SV molecular marker for logarithmic character of pig ribs and application of SV molecular marker

Abstract

The invention relates to the field of animal breeding, in particular to a pig rib logarithmic character SV molecular marker and application. And a primer pair for analyzing the specificity of the sites is provided, so that the early-stage precise breeding of the pigs is realized. The technical scheme has the advantages of simplicity and convenience in operation, low cost, accuracy in detection and the like, and has an important application value in precise molecular breeding of the pig rib logarithmic character.

Description

Technical Field

[0001] The present invention belongs to the field of animal breeding, specifically relates to the field of biological detection technology, and more specifically relates to an SV molecular marker combination for analyzing the number of pairs of pig ribs and its application. Background Art

[0002] China's pig industry has a long history, rich pig breed resources, wide distribution, and huge utilization potential and broad development prospects. Chinese indigenous pigs are an important component of China's pig germplasm resource bank. Through long-term natural and artificial selection, they have formed relatively unique genetic backgrounds and production performances.

[0003] Traditional breeding methods mainly rely on phenotypic selection, with low efficiency, limited to small-scale populations, and vulnerable to the complex interaction of genetic and environmental factors, resulting in limited reliability and application scope of the results. The number of pairs of pig ribs is an important biological trait. There is a direct correlation between the somite development in the embryonic stage and the number of ribs, which affects the body shape and growth performance of pigs. Research shows that for every additional rib in pigs, the carcass length increases by 80 mm and the carcass weight also increases accordingly. Therefore, in modern pig production, the selection of more ribs plays a key role in improving the production efficiency and economic benefits of pigs.

[0004] Genomic genetic variation is an important genetic basis for causing individual trait differences. Genomic variations can be classified into single nucleotide polymorphisms (SNPs), insertions and deletions (Indels), and structural variations (SVs) according to size. Genomic structural variation (SV) refers to a change in the length or orientation of a DNA sequence greater than 50 bp in the genome, including types such as insertions (INS), deletions (DEL), inversions (INV), duplications (DUP), and translocations (TRA). Research shows that SVs have the characteristics of involving more bases, wider coverage, and easier detection compared to SNPs, and the impact they have on the genome far exceeds that caused by SNPs. Therefore, attempting to develop new molecular markers based on SV loci to improve the selection efficiency and accuracy of the number of pairs of pig ribs trait, and thus assist in breeding, is an important technical means to promote the development of the technology in this field. Summary of the Invention

[0005] Aiming at the problems of low efficiency and poor reliability of existing breeding methods, the present invention proposes a molecular marker combination based on structural variation (SV) and its application. The achieved purpose is that the technical solution discovers two significantly related SV loci through genome-wide association analysis (GWAS) and provides specific PCR primer pairs for detecting these loci, thereby achieving early and precise selection of pigs.

[0006] To achieve the above object, the technical solution adopted by the present invention is: an SV molecular marker for detecting the logarithm trait of pig ribs, and the SV molecular marker is SV71 or / and SV284.

[0007] Further, the SV71 is a region of chromosome 7, positions 89528530-89528600 in the pig genome, and the structural variation in this region is the deletion or non-deletion of a 71bp fragment in the genome.

[0008] Further, the SV284 is a region of chromosome 7, positions 91896482-91896765 in the pig genome, and the structural variation in this region is the deletion or non-deletion of a 284bp fragment in the genome.

[0009] Further, the above SV locus positions are determined by aligning with the pig reference genome Sus_scrofa.Sscrofa11.1.

[0010] In particular, the pig is of the plum blossom star pig breed.

[0011] Primers for amplifying the above molecular markers, the upstream primer sequence for amplifying the SV71 locus is as shown in SEQ ID NO.1, and the downstream primer sequence is as shown in SEQ ID NO.2;

[0012] The upstream primer sequence for amplifying the SV284 locus is as shown in SEQ ID NO.3, and the downstream primer sequence is as shown in SEQ ID NO.4.

[0013] A probe combination for detecting the logarithm trait of pig ribs, and the probe combination is used to detect the above SV molecular marker combination.

[0014] A kit for detecting the logarithm trait of pig ribs, which has the above primer combination or the above probe combination.

[0015] A method for detecting the logarithm of pig ribs, using the above primer combination, probe combination, or the above kit to detect a sample to be tested, and determining the logarithm trait of the pig ribs of the sample to be tested according to the detection result.

[0016] Further, when determining the logarithm trait of the pig ribs of the sample to be tested according to the detection result, when there is no deletion of SV71 or / and SV284 in the chromosome of the sample to be tested, it is determined that the sample to be tested can increase the population base of the excellent pig rib logarithm trait in the population and increase the rib logarithm of the population.

[0017] The use of the above molecular marker, or the above probe, or the above kit, which is applied in:

[0018] (1) Detection of pig body size;

[0019] (2) Screening of pig rib logarithmic traits;

[0020] (3) Identification of logarithmic traits of pig ribs;

[0021] (4) Application in pig breeding.

[0022] In summary, the beneficial effects of the present invention are:

[0023] The two SV loci significantly correlated with the logarithmic traits of pig ribs provided by the present invention can quickly and accurately determine whether the pig sample to be tested can improve the group base number of excellent pig rib logarithmic traits in the group, increase the rib logarithm of the group, improve breeding efficiency, and thus improve the production performance and economic benefits of the group; the SV loci and primers, probes, and kits provided by the present invention can be used to complete SV loci detection under ordinary laboratory conditions, which is simple to operate and low-cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a graph showing the agarose gel electrophoresis result when DNA is extracted from a sample in Example 1 of the present invention;

[0025] Figure 2 The Manhattan plot of the logarithmic properties of the pork ribs in Example 1 of the present invention;

[0026] Figure 3 The phenotypic association analysis results of the two SV loci SV72 and SV284 in Application Example 1 of the present invention;

[0027] Figure 4 This is the result of the association analysis between the two SV site combination types and phenotypes in Example 2 of the present invention. DETAILED DESCRIPTION

[0028] The technical solutions in the embodiments of the present invention will be described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without creative work are within the scope of protection of the present invention. The bioinformatics software and products used in the present invention are all commercially available. Various processes and methods not described in detail all adopt conventional methods known to those skilled in the art. The sources of materials used, trade names, and those that need to list their components are all indicated when they first appear. If there is no special explanation, the same reagents used thereafter are the same as those indicated for the first time.

[0029] Example 1 Obtaining SV molecular markers for the logarithmic traits of pig ribs

[0030] The SV molecular markers for detecting the logarithmic trait of the ribs of plum blossom star pigs provided by the present invention, the SV molecular marker combination includes the SV71 structural variation locus and the SV284 structural variation locus.

[0031] Among them, SV71 is the region of 89528530-89528600 on chromosome 7 in the pig genome, and the structural variation in this region is the deletion or non-deletion of a 71bp fragment in the genome;

[0032] SV284 is the region of 91896482-91896765 on chromosome 7 in the pig genome, and the structural variation in this region is the deletion or non-deletion of a 284bp fragment in the genome;

[0033] The positions of the SV loci are determined by aligning with the reference genome Sus_scrofa.Sscrofa11.1 of pigs.

[0034] The following details the complete process from sample collection to data analysis and is described in conjunction with the accompanying drawings.

[0035] 1. Phenotype collection and DNA extraction

[0036] Phenotype statistics were performed on 79 plum blossom star pig populations with different rib logarithms, and the corresponding tissue samples were collected and DNA was extracted.

[0037] Both the phenotype statistics method and the DNA extraction method adopt conventional techniques in the art, and the present invention will not specifically describe them. According to the statistical results, the rib logarithms of the 79 plum blossom star pigs in the present invention are between 13 and 16, as shown in Table 1 specifically:

[0038] Table 1 Descriptive statistics of the rib logarithmic traits of 79 plum blossom star pigs

[0039]

[0040] Since sequencing has certain requirements for the quality of DNA, too low DNA quality will affect the sequencing results. Therefore, in an embodiment of the present invention, 2% agarose gel electrophoresis is used to detect whether the extracted DNA is degraded. The electrophoresis results are as Figure 1 shown. It can be seen from the picture that the DNA sample bands are clear, bright, complete, and there is no trailing phenomenon, indicating that the DNA has not been degraded; the detection results by the ultra-micro spectrophotometer show that the lowest concentration of DNA is 60.65 ng / ul, the highest concentration is 1492.30 ng / ul, and the average concentration is 478.32 ng / ul. The minimum value of OD260 / 280 is 1.87, the maximum value is 2.02, and the average value is 1.95. Through the above detection results, it can be known that the DNA quality of the test population is good and meets the sequencing requirements.

[0041] 2. DNA data processing

[0042] 79 DNA samples with qualified quality were sent to Wuhan Yingzi Gene Technology Co., Ltd. for 20× resequencing (BGI sequencing platform). The original sequencing data were quality controlled using fastp (v0.23.4). The original FASTQ files were then aligned to the reference genome of the pig (Sus_scrofa.Sscrofa11.1) using BWA software, and then the bam files were sorted, deduplicated, deredundant, and indexed using samtools to obtain the preliminarily processed bam files.

[0043] 3. Detection of whole genome SNPs

[0044] To ensure the integrity of the data, in one embodiment of the present invention, whole-genome SNP detection is performed on sequencing data of qualified quality. The specific method is as follows: Haplotype Caller, GenotypeGVCFs and CombineGVCFs tools of GATK are used to detect genetic variations and perform genotyping and merging. The Haplotype Caller tool was used to detect variants and generate respective GVCF files, which recorded the variant information of all sites in preparation for joint typing. The CombineGVCFs tool was used to merge the GVCF files of all samples, and the GenotypeGVCFs tool was used for joint typing to obtain the original VCF file containing SNP variant information. The SelectVariants tool was used to extract SNP variant sites from the original VCF file, and the Variant Filtration tool was used to strictly filter SNPs, with the set filtering parameters of "QD<2.0, QUAL<30.0, SOR>3.0, FS>60.0, MQ<40.0, MQRankSum<-12.5, ReadPosRankSum<-8.0". Variants with coverage>30% and minor allele frequency (MAF)>0.01 were retained, and SNPs within 5bp near InDel were removed. vcftools was used to perform preliminary quality control on SNP loci. The quality control criteria were: the call rate of single-site SNP reached more than 90%; the call rate of individual SNP reached more than 90%; the minimum allele frequency maf threshold was set to 0.05. Finally, 21,692,852 high-quality SNP loci were obtained for subsequent analysis.

[0045] 4. Detection of whole genome SV

[0046] Insertions and deletions were detected using four different tools, Delly (v1.2.6), Manta (v1.6.0), Wham (v1.8.0), and Smoove (v0.2.8). SVs with a deletion rate < 30% and MAF > 0.01 were retained, resulting in 16,645 SV loci.

[0047] 5. Construction of kinship and genome-wide association analysis

[0048] Since population structure and kinship may lead to complex genetic correlations among samples, if not corrected, it may lead to false positive results. Therefore, constructing a kinship matrix can effectively correct these confounding effects and make the association analysis between structural variants (SVs) and phenotypes more accurate. The kinship matrix of the present invention is constructed based on 21,692,852 high-quality SNPs obtained in step 3.

[0049] The present invention uses the single marker regression analysis method in GCTA software for GWAS analysis, and the mixed linear model is as follows:

[0050] y = Xb + Za + e

[0051] In the above model, y represents the phenotypic value; b is the SV effect, a represents the remaining polygenic effect, is the individual additive genetic variance, G is the kinship matrix constructed based on SNPs, X and Z are the association matrices of b and a respectively; e represents the residual effect vector, is the residual variance.

[0052] Using GCTA software, a genome-wide association analysis (GWAS) based on SVs was performed on the rib number trait of 79 Meihuaxing pigs, and the results were visualized using R software (such as Figure 2 ), and 3 SV loci significantly associated with the rib number trait of Meihuaxing pigs were obtained.

[0053] 6. Screening of SVs for pig rib number trait

[0054] The SVs were screened according to the phenotypic data of the pig rib number trait. The screening method can be a conventional method in the art. For example, in one embodiment of the present invention, the phenotypic data was used to verify the SV loci screened in step 5, so as to discover SV loci that can quickly and accurately analyze the pig rib number. After screening, the SV71 structural variation locus located in the region of 89528530 - 89528600 on chromosome 7 with a fragment size of 71 bp, and the SV284 structural variation locus located in the region of 91896482 - 91896765 on chromosome 7 with a fragment size of 284 bp provided by the present invention were obtained.

[0055] For another example, in another embodiment of the present invention, first, transposable element annotation is performed on the SV locus to clarify the cause of SV generation, and it is found that the SV of the present invention is caused by transposons. Subsequently, gene annotation is performed using the pig Susscrofa 11.1 version database on the Ensemble online platform, and functional annotation is performed on each gene using relevant gene information on websites such as NCBI and Ensemble. Combining the above methods, the relevant SVs are defined as candidate loci for the number of pig rib pairs. Finally, gene typing verification of the candidate loci is performed by PCR technology, and the SV71 structural variation locus with a fragment size of 71 bp located in the region of 89528530 - 89528600 on chromosome 7 and the SV284 structural variation locus with a fragment size of 284 bp located in the region of 91896482 - 91896765 on chromosome 7 provided by the present invention are obtained.

[0056] Primers for analyzing the number of pig rib pairs in Example 2

[0057] According to the locus information of SV71 and SV284 provided in Example 1, those skilled in the art can design primers according to conventional biological methods, and the present invention does not make specific limitations. As long as the primers obtained using the loci provided by the present invention belong to the protection scope of the present invention. In one embodiment of the present invention, the primers designed according to the locus information of Example 1 are shown in Table 2:

[0058] Table 2 Primer sequences

[0059]

[0060] Kit for analyzing the number of pig rib pairs in Example 3

[0061] According to the locus information of SV71 and SV284 provided in Example 1 or the primer information of Example 2, those skilled in the art can prepare a kit according to conventional biological methods, and the present invention does not make specific limitations. As long as the kit obtained using the loci provided by the present invention belongs to the protection scope of the present invention. In one embodiment of the present invention, the kit obtained according to the primer information of Example 2 is a PCR kit. Taking a 10 μL amplification system as an example, the reaction process using this kit is as follows:

[0062] The PCR reaction conditions for SV71 are: 1) Pre-denaturation: 94.0 °C for 5 min; 2) Amplification reaction: Denaturation: 94.0 °C for 30 sec, Annealing: 58.0 °C for 30 sec, Extension: 72.0 °C for 1 min, 35 cycles; 72.0 °C for 10 min. 3) Store at 4 °C.

[0063] The PCR reaction conditions for SV284 are as follows: 1) Pre-denaturation: 94.0°C for 5 min; 2) Amplification reaction: Denaturation: 94.0°C for 30 sec, Annealing: 60.0°C for 30 sec, Extension: 72.0°C for 1 min, 35 cycles; 72.0°C for 10 min. 3) Store at 4°C.

[0064] Example 4 Analysis of the logarithmic trait of pig ribs

[0065] Use the locus information provided in Example 1 or the primer information provided in Example 2 or the kit provided in Example 3 to analyze the logarithmic trait of pig ribs. The specific process is as follows:

[0066] Collect tissue samples of the pigs to be tested and extract their DNA to obtain DNA test samples of the pigs to be tested. Use the primer information provided in Example 2 or the kit provided in Example 3 to perform PCR reaction and agarose gel electrophoresis detection on the DNA test samples, determine the genotype of each sample in the population sample, and preferentially retain individuals without deletion of SV71 and SV284 according to the genotype to increase the population base of excellent pig rib logarithmic traits in the population and improve the rib logarithm of the population.

[0067] Apply Example 1 to analyze the logarithmic trait of pig ribs using each SV locus respectively

[0068] The following is the specific implementation process and analysis results of the analysis of the logarithmic trait of pig ribs in the plum blossom star pig population using the loci, primers or kits and methods of Examples 1-3 of the present invention

[0069] 1. Sample collection

[0070] Statistical phenotypic data of 297 plum blossom star pig populations with different rib logarithms, collect ear tissues of 297 plum blossom star pigs and store them in 75% alcohol at -20°C for DNA extraction;

[0071] 2. Extraction and detection of pig ear tissue DNA

[0072] 1) Extract DNA from pig ear tissue;

[0073] 2) Measure the concentration of DNA using an ultraviolet spectrophotometer, and at the same time perform agarose gel electrophoresis to detect the quality of DNA.

[0074] 3. Genotype determination

[0075] Determine the genotypes of two SV loci of qualified DNA, specifically:

[0076] 1). Perform PCR reaction on qualified DNA

[0077] Perform PCR on the qualified DNA samples using the primer pairs of Example 2 (shown in Table 2) to obtain PCR reaction products.

[0078] Among them, the PCR reaction conditions for SV71 are as follows: 1) Pre-denaturation: 94.0°C for 5 min; 2) Amplification reaction: Denaturation: 94.0°C for 30 sec, Annealing: 58.0°C for 30 sec, Extension: 72.0°C for 1 min, 35 cycles; 72.0°C for 10 min. 3) Store at 4°C.

[0079] Among them, the PCR reaction conditions for SV284 are as follows: 1) Pre-denaturation: 94.0°C for 5 min; 2) Amplification reaction: Denaturation: 94.0°C for 30 sec, Annealing: 63.0°C for 30 sec, Extension: 72.0°C for 1 min, 35 cycles; 72.0°C for 10 min. 3) Store at 4°C.

[0080] 2), Detection by agarose gel electrophoresis

[0081] Select 2.0% agarose gel for electrophoresis detection. When loading the sample, use a pipette to add 3 μL of the PCR product into the sample loading well, and at the same time add 3 μL of D2000 DNA Marker as a control. Set the voltage to 100 V and the time to 30 min (50 min for SV71). After electrophoresis is completed, observe the results in a gel imaging system and perform genotype detection.

[0082] 3), Significance test

[0083] Use SAS software to perform a significance test on the genotype and the number of rib pairs trait, and use GraphPad Prism software for result visualization. The results are shown in Table 3 and Figure 3 as follows.

[0084] Table 3 Association analysis of genotypes at two loci and phenotypes

[0085]

[0086] Note: + in the table indicates no deletion, and - indicates deletion.

[0087] According to the results in Table 3, it can be seen that the two SV loci provided by the present invention are significantly correlated with the number of rib pairs (P<0.05). For SV71, the number of rib pairs of the plum blossom star pigs with the + / + genotype is 14.61±0.50, that of the + / − genotype is 14.29±0.50, and that of the − / − genotype is 14.15±0.66. The number of rib pairs of the + / + genotype is significantly greater than that of the + / − and − / − genotypes; for SV284, the number of rib pairs of the plum blossom star pigs with the + / + genotype is 14.70±0.47, that of the + / − genotype is 14.32±0.50, and that of the − / − genotype is 14.13±0.61. The number of rib pairs of the + / + genotype is significantly greater than that of the + / − and − / − genotypes. From the test results of 297 pigs, it can be seen that there are more individuals with the − / − genotype at the two SV loci in the population, and the number of rib pairs of the − / − genotype plum blossom star pigs is significantly lower than that of the + / + genotype, indicating that there is still great potential for improvement in the population.

[0088] Example of application 2 Analyze the trait of the number of rib pairs in pigs using two SV loci together Except for using the detection results of the two SV loci to judge the samples simultaneously, the others are the same as in Example of application 1. The results of the significance test are shown in Table 4 and Figure 4 as shown.

[0089] Table 4 Association analysis of the combined types of two SV loci and phenotypes

[0090]

[0091] Note: In the table, + indicates that the locus is not deleted, and - indicates that the locus is deleted. Different superscripts for the data of the number of rib pairs indicate significant differences (P<0.05); the same superscripts or no superscript annotation indicate no significant differences (P>0.05).

[0092] From Table 4 and Figure 4 it can be seen that the simultaneous action of the two SV loci is also significantly correlated with the number of rib pairs (P<0.05). The number of rib pairs of the ++ / ++ and ++ / +- types of SV71_SV284 is significantly higher than that of the -- / -- and +- / -- types. Generally speaking, the number of rib pairs when neither of the two loci is deleted is significantly higher than that when both loci are deleted.

[0093] It can be seen that in actual breeding, selecting and retaining the plum blossom star pigs with the + / + genotype can increase the population base of excellent traits in the population, increase the number of rib pairs in the population, and specifically improve the trait of the number of rib pairs in the population to achieve the purpose of improving the production performance and economic benefits of the entire population.

[0094] The above are the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. SV molecular markers for detecting the logarithmic traits of pig ribs, characterized in that: The SV molecular markers include SV71 and / or SV284; Among them, SV71 is the region 89528530-89528600 of chromosome 7 in the pig genome, and the structural variation of this region is the deletion or non-deletion of a 71bp fragment in the genome; Among them, SV284 is the region of chromosome 7, 91896482-91896765 in the pig genome, and the structural variation in this region is the deletion or non-deletion of a 284 bp fragment in the genome; The SV site position was determined by comparing with the pig reference genome Sus_scrofa.Sscrofa11.

1.

2. A primer combination for detecting the logarithmic traits of pig ribs, the primer sequences of which are shown in SEQ ID NO.1-SEQ ID NO.

4.

3. A probe combination for detecting the logarithmic traits of pig ribs, which is used to detect the structural variations of SV71 and SV284 in the sample to be tested.

4. A kit for detecting the logarithmic traits of pig ribs, comprising the primer combination of claim 2 or the probe combination of claim 3.

5. A method for detecting the logarithmic traits of pig ribs, comprising: detecting a sample by using the primer combination of claim 2, or the probe combination of claim 3, or the kit of claim 4.

6. The method of claim 5, comprising: The sample to be tested is tested using the primer combination provided in claim 2, the probe combination provided in claim 3, the kit provided in claim 4, or the method provided in claim 5, and the logarithmic properties of the pig ribs of the sample to be tested are determined according to the test results.

7. The method as claimed in claim 6, wherein the test result is used to determine the logarithmic traits of pig ribs of the sample to be tested. When the chromosome of the sample to be tested does not contain the deletion of SV71 and / or SV284, it is determined that the sample to be tested can increase the population base of the excellent logarithmic traits of pig ribs in the population and increase the rib logarithm of the population.

8. Use of the molecular marker according to claim 1, the primer according to claim 2, the probe according to claim 3, or the kit according to claim 4, characterized in that: Apply it in: (1) Detection of pig size; (2) Variety screening for logarithmic traits of pig ribs; (3) Identification of logarithmic traits of pig ribs; (4) Application in pig breeding.

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