Application of cattle LRP5 gene SV marker in identification of varieties of common cattle and tumor cattle

Abstract

The invention discloses a method for rapidly identifying common cattle and tumor cattle by using a deleted large-fragment rapid structure variation marker in a tumor cattle LRP5 gene and an application of the method. Specific high-frequency SV of the common cattle and the tumor cattle is identified through whole genome data analysis. Based on a PCR (Polymerase Chain Reaction) technology, a blood genome DNA (Deoxyribonucleic Acid) pool of Xinjiang brown cattle and Wenshan cattle is used as a template, specific upstream and downstream primers are used for amplifying structural variation of large fragment deletion of cattle LRP5 genes, then agarose gel electrophoresis is carried out, and different individuals are divided into a non-deletion type, a deletion type and a heterozygous type according to electrophoresis results. A correlation analysis result shows that different genotypes of the LRP5 gene are remarkably related to the cattle variety, so that the detection method disclosed by the invention can be applied to variety identification and marker-assisted selection breeding of common cattle and tumor cattle, an excellent cattle genetic resource group is quickly established, and the breeding cost is reduced.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of livestock molecular breeding research, and particularly relates to a method for distinguishing a large fragment deletion structural variation marker of an LRP5 gene related to common cattle and zebu cattle, which utilizes genomic DNA for PCR, followed by agarose gel electrophoresis, and genotyping according to the electrophoresis results. BACKGROUND

[0002] In a broad sense, cattle generally refers to two subspecies of common cattle and zebu cattle, and there is no reproductive isolation between the two, and they can be crossed without obstacles (Lenstra et al. 2012). Zebu cattle and common cattle have significant differences in morphology, and zebu cattle have shoulder peaks while common cattle do not, which is a symbol of the different evolutionary history of the two subspecies (Li Xin-yi, 2025). Chinese cattle are named yellow cattle because most of them are yellow or brownish yellow. China has rich local yellow cattle breeds, which are divided into northern yellow cattle, central yellow cattle obtained by crossing common cattle and zebu cattle, and southern yellow cattle originating from zebu cattle (Zhang Feng-wei, 2024). Zebu cattle originated in India and have excellent qualities such as heat resistance, disease resistance, and mosquito and lice resistance. Many European countries have used European beef cattle to crossbreed with them to breed new excellent breeds, and China has also introduced zebu cattle to improve local yellow cattle breeds. Due to the different proportions of common cattle and zebu cattle bloodlines, Chinese local yellow cattle have rich genetic diversity and a wide gene pool, providing rich breeding materials for new breed development. However, factors such as blind introduction of foreign crossbreeding, reduction of purebred yellow cattle, and insufficient protection of excellent yellow cattle sources pose many challenges and potential threats to the sustainable development and germplasm resource protection of China's local yellow cattle (Wang Qin-qian, 2025). Therefore, developing a method for rapidly identifying common cattle and zebu cattle plays an important role in marker-assisted selection breeding, rapid establishment of excellent cattle genetic resource groups, and protection of excellent local breeds.

[0003] Xinjiang Brown Cattle is the first self-bred milk and meat dual-purpose cattle breed in China, which is developed by using local yellow cattle Kazakh cattle as the female parent and Swiss Brown Cattle, Alatau Cattle, and Kostrom Cattle as the male parent, and has been bred for more than half a century. It has the characteristics of cold tolerance, roughage tolerance, strong adaptability, and suitability for grassland grazing, and is widely distributed in the northern part of Xinjiang (Ma Zhen, 2024; Wang Xiao, 2024). Wenshan Cattle is one of the representative breeds of zebu-origin southern yellow cattle (Zhang Feng-wei, 2024), with a solid body and well-developed muscles, and has good resistance to roughage, heat, and cold conditions. It is famous for its high shoulder peak and large dewlap (Wang Cheng-gao, 2023).

[0004] Structural Variation (SVs) refers to at least 50 bp of large genome sequence changes and positional relationship changes, including long fragment sequence insertion (Insertion, INS) or deletion (Deletion, DEL) with a length of more than 50 bp, duplication (Duplication, DUP), chromosomal inversion (Inversion, INV), sequence translocation between chromosomal internal or chromosomal, copy number variation (CNV) and more complex chimeric variation, compared with single nucleotide polymorphism (SNPs), it is expected to induce larger scale genomic perturbation in genes and regulatory regions, thereby affecting gene expression and phenotype (Cumer T, 2021). Tristan Cumer et al. revealed new regions associated with small ruminant domestication through whole genome detection of structural variation (SVs), and screened SVs associated with production traits, reproduction and immunity, and created the first map of all types of SVs in small ruminant genomes. Xia X et al. genotyped SVs in East Asian cattle, and determined 2610 SVs and 862 related genes enriched in environmental adaptation related pathways. The above shows that SV is a significant variation type in livestock genomes, and mining related SVs in livestock can lay a theoretical foundation for molecular marker assisted breeding, breed identification and protection of cattle. SUMMARY

[0005] The purpose of the present application is to provide an application of a bovine LRP5 gene SV marker in breed identification selection, so as to accelerate the breed identification of zebu and common cattle, and reduce the cost of breeding and conservation.

[0006] In order to achieve the above purpose, the technical scheme adopted by the present application is as follows: By analyzing the whole genome data of 62 cattle of 23 breeds (including 29 zebu and 33 common cattle), the SVs specific to common cattle and zebu were detected, and the high-frequency SVs specific to each were screened by calculating the allele frequency (Allele Frequency, AF). LRP5 is a specific high-frequency deletion mutation detected in 22 zebu individuals, and the mutation is not detected in the common cattle group.

[0007] A detection method of a bovine LRP5 gene large fragment insertion structural variation marker, comprising the following steps The DNA of the Xinjiang Brown Cattle and Wenshan Cattle individuals to be tested is used as a template, the primer pair SEQ ID NO. 1-2 is used as a primer, and the partial fragment of the bovine LRP5 gene containing the insertion / deletion (large fragment insertion structural variation) site is amplified by PCR. The amplification product is electrophoresed, and the genotype of the bovine individual is identified according to the electrophoresis result, so that different bovine individuals are divided into non-deletion type, deletion type and hybrid type: The primer pair is SEQ ID NO. 1-2.

[0008] The LRP5 gene insertion / deletion site is located at 45844973-45845559 bp of the bovine LRP5 gene reference genome sequence NC_037356.1, and has a total length of 586 bp. The genotype of the structural variation site of the bovine LRP5 gene large fragment insertion determined according to the agarose gel electrophoresis result is as follows: the non-deletion type is shown as the presence of a 1111 bp band; the heterozygous type is shown as the presence of two bands of 1111 bp and 525 bp; the deletion type is shown as a 525 bp band; in addition, due to the complexity of the genomic structural variation, most Wenshan cattle individuals have a band near 800 bp.

[0009] The reaction system for the PCR amplification is as follows: 0.1 ng-1 ug of template DNA, 0.8 uL of 10 uMol / L primer pair corresponding to the upper and lower primers of SEQ ID NO. 1-2, 10 uL of 2x Taq Rapid Master Mix, and ddH2O to make up to 20 uL.

[0010] The reaction program for the PCR amplification is as follows: 1) 95℃ pre-denaturation for 3 min; 2) 95℃ denaturation for 15 s, 58℃ annealing for 15 s, 72℃ extension for 30 s, for a total of 35 cycles; and 3) 72℃ terminal extension for 5 min.

[0011] The electrophoresis adopts agarose gel with a mass concentration of 2%.

[0012] The above method for detecting the structural variation marker of the bovine LRP5 gene large fragment insertion can be applied to bovine breed identification and molecular marker assisted selection breeding.

[0013] In bovine, the structural variation marker of the large fragment insertion can be used as a DNA marker for identifying bovine breeds.

[0014] The beneficial effects of the present application are as follows: The present application is directed to a large fragment insertion structural variation marker of bovine LRP5 gene located at 45844973-45845559 bp of chromosome 29 of bovine reference genome, which is identified by PCR amplification and agarose gel electrophoresis technology, and the genotypes of bovine individuals are identified according to the electrophoresis results, so that different bovine individuals are divided into non-deletion type, deletion type and hybrid type. According to the detection of the insertion / deletion polymorphism of the above region and the correlation analysis of the bovine breeds, it is shown that the insertion / deletion polymorphism site can be used as a molecular marker for identifying the breeds of tumor cattle and ordinary cattle. Therefore, the effective DNA marker for simply, quickly, low-cost and accurately identifying the breeds of ordinary cattle and tumor cattle can be used to quickly and accurately establish the population of excellent bovine genetic resources, so as to accelerate the breeding speed of bovine breeds. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 : Agarose gel electrophoresis map of PCR amplification product of large fragment insertion structural variation site of bovine individual LRP5 gene (primer pair SEQ ID NO. 1-2).

[0016] Figure 2 : Sequencing map of PCR amplification product of large fragment insertion structural variation site of bovine LRP5 gene (individuals with genotypes of non-deletion type and deletion type); the part marked by a horizontal line represents the deletion sequence: 45844973-45845559 bp of NC_037356.1. DETAILED DESCRIPTION

[0018] The present application will be described in detail below in combination with specific examples.

[0019] The present application uses PCR to identify the breeds of large fragment insertion structural variation marker of bovine LRP5 gene and for molecular breeding, which usually includes the following steps: (1) According to the start and end positions of the SV, the sequences of 400 bp upstream and downstream are extracted from the corresponding reference genome as reference sequences, the reference sequences are introduced into Primer 5 together with the designed primers according to the Primer-BLAST website of NCBI, it is verified whether the primers are completely matched to the upstream and downstream regions of the SVs, and the primers are detected by PCR.

[0020] (2) The genotyping of the candidate site in the population is detected by ordinary PCR technology and agarose gel electrophoresis, and different individuals are genotyped.

[0021] (3) GraphPrism 9 performs chi-square test and visualization on the test data.

[0022] (4) The breeds of tumor cattle and ordinary cattle are identified according to different types.

[0023] 1. Cattle sample sampling The present application specifically takes 62 cattle as the detection object, wherein 30 blood sample samples of Xinjiang Brown Cattle (code H) are collected from Huoerguosi City of Yili Kazak Autonomous Prefecture, and 32 DNA samples of Wenshan cattle (code W) are from Yunnan Agricultural University.

[0024] 2. Extraction of blood sample DNA ①Take 300 μL of anticoagulant whole blood and add 600 μL of Buffer TBP, mix well, and place at room temperature for 1 min until the red blood cells are completely lysed. At this time, the liquid is transparent red.

[0025] ②Centrifuge at 8,000 rpm for 1 min, discard the supernatant. Resuspend the precipitate with 500 μL of TE Buffer, centrifuge at 8,000 rpm for 1 min, carefully discard the supernatant, invert on a clean absorbent paper for a few seconds, and absorb the residual liquid. The precipitate can be washed once more with TE Buffer until it is white.

[0026] ③Add 180 μL of Buffer Digestion and 20 μL of Proteinase K solution, shake and mix well. Place in a 56°C water bath for 15-30 min until the cells are completely lysed.

[0027] ④Add 60 μL of Buffer PR, mix well by inverting, and place in a -20°C refrigerator for 5 min.

[0028] ⑤Centrifuge at room temperature for 10,000 rpm for 5 min, and transfer the supernatant (200 μL) to a new 1.5 mL centrifuge tube.

[0029] ⑥Add an equal volume of isopropanol, invert 5-8 times to mix well, and place at room temperature for 2-3 min. Centrifuge at room temperature for 10,000 rpm for 5 min, and discard the supernatant.

[0030] ⑦Add 1 mL of 75% ethanol, invert and rinse for 1-3 min, centrifuge at 10,000 rpm for 2 min, and discard the supernatant.

[0031] ⑧Repeat step 7 once ⑨Open the cap and invert at room temperature for 5-10 min until the residual ethanol is completely evaporated.

[0032] ⑩Dissolve the obtained DNA with 50 μL of TE Buffer. The extracted DNA can be immediately subjected to the next experiment or stored at -20°C.

[0033] 2.1 Agarose gel electrophoresis detection of DNA ①Clean the gel electrophoresis tank and insert the comb.

[0034] ② Weigh 1 g of agarose, pour it into a beaker, add 50 mL of 1×TAE to suspend it, heat it in the microwave on medium heat for 2 minutes to completely dissolve the agarose until it is clear and transparent, and when it has cooled to a temperature that is not hot to the touch, add 5 mL of Gold View nucleic acid dye and shake gently to prevent foaming.

[0035] ③ After mixing (60℃), immediately pour the agarose solution into the tank. If air bubbles appear, pop them with a pipette or move it to the end and let it cool completely (20 minutes) before removing the comb.

[0036] ④ Transfer the prepared gel to the electrophoresis apparatus, add 1×TAE to the electrophoresis tank so that the liquid level is 2-5mm higher than the gel level, so that the air in each pore is expelled and filled with liquid.

[0037] ⑤ Add the marker to the first sample, take 5 μL of DNA and load it onto each sample in sequence, electrophoresis at 120 V and 110 A for 30 minutes.

[0038] 2.2 Spectrophotometric Detection of DNA Measure the OD values ​​of the DNA sample at 260 nm and 280 nm using a UV spectrophotometer. Calculate the DNA content and the OD260 / OD280 ratio. For example, if the OD260 / OD280 ratio is less than 1.6, it indicates that the sample contains a large amount of protein or phenol, and purification should be performed; if the ratio is greater than 2.0, RNA removal and purification should be considered.

[0039] 3. PCR amplification conditions The PCR amplification system uses a mixed loading method. That is, based on the amount of each component required for each reaction system and the number of PCR reactions required for one reaction, the total amount of each reaction component is calculated and added to a 1.5 mL centrifuge tube. After thorough mixing, the tube is briefly centrifuged and then aliquoted into each 0.2 mL Eppendorf PCR tube. Template DNA is then added, and the tube is briefly centrifuged again before PCR amplification.

[0040] 3.1 PCR amplification system 0.1 ng-1 μg of template DNA, 0.8 μL each of the upstream and downstream primers corresponding to SEQ ID NO.1-2 (10 μmol / L), 10 μL of 2×Taq PCR StarMix, and ddH2O were added to make up to 20 mL.

[0041] 3.2 PCR reaction procedure 1) Pre-denaturation at 95℃ for 3 min; 2) Denaturation at 95℃ for 15 s, annealing at 58℃ for 15 s, extension at 72℃ for 30 s, for a total of 35 cycles; 3) Final extension at 72℃ for 5 min.

[0042] 4. Agarose gel electrophoresis detection analysis (1) Prepare a 2% agarose gel, stain with Gold View nucleic acid dye, load 5 μL, and then run at 120 V for 30 minutes; (2) When the DNA fragments of different molecular weights are clearly separated, image on a gel imaging system; (3) According to the results of agarose gel electrophoresis, analyze the structural variation polymorphism of large fragment insertion. See Figure 1 , the agarose gel electrophoresis results of the large fragment insertion structural variation site of the LRP5 gene of the cattle individual are as follows: the non-deletion type shows the presence of a 1111 bp band; the heterozygous type shows the presence of two bands of 1111 bp and 525 bp; the deletion type shows a 525 bp band; in addition, due to the complexity of genomic structural variation, most Wenshan cattle individuals have a band near 800 bp. The results were verified by sequencing, see Figure 2 . According to the analysis results, it was found that the large fragment insertion structural variation site (NC_037356.1 45844973-45845559 bp) detected in cattle has a 586 bp deletion.

[0043] Correlation analysis of large fragment structural variation site of LRP5 gene and breed in cattle Table 1. Correlation analysis of large fragment structural variation site of LRP5 gene and tail type traits in cattle As can be seen from Table 1, different genotypes of the insertion / deletion polymorphism site of the LRP5 gene (NC_037356.1 45844973-45845559 bp) have a very significant correlation with cattle breeds (P<0.05), and the non-deletion type of Xinjiang Brown cattle is dominant, and the deletion type and heterozygous type with deletion fragments are dominant in Wenshan cattle. Therefore, the non-deletion type of the above insertion / deletion polymorphism site can be used as a candidate genetic marker for identifying common cattle breeds.

Claims

1. A method for using structural variation markers to distinguish large fragment deletions in the LRP5 gene between ordinary cattle and zebu cattle, characterized in that, Includes the following steps: By analyzing the whole genome data of 62 cattle from 23 breeds (29 zebu cattle and 33 common cattle), unique SVs (segmented variants) were detected in both breeds. Allele frequencies (AFs) were calculated to identify high-frequency SVs specific to each breed. LRP5 was a unique high-frequency deletion mutation detected in 22 zebu individuals, but not in the common cattle group. Using genomic DNA from Xinjiang Brown cattle and Wenshan cattle as templates, partial fragments of the bovine LRP5 gene containing insertion / deletion sites were amplified by PCR using primer pairs labeled with structural variations indicating large deletions in the bovine LRP5 gene, corresponding to the nucleotide sequences shown in SEQ ID NO. 1-2. Agarose gel electrophoresis was then performed, and the different cattle individuals were classified into non-deletion, deletion, and heterozygous types based on the electrophoresis results.

2. The structural variation marker for large-fragment deletions related to the common cattle and zebu breeds according to claim 1, characterized in that, The reference genome version is ARS-UCD2.

0. The structural variant marker for the large fragment insertion is located at 45844973-45845559 bp on chromosome 29 of the bovine reference genome, which is a 586 bp deletion structural variant in the intron region where the bovine LRP5 gene is located.

3. The method for detecting structural variation markers of large fragment deletions in the bovine LRP5 gene according to claim 1, characterized in that: The PCR reaction procedure was as follows: 1) pre-denaturation at 95℃ for 3 min; 2) denaturation at 95℃ for 15 s, annealing at 58℃ for 15 s, extension at 72℃ for 30 s, for a total of 35 cycles; 3) final extension at 72℃ for 5 min; the electrophoresis was performed using a 2% agarose gel.

4. The method for detecting structural variation markers of a 586bp large fragment insertion in the bovine LRP5 gene according to claim 1, characterized in that: In agarose gel electrophoresis, the non-deletion type showed a 1111bp band; the heterozygous type showed two bands, 1111bp and 525bp, at the same time; the deletion type showed a 525bp band. In addition, due to the complexity of genomic structural variations, most Wenshan cattle individuals had a band around 800bp.

5. The application of the method for detecting structural variation markers of large fragment insertions in the bovine LRP5 gene as described in claim 1 in molecular marker-assisted selection breeding for the identification of common cattle and zebu breeds.

6. The application according to claim 5, characterized in that: The non-deletion type can be used as a DNA molecular marker for identifying and breeding ordinary cattle and zebu cattle.

7. A kit for detecting structural variation markers of large fragment insertions in the bovine LRP5 gene, characterized in that: The primer pairs used for PCR amplification of bovine insertion / deletion sites are SEQ ID NO. 1-2.

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