An SNP molecular marker related to the body weight of Nyctereutes procyonoides ussuriensis and its application

Abstract

The present invention belongs to the technical field of molecular genetics, and particularly relates to an SNP molecular marker related to the body weight of Ussuri raccoon dogs and its application. The SNP molecular marker is a C / T base mutation at the 128,151st position on the Nypro681 sequence of the raccoon dog reference genome NYPRO_anot_genome. Individuals of Ussuri raccoon dogs with different genotypes show significant differences in body weight, providing a new marker resource for marker-assisted selection breeding of the body weight trait of Ussuri raccoon dogs. The present invention uses PCR technology and Sanger sequencing method to detect the above SNP molecular marker and applies it to the early selection of the body weight trait of Ussuri raccoon dogs, establishing a molecular marker-assisted selection technology for the body weight trait of Ussuri raccoon dogs to guide its selection and mating, thereby shortening the breeding cycle, accelerating the breeding process, and reducing the breeding cost.

Description

Technical Field

[0001] The present invention relates to the technical field of SNP molecular markers, and particularly relates to an SNP molecular marker related to the body weight of Ussuri raccoon dogs and its application. Background Art

[0002] The raccoon dog is one of the important economically-reared animals, and its economic value mainly comes from its fur. The raccoon dog fur belongs to large hair furs, which have the advantages of being light, warm, and beautiful. Most of them are used to process the cap strips, cuffs, etc. of clothing, and occupy an important position in the international fur market. Therefore, the quality of the pelt directly affects the economic benefits of farmers. In raccoon dog breeding, body type traits such as body weight are one of the most important economic traits, which directly determine the size of the pelt and the classification of fur grades. In raccoon dog breeding work, selecting large-sized raccoon dogs with body weight and other main indicators is the main goal of current raccoon dog breeding.

[0003] With the wide application of molecular biology techniques in livestock and poultry breeding work, SNP molecular markers closely linked to their economic traits have been found in many livestock and poultry, and these molecular markers have been used for assisted selection breeding, greatly accelerating the breeding process. Single nucleotide polymorphism (SNP) mainly refers to the polymorphism of DNA sequences at the genomic level caused by the variation of a single nucleotide, and the forms include the deletion, insertion, transition, and transversion of a single base. As the third-generation genetic marker, SNP is widely used in the breeding of animals and plants. Therefore, in raccoon dog breeding, the development and application of genetic markers are the main research directions of current breeding work, and at the same time, using molecular markers to guide breeding is of great significance for the genetic improvement of raccoon dog economic traits. Summary of the Invention

[0004] The purpose of the present invention is to solve the deficiencies in the prior art and propose an SNP molecular marker related to the body weight of Ussuri raccoon dogs and its application.

[0005] One of the purposes of the present invention is to provide an SNP molecular marker related to the body weight of Ussuri raccoon dogs, which is located at the base of the 128151st point on the Nypro681 sequence of the raccoon dog reference genome, and this SNP molecular marker site has C / T polymorphism.

[0006] GenBank accession number of Nypro681: CAJHUB010000647.1; the information version number of the raccoon dog reference genome is NYPRO_anot_genome, February 2021.

[0007] Preferably, the nucleotide sequence containing the SNP molecular marker is as shown in SEQ ID NO.1, and the SNP molecular marker is located at the 171st position.

[0008] Preferably, the body weight of the CC genotype at the polymorphic site of the SNP molecular marker is significantly higher than that of the CT genotype and the TT genotype, and the body weight of the CT genotype is significantly higher than that of the TT genotype.

[0009] The second object of the present invention is to provide the application of the above SNP molecular marker in the detection of the body weight trait of the Ussuri raccoon dog or the breeding of the Ussuri raccoon dog.

[0010] The third object of the present invention is to provide a primer pair of the above SNP marker, the upstream primer is shown as SEQ ID NO.2, and the downstream primer is shown as SEQ ID NO.3.

[0011] The fourth object of the present invention is to provide the application of the above primer pair in the detection of the body weight trait of the Ussuri raccoon dog or the breeding of the Ussuri raccoon dog.

[0012] The fifth object of the present invention is to provide a kit containing the above primer pair.

[0013] The sixth object of the present invention is to provide a method for predicting the body weight trait of the Ussuri raccoon dog, which detects the genotype of the polymorphic site of the SNP molecular marker as described above.

[0014] Preferably, the above primer pair or the above kit is used for detection.

[0015] Specifically, it includes the following steps:

[0016] (1) Extract the genomic DNA of the Ussuri raccoon dog blood as the template DNA;

[0017] (2) Use the above primer pair to perform PCR amplification on the genomic DNA obtained in step (1) to obtain a PCR amplification product;

[0018] (3) Purify and sequence the PCR amplification product obtained in step (2) to obtain a sequencing result;

[0019] (4) Perform gene typing based on the sequencing result. When the base of the SNP molecular marker is T, the genotype is TT; when the base of the SNP molecular marker is C, the genotype is CC or CT; the body weight of the Ussuri raccoon dog with the CC genotype is extremely significantly higher than that of the CT genotype and the TT genotype (P<0.01), and the body weight of the Ussuri raccoon dog with the CT genotype is significantly higher than that of the TT genotype (P<0.05).

[0020] A seventh objective of the present invention is to provide a method for breeding improvement of Ussuri raccoon dogs, determine the above SNP molecular markers of Ussuri raccoon dogs, and make corresponding selections based on the SNP molecular markers of Ussuri raccoon dogs: for the sub-generation selection of Ussuri raccoon dogs, individuals marked with C at the polymorphic sites of the above SNP molecular markers are referred to, and individuals marked with T at the polymorphic sites of the SNP molecular markers are eliminated.

[0021] That is, detect the base mutation type at the 128,151st position on the Nypro681 (GenBank accession number: CAJHUB010000647.1) sequence of the Ussuri raccoon dog reference genome NYPRO_anot_genome, select Ussuri raccoon dogs with the base C at this position as breeding raccoon dogs, and individuals with the base T are not suitable for breeding.

[0022] The present invention discloses an SNP molecular marker related to the body weight of Ussuri raccoon dogs, which is a C / T base mutation at the 128,151st position on the Nypro681 sequence of the raccoon dog reference genome NYPRO_anot_genome; when the base of the SNP molecular marker is T, the genotype is TT; when the base of the SNP molecular marker is C, the genotype is CC or CT; the body weight of Ussuri raccoon dogs with the CC genotype is extremely significantly higher than that of the TC genotype and TT genotype (P<0.01); the body weight of Ussuri raccoon dogs with the CT genotype is significantly higher than that of the TT genotype (P<0.05).

[0023] Based on the above SNP molecular markers, the present invention has developed a specific primer pair for detecting this molecular marker, and used the PCR technology and Sanger sequencing method to detect the base type, thereby establishing an efficient and accurate molecular marker-assisted breeding technology, which can quickly screen out Ussuri raccoon dog individuals with dominant alleles.

[0024] The present invention applies the above molecular marker detection technology to the genetic improvement of the body weight trait of Ussuri raccoon dog breeding raccoon dogs to guide their selection and mating, which can strengthen the early selection of Ussuri raccoon dogs, improve the accuracy of selection, thereby shortening the generation interval, shortening the breeding cycle, accelerating the breeding process, reducing the breeding cost, and has a positive promoting effect on breeding a Ussuri raccoon dog population with a large body weight. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 It is the PCR amplification result of the DNA fragment where the SNP molecular marker is located in Example 4.

[0026] Figure 2 It is the genotyping result after purification and sequencing of the PCR amplification product in Example 5; where CC, CT, and TT represent the CC genotype, CT genotype, and TT genotype in sequence. DETAILED DESCRIPTION OF THE INVENTION

[0027] The present invention will be further explained below in conjunction with specific embodiments.

[0028] Example 1: Sample collection and phenotype determination

[0029] The applicant used 96 male Ussuri raccoon dogs as the research object, and all the experimental raccoon dog groups were raised under the same breeding environment, feeding conditions, and feeding methods. Before skinning, 5 mL of blood samples from all experimental Ussuri raccoon dogs were collected in EDTA anticoagulant tubes and stored at -20 °C for later use; the weights of the Ussuri raccoon dogs were weighed and the data were recorded.

[0030] Example 2: Genomic DNA extraction and quality detection

[0031] The genomic DNA was extracted from the Ussuri raccoon dog blood samples using the Blood Genomic DNA Extraction Kit (TIANamp Genomic DNA Kit) produced by Beijing Tiangen Biochemical Technology Co., Ltd.

[0032] Take 5 μL of the DNA solution and perform electrophoresis detection on 1% agarose gel. A single band indicates that the DNA is intact and undegraded, and an obvious band indicates that the concentration can meet the PCR requirements; use a spectrophotometer to detect the concentration and purity, take 1 μL to detect the OD value, OD 260 / OD 280 being between 1.7 and 2.0 indicates that the DNA quality is good and meets the experimental requirements.

[0033] Example 3: Primer design

[0034] Taking the sequence of Nypro681 (GenBank accession number: CAJHUB010000647.1) of the Ussuri raccoon dog genome NYPRO_anot_genome version as a reference, a pair of specific primers were designed using Primer Premier 5.0 software, as follows:

[0035] Primer Sequence(5'-3') F tatttgttga atcccatagc cttac R taattatcta agccatggaa aactg

[0036] The amplified product is 447 bp, and the sequence information is as follows:

[0037] tatttgttga atcccatagc cttacagctg aagaatacat ttggatgatt ttcgcctgcc

[0038] tgctgatttg gaagtggaat ggctttctct gtacttcatc tttattgggt aatgatcacg

[0039] gtgtctgcat atttcaagtt cgggaaaata tattccagct ttgttgctgg tactctgagg

[0040] atgatgtttg gtttgtattg tttatcttga gccagtgacc agtgttacac aatatggata

[0041] taattggtag aagtaacaaa gtctgatatg ataagaagaa agactaaaat atcagattta

[0042] ctgaaataat ctctgagtgc tgtgggagag aatcagatac ttggcagtgc acacagtcct

[0043] aagggtggca gttgattcac tcttaattgt ctagagactc aatgaatgac acacatattt

[0044] cacagttttc catggcttag ataatta

[0045] The above amplified fragment contains the SNP molecular marker locus at position 171, that is, C / T at position 128151 on the Nypro681 sequence.

[0046] Example 4 Amplification and Detection of the Target Fragment

[0047] Using the genomic DNA in blood as a template, the target fragment was amplified using the specific primers designed in Step 3.

[0048] PCR Amplification System:

[0049] Reagent Dosage Template DNA 1 μL Forward primer with a concentration of 10 μM 1 μL Reverse primer with a concentration of 10 μM 1 μL Dntp (mix) with a concentration of 10 μM 1 μL <![CDATA[10×Taq Buffer(with MgCl2)]]> 2.5 μL Taq enzyme with a concentration of 5 U / μL 0.2 μL <![CDATA[ddH2O]]> The total volume is made up to 25 μL

[0050] PCR Reaction Program:

[0051]

[0052]

[0053] The obtained reaction product was detected by 1% agarose gel electrophoresis. Electrophoresis parameters: 150V, 100mA, 10 - 20 min; The electrophoresis results are as Figure 1 shown.

[0054] Example 5 Sequencing and Genotyping

[0055] For the purpose of gel extraction and recovery of the PCR bands, Sanger sequencing was used to obtain sequence information, and the sequencing was completed by Sangon Biotech (Shanghai) Co., Ltd. The sequencing results were analyzed using Sequence analysis software, and the SNP markers described in step 3 were genotyped according to the sequencing peak patterns. The 96 samples included three genotypes: CC, CT, and TT, as Figure 2 shown.

[0056] Example 6 Statistical Analysis

[0057] Descriptive statistics of the body weight data of different genotypes of the Ussuri raccoon dog were performed using IBM SPSS Statistics 22 software, and the results are shown in the following table.

[0058]

[0059]

[0060] The number of individuals and body weight (mean ± standard error) of different genotypes of the Ussuri raccoon dog were as follows:

[0061] The number of CC-type individuals was 31, and the body weight was 9.27 ± 0.13 kg;

[0062] The number of TT-type individuals was 23, and the body weight was 8.02 ± 0.14 kg;

[0063] The number of CT-type individuals was 40, and the body weight was 8.55 ± 0.14 kg.

[0064] One-way ANOVA was used to compare the body weight differences among different genotypes of the Ussuri raccoon dog, and the results are shown in the following table:

[0065] Genotype Body weight (kg) CC genotype <![CDATA[9.27±0.13 aA > TT genotype <![CDATA[8.02±0.14 bB > CT genotype <![CDATA[8.55±0.14 cB >

[0066] The body weight of Ussuri raccoon dogs with the CC genotype was extremely significantly higher than that of the TT genotype and the CT genotype (P < 0.01), and the body weight of Ussuri raccoon dogs with the CT genotype was significantly higher than that of the TT type (P < 0.05).

[0067] The results showed that by genotyping the base at position 128151 on the Nypro681 sequence of the Ussuri raccoon dog, it could be used as an important indicator to judge the body weight of the Ussuri raccoon dog, providing a basis for molecular marker-assisted breeding of the Ussuri raccoon dog. By collecting blood samples of the Ussuri raccoon dog, extracting genomic DNA, and applying the specific primers and genotyping methods provided by the present invention, the future body weight could be predicted. By continuously eliminating individuals with the molecular marker T at position 128151 on the Nypro681 sequence of the Ussuri raccoon dog, the body weight of the Ussuri raccoon dog population could be gradually increased.

[0068] The above are only the preferred specific embodiments of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present invention should cover within the protection scope of the present invention according to the technical solution of the present invention and its inventive concept for equivalent replacement or change.

Claims

1. A SNP molecular marker related to the body weight of Nyctereutes ussurienusis, characterized in that, The nucleotide sequence of the SNP molecular marker is shown as SEQ ID NO.1, and the SNP molecular marker has C / T polymorphism at the 171st position; The body weights of the CC genotype and CT genotype at the polymorphic site of the SNP molecular marker are significantly higher than those of the TT genotype.

2. Use of a reagent for detecting the SNP molecular marker as described in claim 1 in the detection of the body weight trait of the Ussuri raccoon dog, characterized in that, The body weights of the CC genotype and CT genotype at the polymorphic site of the SNP molecular marker are significantly higher than those of the TT genotype.

3. Use of a reagent for detecting the SNP molecular marker as described in claim 1 in the breeding of Ussuri raccoon dogs, characterized in that, Detect the SNP molecular marker described in Claim 1 in the Ussuri raccoon dog, and make corresponding selections according to the genotypes of the polymorphic sites of the Ussuri raccoon dog SNP molecular marker: Select individuals with the polymorphic site of the SNP molecular marker described in Claim 1 marked as C for the subsequent selection of the Ussuri raccoon dog, and eliminate individuals with the polymorphic site of the SNP molecular marker marked as T.

4. Use of a primer pair for detecting the SNP molecular marker as described in claim 1 in the preparation of a reagent for detecting the body weight trait of Ussuri raccoon dog, characterized in that, The body weights of the CC genotype and CT genotype at the polymorphic site of the SNP molecular marker are significantly higher than those of the TT genotype; the upstream primer of the primer pair is shown as SEQ ID NO.2, and the downstream primer of the primer pair is shown as SEQ ID NO.

3.

5. Use of a primer pair for detecting the SNP molecular marker as described in claim 1 in the breeding of Ussuri raccoon dogs, characterized in that, Detect the SNP molecular marker described in Claim 1 in the Ussuri raccoon dog, and make corresponding selections according to the genotypes of the polymorphic sites of the Ussuri raccoon dog SNP molecular marker: Select individuals with the polymorphic site of the SNP molecular marker described in Claim 1 marked as C for the subsequent selection of the Ussuri raccoon dog, and eliminate individuals with the polymorphic site of the SNP molecular marker marked as T; the upstream primer of the primer pair is shown as SEQ ID NO.2, and the downstream primer of the primer pair is shown as SEQ ID NO.

3.

6. A method for predicting the body weight trait of Nyctereutes procyonoides ussuriensis, characterized in that, Detect the genotype of the polymorphic site of the SNP molecular marker described in Claim 1; the body weights of the CC genotype and CT genotype at the polymorphic site of the SNP molecular marker are significantly higher than those of the TT genotype.

7. The method according to claim 6, wherein Detect using the primer pair described in the application of Claim 4 or a kit containing the primer pair described in the application of Claim 4.

8. A breeding improvement method for Nyctereutes procyonoides ussuriensis, characterized in that, Detect the SNP molecular marker described in Claim 1 in the Ussuri raccoon dog, and make corresponding selections according to the genotypes of the polymorphic sites of the Ussuri raccoon dog SNP molecular marker: Select individuals with the polymorphic site of the SNP molecular marker described in Claim 1 marked as C for the subsequent selection of the Ussuri raccoon dog, and eliminate individuals with the polymorphic site of the SNP molecular marker marked as T.

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