CN115074449B - A molecular marker related to sheep tail fat deposition and its application - Google Patents

Abstract

The present invention provides a molecular marker associated with sheep tail fat deposition and its application. The present invention performs PCR amplification and sequence analysis on the sheep RAP1GAP gene, and finds that there is an A/G polymorphic site at the 123rd position of the amplified fragment. The polymorphic sites of 1029 Hu sheep are further detected using KASPar primers and a least squares model is established. The genotype and tail fat deposition are analyzed for association, and it is finally determined that the RAP1GAP gene fragment amplified by the present invention can be used as a molecular marker associated with sheep tail fat deposition. By detecting molecular markers, the present invention can select AA homozygous sheep to enter the core group for breeding, which can be used to reduce tail fat deposition in sheep, and help increase economic benefits.

Description

A molecular marker related to sheep tail fat deposition and its application

Technical Field

The invention belongs to the technical field of molecular markers, and particularly relates to a RAP1GAP gene fragment as a molecular marker affecting sheep tail fat deposition and an application thereof.

Background Art

As a common livestock, sheep are important means of production and living in agricultural areas. Their meat and wool products play a vital role in improving and enhancing local production and life. According to historical records, the wild ancestors of sheep were thin-tailed sheep. Later, fat-tailed sheep or fat-hipped sheep were bred through artificial selection (Moradi, Nejati-Javaremi, Moradi-Shahrbabak, Dodds, & McEwan, 2012). Sheep can be divided into five types according to the size of their tails: short-tailed sheep, long-tailed sheep, short fat-tailed sheep, long fat-tailed sheep and fat-hipped sheep. The fat in the tail of sheep can provide enough energy to maintain normal needs when forage is scarce in winter (Ermias, Yami, & Rege, 2002; Kashan, Azar, Afzalzadeh, & Salehi, 2005). However, people are willing to choose to buy low-fat mutton products for a healthier diet. Because a high-fat diet can lead to an increased incidence of cardiovascular disease, diabetes and intestinal cancer. With the commercial breeding of sheep, excessive deposition of tail fat limits the commercial value of mutton. It also reduces feed conversion rate and increases feeding costs. In actual production, it is found that the mating conception rate of large-tailed sheep is lower than that of small-tailed sheep because the fat tail hinders natural mating.

RAP1GAP (RAP1 GTPase activating protein) is a protein-coding gene and the first identified member of the GTPase activating protein (GAP) family (Frisch & Zwartkruis, 2010; Rubinfeld, Munemitsu, Clark, Conroy, & Polakis, 1991), which is relatively well studied in human medicine. RAP1GAP inhibits extracellular signal-regulated kinase activity, cell proliferation, survival, and migration of melanoma cells (Zheng et al., 2009). In vitro, overexpression of RAP1GAP impairs tumor cell proliferation and enhances apoptosis (D'Silva et al., 2005; Mitra et al., 2008; Tsygankova et al., 2007; Zhang & L., 2006; Zheng et al., 2009). Previous studies have found that overexpression of RAP1GAP reduces the proliferation of rat thyroid cells (Tsygankova, Feshchenko, Klein, & Meinkoth, 2004). It is not clear whether the RAP1GAP gene is related to sheep tail fat.

The present invention sequences and analyzes the RAP1GAP gene to explore the association between its different genotypes and the sheep tail fat deposition trait, aiming to provide genetic material for genetic improvement of reducing sheep tail fat deposition and accelerate the breeding process of a new breed of fast-growing small-tailed high-quality mutton sheep with independent intellectual property rights.

Summary of the invention

In order to solve the above technical problems, the present invention provides a molecular marker related to sheep tail fat deposition and its application.

The molecular marker of the present invention is amplified from the sheep RAP1GAP gene, and its specific nucleotide sequence is shown in SEQ ID NO. 1. By amplifying the DNA sequence of the sheep RAP1GAP gene and sequencing, the polymorphic site of the RAP1GAP gene is found, and the correlation between different genotypes and sheep tail fat deposition is analyzed, thereby establishing a detection method for the molecular marker containing the polymorphic site, and the molecular marker can be applied to the breeding of new varieties that reduce sheep tail fat deposition.

To achieve the above object, the present invention adopts the following technical solutions:

On the one hand, the present invention provides a molecular marker associated with sheep tail fat deposition, whose nucleotide sequence is shown in SEQID NO.1, wherein R at the 123bp position represents A or G. Since the above sequence has an A/G mutation at the 123rd base, it leads to the A/G polymorphism of the sheep RAP1GAP gene at this site.

In a second aspect, the present invention provides a primer pair for detecting the above-mentioned molecular marker, the primer pair comprising primer R-F and primer R-R, wherein the sequence of primer R-F is shown in SEQ ID NO.2, and the sequence of primer R-R is shown in SEQ ID NO.3.

In addition, the present invention also provides a KASPar primer pair for detecting the above-mentioned molecular markers. Preferably, the KASPar primer pair includes a forward primer for detecting AlleleG, a forward primer for detecting AlleleA and a universal reverse primer, wherein the sequence of the forward primer for detecting AlleleG is shown in SEQ ID NO.4, the sequence of the forward primer for detecting AlleleA is shown in SEQ ID NO.5, and the sequence of the universal reverse primer is shown in SEQ ID NO.6.

In a third aspect, the present invention provides a detection kit for detecting the above-mentioned molecular markers, which comprises a primer pair for detecting the above-mentioned molecular markers and/or a KASPar primer pair.

In a fourth aspect, the present invention also provides a method for detecting a molecular marker associated with sheep tail fat deposition, wherein the nucleotide sequence of the molecular marker is shown in SEQ ID NO.1, wherein R at position 123 bp represents A or G, and the method comprises using the above-mentioned primer pair or kit to detect the genomic DNA of the sheep, and the specific detection method comprises the following steps:

1) amplifying sheep genomic DNA using the above primer pair, KASPar primer pair or a kit comprising the above primer pair;

2) Typing and identifying the polymorphic sites of the amplified product obtained in step 1).

Wherein, in step 2), the above-mentioned typing and identification methods include but are not limited to direct sequencing method, fluorescent probe method, gene chip method, and high-resolution melting curve method.

When the molecular marker sequence and polymorphic site of the present invention are known, designing corresponding probes for the polymorphic site and detecting the molecular markers and polymorphic sites using the above-mentioned SNP typing method are both relatively common and mature technologies in the field. The probes designed for the polymorphic site may also be included in the kit of the third aspect of the present invention.

More specifically, the method for detecting molecular markers related to sheep tail fat deposition using the above primer pair in the present invention comprises the following steps:

a) extracting genomic DNA from sheep blood as a sample, and performing PCR amplification on the genomic DNA using primers shown in SEQ ID NO.2 and SEQ ID NO.3;

b) sequencing and sequence analysis of the PCR amplification products to determine the genotype based on the base type of the polymorphic site.

In addition, the present invention also relates to a method for detecting molecular markers related to sheep tail fat deposition using KASPar primer pairs, comprising the following steps:

a) extracting genomic DNA from sheep blood as a sample, and performing high-throughput water bath PCR amplification using the primer pairs shown in SEQ ID NO.4-6;

b) After amplification, use the BMG PHERAstar instrument to detect the fluorescence signal and check the typing results.

The present invention detects the above molecular markers by designing KASPar primers required for competitive allele-specific PCR (KASP). This detection method does not need to synthesize specific fluorescent probes for each SNP site, but is based on its own unique ARM PCR principle, so that all site detections are ultimately amplified using universal fluorescent primers, which greatly reduces the cost of reagents and has high accuracy, providing a simple, accurate and low-cost operating method for the detection of molecular markers of the present invention.

In a fifth aspect, the present invention provides the application of the detection method of the molecular marker, PCR primer pair, KASPar primer or kit as described above in the detection of sheep tail fat deposition traits. By detecting the molecular marker of the present invention in the genomic DNA of the sheep to be tested and analyzing the type of polymorphic site, the level of sheep tail fat deposition can be determined, thereby screening out sheep with small tails.

In a sixth aspect, the present invention provides an application of the detection method of the molecular marker PCR primer pair, KASPar primer or kit as described above in sheep breeding. By using the above primer pair or kit to amplify and detect the genomic DNA of the sheep, the genotype of the molecular marker of the sample to be tested is determined, so that a small-tailed sheep breed with less tail fat deposition can be bred therefrom.

Finding the mutation site of the gene and discovering the relationship between the gene and the trait through association analysis with the trait is an important means to study the function of the gene and is also the basis for marker-assisted selection. The present invention performs PCR amplification and sequencing on the RAP1GAP gene of the representative sheep breed Hu sheep, and finds that there is an A/G polymorphic site at the 123rd position of the amplified fragment. By detecting the polymorphism of 1029 Hu sheep and establishing a least squares model, a molecular marker related to the sheep tail fat deposition trait is determined. The molecular marker can be used for the breeding of small tail fat sheep, providing an effective genetic engineering means for the genetic improvement of sheep tail fat, and has great practical application value.

The beneficial effects of the present invention are:

The present invention provides a molecular marker related to sheep tail fat deposition and an A/G polymorphic site thereof, and can effectively identify whether a sheep has a small tail fat type by detecting the genotype of the polymorphism, thereby providing an effective detection means for the breeding of small tail fat sheep.

The present invention can be used to select AA homozygous sheep as breeding sheep through the detection of molecular markers and polymorphic sites, so as to reduce the deposition of fat in the tail and help improve the economic benefits of sheep breeding.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a gel electrophoresis diagram of the sheep RAP1GAP gene fragment in Example 1.

Figure 2 is the sequencing result of the g.13561G>A mutation site of the sheep RAP1GAP gene in Example 1.

FIG. 3 is the KASPar SNP typing result of the amplified fragment of the RAP1GAP gene of sheep in Example 1.

DETAILED DESCRIPTION

The present invention analyzes the relationship between the single nucleotide polymorphism of the RAP1GAP gene of Hu sheep and the tail fat deposition trait. In addition, the expression level of the RAP1GAP gene in the tail fat of small-tailed sheep and large-tailed sheep is also studied. The present invention can provide valuable molecular markers for sheep breeding.

The following examples are used to further illustrate the present invention, but should not be construed as limiting the present invention. Without departing from the spirit and substance of the present invention, modifications or substitutions made to the present invention all belong to the scope of the present invention.

Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art. Unless otherwise specified, the reagents used in the present invention are of analytical grade or above.

Example 1 Amplification of RAP1GAP gene

Using sheep RAP1GAP gene DNA (GenBank accession number: NC_040253.1) as a template, a pair of primers: R-F and R-R were designed using Oligo7.0 software. The primer sequences are as follows:

R-F (SEQ ID NO.2): 5’-CACTCCTCCCACCATCCGTTC-3’

R-R (SEQ ID NO.3): 5’-CCAGCCTCTCTCCTAGAAACC-3’

(2) Amplification and sequencing of the RAP1GAP gene

The total volume of the PCR reaction was 35 μL, including: 2×PCR Master Mix 17.5 μL, upstream primer RF 1 μL at a concentration of 10 μmol/L, downstream primer RR 1 μL at a concentration of 10 μmol/L, DNA template 1.5 μL, ddH ₂ O 14 μL. The DNA template was genomic DNA extracted from sheep blood.

The PCR amplification program was: 94°C pre-denaturation for 3 min, 94°C denaturation for 30 s, 58.5°C annealing for 30 s, 72°C extension for 30 s, 35 cycles, and finally 72°C extension for 10 min.

The PCR amplification reaction product was detected by 1.5% agarose gel electrophoresis, and the result is shown in Figure 1, wherein lane M: molecular weight 2000 marker, lanes 1-10: RAP1GAP gene amplification results. The amplified PCR fragment was sequenced, and the nucleotide sequence of the amplified fragment is shown in SEQ ID NO.1, a total of 937 bp, wherein there is a polymorphic site in the fragment, specifically R at the 123 bp site is A or G, that is, the amplified RAP1GAP gene fragment (SEQ ID NO.1) has A/G polymorphism at the 123 bp site (see Figure 2).

Among them, SEQ ID NO.1:

CACTCCTCCCACCATCCGTTCCCAGGGCCTAGGGACCCTCCAGGAGACTCCCTTTCCAGGAGCTCAGGGTTAGCAGCATCTTAGGTTTGGGTGGGTGCAGCTTGGCTAGAAGGGCCATGAGCRAAGGATGCTTTGAACCTATGAAATTCTCTCAAGGCAAATGTGTTTTTCCGTGATTATGTCTCTAGTCCAGCAAAGTGAGAGTGATGAGTCAACGGAGACCGAGAGAGGAAA ACTGGTTAACATGGGAGGCTGCCTGGCCAGGCAGGAAGCCCAGCACTGCCCTGCCCCCTGCTCAGGATACCGCCCCCGCCACCTGCCCCCAGCAGATAACCTGAATGGGAATGACAGGGGGCAGGGAAAGCATGGAAGGTCACTGGGGATCCACTGGGTTTCCCCAGGGGCTTAGATGGTAAGGAATCTATCTGCAATGCAGGAGACTGCAGTTCGATCCCTGGGATAGGAAGAT GCCCTGGGGGAGGGCATGGCAGCCCACTCCAGTATTCTTGCCTGGAGAATCCCAGAGACAGAGGAGCCCAGCGGGCTATGGCCCACAGGGTCACATAGGATCGGATGTGCAACTAACGCTAAGCCTAGCTGGCGAGAATCGGCCCCACGAGCCCCATCTGGAACCCAGGCTCTTCTCTTCCCAGGAGGCTGCCCCTCCCCGGGGCCCTCAGGTGTCCTGTGGAGTGTTGGGAA GGCAGCACTGGAGGCAGGGGGTGGGACTTTCAGGGTTCAGCCCGGGGTGAACTTGGAGAGGGAGCTCTGCGTCCCGCCACCCCTGTCTGCTTTCCTCCTCCTTCACACTGTCTGGCACCTGTTTGAACAACTCGGGTGCAAAATCAGGGTGGCCCAGAAGAGGAGGCTTCTTCTGAGAAGGAGGGCCTGGTCCCTGGAGGGCAGCCTGGCTCTGGTTTCTAGGAGAGAGGCTGG. DNA sequence homology search and identification:

The DNA sequence obtained after sequencing was compared with the known physiological function genes published in the GenBank database by BLAST (Basic Local Alignment Search Tool) software on the website of the National Center for Biotechnology Information (NCBI, http://www.ncbi.nlm.nih.gov) to identify and obtain the functional information of the DNA sequence. The search results showed that the sequence was 99% homologous to the partial sequence of the sheep RAP1GAP gene DNA (GenBank accession number: NC_040253.1).

Example 2 Establishment of genotyping detection method

1. Primer sequence design

A KASPar primer pair was designed for the G/A polymorphic site of the amplified fragment in Example 1, so as to be used for specific detection of the polymorphic site. The nucleotide sequence of the optimized KASPar primer pair is:

The forward primer A1 for detecting AlleleG is shown in SEQ ID NO.4, wherein:

SEQ ID NO.4: GAAGGTGACCAAGTTCATGCTGAATTTCATAGGTTCAAAGCATCCTTC;

The forward primer A2 used to detect AlleleA is shown in SEQ ID NO.5.

SEQ ID NO.5: GAAGGTCGGAGTCAACGGATTAGAATTTCATAGGTTCAAAGCATCCTTT;

Universal reverse primer C is shown in SEQ ID NO.6,

SEQ ID NO. 6: TGGGTGCAGCTTGGCTAGAAGG.

The above primers were synthesized by Beijing Shenggong Bioengineering Co., Ltd. Each set of primers in the KASPar primer pair was diluted to 10 μmol/L and mixed at a volume ratio of 12:12:30 (primer A1: primer A2: primer C) for later use.

2. Extract genomic DNA and perform quality control

The extraction of genomic DNA from sheep blood can be performed using a DNA extraction kit. The quality of the extracted genomic DNA is tested by 1% agarose electrophoresis and Nanodrop2100, respectively. The qualified DNA requires: (1) agarose electrophoresis shows a single DNA band without obvious diffusion. (2) Nanodrop2100 detection A260/280 is between 1.8-2.0; A260/230 is between 1.8-2.0; there is no obvious light absorption at 270nm. Based on the KASPar detection technology of LGC, UK, and the genome size, the DNA dosage is calculated to be 10-20ng/sample. The extracted genomic DNA is diluted to a concentration of 10-20ng/μL as a DNA template.

3. Perform genotyping

First, use the K-pette dispensing workstation to add 1.5 μL of the diluted test DNA template (10-20 ng/μL) and the blank control (No template control, NTC, using sterile water) into a 384-well reaction plate, and dry it at 60°C for 30 min (drying oven, LGC Company) to turn the DNA into dry powder for use.

Each primer in the above KASPar primer pair was diluted to 10 μmol/L, and mixed at a volume ratio of 12:12:30 for primer A1:A2:C to prepare a primer mixture for later use.

Then, under the Kraken operating system, 1× Master mix (1536 microplate, Part No. KBS-1016-011) and primer mixture were added to each reaction well using the Meridian sample loading workstation. After the Mix was dispensed, the microplate was immediately placed on the Kube heat sealer and the Fusion laser sealer for sealing, and the high-throughput water bath PCR amplification was performed using the Hydrocycler. The PCR reaction was performed in the high-throughput water bath system Hydrocycler, and the specific procedure was as follows:

Pre-denaturation at 94°C for 15 minutes;

94℃, 20 seconds (denaturation) - 61℃-55℃, 1 minute (renaturation & extension), 10 cycles of touch down sequence, decreasing 0.6℃ per cycle;

Amplification was continued for 26 cycles from 94°C, 20 seconds (denaturation) to 55°C, 60 seconds.

After amplification, the BMG PHERAstar instrument was used to detect the fluorescence signal and check the typing situation. The specific results are shown in Figure 3. Each dot in the figure represents a sample of material to be tested, where the red dot near the left side indicates that the site is a homozygous genotype "AA"; the green dot near the middle indicates that the site is a heterozygous genotype "AG" or "GA"; the blue dot near the right side indicates that the site is a homozygous genotype "GG"; the black dot indicates NTC (not shown in Figure 3), that is, water is used as a control.

4. Application of the molecular markers of the present invention in association analysis of sheep tail fat deposition

A total of 1029 Hu sheep were tested for polymorphism, their genotypes were determined, and the least squares model described below was established to conduct association analysis between genotype and growth traits.

Y _ijk =μ+Genotype _i +P _j +S _k +ε _ijk

Among them, _Yik is the observed value of tail fat deposition, μ is the overall mean, Genotype _i is the genotype effect, _Pj is the batch effect, _Sk is the season effect, and _εijk is the random error. It is assumed that _εijk are independent of each other and obey the N(0,σ2) distribution.

The genotype test results showed that among the 1029 individuals, there were 218 individuals with GG genotype, 521 individuals with AG genotype, and 290 individuals with AA genotype. The results of the genotype-trait association analysis are shown in Table 1.

Table 1 Association analysis between sheep RAP1GAP gene polymorphism and tail fat deposition

Note: Data in the same row with different letters in the subscript indicate significant differences (P<0.05), while data with the same letter or no letter in the subscript indicate no significant differences (P>0.05).

The results showed that the G/A polymorphic site of the amplified fragment SEQ ID NO.1 was significantly associated with tail fat deposition in sheep. Sheep carrying the AA genotype had less tail fat deposition than those carrying the GG genotype (P<0.05). This shows that the A allele is the dominant allele. This indicates that the RAP1GAP g.13561G>A mutation site can be used as a potential molecular marker affecting tail fat deposition in sheep (P<0.05). Selecting the AA genotype for seed preservation during breeding can reduce the deposition of fat in the sheep's tail and obtain a dominant flock with less tail fat deposition.

Sequence Listing

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Claims (9)

1. Use of a molecular marker associated with sheep tail fat deposition in sheep breeding, the nucleotide sequence of the molecular marker being shown in SEQ ID No.1, wherein R at 123bp represents a or G, the mutation resulting in a/G polymorphism of the molecular marker, wherein tail fat deposition of sheep carrying the AA genotype is significantly smaller than that of sheep carrying the GG genotype.

2. The application of the primer pair for detecting the molecular marker related to sheep tail fat deposition in sheep breeding is characterized in that the primer pair comprises a primer R-F and a primer R-R, wherein the sequence of the primer R-F is shown as SEQ ID NO.2, and the sequence of the primer R-R is shown as SEQ ID NO. 3; the nucleotide sequence of the molecular marker is shown as SEQ ID NO.1, wherein R at 123bp represents A or G, and the mutation leads to the A/G polymorphism of the molecular marker, and the nucleotide sequence is characterized in that the tail fat deposition of sheep carrying the AA genotype is obviously smaller than that of sheep carrying the GG genotype.

3. Use of a KASPar primer pair for detecting a molecular marker associated with sheep tail fat deposition in sheep breeding, characterized in that the KASPar primer pair comprises a forward primer for detecting AlleleG, a forward primer for detecting AlleleA and a universal reverse primer, wherein the sequence of the forward primer for detecting AlleleG is shown as SEQ ID No.4, the sequence of the forward primer for detecting AlleleA is shown as SEQ ID No.5, and the sequence of the universal reverse primer is shown as SEQ ID No. 6; the nucleotide sequence of the molecular marker is shown as SEQ ID NO.1, wherein R at 123bp represents A or G, and the mutation leads to the A/G polymorphism of the molecular marker, and the nucleotide sequence is characterized in that the tail fat deposition of sheep carrying the AA genotype is obviously smaller than that of sheep carrying the GG genotype.

4. The application of the detection kit for detecting the molecular marker related to sheep tail fat deposition in sheep breeding is characterized by comprising a primer pair with a nucleotide sequence shown as SEQ ID NO.2-3 or/and a KASPar primer pair with a nucleotide sequence shown as SEQ ID NO. 4-6; the nucleotide sequence of the molecular marker is shown as SEQ ID NO.1, wherein R at 123bp represents A or G, and the mutation leads to the A/G polymorphism of the molecular marker, and the nucleotide sequence is characterized in that the tail fat deposition of sheep carrying the AA genotype is obviously smaller than that of sheep carrying the GG genotype.

5. Use of a method for detecting a molecular marker associated with sheep tail fat deposition in sheep breeding comprising the steps of:

1) Amplifying sheep genome DNA by using a primer pair with nucleotide sequences shown as SEQ ID NO.2-3 or a KASPar primer pair with nucleotide sequences shown as SEQ ID NO.4-6, or a kit containing the PCR primer pair or the KASPar primer pair;

2) Carrying out typing identification on specific sites of A or G represented by R at 123bp shown in SEQ ID NO.1 in the nucleotide sequence of the amplification product obtained in the step 1); the deposition of tail fat of sheep carrying the AA genotype is significantly smaller than that of sheep carrying the GG genotype.

6. The method according to claim 5, wherein the typing method in step 2) is a sequencing method, a gene chip method, a fluorescent probe method or a high resolution dissolution profile method.

7. The use according to claim 5, wherein the KASPar primer pair according to claim 3 is used for PCR amplification, and after amplification, the typing result is determined by detecting a fluorescent signal.

8. Use of a molecular marker or a primer pair having a nucleotide sequence shown in SEQ ID No.2-3 or a KASPar primer pair having a nucleotide sequence shown in SEQ ID No.4-6, or a kit comprising the aforementioned primer pair or KASPar primer pair, or a method for detecting a molecular marker associated with sheep tail fat deposition in sheep tail fat deposition trait detection, wherein the nucleotide sequence of the molecular marker is shown in SEQ ID No.1, wherein R at 123bp represents a or G, the mutation resulting in a/G polymorphism of the molecular marker, wherein tail fat deposition of sheep carrying the AA genotype is significantly smaller than sheep carrying the GG genotype.

9. Use according to any one of claims 1 to 7 in sheep breeding for the purpose of selecting small tail sheep.