CN118127186A - Hunan Guangyi black pig SNP molecular marker and application thereof - Google Patents
Hunan Guangyi black pig SNP molecular marker and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of molecular genetics, and discloses a Hunan Guangyi black pig SNP molecular marker and application thereof, wherein the Hunan Guangyi black pig SNP molecular marker consists of 461 SNP molecular markers, and the physical positions of the 461 SNP molecular markers are determined by sequence comparison based on a reference genome Sscroffa 11.1 of a pig; the invention utilizes the SNP molecular markers of the Hunan Guangyi black pigs obtained by screening to design a synthesized chip, can realize large-scale genotyping, provides important tools for identifying and breeding the Hunan Guangyi black pigs, evaluating genetic diversity, identifying germplasm resources and kindred relationship, assisting in breeding by molecular markers and the like, effectively shortens the breeding process of the Hunan Guangyi black pigs, improves the breeding efficiency of the Hunan Guangyi black pigs, and has wide application scenes in a plurality of fields in the breeding of the Hunan Guangyi black pigs.
Description
Technical Field
The invention belongs to the technical field of molecular genetics, and particularly relates to a Hunan Guangyi black pig SNP molecular marker and application thereof.
Background
DNA molecular markers (Molecular markers) are genetic markers based on inter-individual nucleotide sequence variations and are an important basis for molecular breeding. The single nucleotide polymorphism (Single nucleotide polymorphism, SNP) is the most common genetic variation on the genome, has the characteristics of numerous and high detection accuracy, and is the most common molecular marker. At present, the high-throughput SNP detection technology mainly adopts two strategies of a chip and a sequencing, wherein the chip comprises a solid-phase chip and a liquid-phase chip. The liquid phase chip gradually replaces the solid phase chip with the advantages of low parting cost, flexible detection, easy customization and the like, and is widely applied to livestock, poultry and aquatic products at present. The Hunan Guangyi black pig is a high-quality new variety which is cultivated by taking a Duroc pig as a male parent and a Hunan Xie black pig as a female parent through hybridization innovation, cross fixation and subculture breeding and a modern breeding technology combining a BLUP method with whole genome association analysis. As a new variety for breeding, the related research foundation is relatively lacking, and the development of a liquid-phase chip product of Hunan Guangyi black pigs can effectively solve the dilemma of weak research foundation at present.
Disclosure of Invention
The present invention aims to solve at least one of the technical problems in the prior art described above. Therefore, the invention provides the Hunan Guangyi black pig SNP molecular marker which can be used for identifying the germplasm resources and the affinitive relation of the Hunan Guangyi black pigs, provides an important tool for the auxiliary breeding of the Guangyi black pigs and the like, and plays an important role in the research of the Guangyi black pig breeding.
The invention also provides a gene chip with the Hunan Guangyi black pig SNP molecular marker.
The invention also provides a kit with the Hunan Guangyi black pig SNP molecular marker.
The invention also provides application of the Hunan Guangyi black pig SNP molecular marker, a gene chip and a kit.
The invention also provides a breeding method of the Hunan Guangyi black pigs.
According to a first aspect of the invention, there is provided a Hunan Guangyi black pig SNP molecular marker, which consists of 461 Hunan Guangyi black pig SNP molecular markers, wherein the physical positions of the 461 SNP molecular markers are determined by sequence alignment based on a reference genome Srcrofa 11.1 of a pig; the SNP molecular marker locus information is specifically as follows:
。
In some embodiments of the invention, the positional information of the SNP site employs chromosome numbering: the physical location is expressed in the form of a physical location.
In some embodiments of the invention, the 461 SNP molecular markers are as set forth in SNP 001-461; specific site information for SNPs 001-461 is ordered from top to bottom, left to right in the above table.
According to a second aspect of the present invention, there is provided a Hunan Guangyi black pig SNP chip comprising a primer set and/or a liquid phase probe for detecting the above-mentioned Hunan Guangyi black pig SNP molecular markers.
According to a third aspect of the invention, a kit is provided, which comprises a primer set and/or a liquid phase probe for detecting the SNP molecular markers of Hunan Guangyi black pigs.
According to a fourth aspect of the invention, application of the Hunan Guangyi black pig SNP molecular marker, the kit or the Hunan Guangyi black pig SNP chip is provided, wherein the application is application in Hunan Guangyi black pig genotyping.
In some embodiments of the invention, the use is in a hunan guangye black pig whole genome association analysis.
In some embodiments of the invention, the application is in Hunan Guangyi black pig cluster analysis and genetic relationship identification.
In some embodiments of the invention, the use is in the identification of genetic diversity in black pigs in the broad range of hunan.
In some embodiments of the invention, the use is in the breeding or assisted breeding of black pigs in Hunan Guangyi.
In some embodiments of the invention, the breeding or assisted breeding comprises at least one of assisted major gene selection, molecular assisted breeding, whole genome selective breeding, hunan Guangyi black pig breed identification, genetic map construction, gene localization, species evolution analysis, and germplasm resource identification.
According to a fifth aspect of the present invention, there is provided a method for breeding a black pig in Hunan Guangyi, comprising the steps of: detecting DNA of the to-be-detected Hunan Guangyi black pigs by using the Hunan Guangyi black pig SNP molecular markers, the kit or the Hunan Guangyi black pig SNP chip, and selecting the Hunan Guangyi black pigs for subsequent breeding; the Hunan Guangyi black pig breeding method does not aim at diagnosing or treating diseases.
In some embodiments of the invention, the detection is based on targeted capture sequencing technology.
Compared with the prior art, the invention has at least the following beneficial effects:
The Hunan Guangyi black pig specific SNP molecular marker provided by the invention can be used for low-cost and large-scale genotyping of Hunan Guangyi black pigs, is flexible in site based on a liquid chip technology, and can be added with new functional marker sites at any time in the later period. The liquid phase chip based on the specific SNP molecular marker provided by the invention can be used for identifying the germplasm resources and the affinitive relation of the Hunan Guangyi black pigs, provides an important tool for auxiliary breeding of Hunan Guangyi black pigs and the like, and plays an important role in research of Hunan Guangyi black pigs breeding.
Drawings
The invention is further described with reference to the accompanying drawings and examples, in which:
FIG. 1 is a schematic diagram of a flow chart of cGPS liquid phase chip detection in embodiment 2 of the present invention;
FIG. 2 is a graph showing the results of the site detection rate of 16 pieces of the gDNA sample of Hunan local pigs in example 2 of the present invention;
FIG. 3 is a graph showing the average uniformity of genotypes of 4 repetitive gDNA samples according to example 2 of the present invention;
FIG. 4 is a graph showing a group structure analysis of 403 parts of Hunan local pig gDNA samples in example 3 of the present invention.
Detailed Description
The conception and the technical effects produced by the present invention will be clearly and completely described in conjunction with the embodiments below to fully understand the objects, features and effects of the present invention. It is apparent that the described embodiments are only some embodiments of the present invention, but not all embodiments, and that other embodiments obtained by those skilled in the art without inventive effort are within the scope of the present invention based on the embodiments of the present invention.
Example 1
The embodiment designs and prepares a liquid phase chip for identifying Hunan Guangyi black pigs, which comprises the following specific steps:
1) Obtaining a set of SNP candidate positions of whole genome resequencing origin:
In order to obtain rich and various whole genome loci, 51 Guangyi black pigs bred by Hunan Guangyi agricultural development group Co., ltd.) are collected, whole genome re-sequencing is performed, and the library is subjected to on-machine sequencing by adopting a DNBSEQ-T7 high-throughput sequencing platform of MGI.
SNP CALLING the flow was followed using fastp (v0.20.0) filtration, burrows-WHEELER ALIGNER (0.7.12-r 1039) alignment to the porcine reference group (Sscofa 11.1), picard 1.107.107 software (http:// www.psc.edu/index. Php/user-resources/software/picard) ordering and converting the sam files to bam files, and removing PCR duplicates, using GATK software for SNP detection and filtration, using the following filtration criteria based on genome data: QD <2.0, FS >60.0, MQ <35.0, MQRankSum < -12.5, readPosRankSum < -8.0, DP >6950. And filtering to obtain vcf files containing all sample SNP variation information, wherein the sum of the vcf files is 5873685 SNP loci.
2) Further screening of SNP loci:
and calculating the Fst value of each SNP mutation site between Hunan Guangyi black pigs and non-Hunan Guangyi black pig reference groups by using VCFtools-0.1.13, screening the SNP sites to 11527 based on parameters that the Maf is more than or equal to 0.09 and the detection rate is more than or equal to 0.9, and using the sites for probe design.
The design principle of the probe is as follows: probes are designed for all target sites within the range of about 100bp, the length of the probes is generally 100bp, and the GC content is between 20% and 80%. Based on the probe design results, probes that cannot be aligned uniquely on the genome and/or contain repetitive sequences in flanking sequences are removed. Finally, 461 candidate SNP sites with Fst >0.85 were obtained as shown in Table 1.
TABLE 1 Hunan Guangyi black pig specific SNP molecular marker site information
。
3) Preparing a chip:
The system of the Hunan Guangyi black pig liquid phase chip is verified by synthesizing a liquid phase capture probe by Huazhi biotechnology limited company and adopting a precise positioning sequencing typing technology (cGPS) based on the liquid phase capture of a target region genome sequence. cGPS based on an optimized thermodynamic stability algorithm model, carrying out probe design on genome sequences of different target areas, carrying out liquid phase hybridization capture enrichment on a plurality of different target sequences positioned at different genome positions by utilizing a synthesized specific probe, and then carrying out sequencing library construction and high-throughput sequencing on the captured and enriched target genome sequences, thereby obtaining genotypes of all SNP/InDel loci in the target areas.
Example 2
This example uses the SNP molecular marker combination obtained by the screening in example 1 to detect 16 Hunan local pig DNA samples. The 16 local pig samples (including 4 repeated samples and-re) are taken as verification samples of 461 SNP liquid-phase chip development systems, including Guangyi black pig 2 heads (GYHZ-1, GYHZ-36), xiang black pig 1 head (XXHZ-48), temple front pig 1 head (SMQZ-5), dongshan pig 1 head (DSZ-6), sand pig 2 heads (SZLZ-19, SZLZ-20), dafeng pig 2 heads (DWZZ-19, DWZZ-3), ningxiang pig 2 heads (NXZ-10, NXZ-23) and Qianshao pig 1 head (QSHZ-1), and the detection flow is shown in FIG. 1, and the specific steps are as follows:
1) Extracting and detecting the gDNA of the local pig in Hunan: 12 parts of fresh blood of a local pig in Hunan province is collected, and gDNA is extracted from the pig blood by a magnetic bead method. The integrity and purity of gDNA was analyzed by 1% agarose gel electrophoresis, the concentration was accurately quantified using Qubit, and finally 16 Hunan local pig gDNA samples (including 4 replicates) were obtained.
2) CGPS (Genotyping by Pinpoint Sequencing of captured targets) test procedure:
① 200ng of gDNA with qualified quantitative quality inspection is taken, the DNA is cut into fragments with the size of 100-500bp by enzyme cutting reagent, and then Taq enzyme is added for terminal repair;
② Connecting the adaptor fragments to two ends of the DNA by using T4 ligase, purifying the connection products by using fragment sorting magnetic beads and amplifying the library to complete the construction of the library;
③ Placing the qualified library and the mixed solution of the blocking reagent, RNase Block of the RNase inhibitor and liquid phase chip probes of 461 SNP on a PCR instrument for hybridization reaction, and performing hybridization incubation at 55 ℃ overnight (16-24 h);
④ Capturing the hybridization product by using streptavidin, amplifying and enriching the captured library, and sequencing PE150 by using a Huada sequencing platform;
⑤ Raw data after high throughput sequencing is processed by quality control filtration and the like, and Reads containing joint contamination and Reads with low quality are removed by using fastp software. And comparing BWA software with a target genome, and analyzing mutation sites of the sequencing result by using GATK software to obtain a genotyping result of the target site, wherein the detection result is shown in figures 2 and 3.
As shown in FIG. 2, the site detection rate of 16 samples of the local pigs in Hunan province is 98.70% -99.57%, the average detection rate is 99.13%, and the scheme is proved to have high target site detection rate of the liquid phase chip and accurate and reliable typing result. As can be seen from FIG. 3, the consistency of the results obtained by detecting 4 repeated samples is between 99.78% and 100.00%, and the average consistency is 99.83%, which indicates that the liquid phase chip of the scheme has good detection stability and can be applied to actual production on a large scale.
Example 3
In this example, 403 parts of the gDNA samples of pigs in different places in Hunan (the sources of all the samples of pigs are known) were detected and subjected to group structure analysis by using the same detection procedure as in example 2, wherein the detection procedure comprises a Guangyi black pig 51 head, a Hunan black pig 50 head, a Siemens front pig 51 head, an east mountain pig 46 head, a sand mountain pig 52 head, a large girl pig 50 head, a Ningxiang pig 54 head and a Qian Shao Hua pig 49 head, and the analysis results are shown in fig. 4.
As can be seen from FIG. 4, 461 SNP molecular markers and corresponding liquid phase chips provided by the scheme can effectively distinguish Hunan Guangyi black pigs from other Hunan local pig varieties, and the result of FIG. 4 shows that the SNP molecular marker combination provided by the scheme can be applied to the identification of the germplasm resources and the kindred relationship of Hunan Guangyi black pigs as the auxiliary breeding of Guangyi black pig molecular markers.
The embodiments of the present invention have been described in detail with reference to the accompanying drawings, but the present invention is not limited to the above embodiments, and various changes can be made within the knowledge of one of ordinary skill in the art without departing from the spirit of the present invention. Furthermore, embodiments of the invention and features of the embodiments may be combined with each other without conflict.
Claims (10)
1. The SNP molecular marker is characterized by comprising 461 SNP molecular markers of the Guangyi black pigs in Hunan, wherein the physical positions of the 461 SNP molecular markers are determined by sequence comparison based on a reference genome Sscroffa 11.1 of the pigs; the SNP molecular marker locus information is specifically as follows:
。
2. The Hunan Guangyi black pig SNP chip is characterized in that the SNP chip comprises a primer set and/or a liquid phase probe for detecting the Hunan Guangyi black pig SNP molecular marker of claim 1.
3. A kit, which is characterized by comprising a primer group and/or a liquid phase probe for detecting the Hunan Guangyi black pig SNP molecular marker described in claim 1.
4. Use of at least one of the hunan guangying black pig SNP molecular marker of claim 1, the hunan guangying black pig SNP chip of claim 2, and the kit of claim 3 in hunan guangying black pig genotyping.
5. Use of at least one of the hunan guangying black pig SNP molecular marker of claim 1, the hunan guangying black pig SNP chip of claim 2, and the kit of claim 3 in hunan guangying black pig whole genome association analysis.
6. The application of at least one of the Hunan Guangyi black pig SNP molecular marker of claim 1, the Hunan Guangyi black pig SNP chip of claim 2 and the kit of claim 3 in Hunan Guangyi black pig cluster analysis and genetic relationship identification.
7. Use of at least one of the hunan guangying black pig SNP molecular marker of claim 1, the hunan guangying black pig SNP chip of claim 2, and the kit of claim 3 in the identification of genetic diversity of hunan guangying black pigs.
8. Use of at least one of the hunan guangying black pig SNP molecular marker of claim 1, the hunan guangying black pig SNP chip of claim 2, and the kit of claim 3 in hunan guangying black pig breeding or assisted breeding.
9. The use of claim 8, wherein the breeding or assisted breeding comprises at least one of assisted major gene selection, molecular assisted breeding, whole genome selective breeding, hunan guangyne black pig breed identification, genetic map construction, gene localization, species evolution analysis, and germplasm resource identification.
10. A breeding method of Guangyi black pigs in Hunan province is characterized by comprising the following steps: detecting the DNA of the pig to be detected by using at least one of the Hunan Guangyi black pig SNP molecular marker of claim 1, the Hunan Guangyi black pig SNP chip of claim 2 and the kit of claim 3, and selecting the Hunan Guangyi black pig for subsequent breeding.
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