Duroc pig SNP (Single Nucleotide Polymorphism) molecular marker combination, liquid phase chip and application of Duroc pig SNP molecular marker combination

Abstract

The invention discloses a Duroc pig SNP molecular marker combination, a liquid phase chip prepared from the Duroc pig SNP molecular marker combination and application of the Duroc pig SNP molecular marker combination. The Duroc pig SNP molecular marker combination is composed of 18444 SNP molecular markers, and the genome positions of the 18444 SNP molecular markers are determined through sequence alignment on the basis of a pig reference genome Sscrofa11.1. The SNP molecular marker combination disclosed by the invention can be used for specific identification of Hunan Duroc pigs and is used for preparing a liquid phase chip, and the obtained liquid phase chip can realize high-throughput and low-cost genotyping of the Duroc pigs; the method can be applied to Duroc pig variety resource molecular marker fingerprint analysis, hybrid population offspring genotype identification, variety authenticity identification, breeding material genetic background analysis and screening, whole genome correlation analysis, genetic resource genetic diversity analysis and genetic relationship identification. Important technical support is provided for molecular breeding application of Duroc pigs.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of gene chip, in particular to a Duroc pig SNP molecular marker combination, a liquid phase chip prepared therefrom and application thereof. BACKGROUND

[0002] Duroc pigs originated in the northwest of the United States, have strong applicability, wide distribution, fast growth rate, high feed utilization rate, high carcass lean rate and other characteristics, are high-quality paternal breeds, and are currently widely used as one of the breeds of a mating system. Duroc pigs have been domesticated and improved for many years, and the physical structure is basically stable, having the phenotypic characteristics of a typical lean pig breed, such as a wide body, strong muscles, developed hindquarters, and solid limbs.

[0003] The breeding liquid phase chip based on SNP markers has high marker density, strong automation, high detection throughput and other characteristics, can establish a modern breeding system, and promotes the development of Duroc pig breeding. SUMMARY

[0004] The present application aims to at least solve one of the problems in the prior art. To this end, the present application provides a Duroc pig SNP molecular marker combination.

[0005] The present application also provides a Duroc pig liquid phase chip.

[0006] The present application also provides a kit.

[0007] The present application also provides a Duroc pig SNP molecular marker combination, a Duroc pig liquid phase chip or a kit as described above.

[0008] The present application also provides a breeding method for Duroc pigs.

[0009] According to one aspect of the present application, a Duroc pig SNP molecular marker combination is provided, which is composed of 18444 SNP molecular markers, the genomic positions of the 18444 SNP molecular markers are determined based on sequence alignment of a pig reference genome Sscrofa11.1, and the site information is shown in Table 1 as follows:

[0010] Table 1

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[0018]

[0019]

[0020]

[0021]

[0022]

[0023]

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[0030]

[0031]

[0032]

[0033]

[0034]

[0035] In some embodiments of the present application, the position information of the SNP site is represented in the form of chromosome number: physical position.

[0036] In some embodiments of the present application, the 100 SNP molecular markers are as shown in SNP001-18444; the specific site information of SNP001-18444 is sequentially ordered from top to bottom and from left to right in the above table.

[0037] In some embodiments of the present application, in the table 1, Alt is not given, indicating that it has multiple mutation possibilities; the specific mutation possibilities are as follows:

[0038] When ref is A, Alt can be T, G, C or N;

[0039] When ref is T, Alt can be A, G, C or N;

[0040] When ref is G, Alt can be T, A, C or N;

[0041] When ref is C, Alt can be T, A, G or N, wherein N is deletion.

[0042] In a second aspect of the present application, a Duroc pig liquid chip is provided, which comprises a primer set and / or a liquid probe for detecting the above-mentioned Duroc pig SNP molecular marker.

[0043] In a third aspect of the present application, a kit is provided, which comprises a primer set and / or a liquid probe for detecting the Duroc pig SNP molecular marker.

[0044] In some embodiments of the present application, the kit further comprises at least one of a PCR reaction reagent, a nucleic acid extraction reagent.

[0045] In a fourth aspect of the present application, the above-mentioned Duroc pig SNP molecular marker, kit or Duroc pig liquid chip is used in Duroc pig genotyping.

[0046] In some embodiments of the present application, the use is in Duroc pig whole genome association analysis.

[0047] In some embodiments of the present application, the use is in Duroc pig cluster analysis or genetic background detection.

[0048] In some embodiments of the present application, the use is in Duroc pig kinship identification or Duroc pig breed resource molecular marker fingerprinting.

[0049] In some embodiments of the present application, the use is in Duroc pig breeding or assisted breeding.

[0050] In some embodiments of the present application, the use is in Duroc pig genetic map construction.

[0051] In some embodiments of the present application, the use is in Duroc pig germplasm resource identification.

[0052] In a fifth aspect of the present application, a Duroc pig breeding method is provided, comprising the following steps: detecting the DNA of a sample pig by using the above-mentioned Duroc pig SNP molecular marker, kit or Duroc pig liquid chip, and selecting a Duroc pig for subsequent breeding.

[0053] In some embodiments of the present application, the detection is based on a targeted sequencing genotyping technique.

[0054] The present application has at least the following beneficial effects:

[0055] The SNP molecular marker combination of the present application can be used to specifically identify Hunan Duroc pigs, and is used to prepare a liquid phase chip, and the obtained liquid phase chip can realize high-throughput and low-cost genotyping of Duroc pigs, and can be applied to molecular marker fingerprint analysis of Duroc pig breed resources, genotype identification of hybrid population offspring, authenticity identification of breeds, genetic background analysis and screening of breeding materials, whole genome association analysis, genetic diversity analysis of germplasm resources, and kinship identification, thereby providing important technical support for Duroc pig molecular breeding application.

[0056] Other features and advantages of the present application will be illustrated in the following description, and some will become apparent from the description, or will be understood by practicing the present application. BRIEF DESCRIPTION OF DRAWINGS

[0057] The present application will be further described below in combination with the drawings and examples, in which:

[0058] Figure 1 MAF value distribution diagram of Duroc pig 18K site in the embodiment of the present application;

[0059] Figure 2 Distribution diagram of Duroc pig 18K site on Duroc pig chromosome in the embodiment of the present application;

[0060] Figure 3 Cluster analysis diagram of 121 materials in the embodiment of the present application;

[0061] Figure 4 PCA scatter diagram in the test example of the present application.

[0062] Figure 5 Linkage disequilibrium decay analysis diagram in the test example of the present application. DETAILED DESCRIPTION

[0063] The concept and technical effects of the present application will be described clearly and completely in combination with the embodiments below, so as to fully understand the purpose, features and effects of the present application. Obviously, the described embodiments are only some of the embodiments of the present application, but not all the embodiments, and other embodiments obtained by those skilled in the art on the basis of the embodiments of the present application without creative labor all belong to the protection scope of the present application.

[0064] Unless otherwise specified, the examples were carried out under conventional conditions or under the conditions recommended by the manufacturer. Unless otherwise specified, the reagents or instruments used were conventional products that can be obtained commercially.

[0065] Example 1

[0066] The present embodiment provides a Duroc pig SNP molecular marker combination and a liquid phase chip constructed by the Duroc pig SNP molecular marker combination. The Duroc pig SNP molecular marker combination includes 18444 SNP sites, and the physical positions of the 18444 SNP sites are determined based on sequence alignment of a pig reference genome (Sscrofa11.1). The site information is shown in Table 1.

[0067] The screening process of the Duroc pig SNP molecular marker combination A is as follows:

[0068] 1. Duroc pig germplasm resource collection

[0069] In order to ensure the representativeness of the Duroc pig material and the universality of the liquid phase chip, 97 Duroc pig ear sample materials were collected from several breeding enterprises in Hunan Province.

[0070] 2. Duroc pig whole genome resequencing

[0071] The 97 Duroc pig materials collected were subjected to whole genome resequencing, and the specific steps included:

[0072] (1) High-throughput DNA extraction kit was used for sample DNA extraction.

[0073] (2) Library construction was performed using library construction reagents purchased from Huada, and the specific steps were as follows:

[0074] a. 200 ng of quantified and qualified DNA was placed in a 0.2 mL PCR tube, 4 μL End Repair Buffer and 2.6 μL End Repair Enzyme were added to the tube, water was added to 20 μL, and it was placed in an ABI 9700 PCR instrument for incubation at 37°C for 20 min and denaturation at 72°C for 20 min, completing the processes of DNA fragmentation, end repair and A tailing.

[0075] b. 2 μL Ultra DNA Ligase, 8 μL Ultra DNA Ligase Buffer and 4 μL Adapter for ILM, add water to 40 μL, put on ABI 9700 PCR instrument at 22°C for 60 min, complete the connection of sequencing adapter. Then add 48 μL of GenoPrep DNA Clean Beads to purify the connection product, after purification, use 0.68+0.2 times magnetic beads to screen fragments, and retain the connection product with insert fragments of 300-350 bp.

[0076] c. Add 10 μL of sequencing adapter with Barcode sequence, 10 μL of PCR Master Mix to the PCR tube of the previous step, and add water to 20 μL; use ABI 9700 PCR instrument for amplification, and the amplification program is: 98°C pre-denaturation for 2 min, 98°C denaturation for 30 s, 65°C annealing for 30 s, 72°C extension for 40 s, 5 cycles, 72°C extension for 4 min.

[0077] d. Add 20 μL of GenoPrep DNA Clean Beads to the second round of PCR product, place it on the magnetic stand until the solution is clear, discard the supernatant and add 100 μL of 80% ethanol to wash the magnetic beads, and add 35 μL of 10 mM Tris-HCl to obtain the purified DNA library.

[0078] (3) The library that passed the quality inspection was sequenced using the Huada MGI-2000 / MGI-T7 sequencing platform, and the sequencing mode was PE150 mode.

[0079] The raw reads were filtered using the software fastp (version 0.20.0, parameters: -n 10-q 20-u 40), and the data processing steps were as follows:

[0080] 1) Remove adapter sequences;

[0081] 2) When the content of N in the sequencing read exceeds 10% of the length of the read, the paired reads need to be removed;

[0082] 3) When the number of low-quality (Q≤20) bases in the sequencing read exceeds 40% of the length of the read, the paired reads need to be removed. The clean reads after quality control were aligned with the reference genome sequence using the software BWA (mem alignment mode), and the location of the clean reads on the reference genome was located through alignment. According to the alignment results of the clean reads on the reference genome, the HaplotypeCaller module of the software GATK (version 4.0.4.0) was used for variant detection.​

[0083] 3. Whole genome site screening

[0084] 1) Selection of candidate sites: Calculate the quality indicators of multiple sites obtained by resequencing, screen SNP polymorphic sites with heterozygosity less than 50%, site deletion rate less than 10%, minimum allele frequency greater than 0.1, and sequencing depth greater than 5 as candidate SNP sites.

[0085] 2) Probe design: Extract the upstream and downstream sequences of the candidate SNP sites, perform sequence alignment analysis (specificity), and screen sites that can design probes.

[0086] 3) Density screening: According to the principle of uniform distribution of sites, uniformly distributed SNP sites on the chromosome of Duroc pigs are screened, the average interval of the sites is 143Kb, and 18444 SNP sites are included.

[0087] (2) The final site screening index is: site deletion rate (NA_rate) is 7.34%, heterozygosity (Het_alt_rate) is 32.32%, minimum allele mutation frequency (MAF) is 0.289, the average interval between sites is 143Kb, and there are 18444 SNP sites. The site information is shown in Table 1, and the MAF distribution diagram is shown in Figure 1 , and the distribution diagram of 18K sites on the chromosome of Duroc pigs is shown in Figure 2 .

[0088] B Development of Duroc Pig 18K SNP Liquid Chip

[0089] The above selected 18444 candidate SNP sites are prepared into Duroc Pig 18K SNP liquid chip by using targeted sequencing genotyping technology. According to the principle of DNA complementarity, one or more probes covering the target SNP are designed at each site to be tested. These probes modified with biotin can hybridize with the target region in the denatured resequencing library to form double-stranded DNA. Streptavidin-coated magnetic beads are used to adsorb the molecules carrying biotin. After elution, amplification and sequencing, the genotype of the target SNP is obtained.

[0090] The technology involves the following steps:

[0091] (1) Sample DNA extraction and quality control: High-throughput DNA extraction kit is used for sample DNA extraction. The extracted DNA sample is detected by two methods:

[0092] a. Use 1% agarose gel electrophoresis method to analyze the purity and integrity of DNA;

[0093] b. Use Qubit to accurately quantify the concentration of DNA.

[0094] (2) Library construction and quality control:

[0095] a. Take the qualified DNA for quantitative quality control and use the ultrasonic crusher for random physical fragmentation. The peak value of the fragmented fragments is controlled at 200-300 bp. The fragmented DNA is subjected to end repair and then connected to an A tail. The DNA fragments with A added are connected to sequencing adapters using a ligase, and then the library is purified and the fragments are selected using carboxyl-modified magnetic beads, and the connection product with an insertion fragment of 200-300 bp is retained. The connection product is added with a sequencing primer with a Barcode and a high-fidelity PCR reaction system for PCR amplification. Different Barcodes are used to distinguish different samples. After purification by carboxyl magnetic beads, the amplification product can be used for probe hybridization experiment.

[0096] b. Take 500 ng of the constructed sequencing library, freeze-dry, add probes and hybridization reagents, denature, and incubate at 65°C for 2 hours to complete the hybridization reaction. The hybridization product is washed with a washing solution, and then a round of PCR is performed to complete the construction of the hybridization capture library. After the library construction is completed, Qubit 2.0 is used for preliminary quantification, and qPCR is used to accurately quantify the effective concentration of the library to ensure the quality of the library. After the library is qualified, it enters the sequencing stage. The MGI-2000 / MGI-T7 sequencing platform of Huada is used for sequencing, and the sequencing mode is PE150 mode.

[0097] (3) Bioinformatics analysis:

[0098] ①. Sequencing data quality control: Use the software fastp (version 0.20.0, parameters: -n 10-q 20-u 40) to filter the raw reads. The data processing steps are as follows: a. Remove the adapter sequence; b. When the content of N in the sequencing read exceeds 10% of the length of the read, the paired reads need to be removed; c. When the number of low-quality (Q≤20) bases in the sequencing read exceeds 40% of the length of the read, the paired reads need to be removed.

[0099] ②. Reference genome alignment: Use the software BWA (mem alignment mode) to align the clean reads after quality control with the reference genome sequence. Through alignment, the location of the clean reads on the reference genome can be located.

[0100] III. Core SNP analysis: Based on the alignment of Clean Reads to the reference genome, the UnifiedGenotyper module of the software GATK (version v3.5-0-g36282e4) was used for variant calling with the following parameters: "-dcov 1000000 -minIndelFrac 0.15 -glm BOTH -l INFO". The VariantFiltration module was used for filtering with the following parameters: --filterExpression "MQ0 >= 4 && ((MQ0 / (1.0*DP))>0.1)" --filterName "HARD_TO_VALIDATE" --filterExpression "DP<5||QD<2" --filterName "LOW_READ_SUPPORT". When there is an annotation file for the reference genome, the position annotation can be performed based on the SNP dataset.

[0101] Finally, the genotyping results of each target SNP in a specific individual can be obtained in high throughput, and high-throughput SNP genotyping is achieved; that is, the 18444 probe sequences are the information of the developed Duroc pig 18K SNP liquid chip.

[0102] Example 2 Application of Duroc Pig 18K SNP Liquid Chip in Pig Genotyping

[0103] This example provides the application of the Duroc pig 18K SNP liquid chip in pig genotyping in the above-mentioned Example 1, which specifically includes the following contents: based on the targeted sequencing genotyping technology, the Duroc pig 18K SNP liquid chip prepared in Example 1 is used to identify the genotype of 121 Duroc pigs, and the steps are as follows:

[0104] (1) Sample DNA extraction and quality control: high-throughput DNA extraction kit is used to extract sample DNA. The extracted DNA sample is detected by two methods:

[0105] a. The purity and integrity of the DNA are analyzed by using 1% agarose gel electrophoresis method;

[0106] b. The DNA concentration is accurately quantified by using Qubit.

[0107] (2) Library construction and quality control:

[0108] a. The qualified DNA was randomly physically broken by an ultrasonic disruptor, and the peak of the broken fragments was controlled at 200-300 bp. The broken DNA was end-repaired and connected to an A tail. The DNA fragments with A were connected to sequencing adapters by a ligase, and then the library was purified and the fragments were selected by carboxyl-modified magnetic beads to retain the connection products with an inserted fragment of 200-300 bp. The connection products were added with a Barcode sequencing primer and a high-fidelity PCR reaction system for PCR amplification. Different Barcodes were used to distinguish different samples. After purification by carboxyl magnetic beads, the amplification products can be used for probe hybridization experiments.

[0109] b. 500 ng of the constructed sequencing library was taken, and after freeze-drying, the probe and hybridization reagent were added. After denaturation, it was incubated at 65°C for 2 hours to complete the hybridization reaction. The hybridization products were washed with a washing solution, and then a round of PCR was performed to complete the construction of the hybridization capture library. After the construction of the library, Qubit 2.0 was used for preliminary quantification, and qPCR was used to accurately quantify the effective concentration of the library to ensure the quality of the library. After the library was qualified, it entered the sequencing stage. The MGI-2000 / MGI-T7 sequencing platform of Huada was used for sequencing, and the sequencing mode was PE150 mode.

[0110] (3) Bioinformatics analysis:

[0111] ①. Sequencing data quality control: the raw data (raw reads) was filtered by using the software fastp (version 0.20.0, parameters: -n 10-q 20-u 40), and the data processing steps were as follows: a. removing adapter sequences; b. when the content of N in the sequencing read was more than 10% of the length of the read, the paired reads needed to be removed; c. when the number of low-quality (Q≤20) bases in the sequencing read was more than 40% of the length of the read, the paired reads needed to be removed.

[0112] ②. Reference genome alignment: the software BWA (mem alignment mode) was used to align the qualified clean reads with the reference genome sequence, and through the alignment, the position of the clean reads on the reference genome could be located.

[0113] iii. Core SNP analysis: Based on the alignment of Clean Reads to the reference genome, the UnifiedGenotyper module of software GATK (version v3.5-0-g36282e4) was used for variant calling with the following parameters: "-dcov1000000-minIndelFrac 0.15-glm BOTH-l INFO". VariantFiltration module was used for filtering with the following parameters: --filterExpression "MQ0>=4&&((MQ0 / (1.0*DP))>0.1)" --filterName "HARD_TO_VALIDATE" --filterExpression "DP<5||QD<2" --filterName "LOW_READ_SUPPORT". When there is an annotation file for the reference genome, the position annotation can be performed based on the SNP dataset.

[0114] Finally, the genotyping results of each target SNP in a specific individual can be obtained in high throughput.

[0115] Genotype identification was performed on 121 Duroc pig samples, and the genotypes of target sites were extracted. The average detection rate of the samples was 99.49%, and the repeat consistency rate of the same batch of materials was 99.02%, indicating that the liquid chip target site detection rate of this scheme is high, and the genotyping results are accurate and reliable.

[0116] Example 3 Application of Duroc Pig 18K SNP Liquid Chip in Assisting to Distinguish the Genetic Background of Duroc Pigs from Different Pig Farms

[0117] The Duroc pig 18K SNP liquid chip prepared in Example 1 was used to perform cluster analysis on 121 Duroc pig materials from different pig farms in Hunan Province. The experimental process was consistent with the genotype identification method in Example 2. After obtaining the site genotype data, MEGA-X software was used to construct the NJ tree (model: p-distance; bootstrap: 1000 times).

[0118] The results are shown in Figure 3 As can be seen from the figure, the Duroc pig liquid chip prepared in Example 2 can assist in distinguishing the genetic background of Duroc pigs from different pig farms.

[0119] Example 4 Application of Duroc Pig 18K SNP Liquid Chip in Assisting to Distinguish the Genetic Diversity and Population Structure Analysis of Duroc Pigs

[0120] Genotyping of 97 Duroc pigs from different pig farms in Hunan Province was performed using the Duroc 18K SNP liquid chromatography chip prepared in Example 1. The PCA composition of the samples was analyzed using Plink software, and a PCA scatter plot was constructed. Each locus in the scatter plot represents a sample. The greater the distance between two samples, the greater the difference in their genetic background. Individuals with similar genetic backgrounds will cluster together in the plot.

[0121] The results are as follows Figure 4 As shown, statistical analysis revealed that Duroc pig samples were divided into four distinct groups, with samples from the same pig farm clustering together, consistent with known findings. The Duroc 18K SNP liquid phase chip prepared in Example 1 effectively distinguished Duroc pigs from different farms, consistent with expected results, and can achieve accurate inter-breed identification of Duroc pigs. The results indicate that the Duroc 18K SNP liquid phase chip of this invention can achieve excellent genetic diversity and population structure analysis of Duroc pigs.

[0122] Example 5: Application of Duroc pig 18K SNP liquid-phase chip genotyping in linkage disequilibrium decay analysis

[0123] To further evaluate the value of the Duroc 18K SNP liquid-phase chip in actual pig genetic breeding, linkage disequilibrium decay in 97 Duroc pig populations was analyzed. The specific process is as follows:

[0124] The study used DNA extracted from 97 pig samples and employed a designed Duroc pig 18K SNP liquid phase chip to detect the genotype results of the samples for linkage disequilibrium analysis.

[0125] The chain imbalance decay analysis was performed using software, and the results are shown in [link to results]. Figure 5 ,from Figure 5 As can be seen, the markers in this study can determine the marker levels in genome-wide association studies (GWAS), thus better assessing the detection power and accuracy of GWAS. Therefore, the Duroc 18K SNP liquid-phase chip developed in this invention can effectively and accurately determine marker levels, providing better reference value for GWAS and evolutionary and selection analyses.

[0126] The embodiments of the present invention have been described in detail above. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. A Duroc pig SNP molecular marker combinatorial system, characterized in that, It consists of 18,444 SNP molecular markers. The genomic locations of these 18,444 SNP molecular markers were determined by sequence alignment based on the pig reference genome Sscrofa11.

1. The specific site information is shown in Table 1 of the specification.

2. A Duroc pig liquid phase chip, characterized in that, The Duroc pig liquid phase chip includes a primer set and / or liquid phase probe for detecting the Duroc pig SNP molecular marker combination of claim 1.

3. A reagent kit, characterized in that, The kit includes a primer set and / or a liquid-phase probe for detecting the Duroc pig SNP molecular marker combination of claim 1.

4. The application of at least one of the Duroc pig SNP molecular marker combination of claim 1, the Duroc pig liquid phase chip of claim 2, and the kit of claim 3 in Duroc pig genotyping.

5. The application of at least one of the Duroc pig SNP molecular marker combination of claim 1, the Duroc pig liquid phase chip of claim 2, and the kit of claim 3 in the genome-wide association analysis of Duroc pigs.

6. The application of at least one of the Duroc pig SNP molecular marker combination of claim 1, the Duroc pig liquid phase chip of claim 2, and the kit of claim 3 in Duroc pig cluster analysis or genetic background detection.

7. The application of at least one of the Duroc pig SNP molecular marker combination of claim 1, the Duroc pig liquid phase chip of claim 2, and the kit of claim 3 in the identification of kinship of Duroc pigs or molecular marker fingerprinting analysis of Duroc pig breed resources.

8. The application of at least one of the Duroc pig SNP molecular marker combination of claim 1, the Duroc pig liquid phase chip of claim 2, and the kit of claim 3 in Duroc pig breeding or assisted breeding.

9. The application of at least one of the Duroc pig SNP molecular marker combination of claim 1, the Duroc pig liquid phase chip of claim 2, and the kit of claim 3 in the construction of Duroc pig genetic maps or identification of germplasm resources.

10. A method for breeding Duroc pigs, characterized in that, The process includes the following steps: using the Duroc pig liquid phase chip of claim 2 or the kit of claim 3 to detect the pig DNA of the sample to be tested, and selecting Duroc pigs for subsequent breeding.

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