CN112852976B - Molecular marker related to later egg laying characteristics in NCS1 gene of laying hen and application thereof - Google Patents

Abstract

The invention discloses a molecular marker related to later egg laying characteristics in a layer NCS1 gene and application thereof, wherein the molecular marker is positioned in a part of coding region of the NCS1 gene, the nucleotide sequence of the molecular marker is shown as SEQ ID NO:1, the molecular marker totally comprises 3 SNP loci, namely three allele mutation loci of C/T, C/G and A/G exist at 171bp, 481bp and 565bp respectively, and a haplotype combination consisting of the molecular marker can be used as a haplotype molecular marker related to later egg laying characteristics of the layer, wherein the haplotype combination H1H3: the number of eggs laid by individuals with CCG/CGG at the age of 60 weeks is obviously higher than that of other haplotype combinations, and the selection of the haplotype combinations is beneficial to improving the number of eggs laid by the whole later period of the laying hen, namely, the invention provides a novel molecular breeding marker for marker-assisted breeding of the characters laid by the later period of the laying hen, realizes screening of the characters laid by the later period of the laying hen, and has simple and rapid screening method.

Description

Molecular marker related to later egg laying characteristics in NCS1 gene of laying hen and application thereof

Technical Field

The invention relates to the technical field of screening of genetic markers of laying hens, in particular to a molecular marker related to later-period egg production traits in an NCS1 gene of a laying hen and application thereof.

Background

Eggs are rich in protein, fat, lecithin and other various nutrients required by human bodies, and are important components in the dietary structure of residents in China. The later period of laying is the period that the production performance of the chicken flock is continuously reduced, the later period of laying accounts for 50% of the whole period of laying, and the chicken flock which is generally about 500 days old in the breeding production can be eliminated, but the laying rate of the chicken flock is still maintained at 60-70%, and the production performance of all the later periods of laying directly influences the economic benefit of laying hen breeding. The main reasons for the reduction of the laying rate of the laying hens in the later period of laying are aging and hypofunction of ovaries in the later period of laying, which are mainly represented by the reduction of the number of follicles, the increase of the locking rate of the dominant follicles of development, the inhibition of the development of follicles and the like, so that the utilization period of the laying hens in the later period of laying is shortened, and the economic value is reduced, so that the improvement of the laying rate of the laying hens in the later period of laying has important significance for the production of the laying hens.

A single nucleotide polymorphism (Single Nucleotide Polymorphsim, SNP) refers to a polymorphism in a genomic DNA sequence that is caused by mutation of a single nucleotide (A, T, C or G), and includes single base insertions, deletions, transformations, transversions, and the like. The genome-wide association analysis (geome-Wide Association Study, GWAS) is a control analysis and correlation analysis at the genome-wide level using millions of SNPs in the genome as molecular genetic markers. The advent of a large number of SNP markers gradually shifted the single marker-centered association analysis method to a haplotype (haplotype) -based association analysis method. Haplotypes refer to a plurality of SNPs which are closely linked and determine the same character on the same chromosome or in a certain area, and have statistical relevance. The SNP markers and haplotypes formed by the SNP markers play an important role in molecular marker assisted selection (Marker Assisted Selection, MAS) breeding of animals due to the distribution universality and stability, and become a feasible technology for genetic improvement of later-period laying hen egg laying traits, particularly limited traits. Based on the method, SNP markers and haplotype combinations thereof related to the economic traits of the laying hens are further identified, and the SNP markers and haplotype combination sequences thereof are used as screening markers for auxiliary selection of the economic traits of the good laying hens, so that the accuracy of seed selection is greatly improved, and the method has a certain significance for cultivating the laying hens with the good economic traits.

Neuronal calcium sensor 1 (Neuronal Calcium Sensor, ncs 1) is a highly conserved calcium binding protein that is involved in a variety of physiological cellular functions including exocytosis, regulation of calcium permeability channels, neuroplasticity and response to neuronal injury. Studies have shown that NCS1 plays an important role in adipocyte function, NCS1 deficiency leading to obesity and type 2 diabetes in adult mice; altered NCS1 expression is associated with neurodevelopmental and neurodegenerative diseases; can promote invasiveness of tumors by promoting cell survival and metastasis, and the like. However, no report has been made on the research of NCS1 genes on laying hens, and so far, no research has been made on the NCS1 genes of laying hens as molecular markers of later egg laying performance of the laying hens.

Disclosure of Invention

The invention aims to provide a molecular marker related to the later egg laying property in a layer NCS1 gene and application thereof, and the molecular marker related to the later egg laying property in a partial coding region of the layer NCS1 gene is discovered for the first time, so that an important reference basis is provided for auxiliary selection of the later egg laying property of the layer.

The invention aims at providing a molecular marker related to later-period egg laying characteristics in a chicken NCS1 gene, wherein the nucleotide sequence of the molecular marker is shown as a sequence table SEQ ID NO. 1, and the molecular marker comprises:

SNP1 site: a C/T polymorphic site exists at 171bp in the sequence shown in SEQ ID NO. 1;

SNP2 site: the 481bp site of the sequence shown in SEQ ID NO. 1 is provided with a C/G polymorphism site;

SNP3 site: A/G polymorphism site exists at 565bp in the sequence shown in SEQ ID NO. 1.

Further, dominant alleles at 171bp, 481bp and 565bp in the sequence are C, C, G, respectively, and dominant genotypes are CC, CC and GG, respectively.

It is a second object of the present invention to provide a primer set for amplifying the above molecular marker, comprising: the nucleotide sequence of the upstream primer is shown as SEQ ID NO. 2, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 3.

The invention also aims to provide a method for detecting molecular markers related to later-stage egg laying traits in the NCS1 gene of the laying hen, which specifically comprises the following steps: the primer pair is adopted for detection.

Further, the detection method comprises the following steps:

extracting genome DNA from the blood of the laying hen, amplifying by using the primer pair of claim 3 and comparing the sequences of the amplified products by using the genome DNA as a template to obtain 3 SNP loci of claim 1.

The fourth object of the present invention is to provide a haplotype combination comprising the above SNP sites, wherein the haplotype combination is a combination comprising SNP1-SNP3 sites.

The fifth object of the present invention is to provide a method for constructing a haplotype combination, comprising: firstly, detecting 3 SNP loci by adopting the detection method of the molecular marker; six haplotypes, H1: CCG, H2: CCA, H3: CGG, H4: CGA, H5: TGG, H6: TGA; and constructing a haplotype combination according to the haplotype, and correlating with the later egg laying character of the laying hen.

The sixth purpose of the invention is to provide the application of the molecular marker, the primer pair or the haplotype combination in the auxiliary selection of the later egg laying character of the laying hen.

Further, the later egg laying characteristics of the laying hens comprise 40-week-old egg laying number and/or 60-week-old egg laying number

The seventh object of the invention is to provide a breeding method of later-stage high-yield laying hens, which comprises the following steps: the haplotype combination is constructed by adopting the construction method of the haplotype combination, and the haplotype combination is reserved as HIH3: individuals of CCG/CGG.

Compared with the prior art, the invention has the beneficial effects that: the invention firstly discovers a haplotype molecular marker related to the later egg laying performance of the laying hen in a part of coding region of the NCS1 gene of the laying hen, the sequence is shown as SEQ ID NO. 1, the length of the sequence is 1575bp, the sequence totally comprises 3 SNP loci, namely three allele mutation loci of C/T, C/G and A/G exist at the 171bp, 481bp and 565bp positions respectively, and a haplotype combination consisting of the haplotype loci can be used as the haplotype molecular marker related to the later egg laying performance of the laying hen, wherein the haplotype combination H1H3: the number of eggs laid by individuals with CCG/CGG at the age of 60 weeks is obviously higher than that of other haplotype combinations, and the selection of the haplotype combinations is beneficial to improving the number of eggs laid by the whole later period of the laying hen, namely, the invention provides a novel molecular breeding marker for marker-assisted breeding of the characters laid by the later period of the laying hen, realizes screening of the characters laid by the later period of the laying hen, and has simple and rapid screening method.

Drawings

FIG. 1 is a schematic diagram of a technical route of the present invention;

FIG. 2 shows agarose gel detection results of PCR amplification products of NCS1 gene fragments of laying hens, wherein M is Marker, and 1-5 are amplification results of NCS1 gene fragments;

FIG. 3 is a graph showing comparison of individual sequencing results of different genotypes of 3 SNP polymorphic sites of NCS1 gene of a layer chicken in example 1 of the present invention: (a) the presence of a C/T mutation site at 171 bp; (b) the presence of a C/G mutation site at 481 bp; (c) the presence of an A/G mutation site at 565 th bp;

FIG. 4 shows the result of haplotype block analysis of the NCS1 gene in example 4 of the present invention.

Detailed Description

The technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by one of ordinary skill in the art without undue burden on the person of ordinary skill in the art based on embodiments of the present invention, are within the scope of the present invention.

Example 1 acquisition of partial coding region fragment of NCS1 Gene of layer chicken and establishment of SNP detection method

1. Extraction of genomic DNA of laying hen

The test layer breeds of the invention comprise Jiang Han chickens and Luo island red chickens, and the samples are from poultry test sites of the national academy of agricultural sciences of Hubei province. The extraction of the genome DNA of the laying hen adopts a genome DNA kit (operated according to the specification of the kit) produced by Beijing Baitaike biotechnology Co., ltd, and comprises the following specific steps:

(1) About 1ml of blood was withdrawn from the subwing vein using a disposable syringe, poured into a 1.5ml centrifuge tube autoclaved and filled with 200. Mu.L of sterile ACD anticoagulant, gently shaken, wing numbers recorded and stored at-20℃for later use. Absorbing 10 mu L of anticoagulated blood, adding 500 mu L of BB2 and 10 mu L of proteinase K (20 mg/mL), fully mixing, and incubating for 10min at room temperature;

(2) Centrifuging for a short time, adding the whole solution into a centrifugal column, centrifuging for 1min at 12000g, and discarding effluent;

(3) Adding 500 mu L of solution CB3, centrifuging for 30s with 12000g, and discarding effluent;

(4) Adding 500 mu L of solution WB3, centrifuging for 30s with 12000g, and discarding effluent;

(5) Repeating the step 4 once;

(6) Centrifuging for 2min at 12000g to thoroughly remove residual WB3;

(7) Placing the centrifugal column in a clean centrifuge tube, adding 50-200 mu L of preheated EB or deionized water in the center of the column, standing for 1min at room temperature, centrifuging 12000g for 1min, eluting DNA, and preserving the eluted DNA at-20 ℃ for later use.

2. Obtaining of partial nucleotide fragment of NCS1 gene of laying hen

(1) PCR amplification

A primer pair is designed according to the NCS1 Gene sequence (accession number Gene ID: NC_ 006104.5) published by NCBI database, and the sequence of the primer pair is as follows:

an upstream primer: 5'-AGCAGTGGTGAGTAGCAG-3' (SEQ ID NO: 2),

a downstream primer: 5'-GACATTGAACACGAAGGT-3' (SEQ ID NO: 3).

PCR amplification was performed in the layer genomic DNA using the above primers, the PCR reaction system is shown in Table 1, and the PCR reaction conditions are shown in Table 2.

TABLE 1 PCR reaction System

TABLE 2 PCR reaction conditions

The PCR amplified product is subjected to agarose gel electrophoresis detection, the detection result is shown in figure 2, and the fragment size of the product is 1578bp.

(2) PCR amplification product purification

The PCR amplification product was purified using Gel Extraction Kit kit from Shanghai Biotechnology Co., ltd, and the specific procedure was as follows: firstly, cutting gel containing target fragments from agarose gel, putting the gel into a 1.5mL centrifuge tube, adding 400 mu L of sol solution, carrying out water bath at 50-60 ℃ until the sol is thoroughly melted, uniformly mixing every 2min when the sol is heated, and cooling to room temperature; placing the centrifugal column into a collecting pipe, transferring the mixed solution to the centrifugal column, and standing at room temperature for 2min; centrifuging at 12000r/min for 1min, wherein DNA is adsorbed onto the column; pouring out the waste liquid in the collecting pipe, putting the centrifugal column into the same collecting pipe, adding 700 mu L of eluent, and centrifuging for 1min at 12000 r/min; pouring out the waste liquid in the collecting pipe, and centrifuging for 1min at 12000 r/min; placing the column into a sterilized 1.5mL centrifuge tube, adding 40 μl of eluent or double distilled water (pH > 7.0), and standing at room temperature or 37deg.C for 2-3min; centrifuging at 12000r/min for 1min, wherein the liquid in the centrifuge tube is the recovered DNA fragment.

3. Direct sequencing method for detecting molecular markers

And directly sending the purified PCR product to Beijing ao Dingsheng biotechnology Co Ltd for sequencing, and judging the genotype of the locus in the detection group according to the sequencing result. Blast comparison analysis is carried out by DNAStar software, and the analysis result is shown in figure 3. The result shows that three allelic mutations of C/T, C/G and A/G exist at 171bp, 481bp and 565bp in the sequence, the mutations cause polymorphism of NCS1 gene, and the nucleotide sequences of the 3 SNP markers are shown as SEQ ID NO. 1 of the sequence table. The 3 polymorphic sites together form 6 haplotypes, CCG, CCA, CGG, CGA, TGG and TGA, respectively.

EXAMPLE 2 polymorphism distribution detection of molecular markers in laying hens of the present invention

The polymorphism of 3 sites in the sequence shown in SEQ ID NO. 1 was detected, and three genotypes were detected at sites g.171bp C > T (NCS1_SNP1), g.482bp C > G (NCS1_SNP2) and g.567bp A > G (NCS1_SNP3), and the genotype frequencies, allele frequencies and distribution are shown in Table 3.

TABLE 3 genotype frequencies and allele frequencies of layer NCS1 genes

Note that: genotype frequencies the number in parentheses is the number of individuals of the genotype.

As is clear from Table 3, 3 genotypes were detected at all of the 3 mutation sites of the sequence shown in SEQ ID NO. 1, and at the NCS1_SNP1 to NCS1_SNP3 sites, the dominant alleles were C, C, G, respectively, and the dominant genotypes were CC, CC and GG, respectively.

Example 3 correlation analysis and application of molecular markers and later egg laying characters of laying hens

In order to determine whether the detected NCS1_SNT1 to NCS1_SNT3 markers of the laying hens are related to the difference of the later egg laying traits of the laying hens, jiang Han chickens and Luo island red chickens (the total number of samples is 220, wherein 110 chickens are Jiang Han chickens and Luo island red chickens) are selected as test materials, the samples are collected in poultry test sites of livestock and veterinary research institutes of the agricultural sciences of Hubei province, the open-day-old, 40-week-old egg laying number and 60-week-old egg number traits of each individual are recorded, polymorphism detection is carried out by using a direct sequencing method, and the correlation between different genotypes of the gene part coding regions of the NCS1 of the laying hens and the later egg laying traits of the laying hens is analyzed. Correlation analysis between genotype and phenotype was performed using SPSS18.0 software using the following model:

Yij＝u+Gi+Pj+eij

wherein YIj is a property observed value; u is the total average value of the characters; gi is the genotypic effect; pj is the fixation effect; eij is a random error.

Correlation analysis between different genotypes of 3 mutation sites and the later egg-laying character of the laying hen was carried out in Jianghan chicken and Luo island red chicken, and the statistical analysis results are shown in Table 4.

TABLE 4 correlation analysis of 3 mutation site polymorphisms of NCS1 Gene of laying hen and late egg production traits

Note that: the same letters in the above table indicate that the differences are not significant, letters a, b, c indicate that the differences are significant, and n is the number of individuals of that genotype.

From the analysis results of Table 4, it can be seen that the NCS1_SNP1 to NCS1_SNP3 locus polymorphisms of the NCS1 gene have significant correlation with the number of eggs laid at the beginning of the day of production, 40 weeks of age, 60 weeks of age (p < 0.05). Wherein the TT genotype of the NCS1_SNP1 locus, the CC and CG genotypes of the NCS1_SNP2 locus and the AA and GG genotypes of the NCS1_SNP3 locus have higher 40-week-old egg laying numbers; the CT genotype of NCS1_SNP1 locus, the CG genotype of NCS1_SNP2 locus and the GG genotype of NCS1_SNP3 locus have higher egg production number of 60 weeks old.

Example 4 correlation analysis and application of molecular markers and later egg laying characters of laying hens

1. Construction of haplotype and haplotype combinations

And carrying out haplotype analysis on NCS1_SNP1 to NCS1_SNP3 by utilizing Haploview software, inputting genotype data of the NCS1_SNP1 to NCS1_SNP3 loci of all individuals into a PHASE program, calculating the genotype of each individual, and simultaneously calculating the paired linkage disequilibrium degree between loci. The haplotype block analysis results are shown in FIG. 4.

From FIG. 4, it can be seen that 1 haplotype block was found in total according to NCS1_SNP1 to NCS1_SNP3 site linkage disequilibrium analysis, and that haplotype analysis was performed on the haplotype block, and that 6 haplotypes were found in the layer population studied in the present invention, wherein two haplotypes were present in each individual, and the sequence and number statistics of each haplotype are shown in Table 5.

TABLE 5 SNP locus haplotype statistics of NCS1 Gene

The haplotype combinations of each individual of the above haplotype compositions were analyzed to find 11 haplotype combinations in total, as shown in Table 6.

TABLE 6 NCS1 Gene haplotype combinations

Haplotype combinations	Sequence(s)	Quantity (only)
			H1H1	CCG/CCG	81
H1H2	CCG/CCA	46
			H1H3	CCG/CGG	20
H1H4	CCG/CGA	5
			H1H5	CCG/TGG	25
H2H2	CCA/CCA	10
			H2H5	CCA/TGG	6
H3H3	CGG/CGG	5
			H3H5	CGG/TGG	6
H4H4	CGA/CGA	11
			H5H6	TGG/TGA	5

And removing part of haplotype combinations with smaller number in the sample population, and selecting the 5 haplotype combinations with the largest number for correlation analysis.

2. Correlation analysis of haplotype combinations and later egg production traits

Correlation analysis of haplotype combinations and later egg production traits was performed using SPSS18.0 software and the results are shown in table 7.

TABLE 7 correlation analysis results of dominant haplotype combinations and late egg production traits

Note that: the same letters in the above table indicate that the differences are not significant, letters a, b, c indicate that the differences are significant, and n is the number of individuals of that genotype.

From the analysis results of table 7, it can be seen that haplotype combinations H1H2: CCG/CCA and haplotype combination H1H5: the 40 week old egg laying number of CCG/TGG individuals was significantly higher than other haplotype combination individuals (p < 0.05), haplotype combination H1H3: the number of eggs laid by CCG/CGG individuals at 60 weeks of age was significantly higher than those of other haplotype combinations (p < 0.05). From the above analysis results, haplotype combinations H1H3 in the test population of this example: CCG/CGG has the best later egg laying property. Thus in the layer population, by combining the haplotypes H1H3: the selection and the stay of CCG/CGG individuals are beneficial to improving the later egg laying character of the whole laying hen. Namely, the haplotype combination consisting of the mutation sites identified by the invention can be used as a potential genetic marker for improving the later egg laying character of the laying hen for auxiliary selection of the high-yield laying hen.

The present invention is not limited to the above-mentioned embodiments, and any changes or substitutions that can be easily understood by those skilled in the art within the technical scope of the present invention are intended to be included in the scope of the present invention.

Claims (8)

1. Laying henNCS1The molecular marker related to the later egg laying property in the gene is characterized in that the nucleotide sequence of the molecular marker is shown as a sequence table SEQ ID NO. 1, and the molecular marker comprises:

SNP1 site: a C/T polymorphic site at position 171. 171bp in the sequence set forth in SEQ ID NO. 1;

SNP2 site: the 481bp site of the sequence shown in SEQ ID NO. 1 is provided with a C/G polymorphism site;

SNP3 site: A/G polymorphism site exists at 565bp in the sequence shown in SEQ ID NO. 1.

2. The molecular marker of claim 1, wherein the dominant genotype at 171bp in the sequence is CC, the dominant genotype at 481bp in the sequence is CC, and the dominant genotype at 565bp in the sequence is GG.

3. Laying hen according to claim 1NCS1The detection method of the molecular marker related to the later egg production property in the gene is characterized by adopting a primer pair to detect, wherein the primer pair comprises the following steps: the nucleotide sequence of the upstream primer is shown as SEQ ID NO. 2, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 3.

4. A method of detection as claimed in claim 3, wherein the method of detection comprises:

extracting genome DNA from the blood of the laying hen, amplifying by using the primer pair of claim 3 and comparing the sequences of the amplified products by using the genome DNA as a template to obtain 3 SNP loci of claim 1.

5. A method of constructing a haplotype combination, the method comprising: firstly, detecting 3 SNP loci by the method of claim 4; six haplotypes, H1: CCG, H2: CCA, H3: CGG, H4: CGA, H5: TGG, H6: TGA; and constructing a haplotype combination according to the haplotype, and correlating with the later egg laying character of the laying hen.

6. Use of a molecular marker according to any one of claims 1-2, or a haplotype combination according to claim 5, in the auxiliary selection of late egg laying traits in laying hens.

7. The use according to claim 6, wherein the late egg laying traits of the layer comprise 40 week old egg laying number and/or 60 week old egg laying number.

8. A breeding method of a later-stage high-yield laying hen is characterized by comprising the following steps: constructing a haplotype combination by the method of claim 5, retaining the haplotype combination as HIH3: individuals of CCG/CGG.