A SNP molecular marker related to IMF content of ningxiang pig pork and application thereof

Abstract

The application discloses a SNP molecular marker related to the IMF content of Ningxiang pig pork and application thereof, wherein the SNP molecular marker is located at 1001 bp of a nucleotide sequence shown in SEQ ID NO:1, and the polymorphism is C / T. The application provides the molecular marker which is significantly related to the IMF content of Ningxiang pig, has co-dominant inheritance, and is combined with KASP detection technology, so that the designed primer pair can be applied to multiple platforms such as a high-throughput SNP genotyping platform and a high-throughput KASP detection platform, and can be applied to large-scale population breeding and analysis. The application of the molecular marker to Ningxiang pig breeding can accurately and efficiently predict the IMF content and typing, identify and screen pig breeds with high IMF content and excellent meat quality, and improve the breeding efficiency.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to a SNP molecular marker related to the IMF content of Ningxiang pig pork and application thereof. BACKGROUND

[0002] Pork, as the most important component in the meat market, is an important dietary source for human beings. Ningxiang pig is famous for its tender and delicious pork, and the intramuscular fat content is an important indicator affecting flavor and meat quality evaluation.

[0003] With the improvement of people's living standards and social productivity, the demand for pork has changed from quantity to quality. Quality traits are one of the most concerned economic traits by breeders, and the flavor and nutritional value of meat are closely related to IMF content and fatty acid composition. Studies have shown that the IMF content of pork is a key indicator for evaluating pork quality, which will affect the flavor, tenderness and juiciness of pork. At present, finding candidate genes and molecular mechanisms related to IMF deposition has become an important strategy for reasonably improving IMF content.

[0004] As a widely used molecular marker, SNP marker has higher genetic stability compared to SSR molecular marker technology, ISSR molecular marker technology and other polymorphic markers. At the same time, due to the diversity of detection methods, including Taqman method, mass spectrometry, chip method and sequencing method, it can be applied to rapid and large-scale screening. With the progress of sequencing technology and the continuous reduction of sequencing cost, more and more SNP markers of various species are being mined and applied. Therefore, developing a SNP molecular marker related to pork IMF content suitable for high-throughput molecular detection platform can identify and screen pigs with high IMF content and excellent meat quality, and improve breeding efficiency. SUMMARY

[0005] The present application aims to at least solve one of the technical problems existing in the prior art. To this end, the present application provides a SNP molecular marker related to the IMF content of Ningxiang pig pork.

[0006] The present application also provides a primer set for detecting the above-mentioned SNP molecular marker.

[0007] The present application also provides a kit.

[0008] The present application also provides a gene chip.

[0009] The present application also provides the application of the above-mentioned molecular marker, primer set, kit and / or gene chip.

[0010] The present application also provides a detection method of the above-mentioned molecular marker.

[0011] The application further provides a method for breeding a pig strain with high IMF content.

[0012] According to a first aspect of the application, a SNP molecular marker related to the IMF content of Ningxiang pig pork is provided, wherein the SNP molecular marker is located at 1001 bp of the nucleotide sequence shown in SEQ ID NO: 1, and the polymorphism is C / T.

[0013] In some embodiments of the application, the breed of the pig comprises Ningxiang pig.

[0014] According to a second aspect of the application, a primer set for amplifying the above-mentioned SNP molecular marker is provided, wherein the primer set comprises specific primers and universal primers, and the sequence of the specific primers comprises Primer X and Primer Y.

[0015] In some embodiments of the application, the sequence of the specific primers is shown in SEQ ID NO: 2 and SEQ ID NO: 3.

[0016] According to some embodiments of the application, the primer set further comprises a universal primer sequence shown in SEQ ID NO: 4.

[0017] In some embodiments of the application, the specific primers are respectively connected with FAM and HEX fluorescent linker sequences.

[0018] According to a third aspect of the application, a kit is provided, wherein the kit comprises the above-mentioned primer set.

[0019] According to a fourth aspect of the application, a gene chip is provided, wherein the gene chip comprises the above-mentioned primer set.

[0020] According to a fifth aspect of the application, the above-mentioned SNP molecular marker, primer set, kit and / or gene chip are applied to:

[0021] (1) the preparation of a product for detecting or assisting in detecting the IMF content of Ningxiang pig;

[0022] (2) the identification and selection of Ningxiang pigs with different IMF contents;

[0023] (3) the molecular marker assisted breeding of Ningxiang pig;

[0024] (4) the breeding of Ningxiang pig;

[0025] (5) use in preparing a product for breeding Ningxiang pigs.

[0026] According to a sixth aspect of the present application, a method for detecting IMF content of Ningxiang pig meat by using the SNP molecular marker is provided, and the method comprises the following steps:

[0027] S1, extracting genomic DNA of the pig to be tested;

[0028] S2, detecting the polymorphism of the SNP molecular marker on the genomic DNA extracted in step S1, and judging the IMF content of the pig to be tested according to the detection result.

[0029] In some embodiments of the present application, if only the fluorescence signal corresponding to Primer X is detected, the base at the detection site is C, and the IMF content of the test material is low; if only the fluorescence signal corresponding to Primer Y is detected, the base at the detection site is T, and the IMF content of the test material is high; if the fluorescence signals corresponding to Primer X and Primer Y are detected at the same time, the base at the detection site is C:T, and the test material is a hybrid genotype.

[0030] In some embodiments of the present application, in step S1, the genomic DNA of the pig to be tested is extracted by using a magnetic bead method.

[0031] In some embodiments of the present application, in step S2, the SNP molecular marker is detected by using a KASP (Competitive Allele-Specific PCR) technology.

[0032] According to a seventh aspect of the present application, a method for breeding a pig strain with high IMF content is provided, and the method comprises the following steps: using the above method, selecting pigs with a genotype of TT of the SNP molecular marker for subsequent breeding.

[0033] According to some embodiments of the present application, at least the following beneficial effects are achieved: the molecular marker provided by the present application is significantly related to the IMF content of Ningxiang pigs, has co-dominant inheritance, and in combination with the KASP detection technology, the designed primer pair can be applied to multiple platforms such as high-throughput SNP genotyping platforms and high-throughput KASP detection platforms, and can be applied to large-scale population breeding and analysis. The molecular marker can provide guidance for Ningxiang pig breeding, accurately and efficiently predict the content and typing of IMF, identify and screen pigs with high IMF content and excellent meat quality, and improve the breeding efficiency.

[0034] Additional aspects and advantages of the present application will be partially given in the following description, partially will become obvious from the following description, or will be understood by the practice of the present application. BRIEF DESCRIPTION OF DRAWINGS

[0035] The present application is further illustrated in conjunction with the accompanying drawings and examples, in which:

[0036] Figure 1 Manhattan plot for Example 1 of the present application;

[0037] Figure 2 Molecular marker layout developed for the 500k region upstream and downstream of SNP chr06_65261993 in Example 1 of the present application, wherein red represents gene name and black represents molecular marker site;

[0038] Figure 3 Correlation analysis result plot of different allelic genotypes of SNP chr6:65261993 and IMF content in Example 2 of the present application;

[0039] Figure 4 KASP detection plot of 237 materials in Example 2 of the present application;

[0040] Figure 5 KASP detection plot of 11 materials in Example 2 of the present application. DETAILED DESCRIPTION

[0041] The concept and technical effects of the present application will be described below in conjunction with examples to fully understand the purpose, features and effects of the present application. Obviously, the described examples are only a part of the examples of the present application, but not all the examples. Based on the examples of the present application, other examples obtained by those skilled in the art without creative labor are within the protection scope of the present application. The test methods used in the examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the examples are commercially available unless otherwise specified.

[0042] Example 1: Obtaining of SNP molecular markers related to IMF content of Ningxiang pig pork

[0043] 1. Material screening and phenotype recording

[0044] 778 Ningxiang pig purebred materials were collected from Dalong Pig Farm of Hunan Ningxiang Chuwei Xiangnong Farm Co., Ltd., and their IMF phenotype data were collected.

[0045] The specific phenotype detection method is as follows: the test pigs are grown to the slaughter age (180±5 days), sent to the slaughterhouse for unified slaughter, and the longissimus dorsi muscle at the 6th-12th rib of Ningxiang pig is collected, about 25g of chopped longissimus dorsi muscle is weighed and placed in a freeze dryer for dehydration to a constant weight, about 0.50g of dried sample is weighed, sealed into a fat package with filter paper, the fat package is placed in a Soxhlet extractor, petroleum ether is added to immerse the fat package, soaked for 10-15 minutes, connect the condensate water, use a water bath heater (temperature control at 45℃, reflux speed about 2 drops / second), extract for 8-12 hours, then dip a drop of extract on filter paper, if there is no oil spot, then it indicates complete extraction, take out the fat package, recover the petroleum ether, naturally air dry the fat package for 1 hour, then bake at 95-105℃ for 3-4 hours, cool to room temperature in a desiccator, weigh. Dry for another 30 minutes, weigh again after cooling, repeat until the difference between the two weighings is not more than 1mg, measure the IMF. IMF=[m4-(m5-m2)] / m4×(m3 / m1)×100% (fresh meat weight m1, filter paper weight m2, dried sample weight m3 after baking, dried sample weight m4 in the fat package, fat package weight after extraction m5).

[0046] 2. SNP chip detection and genotyping

[0047] (1) DNA sample extraction and quality control

[0048] DNA extraction from pork sample tissue was performed using magnetic bead method. The concentration of the DNA sample was detected by Qubit fluorescence quantifier; the integrity of the DNA sample was detected by 1% agarose gel electrophoresis, and the qualified samples were used for library preparation.

[0049] (2) SNP chip detection

[0050] SNP genotyping was performed using GGP Porcine 50K chip (from Huazhi Biotechnology Co., Ltd.). The chip has 51000 SNP sites, integrates the genetic differences of multiple pig breeds, including Duroc, Landrace and Pietrain pigs, etc., with an average of 50,000 polymorphic sites for each evaluated breed, covering the entire genomic information of pigs. The biochip system is mainly composed of chip, scanner and analysis software. The chip data was visualized and processed using the analysis software Genome Studio, and the genotype data was obtained.

[0051] 3. Genome-wide association analysis, positioning target SNP site

[0052] (1) Genome-wide association analysis

[0053] The material data for chip detection were subjected to whole genome association analysis using rMVP (Yin L, Zhang H, Tang Z et al. 2021), a mixed linear model (MLM) was used to calculate the model, and the principal components were used as covariates to correct the model. The collected pork IMF phenotype traits were subjected to whole genome association analysis, and the Manhattan plot is shown in Figure 1 From the figure, it can be seen that the significance threshold is the Bonferroni corrected P value (p=0.05 / N, N is the number of SNPs), and the significant associated SNP sites are calculated by taking the logarithm-log10.

[0054] (2) Mining significant marker sites

[0055] In the whole genome range, the significant marker sites in the pork IMF phenotype traits were screened, and the most significant SNP chr06_65432163 (rs337141234) was obtained, P<7.0x10 -7 (as shown in Figure 1 The significant SNP site rs337141234 located by GWAS was designed with 20 molecular markers in the upstream and downstream 500 kb regions (as shown in Figure 2 ), and genotyping detection was performed in 236 Ningxiang pig populations, and the association between marker genotypes and phenotypes was tested, and T-test was used for comparison analysis between different genotypes.

[0056] In the 236 Ningxiang pig populations, it was found that LRRC47 The molecular marker of the chr6:65261993 site (rs335878944) within the gene had a significant phenotypic effect. As can be seen from Figure 3 , the chr6:65261993 site exists in three genotypes (TT, TC and CC) in the Ningxiang pig population, the average IMF content of TT type Ningxiang pig is 3.79; the average IMF content of TC type Ningxiang pig is 3.27; the average IMF content of CC type Ningxiang pig is 2.87. Among them, the IMF content of TT type Ningxiang pig has a significant difference compared with the IMF content of TC type Ningxiang pig (p=0.0078).

[0057] Example 2: Primer design and screening test of SNP molecular marker related to pork IMF content of Ningxiang pig

[0058] After screening the target SNP molecular marker (chr06_65432163) in Example 1, the SNP site and flanking sequence were extracted, the primer sequence of the marker was designed and synthesized, and the marker was subjected to screening test, as follows:

[0059] 1. Primer design

[0060] After obtaining the target SNP marker (chr06_65432163), 1000 bp sequences before and after the SNP were extracted, and the reference genome was Sus Scrofa Build11.1. The specific site nucleotide sequence is shown in SEQ ID NO: 1:

[0061]

[0062] KASP primers (Table 1) were designed by BatchPrimer3 (http: / / probes.pw.usda.gov / batchprimer3 / ) for later material validation, and were ordered from Invitrogen. KASP markers were composed of three primers, including two allele-specific primers X (Primer_X) and Y (Primer_Y) and one common primer C (Primer_C). The 5' ends of the specific primers were connected with the fluorescence groups FAM and HEX specific to KASP reaction from LGC. If only FAM fluorescence was detected in the sample, the genotype of the sample was homozygous allele X (Allele_X), and the genotype was CC; if only HEX fluorescence was detected, the genotype of the sample was homozygous allele Y (Allele_Y), and the genotype was TT; if FAM and HEX fluorescence were detected at the same time, the genotype of the sample was heterozygous (with both alleles X and Y), and the genotype was CT.

[0063] Table 1 Allele genotypes (Allele_X, Allele_Y) and primer sequences of KASP markers for detection of IMF markers in Ningxiang pigs

[0064]

[0065] 2 Sample detection

[0066] DNA extraction: Genomic DNA was extracted from Ningxiang pigs using the magnetic bead method.

[0067] Reaction sequencing of KASP markers was performed using the Array Tape system of Douglas Scientific. The Array Tape genotyping platform included NEXAR for PCR amplification system assembly, SOELLEX for PCR amplification, ARAYA for fluorescence signal scanning, and INTELLICS for data analysis.

[0068] PCR reaction system: Automatic assembly of PCR amplification system was performed using NEXAR, and the PCR reaction system is shown in Table 2.

[0069] Table 2 PCR reaction system for KASP marker genotyping

[0070]

[0071] PCR amplification: PCR amplification is carried out by using SOELEX, and the amplification conditions are as follows: 94℃ for 15 minutes; 94℃ for 20 seconds, 65℃-57℃ (the annealing temperature decreases by 0.8℃ for each cycle) for 60 seconds, 10 cycles; 94℃ for 20 seconds, 57℃ for 60 seconds, 30 cycles.

[0072] Signal scanning and genotyping: after the completion of the PCR reaction, the fluorescence signal of the reaction system is scanned by using ARAYA; then, genotyping and data analysis are carried out by using INTELLICS.

[0073] 3 Marker genotyping data

[0074] According to the above detection method, the KASP reaction verification of 237 Ningxiang pig populations with known genotypes and intramuscular fat content IMF is carried out by using the above molecular markers.

[0075] The results are shown in the following table: Figure 4 As can be seen from the figure, the KASP marker is divided into three clusters with obvious and compact genotyping, the upper left circle cluster indicates that the sample contains a homozygous C:C allele at the KASP marker site, the lower right circle cluster indicates that the sample contains a homozygous T:T allele at the KASP marker site, and the middle circle cluster indicates that the sample contains a C and T heterozygous allele at the KASP marker site, there is 1 material containing a homozygous C:C allele; there are 199 materials containing a homozygous T:T allele; there are 37 materials containing a C and T heterozygous allele, which is consistent with the genotyping results in the sequencing reaction, and the intramuscular fat content IMF phenotype result is also consistent with the genotype. The results show that the SNP molecular marker screened by the application has high accuracy and can be used for screening Ningxiang pigs with high IMF content.

[0076] 4. Specificity and utility data

[0077] According to the above detection method, 11 (6 TT, 4 TC, and 1 CC) Ningxiang pig materials with known genotypes and intramuscular fat content IMF completely different from the above 237 Ningxiang pig samples are typed by using the above SNP molecular markers.

[0078] The results are shown in the following table: Figure Five As can be seen from the figure, there is 1 material containing a homozygous C:C allele; there are 6 materials containing a homozygous T:T allele; there are 4 materials containing a C and T heterozygous allele, which is consistent with the actual genotyping and intramuscular fat content IMF phenotype results. The results show that the SNP molecular marker screened by the application has high accuracy and can be used for corresponding breeding of Ningxiang pigs with different IMF contents, which has high accuracy.

[0079] The embodiments of the present application are described in detail above with reference to the drawings, but the present application is not limited to the above-described embodiments, and various changes can be made within the knowledge of those skilled in the art without departing from the spirit of the present application. Furthermore, the embodiments of the present application and the features in the embodiments can be combined with each other without conflict.

Claims

1. Any one of the following applications of a SNP molecular marker, a primer set, a kit comprising the primer set, or a gene chip comprising the primer set: (1) application in detecting IMF content of Ningxiang pigs; (2) application in identifying and screening Ningxiang pigs with different IMF contents. The SNP molecular marker is located at 1001 bp of the nucleotide sequence shown in SEQ ID NO: 1, and the polymorphism is C / T. The primer set comprises primers with sequences shown in SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO:

4.

2. A method for detecting the IMF content of Ningxiang pig meat using molecular markers, characterized in that, The method comprises the following steps: S1, extracting genomic DNA of a pig to be tested; S2, detecting polymorphism of the SNP molecular marker of the genomic DNA extracted in step S1, and judging IMF content of the pig to be tested according to the detection result; The SNP molecular marker is located at 1001 bp of the nucleotide sequence shown in SEQ ID NO: 1, and the polymorphism is C / T.

3. A method for breeding Ningxiang pigs with high IMF content, characterized in that, The method comprises the following steps: selecting Ningxiang pigs with a genotype of TT of the SNP molecular marker by the method of claim 2 for subsequent breeding.

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