A molecular marker, primer pair and application thereof related to the body weight trait of Tibetan chicken

Abstract

The present invention discloses a molecular marker, a primer pair and an application thereof related to the weight trait of Tibetan chickens, and relates to the technical field of molecular marker-assisted selection breeding. The molecular marker is located in 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 the 268th bp, an A / G polymorphic site at the 230th bp, and a C / T polymorphic site at the 234th bp. 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. The molecular marker and primer pair can be used to predict and screen the weight trait of Tibetan chickens. At the same time, when breeding chickens, they can significantly improve breeding efficiency, effectively shorten the breeding cycle, and have high breeding accuracy and low cost.

Description

Technical Field

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

[0002] Single Nucleotide Polymorphism (SNP) refers to genetic polymorphism caused by 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 region 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. It realizes the acquisition of genetic information at the genome level and has become an important means of trait mining and genomic breeding.

[0003] Tibetan chickens have inhabited the Qinghai-Tibet Plateau for generations and are a valuable genetic resource in my country. Due to their long-term semi-pastoral lifestyle at high altitude and in a low-oxygen environment, Tibetan chickens are relatively small. In sampling records of Tibetan chicken populations, the average weight of adult hens ranges from 0.85 to 1.25 kg, and that of adult roosters from 1.5 to 1.9 kg. To meet market demand for broiler-grade Tibetan chickens, genetic improvement for dual-purpose or broiler-grade chickens is in line with the direction of seed industry development. Using whole-genome pooled resequencing and molecular marker-assisted selection techniques, selection is being conducted based on weight-related genes or linked molecular markers. The identification of molecular markers linked to candidate weight loci and their application in weight improvement is of great significance for the development of broiler-grade Tibetan chickens. 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. The molecular marker is located in 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 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 bases 268bp, 230bp, and 234bp 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 described in the first aspect, wherein the primer pair comprises: an upstream primer having a nucleotide sequence as shown in SEQ ID NO. 2, and a downstream primer having a nucleotide sequence as shown in 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 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) Extracting 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 described in claim 4 to obtain a molecular marker having the sequence shown in SEQ ID NO. 1 in claim 1 and purifying the marker;

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

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

[0016] In a preferred embodiment of the present invention, the PCR amplification conditions 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] This study used a Tibetan chicken population from Shannan City to screen for mutation sites based on the interpretation of whole-genome data. The population's adult weight was collected, and the association between SNPs and weight was verified using association analysis. Based on this, the study identified a molecular marker associated with Tibetan chicken weight. This molecular marker can be used to predict and screen Tibetan chicken weight traits, and has the following advantages in chicken breeding: (1) generally improving breeding efficiency and effectively shortening the breeding cycle; (2) improving breeding accuracy; (3) achieving early acquisition of germplasm resources; and (4) simple molecular biology experimental methods that are 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 the weight of three haplotypes of Tibetan chicken. DETAILED DESCRIPTION

[0023] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present invention and are not intended 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. 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 sub-wing vein of the chicken, inject it into a 1.5 mL centrifuge tube with anticoagulant, shake it gently, record the wing number, and store it at -20°C for later use; draw 10 μL of anticoagulated blood, add 500 μL of BB2 and 10 μL of proteinase K (20 mg / mL), mix thoroughly, 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 minute, and discard the effluent;

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

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

[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 to the center of the column, let it stand at room temperature for 1 minute, centrifuge at 12000g for 1 minute to 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 adapter, the Galgal6 reference genome is downloaded for alignment, 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. 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) Blood was collected from a Tibetan chicken population and chicken genomic DNA was extracted. The specific 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] PCR amplification was performed in chicken genomic DNA using the above primer pairs, 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) Detect 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, we can see that GAC haplotype is the dominant haplotype.

[0061] Example 4

[0062] In order to confirm whether the haplotype is associated with the weight of Tibetan chickens at 43 weeks of age, this example used a Tibetan chicken group as the experimental subject and recorded the weight of each chicken at 43 weeks of age. SPSS 18.0 software was used to perform association analysis between haplotype and phenotype, 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 haplotypes 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, and c indicate significant difference.

[0069] As shown in Table 4, the 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 observed Tibetan chicken population, 43-week-old chickens carrying homozygous AGT weighed approximately 449g more than those carrying homozygous GAC. Therefore, the three SNPs in this paper constitute a haplotype locus and can be used as a potential molecular marker for screening or predicting Tibetan chicken weight.

[0070] In summary, the molecular marker provided by the present invention is closely related to the weight of Tibetan chickens. By detecting the genotype of the molecular marker, the weight of individuals can be accurately selected. The present invention further provides a primer pair for specific amplification and constructs a detection method, laying the 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 scope of protection of the present invention.

Claims

1. Application of molecular markers in the prediction and screening of weight traits of Tibetan chickens. The molecular marker is located in 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 the 268th bp, an A / G polymorphic site at the 230th bp, and a C / T polymorphic site at the 234th bp.

2. The use according to claim 1, characterized in that The 268th bp, 230th bp and 234th bp bases are highly linked to form haplotypes AGT and GAC.

3. The use according to claim 1, characterized in that The primer pair used to amplify the molecular marker includes: 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 for predicting and screening the weight traits of Tibetan chickens, characterized in that: The following steps are involved: 1) Extracting Tibetan chicken genomic DNA as a template; 2) Using the genomic DNA obtained in step S1 as a template, PCR amplification is performed using the primer pair shown in SEQ ID NO. 2 and SEQ ID NO. 3 to obtain a molecular marker with the sequence shown in SEQ ID NO. 1 as claimed in claim 1 and purify the marker; 3) Detecting the genotype of the individual to be tested at the 268th, 230th, and 234th bp positions of the sequence shown in SEQ ID NO. 1; 4) Predict or screen chicken weight based on the detected genotype; The Tibetan chickens are a group of Tibetan chicken hens from Shannan City.

5. The method for screening and predicting the weight traits of Tibetan chickens according to claim 4, characterized in that: The PCR amplification conditions were as follows: denaturation at 98°C for 10 seconds, annealing at 58°C for 5 seconds, and extension at 68°C for 5 seconds, for a total of 32 cycles; and the amplification was terminated at 15°C.

6. The method for screening and predicting the weight traits of Tibetan chickens according to claim 4, characterized in that: The PCR amplification system is as follows: 30 μL in total, 15 μL of 2×KOD one mix, 0.5 μL of each of upstream and downstream primers, 2 μL of DNA template, and the balance of ddH2O.

7. The method for screening and predicting the weight traits of Tibetan chickens according to claim 4, 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.

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