SNP genetic marker related to quail egg laying traits and applicationTechnical Field
The invention belongs to the technical field of molecular marker preparation of poultry, and particularly relates to a genetic marker related to the egg laying performance of quails and application thereof. The genetic marker is cloned from a fragment of the EED gene and can be applied to quail marker-assisted selective breeding.
Background
The quail is a special economic poultry, and has extremely high edible value and dietary therapy effect. With the improvement of living standard of people, the demand of quail eggs is more and more. The quail breed for laying eggs at present has the defects of low egg laying performance, uneven egg weight and the like, and the cultivation of the quail breed with high yield performance becomes one of the research focuses in the field of quail breeding and cultivation at present. The egg laying performance is genetically improved by conventional breeding methods such as phenotype selection, the time is long, and the cost is high; with the development of molecular biology technology, the use of molecular marker-assisted selection combined with traditional breeding methods has become one of the main methods for accelerating the breeding of livestock and poultry varieties. Therefore, the screening of the molecular genetic marker linked with the quail egg-laying trait gene locus, the application of the molecular genetic marker in the molecular marker-assisted selection of a new quail variety for egg cultivation, and the selection of the new quail variety for egg cultivation are one of the research focuses in the field of quail molecular biology at present and for a period of time in the future.
Embryonic Ectoderm Development (EED) is one of the members of the PcG protein family. The PcG protein family is an important transcription inhibiting factor, is combined with a regulatory gene promoter region related to cell proliferation (Piunti et al,2011), can be coupled with a DNA specific sequence binding factor, a signal transduction regulatory factor and the like, and dynamically regulates cell cycle, target gene expression, aging and the like (Lai et al, 2013). Although the EED gene plays an important role in the initiation of cell proliferation and sexual maturation as a member of the PcG protein family, few studies on EED genes in livestock and poultry have been made, and only a few results of the studies indicate that EED genes play a role in organogenesis in mammals (Morin-Kensicki et al, 2001), which preliminarily suggested that EED transcription inhibitors are involved in the initiation of sexual maturation in chickens (Zhongwei et al, 2016). However, no report has been made on the further functions of the EED gene and its studies in other poultry.
Comprehensively, the EED gene is closely related to the initiation of poultry sexual maturity, and the research on the regulatory mechanism of the initiation of sexual maturity is of great significance to the breeding of poultry high-yield varieties. One of the important means for exploring molecular markers related to specific traits and studying gene functions is to study polymorphisms of gene mutation sites in a population and perform correlation analysis of traits. In view of the above, the applicant clones a partial sequence of exons 8-9 of the quail EED gene, and performs analysis and research on polymorphism of the sequence and association of the polymorphism with the quail egg laying character, thereby providing important theoretical support for genetic improvement of quails.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to obtain the SNP molecular marker related to the egg laying character of the quails and the application of the SNP molecular marker to the quails. A specific DNA fragment (partial fragment of exons 8-9) is obtained by cloning a quail EED gene, and the relevance of the fragment and the quail egg laying character is analyzed, so the invention provides a new genetic marker for marker-assisted selection breeding of the quail.
In order to achieve the purpose of the present invention, the present inventors have conducted extensive experimental studies and diligent research to finally obtain the following technical solutions:
A SNP genetic marker related to quail egg laying traits is discovered by separating and cloning exon 8-9 regions of a quail EED gene, and has a nucleotide sequence shown as SEQ ID NO:1, the sequence length is 605bp, wherein, R (namely, a base mutation at 187 bp) is A or G (namely, an A/G substitution) and Y (namely, a base mutation at 208 bp) is T or C (namely, a T/C substitution) shown in the sequence, and the site is detected by adopting a direct sequencing method.
The applicant designs a primer pair for amplifying the quail EED gene and detecting the mutation of the gene segment, and the DNA sequence of the primer pair is shown as follows:
a forward primer: 5'-TTGGGTGACCTAATTCTT-3', corresponding to the sequence shown in SEQ ID NO. 2.
Reverse primer: 5'-TTTGACTCACTTGGCTTG-3', corresponding to the sequence shown in SEQ ID NO. 3.
the invention establishes a preparation method of a genetic marker related to the egg laying traits of quails, and the genetic marker is prepared according to the following steps:
Extracting DNA from quail blood, designing primers according to the quail EED gene sequence, wherein the DNA sequence of the primer pair is shown as SEQ ID NO. 2 and SEQ ID NO. 3 of a sequence table, carrying out PCR amplification on the quail genome DNA by using the primers shown as SEQ ID NO. 2 and SEQ ID NO. 3 of the sequence table, and obtaining the nucleotide sequence shown as SEQ ID NO. 1 of the sequence table after purifying, cloning and sequencing a PCR product.
The genetic marker can be used for detecting the egg laying traits of the quails, and the designed primer pair can also be used for detecting the egg laying traits of the quails.
Compared with the prior art, the invention has the following advantages and remarkable progress: (1) by utilizing the molecular marker discovered in the invention, the selection of AT/GC haplotype individuals is increased in the breeding group of the laying quail, the laying rate of the quail can be properly advanced, the selection of the AT/AC haplotype individuals can be increased, the egg yield of the breeding group can be improved, and the individuals of the two haplotypes are simultaneously selected, so that the egg yield of the breeding group can be improved while the day of the laying rate of the breeding group is advanced; (2) the method realizes early selection of the quail egg-laying traits, is quick and accurate, is not influenced by culture environment condition factors, can shorten the breeding period and reduce the breeding cost.
Drawings
FIG. 1: the agarose gel electrophoresis picture of EED gene partial sequence obtained by PCR amplification of the invention, the agarose concentration is 2%, and the marks in the picture indicate that: 1-6 are lane numbers, and M is DSTM2000 Marker.
FIG. 2: the comparison result of the sequencing peak maps of the mutation sites detected by the direct sequencing of the partial sequence of the exon 8-9 region of the quail EED gene is shown in the invention, wherein (a) is the comparison result of the sequencing peak maps of the 187bp mutation sites, and (b) is the comparison result of the sequencing peak maps of the 208bp mutation sites.
Detailed Description
The following are specific examples of the present invention and further describe the technical solutions of the present invention, but the scope of the present invention is not limited to these examples. All changes, modifications and equivalents that do not depart from the spirit of the invention are intended to be included within the scope thereof.
Example 1 acquisition of partial genome sequence of EED Gene and establishment of polymorphism detection method
1. primer design
According to the sequence of the quail EED gene (GenBank access No. NC-029516), the primer sequences of exons 8 to 9 of the gene are designed as follows:
A forward primer F: 5'-TTGGGTGACCTAATTCTT-3'
Reverse primer R: 5'-TTTGACTCACTTGGCTTG-3'
Taking quail for laying eggs as a test population, extracting quail blood genome DNA, and amplifying a partial sequence fragment of the quail EED gene by taking the DNA as a template.
2. PCR amplification conditions
The primers are utilized to carry out PCR amplification in quail genome DNA, the total volume of PCR reaction is 15 mu L, and the concentration of each component in the system is as follows: quail genomic DNA is about 100ng, 1 XTaq Buffer, 1.5mmol/L MgCl2, dNTP 2.5mmol/L, the final concentration of the primer is 0.2 mu mol/L, and 2U Taq DNA polymerase. The PCR amplification procedure was a pre-denaturation at 95 ℃ for 5min, followed by 34 cycles of 94 ℃ for 30s, 53 ℃ for 35s, 72 ℃ for 45s, and a final extension at 72 ℃ for 10 min. The PCR reaction products were detected by 2% agarose gel electrophoresis and photographed, and the results are shown in FIG. 1, lanes 1-6 are the amplification results of individual EED gene exons 8-9 region, M is Marker DS TM2000, and the amplification results can be used for further analysis.
3. purification of PCR products
The PCR product is subjected to gel cutting and purification by using a gel recovery and purification kit produced by Beijing Tiangen Biochemical technology Co., Ltd, and the operation is carried out according to the kit specification, and the specific steps are as follows:
(1) Cutting off the gel containing the target fragment from the agarose gel under an ultraviolet lamp, putting the gel into a 1.5mL centrifuge tube, and weighing;
(2) Adding solution PN with the same volume as the glue block, placing the glue block in a water bath at 50 ℃ for 10 minutes until the glue is completely dissolved, and continuously and gently turning the centrifugal tube up and down to ensure that the glue block is fully dissolved;
(3) adding the solution obtained in the previous step into an adsorption column, standing at room temperature for 2min, centrifuging at 12,000rpm for 30-60 s, discarding the waste liquid, and returning the adsorption column to the collection tube;
(4) Adding 600 μ L of rinsing solution PW into the adsorption column, centrifuging at 12000rpm for 30-60 s, and discarding the waste solution.
(5) Repeating the step 4;
(6) the adsorption column was returned to the collection tube and centrifuged at 12,000rpm for 2 minutes. The column was left to stand at room temperature for several minutes and completely air-dried.
(7) the adsorption column was placed in a clean centrifuge tube, elution buffer was added to the middle of the adsorption column membrane, and after standing at room temperature for 2 minutes, the column was centrifuged at 12,000rpm for 1 minute to collect the purified product.
4. detection of molecular markers by direct sequencing of PCR products
The obtained PCR purified product is directly sent to Beijing Odoku Dingsheng Biotech Co., Ltd for sequencing, and genotype analysis is directly carried out on a sequencing chromatogram (figure 2).
example 2 distribution of genetic markers prepared according to the invention in quail population
The applicant detects the distribution frequency of two alleles in exon 8-9 region of quail EED gene in 2 egg quail populations of yellow feather line and chestnut feather line, the detection result is shown in Table 1, and the result shows that: the G/A polymorphism distribution at 187bp has the advantages that A alleles account for the major dominance in a yellow feather line population, G alleles account for the major dominance in a chestnut feather line population, heterozygote individuals account for the largest proportion, but the difference of the frequency distribution of the alleles in the two populations is not significant; the T/C polymorphism distribution at the 208bp position shows that T allele accounts for the main dominance in both yellow feather and chestnut feather populations, and the ratio of TT genotype accounts for the largest in both populations.
TABLE 1 distribution of polymorphisms in exons 8-9 of the EED Gene in different quail populations
Example 3 correlation analysis and application of genetic marker prepared by the invention and quail egg laying traits
1. Construction of haplotypes
inputting the genotype data of the two polymorphic sites of all individuals obtained by the PCR-direct sequencing method into Haploview software, calculating the haplotype of each individual, and simultaneously calculating the degree of pairwise linkage disequilibrium between the sites, and expressing the degree by a standardized linkage disequilibrium coefficient D'. As a result, it was found that the linkage disequilibrium coefficient D' between the two loci was equal to 1, which is complete linkage disequilibrium. There are 4 haplotypes, AC, AT, GC and GT, accounting for 22.73%, 27.27%, 12.5% and 37.5% of the allele, respectively, making up the 5 haplotype combinations AC/AC, AT/GC, AT/GT, GT/GT.
2. Association analysis of haplotype combination and egg laying character of laying quails
The applicant analyzed the correlation of the genetic markers prepared by the present invention with the quail egg laying traits in 528 individuals with complete production data of quail population for egg (from Hubei Shendan health food Co., Ltd.). The analyzed egg laying traits mainly comprise the age of the laying day, the weight of the laying head, the egg weight, the egg quality, the weight of 20 weeks, the egg laying number of 20 weeks and the like. Genotyping of individuals was performed using the PCR-direct sequencing method established in example 1, and Statistical analysis was performed using SPSS Statistical software (Statistical Package for the Social Sciences, version18.0) general linear model, in which factors other than random effects were fixed effects.
The results of the correlation analysis are shown in Table 2. As can be seen from the table, when the haplotypes are different, the characteristics such as the age of the laying day, the egg weight, the egg laying number in 20 weeks and the like have obvious difference (P is less than 0.05); wherein, the laying date age and the egg weight of the individual with the haplotype of AT/GC are obviously higher than those of other 4 haplotypes (P <0.05), and the egg laying number of the individual with the haplotype of AT/AC AT 20 weeks is obviously higher than that of the individual with the haplotype of AT/AC (P < 0.05). Therefore, the AT/GC haplotype individual has earlier production and larger egg weight, and the AT/AC haplotype individual has the most egg number AT the age of 20 weeks, so that different haplotype individuals can be selected according to different breeding targets to breed in breeding practice.
TABLE 2 Association analysis of exon 8-9 region polymorphism in EED gene and egg-laying traits
Note: marked with different superscript letters a, b, c, indicating significant differences (P < 0.05); n represents the number of individuals.
SEQ ID NO:1
<110> animal husbandry and veterinary institute of agricultural science research institute of Hubei province
<120> SNP genetic marker related to quail egg laying traits and application thereof
<160>3
<210> 1
<211> 605
<212> DNA
<400> 1
ttgggtgacc taattctttc caaggtaaag tgcaagcttt ttcagctacc ttgcttttat 60
ttgctttttt aggctcttaa acagtagtag tttcactaga atacgaattg taggttaact 120
tattatgctg catgaaatga ttttatgtta aagattttgg tgtctgtttg catgtgtaga 180
taagcarctt ttacatcaaa agtaaaayga gttcagtggt cagaactgtg ctagaagtag 240
caacatgtca actacccgtg ttctgcatga ggagtacaga aagagaatga atatatagtt 300
actcatgtag ggtcctgcag ctgaaaggag aaatgaggat tcatgcaatg gagttccttg 360
gatttgtgtg ataacactag cactgcttgc tcggagacat taaactacat ggtggcattg 420
catctttggc agaaattggt gcatgtgtgt tctcctaaaa ctgaatgttg tacgcttttt 480
gttgttgttc attgtttttt tttggttttt ttttttcctc cctagtcttg tgaaaatgcc 540
attgtgtgct ggaaacctgg caaaatggaa gatgatatag ataaaatcaa gccaagtgag 600
tcaaa 605
SEQ ID NO:2
<210> 2
<211> 18
<212> DNA
<400> 2
ttgggtgacc taattctt 18
SEQ ID NO:3
<210> 3
<211> 18
<212> DNA
<400> 3
tttgactcac ttggcttg 18