SV molecular markers for porcine rib traits and their application

Abstract

The application relates to the field of animal breeding, in particular to a SV molecular marker of a pig rib number character and application. Specific primer pairs for analyzing the sites are provided, so that early accurate breeding of pigs is realized. The technical scheme has the advantages of simple operation, low cost, accurate detection and the like, and has important application value in molecular accurate breeding of the pig rib number character.

Description

Technical Field

[0001] This invention belongs to the field of animal breeding, specifically to the field of biological detection technology, and more specifically to the analysis of SV molecular marker combinations for the logarithmic traits of pig ribs and their applications. Background Technology

[0002] my country has a long history of pig farming, abundant and widely distributed pig breeds, and enormous utilization potential and broad development prospects. Local Chinese pigs are an important component of my country's pig germplasm resource bank, having developed unique genetic backgrounds and production performance through long-term natural and artificial selection.

[0003] Traditional breeding methods rely primarily on phenotypic selection, which is inefficient and limited to small populations. They are also susceptible to the complex interactions of genetic and environmental factors, resulting in limited reliability and applicability. The number of rib pairs in pigs is an important biological trait; somatic development during the embryonic period is directly related to the number of ribs, influencing body shape and growth performance. Studies have shown that each additional rib increases carcass length by 80 mm and carcass weight. Therefore, in modern pig production, selective breeding for more ribs plays a crucial role in improving production efficiency and economic benefits.

[0004] Genomic variation is a crucial genetic basis for individual phenotypic differences. Genomic variations can be categorized by size into single nucleotide polymorphisms (SNPs), insertions / deletions (Indels), and structural variations (SVs). Genomic structural variations (SVs) refer to changes in the length or orientation of DNA sequences larger than 50 bp in the genome, including insertions (INS), deletions (DELs), inversions (INVs), duplications (DUPs), and translocations (TRAs). Studies have shown that SVs, compared to SNPs, involve more bases, have a wider coverage, and are easier to detect; their impact on the genome is far greater than that of SNPs. Therefore, developing new molecular markers based on SV sites to improve the efficiency and accuracy of selection for logarithmic traits in pig ribs, thereby aiding breeding, is an important technological means to advance this field. Summary of the Invention

[0005] This invention addresses the problems of low efficiency and poor reliability in existing breeding methods by proposing a combination of structural variation (SV) molecular markers and their applications. The objective is to achieve early precision selection in pigs by using genome-wide association analysis (GWAS) to identify two significantly related SV loci and providing specific PCR primer pairs for detecting these loci.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is: SV molecular markers for detecting the logarithmic traits of pig ribs, wherein the SV molecular markers are SV71 and / or SV284.

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

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

[0009] Furthermore, the locations of the aforementioned SV sites were determined by comparing them with the pig reference genome Sus_scrofa.Sscrofa11.1.

[0010] In particular, the pigs in question are of the Plum Blossom Star Pig breed.

[0011] The primers used to amplify the above molecular markers are shown in SEQ ID NO.1 for the upstream primer sequence and SEQ ID NO.2 for the downstream primer sequence.

[0012] The upstream primer sequence used to amplify the SV284 site is shown in SEQ ID NO.3, and the downstream primer sequence is shown in SEQ ID NO.4.

[0013] A probe array for detecting the logarithmic trait of pig ribs, wherein the probe array is used to detect the SV molecular marker array.

[0014] A kit for detecting the logarithmic traits of pig ribs, which has the above-described primer combination or probe combination.

[0015] The method for detecting the logarithmic number of pig ribs involves using the primer and probe combinations described above, or the kit described above, to test the sample. The logarithmic trait of the pig ribs in the sample is then determined based on the test results.

[0016] Furthermore, the determination of the logarithmic trait of the pig ribs in the test sample based on the test results is that if the chromosome of the test sample does not have the deletion of SV71 or / and SV284, then it is determined that the test sample can increase the population base of the excellent logarithmic trait of pig ribs in the population and increase the number of rib pairs in the population.

[0017] The aforementioned molecular markers, probes, or kits are intended for use in:

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

[0019] (2) Screening of pig breeds with logarithmic traits of ribs;

[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 as follows:

[0023] The two SV loci provided by this invention are significantly associated with the logarithmic trait of pig ribs. They can quickly and accurately determine whether the pig sample to be tested can improve the population base of the excellent logarithmic trait of pig ribs in the population, increase the number of ribs in the population, improve breeding efficiency, and thus improve the production performance and economic benefits of the population. The SV loci, primers, probes and kits provided by this invention can be used to complete the SV locus detection under ordinary laboratory conditions. The operation is simple and the cost is low. Attached Figure Description

[0024] Figure 1 This is an image showing the agarose gel electrophoresis results of DNA extraction from the sample in Example 1 of this invention;

[0025] Figure 2 This is a Manhattan diagram of the logarithmic traits of pig ribs in Embodiment 1 of the present invention;

[0026] Figure 3 This refers to the phenotypic association analysis results of the two SV loci, SV72 and SV284, in Example 1 of this invention.

[0027] Figure 4 This is the result of the association analysis between the combination type of two SV sites and the phenotype in Example 2 of this invention. Detailed Implementation

[0028] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort 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 are all conventional methods known to those skilled in the art. The source of materials used, trade names, and components that need to be listed are indicated when they first appear. Unless otherwise specified, the same reagents used thereafter are the same as those initially indicated.

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

[0030] The present invention provides SV molecular markers for detecting the logarithmic traits of ribs in Plum Blossom Star Pigs, wherein the SV molecular marker combination includes SV71 structural variant sites and SV284 structural variant sites.

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

[0032] SV284 is a region on chromosome 7 of the pig genome, from 91896482 to 91896765. The structural variation in this region is the deletion or non-deletion of a 284bp segment in the genome.

[0033] The location of the SV locus was determined by comparing it with the pig reference genome Sus_scrofa.Sscrofa11.1.

[0034] The following details the complete process from sample collection to data analysis, with accompanying diagrams.

[0035] 1. Phenotyping and DNA extraction

[0036] Phenotypic analysis was performed on 79 Meihuaxing pigs with different numbers of rib pairs, and corresponding tissue samples and DNA were extracted.

[0037] The phenotypic statistical methods and DNA extraction methods used are conventional techniques in the field, and will not be specifically described in this invention. Statistical results show that the number of rib pairs in the 79 Meihuaxing pigs of this invention is between 13 and 16, as shown in Table 1.

[0038] Table 1. Descriptive statistics of the logarithmic traits of ribs in 79 Meihuaxing pigs.

[0039]

[0040] Since sequencing requires certain DNA quality, and low DNA quality can affect sequencing results, in one embodiment of this invention, 2% agarose gel electrophoresis is used to detect whether the extracted DNA has degraded. The electrophoresis results are as follows: Figure 1 As shown in the image, the DNA sample bands are clear, bright, complete, and without tailing, indicating that the DNA has not been degraded. The results of ultra-micro spectrophotometry analysis show that the lowest DNA concentration was 60.65 ng / µl, the highest was 1492.30 ng / µl, and the average was 478.32 ng / µl. The minimum OD260 / 280 was 1.87, the maximum was 2.02, and the average was 1.95. These results indicate that the DNA quality of the experimental population is good and meets the sequencing requirements.

[0041] 2. DNA Data Processing

[0042] Seventy-nine qualified DNA samples were sent to Wuhan Shadow Gene Technology Co., Ltd. for 20× resequencing (BGI sequencing platform). Raw sequencing data were quality controlled using FASTP (v0.23.4). The resulting raw FASTQ files were then aligned to the pig reference genome (Sus_scrofa.Sscrofa11.1) using BWA software. Subsequently, samtools was used to sort, deduplicate, remove redundancy, and index the obtained BAM files, resulting in pre-processed BAM files.

[0043] 3. Detection of whole-genome SNPs

[0044] To ensure data integrity, in one embodiment of the present invention, whole-genome SNP detection is performed on qualified sequencing data. The specific method is as follows: genetic variations are detected and genotyping and merging are performed using GATK's Haplotype Caller, GenotypeGVCFs and CombineGVCFs tools. The Haplotype Caller tool was used to detect variants and generate individual GVCF files. These GVCF files recorded variant information for all loci, preparing for joint genotyping. The CombineGVCFs tool was used to merge the GVCF files of all samples, and the GenotypeGVCFs tool was used for joint genotyping 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 the SNPs with the following parameters: 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 allelic frequency (MAF) > 0.01 were retained, while SNPs within 5 bp of InDel were removed. Preliminary quality control of SNP loci was performed using vcftools. The quality control criteria were: a call rate of over 90% for a single SNP locus; a call rate of over 90% for an individual; and a minimum allele frequency (MAF) threshold of 0.05. A total of 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 of <30% and MAF >0.01 were retained, resulting in 16,645 SV sites.

[0047] 5. Kinship construction and genome-wide association analysis

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

[0049] This invention uses the single-label 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 represents the SV effect; and a represents the residual polygenic effect. Let be the additive genetic variance of individuals, G be the kinship matrix constructed based on SNPs, X and Z be the association matrices of b and a, respectively; e represents the residual effect vector. This represents the residual variance.

[0052] Genome-wide association analysis (GWAS) based on SV was performed on the logarithmic rib trait of 79 Sika Star pigs using GCTA software, and the results were visualized using R software (e.g., Figure 2 Three SV loci were found to be significantly associated with the logarithmic traits of the ribs of the Meihuaxing pig.

[0053] 6. SV screening of logarithmic traits in pig ribs

[0054] SVs were screened based on phenotypic data of the logarithmic traits of pig ribs. The screening method can follow conventional methods in the art. For example, in one embodiment of the present invention, phenotypic data was used to verify the SV loci screened in step 5, thereby discovering SV loci that can quickly and accurately analyze the logarithmic traits of pig ribs. After screening, the SV71 structural variant locus, located in the region 89528530-89528600 of chromosome 7 with a fragment size of 71 bp, and the SV284 structural variant locus, located in the region 91896482-91896765 of chromosome 7 with a fragment size of 284 bp, were obtained, as provided by the present invention.

[0055] For example, in another embodiment of the present invention, transposon elements were first annotated at the SV sites to clarify the cause of SV generation. It was found that the SVs of the present invention are caused by transposons. Subsequently, gene annotation was performed using the porcine Susscrofa version 11.1 database on the Ensemble online platform, and functional annotation was performed on each gene using relevant gene information from websites such as NCBI and Ensembl. Combining the above methods, the relevant SVs were defined as candidate sites for the number of porcine rib logs. Finally, genotyping verification of the candidate sites was performed using PCR technology, obtaining the SV71 structural variant site with a fragment size of 71 bp located in the region 89528530-89528600 of chromosome 7, and the SV284 structural variant site with a fragment size of 284 bp located in the region 91896482-91896765 of chromosome 7, provided by the present invention.

[0056] Example 2: Primers for analyzing the logarithmic traits of pig ribs

[0057] Based on the SV71 and SV284 site information provided in Example 1, those skilled in the art can design primers using conventional biological methods. This invention does not impose specific limitations; any primers obtained using the sites provided by this invention fall within the scope of protection of this invention. In one embodiment of this invention, the primers designed based on the site information of Example 1 are shown in Table 2:

[0058] Table 2 Primer sequences

[0059]

[0060] Example 3: Kit for analyzing the logarithmic traits of pig ribs

[0061] Based on the SV71 and SV284 site information provided in Example 1 or the primer information in Example 2, those skilled in the art can prepare the kit using conventional biological methods. This invention does not impose specific limitations; any kit obtained using the sites provided by this invention falls within the scope of protection of this invention. In one embodiment of this invention, the kit obtained based on the primer information in Example 2 is a PCR kit. Taking 10 μL as an example, the reaction process using this kit is as follows:

[0062] The PCR reaction conditions for SV71 were as follows: 1) Pre-denaturation: 94.0℃ for 5 min; 2) Amplification reaction: denaturation: 94.0℃ for 30 sec, annealing: 58.0℃ for 30 sec, extension: 72.0℃ for 1 min, 35 cycles; 72.0℃ for 10 min. 3) Store at 4℃.

[0063] The PCR reaction conditions for SV284 were as follows: 1) Pre-denaturation: 94.0℃ for 5 min; 2) Amplification reaction: denaturation: 94.0℃ for 30 sec, annealing: 60.0℃ for 30 sec, extension: 72.0℃ for 1 min, 35 cycles; 72.0℃ for 10 min. 3) Store at 4℃.

[0064] Example 4: Analysis of the logarithmic traits of pig ribs

[0065] The logarithmic traits of porcine ribs were analyzed using the site information provided in Example 1, the primer information provided in Example 2, or the kit provided in Example 3. The specific process is as follows:

[0066] Tissue samples were collected from the pigs to be tested, and their DNA was extracted to obtain DNA test samples. The DNA test samples were subjected to PCR reaction and agarose gel electrophoresis using the primer information provided in Example 2 or the kit provided in Example 3 to determine the genotype of each sample in the population. Individuals without SV71 and SV284 deletions were preferentially retained based on genotype to increase the population base of the superior rib logarithmic trait and increase the number of rib pairs in the population.

[0067] Application Example 1: Analysis of logarithmic traits of pig ribs using each SV locus.

[0068] The following is a detailed implementation process and analysis results of the analysis of the logarithmic traits of ribs in a Meihuaxing pig population using the loci, primers or kits and methods described in Examples 1-3 of this invention.

[0069] 1. Sample collection

[0070] Phenotypic data of 297 Meihuaxing pigs with different numbers of rib pairs were collected. Ear tissues of 297 Meihuaxing pigs were collected and stored in 75% alcohol at -20°C for DNA extraction.

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

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

[0073] 2) The concentration of DNA was determined using a UV spectrophotometer, and the quality of DNA was detected by agarose gel electrophoresis.

[0074] 3. Genotype determination

[0075] Genotyping is performed on the two SV loci of the qualified DNA, specifically as follows:

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

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

[0078] The PCR reaction conditions for SV71 were as follows: 1) Pre-denaturation: 94.0℃ for 5 min; 2) Amplification reaction: denaturation: 94.0℃ for 30 sec, annealing: 58.0℃ for 30 sec, extension: 72.0℃ for 1 min, 35 cycles; 72.0℃ for 10 min. 3) Storage at 4℃.

[0079] The PCR reaction conditions for SV284 were as follows: 1) Pre-denaturation: 94.0℃ for 5 min; 2) Amplification reaction: denaturation: 94.0℃ for 30 sec, annealing: 63.0℃ for 30 sec, extension: 72.0℃ for 1 min, 35 cycles; 72.0℃ for 10 min. 3) Storage at 4℃.

[0080] 2) Agarose gel electrophoresis detection

[0081] 2.0% agarose gel electrophoresis was used for detection. During sample loading, 3 μL of PCR product was added to each well using a pipette, along with 3 μL of D2000 DNA Marker as a control. The voltage was set to 100V, and the time was 30 min (50 min for SV71). After electrophoresis, the results were observed using a gel imaging system, and genotyping was performed.

[0082] 3) Significance test

[0083] The significance of genotype and rib logarithmic traits was tested using SAS software, and the results were visualized using GraphPad Prism software. The results are shown in Table 3. Figure 3 As shown.

[0084] Table 3. Association analysis between genotype and phenotype at two loci

[0085]

[0086] Note: In the table, + indicates no missing items, and - indicates missing items.

[0087] As shown in Table 3, the two SV loci provided by this invention are significantly correlated with the number of rib pairs (P<0.05). For SV71, the number of rib pairs in the + / + type of Meihua Star pig is 14.61±0.50, the number of rib pairs in the + / - type of Meihua Star pig is 14.29±0.50, and the number of rib pairs in the - / - type of Meihua Star pig is 14.15±0.66. The number of rib pairs in the + / + type is significantly greater than that in the + / - and - / - types. For SV284, the number of rib pairs in the + / + type of Meihua Star pig is 14.70±0.47, the number of rib pairs in the + / - type of Meihua Star pig is 14.32±0.50, and the number of rib pairs in the - / - type of Meihua Star pig is 14.13±0.61. The number of rib pairs in the + / + type is significantly greater than that in the + / - and - / - types. The results of testing 297 pigs show that there are more individuals with the - / - type of the two SV loci in the population, and the number of rib pairs of the - / - type Plum Blossom Pig is significantly lower than that of the + / + type, indicating that the population still has great potential for improvement.

[0088] Application Example 2 utilizes two SV loci to analyze the logarithmic traits of pig ribs. Except for simultaneously using the detection results of the two SV loci to determine the sample, everything else is the same as in Application Example 1. The significance test results are shown in Table 4. Figure 4 As shown in the figure.

[0089] Table 4. Association analysis between two SV locus combination types and phenotypes

[0090]

[0091] Note: In the table, + indicates that the locus is not missing, and - indicates that the locus is missing. Different letters on the rib logarithmic data indicate significant differences (P < 0.05); the same letter on the rib or no letter indicates no significant differences (P > 0.05).

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

[0093] It is evident that in actual breeding, selecting and retaining + / + type Meihua Star pigs can increase the population base of superior traits and the number of rib pairs in the population, thereby improving the rib pair trait in the population and ultimately enhancing the overall production performance and economic benefits of the population.

[0094] The above description represents the preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for detecting the logarithmic trait of pig ribs, using primers that amplify SV molecular markers, or a kit containing said primers, to detect the sample to be tested; The SV molecular markers are SV71 and / or SV284; wherein... SV71 is a region on chromosome 7 of the pig genome, from 89528530 to 89528600. The structural variation in this region is a 71 bp segment that may or may not be deleted from the genome. SV284 is a region on chromosome 7 of the pig genome, from 91896482 to 91896765. The structural variation in this region is a 284 bp segment that may or may not be deleted from the genome. The positions of the SV molecular markers were determined by comparing them with the pig reference genome Sus_scrofa.Sscrofa11.

1. The upstream primer sequence for amplifying SV71 is shown in SEQ ID NO.1, and the downstream primer sequence is shown in SEQ ID NO.2; the upstream primer sequence for amplifying SV284 is shown in SEQ ID NO.3, and the downstream primer sequence is shown in SEQ ID NO.

4. The pig in question is the Meihuaxing pig breed.

2. The method of claim 1, comprising: The primers or kits are used to test the sample, and the logarithmic trait of the pig ribs in the sample is determined based on the test results.

3. The method as described in claim 2, wherein judging the logarithmic trait of the pig ribs of the test sample based on the test results means that if the chromosome of the test sample does not have the deletion of SV71 or / and SV284, then it is judged that the test sample can increase the population base of the excellent logarithmic trait of pig ribs in the population and increase the number of rib pairs in the population.

4. The use of the molecular marker, primer, or kit according to claim 1, characterized in that, Apply it to: (1) Screening of pig breeds based on the logarithmic trait of ribs; (2) Identification of logarithmic traits of pig ribs; (3) Application of logarithmic traits in pig rib breeding; The pig in question is the Meihuaxing pig breed.

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