CN116855627A - Barley whole genome liquid phase chip and application thereof - Google Patents
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Abstract
The invention discloses a barley whole genome liquid phase chip and application thereof, and belongs to the technical field of gene chips. The invention screens 500SNP loci altogether, comprises 176 SNP loci positioned on the barley 'MorexV 3' version reference genome and 324 SNP loci known in the earlier stage, designs a liquid phase chip, develops and designs a liquid phase chip by utilizing the 500SNP loci, and can realize the genotyping of barley varieties by utilizing a targeted capture sequencing technology. The results show that the liquid chip provided by the invention has good application effects in basic researches or breeding works such as purity identification of barley varieties, population genetic analysis, primary genetic positioning of important agronomic trait regulatory genes and the like.
Description
Technical Field
The invention belongs to the technical field of gene chips, and particularly relates to a barley whole genome liquid phase chip and application thereof.
Background
Barley (Hordeum vulgare l., 2n=2x=14, hh) is the fourth major gramineous grain crop in the world today, the yield per unit of barley in China is greatly improved, the barley is benefited from the breeding of high-yield disease-resistant stress-resistant new varieties and the large-area popularization and use thereof, and the breeding of the new varieties of the barley depends on the excavation and identification of excellent resources and the breeding and utilization of excellent genes.
Molecular marker technology is an important tool for genetic research and is also an important technical method for identifying and utilizing excellent resources and excellent genes of barley. In barley, a variety of molecular marker types have been developed, including amplified fragment length polymorphism (Amplified Fragment Length Polymorphism, AFLP), simple sequence repeat polymorphism (Simple Sequence Repeat, SSR), sequence-Tagged Site polymorphism (STS), single nucleotide polymorphism (Single Nucleotide Polymorphism, SNP), and the like. The low throughput of these traditional molecular marker techniques limits their use in large-scale population samples. With the advent of gene chip technology and high throughput sequencing technology, solid phase chip (SNP array), simplified genome sequencing (GBS), exome-capture sequencing, whole genome re-sequencing (Whole-genome shotgun sequencing, WGS) and other genome research technical methods were derived, and technical support was provided for development and breeding utilization of barley high-density molecular markers. However, the cost of whole genome re-sequencing is high, and the method is not suitable for crops with huge genomes; simplified genome sequencing is low in cost, but has strong randomness of sequence capture and low repeatability and comparability among different batches of experiments.
Amplicon sequencing (mPCR) technology based on multiplex DNA polymerase Chain Reaction (Multiplex Polymerase-Chain-Reaction) is an improvement over traditional simplified genomic sequencing in that the basic principle is to amplify short genomic fragments using sequence-specific primers, followed by genotyping of the sample by sequencing library construction, high throughput sequencing, bioinformatics, etc. The technology has the characteristics of high repeatability, high accuracy, high flux, low cost and the like, and can be used for basic research or breeding work such as variety purity identification, population genetic structure analysis, genetic positioning of important agronomic character regulation genes and the like. The key factors determining the implementation effect of the technology are as follows: 1) Capturing a genome sequence-specific short fragment, i.e., fragment uniqueness, by using sequence-specific oligonucleotide primers; 2) The captured genome sequence-specific short fragments have abundant genetic variation, i.e., fragment polymorphism, in the barley population samples.
Aiming at the two key factors, the invention aims to discover a set of SNP marker loci which can target specific DNA fragments, have abundant nucleotide variation in the fragments and are uniformly distributed in barley genome, design a liquid phase chip by utilizing the set of loci, and test the application effect of the liquid phase chip in aspects of barley variety purity identification, barley population genetic structure analysis, genetic positioning of barley important agronomic trait regulatory genes and the like.
Disclosure of Invention
The invention aims to provide a barley whole genome liquid chip and application thereof, which can be used for genotyping barley, including basic research or breeding work such as purity identification of barley varieties, population genetic analysis, primary genetic positioning of important agronomic trait regulatory genes and the like.
In order to achieve the above purpose, the invention adopts the following technical scheme:
a barley genome-wide liquid chip, the genotyping object of which comprises 500SNP loci, the liquid chip is designed by 176 SNP loci and known 324 SNP loci positioned on barley 'MorexV 3' version reference genome; the site information of the 176 SNP sites is shown below, and the 324 SNP sites are defined by "Exome sequencing of geographically diverse barley landraces and wild relatives gives insights into environmental adaptation. Nature Genetics,2016Sep;48 (9) the SNP locus information screened from the report of 1024-1030.Doi:10.1038/ng.3612 is uniformly distributed on 7 chromosomes of the barley genome;
in the numbering of the physical positions, chr1H, chr H, … … and chr7H represent the chromosome where the site is located, and the subsequent values represent the physical position of the site on the genome of the chromosome.
The liquid phase chip provided by the invention is applied to the purity identification of barley varieties, the genetic structure analysis of barley population, the genetic positioning of barley agronomic characters or the auxiliary selection breeding of barley molecular markers.
The beneficial effects of the invention are as follows:
the invention discovers 176 new SNP loci which are uniformly distributed on 7 chromosomes of a barley genome from large-scale sequencing data, combines with other 324 reported SNP loci of the barley genome, designs a liquid phase chip suitable for barley variety genotyping, and has higher application value in the fields of barley genetics research and breeding application.
The liquid phase chip provided by the invention can be used for typing a target site based on a targeted capture sequencing technology, and SNP in a certain range around the target site can be accurately typed, so that more SNP typing information than an expected marker site can be obtained. Compared with the traditional solid-phase chip, the flexibility is higher, and the marking sites can be added at any time according to application requirements; meanwhile, the liquid phase chip depends on a second generation sequencing platform, so that the typing cost is low, and a technical means is provided for large-scale typing.
Compared with simplified genome sequencing or whole genome re-sequencing, the invention has obvious price advantage and time cost advantage, most research units or application units can economically bear the genotyping of large-scale germplasm materials by utilizing the liquid phase chip, the result is stable and reliable, the detection result can be obtained in a short time, and the invention has obvious promotion effect for promoting the genetic improvement and breeding work of barley.
Drawings
FIG. 1 is a map showing the position of SNP locus on 7 chromosomes of barley genome according to the present invention.
FIG. 2 shows the results of genetic structural analysis of barley varieties using the barley liquid chip designed according to the present invention.
FIG. 3 shows the result of purity identification of barley variety by using the barley liquid chip of the present invention.
FIG. 4 shows the result of genetic mapping of barley grain husk/nude control genes using the barley liquid chip of the present invention.
Detailed Description
The following description of the technical solutions in the embodiments of the present invention will be clear and complete, and it is obvious that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
(1) High density of single nucleotide polymorphism sites was obtained by whole genome resequencing (WGS) of barley cultivars.
For 800 parts of barley local varieties with abundant genetic variation (wherein, 500 parts of Chinese source, 150 parts of Korean source, 20 parts of western and southern Asia source, 100 parts of European source and 30 parts of American source), a high throughput sequencing technology platform (Beijing Berui and Kangbio technology Co., ltd.) was used for the testHiSeq 2000 sequencing platform) for each variety, 0.6Xcoverage of genomic DNA was re-sequenced, and over 28 ten thousand single nucleotide variation Sites (SNPs) were identified on the barley genome by sequence alignment, nucleotide variation site extraction and filtration, and comparison between 800 barley varieties.
(2) A set of single nucleotide polymorphism sites which are uniformly distributed on genome and have high specificity is selected.
The 7 chromosomes of the barley genome were each set to a unit fragment of 10,000,000 base length (10 Mb) from beginning to end, and two SNP sites identified from WGS and flanking sequences of 250bp on each side were arbitrarily extracted from each unit fragment. The barley reference genome "MorexV3" version was downloaded to a local computer, and the randomly extracted 500bp DNA sequences containing SNP sites in each 10Mb unit fragment were aligned and searched in the reference genome, leaving only the sequences in the genome that were specific single copy sites. And (3) extracting the random SNP loci and flanking sequences thereof in a second round of genome comparison search for each unit fragment with the extracted sequences being multiple copy loci until at least one specific single copy locus sequence is extracted from each 10Mb unit fragment in the whole genome (except for the region near the centromere in the middle of each chromosome). The preserved single copy site SNP positions were compared with the existing published SNP position information, and 176 of the novel SNP positions which have not been reported so far were preserved, and their physical position information on the barley reference genome chromosome is shown below.
In addition, the published paper "Exome sequencing of geographically diverse barley landraces and wild relatives gives insights into environmental adaptation. Nature Genetics,2016Sep;48 (9) 1024-1030.Doi:10.1038/ng.3612. "known SNP sites located on single copy fragments of the genome were picked up 324 by the same screening procedure as described above. The position distribution of the 500SNP loci on 7 chromosomes of the barley genome is shown in FIG. 1, wherein (A) in FIG. 1 is the locus position distribution of the 176 SNP loci, and (B) in FIG. 1 is the locus position distribution of the 500SNP loci on 7 chromosomes of the barley genome.
According to the positions of the total 500SNP loci and the sequences at the two sides of the screened SNP loci, taking the sequences extending 250 bases at the two sides of the SNP loci and the sequences formed by the SNP loci as template sequences, developing reverse complementary sequences of the sequences as probes by adopting a conventional design principle through the template sequences, designing primers by adopting a targeted capture sequencing technology by Shimadzu Borui biotechnology Co., ltd, and synthesizing the probes, thereby obtaining the barley 500SNP liquid phase chip "BarPlex v1.0".
(3) The liquid phase chip BarPlex v1.0 is adopted for genotyping detection of barley varieties.
In the test, 973 parts of germplasm resources are selected from 51 parts of wild barley germplasm, 248 parts of Tibet semi-wild barley germplasm, 345 parts of barley local varieties and 329 parts of barley modern bred varieties, genome DNA of each variety is extracted, and the genome DNA (the concentration of working solution is 20 ng/. Mu.L) is used as a template, and the PCR amplification is carried out by using Genoplexs reagent of Shi family Boruidi biotechnology Co. Wherein the amplification primers are mixed solution of 500 pairs of primers designed according to the 500SNP loci, the primers are mixed in an equimolar ratio, the final concentration of each primer is 0.1 mu M, the amplification reaction system is 30 mu L, and specifically, 8 mu L of the mixed solution of primers and 20 ng/mu L of genome DNA 5 mu L, genoPlexs xT MasterMix 10 mu L, ddH 2 O 7μL。
The first round amplification thermal cycling reaction conditions are shown in table 1 below:
TABLE 1 first round amplification procedure
After the amplification reaction is finished, target product fragment purification is carried out by adopting a GenoPrep DNA Clean Beads purification kit of Shijia borrelidi biotechnology Co.
And (3) taking the purified target product fragment as a template, performing a second PCR amplification, connecting a universal sequencing Adaptor sequence (adapter) and a tag sequence (Barcode) on two sides of the fragment, and distinguishing different varieties of samples according to the specific tag sequences. Ginseng radixThe amplification reaction system is designed to be 30 mu L according to the instruction book of the amplification kit, specifically Genoplexs 3 XT MasterMix 10 mu L, I5 Barcode (10 mu M) 1 mu L, I7 Barcode (2 mu M) 5 mu L, ddH 2 O 14μL。
The second round amplification thermal cycling reaction conditions are shown in table 2 below:
TABLE 2 second round amplification procedure
After the second round of amplification reaction, the product fragment was purified, recovered, solubilized and quantified using the GenoPrep DNA Clean Beads purification kit from borrelidin biotechnology limited, shijia.
The product fragment amplified and purified by two rounds of mPCR is adoptedThe X Ten sequencing platform performs high throughput sequencing, and the average sequencing coverage of each variety sample in the test is 750 times. And comparing the obtained short sequence with a corresponding target site sequence in a reference genome MorexV3, and reading genotypes of all varieties at expected SNP sites. Wherein, the probability that each expected SNP site is detected is 99.5% on average. In addition to 500 predicted SNP loci, the captured 500 sequence-specific short fragments also identify 3220 single nucleotide SNP or insertion/deletion (InDel) variation loci in total in 973 barley germplasm resources in the test, which indicates that the liquid phase chip can complete genotyping detection work of barley varieties.
(4) Application of liquid-phase chip "BarPlex v1.0" in genetic structural analysis of barley population.
And (3) carrying out genetic clustering on the variety resources by using the SNP and InDel markers obtained from 973 barley variety resources through a Principal Component Analysis (PCA) method. Wherein, 500 expected SNP loci on the specifically captured 500 nucleotide short sequence can better distinguish genetic differentiation of four types of resources of Wild barley (Wild), tibetan half-Wild barley (Semi-Wild), local variety (Landrace) and recently developed variety (Cultivar), and analysis results are shown in figure 2 by carrying out genetic structure analysis of the barley variety through the liquid chip; wherein, (a) in fig. 2 is a principal component analysis result of variety genetic variation according to the genotyping result of 500SNP sites; the main component analysis result of the variety genetic variation according to the genotyping result of 3220 SNP or InDel loci on 500 short fragments captured by the liquid phase chip is shown in (B) in FIG. 2, which shows that the liquid phase chip can complete the genetic structure analysis of barley.
(5) The application of the liquid-phase chip 'BarPlex v 1.0' in the purity identification of barley varieties.
Three groups of barley varieties were selected, respectively: 1) The same barley variety "Morex" from three different units, named 'Morex-BJ', 'Morex-YC', 'Morex-YZ', respectively; 2) Three barley varieties "ulva barley", "corning dwarf barley" and "ulva yellow", which are named different but suspected to be of the same origin; 3) Three closely related barley varieties, "early maturing No. 3", "salt width short early 3", and "Yu barley No. 1"; the liquid phase chip is used for identifying the purity of barley varieties, and the identification result is shown in figure 3; wherein (a) in fig. 3 is the result of purity identification of the same barley variety "Morex" provided by three different units; FIG. 3 (B) shows three different named but suspected identical source varieties; FIG. 3 (C) shows the results of identifying the genetic similarity levels of three closely related lines.
According to analysis results, the liquid phase chip 'BarPlex v 1.0' is used for carrying out genotyping detection on each sample, so that the similarity between different barley varieties and the purity of each variety can be judged, and the method has remarkable advantages in purity identification of the barley varieties.
(6) Application of liquid-phase chip "BarPlex v1.0" in genetic localization of barley seed coat/naked control gene.
By using the SNP and InDel markers obtained from 973 barley variety resources and carrying out genome-wide association analysis on the grain skin and naked variation of the barley varieties, the result of genetic localization of the barley grain skin/naked control gene is shown in figure 4; wherein (A) in FIG. 4 isCarrying out genome-wide association analysis results of barley grain husk/nude control genes by utilizing 3220 SNP or InDel loci on 500 short fragments captured by the liquid-phase chip, wherein GLM is general linearmodel (simple linear model); MLM is mixed linearmodel (composite linear model); the FarmCPU is Fixed and random model Circulating Probability Unification (fixed effect and random effect alternate application model); FIG. 4 (B) shows a selected one of the varieties "coastal barley" and one of the barleys "white highland barley", which are used to formulate hybrid combinations for creating genetically isolated populations; FIG. 4 (C) shows F of the hybridization combination of "coastal jade barley" and "Bai Qingke" using the liquid phase chip 2 Genotyping the generation genetic segregating population, and constructing 7 chromosome genetic linkage maps of the barley genome; FIG. 4 (D) shows the result of genetic mapping of the barley grain husk/nude control gene using the obtained genetic linkage map. Wherein, the coastal jade barley variety and the naked barley variety are used as parents to carry out hybridization to construct genetic segregation population, thus obtaining F with 95 barks and naked differentiation 2 Generating single plant, combining 2 parts of parent and 95 parts of F 2 The DNA sample of the single plant adopts a barley liquid chip BarPlex v1.0 to carry out genotyping detection, constructs a genetic map, detects a barley grain husk/naked major control gene NUD (the gene ID of the gene is known to be HORVU.MOREX.r3.7HG0719680.1) positioned on a barley 7H chromosome through linkage analysis, and shows that the liquid chip has potential application value in the genetic positioning work of functional genes.
In summary, the liquid phase chip provided by the invention can detect more SNP or InDel sites (for example, at least 500 expected SNPs, and up to thousands of SNPs or InDels can be detected according to the genetic differentiation degree of the test variety) compared with the solid phase chip with the same number of probes. And compared with the traditional solid-phase chip, the liquid-phase chip has flexible design, and the interested marking sites can be added at any time in the later stage. The liquid phase chip provided by the invention is used for genotyping barley, including purity identification of barley varieties, population genetic analysis and the like, and can also be used for primary genetic positioning of important agronomic character regulating genes of barley and the like, so that the liquid phase chip has obvious guiding and promoting significance for genetic breeding of high-quality barley and has obvious application value.
While the present invention has been described in detail through the foregoing description of the preferred embodiment, it should be understood that the foregoing description is not to be considered as limiting the invention. Many modifications and substitutions of the present invention will become apparent to those of ordinary skill in the art upon reading the foregoing. Accordingly, the scope of the invention should be limited only by the attached claims.
Claims (5)
1. The liquid phase chip of the whole genome of the barley is characterized in that the genotyping object of the liquid phase chip comprises 500SNP loci, and the liquid phase chip is designed by 176 SNP loci and known 324 SNP loci positioned on the reference genome of the 'MorexV 3' version of the barley; the site information of the 176 SNP sites is as follows:
in the numbering of the physical positions, chr1H, chr H, … … and chr7H represent the chromosome where the site is located, and the subsequent values represent the physical position of the site on the genome of the chromosome.
2. The use of the liquid-phase chip of claim 1 for the identification of barley variety purity.
3. Use of the liquid phase chip of claim 1 in genetic structural analysis of barley population.
4. Use of the liquid chip of claim 1 in genetic mapping of agronomic traits in barley.
5. The use of the liquid-phase chip of claim 1 in barley molecular marker-assisted selective breeding.
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