A snp molecular marker related to the pipe surrounding character of ningxiang pig and application thereof

Abstract

The application relates to the field of biotechnology, and particularly relates to a SNP molecular marker related to a Ningxiang pig girth character and application thereof. The molecular marker comprises at least one of SNP1, SNP2, SNP3 and SNP4; the SNP1 molecular marker corresponds to a 29911235th site from a 5' end on a chromosome 7 of a reference genome Sus Scrofa Build11.1, and is T or C; the SNP2 molecular marker corresponds to a 30014819th site, and is T or A; the SNP3 molecular marker corresponds to a 30176519th site, and is G or insA; and the SNP4 molecular marker corresponds to a 30404180th site, and is A or G. The application obtains a molecular marker significantly related to a Ningxiang pig girth size, provides guidance for Ningxiang pig breeding by using the molecular marker, can accurately and efficiently predict the size and typing of the girth, and identifies and screens pig breeds with excellent girth characters.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a SNP molecular marker related to the circumference trait of Ningxiang pigs and its application. Background Technology

[0002] Bone circumference (PC) is an important component of livestock and poultry body shape and appearance, possessing unique biological significance and application value in breeding. Studies have shown that PC is positively correlated with growth rate, body size (such as body length and height), and overall skeletal development in pigs. It can serve as an early indicator for assessing growth potential, helping to screen for individuals with superior growth and reducing breeding costs and time. PC is related to limb muscle mass, and the development of limb muscles may have a synergistic effect with the growth of trunk muscles (such as the longissimus dorsi). Research on the PC trait of Ningxiang pigs can indirectly assist in assessing their muscle quality (such as tenderness and juiciness) and fat deposition capacity (affecting flavor). As a fat-type local breed, the PC trait of Ningxiang pigs may be related to their unique body structure (such as compact body and short limbs) and adaptability. In commercial breeding, in-depth research on the PC trait can better grasp breed characteristics, thereby preserving the original breed advantages while pursuing growth rate and avoiding the loss of breed characteristics.

[0003] SNP markers are widely used molecular markers. Compared with SSR molecular marker technology, ISSR molecular marker technology and other polymorphic markers have higher genetic stability. At the same time, due to the diversity of detection methods applicable to them, including TaqMan method, mass spectrometry, microarray method, sequencing method, etc., they can be used for rapid and large-scale screening. With the advancement of sequencing technology and the continuous reduction of sequencing costs, more and more SNP markers of species are being explored and applied.

[0004] Therefore, developing a PC-related SNP molecular marker suitable for a high-throughput molecular detection platform for Ningxiang pigs can identify and screen pig breeds with excellent PC traits, thereby improving the health, production efficiency, and breeding efficiency of Ningxiang pig herds. Summary of the Invention

[0005] To address the aforementioned shortcomings in the existing technology, the present invention aims to provide an SNP molecular marker related to the canal circumference trait of Ningxiang pigs and its application. This marker can assist in the identification or screening of PC size in Ningxiang pigs, thereby improving the breeding of superior traits in Ningxiang pigs.

[0006] To achieve the above-mentioned objectives, the technical solution adopted by this invention is as follows:

[0007] In the first aspect, a SNP molecular marker related to the circumference trait of Ningxiang pigs is provided, the molecular marker including at least one of SNP1, SNP2, SNP3 and SNP4;

[0008] The sequence of the nucleic acid containing the SNP1 molecular marker is shown in SEQ ID No. 1. The SNP1 molecular marker corresponds to the 29911235th position from the 5' end on chromosome 7 of the reference genome Sus Scrofa Build11.1, and is either T or C.

[0009] The sequence of the nucleic acid containing the SNP2 molecular marker is shown in SEQ ID No. 2. The SNP2 molecular marker corresponds to the 30014819th position from the 5' end on chromosome 7 of the reference genome Sus Scrofa Build11.1, and is either T or A.

[0010] The sequence of the nucleic acid containing the SNP3 molecular marker is shown in SEQ ID No. 3. The SNP3 molecular marker corresponds to the 30176519th site from the 5' end on chromosome 7 of the reference genome Sus Scrofa Build11.1, and is either G or insA.

[0011] The sequence of the nucleic acid containing the SNP4 molecular marker is shown in SEQ ID No. 4. The SNP4 molecular marker corresponds to the 30404180th position from the 5' end on chromosome 7 of the reference genome Sus Scrofa Build 11.1, and is either A or G.

[0012] Secondly, the application of the aforementioned SNP molecular markers in detecting the canal girth size of Ningxiang pigs is provided.

[0013] Thirdly, the application of the above-mentioned SNP molecular markers in early screening of growth and development performance of Ningxiang pigs is provided.

[0014] Fourthly, the application of the aforementioned SNP molecular markers in marker-assisted breeding of Ningxiang pigs is provided.

[0015] Fifthly, the application of the aforementioned SNP molecular markers in the screening of Ningxiang pig breeds is provided.

[0016] Furthermore, the four loci SNP1, SNP2, SNP3, and SNP4 have a linkage effect, and the TT / TT / GG / GG genotype combination is a favorable genotype.

[0017] The beneficial effects of this invention are as follows:

[0018] (1) This invention has obtained a molecular marker that is significantly related to the size of the canal girth of Ningxiang pigs. Using this molecular marker, we can provide guidance for the breeding of Ningxiang pigs, accurately and efficiently predict the size and type of the canal girth, identify and screen pig breeds with excellent canal girth traits, and improve the health level, production efficiency and breeding efficiency of Ningxiang pig herds.

[0019] (2) The present invention provides sequencing primer pairs and KASP primer pairs for detecting SNP molecular markers related to canal girth in Ningxiang pigs. These primer pairs are highly specific and accurate, and can accurately obtain sequences containing SNP molecular marker sites of the present invention. They can be applied to the selection of superior traits in Ningxiang pigs and efficiently identify the size of canal girth.

[0020] (3) The molecular markers of the present invention have codominant inheritance, and the designed primer pairs are applicable to multiple platforms such as high-throughput SNP genotyping platform and high-throughput KASP detection platform, and can be applied to large-scale population selection and analysis. Attached Figure Description

[0021] Figure 1 Manhattan plot for genome-wide association analysis of PC phenotype in Ningxiang pigs;

[0022] Figure 2 Molecular markers developed for the 500 kb region upstream and downstream of SNP chr7:30404180 (red indicates gene name, black indicates molecular marker site).

[0023] Figure 3 Association analysis of different alleles and PC size for SNPs chr7:29911235, chr7:30014819, chr7:30176519 and chr7:30404180;

[0024] Figure 4 KASP test charts for 207 materials labeled chr7:29911235;

[0025] Figure 5 KASP test charts for 207 materials labeled chr7:30014819;

[0026] Figure 6 KASP test charts for 207 materials labeled chr7:30176519;

[0027] Figure 7 KASP test results for 207 materials labeled chr7:30404180. Detailed Implementation

[0028] The specific embodiments of the present invention are described below to enable those skilled in the art to understand the present invention. However, it should be understood that the present invention is not limited to the scope of the specific embodiments. For those skilled in the art, various changes are obvious as long as they are within the spirit and scope of the present invention as defined and determined by the appended claims. All inventions utilizing the concept of the present invention are protected.

[0029] Example

[0030] 1. Material screening and phenotypic recording

[0031] At the Ningxiang Pig National Breeding Farm, the breeding farm of Hunan Liushahe Flower Pig Ecological Animal Husbandry Co., Ltd., the production performance of the Ningxiang pig herd was measured using 10 sets of automatic feeding systems from Shenzhen Runnong Company over a period of 90 days. Upon leaving the farm, the length of the circumference of the pig's ankle was measured, obtaining PC phenotypic data for these 118 Ningxiang pigs.

[0032] 2. Resequencing and Genotyping

[0033] 2.1 DNA Sample Extraction and Quality Control

[0034] One week before Ningxiang pigs entered the testing station, right ear tissue was collected from 118 purebred Ningxiang pigs using ear clippers. The tissue was placed in 1.5 mL sterile, enzyme-free centrifuge tubes containing 75% ethanol and stored at -20℃ for whole-genome resequencing. DNA was extracted from the tissue samples using the CTAB method, and the concentration of the DNA samples was detected using a Qubit fluorescence quantitative quantitation system. Purity was determined using a Nanodrop spectrophotometer based on the absorbance ratios of 260 / 280 and 260 / 230. Samples that passed quality control were used for library preparation.

[0035] 2.2 Resequencing

[0036] DNA samples that passed inspection were randomly fragmented into 350 bp fragments using a Covaris shredder. Library construction was performed using the TruSeq Library Construction Kit. The DNA fragments underwent end repair, polyA tailing, sequencing adapter addition, purification, and PCR amplification to complete the entire library preparation process. After library construction, preliminary quantification was performed using Qubit 3.0, diluting the library to 1 ng / μL. Subsequently, the insert size of the library was detected using an Agilent 2100. Once the insert size met expectations, the effective concentration of the library was accurately quantified using q-PCR (effective concentration >2 nM). After passing the library inspection, sequencing was performed on the Illumina platform using PE150 sequencing, based on the effective concentration and data output requirements. The sample genome resequencing depth was 10X. Library preparation and sequencing were professionally supported by Beijing Novogene Technology Co., Ltd. The resequencing data was visualized using bioinformatics analysis software to obtain high-quality genotyping data.

[0037] 3. Genome-wide association analysis to locate target SNP loci.

[0038] 3.1 Genome-wide association analysis

[0039] Genome-wide association analysis (GWA) was performed on 118 Ningxiang pig resequencing data files using rMVP (Yin L, Zhang H, Tang Z et al. 2021). A mixed linear model (MLM) was used to calculate the model, and principal components were added as covariates to the model for correction. GWA analysis was then performed on the collected PC phenotypic traits. Figure 1 The significance threshold is calculated by taking the logarithm of the Bonferroni-corrected P-value (p = 0.05 / N, where N is the number of SNPs) and then subtracting log10. This yields the significantly associated SNP sites.

[0040] 3.2 Discovering significant marker sites

[0041] Significant marker sites for the PC phenotypic trait were identified across the entire genome, including the significant SNP rs3474141187 located by GWAS. Seven molecular markers were designed within a 500 kb region upstream and downstream of the site. Figure 2 Using Ningxiang pigs as experimental material, molecular markers at the loci rs318274564 (chr7:29911235), rs318821534 (chr7:30014819), rs80983858 (chr7:30176519) and rs3474141187 (chr7:30404180) showed significant phenotypic effects.

[0042] 4. Design of SNP site sequencing primers and KASP primers

[0043] After obtaining significant SNP markers from genome-wide association analysis, 1000 bp sequences before and after the SNP were extracted (Table 1). The reference genome was Sus Scrofa Build 11.1. Sequencing primers (Table 2) and KASP primers (Table 3) were designed using BatchPrimer3 (http: / / probes.pw.usda.gov / batchprimer3 / ) for later material validation. The primers were synthesized by Invitrogen.

[0044] The sequencing primers consist of two primers: the forward amplification primer Primer_F and the reverse amplification primer Primer_R.

[0045] The KASP marker consists of three primers: two allele-specific primers, X (Primer_X) and Y (Primer_Y), and one universal primer, C (Primer_C). The 5' ends of the allele-specific primers are connected to the LGC KASP reaction-specific fluorescent groups FAM and HEX, respectively. If only FAM fluorescence is detected in the sample, the genotype is homozygous allele X (Allele_X); if only HEX fluorescence is detected, the genotype is homozygous allele Y (Allele_Y); if both FAM and HEX fluorescence are detected, the genotype is heterozygous (carrying both alleles X and Y).

[0046] Table 1. Site Information Table

[0047]

[0048] Table 2. Sequence information of sequencing primers for PC marker detection in Ningxiang pigs.

[0049]

[0050] Table 3. Alleles (Allele_X, Allele_Y) and primer sequences of KASP markers detected by PC markers in Ningxiang pigs.

[0051]

[0052] 5. Material validation (next-generation sequencing validation, KASP validation)

[0053] 5.1 Sequencing Validation

[0054] The target SNP markers were detected using 207 Ningxiang pig samples, and PC phenotype data of 207 Ningxiang pig samples were collected. The association between marker genotype and phenotype was tested, and the T-test was used to compare and analyze different genotypes.

[0055] The results showed that the Chr7:29911235 locus represented three genotypes (TT, TC, and CC) in the Ningxiang pig population. The average PC value for TT-type Ningxiang pigs was 15.92; for TC-type Ningxiang pigs, it was 15.32; and for CC-type Ningxiang pigs, it was 13.75. The average PC value for CC-type Ningxiang pigs was significantly different from that for TT-type Ningxiang pigs. p <0.01).

[0056] The Chr7:30014819 locus identified three genotypes (TT, TA, and AA) in the Ningxiang pig population. The average PC (probability and criterion) for TT-type Ningxiang pigs was 15.92; for TA-type, it was 15.32; and for AA-type, it was 13.75. The average PC of TT-type Ningxiang pigs was significantly different from that of AA-type Ningxiang pigs. p <0.01).

[0057] At the Chr7:30176519 locus, three genotypes (GG, G / insA, and insA) were identified in the Ningxiang pig population. The average PC (probability and criterion) for GG-type Ningxiang pigs was 15.92; for G / insA-type Ningxiang pigs, it was 15.32; and for insA-type Ningxiang pigs, it was 13.75. The average PC of GG-type Ningxiang pigs was significantly different from that of insA-type Ningxiang pigs. p <0.01).

[0058] At the Chr7:30404180 locus, three genotypes (GG, AG, and AA) were identified in the Ningxiang pig population. The average PC (probability and criterion) for GG-type Ningxiang pigs was 15.92; for AG-type, it was 15.32; and for AA-type, it was 13.75. The average PC for GG-type Ningxiang pigs was significantly different from that for AA-type Ningxiang pigs. p <0.01).

[0059] The sample numbers corresponding to the TT genotype of chr7:29911235, the TT genotype of chr7:30014819, the GG genotype of chr7:30176519, and the GG genotype of chr7:30404180 are consistent; the sample numbers corresponding to the TC genotype of chr7:29911235, the TA genotype of chr7:30014819, the G / insA genotype of chr7:30176519, and the AG genotype of chr7:30404180 are consistent; chr7:29911 The sample numbers corresponding to the CC genotype of 235, the AA genotype of chr7:30014819, the insA genotype of chr7:30176519, and the AA genotype of chr7:30404180 are consistent. Due to the strong association of this genotype and its significant correlation with the phenotype, it is speculated that chr7:29911235, chr7:30014819, chr7:30176519, and chr7:30404180 have a linkage effect, and the TT / TT / GG / GG genotype combination is a favorable genotype.

[0060] 5.2 KASP Tag Validation

[0061] 5.2.1 KASP Reaction Procedure

[0062] KASP-tagged reactive sequencing was performed using the Douglas Scientific ArrayTape system. The ArrayTape genotyping platform includes NEXAR for PCR amplification system assembly, SOELLEX for PCR amplification, ARAYA for fluorescence signal scanning, and INTELLICS for data analysis.

[0063] PCR reaction system: The PCR amplification system was automatically assembled using NEXAR, and the PCR reaction system is shown in Table 4 below.

[0064] Table 4. PCR reaction system for KASP marker genotyping

[0065]

[0066] PCR amplification: PCR amplification was performed using SOELLEX under the following conditions: 94℃ for 15 minutes; 94℃ for 20 seconds, 65℃-57℃ (annealing temperature decreased by 0.8℃ per cycle) for 60 seconds, 10 cycles; 94℃ for 20 seconds, 57℃ for 60 seconds, 30 cycles.

[0067] Signal scanning and genotyping: After the PCR reaction was completed, the fluorescence signal of the reaction system was scanned using ARAYA; then genotyping and data analysis were performed using INTELLICS.

[0068] 5.2.1 KASP Test Results

[0069] To test the specificity and practicality of the markers in this invention, the target SNP markers were detected using 207 samples of Ningxiang pigs.

[0070] Validation showed that the KASP markers divided the cells into three distinct and compact clusters. The KASP marker genotyping diagram for chr7:29911235 is shown below. Figure 4 As shown in the figure, the upper left circle cluster indicates that the sample contains a homozygous T:T allele at this KASP marker site, the lower right circle cluster indicates that the sample contains a homozygous C:C allele at this KASP marker site, and the middle circle cluster indicates that the sample contains a heterozygous T and C allele at this KASP marker site. The results showed that 112 samples contained a homozygous T:T allele; 14 samples contained a homozygous C:C allele; and 81 samples contained a heterozygous T and C allele, consistent with the genotyping results in the sequencing reaction.

[0071] The KASP marker genotyping diagram for chr7:30014819 is shown below. Figure 5As shown in the figure, the upper left circle cluster indicates that the sample contains a homozygous T:T allele at this KASP marker site, the lower right circle cluster indicates that the sample contains a homozygous A:A allele at this KASP marker site, and the middle circle cluster indicates that the sample contains a heterozygous T and A allele at this KASP marker site. The results showed that 112 samples contained a homozygous T:T allele; 14 samples contained a homozygous A:A allele; and 81 samples contained a heterozygous T and A allele, consistent with the genotyping results in the sequencing reaction.

[0072] The KASP marker genotyping diagram for chr7:30176519 is shown below. Figure 6 As shown in the figure, the upper left circle cluster indicates that the sample contains a homozygous G:G allele at this KASP marker site, the lower right circle cluster indicates that the sample contains a homozygous insA allele at this KASP marker site, and the middle circle cluster indicates that the sample contains a heterozygous G and insA allele at this KASP marker site. The results showed that 112 samples contained a homozygous G:G allele; 14 samples contained a homozygous insA allele; and 81 samples contained a heterozygous G and insA allele, consistent with the genotyping results in the sequencing reaction.

[0073] The KASP marker genotyping diagram for chr7:30404180 is shown below. Figure 7 As shown in the figure; the upper left circle cluster indicates that the sample contains a homozygous G:G allele at this KASP marker site, the lower right circle cluster indicates that the sample contains a homozygous A:A allele at this KASP marker site, and the middle circle cluster indicates that the sample contains a heterozygous A and G allele at this KASP marker site. The results showed that 112 samples contained a homozygous G:G allele; 14 samples contained a homozygous A:A allele; and 81 samples contained a heterozygous A and G allele, consistent with the genotyping results in the sequencing reaction. This indicates that chr7:29911235, chr7:30014819, chr7:30176519, and chr7:30404180 have a linkage effect.

[0074] In summary, this invention, through genome-wide association analysis of resequencing data from 118 purebred Ningxiang pigs and the PC phenotype, located linkage SNP markers (chr7:29911235, chr7:30014819, chr7:30176519, and chr7:30404180) significantly associated with PC size in Ningxiang pigs. Sequencing primer pairs and KASP primer pairs were designed for identification analysis and large-scale detection. This allows for efficient and accurate identification and screening of Ningxiang pig individuals with PC size meeting the requirements.

[0075] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within the present invention.

[0076] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. The application of a SNP molecular marker associated with the canine girth trait of Ningxiang pigs in the detection of canine girth size in Ningxiang pigs, characterized in that, The molecular markers include at least one of SNP1, SNP2, SNP3 and SNP4; The SNP1 molecular marker corresponds to the 29911235th site from the 5' end on chromosome 7 of the reference genome Sus Scrofa Build11.1, and is either T or C; The SNP2 molecular marker corresponds to the 30014819th site from the 5' end on chromosome 7 of the reference genome Sus Scrofa Build11.1, and is either T or A; The SNP3 molecular marker corresponds to the 30176519th site from the 5' end on chromosome 7 of the reference genome Sus Scrofa Build11.1, and is either G or insA; The SNP4 molecular marker corresponds to the 30404180th site from the 5' end on chromosome 7 of the reference genome Sus Scrofa Build11.1, and is either A or G.

2. The application of a SNP molecular marker associated with the canine girth trait of Ningxiang pigs in assisted breeding of Ningxiang pigs with superior canine girth size, characterized in that, The molecular markers include at least one of SNP1, SNP2, SNP3 and SNP4; The SNP1 molecular marker corresponds to the 29911235th site from the 5' end on chromosome 7 of the reference genome Sus Scrofa Build11.1, and is either T or C; The SNP2 molecular marker corresponds to the 30014819th site from the 5' end on chromosome 7 of the reference genome Sus Scrofa Build11.1, and is either T or A; The SNP3 molecular marker corresponds to the 30176519th site from the 5' end on chromosome 7 of the reference genome Sus Scrofa Build11.1, and is either G or insA; The SNP4 molecular marker corresponds to the 30404180th site from the 5' end on chromosome 7 of the reference genome Sus Scrofa Build11.1, and is either A or G.

Images