CN108707676B - SNP (single nucleotide polymorphism) marker related to chicken death and application thereof, detection primer and detection kit - Google Patents

Abstract

The invention relates to a chicken death-related SNP marker and application thereof, a detection primer and a detection kit, belonging to the technical field of biological breeding. The invention discovers an SNP marker related to chicken lethality for the first time, the sequence of the SNP marker is shown as SEQ ID NO.1, wherein the 267 th base has T & gtG mutation, the homozygous mutation of GG can cause the chicken to be lethal to early embryos, and only TT genotype or TG genotype exists at the site in a chicken population. The invention designs a specific primer according to the SNP marker, is used for amplifying target fragments containing the site, can detect the lethal mutation genotype of chicken individuals, eliminates heterozygous chicken individuals of TG genotype, can quickly homozygously breed non-lethal allele, and can improve the reproductive performance of chicken flocks.

Description

SNP (single nucleotide polymorphism) marker related to chicken death and application thereof, detection primer and detection kit

Technical Field

The invention relates to a chicken death-related SNP marker and application thereof, a detection primer and a detection kit, belonging to the technical field of biological breeding.

Background

In the genetic breeding of livestock and poultry, the modern breeding technology applying molecular markers can improve the accuracy of seed selection and rapidly improve and improve the production performance of livestock and poultry, and is an effective method for the genetic breeding of livestock and poultry. The molecular marker breeding is firstly to screen the genetic markers closely related to the economic traits of the livestock and poultry on the DNA level, and secondly to establish a rapid detection method of the gene polymorphism, thereby realizing early selection and genetic marker-assisted selection.

The chicken is an important economic poultry and an important model organism in scientific research, and the reproductive performance is a main index for evaluating the production performance of the laying hens. The domestic high-quality local chickens generally have the problems of poor reproductive performance (including poor egg laying performance, low hatching rate and the like), so that the economic benefit is influenced. Reproductive performance is a complex trait determined by multiple genes, is jointly regulated and controlled by heredity, environment and interaction of the two, and discussion and research on the formation mechanism of the complex trait are always the focus of attention in the field of animal genetic breeding. Therefore, the molecular marker related to the reproductive performance is developed, the molecular auxiliary selection of the reproductive performance can be realized, the genetic progress of the reproductive performance of the chicken is accelerated, and the application prospect is wide.

Disclosure of Invention

The invention aims to provide a SNP marker related to chicken death, and homozygous mutation of the marker can cause the death of chicken.

The invention also provides application of the SNP marker.

The invention also provides a detection primer and a detection kit for detecting the lethal genotype of the chicken.

In order to achieve the purpose, the invention adopts the technical scheme that:

a SNP marker related to chicken lethality has a nucleotide sequence shown in SEQ ID NO.1, wherein the 267 th base from the 5' end has a T & gtG mutation.

The invention discovers the SNP locus for the first time, the molecular marker locus corresponds to the 100471368 th deoxynucleotide of the positive strand of the 3 rd chromosome of the sequence information of the chicken reference genome Gallus _ Gallus-5.0 version published by NCBI, the SNP locus has not been reported before, and is a newly discovered molecular marker, and the homozygous mutation of the marker can cause the death of chicken. Therefore, only TT genotype or TG genotype exists at the site in the chicken population, and the GG homozygous mutant can cause death of the chickens, so that individuals with the genotype do not exist.

The genotype of the sample to be tested can be judged by PCR amplification of the marker and then digestion or sequencing with PvuII endonuclease.

The SNP marker is applied to chicken breeding. In particular to application in breeding selection of chicken breeding traits. More specifically, the method is applied to detecting and eliminating heterozygous individual chickens carrying lethal alleles.

In practical application, the type of 267 th nucleotide from the 5' end of the sequence shown in SEQ ID NO.1 is detected, chicken individuals with the TT homozygous site are selected, and chicken individuals with TG genotype at the site are discarded.

Detecting the 267 th nucleotide species by PCR amplification using the following primers:

SNP-F：5'-CCTTATGGGGGAGAGGAGACA-3'；

SNP-R：5'-CTGCACATAGCAGAGGGGTT-3'。

after PCR amplification, the genotype of the SNP site of the PCR amplified product can be detected by using a sequencing or enzyme digestion method, and then the genotype of the SNP site of the detected sample can be determined.

Preferably, the type of the 267 th nucleotide is detected by digesting the PCR amplification product with PvuII endonuclease. After PCR amplification, the PCR amplified product can be digested by PvuII endonuclease, and then the digested product is separated by agarose gel electrophoresis, and the kind of the 267 th nucleotide is detected by the size of the digested fragment. If only one 626bp fragment is obtained after the PCR product is digested by PvuII, the sample to be detected is the TT genotype; if 3 fragments of 626bp, 267bp and 359bp are generated, the sample to be detected is the TG genotype. Because PvuII can digest it when the base at this site in the PCR product is G, yielding 3 fragments of 626bp, 267bp and 359bp in length, while PvuII cannot digest it and remains 626bp in length when the base at this site in the PCR product is T.

The newly discovered SNP marker can be applied to early selection of chicken non-lethal genotypes, is assisted in chicken breeding, improves the chicken reproductive performance, is beneficial to saving the production cost and accelerating genetic breeding progress, and has great economic application value and scientific research value.

A detection primer for detecting a lethal genotype of chicken is designed according to a sequence shown as SEQ ID NO.1, and an amplification fragment of the detection primer comprises 267 th site from the 5' end of the sequence. The primer is designed by a common design method in the prior art, and can also be designed according to different detection methods.

Specifically, the primer sequences are shown as follows:

SNP-F：5'-CCTTATGGGGGAGAGGAGACA-3'；

SNP-R：5'-CTGCACATAGCAGAGGGGTT-3'。

a detection kit comprising the detection primer. Specifically, the kit also comprises one or more of dNTPs, PCR reaction buffer solution, DNA polymerase and PvuII endonuclease. Preferably, dNTPs, PCR reaction buffer, DNA polymerase and PvuII endonuclease are included.

The detection primer and the detection kit are applied to chicken breeding. In particular to application in breeding selection of chicken breeding traits. More specifically, the method is applied to detecting and eliminating heterozygous individual chickens carrying lethal alleles.

The detection primer and the kit have accurate and reliable detection results and strong operability, and provide a method for rapidly and efficiently distinguishing heterozygous individual breeding hens carrying mutant homozygous lethal alleles for the field. The key technology of the invention is that a mutation SNP locus related to chicken embryo lethality is found, and whether the individual is a heterozygous individual carrying the lethal allele can be judged by detecting the genotype of the allele of the locus on the genome of the individual, so that the individual is eliminated, and the non-lethal allele can be quickly homozygous by using the method.

Drawings

FIG. 1 is a band diagram of PCR products PvuII of different breeder chickens cut by n.267T > G SNP site;

FIG. 2 is a sequence diagram of PCR products of different genotypes.

Detailed Description

The present invention will be described in further detail with reference to specific examples. The equipment and reagents used in the examples were, except where specifically indicated, conventionally commercially available.

Example 1

The SNP marker related to chicken lethality in this example is shown in SEQ ID NO.1, the 267 th site of the sequence is T or G from the 5' end, and the SNP site corresponds to the 100471368 th deoxynucleotide of the positive strand of chromosome 3 of the sequence information version 3 of the chicken reference genome Gallus Gallus-5.0 published by NCBI.

Example 2

The detection primers used in this example to detect lethal chicken SNP are shown below:

SNP-F：5'-CCTTATGGGGGAGAGGAGACA-3'；

SNP-R：5'-CTGCACATAGCAGAGGGGTT-3'。

example 3

The kit for detecting the lethal SNP of the chicken in the embodiment comprises the primers shown in the embodiment 2, and also comprises dNTPs, PCR reaction buffer solution, DNA polymerase and Pvu II endonuclease.

Example 4

The chicken population to be tested in the embodiment is from a chicken breed quality resource field of the university of Henan agriculture, and comprises 96 Hiland brown chickens, 60 Roman chickens, 188 Lushi green-shell laying hens, 136 dark-river black-bone chickens, 180 immobilized chickens, 144 silky black-bone chickens, 60 Anka chickens and 736 Anka chickens, and F2 generation individuals generated by positive and negative crossing of the Anka chickens and the immobilized chickens.

In this example, chickens were bred using the SNP markers of example 1. Specifically, heterozygous individual chickens carrying lethal alleles are detected, and then chickens of the genotype are eliminated, so that the reproductive performance of chicken flocks is improved.

The method for detecting the lethal genotype of the chicken comprises the following steps:

1. genomic DNA extraction

Collecting blood of wing vein of chicken to be detected, treating with anticoagulant, cracking, digesting with protease, extracting genome by phenol-imitation method, and dissolving with sterilized double distilled water for use.

Specifically, the chicken wings are subjected to venous blood collection, ACD anticoagulant (1.32% (m/v) sodium citrate, 0.48% (m/v) citric acid and 1.47% (m/v) glucose) is subjected to anticoagulation and then is subjected to cracking, protease (purchased from Biotechnology engineering (Shanghai) GmbH) is subjected to digestion treatment, DNA is extracted by adopting a phenol-imitation method, and the extracted DNA is dissolved by sterilizing double-distilled water.

2. PCR amplification of SNP sites

PCR amplification is carried out on chicken genome DNA by using PCR primers, wherein the PCR reaction system is 10 mu L, and comprises 2 xEasyTaq PCR Supermix 5 mu L, and upstream and downstream primers SNP-F (shown as SEQ ID NO.2) and SNP-R (shown as SEQ ID NO.3) are respectively 0.5 mu L (10 umol. L)^-1) Genomic DNA (50 ng. mu.L)^-1) 0.5. mu.L, made up to 10. mu.L with double distilled water.

And (3) PCR reaction conditions: pre-denaturation at 95 ℃ for 5 min; pre-denaturation at 95 ℃ for 30s, annealing at 60 ℃ for 30s, and extension at 72 ℃ for 30s for 35 cycles; extending for 10min at 72 ℃; storing at 4 ℃. The length of the PCR amplified fragment is 626 bp.

3. Genotyping

1) Detecting the genotype of the chicken to be detected by adopting an enzyme cutting method: digesting the amplified PCR product by using a restriction enzyme PvuII (the site cut by PvuII is CAG ^ CTG, and the 265 th and 270 th sites of SEQ ID NO.1 are correspondingly cut).

Taking 10. mu.L of PCR product, adding 0.2. mu.L of restriction enzyme PvuII and 1.5. mu.L of reaction buffer (10X), and supplementing to 15. mu.L with ultrapure water; digestion was carried out in a 37 ℃ incubator for 6 hours. PvuII is a product of Takara, Japan, and its product designation is D1076B.

2) The digested PCR products were electrophoretically separated on a 1.5% agarose gel, and the TT genotype was obtained if there was only one 626bp fragment, and the TG genotype was obtained if there were 3 fragments of 626bp, 267bp and 359bp (as shown in FIG. 1).

PCR products identified as TG and TT by enzyme digestion are respectively selected for sequencing, the results are shown in figure 2, one characteristic band is TT genotype, three characteristic bands are TG genotype, the sequencing genotype detection result is consistent with the enzyme digestion detection genotype result in different breeder breeders, and the enzyme digestion detection result is accurate. The sequencing results for the TT genotype and the TG genotype are shown in FIG. 2.

3) The results of the genotype test statistical analysis of the T > G mutation site at 267 in the chicken of different breeds as described in SEQ ID NO.1 are shown in Table 1.

TABLE 1 statistical results of typing of n.267T > G SNP loci in different breeds of chicken populations

As can be seen from the results in Table 1, only TT genotype exists in experimental groups of high-yield laying hens, kalanchoe brown chickens and roman chickens; TT and TG genotypes were present in all but the high producing layer group, whereas GG genotype was absent in all tested groups.

Example 5

The application of the SNP marker in the embodiment comprises the following steps:

1) extracting chicken genome (same as the conventional extraction method or the extraction method of example 4).

2) PCR amplification (same PCR amplification method as in example 4).

The PCR reaction system is 10 μ L, including 2 × EasyTaq PCR Supermix 5 μ L, upstream and downstream primers SNP-F (shown as SEQ ID NO.2) and SNP-R (SEQ ID NO.3) each 0.5 μ L (10 umol. mu.L-1), genomic DNA (30-50 ng. mu.L-1) 0.5 μ L, and is made up to 10 μ L with pure distilled water.

And (3) PCR reaction conditions: pre-denaturation at 95 ℃ for 5 min; pre-denaturation at 95 ℃ for 30s, annealing at 60 ℃ for 30s, and extension at 72 ℃ for 30s for 35 cycles; extending for 10min at 72 ℃; storing at 4 ℃. The length of the PCR amplified fragment is 626 bp.

3) The amplified PCR product was digested with the restriction enzyme PvuII.

Taking 10. mu.L of PCR product, adding 0.2. mu.L of restriction enzyme PvuII and 1.5. mu.L of reaction buffer (10X), and supplementing to 15. mu.L with ultrapure water; digested in a water bath at 37 ℃ for 6 hours.

And (3) carrying out electrophoretic separation on the digested PCR product by using 1.5% agarose gel, wherein if only one 626bp fragment exists, the TT genotype is determined, and if three fragments of 626bp, 267bp and 359bp exist, the TG genotype is determined.

4) TT genotype individuals are selected for breeding, and the chicken individuals of TG genotypes are discarded, so that the dominant allele can be quickly homozygous, and chicken flocks with good reproductive performance are obtained.

<110> Henan university of agriculture

<120> SNP marker related to chicken death and application thereof, detection primer and detection kit

<160> 3

<170> SIPOSequenceListing 1.0

<211> 626

<212> DNA

<213> Chicken

<400> 1

ccttatgggg gagaggagac aactaggttg gccctccagg tgcctgagag caagcaagcc 60

acacgaggct tttagaggca gcaaggactg tccagctttc tggaggtctt tgcatactac 120

agctaagttt taggtgtggg aacagagtgg gccctttaga acaagtggca gagacaaatg 180

agggtgggag tcagccatgg gacagagaca gccacgcctg ttcttttgct tgtgggcagt 240

gacccttgga ataacagaca gatgcatctg cgggcaccac ctctccttct tgctctggga 300

cgggatgtat ctatctcccc cagaacccct gcctctccct ttcaagggca gtaaggattg 360

tgccaaagtg gtgacctgag ggccctggga ggctggcagg atcccagtcc tgtgctctgg 420

ggctcacatg gagctctacc tgcacaacta tgtcctagga tgagaaggtg gtcaaggagg 480

aggagatgaa cagaatccct aagtaggcag ggcagtgatg gcctttgccg gtcctactgc 540

ttctcattac tcatagaatc atagaatggc ccgggttgaa aaggacctca aagatcatct 600

agtttcaacc cctctgctat gtgcag 626

<211> 21

<212> DNA

<213> Artificial sequence

<221> SNP-F

<400> 2

ccttatgggg gagaggagac a 21

<211> 20

<212> DNA

<213> Artificial sequence

<221> SNP-R

<400> 3

aacccctctg ctatgtgcag 20

Claims (4)

1. A SNP marker related to chicken lethality, characterized in that: its nucleotide sequence is as shown in SEQ ID NO. 1, and a T>G mutation occurs at the 267th base from the 5' end. 2. the application of SNP marker as claimed in claim 1 in chicken breeding, described application refers to the application in detecting and eliminating the application in the heterozygous individual chicken carrying lethal allele: detect as shown in SEQ ID NO.1 The species of the 267th nucleotide from the 5' end of the sequence is shown, the chicken individuals with the TT homozygous at this site are selected, and the chicken individuals with the TG genotype at this site are discarded. 3. application according to claim 2 is characterized in that: the kind of the 267th nucleotide is detected by PCR amplification, and the primers used during described PCR amplification are as follows: SNP-F: 5'-CCTTATGGGGGAGAGGAGACA-3'; SNP-R: 5'-CTGCACATAGCAGAGGGGTT-3'. 4 . The application according to claim 3 , wherein the type of the 267th nucleotide is detected by the method of digesting the PCR amplification product with PvuII endonuclease. 5 .

Images