CN117512121A - Jia Ji duck SNP molecular marker and application thereof - Google Patents
Jia Ji duck SNP molecular marker and application thereof Download PDFInfo
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Abstract
The invention discloses a Jia Ji duck SNP molecular marker and application thereof, wherein the Jia Ji duck SNP molecular marker comprises at least one of 20327 SNP molecular markers, and the physical positions of the 20327 SNP molecular markers are determined by sequence comparison based on a reference genome of a Muscovy duck; the invention utilizes the SNP molecular markers of the selected Jia-Ji ducks to design a synthesized chip, can realize genotyping, is used as a first-money whole genome liquid-phase chip of the Jia-Ji ducks, provides important tools for whole genome association analysis, whole genome selection, variety identification and breeding, genetic diversity assessment, germplasm resource and kindred relation identification, genetic map construction, gene positioning, molecular marker assisted breeding and the like of the Jia-Ji ducks, effectively shortens the breeding process of the Jia-Ji ducks, improves the breeding efficiency of the Jia-Ji ducks, and has wide application scenes in a plurality of fields of Jia-Ji duck breeding.
Description
Technical Field
The invention belongs to the technical field of poultry genetic breeding, and particularly relates to a Jia Ji duck SNP molecular marker and application thereof.
Background
The DNA molecular marker technology is an important tool in modern molecular breeding, and the traditional molecular marker plays an important role in the fields of animal and plant genetic improvement and the like. Such as DNA labeling techniques based on molecular hybridization (restriction fragment length polymorphism markers-RFLPs) and various DNA fingerprinting techniques based on polymerase chain reaction (random amplified polymorphic DNA markers-RAPD, simple sequence repeat markers-SSRs, sequence marker sites-STSs, etc.). However, the two traditional molecular markers have small distribution quantity in genome and complex experimental operation, so that large-scale commercial breeding application is difficult to realize. Single nucleotide polymorphism SNP mainly refers to DNA sequence polymorphism at genome level due to variation of single nucleotide, and is diverse in form, including deletion, insertion, transition, transversion, etc. of single base. SNP markers are used as the most widely used molecular markers at present, have the advantages of high genetic stability, abundant sites, wide distribution, representativeness, rapid detection, easy realization of automatic analysis and the like, are new generation molecular markers widely used at present, and are also ideal genotyping targets for high-flux molecular marker development.
In recent years, the main techniques for SNP locus typing include whole genome resequencing and gene chip. Whole genome resequencing is by genomic sequencing of different individuals of a species for which reference sequences are present, and based thereon, genetic differential analysis at the individual or population level is performed. Although sequencing costs have been significantly reduced in recent years, there are still significant challenges in the cost-prohibitive and massive data storage analysis for hundreds of sample sizes. It is particularly important to establish novel hybridization and sequencing methods to allow for efficient and rapid detection and analysis of large amounts of genetic information. In order to solve the technical problems, a technology based on genotyping target sites and regions, namely a gene chip, is developed and widely applied, and high-density molecular marker detection covering the whole genome is realized with smaller sequencing quantity. The existing gene chip mainly comprises a solid-phase chip and a liquid-phase chip, wherein the solid-phase chip is used for hybridizing a plurality of probe molecules on a support and then marking sample molecules, and the genotyping result of a sample is obtained by detecting the hybridization signal intensity of each probe molecule. The liquid phase chip is characterized in that a biotin marked probe is designed near a target site or region, then hybridization capture is carried out on the target site or region and a sample gDNA, amplification is carried out, and finally, the genotype of the target site is obtained after second-generation sequencing and data analysis. Compared with the solid phase chip, the liquid phase chip has the advantages of flexible site detection, short detection period, low cost and the like besides high detection rate and high accuracy; in addition, based on the amplification principle, the liquid-phase chip can be used for typing not only the target site, but also other SNP sites in the coverage area of the probe around the target site.
The Jia Ji duck belongs to a tumor head duck and is commonly called a Muscovy duck; is one of four famous dishes in Hainan province, and is defined as one of species for protecting world genetic diversity by united nations. The interaction of the excellent genetic background and the long-term tropical island climate condition and the unique feeding mode of the preserved duck has the excellent quality characteristics of big meat, white and smooth skin, tender meat, soft bone, less fat and the like. However, due to the lack of molecular breeding tools of the Jia-Ji ducks, the protection and breeding work is relatively extensive and has no sustainability, so that the variety of the Jia-Ji ducks is mixed, the excellent characters are seriously degraded, and the economic benefit is directly influenced. At present, no relevant report of a breeding chip is found in the field of Jia Ji duck breeding at home and abroad, and along with successful assembly of a reference genome of Jia Ji duck by Kunming animal science institute of China academy of sciences, tropical crop variety resource institute of China Tropical agricultural science institute and livestock veterinary institute of Hainan agricultural science institute, in order to shorten the breeding process of Jia duck, improve the selective breeding efficiency of Jia duck and accelerate the breeding pace of new varieties, the development of a SNP liquid chip of the whole genome of Jia Ji duck, which covers the whole genome, has high flux, strong flexibility and low single tag data cost, is needed to further improve the international competitiveness of Jia duck industry in China.
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 a SNP molecular marker for Jia Ji ducks.
The invention also provides a gene chip with the Jia Ji duck SNP molecular marker.
The invention also provides a kit with the Jia Ji duck SNP molecular marker.
The invention also provides application of the Jia-Ji duck SNP molecular marker, a gene chip and a kit.
According to one aspect of the invention, a Jia Ji duck SNP molecular marker is provided, the Jia Ji duck SNP molecular marker comprises at least one of 20327 SNP molecular markers, the physical positions of the 20327 SNP molecular markers are determined based on sequence alignment of a reference genome of a Muscovy duck, and the site information is specifically as follows:
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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 20327 SNP molecular markers are as set forth in SNPs 00001-20327; the specific site information of SNPs 00001-20327 is ordered from top to bottom, and from left to right as indicated in the above table.
According to a second aspect of the present invention, there is provided a Jia Jie duck SNP chip comprising a primer set and/or a liquid phase probe for detecting the above mentioned Jia Jie duck SNP molecular markers.
According to a third aspect of the present invention, a kit is provided, which comprises a primer set and/or a liquid phase probe for detecting the aforementioned Jia Ji duck SNP molecular markers.
According to a fourth aspect of the invention, the application of the Jia-Ji duck SNP molecular marker, the kit or the Jia-Ji duck SNP chip is provided.
In some embodiments of the invention, the use is in the genotyping of the gali duck.
In some embodiments of the invention, the use is in a whole genome association analysis of a japroduct duck.
In some embodiments of the invention, the application is in the analysis of the Jiaji duck clusters and the identification of the genetic relationship.
In some embodiments of the invention, the use is in the identification of genetic diversity of the jaboticaba ducks.
In some embodiments of the invention, the use is in breeding or assisted breeding of a Jia Ji duck.
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, jalousie duck variety 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 Jiaji ducks, comprising the steps of: detecting DNA of a sample to be detected by using at least one of the Jia-Ji duck SNP molecular markers, the kit or the Jia-Ji duck SNP chip, and selecting a proper Jia-Ji duck for subsequent breeding; the Jia Ji duck breeding method is not aimed at diagnosing or treating diseases.
In some embodiments of the invention, the detection is based on targeted capture sequencing technology.
According to some embodiments of the invention, at least the following benefits are provided: according to the invention, 18942 high-polymorphism whole genome loci and 1385 loci with high population differentiation index are obtained by mining and screening 165 sequencing data, genotyping can be realized by utilizing the loci to design and synthesize the loci, and the loci are used as a first whole genome liquid phase chip of the Jia-long ducks, so that important tools are provided for the whole genome association analysis, whole genome selection, variety identification and breeding, genetic diversity evaluation, germplasm resource and genetic relationship identification, genetic map construction, gene positioning, molecular marker assisted breeding and the like of the Jia-long ducks, the breeding process of the Jia-long ducks is effectively shortened, the breeding efficiency of the Jia-long ducks is improved, and the loci have wide application scenes in a plurality of fields of Jia-long ducks breeding. Meanwhile, the Jia Ji duck 20K liquid phase chip disclosed by the invention 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 other SNP sites in the coverage range of a probe around the target site, so that more SNP typing information can be obtained than the target site. Compared with a solid-phase chip, the method has the advantages of strong flexibility in site selection and sample detection quantity, low typing cost and the like, and provides a technical means for large-scale typing by depending on a second-generation sequencing platform.
Drawings
The invention is further described with reference to the accompanying drawings and examples, in which:
FIG. 1 is a distribution diagram of SNP loci of a 20K cGPS liquid phase chip of Jia Ji duck in example 2 of the invention on a chromosome;
FIG. 2 is a schematic diagram of the flow detection of a cGPS liquid phase chip in embodiment 3 of the present invention;
fig. 3 is a graph of the detection rate of a sample of a 20K cGPS liquid-phase breeding chip of a jia duck in embodiment 4 of the invention;
FIG. 4 is a graph showing the genotype consistency rate of repeated samples of the 20K cGPS liquid phase breeding chip of Jia Ji duck in the embodiment 4 of the invention;
fig. 5 is a liquid phase chip cluster analysis chart of jia duck 20K cGPS in example 5 of the present invention;
fig. 6 is a graph of the results of application of the GWAS liquid chip of 20K cGPS on the jia duck in example 6 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 provides a Jia Ji duck SNP molecular marker, which comprises 20327 SNP loci, wherein the physical positions of the 20327 SNP loci are determined based on sequence alignment of a reference genome (sequence reference article Chromosome-level genome assembly of the Muscovy duck provides insight into fatty liver susceptibility, biological item accession number: PRJCA009398 (accession number: GWHBKBF 0000000)) of Muscovy ducks, and the 20327 SNP molecular markers are shown in an invention content part (specific information is shown in an invention content table). The screening process is specifically as follows:
1. whole genome resequencing of Jia Ji duck
Collecting a diversity sample of the Jialong ducks and carrying out whole genome resequencing: the different Jia Ji duck populations 165 are selected to carry out whole genome re-sequencing, DNA is extracted from duck blood by using a magnetic bead method, and the integrity and concentration of the DNA are determined and analyzed by using 1% agarose gel electrophoresis and Qubit. Each gDNA sample was randomly broken, added with a sequencing adapter, and detected, and the library was sequenced on-machine using the DNBSEQ-T7 high throughput sequencing platform of MGI.
After the original data are obtained after sequencing, the genome comparison software BWA is utilized, and the comparison algorithm is the default parameters of BWA mem. Comparing the filtered clear Reads of each sample to a reference genome, and counting the comparison result. Based on the compared BAM file; then, performing de-duplication by using Picard-Mark Duplex; detecting all potential polymorphic SNP loci and INDEL loci in the whole genome of each sample through GATK to obtain GVCF of a single sample; finally, all samples GVCF were pooled using GATK-combineGVCFs. The pooled SNP sites were then subjected to preliminary filtration by the following criteria: "QD < 2.0|FS > 60.0|MQ < 35.0|MQRankSum < -12.5|read PosRankSum < -8.0|DP >6950"; and filtering to obtain a vcf file containing all sample SNP variation information.
2. Site selection
Calculating and counting MAF values, detection rates, heterozygosity rates and sequencing depths of loci from a vcf file containing SNP variation information of all samples, and screening 648895 target loci (including 1385 loci with high population differentiation indexes) according to MAF > 0.05, SNP locus detection rates of more than or equal to 90%, heterozygosity rates of less than or equal to 35% and sequencing depths of more than or equal to 10X.
And (3) designing probes for the target site set obtained by screening, wherein the principle of probe design is to design probes within the range of about 100bp aiming at all target sites, the length of the probes is generally 100bp, and the GC content is 20% -80%. According to the design result of the probe, the probe which cannot be uniquely aligned on the genome and contains the repetitive sequence in the flanking sequence is removed. Based on the principle of uniform distribution, 20327 SNP loci are preferably selected as target loci for synthesis, and the average interval is 45Kb.
Example 2
The embodiment provides a Jia Ji duck whole genome liquid-phase gene chip.
According to the probe prepared in example 1, a Jia-Ji duck whole genome liquid phase gene chip (20K chip) is prepared, a distribution diagram of SNP loci of the 20K cGPS liquid phase chip of the Jia-Ji duck on a chromosome is shown in figure 1, and the average interval is 45Kb.
Example 3 application of 20K SNP liquid phase chip in detection of DNA sample of Jia Ji duck
To verify the genotyping effect of the 20K SNP liquid chip, genotyping detection was performed on 51 parts of jia long duck material using the 20K SNP liquid chip obtained in example 2, 7 replicates were set in the experiment, and 58 parts of data were generated in total. The method comprises the following specific steps:
1. gDNA extraction and detection of Jia Ji duck
51 parts of fresh blood of Jia Ji duck is collected, and gDNA is extracted from duck blood by a magnetic bead method. The integrity and purity of gDNA was analyzed by 1% agarose gel electrophoresis, and the concentration was accurately quantified using Qubit.
2. cGPS experimental 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 library qualified in quality inspection, a blocking reagent, an RNase Block, and a 20K liquid chip probe on a PCR instrument for hybridization reaction, and performing hybridization incubation at 55 ℃ for 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) The raw data after high-throughput sequencing is subjected to quality control filtering and other treatments, and the fastp software is used for removing the Reads containing the joint pollution and the low-quality Reads. And (3) comparing the BWA software with a target genome, and then analyzing the mutation site of the sequencing result by using GATK software to obtain the genotyping result of the target site, wherein the flow chart is shown in figure 2.
The results of sequencing and data analysis are shown in fig. 3, and it can be seen from the graph that the detection rate of 58 sample sites is between 99.65% and 99.85%, and the average detection rate is 99.82%. The genotype results of 7 repeated samples are shown in fig. 4, and the consistency ratio is between 99.39% and 99.75% and the average consistency ratio is 99.59% as can be seen from the graph. The genotyping effect evaluation result of the 20K SNP liquid phase chip shows that the 20K SNP liquid phase chip prepared by the invention has good detection stability, high target site detection rate and accurate and reliable genotyping result.
Example 4 Jia Jie duck 20K SNP liquid phase chip population Structure analysis and Whole genome correlation analysis
(1) Group structure analysis
The analysis results of the colony structure analysis of the Jia Ji duck 20K SNP liquid chip prepared in the embodiment 2 are shown in the figure 5, and the Jia Ji duck 20KSNP liquid chip prepared in the embodiment 2 can effectively distinguish the Jia Ji duck varieties from different sources, and the results are consistent with expectations.
(2) Whole genome association analysis
The experimental method comprises the following steps: the whole genome correlation analysis is carried out on 236 parts of Jia Ji duck (variety is known) by using the liquid-phase chip of Jia Ji duck 20K SNP prepared in example 2, and the analysis result is shown in figure 6, and from the figure, it can be seen that the liquid-phase chip of Jia Ji duck 20KSNP prepared in example 2 can effectively distinguish local Jia Ji duck varieties from different sources, and the result accords with expectations.
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. A jia long duck SNP molecular marker, comprising: at least one of 20327 SNP molecular markers is included, and the physical positions of the 20327 SNP molecular markers are determined based on sequence alignment of a reference genome of the Muscovy duck; the SNP molecular marker locus information is specifically as follows:
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2. a Jia Jie duck SNP chip, wherein the SNP chip comprises a primer set and/or a liquid phase probe for detecting the Jia Jie duck SNP molecular marker according to claim 1.
3. A kit comprising a primer set and/or a liquid phase probe for detecting the jia long duck SNP molecular markers described in claim 1.
4. Use of at least one of the jia duck SNP molecular markers as set forth in claim 1, the jia duck SNP chip as set forth in claim 2 and the kit as set forth in claim 3 in genotyping of jia ducks.
5. Use of at least one of the jia duck SNP molecular markers described in claim 1, the jia duck SNP chip described in claim 2 and the kit described in claim 3 in whole genome association analysis of jia ducks.
6. Application of at least one of the Jia Ji duck SNP molecular marker of claim 1, the Jia Ji duck SNP chip of claim 2 and the kit of claim 3 in Jia Ji duck cluster analysis and genetic relationship identification.
7. Use of at least one of the jia duck SNP molecular markers as defined in claim 1, the jia duck SNP chip as defined in claim 2 and the kit as defined in claim 3 for genetic diversity identification of jia ducks.
8. Use of at least one of the Jia Ji duck SNP molecular markers of claim 1, the Jia Ji duck SNP chip of claim 2 and the kit of claim 3 in Jia Ji duck breeding or auxiliary 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, jalousie duck variety identification, genetic map construction, gene localization, species evolution analysis, and germplasm resource identification.
10. The breeding method of Jiajia ducks is characterized by comprising the following steps of: detecting DNA of a sample to be detected by using at least one of the Jia Ji duck SNP molecular marker in claim 1, the Jia Ji duck SNP chip in claim 2 and the kit in claim 3, and selecting proper Jia Ji duck for subsequent breeding.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311325462.6A CN117512121A (en) | 2023-10-12 | 2023-10-12 | Jia Ji duck SNP molecular marker and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311325462.6A CN117512121A (en) | 2023-10-12 | 2023-10-12 | Jia Ji duck SNP molecular marker and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN117512121A true CN117512121A (en) | 2024-02-06 |
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Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202311325462.6A Pending CN117512121A (en) | 2023-10-12 | 2023-10-12 | Jia Ji duck SNP molecular marker and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN117512121A (en) |
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2023
- 2023-10-12 CN CN202311325462.6A patent/CN117512121A/en active Pending
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