID NO. 3, and the position has CC and CT genotypes, wherein the concentrated egg protein height of a CC type individual is the highest;

the chr 1-68526344 site is positioned at the 101 th site of the nucleotide sequence shown in SEQ ID NO. 4, and the position has two genotypes of CC and CA, wherein the concentrated egg protein height of a CC type individual is the highest;

the chr 1-85290991 site is positioned AT the 101 th site of the nucleotide sequence shown in SEQ ID NO. 5, and AA and AT genotypes are positioned AT the position, wherein the egg yolk of an AT individual has the darkest color;

the chr 1-65234293 site is positioned at the 101 th site of the nucleotide sequence shown in SEQ ID NO. 6, and the position has two genotypes of CC and CG, wherein the egg yolk of CG individuals has the darkest color;

the chr 1-65258207 site is positioned AT the 101 th site of the nucleotide sequence shown in SEQ ID NO. 7, and AA and AT genotypes are positioned AT the position, wherein the egg yolk of an AT individual has the darkest color;

the chr4_16562070 site is located at the 101 th site of the nucleotide sequence shown in SEQ ID NO. 8, and two genotypes of GG and GA are arranged at the position, wherein the egg thick albumen height of GG individuals is the highest, and the yolk color is the deepest.

The 8 SNP molecular markers related to the height of the concentrated egg protein of the Nandan Yao chicken and the color of the egg yolk can be used for breeding the Nandan Yao chicken.

The 8 SNP loci are applied to the preparation of a kit or a detection method; the kit or the detection method is used for breeding the Nandan Yao chicken.

Through the technical scheme, the invention has the following beneficial effects:

the screening method of the SNP molecular marker associated with the concentrated egg white height and the yolk color of the Nandan Yao chicken eggs simplifies the whole genome association analysis, directly screens the SNP marker of the character related genes, has lower cost than whole genome sequencing, and is suitable for the quantitative character breeding of high heritability.

The SNP molecular marker obtained by screening can be used for molecular assisted breeding, individuals can be directly selected and bred on the genome level in the early stage, phenotype information is not relied on, the selection efficiency can be obviously improved for the characters with low heritability and difficult measurement, the breeding process is accelerated, the intermediate breeding cost is saved, the breeding efficiency is improved, the breeding cost is reduced, and the application prospect is wide.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. It is intended that all modifications or alterations to the methods, procedures or conditions of the present invention be made without departing from the spirit or essential characteristics thereof.

Animals, instruments, reagents, and kits used in the following examples are commercially available;