Application of SV marker of sheep PDGFD gene in early selection of tail type character

Abstract

The detection method comprises the following steps: based on a PCR (Polymerase Chain Reaction) technology, by taking a sheep blood genome DNA (Deoxyribonucleic Acid) pool as a template, amplifying the large-fragment-inserted structural variation of the sheep PDGFD gene by utilizing specific upstream and downstream primers SEQ ID NO.1-2, then carrying out agarose gel electrophoresis, and finally detecting the large-fragment-inserted structural variation of the sheep PDGFD gene. And classifying different individuals into an insertion type, a deletion type and a heterozygous type according to electrophoresis results. A correlation analysis result shows that different genotypes of the PDGFD gene are remarkably related to the sheep tail type character, and fat is deposited on the tail buttocks to directly influence the meat growth performance and carcass quality of the sheep, so that the sheep carcass lean meat percentage and the feed conversion efficiency can be improved by excavating the DNA marker of the sheep tail type character and applying the DNA marker to breeding; therefore, the detection method disclosed by the invention can be applied to marker-assisted selective breeding of sheep, and an excellent sheep genetic resource group can be quickly established.

Description

Technical Field

[0001] The invention belongs to the research field of livestock breeding, and specifically relates to a method for structural variation marking of a large segment of a PDGFD gene associated with sheep tail type traits. The method uses genomic DNA for PCR, then performs agarose gel electrophoresis, and performs typing according to the electrophoresis result. Background Art

[0002] my country has a vast territory and rich sheep germplasm resources. The tail shape of sheep is the product of long-term adaptive evolution. Fat is deposited in the tail of sheep, which can be used as energy storage to provide guarantee for the mobilization of local sheep. However, in the intensive and semi-stalled breeding mode, excessive fat deposition in the tail and buttocks of sheep will reduce feed conversion rate and affect the quality of meat. Therefore, it is urgent to cultivate new breeds with less tail fat deposition suitable for stalling to adapt to the development of the modern sheep industry.

[0003] PDGFD is an important member of the platelet growth factor family and belongs to the vascular endothelial growth factor (VEGF) family. PDGFD is a growth factor that can regulate cell growth and division. It is derived from platelets, smooth muscle cells, and activated macrophages. It participates in the regulation of embryonic development and plays an important regulatory role in the migration and proliferation of mesenchymal cells (Hannink and Donoghue, 1989). In human studies, the PDGFD gene is associated with human fat deposition. The gene is highly expressed in adipose-derived stem cells (ASCs). It can regulate the proliferation and migration of ASCs through the PI3K / Akt pathway, mitochondrial fission, and the generation of mitochondrial reactive oxygen species. However, it gradually decreases as ASCs develop into mature adipocytes. A large number of research reports have shown that the PDGFD gene is associated with fat deposition in the sheep tail and can be used as a key candidate gene for sheep tail type traits. For example, Han et al. conducted transcriptome analysis on developing sheep fat tail tissue and identified 17 genes related to early adipose tissue development, including the PDGFD gene; Zhu et al. conducted genome-wide association analysis on large-tailed Han sheep, Altay sheep and Tibetan sheep and found that there was a SNP site on the PDGFD gene of Altay sheep and Tibetan sheep, which was significantly associated with sheep tail type. Dong et al. found that the gene with the highest genetic differentiation between fat-tailed and thin-tailed sheep has a 6.8 kb region in its intron that may contain positive selection mutations that regulate fat deposition. Our previous studies revealed that there are many structural variations in the PDGFD gene that are associated with fat deposition in the sheep tail buttocks. In 2020, Lv et al. found through whole-genome resequencing analysis that the PDGFD gene is strongly positively selected in sheep tail fat selection, indicating that it is of great significance in the process of sheep domestication and breeding. The above results show that the PDGFD gene is a key selected region related to fat deposition in the sheep's tail. Exploring the mutations and variations of the PDGFD gene can lay a theoretical foundation for molecular marker-assisted breeding of sheep tail type traits. Summary of the invention

[0004] The purpose of the present invention is to provide an application of a sheep PDGFD gene SV marker in early selection of tail type traits, thereby accelerating the breeding speed of sheep.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions: A method for detecting structural variation markers of large fragment insertion of sheep PDGFD gene, comprising the following steps Using the DNA of the sheep individual to be tested as a template and the primer pair SEQ ID NO.1-2 as primers, PCR was used to amplify the partial fragment of the sheep PDGFD gene containing the insertion / deletion (structural variation of large fragment insertion) site, and the amplified product was subjected to electrophoresis. The sheep individual genotype was identified according to the electrophoresis results, so that different sheep individuals were divided into insertion type, deletion type and heterozygous type: The primer pair is SEQ ID NO.1-2.

[0006] The PDGFD gene insertion / deletion site is located at 3952061-3952192 bp of the sheep PDGFD gene reference genome sequence NC_056068.1, totaling 131 bp.

[0007] The genotype of the structural variation site where the sheep PDGFD gene large fragment is inserted is determined according to the agarose gel electrophoresis result. Specifically, the insertion type is shown as a band of 662 bp in the agarose gel electrophoresis result; the heterozygous type is shown as two bands of 531 bp and 662 bp; and the deletion type is shown as a band of 531 bp.

[0008] The reaction system used for the PCR amplification is: 2 μL of 200 ng / μL template DNA, 0.5 μL of each of the upstream and downstream primers corresponding to the primer pair SEQID NO.1-2 at 10 μmol / L, 10 μL of 2×Taq PCR StarMix and ddH2O.

[0009] The reaction procedure used in the PCR amplification is: 1) pre-denaturation at 94°C for 3 minutes; 2) denaturation at 94°C for 30 seconds, annealing at 58°C for 30 seconds, and extension at 72°C for 1 minute, for a total of 35 cycles; 3) extension at 72°C for 5 minutes.

[0010] The electrophoresis uses agarose gel with a mass concentration of 1.5%.

[0011] The above method for detecting structural variation markers of large fragment insertion in sheep PDGFD gene is applied in sheep molecular marker-assisted selection breeding.

[0012] In sheep, structural variation markers with large insertion fragments can be used as DNA markers to screen for sheep thin tails.

[0013] The beneficial effects of the present invention are embodied in: The structural variation marker for the insertion of a large fragment of the sheep PDGFD gene of the present invention is located at 3952061-3952192bp of chromosome 15 of the sheep reference genome. The genotype of the individual sheep is identified according to the electrophoresis results by PCR amplification and agarose gel electrophoresis identification technology, thereby different sheep individuals are divided into insertion type, deletion type and heterozygous type. According to the detection of the insertion / deletion polymorphism in the above-mentioned region and the association analysis with the important growth traits of the sheep, it is shown that the insertion / deletion polymorphic site can be used as a molecular marker for the selection of sheep tail type traits in molecular breeding. Therefore, the purpose of quickly and accurately establishing a population of excellent sheep genetic resources can be achieved by simply, quickly, low-cost and accurately detecting effective DNA markers related to sheep growth traits, thereby accelerating the speed of sheep breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 : Agarose gel electrophoresis of the product of PCR amplification (primer pair SEQ ID NO.1-2) of the structural variation site where a large fragment of the sheep PDGFD gene was inserted.

[0015] Figure 2 : Sequencing diagram of PCR amplification products of structural variation sites with large insertion fragments of sheep PDGFD gene (individuals with insertion type and deletion type genotypes); the part marked with horizontal lines represents the missing sequence: 3952061-3952192bp of NC_056068.1.

[0016] Figure 3 : Comparison of PDGFD sequencing results with genome sequences of different species retrieved by NCBI. DETAILED DESCRIPTION

[0017] The present invention is described in detail below with reference to specific embodiments.

[0018] The present invention uses PCR to detect structural variation markers of large fragment insertion of sheep PDGFD gene and uses it for molecular breeding, which generally includes the following steps: (1) Use the NCBI database to find the PDGFD gene sequence, then use Primer5 software to design primers, and use PCR to detect primers: (2) Use common PCR technology and agarose gel electrophoresis to detect the typing of candidate loci in the population and perform typing marking on different individuals: (3) Using SPASS 20.0 software, we conducted an association analysis between different genotypes and sheep tail traits: (4) Select and breed sheep with excellent tail traits according to different types.

[0019] 1. Sheep sampling The present invention specifically uses 152 sheep as detection objects, wherein blood samples of the 152 sheep are collected from the Animal Husbandry Teaching Experimental Station of Shihezi University in Xinjiang Uygur Autonomous Region, Mingyang Bashibai Animal Husbandry Co., Ltd. in Yumin County, Tacheng, Yili Agriculture and Animal Husbandry Technology Development Co., Ltd., and professional breeders in Sunan Yugur Autonomous County, Zhangye City, Gansu Province.

[0020] ① Take 300 μL of anticoagulated whole blood and add 600 μL of Buffer TBP, mix thoroughly, and leave at room temperature for 1 min until the red blood cells are completely lysed. At this time, the liquid is transparent red.

[0021] ②8,000rpm, centrifuge for 1min, discard the supernatant. Resuspend the precipitate with 500 μL TE Buffer, centrifuge for 1min at 8,000rpm, carefully discard the supernatant, invert on a clean absorbent paper for a few seconds, and absorb the remaining night. You can wash it again with TE Buffer until the precipitate is white.

[0022] ③Add 180 μL Buffer Digestion and 20 μL Proteinase K solution, shake and mix. Incubate in a 56℃ water bath for 15-30 min until cells are completely lysed.

[0023] ④ Add 60 μL Buffer PR, mix thoroughly by inversion, and place in a -20℃ refrigerator for 5 min.

[0024] ⑤ Centrifuge at 10,000 rpm for 5 min at room temperature and transfer the supernatant (200 μL) to a new 1.5 mL centrifuge tube.

[0025] ⑥Add an equal volume of isopropanol, invert 5-8 times to mix thoroughly, and leave at room temperature for 2-3 minutes. Centrifuge at 10,000 rpm for 5 minutes at room temperature and discard the supernatant.

[0026] ⑦ Add 1 mL of 75% ethanol, invert and rinse for 1 to 3 minutes, centrifuge at 10,000 rpm for 2 minutes, and discard the supernatant.

[0027] ⑧Repeat step 7 once ⑨Open the lid and invert at room temperature for 5 to 10 minutes until the remaining ethanol is completely evaporated.

[0028] ⑩ The obtained DNA was dissolved in 50μ TE Buffer. The extracted DNA can be used for the next experiment immediately or stored at -20℃.

[0029] 2.1 DNA detection by agarose gel electrophoresis ① Clean the gel electrophoresis tank and insert the comb.

[0030] ② Weigh 0.75 g of agarose, pour it into a beaker, add 50 mL of 1×TAE to suspend it, and heat it on medium heat in a microwave for 2 minutes to completely dissolve the agarose until it is clear and transparent. When it cools down to a level that is not too hot to touch, add 5 mL of Gold View Nucleic Acid Dye and shake gently to prevent foaming.

[0031] ③After mixing (60℃), pour the agarose solution into the tank immediately. If bubbles appear, puncture them with a pipette or move them to the tail. Wait for it to cool completely (20 minutes) and remove the comb.

[0032] ④ Move 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 surface, and discharge the air in each hole and fill it with liquid.

[0033] ⑤ Add the marker to the first sample, take 5 μL of DNA and load them one by one in sequence, and perform electrophoresis at 120 V and 110 A for 30 minutes.

[0034] 2.2 Spectrophotometric detection of DNA Use a violet photometer to measure the OD value of the DNA sample at 260nm and 280nm. Calculate the DNA content and the ratio of OD260 / OD280. For example, if the OD260 / OD280 ratio is less than 1.6, it means that the sample contains more protein or phenol, and purification should be performed; if the ratio is greater than 2.0, RNA purification should be considered.

[0035] 3. PCR Amplification Conditions The PCR amplification system adopted the mixed addition method, that is, according to the quantity of various components required for each reaction system and the number of PCR reactions required for one reaction, the total amount of various reaction components was calculated, added to a 1.5 mL centrifuge tube, mixed thoroughly and centrifuged instantly, and then dispensed into each 0.2 mL Eppendorf PCR tube, and then template DNA was added, and PCR amplification was performed after instant centrifugation.

[0036] 3.1 PCR amplification system 2 μL of 200 ng / μL template DNA, 0.5 μL of each of the upstream and downstream primers corresponding to the primer pair SEQ ID NO.1-2 at 10 μmol / L, 10 μL of 2×Taq PCR StarMix and ddH2O; a total of 20 mL.

[0037] 3.2 PCR reaction procedure Pre-denaturation at 94°C for 3 min; denaturation at 94°C for 30 s, annealing at 58°C for 30 s, extension at 72°C for 1 min, for a total of 35 cycles; extension at 72°C for 5 min.

[0038] 4. Agarose gel electrophoresis analysis 1) Prepare a 1.5% agarose gel, stain with Gold View nucleic acid dye, apply 6 μL, and then electrophoresed at 120V110 for 30 minutes; 2) When DNA fragments of different molecular weights are clearly separated, image them on a gel imaging system; 3) Analysis of structural variation polymorphism of large fragment insertions based on agarose gel electrophoresis results See also Figure 1 The agarose gel electrophoresis results of the structural variation site of the large fragment insertion of the sheep PDGFD gene are: the insertion type shows a band of 662bp; the heterozygous type shows two bands of 531bp and 662bp; the deletion type shows a band of 531bp. The results were verified by sequencing, see Figure 2 According to the analysis results, it was found that in sheep, there was a 131bp deletion at the structural variation site where the large fragment was inserted (3952061-3952192bp of NC_056068.1). Further analysis found that the non-deletion of the insertion type NC_056068.1 was the main effect mutation, and the base 131bp at this position was highly conserved in animals.

[0039] 5. Statistical analysis of the frequency of structural variation sites with large fragment insertion in sheep PDGFD gene 1) Gene and genotype frequencies Genotype frequency refers to the ratio of the number of individuals with a certain genotype of a trait in a population to the total number of individuals. The calculation formula can be written as: Pu = NDD / N In the formula, Pu represents the DD genotype frequency of a certain site; NDD represents the number of individuals with DD genotype in the population; and N is the total number of the tested population.

[0040] Gene frequency refers to the relative ratio of the number of a gene in a population to the total number of its alleles. The calculation formula can be written as: PD = (2ND + NDa1 + NDa2 + NDa3 + NDa4 + ... + NDa n ) / 2D In the formula, PD represents the frequency of allele D, NDD represents the number of individuals with DD genotype in the population, NDai represents the number of individuals with Dai genotype in the population, and ai=al,…,an are n different multiple alleles of allele D.

[0041] The genotype frequency and allele frequency of the structural variation site (3952061-3952192 bp of NC_056068.1) where the large fragment of the PDGFD gene was inserted in sheep individuals are shown in Table 1.

[0042] Table 1 Genotype statistics and population genetic diversity parameters As shown in Table 1, the structural variation site of the large fragment insertion of the PDGFD gene in sheep individuals (3952061-3952192bp of NC_056068.1) is II in the thin tail (Tibetan sheep, high Merino sheep, black-bone sheep), and the dominant genotype is I, with frequencies of 0.79, 0.79 and 0.60 respectively; in the fat tail (Hu sheep, Yemul sheep, Bashibai sheep, Kazakh sheep), the dominant genotype is DD, the dominant allele type is D, and the frequency is 0.69 , 0.80, 0.73, 0.87; because the dominant allele type of this SV in thin-tailed sheep (Tibetan sheep, high Merino sheep, black-bone sheep) is II, the tail fat deposition ability is weak; while the dominant genotype of this SV in fat-tailed sheep (Hu sheep, Yemul sheep, Bashbai sheep, Kazakh sheep) is DD, and the tail fat deposition ability is strong. Therefore, in actual production, local fat-tailed sheep can be bred according to this SV genotype, and the breeding of thin-tailed sheep can be accelerated to improve the feed conversion rate of sheep and increase the lean meat rate of carcass.

[0043] 6. Analysis of the association between large fragment structural variation sites of sheep PDGFD gene and tail type Table 2. Correlation analysis between large fragment structural variation sites of PDGFD gene and sheep tail type traits It can be seen from Table 2 that different genotypes of the insertion / deletion polymorphic site of the PDGFD gene (NC_056068.1 3952061-3952192bp) have a very significant effect on the tail type traits of individual sheep (P<0.05). The thin tail trait of the insertion type is better than that of the deletion type. Therefore, the insertion type of the above insertion / deletion polymorphic site can be used as a candidate genetic marker to improve the tail type trait (thin tail) of sheep.

Claims

1. A method for detecting structural variation markers of large fragment insertions of the sheep PDGFD gene, characterized in that: The following steps are involved: Using sheep genomic DNA as a template, a primer pair with a structural variation marker inserted into a large fragment of the sheep PDGFD gene was used as the nucleotide sequence shown in SEQ ID NO.1-2, and PCR amplification was performed to obtain a partial fragment of the sheep PDGFD gene containing an insertion / deletion site, and then agarose gel electrophoresis was performed. Different sheep individuals were divided into insertion type, deletion type and heterozygous type according to the agarose gel electrophoresis results.

2. A structural variation marker for a large insertion related to a sheep tail type trait according to claim 1, characterized in that: The structural variation marker for the large fragment insertion is located at 3952061-3952192 bp of chromosome 15 of the sheep reference genome, and is located at a 131 bp structural variation upstream of the location of the sheep PDGFD gene.

3. The structural variation of the large fragment insertion according to claim 1 is located at 3952061-3952192bp of chromosome 15 of the sheep reference genome and is annotated as a transposable element of a repeated fragment.

4. A method for detecting structural variation markers of a sheep PDGFD gene large fragment insertion according to claim 1, characterized in that: The reaction procedure used in the PCR was: 1) pre-denaturation at 94°C for 3 min; 2) denaturation at 94°C for 30 s, annealing at 58°C for 30 s, and extension at 72°C for 1 min, for a total of 35 cycles; 3) extension at 72°C for 5 min; the electrophoresis used agarose gel with a mass concentration of 1.5%.

5. A method for detecting structural variation markers of a 131 bp large fragment insertion in the sheep PDGFD gene according to claim 1, characterized in that: The insertion type showed a band of 662 bp in agarose gel electrophoresis results; the heterozygous type showed two bands of 531 bp and 662 bp; the deletion type showed a band of 531 bp.

6. Application of a method for detecting structural variation markers of large fragment insertion of sheep PDGFD gene as claimed in claim 1 in molecular marker-assisted selection breeding of sheep tail type traits.

7. The use according to claim 5, characterized in that: The insertion type can be used as a Central Asian DNA molecular marker for breeding sheep with thin tail phenotype.

8. A kit for detecting structural variation markers of large fragment insertions of sheep PDGFD gene, characterized in that: The primer pair included for PCR amplification of the sheep insertion / deletion site is SEQ ID NO. 1-2.

Images