Molecular marker related to weight character of Tibetan chicken, primer pair and application of molecular marker

Abstract

The invention discloses a molecular marker related to Tibetan chicken weight traits, a primer pair and application of the molecular marker and the primer pair, and relates to the technical field of molecular marker-assisted selective breeding. The molecular marker is located at a 5'flanking sequence of a GPR75 gene, the sequence of the molecular marker is shown as SEQ ID NO.1, in the sequence shown as SEQ ID NO.1, a G / A polymorphic site exists at the 268bp position, an A / G polymorphic site exists at the 230bp position, and a C / T polymorphic site exists at the 234bp position. The primer pair comprises an upstream primer and a downstream primer, wherein 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 molecular marker and the primer pair can be used for predicting and screening body weight characters of Tibetan chicken, can remarkably improve the breeding efficiency and effectively shorten the breeding period during chicken breeding, and are high in breeding accuracy and low in cost.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular marker-assisted selection breeding, and specifically relates to a molecular marker related to the body weight trait of Tibetan chickens, a primer pair and an application thereof. Background Art

[0002] Single Nucleotide Polymorphism (SNP) refers to the genetic polymorphism caused by the mutation of a single nucleotide in the genomic DNA sequence. Due to the wide distribution and stability of SNP, it plays an important role in animal molecular marker-assisted selection (MAS) breeding. With the rich development of MAS, haplotype-based molecular markers have become a feasible technology to accelerate the genetic improvement of important economic traits of livestock and poultry. Haplotypes are multiple SNP sites associated with a certain area on a chromosome, which can effectively improve the accuracy of selection. In addition, genome resequencing technology is a high-throughput data detection technology developed on the basis of single nucleotide detection. At the genome level, it realizes the acquisition of genetic information and has become an important means of trait mining and genome breeding.

[0003] Tibetan chickens have lived on the Qinghai-Tibet Plateau for generations and are a valuable genetic resource in my country. Due to their long-term semi-pastoral life in a high-altitude, low-oxygen environment, Tibetan chickens are slightly smaller in size. In the sampling records of Tibetan chicken populations, the average weight of adult hens is 0.85-1.25 kg, and that of adult roosters is 1.5-1.9 kg. In order to meet the market demand for meat-type Tibetan chickens, genetic improvement for dual-purpose or meat-type chickens is in line with the direction of seed industry development. The use of whole genome mixed pool resequencing technology, molecular marker-assisted selection technology, etc., through the selection of weight-related genes or molecular markers linked to them, to find molecular markers linked to the weight candidate loci, and to apply them to the improvement of weight, is of great significance to the development of Tibetan meat-type resources. Summary of the invention

[0004] In view of this, the purpose of the present invention is to provide a molecular marker that can determine the weight of Tibetan chickens at 43 weeks of age, and to predict the adult weight of individuals by detecting the genotype of the molecular marker, thereby improving breeding efficiency.

[0005] In order to achieve the above object, the technical solution of the present invention is as follows:

[0006] In the first aspect, the present invention discloses a molecular marker related to the weight trait of Tibetan chickens, wherein the molecular marker is located in the 5' flanking sequence of the GPR75 gene, and the sequence of the molecular marker is shown in SEQ ID NO.1. In the sequence shown in SEQ ID NO.1, there is a G / A polymorphic site at the 268bp, an A / G polymorphic site at the 230bp, and a C / T polymorphic site at the 234bp.

[0007] In a preferred embodiment of the present invention, the 268 bp, 230 bp and 234 bp bases are highly linked to form haplotypes AGT and GAC, which are mainly divided into three genotypes, AGT / AGT, GAC / GAC and AGT / GAC.

[0008] In a second aspect, the present invention discloses an application of the molecular marker described in the first aspect in the prediction and screening of body weight traits of Tibetan chickens.

[0009] In a third aspect, the present invention discloses a primer pair for amplifying the molecular marker as described in the first aspect, the primer pair 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. .

[0010] In a fourth aspect, the present invention discloses an application of the primer pair described in the third aspect in the prediction and screening of body weight traits of Tibetan chickens.

[0011] In a fifth aspect, the present invention discloses a method for predicting and screening the weight traits of Tibetan chickens, comprising the following steps:

[0012] 1) Extract Tibetan chicken genomic DNA as a template;

[0013] 2) using the genomic DNA obtained in step S1 as a template, performing PCR amplification using the primer pair as claimed in claim 4 to obtain a molecular marker of the sequence shown in SEQ ID NO. 1 in claim 1 and purifying the marker;

[0014] 3) Detecting the genotype of the 268bp, 230bp, and 234bp positions of the sequence shown in SEQ ID NO.1 of the individual to be tested;

[0015] 4) Predict or screen chicken weight based on the detected genotype.

[0016] In a preferred embodiment of the present invention, the conditions for PCR amplification are: denaturation at 98°C for 10s, annealing at 58°C for 5s, and extension at 68°C for 5s, for a total of 32 cycles; and finally ending at 15°C.

[0017] In a preferred embodiment of the present invention, the PCR amplification system is: 30 μL in total, 15 μL of 2×KOD one mix, 0.5 μL of upstream and downstream primers, 2 μL of DNA template, and the balance of ddH2O.

[0018] In a preferred embodiment of the present invention, in step 4), through detection and genotyping, 43-week-old Tibetan chickens carrying homozygous AGT are heavier than those carrying homozygous GAC.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] The present invention uses the Tibetan chicken population in Shannan City, screens mutation sites based on the interpretation of the chicken genome data information, collects the adult weight of the population, and uses association analysis to verify the correlation between SNP and weight. Based on this, the present invention determines a molecular marker related to the weight of Tibetan chickens, which can be used to predict and screen the weight traits of Tibetan chickens. At the same time, it has the following advantages when breeding chickens: (1) generally improves breeding efficiency and effectively shortens the breeding cycle; (2) improves breeding accuracy; (3) achieves early acquisition of germplasm resources; (4) the molecular biology experimental method is simple and easy to implement. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is a diagram showing the 5' flanking SNP mutation of the Tibetan chicken GPR75 gene;

[0022] Figure 2 This is a bar chart of body weight of three haplotypes of Tibetan chicken. DETAILED DESCRIPTION

[0023] The technical solution of the present invention will be described clearly and completely below in conjunction with the embodiments. It should be understood that the specific implementation methods described herein are only used to illustrate and explain the present invention and are not used to limit the present invention.

[0024] In the following examples, unless otherwise specified, all methods are conventional methods; the reagents and materials described, unless otherwise specified, can be obtained from commercial sources.

[0025] Example 1

[0026] This example uses a Tibetan chicken hen population in Shannan City to identify specific SNPs by resequencing the Tibetan chicken genome pool. The specific process is as follows:

[0027] (1) Extraction of chicken genomic DNA: This example uses a genomic DNA kit produced by Beijing Biotech Biotechnology Co., Ltd. (operated according to the kit instructions), and the specific steps are as follows:

[0028] ① Use a disposable syringe to draw about 1 mL of blood from the vein under the chicken wing, inject it into a 1.5 mL centrifuge tube with anticoagulant, shake it gently, record the wing number, and store it at -20℃ for later use; draw 10 μL of anticoagulated blood, add 500 μL BB2 and 10 μL proteinase K (20 mg / mL), mix well, and incubate at room temperature for 10 min;

[0029] ② Briefly centrifuge, add all the solution to the centrifuge column, centrifuge at 12000g for 1 min, and discard the effluent;

[0030] ③ Add 500 μL of solution CB3, centrifuge at 12000 g for 30 s, and discard the effluent;

[0031] ④ Add 500 μL of WB3 solution, centrifuge at 12000 g for 30 seconds, and discard the effluent;

[0032] ⑤Repeat step ④ once;

[0033] ⑥ Centrifuge at 12000g for 2 minutes to completely remove residual WB3;

[0034] ⑦ Place the centrifuge column in a clean centrifuge tube, add 50-200 μL of preheated EB or deionized water in the center of the column, let it stand at room temperature for 1 min, centrifuge at 12000g for 1 min, elute the DNA, and store the eluted DNA at -20℃ for later use.

[0035] (2) Pooled resequencing and SNP screening. The specific steps are as follows:

[0036] ① Send the Tibetan chicken DNA to Wuhan Yingzi Gene Technology Co., Ltd. for sequencing, with a sequencing depth of at least 30×;

[0037] ② The raw data is filtered by the connector, the Galgal6 reference genome is downloaded for comparison, and SNPs are called by GATK to obtain a vcf file;

[0038] ③ In the screening results, base mutations were found at positions 3015531, 3015533, and 3015537 on chromosome 3, and the annotation information showed that they were located in the 5' flanking sequence of the GPR75 gene (the sequence is shown in SEQ ID NO.1).

[0039] SEQ ID NO.1:

[0040] .

[0041] Example 2

[0042] In this example, the 5' flanking sequence of the chicken GPR75 gene (sequence shown in SEQ ID NO.1) was cloned, and the genotyping of three SNP sites was analyzed. The specific process is as follows:

[0043] (1) Collect blood from a Tibetan chicken population and extract chicken genomic DNA. The specific operation steps are shown in Example 1.

[0044] (2) A primer pair was designed based on the 5' flanking upstream sequence of the GPR75 gene sequence published in the NCBI database (Gene ID: NM_001389511.2). The sequence of the primer pair is as follows:

[0045] Upstream primer: 5'-CATTTCTATATGGATTTCTAT-3' (as shown in SEQ ID NO.2),

[0046] Downstream primer: 5'-TTATGTGGGTGAGCCCTCAGT-3' (as shown in SEQ ID NO. 3).

[0047] The above primer pairs were used to perform PCR amplification in chicken genomic DNA, wherein the PCR reaction system is shown in Table 1, and the PCR reaction conditions are shown in Table 2.

[0048] Table 1

[0049]

[0050] Table 2

[0051]

[0052] The PCR amplification products were detected by agarose gel electrophoresis.

[0053] (3) Detection of haplotype markers using direct sequencing of PCR products:

[0054] The PCR purified products obtained above were directly sent to Beijing Aoke Dingsheng Biotechnology Co., Ltd. for sequencing, and the genotype of the site in the test population was determined based on the sequencing results.

[0055] Example 3

[0056] In this example, the polymorphism of the rs313381871 locus was detected, and three haplotype combinations were detected in the F2 population. The frequencies and distributions are shown in Table 3.

[0057] Table 3

[0058]

[0059] Note: The numbers in brackets in the haplotype (combination) frequency column are the numbers of individuals.

[0060] As shown in Table 3, the frequency of AGT haplotype is 0.37, and the frequency of GAC haplotype is 0.63. From the frequency distribution, it can be seen that GAC haplotype is the dominant haplotype.

[0061] Example 4

[0062] In order to confirm whether the haplotype is related to the weight of Tibetan chickens at 43 weeks of age, this example uses a Tibetan chicken group as the experimental object, and records the weight of the chickens at 43 weeks of age one by one. SPSS18.0 software was used to perform association analysis between haplotypes and phenotypes, and the model used was as follows:

[0063] Y ij =u+G i +e ij

[0064] Among them, Y ij is the observed value of the trait, u is the average value of the trait, G i is the haplotype effect, e ij is a random error.

[0065] The results of the association analysis between haplotype and body weight at 43 weeks of age are shown in Table 4.

[0066] Table 4

[0067]

[0068] Note: The same letters in the above table indicate no significant difference, while letters a, b, c indicate significant difference.

[0069] As can be seen from Table 4, haplotype polymorphism is significantly associated with the weight of 43-week-old Tibetan chickens (P<0.05); in actual production, we found that in the Tibetan chicken observation group, the 43-week-old Tibetan chickens carrying homozygous AGT individuals weighed about 449g more than those carrying homozygous GAC individuals. Therefore, the three SNPs in the present invention constitute haplotype sites, which can be used as potential molecular markers for screening or predicting the weight of Tibetan chickens.

[0070] In summary, the molecular marker provided by the present invention is closely related to the weight of Tibetan chickens. The individual weight can be accurately selected by detecting the genotype of the molecular marker. The present invention further provides a primer pair for specific amplification and constructs a detection method, which lays a foundation for further breeding applications of the molecular marker.

[0071] The above description is a preferred embodiment of the present invention, which cannot be used to limit the scope of rights of the present invention. It should be pointed out that for ordinary technicians in this technical field, any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A molecular marker related to the body weight trait of Tibetan chicken, characterized in that: The molecular marker is located at the 5' flanking sequence of the GPR75 gene. The sequence of the molecular marker is shown in SEQ ID NO.

1. In the sequence shown in SEQ ID NO.1, there is a G / A polymorphic site at 268bp, an A / G polymorphic site at 230bp, and a C / T polymorphic site at 234bp.

2. The molecular marker associated with the body weight trait of Tibetan chicken according to claim 1, characterized in that: The 268 bp, 230 bp and 234 bp bases are highly linked to form haplotypes AGT and GAC.

3. Application of the molecular marker as described in any one of claims 1 or 2 in the prediction and screening of body weight traits of Tibetan chickens.

4. A primer pair for amplifying the molecular marker according to claim 1, characterized in that: The primer pair includes: the nucleotide sequence of the upstream primer is shown in SEQ ID NO.2, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.

3.

5. Application of the primer pair as claimed in claim 4 in prediction and screening of body weight traits of Tibetan chickens.

6. A method for predicting and screening the weight traits of Tibetan chickens, characterized in that: The following steps are involved: 1) Extract Tibetan chicken genomic DNA as a template; 2) using the genomic DNA obtained in step S1 as a template, performing PCR amplification using the primer pair as claimed in claim 4 to obtain a molecular marker having a sequence as shown in SEQ ID NO. 1 in claim 1 and purifying the marker; 3) Detecting the genotype of the 268bp, 230bp, and 234bp positions of the sequence shown in SEQ ID NO.1 of the individual to be tested; 4) Predict or screen chicken weight based on the detected genotype.

7. The method for screening and predicting the weight traits of Tibetan chickens according to claim 6, characterized in that: The PCR amplification conditions are: denaturation at 98°C for 10s, annealing at 58°C for 5s, extension at 68°C for 5s, for a total of 32 cycles; and end at 15°C.

8. The use according to claim 6, characterized in that: The PCR amplification system is: 30 μL in total, 15 μL of 2×KODone mix, 0.5 μL of upstream and downstream primers, 2 μL of DNA template, and the balance of ddH2O.

9. The use according to claim 6, characterized in that: In step 4), through detection and genotyping, the 43-week-old Tibetan chickens carrying homozygous AGT were heavier than those carrying homozygous GAC.

Images