CN118086527A - Egg duck 20K low-density SNP chip based on targeted capture sequencing and application thereof - Google Patents
Egg duck 20K low-density SNP chip based on targeted capture sequencing and application thereof Download PDFInfo
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Abstract
The invention discloses a 20K low-density SNP chip for laying ducks based on targeted capture sequencing and application thereof, and relates to the technical fields of gene chip technology and gene molecular breeding. The low-density SNP locus combination of the whole genome of the laying duck consists of 20000 SNP molecular markers, and the positions of the SNP molecular markers on the ZJU1.0 of the reference genome of the laying duck are shown in the table 1 in the specification. The SNP locus combination provided by the invention can realize the genotyping of the laying ducks by a targeted capturing sequencing technology, and the result can be used for the application of germplasm resource evaluation, genetic relationship identification and genome selection of the laying ducks; secondly, the target SNP locus can be adjusted by directly increasing or decreasing the probes, so that the method has better flexibility compared with a solid-phase chip; in addition, the method can break through the key technical evaluation restricting the selective breeding of the laying duck genome, lay a foundation for building a sharing platform of the laying duck genome selection, and accelerate the crossing development of the laying duck breeding from 'phenotypic selection' to 'genome selection'.
Description
Technical Field
The invention relates to the technical field of gene chip technology and genetic molecular breeding, in particular to a 20K low-density SNP chip for laying ducks based on targeted capturing and sequencing and application thereof.
Background
SNP has the characteristics of large number, wide distribution, easy rapid large-scale screening, convenient genotyping and the like, and is a third generation genetic marker following a first generation restriction fragment length polymorphic marker and a second generation microsatellite, namely a simple tandem repeat marker. With the rapid development of the sequencing technology and the reduction of the cost, more livestock and poultry samples are sequenced to generate massive sequencing data, so that a data source is provided for mining SNP loci related to important characters, and unprecedented convenience is brought for the development of SNP chips. SNP chip technology has been widely used in chicken research as a new generation molecular genetic variation detection tool. The conventional commercial chicken SNP chip mainly comprises Illumina 60K, axiom K, pheno ixChip-I (Illumina 50K), IASCHICK (Illumina 50K), chicken core No. 1 (50K) and Beijing core No. 1 (55K), has been widely adopted by breeding enterprises and scientific research institutions, and is mainly applied to chicken genome selection, germplasm resource genetic diversity analysis, genetic relationship identification, genome association analysis and the like. The SNP information in the genome range is flexibly applied, and the SNP information can play an important role in the aspects of local variety protection, variety approval, group construction by selecting pure individuals, potential error pedigree correction, accurate pedigree system establishment, breeding value estimation accuracy improvement and the like. However, the molecular marking technology based on the sequencing technology has high cost and technical threshold, is not suitable for practical production and application, and lacks a special SNP chip for the laying ducks in the market at present.
SNP chips are classified into solid-phase chips and liquid-phase chips according to the difference of SNP locus dependent carriers. The solid phase chip performs typing through fluorescent signals of the markers based on complementary hybridization of the probes and the DNA sequences, has the advantages of high typing accuracy, short period and the like, but also has the defects of high single-site typing cost, difficult customization and the like. The liquid phase chip is a novel molecular detection technology following gel electrophoresis, fluorescence detection and solid phase chip, and the technology is based on targeted sequencing genotype detection (Genotyping By Target Sequencing, GBTS), namely, after a specific target point is selected from a genome, a target probe and a targeted sequence are complementarily combined to perform fixed-point capture, so that a marker with a relatively fixed physical position is obtained. The kit capable of simultaneously and rapidly completing hybridization reactions of thousands of probes in a liquid phase is visually called as a liquid phase chip. Compared with the solid-phase SNP chip technology, the liquid-phase SNP chip technology has the advantages of wide platform adaptability, simple data analysis, short detection period, flexible marking sites, flexible information addition and application at any time, and the like. Different mark forms (mSNP, SNP and haplotype) can be obtained by the same SNP chip site set, and various mark densities can be obtained by controlling the sequencing depth according to the requirements of application scenes.
At present, no SNP chip related to the laying duck exists in China, so that the selection of a proper SNP to manufacture a low-density liquid-phase chip by taking the genomic information of the variety of the laying duck in China as the cost is one of the problems to be solved urgently in the industry of the laying duck in China.
Disclosure of Invention
The invention aims to provide a 20K low-density SNP chip for laying ducks based on targeted capturing and sequencing, which aims to solve the problems of slow breeding progress and long breeding period in the breeding process of the laying ducks. The whole genome low-density SNP liquid-phase chip related to the important traits of the laying ducks can be used for genome selective breeding, genetic diversity analysis, variety identification, genetic relationship identification and whole genome association analysis of the laying ducks.
In order to achieve the technical purpose, the invention mainly adopts the following technical scheme:
In a first aspect, the invention discloses a low-density SNP locus combination of a whole genome of an laying duck, wherein the SNP locus combination consists of 20000 SNP molecular markers, and the positions of the SNP molecular markers on a ZJU1.0 reference genome of the laying duck are shown in a table 1 in the specification.
Table 1 20000 SNP molecular marker positions
In a second aspect, the invention discloses a probe for targeting the whole genome low-density SNP locus combination of the laying duck in the first aspect.
In a third aspect, the invention discloses a whole genome 20K low-density SNP chip of an egg duck, and the raw materials comprise the probe in the second aspect.
Preferably, the chip is a liquid phase chip.
In a fourth aspect, the invention discloses an application of the whole genome 20K low-density SNP chip of the laying duck in the third aspect in the analysis of important traits of the laying duck.
Further, the important characteristics of the laying duck comprise 40-week-old egg yield, 40-week-old weight, egg weight, eggshell color L, eggshell color a, eggshell color b, eggshell strength, eggshell thickness, eggshell weight and eggshell proportion.
In a fifth aspect, the invention discloses an application of the whole genome 20K low-density SNP chip of the laying ducks in the third aspect in genome selection and seed selection breeding of different varieties of laying ducks.
In a sixth aspect, the invention discloses an application of the whole genome 20K low-density SNP chip of the laying duck in the third aspect in filling into a high-density chip.
In a seventh aspect, the invention discloses an application of the whole genome 20K low-density SNP chip of the laying duck in the third aspect in genetic diversity analysis, genetic relationship identification, variety identification and whole genome association analysis of the laying duck and SNP locus typing detection of the laying duck.
Compared with the prior art, the invention has the following beneficial effects:
The liquid phase chip provided by the invention is used for carrying out whole genome resequencing on the egg-laying duck variety, has rich polymorphism in the egg-laying duck population, and has stronger pertinence, lower detection cost and higher speed compared with high-throughput sequencing;
The SNP locus contained in the liquid phase chip is excavated aiming at the important characteristics of the laying ducks, so that the liquid phase chip is suitable for breeding improvement of the important characteristics of the laying ducks and the like;
The 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 SNP (single nucleotide polymorphism) of which the target site is close to a certain range, so that more typing information can be obtained;
the liquid phase chip can adjust target SNP loci in a mode of directly increasing or decreasing probes, and has better flexibility compared with a solid phase chip;
the liquid phase chip provided by the invention can be used for rapidly detecting relevant SNP markers with low cost, has an pioneering significance in the aspect of breeding and breeding of the laying ducks, enables the popularization of molecular breeding of the laying ducks through the whole genome 20K low-density SNP liquid phase chip of the laying ducks to be possible, and greatly improves the breeding and breeding process of the laying ducks in China.
Drawings
FIG. 1 is a map of SNP site distribution on each chromosome;
FIG. 2 shows the results of three-variety phylogenetic tree analysis.
Detailed Description
Embodiments of the technical scheme of the present application will be described in detail below. The following examples are only for more clearly illustrating the technical aspects of the present application, and thus are merely examples, which should not be construed as limiting the scope of the present application. It is noted that unless otherwise indicated, technical or scientific terms used herein should be given the ordinary meaning as understood by one of ordinary skill in the art to which this application belongs.
Example 1 design and preparation of 20K Low Density SNP liquid phase chip for laying ducks
1. Establishment of SNP background database of laying duck
The invention obtains highly reliable SNP loci for subsequent screening by sequencing analysis by utilizing full genome re-sequencing data of 483 individuals of local varieties of 5 laying ducks.
The test population was derived from Jingjiang individuals of the 5 local egg-laying duck breeds of the duck, shaoxing duck, shanxing duck, shanjing duck, jinding duck and Duck at the county, wherein Jingjiang ducks 128, shaoxing duck 104, shanxing duck 97, jinding duck 83 and Duck at the county 71. First, 483 individuals were subjected to whole genome resequencing using DNBSEQ-T7 sequencer (Huada gene), and the average genome coverage of each sample was 10X or more. After preliminary quality control is carried out on the off-machine data, sequencing data are compared to a duck reference genome (ZJU 1.0) through BWA software, SNP mutation identification is carried out by utilizing a Haplotype cam module of GATK4 software, and 20,352,191 mutation sites are obtained in total. SNP of chromosome with site deletion rate >10%, minimum allele frequency <0.05, hardy-Winberg test P value less than 0.000001 and Z, W is filtered, and 12,142,344 SNPs are reserved for subsequent analysis through quality control.
2. Screening of functional loci
And screening and obtaining SNP loci related to the important traits of the laying ducks by adopting a whole genome association analysis strategy.
And (3) carrying out full genome association analysis by utilizing the whole genome resequencing data of 5 local varieties of the laying ducks according to the phenotype data measurement and statistics results of each variety, and utilizing a GLM model of PLINK software to obtain 1790 SNP (single nucleotide polymorphism) which are obviously related to the phenotype index association after quality control, and adding the SNP serving as a functional site into a chip design.
3. Screening of background sites
And supplementing background SNP loci on the chip based on resequencing data of 5 laying duck varieties according to the principle that the selected SNP loci are uniformly distributed in the genome. The genome is divided into intervals of 200kb as a standard, and if the intervals have functional sites, the sites are not supplemented, and if the intervals lack functional sites, 1 background site is supplemented. The screening principle of the background site is that MAF >0.05, genotype deletion rate <0.2, heterozygosity rate <0.2, and Hardy-Wenberg test P value is less than 0.000001.
Through the steps, the total number of functional sites and background sites is 20,099, sites which cannot be compared uniquely on the genome, sites containing repeated sequences in flanking sequences and sites with adjacent intervals smaller than 500bp are removed, and finally 20,000 SNP sites uniformly distributed on the duck genome are obtained, wherein the specific genome coordinates are shown in table 1. The distribution of all loci on each chromosome of the duck genome is shown in FIG. 1. According to the position and two-side sequence information of 20,000 SNP loci, a primer is designed and probe synthesis is carried out by Beijing Kang Pusen agricultural science and technology Co., ltd. By adopting a targeted capture sequencing technology, so that the 20K SNP liquid-phase chip of the laying duck is obtained.
Example 2 application of egg-laying duck high-density SNP liquid phase chip in egg-laying duck DNA sample detection
1. DNA extraction
The blood sample of the laying duck is subjected to DNA extraction by using a CWE9600 Magbead Blood DNA Kit kit of century through a magnetic bead method.
2. DNA quality inspection
Agarose gel electrophoresis to detect genome integrity: the DNA sample to be detected is sampled and spotted on 1.5% agarose gel and electrophoresed for 25min at 150V.
NanoDrop detection of DNA purity: the purity of the DNA is detected by using a Nanodrop 2000 nucleic acid protein tester, the A260/A280 ratio is in the range of 1.7-2.1, and the A260/A230 ratio is in the range of 1.8-2.2, so that the requirements of library construction are met.
Qubit carries out accurate quantification to the DNA concentration: samples were precisely quantified using DSDNA HS ASSAY KIT for Qubit kit.
3. Library construction
Qualified genomic DNA was detected using the universal library construction kit (for MGI)Library construction, wherein DNA is randomly interrupted by a breaker covaries TM, repaired by the end, A-tailed and connected with a sequencing linker, and thenSP Beads screen out fragments of about 300-350bp, PCR amplification is performed, andThe SP beams purified the PCR product, eventually obtaining a sequencing library.
4. Liquid phase probe capture
The constructed library is quantified by Qubit2.0 and is(Agilent) detection of the inserts of the library, and after the library is qualified, the probes with biotin labels are specifically bound to the DNA library with the linker sequence already on, i.e. the library hybridizes. The hybridization system consists of a probe, a hybridization buffer solution, a blocking reagent and a DNA library, and the whole hybridization reaction is carried out on a PCR instrument, so that the temperature is required to be precisely controlled, and the system is prevented from evaporating. After the hybridization reaction is finished, adding streptavidin magnetic beads into a reaction system to carry out hybridization capture. Streptavidin magnetic beads adsorb the double-stranded complex of the probe with biotin label and DNA. And (3) adsorbing the magnetic beads by using a magnetic frame, thereby obtaining a hybridization capture library. The library product was eluted and the capture was completed and nonspecific hybridization was removed using an eluent. The high temperature and low salt environment makes the existence of a large amount of nonspecific hybridization complex unstable, and the complex can be removed by multiple cleaning. The captured product was enriched and the eluted product was amplified by post-PCR and taken out of stock. The amplification cycle number is reasonably adjusted according to the size of the liquid phase probe.
5. Sequencing on machine
Sequencing by sequencing protocol using DNBSEQ-T7 sequencer, PE150 was selected.
6. Genotype data analysis
The raw data obtained were quality controlled using FastQC (www.bioinformatics.babraham.ac.uk/project), after which the sequencing data were aligned to the duck reference genome ZJU1.0 using the default parameters of BWA software (bio-BWA. Sourceforge. Net), and SNPs were genotyped using the standard procedure of GATK (software. Broaddetect. Org/GATK) software.
Example 3 reliability analysis of 20K chip test designed
10 Ducks, male and female halves, were randomly extracted from 5 varieties of sequencing samples, 50 samples were taken in total, blood DNA was extracted, a liquid chip probe was synthesized according to the designed chip information, and 50 samples were detected by using a liquid chip, and the results are shown in Table 2.
TABLE 2 detection rate of 20K liquid phase chip
As can be seen from table 2, the sample site capture efficiency is between 99.48% and 99.76%, with an average detection rate of 99.63%.
Example 4 identification of local variety of laying ducks Using chip
1. Collecting 299 local chicken blood of 3 local chicken species, extracting DNA, constructing a library for each qualified sample by using a standard program, synthesizing a liquid phase chip probe according to designed chip information, and carrying out SNP typing detection on the 299 samples by using the liquid phase chip.
2. And (5) detecting reliability analysis. The detection rate of 299 samples is between 99.14% and 99.88%, and the average detection rate is 99.635%.
3. Principal Component Analysis (PCA).
The obtained SNP was subjected to Principal Component Analysis (PCA) using Plink (version 1.9) to analyze the population structure. And constructing a sequenced variety phylogenetic tree according to the detected SNP and the corresponding genotype by utilizing TASSEL 5.0.0 software. As shown in FIG. 2, the colony structure of 7 Shandong local chickens can be distinguished based on the liquid phase chip, and the result of comparative example 1 can be consistent with the result of genome resequencing by adopting different sample DNA information.
While particular embodiments of the present invention have been illustrated and described, it will be appreciated that various other changes and modifications can be made without departing from the spirit and scope of the invention. It is therefore intended to cover in the appended claims all such changes and modifications that are within the scope of this invention.
Claims (9)
1. The whole genome low-density SNP locus combination of the laying duck is characterized by comprising 20000 SNP molecular markers, wherein the positions of the SNP molecular markers on a ZJU1.0 reference genome of the laying duck are shown in table 1 of the specification.
2. A probe targeting the whole genome low-density SNP locus combination of an laying duck of claim 1.
3. A whole genome 20K low-density SNP chip for laying ducks, characterized in that the raw material comprises the probe of claim 2.
4. The whole genome 20K low-density SNP chip of claim 3, wherein the chip is a liquid phase chip.
5. Use of the 20K low-density SNP chip of the whole genome of an laying duck as set forth in any one of claims 3-4 for analysis of importance traits of the laying duck.
6. The use of claim 5, wherein the egg duck importance traits comprise 40 week old egg yield, 40 week old body weight, egg weight, eggshell color L, eggshell color a, eggshell color b, eggshell strength, eggshell thickness, eggshell weight, and eggshell ratio.
7. The use of the whole genome 20K low-density SNP chip of laying ducks as set forth in any one of claims 3-4 for genome selection and seed selection and breeding of different varieties of laying ducks.
8. Use of the 20K low-density SNP chip of the whole genome of an laying duck as set forth in any one of claims 3-4 for filling into a high-density chip.
9. The use of the whole genome 20K low-density SNP chip of any one of claims 3-4 in genetic diversity analysis of laying ducks, genetic relationship identification of laying ducks, variety identification of laying ducks, and whole genome association analysis and SNP locus typing detection of laying ducks.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105331744A (en) * | 2015-12-08 | 2016-02-17 | 山东出入境检验检疫局检验检疫技术中心 | Detection method of duck flavivirus nucleic acid liquid phase chip |
| CN117512121A (en) * | 2023-10-12 | 2024-02-06 | 中国热带农业科学院热带作物品种资源研究所 | Jia Ji duck SNP molecular marker and application thereof |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105331744A (en) * | 2015-12-08 | 2016-02-17 | 山东出入境检验检疫局检验检疫技术中心 | Detection method of duck flavivirus nucleic acid liquid phase chip |
| CN117512121A (en) * | 2023-10-12 | 2024-02-06 | 中国热带农业科学院热带作物品种资源研究所 | Jia Ji duck SNP molecular marker and application thereof |
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| "Genome assembly ZJU1.0", NCBI, 20 December 2020 (2020-12-20), pages 1 - 6 * |
| 黄银花;李宁;D.W.BURT;吴非;: "鸭基因组研究现状和未来展望", 中国家禽, vol. 31, no. 09, 5 May 2009 (2009-05-05), pages 4 - 9 * |
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