Primer pair for detecting multiple birth trait of mongolian sheep, and detection method and application thereof

Abstract

The application provides a primer pair for detecting a multiple birth trait molecular marker of Mongolian sheep, and a detection method and application thereof, and belongs to the technical field of molecular marker assisted breeding. The primer pair for detecting the multiple birth trait molecular marker of the Mongolian sheep comprises primer pair 1 or primer pair 2. The primer pair 1 can detect a polymorphic site at the 7828531th position of exon 6 of an MSI2 gene, the nucleotide of the site is A or G, and a Mongolian sheep with a genotype AA has a multiple birth trait. The primer pair 2 can detect a polymorphic site at the 8144512th position of exon 11 of the MSI2 gene, the nucleotide of the site is C or G, and a Mongolian sheep with a genotype CC has a multiple birth trait. In the process of breeding and cultivating the Mongolian sheep, the AA type or the CC type individual with a twin birth trait can be selected through the SNP molecular marker assisted selection, so that the breeding process of the reproductive performance of the Mongolian sheep is accelerated.

Description

Technical Field

[0001] This invention relates to the field of molecular marker-assisted breeding technology, and in particular to primer pairs for detecting molecular markers of polyfertility in Mongolian sheep, as well as their detection methods and applications. Background Technology

[0002] For breeding livestock, reproductive capacity is productivity, and reproductive performance directly affects the economic impact of the sheep industry. Reproductive performance mainly includes indicators such as total number of lambs born, number of live lambs born, birth weight, litter weight at birth, and weaning litter weight. Low lambing rate is a major bottleneck restricting the development of high-quality and efficient sheep farming. Conventional breeding methods are unlikely to quickly improve the reproductive performance of sheep populations. Researching breeding methods to improve lambing rate from a genetic perspective is a technical challenge that sheep farming scientists both domestically and internationally are striving to solve. Marker-assisted selection (MAS) is a breeding technology that can directly select genotypes for lambing traits at the molecular level, overcoming the drawbacks of conventional breeding, such as long time consumption and poor results, and rapidly improving breeding efficiency. MAS methods can be used to improve traits that are difficult or costly to measure in vivo, such as disease resistance and meat quality, as well as limited traits such as reproduction (e.g., number of lambs born), which are expressed relatively late in the life cycle. MAS allows for early selection of these traits, thereby shortening generation intervals and accelerating the breeding process. Therefore, if MAS technology can be used to improve the lambing trait of breeding sheep, it will have broad application prospects. It is evident that finding molecular helper marker genes closely linked to lambing traits and screening functional genes that regulate lambing traits are beneficial means to organically combine modern molecular breeding techniques with conventional breeding techniques and improve reproductive efficiency. However, finding molecular genetic markers associated with these quantitative trait loci is a prerequisite for realizing MAS.

[0003] Mongolian sheep (Xingan Black Sheep) are offspring of the Zasaktu sheep from Inner Mongolia and the Suffolk Black-headed sheep introduced from abroad. They inherit the genetic advantages of Suffolk sheep in terms of body shape and growth rate, without sacrificing their ability to thrive in confinement. They also possess the traits of high fertility or multiple lambing, effectively alleviating common problems such as poor adaptability of imported meat sheep and insufficient utilization of superior traits in local breeds. Therefore, market demand is very strong. Currently, no research has reported that Samurai protein 2 (MSI2) is a molecular marker associated with the fertility trait of Mongolian sheep. Summary of the Invention

[0004] In view of this, the present invention provides primer pairs for detecting molecular markers of polyfertility in Mongolian sheep, as well as their detection methods and applications. The method of the present invention can realize the detection of polyfertility in Mongolian sheep and accelerate the breeding process of Mongolian sheep reproductive performance.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0006] This invention provides primer pairs for detecting molecular markers of polyfertility traits in Mongolian sheep, wherein the primer pairs include primer pair 1 or primer pair 2;

[0007] The nucleotide sequence of the upstream primer of primer pair 1 is shown in SEQ ID NO.1, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.2;

[0008] The nucleotide sequence of the upstream primer of primer pair 2 is shown in SEQ ID NO.3, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.4;

[0009] The SNP site detected by primer pair 1 is position 7828531 of exon 6 of the MSI2 gene, and the nucleotide at this site is A or G; the SNP site detected by primer pair 2 is position 8144512 of exon 11 of the MSI2 gene, and the nucleotide at this site is C or G.

[0010] The present invention also provides the application of the primer pair in the detection of twins in Mongolian sheep.

[0011] The present invention also provides a kit for detecting molecular markers of polyfertility in Mongolian sheep, the kit comprising the primer pair described above.

[0012] This invention also provides a method for detecting the polyfertility trait in Mongolian sheep, comprising the following steps:

[0013] (1) Using the genomic DNA of the Mongolian sheep to be tested as a template, PCR amplification was performed using the primer pair described above to obtain the amplification product;

[0014] (2) Sequencing the amplified products to obtain genotyping information of SNP molecular marker sites, and determining the twinning characteristics of Mongolian sheep based on the genotyping results;

[0015] If the test result for position 7828531 of exon 6 of the MSI2 gene is AA genotype, then the Mongolian sheep to be tested has the twinning trait.

[0016] If the test result for position 8144512 of exon 11 of the MSI2 gene is CC genotype, then the Mongolian sheep being tested has the twinning trait.

[0017] Preferably, the PCR amplification reaction system is as follows: 1 μL template DNA, 1 μL each of upstream and downstream primers, 1 μL dNTP Mixture, 2.5 μL Taq Buffer, 0.2 μL Taq enzyme, and RNase-free ddH2O to a final volume of 25 μL.

[0018] Preferably, the concentrations of the upstream and downstream primers are 10 μM.

[0019] Preferably, the PCR amplification program is as follows: 95℃ for 5 min; 94℃ for 30 s; 63℃, decreasing by 0.5℃ for 30 s per cycle. 72 ℃ for 30s, 10 cycles; 95℃ for 30s, 58℃ for 30s, 72℃ for 30s, 30 cycles; 72℃ for 10min.

[0020] By adopting the above technical solution, the present invention has the following beneficial effects: The primer pairs for detecting molecular markers of polygamy in Mongolian sheep include primer pair 1 or primer pair 2. Primer pair 1 can detect the polymorphic site at position 7828531 of exon 6 of the MSI2 gene, where the nucleotide is A or G. Mongolian sheep with the genotype AA have the polygamy trait. Primer pair 2 can detect the polymorphic site at position 8144512 of exon 11 of the MSI2 gene, where the nucleotide is C or G. Mongolian sheep with the genotype CC have the polygamy trait. In the process of breeding and raising Mongolian sheep, this SNP molecular marker can be used to assist in the selection of AA or CC individuals with the twinning trait, thereby accelerating the breeding process of Mongolian sheep reproductive performance. Attached Figure Description

[0021] Figure 1 These are single and double luteal ovaries of the Mongolian sheep hybrid breed (Xingan Black Sheep).

[0022] Figure 2 The first image shows an electrophoresis image of the PCR amplification products; the left image shows the electrophoresis image of the PCR amplification products of primer pair 1, and the right image shows the electrophoresis image of the PCR amplification products of primer pair 2.

[0023] Figure 3 Sequencing peak diagram of SNPs in the MSI2 gene of Mongolian sheep. Detailed Implementation

[0024] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0025] The DNA extraction kit used in this embodiment of the invention was purchased from Inner Mongolia Jinmaishi Biotechnology Co., Ltd., and the PCR amplification kit was purchased from Sangon Biotech (Shanghai) Co., Ltd.

[0026] Example 1

[0027] During the estrus season in Inner Mongolia in September, healthy, non-pregnant Mongolian crossbred sheep (Xingan Black sheep) of 1 year old were selected from the Haoren Li Yongqiang Meat Sheep Farm in Kezuoqian Banner, Xingan League, northeastern Inner Mongolia for estrus synchronization. Figure 1(AC). All ewes were growing and developing well, and were of similar age and weight.

[0028] All ewes in the experiment had progesterone silicone plugs implanted and were injected with pregnant mare serum emerald and ovulation-inducing hormone 3 (LRH-A3). Rams were selected after estrus detection, and the state and number of corpus luteum formation were observed surgically. Ovaries with a single corpus luteum (single-fetal trait control group) were removed. Figure 1 Thirty ovaries from the middle D) were selected, with unilateral double corpus luteum and bilateral single corpus luteum forming the experimental groups (twin trait experimental group). Figure 1 Thirty samples each of E and F were collected from the corpus luteum of the ovary for single nucleotide polymorphism (SNP) sequencing.

[0029] DNA was extracted from samples of mono- and di-luteal bodies of Mongolian sheep using a DNA extraction kit. The amplified PCR products were then detected and purified through PCR amplification, agarose gel electrophoresis, and gel recovery. The PCR products were sequenced using an ABI 3730XL sequencer.

[0030] 1. Primer design and PCR amplification

[0031] Based on the MSI2 gene reference sequence (GeneBamk No. 101102321) in NCBI, primers were designed and synthesized for the exons of the candidate gene MSI2. Primer information is shown in Table 1, and the PCR reaction procedure and reaction system are shown in Tables 2 and 3.

[0032] Table 1. MSI2 gene primer information

[0033]

[0034] Table 2 PCR amplification reaction system

[0035]

[0036] Table 3 PCR amplification reaction procedure

[0037]

[0038]

[0039] 2. Identification of amplification products by gel electrophoresis

[0040] After PCR amplification, four samples were randomly selected for gel electrophoresis. 5 μL of the PCR amplification product was taken, and using 5 μL of DNA Marker DL2000 as a reference, the voltage was set to 220V. The gel was run in a 1.5% agarose gel for approximately 30 minutes. The size of the PCR products was determined using a gel imaging system. The results are shown below. Figure 2 As shown.

[0041] 3. Sequencing of PCR products

[0042] The amplified PCR products were recovered by gel extraction, detected, and purified. The PCR products were then sequenced using an ABI 3730XL sequencer.

[0043] Analysis revealed two SNP sites in the MSI2 gene of Mongolian sheep: g.7828531A>G in exon 6 and g.8144512C>G in exon 11. These two SNP sites produce two genotypes, such as... Figure 3 As shown in Table 4.

[0044] Table 4. Analysis of the genetic characteristics of the MSI2 gene in Mongolian sheep.

[0045]

[0046]

[0047] The results showed that when primer pair 1 was used, the genotype of Mongolian sheep with unilateral double corpus luteum and bilateral single corpus luteum was AA, while the genotype of Mongolian sheep with unilateral single corpus luteum ovary was AG. When primer pair 2 was used, the genotype of Mongolian sheep with unilateral double corpus luteum and bilateral single corpus luteum was CC, while the genotype of Mongolian sheep with unilateral single corpus luteum ovary was CG. Therefore, the molecular marker primers of this invention can be used to detect twinning in Mongolian sheep. During the breeding and rearing of Mongolian sheep, this SNP molecular marker can be used to assist in the selection of AA or CC individuals with twinning traits, thereby accelerating the breeding process of Mongolian sheep reproductive performance.

[0048] As can be seen from the above embodiments, the present invention provides primer pairs for detecting molecular markers of polyfertility in Mongolian sheep, as well as their detection methods and applications. The primer pairs of the present invention for detecting molecular markers of polyfertility in Mongolian sheep can realize the detection of polyfertility in Mongolian sheep.

[0049] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The application of primer pairs in the detection of twins in Mongolian sheep, characterized in that, The primer pair includes primer pair 1 or primer pair 2; The nucleotide sequence of the upstream primer of primer pair 1 is shown in SEQ ID NO.1, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.2; The nucleotide sequence of the upstream primer of primer pair 2 is shown in SEQ ID NO.3, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.4; The SNP site detected by primer pair 1 is position 7828531 on chromosome 11, located at... MSI2 The third position of the CCAAGCC sequence on exon 6 of the gene, where the nucleotide is either A or G; the SNP site detected by primer pair 2 is position 8144512 on chromosome 11, located at... MSI2 The third position of the sequence GGCTACCC on exon 11 of the gene, where the nucleotide is either C or G.

2. A method for detecting the polyfertility trait in Mongolian sheep, characterized in that, Includes the following steps: (1) Using the genomic DNA of the Mongolian sheep to be tested as a template, PCR amplification was performed using the primer pair described in claim 1 to obtain the amplification product; (2) Sequencing the amplified products to obtain genotyping information of SNP molecular marker sites, and determining the twinning characteristics of Mongolian sheep based on the genotyping results; For position 7828531 of chromosome 11 MSI2 If the third position of the sequence CCAAGCC on exon 6 of the gene indicates the AA genotype, then the Mongolian sheep being tested has the twinning trait. For position 8144512 of chromosome 11 MSI2 If the third position of the sequence GGCTACCC on exon 11 of the gene shows a CC genotype, then the Mongolian sheep being tested has the trait of twinning.

3. The method according to claim 2, characterized in that, The PCR amplification reaction system consisted of: 1 μL template DNA, 1 μL each of upstream and downstream primers, 1 μL dNTP Mixture, 2.5 μL Taq Buffer, 0.2 μL Taq enzyme, and RNase-free ddH2O to a final volume of 25 μL.

4. The method according to claim 2, characterized in that, The concentrations of the upstream and downstream primers were 10 μM.

5. The method according to claim 4, characterized in that, The PCR amplification program was as follows: 95℃ for 5 min; 94℃ for 30 s; 63℃ for 30 s, decreasing by 0.5℃ per cycle; 72℃ for 30 s; 10 cycles; 95℃ for 30 s; 58℃ for 30 s; 72℃ for 30 s; 30 cycles; 72℃ for 10 min.

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