A PAPPA gene and its application in screening sheep with short lambing interval

Abstract

The present invention provides a PAPPA gene and its application in screening sheep with short lambing interval, belonging to the field of SNP detection. The present invention provides a screening kit for sheep with short lambing interval, which includes a reagent for detecting the 7400929th site of chromosome 2 of the sheep reference genome. The present invention also provides a method for screening sheep with short lambing interval. The kit and method of the present invention effectively screen sheep with short lambing interval, quickly determine sheep populations with excellent lambing interval traits, shorten the lambing interval, greatly improve the production efficiency of sheep, provide convenience for breeding of high-performance sheep (especially Tibetan sheep), and have good application prospects.

Description

Technical Field

[0001] The invention belongs to the field of SNP technology, and in particular relates to a PAPPA gene and application thereof in screening sheep with short lambing interval. Background Art

[0002] Tibetan sheep provide wool, fur, meat and milk for pastoral people, so they have important economic and agricultural value to the local area. Lambing interval is one of the most important reproductive traits, which directly affects the reproductive efficiency and profitability of animal husbandry (Abdoliet al., 2019). The shorter the lambing interval, the stronger the reproductive efficiency and profitability of animal husbandry. In local rare sheep breeds, improving reproductive traits can protect genetic diversity and increase profitability. Lambing interval is an important reproductive trait that affects the life span and final production of sheep (Schoeman et al., 1991). Shortening the lambing interval can increase meat production more than increasing growth rate and reduce the economic cost of raising sheep. Sheep lambing interval is a complex reproductive trait with low heritability and controlled by multiple genes, so it is difficult to quickly improve it using conventional breeding techniques. Molecular marker-assisted selection can overcome the selection difficulty of such low heritability traits. Therefore, studying the molecular markers that affect the lambing interval of Tibetan sheep is of great significance for the sustainable, green and efficient development of animal husbandry in the Qinghai-Tibet Plateau.

[0003] Pregnancy-associated plasma protein-A (PAPPA) plays a vital role in various reproductive processes, such as stimulating granulosa cell proliferation and steroidogenesis, follicle development and ovulation (Mazerbourg et al., 2001). In the study of bovine follicles, it was found that cattle carrying high fertility alleles acquired the dominant marker (PAPPA) and the ability to ovulate with smaller follicle size. PAPPA plays an important role in the growth, development and steroidogenesis of ovarian follicles. This shows that PAPPA is related to reproductive traits.

[0004] There are currently reports on the relationship between PAPPA and the number of lambs born in sheep, but there are no reports on the relationship between PAPPA and the interval between lambing in sheep. Summary of the invention

[0005] The object of the present invention is to provide a new SNP of PAPPA gene and its application in screening sheep with short lambing interval.

[0006] The invention provides a gene, which is a sheep PAPPA gene with A at the 7400929th position of chromosome 2 of the sheep reference genome.

[0007] The present invention also provides a sheep screening kit with a short lambing interval, which comprises a reagent for detecting the 7400929th site of chromosome 2 of the sheep reference genome.

[0008] Among them, the reagents include relevant reagents for detecting G>A at position 7400929 of chromosome 2 of the sheep reference genome.

[0009] Wherein, the reagent is a sequencing reagent, a PCR detection reagent, a reagent for a restriction fragment length polymorphism method or a reagent for single-stranded conformation polymorphism analysis.

[0010] Preferably, the reagent is a competitive allele-specific PCR detection reagent, and further preferably includes primers shown in SEQ ID NOs. 1 to 3.

[0011] The present invention also provides use of a reagent for detecting site 7400929 of chromosome 2 of a sheep reference genome in preparing a reagent for screening sheep with a short lambing interval.

[0012] Wherein, the reagent includes a related reagent for detecting G>A at position 7400929 of chromosome 2 of the sheep reference genome; or, the screening reagent is a screening reagent for Tibetan sheep with a short lambing interval.

[0013] Wherein, the reagent is a sequencing reagent, a PCR detection reagent, a reagent for a restriction fragment length polymorphism method or a reagent for single-stranded conformation polymorphism analysis.

[0014] Preferably, the reagent is a competitive allele-specific PCR detection reagent, and further preferably includes primers shown in SEQ ID NOs. 1 to 3.

[0015] The present invention also provides a method for screening sheep with a short lambing interval, comprising the following steps:

[0016] (1) Extracting total genomic DNA from the sheep sample to be tested;

[0017] (2) Detection of position 7400929 of chromosome 2 of the sheep reference genome;

[0018] Preferably, the sheep are Tibetan sheep.

[0019] The present invention first discovered that the polymorphism of the 7400929th site of chromosome 2 (NC_056055.1) in the PAPPA gene located in the sheep reference genome (ARS-UI_Ramb_v2.0) has a very strong correlation with the lambing interval of sheep (especially Tibetan sheep), and the site can be used as a molecular genetic marker for the interval trait of sheep. Sheep with a short lambing interval can be screened by detecting the SNP site, and can be used to quickly screen sheep populations with excellent interval traits, thereby shortening the lambing interval in Mianyang, accelerating the breeding process, and saving breeding costs.

[0020] The key to the present invention is to find that the polymorphism of the 7400929th site of the PAPPA gene located on chromosome 2 of the sheep reference genome has a strong correlation with the lambing interval of sheep (especially Tibetan sheep), and sheep with a short lambing interval can be screened by detecting the 7400929th site of chromosome 2 of the sheep reference genome. Any method and reagent capable of detecting the 7400929th site of chromosome 2 of the sheep reference genome can be used for screening sheep with a short lambing interval, and the transformation prospect is very broad.

[0021] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0022] The above contents of the present invention are further described in detail below through specific implementation methods in the form of embodiments. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is the overall technical flow chart for detecting single nucleotide polymorphism of sheep PAPPA gene.

[0024] Figure 2 This is the electrophoresis diagram of the quality inspection of the Tibetan sheep sample genome DNA (in the figure: 001-024 is the sample number, and M is the schematic diagram of the marker band).

[0025] Figure 3 This is the KASP typing result of chromosome 2, position 7400929 of the Tibetan sheep sample (GG, AG, AA genotypes) (in the figure: NTC is the negative control, and X is Undetermined). DETAILED DESCRIPTION

[0026] In order to understand the present invention more clearly, the present invention is further described with reference to the following examples and drawings. The examples are only used for explanation and are not intended to limit the present invention in any way.

[0027] The experimental methods without specific conditions in the examples are conventional methods and conventional conditions well known in the art, or according to the conditions recommended by the manufacturer; the various chemical reagents used in the examples are commercially available, and the primers used are commissioned for synthesis.

[0028] Example 1. Kit and method for screening sheep with short lambing interval of the present invention

[0029] 1. The components of the KASP kit of the present invention include:

[0030] (1) KASP reaction primer:

[0031] PAPPA-Primer1-FAM (upstream primer F1, SEQ IN NO.1):

[0032] 5'-gaaggtgaccaagttcatgctGAAGGTGACCAAGTTCATGCTTGAATTTGAACATTTCTAGGAGGCAA-3' (lowercase letters represent FAM linker)

[0033] PAPPA-Primer2-HEX (upstream primer F2, SEQ IN NO.2):

[0034] 5'-gaaggtcggagtcaacgattGAAGGTCGGAGTCAACGGATTTGAATTTGAACATTTCTAGGAGGCAG-3' (lowercase letters represent HEX linker)

[0035] PAPPA-Common-Primer (downstream primer, SEQ IN NO.3):

[0036] 5'-AAACACTTTAAGGCTCTGAATGGC-3'

[0037] (2) Other reagents for KASP reaction: KASP Master Mix; Primer Mix; water.

[0038] 2. Detection method

[0039] (1) Extracting total genomic DNA from the sheep sample to be tested;

[0040] (2) Use the KASP kit to detect the genotype of site 7400929 of chromosome 2 of the sheep reference genome.

[0041] Example 2: Verification of the correlation between SNP loci and Tibetan sheep fecundity traits

[0042] (1) Extraction of genomic DNA

[0043] Tibetan sheep were selected as the experimental material. Ear edge tissue the size of mung bean was collected using ear picking forceps and stored in 75% alcohol for subsequent extraction of genomic DNA.

[0044] (2) Extraction of genomic DNA

[0045] DNA was extracted from the ear margin tissue of Tibetan sheep using a genomic DNA kit ( TIANamp Genomic DNA Kit) was used for extraction, and the extraction was performed strictly according to the operating instructions.

[0046] (3) Sample DNA quality testing

[0047] The concentration of the extracted genomic DNA was measured using Thermo Scientific's NanoDrop 8000 ultra-micro spectrophotometer. The results showed that the sample concentrations were all above 100 ng / μL, and the A260 / A280 values ​​were between 1.8-2.0. Good DNA electrophoresis results should be: ① The main band is bright and clear, without dragging, and the main band size is around 10 kb; ② If the main band becomes wider and darker, it indicates that the DNA is slightly degraded. If it becomes a diffuse DNA band, it indicates that the DNA is severely degraded; ③ If there is no band in the electrophoresis, it indicates that the total amount and concentration of the extracted DNA are extremely low, which is likely to cause PCR amplification failure. The electrophoresis results show that the genomic DNA bands are clear and free of impurities ( Figure 2 ), indicating that the extracted genomic DNA had good integrity and met the standards for subsequent experiments.

[0048] (4) KASP primer design

[0049] Using the partial fragment of the PAPPA gene at position 7400929 of chromosome 2 of the sheep reference genome ARS-UI_Ramb_v2.0 as a template, the primer design tool snpway was used to design the KASP reaction primers:

[0050] PAPPA-Primer1-FAM (upstream primer F1, SEQ IN NO.1):

[0051] 5'-gaaggtgaccaagttcatgctGAAGGTGACCAAGTTCATGCTTGAATTTGAACATTTCTAGGAGGCAA-3' (lowercase letters represent FAM linker)

[0052] PAPPA-Primer2-HEX (upstream primer F2, SEQ IN NO.2):

[0053] 5'-gaaggtcggagtcaacgattGAAGGTCGGAGTCAACGGATTTGAATTTGAACATTTCTAGGAGGCAG-3' (lowercase letters represent HEX linker)

[0054] PAPPA-Common-Primer (downstream primer, SEQ IN NO.3):

[0055] 5'-AAACACTTTAAGGCTCTGAATGGC-3'

[0056] (5) KASP amplification

[0057] The extracted genomic DNA was used as a template, and the synthesized upstream primers PAPPA-Primer1-FAM, PAPPA-Primer2-HEX and the common primer PAPPA-Common-Primer were used as the experimental primer set for PCR amplification. The specific reaction system and reaction procedure are as follows:

[0058] The reaction system was 5 μL: including 1.0 μL of genomic DNA (20 ng / μL), 2.5 μL of 2×KASP Master Mix, 0.15 μL of Primer Mix (PAPPA-Primer1-FAM concentration was 10 μM, PAPPA-Primer2-HEX concentration was 10 μM and PAPPA-Common-Primer concentration was 20 μM) and 1.35 μL of water.

[0059] The reaction procedure was as follows: ① pre-denaturation at 94.0°C for 15 min, 1 cycle; ② denaturation at 94.0°C for 20 sec, annealing and extension at 65°C for 60 sec, 10 cycles (annealing temperature decreased by 0.7°C in each cycle); ③ denaturation at 94.0°C for 20 sec, annealing and extension at 57°C for 60 sec, 30 cycles; ④ fluorescence reading at 30°C for 60 sec, 1 cycle.

[0060] (6) Genotyping

[0061] Using QuantStudio TM 5Real-Time PCR System was used for genotyping. The genotype of the PAPPA gene located at the 7400929th site of chromosome 2 of the sheep reference genome was determined according to the analysis results. The typing results are as follows: Figure 3 shown.

[0062] (7) Correlation analysis model between genotype and lambing interval traits

[0063] One-way ANOVA in SPSS software was used to conduct the association analysis between genotype and lambing interval traits. The specific linear analysis model is as follows:

[0064] Y ij =μ+G i +e ij

[0065] Where: Y ij is the individual phenotypic record; μ is the population mean; G i is the genotype effect of each site; e ij is a random error.

[0066] (8) Analysis of the significance of genotype and lambing interval

[0067] The experiment tested the genotype of chromosome 2 at position 7400929 of 366 Tibetan sheep and recorded the lambing interval data. The correlation analysis between genotype and lambing interval trait was as follows:

[0068] The KASP typing results showed that among the 366 individuals, there were 135 with GG genotype, 153 with AG genotype, and 78 with AA genotype.

[0069] Table 1 Correlation between different genotypes of PAPPA gene and lambing interval

[0070]

[0071] Note: Different lowercase letters in the same column indicate significant differences (p<0.05).

[0072] The results showed (Table 1) that the 7400929th site of chromosome 2 of the sheep reference genome was significantly correlated with the lambing interval of Tibetan sheep. The lambing interval of individuals with AA genotype was significantly lower than that of individuals with GG and AG genotypes, and the lambing interval of individuals with AG genotype was significantly lower than that of individuals with GG genotype. Individuals whose lambing interval contained A at the 7400929th site of chromosome 2 of the sheep reference genome, especially those with homozygous AA genotype, had a short lambing interval. Tibetan sheep with a short lambing interval can be screened by detecting the 7400929th site of chromosome 2 of the sheep reference genome, thereby improving the production efficiency of the Tibetan sheep population.

[0073] In summary, the present invention provides a kit and method for effectively screening sheep (especially Tibetan sheep) with a short lambing interval, which can be used to quickly screen sheep populations with excellent lambing interval traits, shorten the lambing interval, greatly improve the production efficiency of sheep, and provide convenience for the breeding of high-performance sheep (especially Tibetan sheep), and has good application prospects.

Claims

1. Use of a reagent for detecting locus 7400929 of chromosome 2 of a sheep reference genome in the preparation of a sheep screening reagent with a short lambing interval, characterized in that: The version of the sheep reference genome is ARS-UI_Ramb_v2.0; When the genotype of the 7400929th locus is AA, the lambing interval is short; The sheep are Tibetan sheep.

2. The use according to claim 1, characterized in that: The reagent for detecting the 7400929th site of chromosome 2 of the sheep reference genome includes a related reagent for detecting G>A at the 7400929th site of chromosome 2 of the sheep reference genome.

3. The use according to claim 1 or 2, characterized in that: The reagent for detecting the 7400929th site of chromosome 2 of the sheep reference genome is a sequencing reagent, a PCR detection reagent, a reagent for a restriction fragment length polymorphism method, or a reagent for single-stranded conformation polymorphism analysis.

4. The use according to claim 3, characterized in that: The reagent for detecting the 7400929th site of chromosome 2 of the sheep reference genome is a competitive allele-specific PCR detection reagent.

5. The use according to claim 4, characterized in that: The reagent for detecting site 7400929 of chromosome 2 of the sheep reference genome includes primers shown in SEQ ID NOs. 1 to 3.

6. A method for selecting sheep with a short lambing interval, characterized in that: The steps include: (1) Extracting total genomic DNA from the sheep sample to be tested; (2) Detection of position 7400929 of chromosome 2 of the sheep reference genome; The version of the sheep reference genome is ARS-UI_Ramb_v2.0; When the genotype of the 7400929th locus is AA, the lambing interval is short; The sheep are Tibetan sheep.

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