Specific SVs and SNPs molecular marker combinations for detecting genes in the longissimus dorsi muscle of Wuzhishan pigs, screening methods, and applications

Abstract

The present invention discloses a specific combination of SVs and SNPs molecular markers within the region for detecting genes in the longissimus dorsi muscle of Wuzhishan pigs, comprising 78 SVs markers and 104 SNPs molecular markers. The SVs screened in the present invention have breed specificity themselves, and when combined with SNPs within the SVs region, they exhibit stronger specificity and accuracy, which is conducive to in-depth analysis of the effects of mutation sites on muscle growth, development and function, and provides theoretical support for the directional breeding of gene structure.

Description

Technical Field

[0001] The present invention belongs to the field of molecular biology, and particularly relates to specific SVs of genes in the longissimus dorsi muscle of Wuzhishan pigs, SNP molecular marker combinations within the regions thereof, screening methods and applications. Background Art

[0002] Wuzhishan pigs are a local small pig breed in Hainan Province, and have characteristics such as being resistant to roughage, having a high lean meat rate, and tender meat quality. [1] In addition, due to the high similarity between Wuzhishan pigs and humans in terms of anatomy, physiological structure, genetics, etc., they are an ideal animal model for translational medicine research on human diseases. [2-3] However, due to the slow growth rate and low feed-to-meat ratio of Wuzhishan pigs, it directly affects their market scale and economic benefits. Farms usually introduce pig breeds with excellent performance cultivated abroad, including Large White pigs, Landrace pigs, Duroc pigs, etc., and obtain heterosis through cross-breeding to improve the growth rate of Wuzhishan pigs. However, heterosis cannot be stably inherited, and there may be significant differences in production performance. Therefore, it is necessary to compare the genetic and variation differences between Wuzhishan pigs and other pig breeds at the genomic level, screen out specific structural mutations and other variation characteristics of genes in the longissimus dorsi muscle of Wuzhishan pigs, further analyze the effects and functions of mutations on muscle growth, and provide theoretical support for the directional breeding of gene structures.

[0003] Genomic structural variations (abbreviated as SVs), usually refer to sequence changes and positional relationship changes of relatively long genomic lengths. It includes long fragment sequence insertions or deletions (Big Indel) with a length of more than 50bp, tandem repeats, chromosomal inversions, sequence translocations within or between chromosomes, copy number variations (CNV), and more complex chimeric variations. Most SVs are located in the upstream and downstream regions or intron regions of genes, mainly having regulatory effects, and SVs in gene coding regions are extremely rare. Comparative analysis of Ningxiang pigs and Duroc pigs found that some SVs in Ningxiang pigs are related to fat metabolism, immune response and nervous system function. [4] By detecting domestic pig populations, it was found that some gene (Fibronectin Type III And SPRY Domain Containing2, (FSD2); WASP Like Actin Nucleation Promoting Factor, (WASL)) SVs are associated with meat quality and muscle fiber volume. [5] These results reflect the growth state differences of different pig breeds in different regions of China.

[0004] Single nucleotide polymorphisms (SNPs) in next-generation sequencing variant detection are numerous and are the most abundant type of variation. Although most SNPs are located in the upstream and downstream regions of genes, 5'UTR, 3'UTR regions or intron regions, the detected coding sequence SNPs are still the most numerous type of variation. The gene structure is very complex. Exons may overlap with 5'UTR or alternative splicing may produce different transcripts. However, the coding sequence is always located between the 5'UTR and 3'UTR regions and functions as a gene through processes such as transcription and translation. Moreover, most SNPs located in the coding sequence only result in changes in the codons encoding amino acids and do not cause amino acid changes, that is, synonymous codons. Whole-genome resequencing analysis found that some genes in Wuzhishan pigs are related to immune response and meat quality. [6] ; Copy number variations in wild boars and domesticated pigs are related to meat quality, growth and development, and immune response. [7] These research results indicate that short sequence mutations such as SNPs may be related to muscle growth.

[0005] From an evolutionary perspective, generally speaking, variation is more harmful than beneficial to a stable species population. Except in a few cases where the earth undergoes drastic geological changes or the environmental climate undergoes great turmoil, and the original habitat is not suitable for the survival of the species, and only the traits derived from new variations can help the species survive, most variations are harmful. This view is consistent with the phenomenon that different types of variations are mostly located in regulatory regions and synonymous mutations account for a large proportion. Although the number of SVs is small, they belong to large fragment variations and have a greater impact on gene function. They are usually located in regulatory regions such as upstream and downstream regions of genes, promoter regions, and introns, and there are fewer coding region SVs; relatively speaking, the number of SNPs is large and they are widely distributed. SNPs exist in almost all gene structures, but they lack specificity. According to the currently known research results, except for the halothane gene as the major gene causing porcine stress syndrome, in most other cases, there are multi-gene and multi-locus effects, and it is difficult to accurately locate the SNPs sites that play a role. Therefore, different types of variations such as SVs and SNPs have different effects on gene function and each has its own advantages and limitations. In view of the above problems, the present invention combines the characteristics of different types of variations of SVs and SNPs, analyzes the differential mutation sequences of genes related to muscle growth and development in Wuzhishan pigs and Large White pigs, and screens specific combinations of SVs and SNPs in the longissimus dorsi muscle genes of Wuzhishan pigs, providing a reference for the direction of gene-directed breeding in genetic breeding. Summary of the Invention

[0006] In view of the deficiencies in the above problems, the present invention provides a specific SVs molecular marker combination for detecting the longissimus dorsi muscle gene of Wuzhishan pigs, and the sequence information of the SVs markers is shown in Table 1, numbered 1 to numbered 78.

[0007] The present invention also provides a specific SNPs molecular marker combination for detecting the longissimus dorsi muscle gene of Wuzhishan pigs. The SNPs molecular markers are located within the above-mentioned SVs region, and the locus information of the SNPs markers is shown as No. 1 to No. 104 in Table 2.

[0008] The present invention also provides a specific SVs and the SNPs molecular marker combination within its region for detecting the longissimus dorsi muscle gene of Wuzhishan pigs, including the specific SVs of the longissimus dorsi muscle gene of Wuzhishan pigs and the SNPs molecular markers within its region. The sequence information of the specific SVs and the SNPs within its region of the longissimus dorsi muscle gene of Wuzhishan pigs is shown as No. 1 to No. 39 in Table 3.

[0009] The present invention also provides a detection substance, and the detection substance includes probes with the following functions.

[0010] The above-mentioned SVs molecular marker combination can be detected, and the sequence information of the SVs probes is shown as No. 1 to No. 78 in Table 4.

[0011] Table 4 Probe sequences for detecting specific SVs markers of the longissimus dorsi muscle gene of Wuzhishan pigs

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[0024] and / or, (2) the probe can detect the molecular marker combination according to the above SNPs, and the probe sequence information of the SNPs is shown in Nos. 1 to 104 in Table 5.

[0025] Table 5 Probe sequences for detecting specific SNPs markers of the longissimus dorsi muscle gene in Wuzhishan pigs

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[0031] The present invention also provides a detection product containing the above detection substances, and the detection product can be a kit and / or a chip.

[0032] The present invention also provides a screening method for detecting specific SVs and SNP marker combinations in the longissimus dorsi muscle gene of Wuzhishan pigs, comprising the following steps:

[0033] Step 1: Analyze the third-generation variant sequencing data of the genomes of Wuzhishan pigs and Large White pigs, and extract SVs; filtering conditions: 50bp ≤ SVs ≤ 1Mb, score ≥ 10;

[0034] Step 2: Screen specific SVs of the longissimus dorsi muscle gene in Wuzhishan pigs, and design and synthesize SVs probes;

[0035] Step 3: Analyze the second-generation sequencing data of the whole genome of Wuzhishan pigs, and extract SNPs;

[0036] Step 4: Screen SNPs within the range of specific SVs regions of the longissimus dorsi muscle gene in Wuzhishan pigs according to Step 2 and Step 3;

[0037] Step 5: Count the gene positions and structural variant types of the SVs in Step 4;

[0038] Step 6: Retain the SVs and SNPs in the 5'UTR to 3'UTR regions of the gene in Step 4, and combine the specific SVs of the longissimus dorsi muscle gene in Wuzhishan pigs and the SNPs within the range of the SVs regions, which are the specific SVs and SNP marker combinations of the longissimus dorsi muscle gene in Wuzhishan pigs; design and synthesize SVs and SNPs probes;

[0039] As a further improvement of the present invention, the information of specific SVs of the longissimus dorsi muscle gene in Step 2 is shown in Nos. 1 to 78 in Table 1 of the specification.

[0040] As a further improvement of the present invention, the method for designing SVs probes in step 2 is divided into three types: design one probe on each side outside (excluding SVs) near the SVs junction point (a total of 2 probes); design one probe on each side inside (SVs region) near the SVs junction point (a total of 2 probes); if the length of SVs is less than 120 bp, design 1 probe at one end on each side outside (excluding SVs) of the SVs junction point, and design 1 probe inside the SVs region (a total of 2 probes). The SVs probe is a single-stranded DNA with a length of 120 bp. Design 2 probes to simultaneously capture the target sequences near the SVs junction point and detect the SVs sequence.

[0041] As a further improvement of the present invention, the sequence information of SNPs markers in the gene-specific SVs region of the longissimus dorsi muscle in step 4 is as shown in Nos. 1 to 104 in Table 2 of the specification; the designed SNPs probe is a single-stranded DNA with a length of 120 bp. Moreover, the SNPs markers in the gene-specific SVs region of the longissimus dorsi muscle of Wuzhishan pigs are placed in the middle position of the probe sequence. The SNPs probe detects SNPs by capturing the target sequences near the SNPs. The detection method is similar to the method for the probe to detect the SVs sequence.

[0042] As a further improvement of the present invention, the range for screening SVs and SNPs in step 6 is within the 5' UTR to 3' UTR region of the gene.

[0043] As a further improvement of the present invention, the specific steps in step 1 are as follows: Collect the longissimus dorsi muscle tissues of 3 Wuzhishan pigs and 3 Large White pigs, send them to Novogene Co., Ltd. in Beijing by dry ice, construct a library and sequence on the PacBio third-generation sequencing platform with a sequencing depth of approximately 15×. Align the raw data with the reference genome (Sscrofa 11.1). Use the default parameters of the Sniffles and PBSV software to detect SVs, including deletions (DELs), insertions (INSs), duplications (DUP), inversions (INVs), and translocations (TRAs). Combine the SVs of the 3 replicate samples of the two breeds respectively to obtain the SVs of the two breeds of Wuzhishan pigs and Large White pigs.

[0044] As a further improvement of the present invention, the specific steps in the second step are as follows: Compare the SVs of the two breeds obtained in the first step, and screen for the longissimus dorsi muscle-specific SVs of Wuzhishan pigs. The SVs are divided into four categories: First, the SVs with the same position, the same mutation type, and the same mutation sequence in the two breeds; second, the SVs with the same position and the same mutation type in the two breeds but with different sequences; third, the SVs with different sequence positions in the two breeds; fourth, the SVs with different types of structural variations in the two breeds. Retain the second, third, and fourth categories, that is, the SVs specific to the longissimus dorsi muscle gene position or mutation type of Wuzhishan pigs, and the SVs with sequence differences between Wuzhishan pigs and Large White pigs.

[0045] As a further improvement of the present invention, the specific steps in the sixth step are as follows: Retain the SVs and SNPs within the 5'UTR to 3'UTR regions of the genes screened in the fourth step, and separately combine the longissimus dorsi muscle gene-specific SVs of Wuzhishan pigs and the SNPs within the SVs regions, which are the longissimus dorsi muscle gene-specific SVs and SNP marker combinations of Wuzhishan pigs.

[0046] The present invention also provides the application of the above SVs molecular marker combinations and / or the SNPs molecular marker combinations within the SVs regions in the molecular marker genetic breeding of Wuzhishan pigs.

[0047] Beneficial effects of the present invention compared with the prior art:

[0048] The present invention discloses a specific SVs and SNPs molecular marker combination within the SVs region for detecting the longissimus dorsi muscle gene of Wuzhishan pigs. The SVs screened by the present invention itself has breed specificity, and combined with the SNPs within the SVs region, it shows stronger specificity and accuracy, which is conducive to in-depth analysis of the effects of mutation sites on muscle growth, development, and function, and provides theoretical support for the directional breeding of gene structures. Brief Description of the Drawings

[0049] Figure 1 Flow chart for screening the specific SVs and SNP marker combinations of the longissimus dorsi muscle gene of Wuzhishan pigs;

[0050] Figure 2 Position map of the specific SVs of the longissimus dorsi muscle gene of Wuzhishan pigs and the SNPs within the SVs region on the chromosome. The vertical lines in the outer chromosomal box represent the regions where the SVs are located; the vertical lines in the inner chromosomal box represent the positions where the SNPs are located;

[0051] Figure 3This is the region for designing gene-specific SVs probes of the longissimus dorsi muscle of Wuzhishan pigs. The middle black region represents SVs, and the two gray regions on both sides represent the regions without mutations. There are three methods for designing SVs probes: Design one probe (probe 1 and 2) near the outer sides (excluding SVs) on both sides of the SVs junction point; Design one probe (probe 3 and 4) near the inner sides (SVs region) on both sides of the SVs junction point; If the length of SVs is less than 120bp, select one end on the outer sides (excluding SVs) on both sides of the SVs junction point to design 1 probe (probe 1 or 2), and design 1 probe inside the SVs region. Detailed implementation mode

[0052] The following will describe the implementation plan of the present invention in detail in combination with embodiments. However, the following embodiments are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention. For those not specified in the embodiments, they are carried out according to conventional conditions or the conditions recommended by the manufacturer. For reagents or instruments without indicating the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0053] Example 1 - Screening SVs of the longissimus dorsi muscle gene of Wuzhishan pigs and Large White pigs

[0054] Collect the longissimus dorsi muscle tissues of 3 Wuzhishan pigs and 3 Large White pigs, and send them to Novogene Co., Ltd. in Beijing by dry ice for library construction and sequencing on the PacBio third-generation sequencing platform, with a sequencing depth of approximately 15×.

[0055] By analyzing the third-generation variant detection data of the above 6 pigs (3 Wuzhishan pigs and 3 Large White pigs) (the sequencing data has been uploaded to the NCBI database (PRJNA1030798)), and aligning with the reference sequence (Sscrofa11.1), screen the SVs of the two breeds of Wuzhishan pigs and Large White pigs. Use the default parameters of Sniffles and PBSV software to extract SVs, and the filtering conditions are: 50bp ≤ SVs ≤ 1Mb for the length of SVs, and the sequence quality score ≥ 10. SVs include deletions (DELs), insertions (INSs), duplications (DUP), inversions (INVs), and translocations (TRAs). Combine the SVs extracted from the 3 repeated samples of the above two breeds respectively to obtain the SVs of the two breeds of Wuzhishan pigs and Large White pigs.

[0056] A total of 78 SVs of the longissimus dorsi muscle gene of Wuzhishan pigs were screened, corresponding to 24 genes; a total of 40 SVs of the genes of Large White pigs were screened, corresponding to 14 genes.

[0057] Example 2 - Screening gene-specific SVs of the longissimus dorsi muscle of Wuzhishan pigs

[0058] Compare the SVs of the two breeds in Example 1 and screen for specific SVs in Wuzhishan pigs. The SVs are divided into four categories: one is the SVs with the same position, the same mutation type, and the same mutation sequence in the two breeds; the second is the SVs with the same position and the same mutation type in the two breeds but with different sequences; the third is the SVs with different sequence positions in the two breeds; the fourth is the SVs with different structural variation types in the two breeds. Retain the second, third, and fourth categories, that is, the specific SVs of the longissimus dorsi muscle genes in Wuzhishan pigs, and the SVs with sequence differences between Wuzhishan pigs and Large White pigs. A total of 78 specific SVs of the longissimus dorsi muscle genes were screened out in Wuzhishan pigs, as shown in Table 1.

[0059] Example 3 - Screening for specific SNPs in the longissimus dorsi muscle genes of Wuzhishan pigs

[0060] Based on the SVs screened in Example 2, further screen for SNPs within the SVs region. The samples for second-generation sequencing of Wuzhishan pigs are the same as those for third-generation sequencing (the sequencing data has been uploaded to the NCBI database (PRJNA378496)). The raw data is first quality controlled. The fastqc software is used to check the data quality, and the fastp software is used to remove adapters and low-quality sequences [8] . The filtered high-quality sequences are aligned to the genome using the bwa software [9] , and the GATK4 software is used to extract

[10] . Extract the SNPs within the range from the start to the end of the SVs sequence to obtain the SNPs within the specific SVs region of the longissimus dorsi muscle genes in Wuzhishan pigs. A total of 104 SNPs were detected in 8 genes in Wuzhishan pigs, as shown in Table 2.

[0061] Example 4 - Design and synthesis of probes for detecting specific SVs markers and SNPs markers in the longissimus dorsi muscle of Wuzhishan pigs

[0062] 1. Method for designing and synthesizing probes for detecting specific SVs markers in the longissimus dorsi muscle of Wuzhishan pigs

[0063] As shown in the appendix Figure 3 , design one SVs probe (Probe 1 and 2) respectively near the outside of both sides of the SVs marker junction point (excluding the SVs); design one SVs probe (Probe 3 and 4) respectively near the inside of both sides of the SVs marker junction point (SVs region); if the length of the SVs marker is less than 120bp, select one end near the outside of both sides of the SVs marker junction point (excluding the SVs) to design 1 SVs probe (Probe 1 or 2), and design 1 marker probe inside the SVs marker region, and design 2 probes to capture the target sequence near the SVs marker junction point simultaneously to detect the SVs marker. The above SVs probes are single-stranded DNAs with a length of 120bp, and the sequence information of the SVs probes is shown in Table 4.

[0064] 2. Method for Detecting Specific SVs Markers in the Longissimus Dorsi Muscle of Wuzhishan Pigs Using the Above SVs Probes

[0065] Extract porcine genomic DNA by conventional methods. After sonication, small DNA fragments are obtained. These small DNA fragments are then subjected to end repair, tailing, and pre-PCR reactions to construct a small-fragment DNA library. The above DNA library is mixed with the target probe and hybridization buffer for hybridization capture reaction, and then sequenced on a machine to detect the sequences containing specific SVs markers in the longissimus dorsi muscle of Wuzhishan pigs. When two probes can simultaneously capture the target sequences near the SVs marker junction point, further analyze the sequencing sequences to detect the integrity of the sample SVs marker sequences; if only one probe can capture the target region and the captured region is within the SVs marker region, it means that only a part of the SVs region can be detected, that is, the detected SVs markers are incomplete; if only one probe can capture the target region and the captured region is outside the SVs marker region, or if neither of the two probes can capture the target region, it means that the sample does not have the detected SVs markers. 3. Method for Designing and Synthesizing Probes for Detecting Specific SNPs Markers in the Longissimus Dorsi Muscle of Wuzhishan Pigs

[0066] Place the SNPs markers located in the specific SVs region of the longissimus dorsi muscle gene of Wuzhishan pigs in the middle position of the probe sequence to obtain SNPs probes, and their sequence information is shown in Table 5. The SNPs probes detect SNPs markers by capturing the target sequences near the SNPs.

[0067] 4. Method for Detecting Specific SVs Markers in the Longissimus Dorsi Muscle of Wuzhishan Pigs Using the Above SVs Probes

[0068] Extract porcine genomic DNA by conventional methods. After sonication, small DNA fragments are obtained. These small DNA fragments are then subjected to end repair, tailing, and pre-PCR reactions to construct a small-fragment DNA library. The above DNA library is mixed with the target probe and hybridization buffer for hybridization capture reaction, and then sequenced on a machine to detect the sequences containing specific SNPs markers in the longissimus dorsi muscle of Wuzhishan pigs.

[0069] Example 5 - Merging Specific SVs and SNPs in the Longissimus Dorsi Muscle of Wuzhishan Pigs

[0070] Retain the SVs and SNPs in the gene 5'UTR to 3'UTR region in Step 4, and merge the SVs of Wuzhishan pigs and the SNPs within the SVs region screened in Example 2 and Example 3. The content includes gene name, chromosome number, start to end position of SVs, SVs mutation type, and the number of SNPs within the SVs region, which is the specific SVs and SNPs marker combination of the longissimus dorsi muscle gene of Wuzhishan pigs, as shown in Table 3 specifically.

[0071] Table 1 Specific SVs markers for detecting the longissimus dorsi muscle gene of Wuzhishan pigs

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[0076] Table 2 Specific SNPs molecular markers for detecting the longissimus dorsi muscle gene of Wuzhishan pigs

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[0082] Table 3 Specific SVs markers and SNPs marker combinations for detecting the longissimus dorsi muscle gene of Wuzhishan pigs

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[0088] Table 4 Specific SVs probe sequences for the longissimus dorsi muscle gene of Wuzhishan pigs

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[0102] Table 5 Probe sequences of SNPs markers in the specific SVs regions of genes in the longissimus dorsi muscle of Wuzhishan pigs

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[0108] References:

[0109] [1] Cao Ting, Zhou Hanlin, Yu Ping, et al. Current situation, protection, development and utilization of Wuzhishan pigs [J]. Acta Ecologiae Animalis Domastici, 2017, 38(07): 79 - 83.

[0110] [2] Chao Zhe, Sun Ruiping, Liu Hailong, et al. Research progress and application prospects of experimental animalization of Wuzhishan pigs [C]. Proceedings of the 2015 (5th) Annual Conference on Drug Toxicology. 2015: 304 - 305.

[0111] [3] VINEREAN H, GAZDA L, HALL R, et al. Streptozotocin is responsible for the induction and progression of renal tumorigenesis in diabetic Wistar - Furth rats treated with insulin or transplanted with agarose encapsulated porcine islets [J]. Islets, 2011, 3(4): 196 - 203.

[0112] [4] Ma H, Jiang J, He J, et al. Long-read assembly of the Chinese indigenous Ningxiang pig genome and identification of genetic variations in fat metabolism among different breeds[J]. Mol. Ecol. Resour. 2022, 22, 1508 - 1520.

[0113] [5] Li X, Liu Q, Fu C, et al. Characterizing structural variants based on graph - genotyping provides insights into pig domestication and local adaption[J]. J. Genet. Genomics. 2023, S1673 - 8527(23)00241 - 2.

[0114] [6] Ren Y, Wang F, Sun R, et al. The Genetic Selection of HSPD1 and HSPE1 Reduce Inflammation of Liver and Spleen While Restraining the Growth and Development of Skeletal Muscle in Wuzhishan Pigs[J]. Animals, 2024, 14, 174.

[0115] [7] Fan S, Kong C, Chen Y et al. Copy number variation analysis revealed the evolutionary difference between Chinese indigenous pigs and Asian wild boars. Genes 2023, 14, 472.

[0116] [8]Chen S, Zhou Y, Chen Y, et al. fastp: an ultra-fast all-in-one FASTQ preprocessor. Bioinformatics, 2018, 34(17): i884-i890.

[0117] [9]Jung Y, Han D. BWA-MEME: BWA-MEM emulated with a machine learning approach. Bioinformatics, 2022, 38(9): 2404-2413.

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[10] Heldenbrand JR, Baheti S, Bockol MA, et al. Recommendations for performance optimizations when using GATK3.8 and GATK4. BMC Bioinformatics, 2019, 20(1): 557.

Claims

1. A detection substance, characterized in that: The detection substance includes a probe having the following functions: (1) the probe can detect the SVs molecular marker combination specific to the longissimus dorsi muscle gene of Wuzhishan pigs, and the sequence information of the probe is shown in the table numbered 1 to numbered 78 in the following table, and / or, (2) the primers and / or probes are capable of detecting a specific SNPs molecular marker combination of the Wuzhishan pig longissimus dorsi muscle gene, and the sequence information of the primers and / or probes is shown in the table numbered 1 to numbered 104 below, The Wuzhishan pig longissimus dorsi muscle gene-specific SVs molecular marker locus refers to the pig genome Sscrofa11.1, and the sequence information is shown in the table numbered 1 to numbered 78 as follows: The Wuzhishan pig longissimus dorsi gene-specific SNPs molecular marker is located within the SVs molecular marker region, and the site information of the SNPs molecular marker is referenced to the pig genome scrofa11.1, as shown in the following table from number 1 to number 104, 2. A detection product comprising the detection substance according to claim 1, characterized in that: The detection product may be a kit and / or a chip.

3. Use of the detection substance according to claim 1 and the detection product according to claim 2 in the molecular marker genetic breeding of the longissimus dorsi muscle of Wuzhishan pig.

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