Primer pair of snp marker related to number of teats trait on pig chromosome 18 and application thereof

Abstract

The application relates to a SNP marker primer pair related to a pig chromosome 18 and a teat number character and application thereof. The SNP marker site is a molecular marker containing an rs328937344 nucleotide site of an international pig genome 11.1 version reference sequence pig chromosome 18, and has T / A polymorphism, and the SNP marker is significantly related to a pig teat number character. A primer pair for detecting the SNP marker is as follows: an upstream primer is SEQ ID NO: 2, and a downstream primer is SEQ ID NO: 3. The SNP marker provided by the application is significantly related to a pig teat number character, and high teat number pig strains can be screened by identifying the SNP marker, and the obtained high teat number pig strains have important economic benefits and social values.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of molecular biology, and relates to a SNP marker primer pair related to a pig teat number trait and application thereof. BACKGROUND

[0002] The pig teat number trait is an important economic trait in pig production, which is mainly responsible for secreting milk for nursing piglets. A sow with enough and well-developed teats can nurse more piglets. In fact, in the breeding goals of many lean-type pig breeds, the weight of the farrowing performance has been placed at a higher level, promoting the increase in the number of piglets per litter, but due to the lack of corresponding systematic selection of teat number, this leads many sows to give birth to more live piglets than their own teats can nurse. The competition for teats will lead to an increase in mortality before weaning, and the main causes of death are being crushed and starvation, as well as the decrease in disease resistance caused by the lack of breast milk. Therefore, systematic selection of teat number to ensure that sows can nurse all piglets is increasingly valued in current pig production.

[0003] In addition, the pig teat number trait also has a strong genetic correlation with growth and development traits such as body length and birth weight, so the selection and promotion of the pig teat number trait have high economic value. In fact, internationally renowned breeding companies such as PIC, Cooperl, etc. have included the teat number trait in their comprehensive index, and the selection of the teat number of the population has already made certain genetic progress. However, due to the small mean value of the total teat number trait of the commercial pig population and the low degree of variation within the population, most western commercial pigs use independent culling to select the teat number trait, that is, culling individuals with a total teat number less than 14. Although this selection method can improve the total teat number of the population, it is far lower than the number of piglets, and the mismatch between the number of piglets and the number of teats is very common in real production. The teat number belongs to a medium heritability trait, and the degree of variation within the population is low, so the conventional breeding progress is slow. Therefore, it is very important to identify the key mutation sites of the teat number and use molecular marker-assisted selection to select the teat number trait and accelerate the breeding progress.

[0004] According to the pig QTL database website (http: / / www.animalgenome.org / cgi-bin / QTLdb / SS / index), there are a total of 623 QTLs related to pig teat number, which are widely distributed on chromosomes 1-18 and X sex chromosome, indicating that the pig teat number trait is a complex quantitative trait controlled by multiple genes with small effects. However, most of these QTLs are QTLs located by microsatellite markers, with confidence intervals mostly within 10-20 cM, which cannot determine the true major genes and key mutation sites. Therefore, it is difficult to directly apply to pig breeding improvement. In addition, at present, regarding the pig teat number trait, many studies have identified significant signal sites near the ABCD4-VRTN gene interval on pig chromosome 7 by whole genome association analysis and other methods. In addition to this region, whether there are still key genes or mutation sites affecting the pig teat number trait is still unknown. In order to solve the above problems, through population quantitative genetics and other research means, it is found that the rs328937344 nucleotide site located on pig chromosome 18 is significantly associated with the teat number trait of Landrace pigs, which can be used as a reliable molecular marker for genetic improvement of pig teat number trait. SUMMARY

[0005] The purpose of the present application is to provide a SNP marker related to the pig teat number trait and develop it as a molecular marker in view of the slow progress and poor breeding effect of traditional pig teat number selection.

[0006] Another purpose of the present application is to provide a primer pair and detection method for detecting the above-mentioned SNP marker.

[0007] Another purpose of the present application is to provide the use of the above-mentioned SNP marker.

[0008] A SNP marker related to the teat number trait of Landrace pigs, the SNP marker is located at the rs328937344 nucleotide site of pig chromosome 18 in the international pig genome 11.1 version reference sequence, the site has T / A polymorphism, the SNP marker is significantly related to the total teat number trait of Landrace pigs, the total teat number of TT type Landrace pigs is significantly more than that of TA type and AA type Landrace pigs.

[0009] A method for developing a molecular marker related to the teat number trait of Landrace pigs on pig chromosome 18, taking the sequence containing the SNP site on chromosome 18 in the pig genome as the basic sequence, designing a primer pair, and performing PCR amplification with pig genome DNA as the template to convert the rs328937344 nucleotide site of pig chromosome 18 in the international pig genome 11.1 version reference sequence into a molecular marker.

[0010] As a preferred embodiment of the present application, the primer pair sequence is upstream primer: SEQ ID NO: 2, downstream primer: SEQ ID NO: 3.

[0011] The molecular marker obtained by the method.

[0012] As a preferred embodiment of the present application, the molecular marker sequence is shown as SEQ ID NO: 1, and the international pig genome 11.1 version reference sequence pig chromosome 18 rs328937344 nucleotide site is located at position 216 in SEQ ID NO: 1, and there is a T / A polymorphism.

[0013] A primer pair for detecting a SNP marker related to the number of teats of a long white pig on chromosome 18 of a pig, wherein the upstream primer is SEQ ID NO: 2, and the downstream primer is SEQ ID NO: 3; the SNP marker is a molecular marker of the international pig genome 11.1 version reference sequence pig chromosome 18 rs328937344 nucleotide site, and has a T / A polymorphism, and the SNP marker is significantly related to the total number of teats of a long white pig (P<0.05).

[0014] A method for detecting a SNP marker related to the number of teats of a pig, comprising PCR amplifying a sequence of the international pig genome 11.1 version reference sequence pig chromosome 18 rs328937344 nucleotide site, and sequencing the amplification product to determine the T / A polymorphism of the site.

[0015] As a preferred embodiment of the present application, the specific method comprises the following steps:

[0016] (1) Extract total DNA from a long white pig tissue sample;

[0017] (2) Using the extracted pig genome DNA as a template, PCR amplification is performed using the primer pair;

[0018] (3) The amplification product is sequenced, and the sequencing result is analyzed to determine the T / A polymorphism at position 216 of SEQ ID NO: 1.

[0019] The molecular marker and the primer pair of the present application are used for screening a long white pig population with a high number of teats.

[0020] A method for screening a long white pig population with a high number of teats, comprising detecting the genotype of the international pig genome 11.1 version reference sequence pig chromosome 18 rs328937344 nucleotide site, and breeding individuals with TT type of rs328937344 nucleotide site as breeding pigs.

[0021] Beneficial effects:

[0022] The SNP marker provided by the application is related to the number of teats of a pig, and a molecular marker and a primer developed based on the SNP can be used for detection of the SNP. Therefore, a high-teat-number Landrace pig strain can be screened by identifying the SNP marker, and the obtained high-teat-number pig strain has important economic benefits and social values. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 Gel map of amplification of rs328937344 site on chromosome 18 of Landrace pig

[0024] Figure 2 Example of genotyping map of rs328937344 site on chromosome 18 of Landrace pig DETAILED DESCRIPTION

[0025] The following examples are used to illustrate the application, but are not used to limit the scope of the application. Modifications or replacements of the methods, steps or conditions of the application, without departing from the spirit and essence of the application, all belong to the scope of the application.

[0026] Example 1:

[0027] 1. Source of test animals

[0028] Jiangxi Jiada Group Co., Ltd.

[0029] 2. Extraction of pig genomic DNA

[0030] 841 American Landrace pigs were collected for 1 tissue sample per pig for individual DNA extraction;

[0031] According to the instructions of the tissue DNA extraction kit of Tiangeng Biological Technology Co., Ltd., the extraction was performed in the following order:

[0032] ① First, add 68 mL and 200 mL of anhydrous ethanol to the buffer GD and rinse PW, respectively, and mix thoroughly.

[0033] ② Collect about 100 mg of tissue sample in a 2 mL EP tube, cut it completely, add 200 μL of buffer GA, and shake until completely suspended.

[0034] ③ Add 20 μL of proteinase K solution, mix well, and place in a 56°C metal bath for digestion overnight until the tissue sample is dissolved. Briefly centrifuge to remove water droplets on the inner wall of the tube cap.

[0035] ④ Add 200 μL of buffer GB, mix well by inverting, and place in a 70°C metal bath for 10 min. The solution should be clear, and briefly centrifuge to remove water droplets on the inner wall of the tube cap.

[0036] (5) Add 200 μL of absolute ethanol, shake well to mix for 15 seconds. At this point, a flocculent precipitate may appear. Briefly centrifuge to remove water droplets from the inside of the tube cap.

[0037] (6) Add both the solution and the flocculent precipitate from the previous step to an adsorption column CB3, which is placed in a collection tube. Then centrifuge at 12,000 rpm for 30 seconds, discard the waste, and place the adsorption column CB3 back into the collection tube.

[0038] (7) Add 500 μL of buffer GD to the adsorption column CB3, centrifuge at 12,000 rpm for 30 seconds, discard the waste, and place the adsorption column CB3 into the collection tube.

[0039] (8) Add 600 μL of rinse solution PW to the adsorption column CB3, centrifuge at 12,000 rpm for 30 seconds, discard the waste, and place the adsorption column CB3 into the collection tube.

[0040] (9) Repeat step (8).

[0041] (10) Place the adsorption column CB3 back into the collection tube, centrifuge at 12,000 rpm for 2 minutes, discard the waste, and place the adsorption column CB3 at room temperature for several minutes to completely dry the residual rinse solution in the adsorption material.

[0042] Transfer the adsorption column CB3 into a clean centrifuge tube, and add 100 μL of elution buffer TE dropwise to the middle of the adsorption membrane, place at room temperature for 2-5 minutes, centrifuge at 12,000 rpm for 2 minutes, collect the solution into a centrifuge tube, and add the solution obtained by centrifugation back into the adsorption column CB3, place at room temperature for 2 minutes, centrifuge at 12,000 rpm for 2 minutes, and collect the solution into a centrifuge tube.

[0043] After detecting the quality and concentration by a Nanodrop-100 spectrophotometer, dilute the same to 50 ng / μL, and store at -20°C for standby use.

[0044] 3. PCR amplification and sequencing of the target fragment

[0045] Using the extracted DNA as a template, perform PCR amplification according to the designed primers: take 1 μL of the DNA template, 1 μL of each of the primers shown in SEQ ID NO: 2 and SEQ ID NO: 3, 12.5 μL of PCR Mix reagent, and 9.5 μL of double-distilled water; set the PCR amplification system as follows:

[0046]

[0047] Detect the PCR product by electrophoresis in a 1.2% agarose gel. The size of the amplified target fragment is about 481 bp. The electrophoresis chart is shown in Figure 1The remaining amplified products were sequenced, and the sequencing results were compared and analyzed with the relevant gene fragment sequences of pigs in GenBank to determine the genotypes of the rs328937344 site.

[0048] 4 Statistical analysis

[0049] The SAS software general linear model was used to analyze the effect of genotypes on phenotypes. The analysis model was

[0050] Y ijnk = u i + G j + S n + D k + e jnk

[0051] wherein: Y ijnk is the total teat number trait of pigs; G j represents the genotype fixed effect of the jth SNP; S n represents the fixed effect of gender; D k represents the fixed effect of the birth year season; and e jnk is the residual.

[0052] 5 Results

[0053] Table 1 shows the effect of the rs328937344 variation site T / A on the total teat number trait in the Landrace pig population. As shown in Table 1, there is a significant difference (P<0.05) in the total teat number trait among the three genotypes of the rs328937344 site. The average total teat number of the superior genotype TT type individual is 0.4 more than that of the inferior genotype AA type individual (P<0.05). The average total teat number of the superior genotype TA type individual is 0.13 more than that of the inferior genotype AA type individual (P<0.05). Therefore, in the Landrace pig population in the United States, the TT type individual of the rs328937344 site can gradually improve the total teat number trait of the Landrace pig population through subsequent selection and breeding.

[0054] Table 1 Association analysis of the rs328937344 site on chromosome 18 of pigs and the total teat trait of Landrace pigs

[0055]

[0056] Note: The same row numbers with different letters represent significant differences (P<0.05).

Claims

1. Use of a detection reagent for a SNP marker in screening a population of long white pigs with a high number of teats, characterized in that, The SNP marker site is the nucleotide site rs328937344 of the international pig genome 11.1 version reference sequence pig chromosome 18, which has T / A polymorphism, the SNP marker is significantly related to the total teat number of Landrace, and the total teat number of Landrace with TT type is significantly more than that of Landrace with TA type and AA type.

2. Use according to claim 1, characterized in that, The detection reagent of the SNP marker is a primer pair for detecting the SNP marker, and the sequence of the primer pair is shown in SEQ ID NO: 2 and SEQ ID NO:

3.

3. A method of selecting a population of high teat number Large White pigs, characterized in that The method comprises detecting the genotype of the nucleotide site rs328937344 of the international pig genome 11.1 version reference sequence pig chromosome 18 of Landrace, and breeding individuals with TT type of the nucleotide site rs328937344 as breeding pigs.

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