Application of method for detecting cattle SRSF12 gene copy number variation in cattle molecular marker-assisted selective breeding and kit

Abstract

The invention discloses application of a method for detecting cattle SRSF12 gene copy number variation in cattle molecular marker-assisted selective breeding and a kit, and belongs to the technical field of molecular genetics. Based on a real-time fluorescent PCR (polymerase chain reaction) technology, cattle genome DNA (deoxyribonucleic acid) is taken as a template, a cattle SRSF12 gene copy number variation region is amplified, and the cattle SRSF12 gene copy number variation region is amplified; meanwhile, a cattle general transcription factor BTF3 gene is amplified as an internal reference, and finally, the copy number variation type of an individual is calculated and judged by using a 2-delta delta Ct method. The method provided by the invention is beneficial to correlation analysis between the cattle SRSF12 gene CNV and cattle growth traits, expands application of molecular marker-assisted selection in cattle genetic breeding, has the characteristics of convenience and rapidness in operation, and is convenient for rapid popularization and application.

Description

Technical Field

[0001] The present invention belongs to the field of molecular genetics research, and specifically relates to a method for detecting copy number variation of the SRSF12 gene in cattle, and its application in molecular marker-assisted selection breeding of cattle, and a kit. Background Art

[0002] As an important livestock breed in my country, cattle's economic traits, such as growth performance and meat quality, have a significant impact on livestock profitability and market demand. Traditional breeding methods, which primarily rely on phenotypic selection, suffer from long selection cycles and low accuracy, making them inadequate for the rapidly developing modern livestock industry. To accelerate genetic improvement, molecular marker-assisted selection (MAS) has been widely applied in livestock and poultry breeding. Single nucleotide polymorphisms (SNPs) and insertion-deletions (InDels) are currently the primary marker types used in MAS breeding. Copy number variations (CNVs), as an important form of genetic variation, are increasingly gaining attention. CNVs provide genomic structural information that complements SNP data and profoundly influences gene structure, dosage, regulation, and exposure of recessive alleles. However, the widespread application of novel molecular markers, such as CNVs, has limited the further development of breeding technology.

[0003] The SRSF12 gene (Serine / Arginine-Rich Splicing Factor 12) encodes a splicing factor that plays a crucial role in regulating pre-mRNA splicing. In recent years, with the continuous advancement of genomics and transcriptomics, the potential value of SRSF12 in animal genetics and breeding has gradually attracted attention. While this gene has been extensively studied in humans, its application in cattle breeding, particularly the use of its copy number variants in molecular marker-assisted selection breeding, has not yet been systematically reported. Summary of the Invention

[0004] In view of the current technical status that traditional cattle breeding methods rely on phenotypic selection, resulting in long breeding cycles, low accuracy, and lack of effective molecular marker-assisted selection, the present invention aims to provide a method for detecting copy number variation of the cattle SRSF12 gene and its application in cattle molecular marker-assisted selection breeding. By revealing the association between the SRSF12 gene and cattle traits, molecular markers are developed to improve breeding efficiency and accuracy, thereby promoting the advancement of cattle breeding technology.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions: The present invention provides an application of a method for detecting copy number variation of a cattle SRSF12 gene in molecular marker-assisted selection breeding of cattle.

[0006] The SRSF12 gene copy number variation site serves as a molecular genetic marker site for cattle growth traits.

[0007] Using the cattle gene DNA as the template and primer pair P1 and primer pair P2 as primers, the copy number variation region of the SRSF12 gene and a partial fragment of the BTF3 gene as a control were amplified by real-time quantitative PCR. The copy number variation type of the cattle SRSF12 gene was then identified based on the quantitative results. The copy number variation region of the SRSF12 gene is located at Chr9: 62199601bp~62201200bp.

[0008] The copy number variation types are categorized into three types based on the quantitative results of 2-ΔΔCt: deletion type, i.e., -ΔΔCt<-0.5; normal type, i.e., -0.5≤-ΔΔCt≤0.5; and duplication type, i.e., -ΔΔCt>0.5.

[0009] The primer sequences of the primer pair P1 and the primer pair P2 are shown as SEIQ NO.1 to SEIQ NO.4.

[0010] Furthermore, the primer pair SRSF12 gene-CNV (primer pair P1) is: Upstream primer F1: 5'-CATAAACCGAGCAGGCGAGA -3' (SEIQ NO. 1) Downstream primer R1: 5'-AGTCTCAACCTGGTGTTGGC-3' (SEIQ NO. 2) The primer pair BTF3-CNV (primer pair P2) is: Upstream primer F2: 5'-AACCAGGAGAAACTCGCCAA-3' (SEIQ NO. 3) Downstream primer R2: 5'-TTCGGTGAAATGCCTCTCG-3' (SEIQ NO. 4) The amplification system used in the real-time quantitative PCR includes: 1 μL of 10 ng / μL template DNA, 0.5 μL of each of the upstream and downstream primers corresponding to 10 μmol / L primer pair P1 or primer pair P2, 5 μL of SYBR® PreMix Ex TaqTM II, and 3 μL of ddH2O.

[0011] The reaction procedure used in the real-time quantitative PCR is as follows: (1) pre-deformation at 95°C for 1 min; (2) denaturation at 95°C for 10 s, annealing at 60°C for 30 s, for a total of 40 cycles.

[0012] Among the copy number variation types, the repeat copy number variation type is superior to the cattle individuals of the deletion type and normal copy number variation types in growth traits.

[0013] Furthermore, the growth traits are one or more of cross height, body height, body length, waist and foot width, and ischium width.

[0014] Furthermore, among the copy number variation types, repeat-type copy number variation significantly predominated in Qinchuan and Yunling cattle, with significant differences in copy number. Qinchuan cattle with repeat-type copy number variation also outperformed those with deletion-type copy number variation in growth traits, while Yunling cattle with repeat-type copy number variation outperformed those with deletion-type and normal copy number variation in growth traits.

[0015] The present invention also provides a real-time quantitative PCR detection kit for copy number variation of the SRSF12 gene related to the growth traits of yellow cattle. The kit includes a primer pair for amplifying the copy number variation region of the SRSF12 gene. The copy number variation region of the SRSF12 gene is located at Chr9: 62199601bp~62201200bp, and the primer pair sequence is shown as SEIQNO.1~SEIQ NO.2.

[0016] The kit also includes amplification of internal reference genes BTF3 Primer pairs for partial gene fragments, the primer pair sequences are shown as SEIQ NO.3~SEIQ NO.4.

[0017] Compared with the prior art, the present invention has the following beneficial effects: The method for detecting copy number variation of the SRSF12 gene of cattle provided by the present invention is applied in molecular marker-assisted selection breeding of cattle. Through real-time fluorescence quantitative PCR technology, the copy number variation of the SRSF12 gene can be detected quickly and accurately, avoiding the problems of long time consumption and low efficiency of traditional phenotypic selection, and significantly shortening the breeding cycle. Based on the molecular marker of SRSF12 gene copy number variation, it can be directly linked to the growth, meat quality and other traits of cattle, realizing early selection, reducing selection errors caused by interference from environmental factors, and improving the accuracy of breeding selection. It provides a new molecular marker for cattle breeding, helps to improve cattle breeding efficiency, and promotes the sustainable development of the cattle industry.

[0018] Furthermore, by designing specific primers for the SRSF12 gene and using internal reference genes (such as the BTF3 gene) as a reference, the copy number variation of the SRSF12 gene can be distinguished more accurately, avoiding misjudgment and missed judgment; compared with high-throughput sequencing methods, gene chips and other methods, it is fast, simple, low-cost, and can accurately identify the copy number type of an individual.

[0019] The real-time quantitative PCR (qPCR) detection kit for detecting copy number variation in the SRSF12 gene, which is associated with growth traits in cattle, uses specific primers targeting the SRSF12 gene and uses the internal reference gene BTF3 for standardization. This effectively avoids interference from nonspecific amplification or background noise, enhancing detection specificity. Real-time quantitative PCR (qPCR) technology accurately quantifies copy number variation in the SRSF12 gene through real-time monitoring of fluorescent signals. Compared to traditional PCR or sequencing methods, qPCR offers greater sensitivity and can accurately distinguish single-copy differences within the genome, ensuring the reliability of test results. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is the amplification curve drawn by qPCR (SRSF12 gene) in the embodiment of the present invention; Figure 2 This is the melting curve drawn by qPCR (SRSF12 gene) in the embodiment of the present invention; Figure 3 This is the frequency distribution of the SRSF12 gene copy number variation types in 6 cattle breeds. DETAILED DESCRIPTION

[0021] In order to enable those skilled in the art to better understand the solutions of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts should fall within the scope of protection of the present invention.

[0022] In the cattle genome resequencing study, the CNV region of the SRSF12 gene in the Chinese cattle genome was detected: Chr9: 62,199,601bp~62,201,200bp, and Primer v5.0 software was used to design a CNV in this region. Qinchuan cattle, Pinan cattle, Yunling cattle, Xianan cattle, Jiaxian red cattle, and Guyuan cattle were used as experimental subjects. Their genomic DNA was used as template and BTF3 was used as the internal reference gene. The copy number variation of the SRSF12 gene was amplified by qPCR technology. -ΔΔCtMethods The copy number variation type of the SRSF12 gene was determined. Based on the physiological role of the SRSF12 gene and the regulatory mechanism of CNV, the association between the SRSF12 gene CNV and growth traits was studied, providing a scientific basis for the application of SRSF12 gene CNV as a molecular marker for assisted selection in molecular breeding of yellow cattle.

[0023] 1. Scalper sample collection In the present invention, the collection method is venous blood collection. The Qinchuan cattle collected are from the Qinchuan Beef Cattle Breeding Center in Shaanxi Province (collection time: January 2013), the Pinan cattle are from Xinye County, Nanyang City, Henan Province (collection time: January 2016), the Yunling cattle are from the Demonstration Ranch of the Yunnan Grassland Animal Science Research Institute (collection time: September 2018), the Xianan cattle are from Xianan Cattle Technology Development Co., Ltd. in Biyang County, Zhumadian City, Henan Province (collection time: June 2015), the Jiaxian Honghong cattle are from the Jiaxian Honghong cattle conservation area in Pingdingshan City, Henan Province (collection time 2008), and the Guyuan yellow cattle are from Limin Village, Ying Town, Yuanzhou District, Guyuan (collection time: June 2019). Their growth trait data, such as body height, body length, chest circumference, rump length, ischium tip width, and cruciate height, were recorded for subsequent correlation analysis.

[0024] 2. Blood DNA Extraction The phenol-chloroform method was used as follows: (1) Preliminary processing Frozen blood samples were thawed in a room temperature water bath; Transfer 2 mL of whole blood into a sterile 2 mL centrifuge tube; Centrifuge at 12000 rpm for 10 min at 4°C to separate the cellular components of the blood from the plasma; Discard the supernatant and retain the precipitate. Add 1.5 mL of PBS buffer, vortex to suspend the precipitate, and gently shake on ice for 15 min. Centrifuge at 12000 rpm for 10 min at 4°C, discard the supernatant, and retain the precipitate; Repeat steps 4 and 5 once to further purify the cell pellet and improve the purity of DNA extraction; (2) Cell lysis and digestion Use a blue gun tip to crush the precipitate into flocculent form; Add 500 μL of DNA extraction solution and 6 μL of proteinase K to the centrifuge tube; Incubate in a constant temperature water bath at 37°C overnight (about 16 h) until the cell pellet is completely digested and the solution is clear.

[0025] (3) DNA extraction and purification Add 1 mL of Tris-saturated phenol and place on ice with gentle shaking for 20 min; Centrifuge at 12,000 rpm for 10 min at 4°C. Use a pipette to transfer the upper aqueous phase to another 2.0 mL sterile centrifuge tube; Add 0.5 mL of saturated phenol and 0.5 mL of chloroform, place on ice and shake gently for 20 min; Centrifuge at 12,000 rpm for 10 min at 4°C. Use a pipette to transfer the upper aqueous phase to another 2.0 mL sterile centrifuge tube; Add 1 mL of chloroform and place on ice with gentle shaking for 20 min; Centrifuge at 12,000 rpm for 10 min at 4°C. The upper aqueous phase was transferred to a 1.5 mL centrifuge tube using a pipette.

[0026] (4) DNA precipitation and washing Add 1 mL of pre-cooled anhydrous ethanol (-20°C), shake gently several times until the DNA precipitates, and then place at -20°C for 30 minutes; Centrifuge at 12,000 rpm for 10 min at 4°C and discard the ethanol. Add 1 mL of 70% ethanol and shake gently for 10 min; Centrifuge at 12,000 rpm for 10 min at 4°C, discard the ethanol, and repeat the rinse (use a pipette to remove the remaining alcohol at the bottom of the tube); (5) DNA drying and preservation Place at room temperature for 30 minutes, and then dry in a 60°C oven for 30 seconds to evaporate the ethanol. Add 50 μL of ultrapure water and store at 4°C until the DNA is completely dissolved. Measure the concentration using a spectrophotometer and store at -80°C.

[0027] 3. Primer Design The CNV region of the SRSF12 gene in the Chinese cattle genome was detected to be located at Chr9: 62,199,601 bp to 62,201,200 bp (length 1600 bp). Primers were designed in this region using Primer v5.0 software (PREMIER Bio Soft International, California, UFA). The sequences of primer pair P1 are as follows: Upstream primer F1: 5'-CATAAACCGAGCAGGCGAGA-3' (SEQ ID NO. 1) Downstream primer R1: 5'-AGTCTCAACCTGGTGTTGGC-3' (SEQ ID NO. 2) At the same time, using the bovine BTF3 gene (AC000177.1) published by NCBI as a reference sequence, primers were designed to amplify a specific fragment (166 bp) of the BTF3 gene (internal reference gene). The sequences of primer pair P2 are as follows: Upstream primer F2: 5'-AACCAGGAGAAACTCGCCAA-3' (SEQ ID NO. 3) Downstream primer R2: 5'-TTCGGTGAAATGCCTCTCG-3' (SEQ ID NO. 4) 3. Real-time Quantitative PCR The qPCR reaction system is shown in Figure 1.

[0028] Table 1: qPCR reaction system

[0029] The qPCR reaction procedure was as follows: (1) pre-denaturation at 95°C for 1 min; (2) denaturation at 95°C for 10 s and annealing at 60°C for 30 s, for a total of 40 cycles.

[0030] 4. Analysis of CNV types of SRSF12 gene in cattle individuals The cycle threshold (Ct) is used as a parameter to draw a standard curve to detect the initial copy number of the template in the sample to be tested. Each sample was amplified using primers (P1 and P2) of the target sequence and the internal reference sequence, and three parallel replicates were set up at the same time. -ΔΔCt Statistical methods were used to analyze copy number (ΔCt = Ct target gene - Ct reference gene). Based on the criteria, the copy number variation types of the SRSF12 gene were divided into three categories: deletion type, normal type, and duplication type.

[0031] Judgment criteria: when -ΔΔCt<-0.5, it is a deletion type; when -0.5≤-ΔΔCt≤0.5, it is a normal type; and when -ΔΔCt>0.5, it is a duplication type.

[0032] 5. Data Processing Count the number of individuals of each type (Deletion, Median, and Duplication) in the detection group, and count the frequency of each type. The calculation formula is as follows: P C =N C / N Among them, P C Represents the frequency of a certain copy number variation type; N Crepresents the number of individuals with CNV type C in the population; N represents the total number of tested populations SPSS software (20.0) was used to conduct a univariate analysis of the correlation between SRSF12 gene copy number variation types and growth traits. Based on the different factors affecting body shape traits, age and genetic effects were considered, and a fixed model was used for analysis. The simplified model was then simplified according to the actual situation. Y ijk =μ + A i +G j + E ijk Among them, Y ijk is the phenotypic record of individual growth traits; μ is the population mean; A i is the age effect; G j is the copy number effect of each sample; E ijk It is a random error.

[0033] 6. Test results (1) qPCR primer specificity detection To confirm the specificity of the designed primers, qPCR was used for detection. The results showed that the amplification curve was smooth and presented an "S" curve, and the Ct value was between 20-30, indicating that the genomic DNA was in a normal amplification state ( Figure 1 The melting curve showed a single peak, and the melting temperature was around 80°C, indicating that the amplified product had no interfering products and was single ( Figure 2 ). The above results demonstrate that the primers have good specificity and amplification efficiency.

[0034] (2) Frequency distribution of SRSF12 gene copy number variation Six different breeds of cattle (a total of 635 heads) were selected to study the distribution frequency of SRSF12 gene copy number variation in different cattle breeds. The analysis results showed that ( Figure 3 (See Table 2 for details). Duplication patterns were significantly more common in all six cattle breeds than deletion patterns and normal patterns, showing a consistent trend, suggesting that multiple copies of the SRSF12 gene are more likely to occur in genomic structural variation. Furthermore, with the exception of GY cattle, where deletion patterns were significantly less common than normal and duplication patterns, the normal patterns in the remaining five cattle breeds (QC, PN, YL, XN, and JX) were all less common than deletion patterns to varying degrees.

[0035] Table 2: Frequencies of different CNV types in six cattle breeds

[0036] (3) Association analysis between SRSF12-CNV and growth traits of cattle Association analysis between SRSF12-CNV and growth traits of Qinchuan cattle An association analysis was conducted between SRSF12 CNVs and ten growth traits in Qinchuan cattle, using a sample size of 109 cattle. The results, shown in Table 3, show a significant correlation between SRSF12 CNVs and ischial width (P < 0.05). Of the three CNV types, the duplication type performed best, with a mean of 24.827 ± 0.459 cm, suggesting a trend toward becoming a dominant gene. Therefore, the duplication type of the SRSF12 gene could serve as a marker for Qinchuan cattle breeding, accelerating the development of superior traits.

[0037] Table 3: Association analysis between SRSF12-CNV and growth traits of Qinchuan cattle

[0038] Note: a and b indicate significant differences at P < 0.05.

[0039] Association analysis between SRSF12-CNV and growth traits in Pinan cattle Association analysis was conducted between the SRSF12-CNV and six growth traits in Pinan cattle, using a sample size of 112. The results, shown in Table 4, showed no significant correlation between the SRSF12-CNV and any of the traits (P>0.05). However, the deletion variant was superior to the normal and duplication variants for each trait, suggesting a trend toward becoming a dominant gene.

[0040] Table 4: Association analysis between SRSF12-CNV and growth traits of Pinan cattle

[0041] Note: a and b indicate significant differences at P < 0.05.

[0042] Association analysis between SRSF12-CNV and growth traits of Yunling cattle An association analysis was conducted between SRSF12 CNV and 15 growth traits in Yunling cattle, using a sample size of 104. The results, shown in Table 5, indicate that the normal genotype for rump length is superior to both the deletion and duplication genotypes and appears to be a dominant gene. SRSF12 CNV showed a significant correlation with rump length (P < 0.05). Therefore, the normal genotype of the SRSF12 gene can be used as a marker to accelerate the selection and breeding of superior traits in Yunling cattle.

[0043] Table 5: Association analysis between SRSF12-CNV and growth traits of Yunling cattle

[0044] Note: a and b indicate significant differences at P < 0.05.

[0045] Association analysis between SRSF12-CNV and growth traits of Xianan cattle Association analysis was conducted between the SRSF12 CNV and seven growth traits in a sample of 106 Xianan cattle. The results, shown in Table 6, showed no significant correlation between the SRSF12 CNV and any of the traits (P>0.05). However, the duplication variant significantly outperformed the deletion and normal variants in body weight, with a mean of 559.942±7.192 kg. Furthermore, there was little difference in body height and cross-body height between the different CNV types.

[0046] Table 6: Association analysis between SRSF12-CNV and growth traits of Xianan cattle

[0047] Note: a and b indicate significant differences at P < 0.05.

[0048] In summary, among the four types of cattle (Qinchuan cattle, Pinan cattle, Yunling cattle, and Xianan cattle), choose 2×2 -ΔΔCt Using a genetic analysis method, SRSF12-CNV-type growth traits were associated with an analysis. CNVs in the SRSF12 gene do affect the growth traits of Chinese cattle. In all four cattle breeds, the duplication type was more common than the deletion and normal types, and the copy number differences were significant, indicating that the SRSF12 gene has rich genetic copy number diversity. In Qinchuan cattle, SRSF12 gene copy number variation was significantly associated with ischial tip width; individuals with duplication-type copy number variation had better growth traits than individuals with deletion-type copy number variation. In Yunling cattle, SRSF12 gene copy number variation was significantly associated with rump length; individuals with duplication-type copy number variation had better growth traits than individuals with deletion-type and normal-type copy number variation. Therefore, CNVs in the SRSF12 gene can be used as a molecular marker for early breeding of Chinese cattle.

[0049] The above content is only for explaining the technical idea of ​​the present invention and cannot be used to limit the protection scope of the present invention. Any changes made on the basis of the technical solution in accordance with the technical idea proposed by the present invention shall fall within the protection scope of the claims of the present invention.

Claims

1. Application of a method for detecting copy number variation of the SRSF12 gene in cattle in molecular marker-assisted selection breeding of cattle.

2. The use according to claim 1, characterized in that Using the cattle gene DNA as the template and primer pair P1 and primer pair P2 as primers, the copy number variation region of the SRSF12 gene and a partial fragment of the BTF3 gene as a control were amplified by real-time quantitative PCR. The copy number variation type of the cattle SRSF12 gene was then identified based on the quantitative results. The copy number variation region of the SRSF12 gene is located at Chr9: 62199601bp~62201200bp.

3. The use according to claim 2, characterized in that The copy number variation types are categorized into three types based on the quantitative results of 2-ΔΔCt: deletion type, i.e., -ΔΔCt<-0.5; normal type, i.e., -0.5≤-ΔΔCt≤0.5; and duplication type, i.e., -ΔΔCt>0.

5.

4. The use according to claim 2, characterized in that The primer sequences of the primer pair P1 and the primer pair P2 are shown as SEIQ NO.1 to SEIQ NO.

4.

5. The use according to claim 2, characterized in that The amplification system used in the real-time quantitative PCR includes: 1 μL of 10 ng / μL template DNA, 0.5 μL of each of the upstream and downstream primers corresponding to 10 μmol / L primer pair P1 or primer pair P2, 5 μL of SYBR® PreMix Ex TaqTM II, and 3 μL of ddH2O.

6. The use according to claim 2, characterized in that The reaction procedure used in the real-time quantitative PCR is as follows: (1) pre-denaturation at 95°C for 1 min; (2) denaturation at 95°C for 10 s, annealing at 60°C for 30 s, for a total of 40 cycles.

7. The use according to claim 2, characterized in that Among the copy number variation types, the repeat copy number variation type is superior to the cattle individuals of the deletion type and normal copy number variation types in growth traits.

8. The use according to claim 7, characterized in that The growth traits are cross height, body height, body length, waist and foot width or ischium width.

9. A real-time quantitative PCR detection kit for SRSF12 gene copy number variation related to cattle growth traits, characterized in that: The kit includes a primer pair for amplifying the copy number variation region of the SRSF12 gene, the copy number variation region of the SRSF12 gene is located at Chr9: 62199601bp~62201200bp, and the primer pair sequence is shown as SEIQ NO.1~SEIQNO.

2.

10. A real-time quantitative PCR detection kit for detecting SRSF12 gene copy number variation related to cattle growth traits according to claim 9, characterized in that: The kit also includes amplification of internal reference genes BTF3 Primer pairs for partial gene fragments, the primer pair sequences are shown as SEIQ NO.3~SEIQ NO.4.

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