CN118086519A - Pig liquid phase chip and preparation method and application thereof - Google Patents
Pig liquid phase chip and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a pig liquid phase chip, a preparation method and application thereof, wherein the liquid phase chip designed by utilizing a targeted capture sequencing technology can realize pig genotyping. Most of the existing chips are chips prepared based on analysis and detection of a plurality of varieties of big white pigs, long white pigs, duroc pigs and the like, and different from varieties of other chips, the chips comprise effect SNP loci of a plurality of varieties of Anhui local pigs, and the chips contain locus information which is screened by the Anhui local pigs and is obviously related to important economic characters such as growth characters, propagation characters, meat quality characters and the like, so that the chips are more suitable for application in local pig varieties. In particular to application in the fields of Anhui local pig genetic breeding, germplasm resource genetic improvement, genetic relationship identification, variety identification, genetic diversity analysis, whole genome association analysis, genome selective breeding and the like.
Description
Technical Field
The invention relates to the fields of molecular biology and animal breeding, in particular to a pig liquid phase chip and a preparation method and application thereof.
Background
Molecular markers (Molecular Markers), also called genetic markers, refer to means for identifying organisms by polymorphisms in the genomic DNA sequence of an individual. It uses inter-individual genetic sequence variation to reveal differences between individuals, such as genetic diversity, species relationships, and the like. The molecular marker has the advantages of polymorphism, co-dominance, inheritability and the like, and is widely applied to the fields of biodiversity protection, germplasm resource identification, genetic breeding and the like. Wherein, single nucleic acid polymorphism (SingleNucleotidePolymorphism, SNP) refers to a polymorphism of a DNA sequence at a genomic level caused by variation of a single nucleotide, including a transversion, a transition, an insertion or a deletion of a base. As a third generation molecular marker technology, SNP has more advantages than the first two generation technologies, including huge quantity, wide distribution, stable inheritance and convenient automatic detection. Currently, various means have been developed for SNP detection methods, such as conventional Sanger sequencing, taqMan probe technology, and gene chip technology.
The gene chip is a technology based on nucleic acid molecular hybridization development, and can be used for rapidly and efficiently detecting sequences of biological samples by the intensity of hybridization signals, and simultaneously processing and analyzing a large number of DNA fragments, and has the advantages of simple technical operation, multiple sample parallel processing, high degree of automation, high detection efficiency, wide application range, high analysis speed and the like, and overcomes a plurality of defects of traditional nucleic acid imprinting hybridization to a great extent. SNP (Single nucleotide polymorphism) chips related to the detection technology of the gene chip method are mainly based on an Illumina platform and an Affymetrix platform. The existing solid-phase gene chip has more parting sites in the detection flow and application; the SNP loci which are contained on the chip can be typed, the SNP loci around the loci cannot be typed, and once the solid-phase chip is designed, the loci which can be detected are fixed, the loci cannot be added and deleted, and the flexibility is poor. If a new solid state chip needs to be newly manufactured, special detection equipment needs to be used, and the requirement on the storage processing of data is high, so that the manufacturing cost is high. Therefore, a new genotyping technology is urgently needed to reduce the manufacturing cost of high-throughput SNP chips and flexibly adjust SNP markers according to actual population conditions.
Unlike solid-phase chips, liquid-phase chips are novel molecular detection techniques following gel electrophoresis, fluorescence detection, solid-phase chips, which are based on targeted sequencing genotype detection (GBTS) techniques, allowing complementary binding of target probes to target sequences for site-directed capture. The targeted sequencing genotyping technology system consists of two unique but mutually crossing technologies: genoBaits and GenoPlexs. Both techniques can realize accurate capture of any position and any length section of genome, and can detect various genetic variations such as SSR, SNP and the like at the same time. Compared with the traditional genotype detection technology and a solid-phase chip, the liquid-phase chip has the advantages of wide platform adaptability, flexibility of marking, high detection efficiency, information additivity and the like.
The Anhui province local pigs have abundant genetic resources and are provided with Anqing white pigs, anhui nan black pigs, anhui Zhejiang Hua pigs, dingyuan pigs, huo Shouhei pigs, anhui North pigs, polder pigs and other local pig species. However, the pig gene chip mainly used in China is developed mainly aiming at foreign pig varieties and combined multiple varieties, so that the situation that a plurality of SNP loci are low in quality and functional gene coverage is low when analyzing a certain variety in China is caused. Therefore, in order to accelerate the genetic progress of Anhui local pigs in China, a liquid phase chip with good use effect for covering the whole genome and molecular breeding of Anhui local pigs needs to be developed so as to comprehensively improve the protection efficiency and development and utilization efficiency of Anhui local pig resources and accelerate the breeding work of new varieties of Anhui local pigs and the progress of pig genetic breeding improvement.
Disclosure of Invention
The invention aims to provide a pig liquid phase chip and a preparation method and application thereof, so as to solve the problems.
According to a first aspect of the present invention, there is provided a swine liquid phase chip having single nucleotide core site information as shown in Table 1. Therefore, the pig liquid phase chip is designed by utilizing a targeted capture sequencing technology, and can realize pig genotyping. Most of the existing chips are chips prepared based on analysis and detection sites of several varieties such as big white pigs, long white pigs and Duroc pigs, and are different from the varieties of other chips, the chips comprise effect SNP sites of a plurality of varieties of Anhui local pigs, and the chips contain site information which is obviously related to important economic characters such as growth characters, propagation characters and meat quality characters and is screened by the Anhui local pigs, so that the chips are more suitable for application in local pig varieties. In particular to application in the fields of Anhui local pig genetic breeding, germplasm resource genetic improvement, genetic relationship identification, variety identification, genetic diversity analysis, whole genome association analysis, genome selective breeding and the like.
In certain embodiments, the sites of the liquid phase chip include the PHR0101_ Sus40K@V1.0 product site of Shimadzu borrelia biotechnology Co., ltd and the single nucleotide core site described in Table 1.
In certain embodiments, the liquid phase chip is composed of a probe mixture and a hybridization capture reagent designed and synthesized according to PHR0101_ Sus40K@V1.0 product site of Shimadzu borrelia biotechnology Co., ltd. And the single nucleotide core site described in Table 1.
According to a second aspect of the present invention, there is provided a method for preparing the above pig liquid phase chip, comprising the steps of:
S1: target SNP locus information acquisition and screening: carrying out resequencing data acquisition on a target population, and comparing resequencing results to a porcine reference genome to screen SNP loci, wherein the SNP locus screening conditions are as follows: SNP loci were selected with MissRate < 0.1, hetRate < 0.3, MAF > 0.05;
S2: and (3) probe design synthesis and test adjustment: designing a nucleotide sequence according to the SNP locus screened in the step S1, designing a primer by adopting a targeted capture sequencing technology, and synthesizing a DNA double-stranded probe, wherein the selection standard of the probe is as follows: selecting all probe lengths of the coverage areas to be 110bp, ensuring that the coverage degree of each section is relatively uniform, the Tm value is between 68 ℃ and 70 ℃, the GC content is between 30% and 70%, the content of base C is higher than the content of G, the number of the selected homologous areas is less than or equal to 5, screening out the probe sequences which finally meet the requirements through setting conditions, further confirming the coverage condition of target sites, and finally synthesizing the probe sequences;
S3: determining the final probe and capture site conditions: and (2) detecting and adjusting the SNP loci and the synthesized probes obtained in the step (S2) by a certain number of samples, and removing loci with low efficiency through the capture efficiency of the test loci to obtain the final pig liquid phase chip probes and hybridization capture reagents to form the pig liquid phase chip.
According to a third aspect of the present invention there is provided the use of a swine liquid phase chip as described above in genotyping swine DNA samples.
According to a fourth aspect of the invention, there is provided the use of a pig liquid phase chip as described above in pig genetic relationship analysis.
According to a fifth aspect of the invention, there is provided the use of a swine liquid phase chip as described above in the analysis of linkage disequilibrium attenuation of swine genotypes.
According to a sixth aspect of the invention, there is provided the use of a pig liquid phase chip as described above in whole genome association analysis or molecular assisted breeding or whole genome breeding or genetic modification of pig germplasm resources.
According to a seventh aspect of the present invention, there is provided a gene related to meat quality traits including any one of flesh color, pH, intramuscular fat, drip loss, which is screened by using the above-mentioned swine liquid phase chip;
the gene related to the flesh color property is any one of the following genes SPOCK1、CEPT1、NOX3、RPL10L、YAP1、BNIP3L、PNMA2、ENSSSCG00000049953、ENSSSCG00000051043、ENSSSCG00000043847、ENSSSCG00000044646、ENSSSCG00000043544、ENSSSCG00000051403;
The gene related to the pH value character is any one of the following genes GFRA2, ENSSSCG00000051172 and ENSSSCG 00000051623;
the gene related to the intramuscular fat trait is any one of the following genes AFF1,GPR139,TLE3,TMEM106A、ENSSSCG00000049537、ENSSSCG00000049162、ENSSSCT00000062070、ENSSSCT00000023596;
the gene related to the drip loss trait is any one of the following genes ALPK2、NOS3、KCNH2、PAXBP1、C21orf62、PITPNM2、ENSSSCG00000040330、ENSSSCG00000045739、ENSSSCG00000037885、ENSSSCG00000043855、ENSSSCG00000049372、ENSSSCG00000049537、ENSSSCG00000049162、ENSSSCT00000062070、ENSSSCT00000023596.
According to an eighth aspect of the present invention, there is provided a SNP molecular marker related to meat quality traits including any one of flesh color, pH, intramuscular fat, and drip loss, screened using the above-mentioned swine liquid phase chip;
SNP molecular markers related to flesh color characters are base mutation at 139503023bp position of a Sus_scrofa Sscoffa 11.1 version 4 chromosome, or base mutation at 139503023bp, 111068635bp and 112338624bp position of a2 chromosome, or base mutation at 11462282bp and 175742704bp positions of a1 chromosome, or base mutation at 32964891bp position of a 9 chromosome, or base mutation at 10378528bp position of a 14 chromosome;
The SNP molecular marker related to the pH value character is a base mutation at 10030031bp and 5467506bp of chromosome 14 of the Sus-scrofa Sscoffa 11.1 version of the pig genome;
The SNP molecular marker related to intramuscular fat property is a base mutation at 25623186bp position of a Sus_scrofa Srcrofa 11.1 version 3 chromosome, a base mutation at 167879482bp position of a1 chromosome, a base mutation at 131908789bp position of a 8 chromosome or a base mutation at 19747123bp position of a 12 chromosome;
SNP molecular markers related to the drip loss property are the base mutation at the 27042063bp and 29794808bp positions of chromosome 14 of Sus_scrofa Sscoffa 11.1 version of pig genome, the base mutation at the 162242993bp position of chromosome 1, the base mutation at the 7734440bp and 6239722bp positions of chromosome 18, the base mutation at the 196377418bp position of chromosome 13, or the base mutation at the 130958978bp position of chromosome 3.
The invention has the beneficial effects that:
1. the liquid phase chip has rich site polymorphism, and the chip contains sites selected by local pigs aiming at Anhui, so that the detection effectiveness can be improved;
2. The liquid phase chip is based on a targeted capture sequencing technology, not only can be used for typing a target site, but also can be used for typing SNP (single nucleotide polymorphism) of which the target site is close to a certain range, and more SNP sites can be detected compared with solid chips with the same density. The chip has flexibility in design, and new site information can be added at any time in a liquid reaction system, so that the detection accuracy is improved;
3. The liquid phase chip can rapidly detect related SNP molecular markers, especially SNP molecular markers of important economic characters such as growth characters, meat quality characters, propagation characters, disease resistance characters and the like of Anhui local pigs, so that important foundation is provided for cultivating various special local pig new strain and mating line combinations, and the breeding work of Anhui local pigs and the progress of pig genetic breeding improvement are accelerated;
4. The chip is a liquid phase chip prepared by combining the positions (position information in table 1) analyzed and detected by the Anhui local pig breeds based on a universal pig 40K liquid phase chip, and different from other existing chips, the chip comprises effect SNP positions of the Anhui local pig and screens the remarkably relevant functional positions of the researched growth characters, meat quality characters, propagation characters, disease resistance characters and the like. Therefore, the method is more suitable for being applied to Anhui local pig breeds, and plays an important role in the aspects of Anhui local pig genetic breeding, pig germplasm resource genetic improvement, pig genetic relationship identification and the like;
5. The chip is not only used for genotyping of pig DNA samples, pig genetic relationship analysis, linkage disequilibrium attenuation analysis of pig genotypes, pig whole genome association analysis, molecular marker assisted breeding, whole genome breeding, genetic improvement of pig germplasm resources and the like, but also can screen genes or molecular markers related to economic characters, such as high-efficiency screening of new genes or new molecular markers related to meat quality characters, and has important economic value for molecular marker assisted breeding of pigs, especially for genetic breeding of Anhui local pig species.
In summary, compared with the prior art, the invention combines the characteristics of the genome of the Anhui local pig on the basis of the developed pig 40K liquid phase chip, and utilizes the targeted capture sequencing technology to develop the liquid phase chip, compared with the existing liquid phase chip, the SNP loci contained in the liquid phase chip are derived from the whole genome re-sequencing data of 7 representative Anhui local pig varieties and functional loci found by early-stage research, so that the invention has rich polymorphism in the Anhui local pig population, and is more suitable for genetic improvement and resource evaluation of the Anhui local varieties. In addition, the liquid phase chip can adjust the target SNP locus in a mode of directly increasing or decreasing probes, so that the liquid phase chip has better flexibility. The liquid phase chip is based on a targeted capture sequencing technology, not only can be used for typing a target site, but also can be used for typing SNP (single nucleotide polymorphism) of the target site adjacent to a certain range, and more typing information can be obtained. Furthermore, the chip is not only used for genotyping of pig DNA samples, pig genetic relationship analysis, linkage disequilibrium attenuation analysis of pig genotypes, pig whole genome association analysis, molecular marker assisted breeding, whole genome breeding, genetic improvement of pig germplasm resources and the like, but also can screen genes or molecular markers related to economic characters, such as high-efficiency screening of new genes or new molecular markers related to meat quality characters, and has important economic value for molecular marker assisted breeding of pigs, especially genetic breeding of Anhui local pig species.
Drawings
FIG. 1 is a distribution diagram of SNP markers on different chromosomes in the first embodiment;
FIG. 2 is a uniform distribution diagram of SNP markers on different chromosomes in the first embodiment;
FIG. 3 is a map of Gap analysis of a target site on a genome in accordance with the first embodiment;
FIG. 4 is a graph showing MAF distribution statistics in accordance with a first embodiment;
FIG. 5 is a population PCA profile for all sites within a target segment in accordance with one embodiment;
FIG. 6 is a GenoBaits flow chart based on a targeted sequencing genotype detection technique in example two;
FIG. 7 is a statistical chart of reference sequence alignment results in the second embodiment: wherein, align_rate: alignment, calculation formula = number of reads aligned to reference genome/number of original sequencing sequence reads after filtering;
Fig. 8 is a sample statistical chart in the second embodiment: wherein, sample: sample name, na_rate: loss rate, hetalt rate: ratio of number of heterozygous sites to total number of non-deleted sites, hom_alt_rate ratio of number of homozygous sites to total number of non-deleted sites, ref_rate: referring to the ratio of the consistent number to the total number of non-missing sites, wherein the abscissa is the name of the sample, and the ordinate is the percentage;
FIG. 9 is a statistical diagram of SNP annotation in example two: wherein UTR3: the variation is located in the region of the 3' UTR of the gene, UTR5: the variation is located in the region of the 5' UTR of the gene, UTR3: mutation is located in the 5 'UTR region of the gene, while mutation is located in the 3' UTR region of the gene, downstream: the variation is located 2kbp downstream of the gene, exonic: the variation is located in the exon coding region intergenic: the variation is located in the intergenic region, intronic: the variation is located in the intron region, ncrna_ exonic: the variation is located in the ncRNA exon region, ncrna_intronic: the variation is located in the intron region of ncRNA, ncrna_ splicing: the variation is located at the ncRNA splice site, splicing: the mutation is located at the splice site, upstream: the mutation is located 2kbp upstream of the gene, downstream: the mutation is located 2Kbp upstream of the gene, and the mutation is located 2Kbp downstream of the gene;
FIG. 10 is a relationship heat map in the third embodiment;
Fig. 11 is an LD attenuation chart in the fourth embodiment: wherein the abscissa is the physical distance (kbp) and the ordinate is the r 2 value;
FIG. 12 is a Manhattan diagram and a QQ diagram of a genome-wide association analysis in example five: wherein A, B, C, D, E, F, G, H, I, J, K represents a Manhattan diagram and a QQ diagram of a 45min flesh color a value, a 45min flesh color b value, a 45min flesh color L value, a 24h flesh color a value, a 24h flesh color b value, a 24h flesh color L value, pH 45min、pH24h, a 24h drip loss rate, a 48h drip loss rate property and an intramuscular fat content property respectively.
Detailed Description
The invention is described in further detail below with reference to the accompanying drawings.
Example one preparation of pig liquid phase chip
The liquid phase chip design is a targeted genotyping technology based on liquid phase probe hybridization, which is a targeted genotyping technology based on the principle of nucleic acid complementary pairing. The principle is based on designing a specific probe according to the sequence of a target gene, carrying out complementary combination of the designed probe and a DNA sample to be detected on a targeting sequence, and obtaining the probe-target gene complex by utilizing a complementary pairing principle.
1.1 Acquisition and screening of target SNP locus information.
SNP locus information acquisition: the target SNP locus information is derived from genotyping results of 62 sample resequencing data and 40 sample resequencing vcf files of the local Anhui pigs including Anqing white pigs, southern Anhui black pigs, zhejiang Anhui Hua pigs, dead pigs, huo Shouhei pigs, northern Anhui pigs, and 7 pig breeds of polder pigs, and then all obtained candidate locus sets and sample information are subjected to the condition that the coverage is not less than 5 multiplied.
Site screening procedure: the method comprises the steps of performing site evaluation design on a core SNP site which is found by researching Anhui local pigs and is obviously related to important economic characters (including growth characters, reproduction characters, meat quality characters, body type characters, disease resistance characters and the like) and a PHR0101_ Sus40K@V1.0 product site of Shijia borrelidin biotechnology limited company according to a pig reference genome version Sus_scrofa Sscoffa 11.1, and screening the SNP site by comparing the re-sequencing result of the pig to a pig reference genome, wherein the screening conditions of the SNP site are as follows: sites were selected with MissRate < 0.1, hetrate < 0.3, MAF > 0.05. And ensures that SNP loci are uniformly distributed on each chromosome and have good polymorphism, and then the SNP loci after screening need to be subjected to duplication removal treatment. And 68,677 available sites for design completion are finally obtained.
1.2 Synthesis of Probe design and test adjustment.
Designing a nucleotide sequence according to the screened SNP locus, designing a primer by adopting a targeted capture sequencing technology, and synthesizing a DNA double-stranded probe. The probe design is to select all the probes of the coverage area to be 110bp in length, so that the coverage degree of each section is relatively uniform; tm values between 68 ℃ and 70 ℃; GC content is between 30% and 70% and the content of base C is higher than the content of G; the number of the selected homologous regions is less than or equal to 5; and screening out a probe sequence which finally meets the requirements through setting conditions, further confirming the coverage condition of a target site, and finally synthesizing the probe sequence.
1.3 Determination of final Probe and Capture site conditions
Adding EDTA and Tris-HCl mixed solution into the synthesized probe sequence to prepare a probe mixed solution, testing 68,677 sites meeting the evaluation requirement by adopting 200 Anhui local pig samples in the synthesized probe mixed solution, and removing sites with lower efficiency through the capture efficiency of the test sites; and finally, 55,880 sites (PHR 0101_ Sus40K@V1.0 product sites of 40K and 13917 sites screened by pigs in Anhui) are obtained, wherein the 4-sample experiment is failed, the site capture efficiency of the residual sample is 98.83% -99.81%, the average capture efficiency is 99.69%, and the average detection rate is 99.69%. The liquid phase chip for pig is composed of probe mixture and hybridization capture reagent, and the hybridization capture reagent comprises individually packaged GenoBaits Block I、GenoBaits Block II、GenoBaits 2×Hyb Buffer、GenoBaits Hyb Buffer Enhancer、GenoBaits 2×Beads Wash Buffer、GenoBaits 10×Wash Buffer I、GenoBaits 10×Wash Buffer II、GenoBaits 10×Wash Buffer III、GenoBaits 10×StringentWash Buffer.
The data test is carried out on the 68,677 sites which are preliminarily screened by adopting 200 Anhui local pig samples, and the distribution evaluation result of the tested SNP markers is shown in the figures 1-3, so that the distribution condition that each chromosome is covered by the SNP markers screened by using a pig liquid phase chip according to 200 test samples can be shown. Wherein, figure 1 shows the statistics result of the number of target sites on different chromosomes, and a histogram drawn by taking the abscissa as the chromosome ID and the ordinate as the site number; FIG. 2 shows the distribution of target sites on a chromosome, using the starting position of a target segment for the drawing of a uniform distribution map; FIG. 3 is a map of the Gap analysis of the target site on the genome, wherein all sections are adopted for analysis, the distance between the two sections is obtained from the starting position of the next section to the ending position of the previous section, the map of the Gap analysis of the target site on the genome is drawn, and the prepared liquid phase chip SNP interval is reasonable as can be shown from FIGS. 1 to 3. And finally, selecting 55,880 final target sites according to site capturing efficiency, wherein the number of core sites after screening for Anhui local pigs is 13,917.
The mononucleotide site of the pig liquid phase chip is a final site obtained by combining 13917 sites obtained by screening from Anhui local pig sites based on PHR0101_ Sus40K@V1.0 product of Shijia Boruidi biotechnology limited company, and the information of the mononucleotide core site related to the economic characters of the Anhui local pig is shown in table 1, wherein: site numbers from Chr 01-ChrX on the left indicate the chromosome where the site is located, the middle number represents the position of the site on the reference genome (version sus_scrofa sscroffa 11.1), and the last letter indicates the base of the site on the reference genome/the mutated base of the site. For example, the first locus Chr01_853058_C/T represents a C/T base mutation at position 853058 of chromosome 01.
1.4 Detection of prepared pig liquid phase chip
And further detecting the prepared liquid phase chip: minimum Allele Frequency (MAF) analysis is performed on 200 Anhui local pig samples, wherein MAF refers to the unusual allele occurrence frequency of a certain site in a specific population, and false positives are caused by the fact that the result is generated due to the fact that less available information is obtained by the smaller MAF in the population analysis, and MAF is often required to be larger than 0.05. As can be seen from the MAF distribution statistical chart shown in FIG. 4, the Minimum Allele Frequency (MAF) of the liquid phase chip is 0.2997, which meets the design requirements.
And (3) carrying out group Principal Component Analysis (PCA) on all sites in a target section tested by 200 Anhui local pig samples, adopting GAPTI analysis software, and drawing a visual three-dimensional graph by utilizing the first three principal components, wherein the result is shown in FIG. 5. In the PCA profile, each site represents a different sample, with a greater difference in genetic background being represented by the farther distance location between the sites. From fig. 5, it can be seen that the genetic background difference between different populations of the test sample is obvious, the population diversity is high, and the test sample information is met.
Further, the average distance between adjacent SNP marker loci of the swine liquid phase chip was 74.73kb. The degree of linkage disequilibrium r 2 between adjacent SNP sites was 0.55. The linkage disequilibrium degree of SNP markers is the guarantee of the whole genome association analysis and genome selection accuracy, and the genome selection accuracy is greatly reduced as the linkage disequilibrium is lower.
Example two application of pig liquid phase chip in genotyping of Anhui local pig DNA sample
The genotyping of the pig liquid phase chip relative to the Anhui local pig DNA sample prepared in the first embodiment is a targeted genotyping technology based on liquid phase probe hybridization, and the accurate detection technology means is carried out by the DNA base complementary pairing principle. This technique designs specific probes for each site to be tested, which cover the target SNP and are labeled with biotin. Under a liquid state environment, the probes can be complementarily matched with a target region of a genome to form a double-stranded structure. The streptavidin-coated magnetic beads can be used for effectively adsorbing molecules carrying biotin. Through a series of elution, amplification, pooling and sequencing, the genotyping status of the target SNP in a particular individual can be ultimately determined. The whole test flow is completed based on GenoBaits technology, the GenoBaits technology flow is shown in fig. 6, and the specific genotyping detection flow is as follows:
2.1 construction of sample libraries based on GenoBaits technology
The research group is selected from a huidinhua seed-retaining field in Taihu county of Anqing city in Anhui province, 78 head collection ear tissue samples of six white pigs Anqing in Anhui province are selected, a CTAB method is adopted for extracting DNA samples, the extracted sample DNA is subjected to 1% agarose gel electrophoresis to detect the purity and the integrity of the DNA, and the Qubit is used for accurately quantifying the DNA concentration. And (3) fragmenting the sample DNA qualified in quantitative quality inspection by using an ultrasonic fragmenting instrument, controlling the fragmentation peak value of the DNA fragment to be 200-300 bp, and then carrying out terminal repair on the DNA fragment and connecting the A tail. And (3) connecting the DNA fragment added with A with a sequencing joint by using ligase, purifying the library by using carboxyl modified magnetic beads, selecting the fragment, and retaining a connection product of the inserted fragment in 200-300 bp. And adding the connection product into a sequencing primer with Barcode and a high-fidelity PCR reaction system to carry out PCR amplification. The amplified product can be used for probe hybridization experiments after being purified by carboxyl magnetic beads. Then 500ng of the sequencing library which is constructed is taken, the probe and the hybridization reagent are added after freeze-drying, and the hybridization reaction is finished after denaturation and 2 hours incubation at 65 ℃. Washing the hybridization product, performing a round of PCR, completing the construction of a hybridization capture library, performing preliminary quantification by using Qubit2.0 after the construction of the library, and accurately quantifying the effective concentration of the library by using a qPCR method to ensure the quality of the library. The total amount of the library detected is more than 150ng, thereby meeting the sequencing requirement.
2.2 Detection and filtration of genotyping
And after the library is detected to be qualified, entering an on-machine sequencing stage. The resulting original sequenced sequence (Sequenced Reads) was sequenced, which contained low quality reads with adaptors. In order to ensure the quality of information analysis, the software fastp is used for filtering the raw reads, and the processing conditions are as follows: removing the linker sequence (adapter); when the amount of N contained in the sequencing read exceeds 10 bases, this pair PAIRED READS needs to be removed; when the number of low-mass (Q.ltoreq.20) bases contained in a sequencing read exceeds 40% of the length proportion of the strand, the pair pairedreads needs to be removed. Cleanreads is finally obtained and then used CLEAN READS for subsequent analysis.
2.3 Reference sequence alignment analysis
And comparing CLEAN READS after quality control with a reference genome sequence by using software BWA, and positioning CLEAN READS on the reference genome by the comparison to reflect the similarity of sample sequencing data and the reference genome. The statistics of specific reference sequence comparison results are shown in fig. 7, and the comparison rate is 97.71% -99.21% as can be seen from fig. 7, which shows that the similarity between the quality-controlled reference sequence and the reference genome sequence is higher.
2.4 Genotyping and site SNP analysis
Mutation detection is performed based on the genomic analysis tool GATK, and position annotation can be performed on the basis of the SNP dataset. After the annotation file of the reference genome is obtained, the comparison result of the target region is combined with the mutation result through a specific script, and the genotyping information of the SNP locus is extracted from the comparison result. To ensure the accuracy of genotyping results, coverage depth of a certain SNP site less than 5X is considered to be a deletion and expressed by NA. For heterozygous genotypes, at least 4 reads need to be supported per allele, otherwise the locus is equally considered deleted. Finally, counting the site deletion and mutation conditions of each sample, wherein the specific sample statistics are shown in fig. 8, and the sample detection deletion rate meets the requirements when the deletion rate is below 10% according to the test standard. As can be seen from FIG. 8, the deletion rates are all below 5%, which means that the number of undetected sites of the sample is small, and the detection accuracy is high, i.e. the test result meets the requirements.
2.5 Core SNP site annotation
And carrying out functional annotation based on the detected genetic variation information, and obtaining the region where the variation site occurs in the genome and the influence of variation according to the position of the variation site on the reference genome and the genetic position information on the reference genome by adopting an ANNOVAR software tool. The statistics of SNP annotation are shown in FIG. 9, from which it can be seen that the mutated regions are mainly in the intron and exon coding regions, accounting for 44.08% and 42.68% respectively, and a small portion in the other regions.
The reference genome locus is compared with the chip detection locus, the number of detection loci is 55019 for 78 samples, the average detection rate is 98.03%, and the sample deletion rate of each SNP locus is 2.29%. The results show that the SNP state of the target gene locus can be accurately detected by using the genotype of the designed pig liquid phase chip detection sample, and the method has the characteristics of high detection rate, strong specificity, high accuracy, good polymorphism and the like.
Embodiment III, application of pig liquid phase chip in pig genetic relationship analysis in Anhui places
In order to further evaluate the value of the pig liquid phase chip in actual pig genetic breeding, the pig liquid phase chip is applied to pig genetic relationship analysis in Anhui places, and the application effect of the pig liquid phase chip is shown. The specific procedure and results are as follows.
Firstly, collecting 139 head ear tissue samples of Anhui Yue black pigs with the weight of 20 kg-30 kg, wherein the Anhui Yue black pig group is selected from Anhui Mingshan agricultural Co. And then extracting DNA from the collected sample, and then adopting the pig liquid phase chip to genotype the quality-checked qualified DNA sample, wherein the purity OD 260/OD280 value of the qualified DNA sample is 1.6-1.8, so as to obtain 55,879 SNP markers covering the whole genome.
Quality control is carried out on the obtained genotype, plink software is utilized for quality control, and the quality control filtering standard is as follows: removing loci with Minor Allele Frequencies (MAFs) less than 0.05; removing sites with deletion rate of more than 20%; removing sites with a hybridization ratio of more than 50%; removing non-binary alleles; and filtering to obtain 42,686 SNP loci, and performing genetic relationship analysis by using the SNP loci after quality control.
The genetic relationship analysis uses GCTA software (v1.92.4) to analyze, based on SNP markers obtained after screening, a genetic relationship matrix between every two samples is obtained, a heat diagram drawn by the genetic relationship matrix is shown in fig. 10, the genetic relationship between every two samples can be represented in fig. 10, and an individual with a general diagonal less than 0.8 or greater than 1.2 can be clearly judged to be an outlier sample. Therefore, the genetic relationship between samples can be accurately judged by detecting the genotypes of the samples through the designed pig liquid phase chip.
Application of swine liquid phase chip genotype in linkage disequilibrium attenuation analysis
In order to further evaluate the value of the pig liquid phase chip in actual pig genetic breeding, the pig liquid phase chip is used for analyzing the linkage disequilibrium attenuation of an Anhui local pig group. The specific process is as follows
DNA is extracted from 78 head collected ear tissue samples of Anhui local square pigs Anqing and white pigs in the second embodiment, and the genotype results of the samples are detected by using a designed pig liquid phase chip for linkage disequilibrium analysis.
Linkage disequilibrium (linkage disequilibrium, LD), which refers to a non-random combination of alleles at different loci within a population, that is, linkage disequilibrium is exhibited to some extent as long as the two genes are not inherited completely independently. The study used PopLDdecay software for linkage disequilibrium analysis, analysis conditions: maxdis is equal to 1000, minMAF is equal to 0.01, bin1 is equal to 10, break is equal to 100, bin2 is equal to 500. The change with increasing distance is plotted and the LD decay distance is compared with the average distance between markers to determine if the markers have adequate coverage for the whole genome.
Linkage disequilibrium analysis is the basis of whole genome association analysis. By utilizing the correlation between the molecular markers and the functional mutations, i.e., linkage disequilibrium, the location of the functional mutation associated with a particular trait can be located. In general, when the linkage disequilibrium coefficient is greater than 0.8, a strong correlation can be considered to exist. And when the linkage disequilibrium coefficient is less than 0.1, it is considered that there is no correlation. If the marker coverage is not present in an interval as large as 0.1, no correlation signal is detected even if there is a functional mutation with a strong effect in this interval. Therefore, comparing the distance that linkage disequilibrium decays to 0.1 to the average distance between markers is critical to determine the extent of coverage of the whole genome by the markers.
By software analysis and drawing of a linkage disequilibrium attenuation analysis chart, the specific result is shown in fig. 11, and it can be seen from fig. 11 that the interval of LD attenuation to 0.2 is substantially covered by the mark. Therefore, the research mark can judge the marking quantity of the whole genome association analysis, and can better judge the detection effectiveness and accuracy of the whole genome association analysis; in addition, the method can also assist in analyzing evolution and selection, and LD attenuation slowly indicates that the population is selected on the same linkage group. Therefore, the swine liquid phase chip developed by the invention can effectively and accurately judge the marking quantity, and provides a better reference value for whole genome association analysis, evolution and selection analysis.
Fifth embodiment, application of pig liquid phase chip in Anhui local pig whole genome correlation analysis
In order to further evaluate the value of the pig liquid phase chip in actual pig genetic breeding, the whole genome correlation analysis of important economic characters of Anhui local pigs is utilized. The specific process is as follows:
5.1 phenotype data acquisition and quality control
The study population was selected from the hueing lake seed retention field of Taihu county, anqing city, anhui province. Collecting Anqing white pigs with average weight of about 95kg for uniform slaughter, collecting DNA extracted from ear tissue and longus dorsi muscle of each sample for meat quality property measurement, comprising: pH, flesh color, intramuscular fat, drip loss. And selecting genotype data with effective recorded phenotype data and detected by a pig liquid phase chip after quality control.
5.2 Genotyping and quality control
Quality control is carried out by using Plink software, genotyping detection is carried out on 190 Anqing six white pigs, and the quality control standard is as follows: individuals with SNP detection rate less than 90% and minimum allele frequency less than 90% were removed, and loci with minimum allele MAF less than 5% were removed. Finally 189 samples and 49,330 SNP loci were retained, and the total genotyping rate was 0.98833.
5.3 Whole genome correlation analysis
The study used Mixed Linear Model (MLM) for genome-wide association analysis of each trait. For different traits, adding the effect with obvious influence on the traits into a model as a fixed effect, wherein the MLM mixed linear model is as follows: y=xb+kμ+zα+sβ+e
Wherein y is a vector of phenotype values; x, K, Z, S are the correlation matrices of b, μ, α, β, respectively; b is a vector comprising sex and weight fixing effects; mu is a random effect vector conforming to normal distribution; alpha is a random additive genetic effector vector; beta is a population structure effect vector; e is a random residual vector conforming to a normal distribution.
Then, according to the Bonferroni correction threshold, the significant SNPs affecting the multiple traits of the test population were located with 0.05/SNP number as the whole genome significant level threshold and 1/SNP number as the chromosome significant level threshold, so as to determine the significant SNP physical locations, and the QQ map and manhattan map were drawn using R software (the results are shown in fig. 12), and the significant SNP sites were located. It can be seen from fig. 12 that the initial sites are consistent with the uniform distribution, which is caused by random drift, and after-log 10 is greater than 3, the uniform distribution remains on a straight line, but because of random drift and the presence of true sites, the start deviates from a straight line, where upturned sites are interesting sites and the analytical model is reasonable. From the manhattan diagram of fig. 12, distinct signal peaks are seen, these being the sites of interest.
And carrying out gene function annotation on the significant SNP locus genes by utilizing Ensembl database information, and primarily screening candidate genes possibly related to important economic traits through annotated gene functions and related researches. Through whole genome association analysis, meat color, pH, intramuscular fat, drip loss all detected a number of significant SNPs. Wherein the significant loci related to the flesh color characters are respectively located at 139503023bp on chromosome 4; 139503023bp,111068635bp,112338624bp on chromosome 2; chromosome 1 11462282bp and 175742704bp; 32964891bp on chromosome 9; at 10378528bp on chromosome 14 (the above sites were mapped with reference to the swine genome as Sscofa 11.1 version, the following are the same), candidate genes were identified as SPOCK1, CEPT1, NOX3, RPL10L, YAP1, BNIP3L, PNMA2 and ENSSSCG00000049953、ENSSSCG00000051043、ENSSSCG00000043847、ENSSSCG00000044646、ENSSSCG00000043544、ENSSSCG00000051403,6 new genes were found by gene annotation. The GFRA2 gene and novel genes ENSSSCG00000051172 and ENSSSCG00000051623 are discovered through gene annotation by locating the sites which are obviously related to the pH value on the chromosome 14 at 10030031bp and 5467506bp through chip detection and analysis. The sites with significant correlation of intramuscular fat are located at 25623186bp on chromosome 3, 167879482bp on chromosome 1, 131908789bp on chromosome 8 and 19747123bp on chromosome 12, and the corresponding genes detected by gene annotation are AFF1, GPR139, TLE3, TMEM106A and 4 new genes ENSSSCG00000049537, ENSSSCG00000049162, ENSSSCT00000062070 and ENSSSCT00000023596. In addition, SNP loci which are obviously related to water dripping loss exist at 27042063bp and 29794808bp on chromosome 14, 162242993bp on chromosome 1, 7734440bp and 6239722bp on chromosome 18, 196377418bp on chromosome 13 and 130958978bp on chromosome 3, candidate genes are identified as ALPK2, NOS3, KCNH2, PAXBP, C21orf62, PITPNM2 and 9 new genes ENSSSCG00000040330、ENSSSCG00000045739、ENSSSCG00000037885、ENSSSCG00000043855、ENSSSCG00000049372、ENSSSCG00000049537、ENSSSCG00000049162、ENSSSCT00000062070、ENSSSCT00000023596. healthily by gene annotation, genes near the prominent sites were found based on the start and end positions, and candidate genes possibly related to meat quality traits as above were found together.
Compared with solid-phase chip detection, the pig liquid-phase chip can detect different obvious sites and candidate genes, and more new candidate genes are found. Therefore, compared with a solid-phase chip, the pig liquid-phase chip provided by the invention can detect more SNP loci, and new locus information which is newly discovered and verified can be added into the liquid-phase chip at any time, so that the detection accuracy is better improved. The results show that more SNPs can be obtained by using the pig liquid phase chip of the invention without filling by adopting targeted capture sequencing, and more accurate genotyping and whole genome correlation analysis can be performed. In addition, the whole genome correlation analysis application is carried out on the liquid phase chip of the pig. The chip has the characteristics of high stability, high detection efficiency, good genotype quality, high throughput and the like. The technical progress not only reduces the detection cost and improves the breeding efficiency, but also is helpful to comprehensively improve the protection efficiency and the development and utilization efficiency of local pig resources, and further accelerates the breeding work of new local pig varieties and the progress of pig genetic breeding improvement.
Through the effect evaluation of the liquid-phase SNP chip in the third embodiment to the fifth embodiment in the application of the genetic breeding of Anhui local pigs, the invention can be widely applied to the researches such as LD attenuation analysis, genetic relationship analysis, whole genome association analysis and the like of Anhui local pigs such as Anqing white pigs, anhui Yue black pigs and the like. The chip can be widely popularized and applied to the genetic breeding of Anhui local pigs, and the efficiency of molecular breeding is comprehensively improved.
TABLE 1 pig liquid phase chip mononucleotide core site information Table
What has been described above is merely some embodiments of the present invention. It will be apparent to those skilled in the art that various modifications and improvements can be made without departing from the spirit of the invention.
Claims (10)
1. Pig liquid phase chip, wherein the single nucleotide core site information of the liquid phase chip is shown in the following table 1:
2. The chip of claim 1, wherein the sites of the liquid phase chip comprise the PHR0101_ Sus40K@V1.0 product site of borrelia biotechnology limited, borrelia, shijia, and the single nucleotide core site described in table 1.
3. The chip of claim 2, wherein the liquid phase chip is composed of a probe mixture and a hybridization capture reagent designed and synthesized according to the PHR0101_ Sus40K@V1.0 product site of borrelidin biotechnology limited, stone family and the single nucleotide core site described in table 1.
4. A method of manufacturing a chip as claimed in any one of claims 1 to 3, wherein the method comprises the steps of:
S1: target SNP locus information acquisition and screening: carrying out resequencing data acquisition on a target population, and comparing resequencing results to a porcine reference genome to screen SNP loci, wherein the SNP locus screening conditions are as follows: SNP loci were selected with MissRate < 0.1, hetRate < 0.3, MAF > 0.05;
S2: and (3) probe design synthesis and test adjustment: designing a nucleotide sequence according to the SNP locus screened in the step S1, designing a primer by adopting a targeted capture sequencing technology, and synthesizing a DNA double-stranded probe, wherein the selection standard of the probe is as follows: selecting all probe lengths of the coverage areas to be 110bp, ensuring that the coverage degree of each section is relatively uniform, the Tm value is between 68 ℃ and 70 ℃, the GC content is between 30% and 70%, the content of base C is higher than the content of G, the number of the selected homologous areas is less than or equal to 5, screening out the probe sequences which finally meet the requirements through setting conditions, further confirming the coverage condition of target sites, and finally synthesizing the probe sequences;
S3: determining the final probe and capture site conditions: detecting and adjusting the SNP loci and the synthesized probes obtained in the step S2 by a certain number of samples, removing loci with lower efficiency through the capture efficiency of the test loci, and obtaining the final pig liquid phase chip probes and hybridization capture reagents to form the pig liquid phase chip according to any one of claims 1-3.
5. Use of the chip of any one of claims 1-3 for genotyping a porcine DNA sample.
6. Use of the chip of any one of claims 1-3 in swine genetic analysis.
7. Use of the chip of any one of claims 1-3 in linkage disequilibrium attenuation analysis of porcine genotypes.
8. Use of the chip of any one of claims 1-3 in porcine whole genome association analysis or molecular marker assisted breeding or whole genome breeding or genetic modification of porcine germplasm resources.
9. A gene related to meat quality traits selected using the chip of any one of claims 1-3, wherein the meat quality traits comprise any one of flesh color, pH, intramuscular fat, drip loss;
The gene related to the flesh color trait is any one of the following genes SPOCK1、CEPT1、NOX3、RPL10L、YAP1、BNIP3L、PNMA2、ENSSSCG00000049953、ENSSSCG00000051043、ENSSSCG00000043847、ENSSSCG00000044646、ENSSSCG00000043544、ENSSSCG00000051403;
The gene related to the pH value character is any one of the following genes GFRA2, ENSSSCG00000051172 and ENSSSCG 00000051623;
the gene related to the intramuscular fat trait is any one of the following genes AFF1,GPR139,TLE3,TMEM106A、ENSSSCG00000049537、ENSSSCG00000049162、ENSSSCT00000062070、ENSSSCT00000023596;
The gene related to the drip loss trait is any one of the following genes ALPK2、NOS3、KCNH2、PAXBP1、C21orf62、PITPNM2、ENSSSCG00000040330、ENSSSCG00000045739、ENSSSCG00000037885、ENSSSCG00000043855、ENSSSCG00000049372、ENSSSCG00000049537、ENSSSCG00000049162、ENSSSCT00000062070、ENSSSCT00000023596.
10. A SNP molecular marker related to meat quality traits selected using the chip of any one of claims 1-3, wherein the meat quality traits comprise any one of flesh color, pH, intramuscular fat, drip loss;
SNP molecular markers related to the flesh color characters are a base mutation at the 139503023bp position of a Sus-scrofa Sscoffa 11.1 version 4 chromosome, a base mutation at the 139503023bp, 111068635bp and 112338624bp position of a2 chromosome, a base mutation at the 11462282bp and 175742704bp positions of a1 chromosome, a base mutation at the 32964891bp position of a 9 chromosome, or a base mutation at the 10378528bp position of a 14 chromosome;
SNP molecular markers related to the pH value character are base mutations at 10030031bp and 5467506bp positions of chromosome 14 of Sus_scrofa Srcrofa 11.1 version of the pig genome;
The SNP molecular marker related to the intramuscular fat trait is a base mutation at the 25623186bp position of chromosome 3 or a base mutation at the 167879482bp position of chromosome 1 or a base mutation at the 131908789bp position of chromosome 8 or a base mutation at the 19747123bp position of chromosome 12 of the Sus-scrofa Sscoffa 11.1 version of the pig genome;
The SNP molecular markers related to the drip loss property are the base mutation at the 27042063bp and 29794808bp positions of chromosome 14 of Sus_scrofa Sscoffa 11.1 version of pig genome, the base mutation at the 162242993bp position of chromosome 1, the base mutation at the 7734440bp and 6239722bp positions of chromosome 18, the base mutation at the 196377418bp position of chromosome 13, or the base mutation at the 130958978bp position of chromosome 3.
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