CN102747138A - Rice whole genome SNP chip and application thereof - Google Patents
Rice whole genome SNP chip and application thereof Download PDFInfo
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Abstract
The present invention discloses a rice whole genome SNP chip and an application thereof. A method for preparing the chip comprises: (1) obtaining a first class of probes on a chip, wherein sequencing is performed to obtain a parental genome sequence, resequencing data of other rice varieties in a public database are combined, a Nipponbare genome is adopted as a reference sequence, a MAQ software is adopted to match and analyze all the sequencing data, and finally a SNP marker is screened; (2) obtaining a second class of probes on the chip, wherein a rice function gene is obtained from the public database, sequence difference reflecting gene function is searched, and a SNP/INDEL probe is designed according to the sequence difference; (3) adopting an infinium chip manufacturing technology to produce a SNP chip; and (4) testing accuracy and application efficiency of the chip. The chip of the present invention can be applicable for rice germplasm resource molecule marker fingerprint analysis, seed authenticity detection, filial generation genotyping, and other related researches.
Description
Technical field
The present invention relates to molecular biology, functional genomics, information biology and genome breeding field, more specifically relate to a kind of paddy rice full genome SNP chip and preparation method, also relate to the purposes of this paddy rice SNP chip simultaneously.
Background technology
Molecule marker (Molecular marker) is meant the application macromolecular cpd, protein and DNA, and the difference between biont identifies the technological method of heritable variation.Because the difference of protein between individuality is little than DNA, and be difficult to detect, therefore, most of molecule marker is the genetic polymorphism of utilizing on the dna level, is also referred to as dna molecular marker.Molecular marking technique (Molecular marker technology) mainly is meant the Protocols in Molecular Biology that detects the DNA variation.Molecular marking technique mainly contains the purposes aspect following 4 in crop investigations: the structure and the assignment of genes gene mapping of (1) genetic linkage map; (2) germ plasm resource research and cultivar identification; (3) biological character association analysis and phyletic evolution research; (4) molecular breeding and crop genetic improvement.Along with the development of molecular biology, genomics and information biology, molecular marking technique also develops to low cost, high-throughput, high precision direction gradually.The tradition molecular marking technique; Like RFLP (Restriction Fragment Length Polymorphism; Restriction fragment length polymorphism) and in the past 30 years of SSR (Simple Sequence Repeat, simple sequence repeat) technology bringing into play important effect.But there are many limitation in traditional molecular marking technique, and is low like flux, quantity is few, operating process is loaded down with trivial details, can not satisfy the demand of functional genome research.In addition; Modern molecular breeding technology requires increasingly highly to molecule marker, requires gene is accurately operated, and requires full genome genetic background is accurately controlled; Requirement precisely improves specific trait, presses for extensive high-throughout molecular marking technique.Therefore, development and utilization novel molecular labeling technique has important theory and practice significance for functional genome research and crop genetic improvement.
SNP (Single Nucleotide Polymorphism, SNP) is meant the polymorphum on the dna sequence dna that the variation of single Nucleotide on the genome forms.Theoretically; Any one Nucleotide all has four kinds of possible based composition forms on the genomic dna; But in most cases has only the variation of two kinds of bases; Changed (Transitions) or transversion (Transversion) and become another base by a base, the frequency that takes place of conversion will be higher than transversion usually, so the SNP mark is generally diallele (Vignal etc.; A review on SNP and other types of molecular markers and their use in animal genetics.Genet Sel Evol.2002,34:275-306.).SNP mark quantity is many, and it is wide on genome, to distribute.Novel high-throughput molecular marking technique based on SNP mainly contains two big types: one type of high-throughput molecular marking technique that is based on the new-generation sequencing technology; The another kind of molecular marking technique that is based on biochip technology.In paddy rice, obtained extensive application based on s-generation sequencing technologies exploitation molecular marker method; Utilize the Illumina/Solexa sequencing technologies to RIL (Recombinant Inbred Line who comprises 150 familys in the paddy rice like Huang etc.; RIL) colony has carried out full genome low cover degree order-checking acquisition SNP mark; RIL is carried out gene type and makes up high-density genetic linkage map (Huang etc.; High-throughput genotyping by whole-genome resequencing.Genome Res.2009,19:1068-1076); Xie etc. have invented a kind of parent's of not relying on RIL colony methods of genotyping; Utilize the most brief reorganization (Maximum Parsimony of Recombination; MPR) principle obtains SNP mark supposition parent genotype from low cover degree order-checking RIL colony genome sequence column information; Greatly reduce order-checking cost (Xie etc.; Parent-independent genotyping for constructing an ultrahigh-density linkage map based on population sequencing.Proc Natl Acad Sci USA.2010,107:10578-10583); Yu etc. compare the SNP mark based on the exploitation of the s-generation sequencing technologies genetic linkage map that makes up and the linkage map that makes up with traditional molecule marker RFLP/SSR; The SNP that has set forth based on order-checking makes up the advantage (Yu etc. of genetic linkage map in the assignment of genes gene mapping; Gains in QTL detection using an ultra-high density SNP map based on population sequencing relative to traditional RFLP/SSR markers.PLoS One.2011,6:e17595).Recently; Utilize s-generation sequencing technologies that a large amount of rice varieties are carried out genome sequencing exploitation SNP mark; With full genome association analysis (Genome-Wide Association Study; GWAS) many sites relevant (Huang etc., Genome-wide association studies of 14 agronomic traits in rice landraces.Nat Genet.2010,42:961-967 have been obtained with Main Agronomic Characters; Huang etc., Genome-wide association study of flowering time and grain yield traits in a worldwide collection of rice germplasm.Nat Genet.2011).But; This method also has some shortcomings; The sequencing data of low cover degree can not make mark cover each individuality; Have order-checking randomness, most of individual SNP loci gene types calculate through information biology, and the single SNP gene type data that check order between the Different Individual are difficult to directly compare; And directly carry out degree of depth order-checking, even the minigene group as the paddy rice, the cost that checks order at present is still very high, is difficult to satisfy extensive breeding demand.The another kind of way of utilizing order-checking to obtain the SNP mark is genomic dna to be handled through enzyme method such as cut earlier reduce genomic complicacy and make up the genome dna library that complicacy reduces; Utilize s-generation sequencing technologies to carry out degree of depth order-checking then; Like RAD (Restriction site Associated DNA; The related DNA of restriction enzyme site) label sequencing (Baird etc.; Rapid SNP discovery and genetic mapping using sequenced RAD markers.PLoS One.2008,3:e3376).Though the molecular marking technique flux based on s-generation sequencing technologies is high, flexible and efficient; But very difficult the detection and analysis in zone that short segments checked order and depends on reference gene group sequence, was positioned at the Tumor-necrosis factor glycoproteins zone or on the reference gene group, do not have; The complex processes such as calculating of the processing of sequencing data, sequence gene group location and gene type are had relatively high expectations to data analysis, need professional information biology personnel to accomplish.These shortcomings to a certain degree limit being widely used of this method, particularly in molecular breeding, use on a large scale.
Another kind of high-throughput molecular marking technique is based on the technology of gene chip.Gene chip (gene chip) is claimed DNA chip (DNA chip) or DNA array (DNA array) again, refers to be fixed on the lattice array that nucleic acid molecular probe constituted (array) on the supporting dielectrics such as slide, silicon chip, nylon membrane.Biochip technology is to put forward the eighties in 20th century, at the beginning of 21 century, gets into develop rapidly period.Gene chip is mainly used in research (Birnbaum etc., Agene expression map of the Arabidopsis root.Science.2003, the 302:1956-1960 of gene expression profile and regulated and control network at the research and development initial stage; Wang etc., A dynamic gene expression atlas covering the entire life cycle of rice.Plant J.2010,61:752-766; West etc., Global eQTL mapping reveals the complex genetic architecture of transcript-level variation in Arabidopsis.Genetics.2007,175:1441-1450; Wang etc., Aglobal analysis of QTLs for expression variations in rice shoots at the early seedling stage.Plant J.2010,63:1063-1074).Now be used for the research of genotyping and functional genomics more.Molecular marking technique based on gene chip mainly contains: SNP gene chip (McNally etc.; Genomewide SNP variation reveals relationships among landraces and modern varieties of rice.Proc Natl Acad Sci USA.2009; 106:12273-12278), SFP (Single Feature Polymorphism; The single slice polymorphum) (Borevitz etc.; Large-scale identification of single-feature polymorphisms in complex genomes.Genome Res.2003; 13:513-523), DArT technology (Diversity Array Technology; The variety chip technology) (Jaccoud etc., Diversity arrays:a solid state technology for sequence information independent genotyping.Nucleic Acids Res.2001,29:E25), RAD technology (Miller etc.; RAD marker microarrays enable rapid mapping of zebrafish mutations.Genome Biol.2007,8:R105; Miller etc.; Rapid and cost-effective polymorphism identification and genotyping using restriction site associated DNA (RAD) markers.Genome Res.2007,17:240-248.) etc.The chip gene expression profile of initial exploitation just can be applied to detect SFP; The nucleotide probe hybridization of living alone as a widow with on genomic dna or RNA and the chip gene expression profile get final product, does not need to develop specially the marker gene chip, but the marker detection that this method obtains false positive height (Kumar etc. as a result; Single feature polymorphism discovery in rice.PLoS One.2007,2:e284; Luo etc., SFP genotyping from affymetrix arrays is robust but largely detects cis-acting expression regulators.Genetics.2007,176:789-800.).Though DArT and RAD technology does not rely on genome sequence, making up genomic library wastes time and energy, and can only reach intermediate density and flux, is difficult to satisfy in the molecular breeding on a large scale, high-density and high-precision requirement.The RAD chip technology developed into afterwards and utilized s-generation sequencing technologies exploitation SNP mark (Hohenlohe etc.; Population genomics of parallel adaptation in threespine stickleback using sequenced RAD tags.PLoS Genet.2010,6:e 1000862; Emerson etc., Resolving postglacial phylogeography using high-throughput sequencing.Proc Natl Acad Sci USA.2010,107:16196-16200).The SNP chip is the genotyping technique that is suitable for extensive breeding in the biochip technology most.At present; Illumina Infinium MaizeSNP50 chip is used for germ plasm resource evaluation and association analysis (Ganal etc. in the corn; A large maize (Zea mays L.) SNP genotyping array:development and germplasm genotyping; And genetic mapping to compare with the B73 reference genome.PLoS One.2011,6:e28334; Cook etc.; Genetic architecture of maize kernel composition in the nested association mapping and inbred association panels.Plant physiology.2011); Affymetrix GeneChip Rice 44K gene chip is used for rice germplasm resource genetic diversity somatotype and full genome association analysis (Zhao etc. in the paddy rice; Genome-wide association mapping reveals a rich genetic architecture of complex traits in Oryza sativa.Nat Commun.2011; 2:467); And the Illumina GoldenGate SNP gene chip of different densities has been applied to rice molecular breeding (Zhao etc.; Genomic diversity and introgression in O.sativa reveal the impact of domestication and breeding on the rice genome.PLoS One.2010,5:e10780; Chen etc.; Development and application of a set of breeder-friendly SNP markers for genetic analyses and molecular breeding of rice (Oryza sativa L.) .Theor Appl Genet.2011,123:869-879; Thomson etc., High-throughput single nucleotide polymorphism genotyping for breeding applications in rice using the BeadXpress platform.Mol Breeding.2011:1-12).Though GoldenGate SNP chip is a lot of in the paddy rice; But because technology limitation; Reference numerals on chip generally is no more than 3000, is difficult to satisfy in the breeding the accurately demand of control of full genome background, presses for the rice breeding chip of high-throughput, good reproducibility, technology maturation.
The Infinium SNP chip technology of Illumina company is present comparative maturity and widely used full genome SNP detection platform.It uses laser co-focusing optical fiber beadchip technology can carry huge microballon with the technological chip of producing of unique micropearl array BeadArray---the SNP number.At present the human SNP chip produced of the said firm can hold at most 1,100,000 SNP marks (
Http:// www.illumina.com).When chip manufacturing; Each comprises the SNP probe sequence of 50 deoxynucleotides and specific microballon coupling joins; The microballon kind is according to the SNP number decision of carrying, and from several thousand to more than 1,000,000, every type of microballon is encoded by its specific address sequence and SNP probe sequence and detected.Every type microballon is average the repetition 30 times on every chip, thereby guarantees success ratio and the repeatability that each SNP is to be detected.Illumina Infinium SNP chip has been used widely in the genome mutation research of species such as the mankind, mouse, corn, but in paddy rice, does not also have the report of this type chip.In order to make paddy rice functional genome research achievement obtain utilizing, constantly satisfy paddy rice large-scale commercial applications breeding demand, the applicant has designed and produced this SNP gene chip.
Summary of the invention
The object of the present invention is to provide the full genome SNP of a kind of paddy rice chip, called after RICE6K paddy rice SNP chip.SNP site on this chip comprises two types; One type is the good and representative SNP site designing probe (this paper is called first kind probe) of polymorphum that from the rice varieties sequencing data, filters out, and another kind is the SNP/INDEL site designed probe relevant with function (this paper is called second type of probe) according to the paddy rice critical function gene of having cloned of bibliographical information.Wherein, First kind probe comprise from core parental gene group sequence and 520 rice varieties are resurveyed the order sequenced data comparative analysis, filter out 5; 556 SNP sites; Second type of probe comprises 80 the SNP/INDEL sites relevant with the functional site of 40 paddy rice functional genes, detects 5,636 SNP/INDEL sites altogether.RICE6K paddy rice SNP chip is the optical fiber beadchip that adopts Illumina Infinium patent system technology for making (USP, US Patent#US6,429,027) to make, and every chip can detect 24 samples simultaneously.
Another object of the present invention has provided a kind of paddy rice full genome SNP chip and preparation method, is used for the specific probe of paddy rice SNP detection chip of the present invention.5,636 SNP/INDEL sites that the present invention obtains have dna sequence dna and the characteristic shown in SEQID No.0001～SEQ ID No.5636.The full genome SNP of the said RICE6K paddy rice of the present invention chip is meant the chip that utilizes Infinium patent design and manufacturing technology (USP, US Patent#US 6,429,027) to make according to these 5,636 sequences.SNP/INDEL site on the full genome SNP of the said RICE6K paddy rice of the present invention chip is meant the Nucleotide in the square brackets ([]) in the sequence shown in SEQ ID No.0001～SEQ ID No.5636.
A further object of the present invention has been to provide the application of the full genome SNP of a kind of paddy rice chip in detecting paddy DNA sample (fingerprinting of rice germplasm resource molecule marker, long-grained nonglutinous rice and japonica rice hybridization colony genotype, the genetic background of breeding material, rice paddy seed); Owing to tend to select the SNP site that has polymorphum at long-grained nonglutinous rice and two subspecies of japonica rice during this chip design; So the hybridization colony of preferred detection long-grained nonglutinous rice and two subspecies of japonica rice also is applicable to the hybridization colony of detecting in long-grained nonglutinous rice or the japonica rice subspecies simultaneously.This chip can carry out the molecule marker fingerprinting, the hybridization offspring of colony is carried out genotype identification, the critical function gene of concrete material is identified and the phenotype prediction variety resources of rice.
The full genome SNP of a kind of paddy rice chip production method may further comprise the steps:
1, the acquisition of the first kind probe on the RICE6K SNP chip
The applicant utilizes Illumina new-generation sequencing technology that paddy rice core parent material has been carried out gene order-checking.In addition; The end of the year in 2010; Huang etc. announced the genome of 520 paddy rice local variety resurvey order sequenced data (Huang etc., Genome-wide association studies of 14 agronomic traits in rice landraces.Nat Genet.2010,42:961-967).The applicant has downloaded this data from public database, and with the fine genome of Japan (MSU the 6.1st edition,
Http:// rice.plantbiology.msu.edu/) be reference sequences, use MAQ software (
Http:// sourceforge.net/projects/maq) these genomic sequence datas are carried out The matching analysis.The concrete steps of probe design are following:
(1) obtained 4,236,029 high quality SNP according to the criterion evaluation earlier:
Only when the matching fractional (mapping quality) of sequencing sequence on genome when being at least 20, this sequence just is used to identify SNP;
Calculate the summation of the sequencing quality mark (base quality) of the different bases in every site on the genome, the SNP site must have only the sequencing quality mark summation of two kinds of bases greater than 100, and these two kinds of bases all have at least 10 sequencing sequence supports for every kind; Two kinds of bases in SNP site have at least 5 matching fractional to surpass 40 sequence support for every kind, and corresponding base sequencing quality all surpasses 20; The total order-checking coverage in this SNP site is more than or equal to 50 and be less than or equal to 1000.
(2) further use stricter standard to obtain 1,559,745 candidate SNP:
Remove the SNP site that at least 5 kinds, presents heterozygous state;
Remove the order-checking coverage less than 80 or greater than 800 SNP site;
All kinds are divided into long-grained nonglutinous rice and japonica rice, and calculate the frequency of less important allelotrope in these two subspecies of SNP, remove in all kinds, indica and japonica subspecies less important gene frequency all less than 0.2 SNP;
Remove still with left and right sides SNP distance all less than the SNP of 50bp.
(3) because probe length is 50bp, need select during design, in order to avoid influence hybridization than conserved sequence.In order to obtain suitable probe, the applicant has also done following analysis:
Extract SNP left side and right side 50bp sequence (, then not extracting) respectively, use BLAT program (Kent etc., BLAT-the BLAST-like alignment tool.Genome Res.2002,12:656-664.) comparison genome if 50bp contains other SNP.If the both sides sequence all exists two or above identity (identity) to surpass 85% coupling, then remove this SNP on genome;
Extract the precious Shan 97 of rice variety the SNP left side and right side 50bp sequence corresponding respectively with bright extensive 63 (seeing genetic resources table 1), and with the Japanese fine canonical sequence of japonica rice (MSU the 6.1st edition,
Http:// rice.plantbiology.msu.edu/) compare, if the both sides sequence is all inconsistent, remove this SNP.
Through above-mentioned analysis; The applicant has obtained 105; 5959 meet the SNP that probe design requires, and 35.5% SNP fixing in indica and japonica subspecies basically (in the highest flight allelic gene type is different in the indica and japonica subspecies, and occupies the ratio more than 90% respectively) is wherein arranged; 42.1% SNP has polymorphum in the long-grained nonglutinous rice subspecies, and 16.9% SNP has polymorphum in the japonica rice subspecies.
The applicant has further analyzed the linkage disequilibrium between the SNP.Karyomit(e) is divided into section of every 100kb, all SNP r between any two in the calculation of sector
2(r is Pearson's relation conefficient, Pearson correlation coefficient).Through trial, the applicant is with r
2=0.64 as threshold value; Utilize a kind of greedy algorithm (Carlson etc.; Selecting a maximally informative set of single-nucleotide polymorphisms for association analyses using linkage disequilibrium.Am J Hum Genet.2004; 74:106-120.), with mutual r
2>=0.64 SNP is divided into one group, obtains 86,075 groups of SNP altogether.Because the height of the SNP in group linkage disequilibrium, only need get a representational SNP can provide the most information of SNP on the same group.Maximum 3 SNP of every group selection obtain 187,284 " label SNP (tag SNP) " altogether.These SNP by Illumina company (
Http:// www.illumina.com/) give a mark, remove the Score score value less than 0.6 SNP, obtain altogether 115,740 can supply custom chip SNP.
Because this chip design is primarily aimed at the evaluation and the somatotype of long-grained nonglutinous rice and two inter-subspecies hybrid colonies of japonica rice; Therefore the applicant is according to the distribution situation of SNP in indica and japonica subspecies; Karyomit(e) is divided into interval of 100kb; Each interval preferential SNP that selects two subspecies basic fixed of Xian round-grained rice, if not enough two, the SNP of other types then selected.Because the Infinium technology, the SNP that A/T, G/C change need use two probe in detecting (Infinium I), and the SNP of other types only needs a probe (Infinium II).In order on chip, to place SNP as much as possible, the applicant has defined a cover marking system.The result who rule of thumb attempts, definition: total points S=MAF*100+ (SNP that non-A/T, G/C change) * 3.5 (wherein MAF is Minor allele frequency, minimum gene frequency).Remove total points S less than 33 SNP, obtain 5,556 SNP sites at last altogether.
2, the acquisition of second type of probe on the RICE6K SNP chip:
To the end of the year in 2010; Successfully cloned more than 600 gene in the paddy rice; Wherein number of genes is controlled Main Agronomic Characters such as output, quality, antibiont and abiotic stress, nutritional utilization efficient etc.; These genes have very big breeding potentiality (Jiang etc., Rice functional genomics research:Progress and implications for crop genetic improvement.Biotechnol Adv.2011).The applicant hopes and can disposable the partial function gene be identified, so designed gene function property SNP/INDEL probe.Concrete steps are following:
At first, the inquiry lot of documents obtains the different allelic forms of functional gene from public database (http://www.ncbi.nlm.nih.gov), particularly between rice varieties, can reflect the sequence difference of gene function.If the functional aberrancy not between the isoallele is the difference (SNP) of single base and one-sided 50 bases are arranged is identical, the conserved sequence of so directly using these 50 bases is as the candidate probe site; If functional site is a difference of inserting or lack (INDEL); Then use two kinds of method designing probes; A kind of is to convert INDEL into SNP to detect; Promptly according to the one-sided conserved sequence designing probe of INDEL, first base of insertion sequence and the next base difference of deletion sequence are as SNP to be measured (codominant marker), and another kind of method is that probe directly designs on the INDEL sequence; The allelotrope that make to insert can be hybridized and detected signal, and the allelotrope of disappearance is not hybridized and had only extremely low detection signal (dominant marker).Design 80 SNP/INDEL candidate probe sites in this way altogether, can detect 40 paddy rice functional genes.
More than totally 5,636 in the candidate probe sequence site that obtains of two kinds of methods, require designing probe to make chip according to Illumina Infinium iSelectHD.The probe sequence site characteristic of two kinds of method acquisitions does not have notable difference, in the chip manufacturing process, does not distinguish, and the chip detection experimental implementation of two types of probes was also identical after chip was processed, and was regarded as same set of probe groups.First chip manufacturing obtains the significant digit of coincidence detection requirement and counts 5,102, comprises 68 functional sites of more than 30 gene.Following laboratory test results all refers to the detected result of first chip.
The application of a kind of RICE6K paddy rice SNP chip in detecting the paddy DNA sample comprises the following steps:
1. oryza sativa genomic dna extracts: organize the extracting genomic dna according to the detection needs from rice paddy seed, blade etc.Wherein paddy rice young leaflet tablet DNA extracting recommends to adopt Promega Plant Genome extraction agent box according to the normal process extracting.
2.DNA sample quality detects: the use massfraction is 1% agarose gel electrophoresis detection, judges electrophoresis result with gel imaging system, guarantee the genomic dna good in integrity, and this genomic DNA fragment length is greater than 10kb; Measure protein and organic substance pollution level in the genomic dna with ultraviolet spectrophotometer, genomic dna A260/280 ratio should be between 1.8-2.0, and A260/230 ratio should be between 1.8-2.2.With the DNA concentration dilution to working concentration 50ng/ul.
3. gene chip detects: according to Illumina Infinium gene chip examination criteria flow operations (Infinium HD Assay Ultra Protocol Guide, http://www.illumina.com/).Chip scanning uses Illumina HiScan chip scanner.
4. data analysis: Illumina HiScan scanning result is with GenomeStudio software analysis genotype.
The present invention compares with other molecular marking techniques, has the following advantages and effect:
1) flux is high.Comprise more than 5000 SNP site on the chip; Just can obtain the genotype that is distributed in complete genomic about 4500 sites of a sample in 3 days according to Illumina Infinium examination criteria flow process; 1 chip can detect 24 samples simultaneously; A flow process can be done 8 chips, can obtain 192 samples totally 86.4 ten thousand data points (4500*24*8) in promptly 3 days;
2) good reproducibility.Different batches detects same duplicate samples technology and repeats to reach more than 99.9%, and its repeatability and accuracy are better than similar chip technology;
3) suitability is wide.Because most of SNP site is the sequencing data that comes from more than 520 rice varieties, these SNP sites have representative widely.According to the applicant to more than 100 parts of rice germplasm resource Preliminary detection results; Between the long-grained nonglutinous rice, between the japonica rice, can on average be about 800,1000 and 2600 respectively in detected polymorphum SNP site between long-grained nonglutinous rice and the japonica rice, can be adaptable across the dissimilar rice varieties gene type of long-grained nonglutinous rice and two inter-subspecies hybrid colonies of japonica rice particularly;
4) gene function prediction.80 functional SNP/INDEL sites that comprise 40 paddy rice critical function genes of having cloned in the chip; Detect these sites and just know the function of these genes in the material; Infer its phenotype according to gene function, common molecular marking technique is only to indivedual gene development functionality marks.
Description of drawings
Fig. 1. be a kind of synoptic diagram of gene function property SNP/INDEL probe design example.
That show among the figure is two kinds of allelotrope of " Green Revolution gene " Sd-1---the high stalk genotype (Nipponbare in the paddy rice; Japan is fine) and DGWG (Dee-geo-woo-gee; Short source low pin crow point) sequence alignment of type semi-short-stalked genotype (Milyang 23, close positive 23).With respect to wild-type Sd-1; DGWG class mutant sd-1 has the disappearance of 383 bp from first exon middle; Comprise 278 bp sequences of exons 1 and 2 and the intron (Monna etc. of 105 bp; Positional cloning of rice semidwarfing gene, sd-1:rice " green revolution gene " encodes a mutant enzyme involved in gibberellin synthesis.DNA Res.2002,9:11-17.).3 probe: probe I D01g00SD1.1 have been designed and ID01g00SD1.2 is the INDEL probe to the INDEL applicant of this 383bp; Probe sequence is positioned at INDEL inside; Wild type gene group DNA and probe hybridization extend a base A and C respectively and detect high signal, and DGWG class mutant is owing to the corresponding genome sequence of disappearance probe can not have only extremely low signal with probe hybridization; Os01g66100.1 is the SNP probe, and probe sequence is positioned at the INDEL border, and just in time there is the difference (T/A) of individual SNP on the border, can be used as common SNP probe in detecting, and detecting this site is that T then is wild-type, and A is a mutant.Box indicating Sd-1 gene start codon ATG, arrow is represented probe sequence position and direction, the base of triangular form indication is the base that extend probe and genomic dna hybridization back.
Fig. 2. be a kind of Sd-1 gene function property SNP and INDEL probe in detecting effect synoptic diagram.
The functional SNP/INDEL probe design of paddy rice plant height gene Sd-1 is seen Fig. 1. this figure shows 71 paddy DNA test result of samples.SNP probe Os01g66100.1 detected result shows that 26 samples are wild-type (AA genotype), and 21 is DGWG (Dee-geo-woo-gee, short source low pin crow point) type (BB genotype), and other 21 is heterozygosis (AB genotype); INDEL probe I D01g00SD1.1 detected result shows that the Sd-1 gene of 50 samples has not disappearance of allelotrope 383 bp at least, and consistent with wild-type, other sample is consistent with the DGWG class.
Fig. 3. be the distribution schematic diagram on full genome of all probe site on a kind of RICE6K paddy rice SNP chip.
The reference gene group is Japan's fine (rice genome MSU note the 6.1st edition), and each the bar short-term on the karyomit(e) is represented 1 SNP site, and trilateral is indicated centric position.
Fig. 4. be polymorphum SNP frequency distribution synoptic diagram between a kind of long-grained nonglutinous rice, between the japonica rice and between long-grained nonglutinous rice and the japonica rice.
Between any two comparison after 106 parts of rice germplasm resources of RICE6K SNP chip detection (18 japonica rice, 88 long-grained nonglutinous rices).4a is between the long-grained nonglutinous rice (indica), and 4b is between the japonica rice (japonica), and 4c is between long-grained nonglutinous rice and two subspecies of japonica rice.
Fig. 5. long-grained nonglutinous rice and japonica rice are hybridized the synoptic diagram as a result of a F2 plant detection that obtains for a kind of RICE6K paddy rice SNP chip.
The full gene genotype of japonica rice Barilla (Balilla) and long-grained nonglutinous rice Nanjing 11 (Nanjing 11) hybridization F2 plant.AA is the Barilla homozygous genotype, and BB is Nanjing 11 homozygous genotypes, and AB is the heterozygous genes type, and round dot is represented centromere position.
Fig. 6. be a kind of context analyzer synoptic diagram of back cross breeding orderly improvement material.
This figure shows that B parent donor gene imports two family genotype that the A parent backcrosses BC4F1.Short-term on the karyomit(e) representes to contain the donor parents genotype, is heterozygous genes type AB, and round dot is represented the position at target gene place, trilateral indication centromere position.
Fig. 7. deriving for rice variety 93-11 and its is that peace is selected difference site distribution schematic diagram between No. 6.
Every black short-term is represented a difference SNP position on the 8th karyomit(e).Trilateral indication centromere position.
Embodiment
Embodiment 1:RICE6K SNP chip preparation method
1, the acquisition of the first kind probe on the RICE6K SNP chip
The applicant utilizes Illumina new-generation sequencing technology that paddy rice core parent material has been carried out gene order-checking.In addition; The end of the year in 2010; Huang etc. announced the genome of 520 paddy rice local variety resurvey order sequenced data (Huang etc., Genome-wide association studies of 14 agronomic traits in rice landraces.Nat Genet.2010,42:961-967).The applicant has downloaded this data from public database, and with the fine genome of Japan (MSU the 6.1st edition,
Http:// rice.plantbiology.msu.edu/) be reference sequences, use MAQ software (
Http:// sourceforge.net/projects/maq) all kinds order sequenced data of resurveying is matched reference sequences.The concrete steps of probe design are following:
(2) obtained 4,236,029 high quality SNP according to the criterion evaluation earlier:
Only when the matching fractional (mapping quality) of sequencing sequence on genome when being at least 20, this sequence just is used to identify SNP;
Calculate the summation of the sequencing quality mark (base quality) of the different bases in every site on the genome, the SNP site must have only the sequencing quality mark summation of two kinds of bases greater than 100, and these two kinds of bases all have at least 10 sequencing sequence supports for every kind; Two kinds of bases in SNP site have at least 5 matching fractional to surpass 40 sequence support for every kind, and corresponding base sequencing quality all surpasses 20; The total order-checking coverage in this SNP site is more than or equal to 50 and be less than or equal to 1000.。
(2) further use stricter standard to obtain 1,559,745 candidate SNP:
Remove the SNP site that at least 5 kinds, presents heterozygous state;
Remove the order-checking coverage less than 80 or greater than 800 SNP site;
All kinds are divided into long-grained nonglutinous rice and japonica rice, and calculate the frequency of less important allelotrope in these two subspecies of SNP, remove in all kinds, indica and japonica subspecies less important gene frequency all less than 0.2 SNP;
Remove through after the last step still with left and right sides SNP distance all less than the SNP of 50bp.
(3) because probe length is 50bp, need select during design, in order to avoid influence hybridization than conserved sequence.In order to obtain suitable probe, the applicant has also done following analysis:
Extract SNP left side and right side 50bp sequence (, then not extracting) respectively, use BLAT program (Kent etc., BLAT-the BLAST-like alignment tool.Genome Res.2002,12:656-664.) comparison genome if 50bp contains other SNP.If the both sides sequence all exists two or above identity (identity) to surpass 85% coupling, then remove this SNP on genome;
Extract the precious Shan 97 of rice variety the SNP left side and right side 50 bp sequences corresponding respectively with bright extensive 63 (seeing genetic resources table 1), and with the Japanese fine canonical sequence of japonica rice (MSU the 6.1st edition,
Http:// rice.plantbiology.msu.edu/) compare, if the both sides sequence is all inconsistent, remove this SNP.
Through above-mentioned analysis; The applicant has obtained 105; 5959 meet the SNP that probe design requires, and 35.5% SNP fixing in indica and japonica subspecies basically (in the highest flight allelic gene type is different in the indica and japonica subspecies, and occupies the ratio more than 90%) is wherein arranged; 42.1% SNP has polymorphum in the long-grained nonglutinous rice subspecies, and 16.9% SNP has polymorphum in the japonica rice subspecies.
The applicant has further analyzed the linkage disequilibrium between the SNP.Karyomit(e) is divided into section of every 100kb, all SNP r between any two in the calculation of sector
2(r is Pearson's relation conefficient, Pearson correlation coefficient).Through trial, the applicant is with r
2=0.64 as threshold value; Utilize a kind of greedy algorithm (Carlson etc.; Selecting a maximally informative set of single-nucleotide polymorphisms for association analyses using linkage disequilibrium.Am J Hum Genet.2004; 74:106-120.), with mutual r
2>=0.64 SNP is divided into one group, obtains 86,075 groups of SNP altogether.Because the height of the SNP in group linkage disequilibrium, only need get a representational SNP can provide the most information of SNP on the same group.Maximum 3 SNP of every group selection obtain 187,284 " label SNP (tag SNP) " altogether.These SNP are given a mark by Illumina company (http://www.illumina.com/), remove the Score score value less than 0.6 SNP, obtain altogether 115,740 can supply custom chip SNP.
Because the evaluation and the somatotype that are primarily aimed at long-grained nonglutinous rice and two inter-subspecies hybrid colonies of japonica rice of this chip design; Therefore the applicant is according to the distribution situation of SNP in indica and japonica subspecies; Karyomit(e) is divided into interval of 100kb; Each interval preferential SNP that selects basic fixed between two indica and japonica subspecies, if not enough two, the SNP of other types then selected.Because the Infinium technology, the SNP that A/T, G/C change need use two probe in detecting (Infinium I), and the SNP of other types only needs a probe (Infinium II).In order on chip, to place SNP as much as possible, the applicant has defined a cover marking system.The result who rule of thumb attempts, definition: total points S=MAF*100+ (SNP that non-A/T, G/C change) * 3.5 (wherein MAF is Minor allele frequency, minimum gene frequency).Remove total points S less than 33 SNP, obtain 5,556 SNP sites at last altogether.
2, the acquisition of second type of probe on the RICE6K SNP chip
To the end of the year in 2010; Successfully cloned more than 600 gene in the paddy rice; Wherein number of genes is controlled Main Agronomic Characters such as output, quality, antibiont and abiotic stress, nutritional utilization efficient etc.; These genes have very big breeding potentiality (Jiang etc., Rice functional genomics research:Progress and implications for crop genetic improvement.Biotechnol Adv.2011).The applicant hopes and can disposable the partial function gene be identified, so designed gene function property SNP/INDEL probe.Concrete steps are following:
At first, the inquiry lot of documents obtains the different allelic forms of functional gene from public database (http://www.ncbi.nlm.nih.gov), particularly between rice varieties, can reflect the sequence difference of gene function.If the functional aberrancy not between the isoallele is the difference (SNP) of single base and one-sided 50 bases are arranged is identical, the conserved sequence of so directly using these 50 bases is as the candidate probe site; If functional site is a difference of inserting or lack (INDEL); Then use two kinds of method designing probes; A kind of is to convert INDEL into SNP to detect; Promptly according to the one-sided conserved sequence designing probe of INDEL, first base of insertion sequence and the next base difference of deletion sequence are as SNP to be measured (codominant marker), and another kind of method is that probe directly designs on the INDEL sequence; The allelotrope that make to insert can be hybridized and detected signal, and the allelotrope of disappearance is not hybridized and had only extremely low detection signal (dominant marker) (Fig. 1 and Fig. 2).Design 80 SNP/INDEL candidate probe sites in this way altogether, can detect 40 paddy rice functional genes.
More than totally 5,636 in the candidate probe sequence site that obtains of two kinds of methods, require designing probe to make chip according to Illumina Infinium iSelectHD.Effective SNP/INDEL site that first chip manufacturing obtains the coincidence detection requirement is 5,102, and the distribution of these sites on full genome is as shown in Figure 3.
The application of embodiment 2:RICE6K paddy rice SNP chip in detecting the paddy DNA sample
1. oryza sativa genomic dna extracts: organize the extracting genomic dna according to the detection needs from rice paddy seed, blade etc.Wherein paddy rice young leaflet tablet DNA extracting adopts Promega Plant Genome extraction agent box according to normal process extracting (Wizard Magnetic 96 DNA Plant System Kit, article No. FF3760 or FF3761, U.S. Promega company).
2.DNA sample quality detects: using massfraction is the agarose gel electrophoresis detection of 1% (W/W); With gel imaging system (Gel Doc XR System; U.S. Bio-Rad company) judges electrophoresis result, guarantee the genomic dna good in integrity, and this genomic DNA fragment length is greater than 10kb; With ultraviolet spectrophotometer (NanoDrop2000; U.S. Thermo Scientific company) measures protein and organic substance pollution level in the genomic dna; Genomic dna A260/280 ratio should be between 1.8-2.0, and A260/230 ratio should be between 1.8-2.2.With the DNA concentration dilution to working concentration 50ng/ul.
3. gene chip detects: according to Illumina Infinium gene chip examination criteria flow operations (Infinium HD Assay Ultra Protocol Guide, http://www.illumina.com/).Chip scanning uses Illumina HiScan chip scanner (HiScan, American I llumina company).
4. data analysis: Illumina HiScan scanning result is with GenomeStudio software (http://www.illumina.com/) analyzing gene type
The application of embodiment 3:RICE6K paddy rice SNP chip in the fingerprinting of rice germplasm resource molecule marker
Utilize RICE6K SNP chip, the applicant carries out the molecule marker fingerprinting to 106 parts of micro core rice germplasm resources.These 106 parts of rice varieties genomic dnas of RICE6K chip detection; 5; In 102 effective detection site; Having 636 sites in greater than 3 kinds, to detect genotype is the heterozygosis site, and the applicant thinks that the detectivity in these sites is relatively poor, remains the gene type that 4466 high quality sites are used for 106 kinds.According to the gene type result these 106 parts of rice varieties are carried out cluster analysis, the result finds that it is one type that whole 18 portions of japonica rice gather, and other 88 portions of long-grained nonglutinous rices gather for another kind of.The applicant analyzed this batch RICE6K SNP chip can detected any two kinds between polymorphum SNP number of sites.The result shows that between japonica rice and the japonica rice, between long-grained nonglutinous rice and the long-grained nonglutinous rice, polymorphum SNP ratio on average is respectively 18.2%, 23.4% and 58.9% (Fig. 4) between long-grained nonglutinous rice and japonica rice two subspecies, and corresponding number of sites is 813,1046 and 2630.
This result shows, RICE6K SNP chip can be preferably applied in to detect between long-grained nonglutinous rice and japonica rice two subspecies hybridizes colony's gene type, also can be used between japonica rice and the japonica rice, hybridizes colony between long-grained nonglutinous rice and the long-grained nonglutinous rice, has flexibility widely.
The application of embodiment 4:RICE6K paddy rice SNP chip in detecting long-grained nonglutinous rice and japonica rice hybridization F2 colony genotype
The seed of gathering in the crops on japonica rice Barilla (Balilla) and long-grained nonglutinous rice Nanjing 11 (Nanjing 11) (seeing genetic resources table 2) the hybridization F1 plant is in indoor germination; The young leaflet tablet extracting genomic dna of getting the week of germinateing carries out gene chip experiment; Be contrast (parent once repeats, twice repetition of hybrid) with parent's Barilla, Nanjing 11 and hybrid F1 simultaneously.Through analyzing; Variant between parent's Barilla and the Nanjing 11; And in parents, be judged as homozygous genotype, have 3 in the site that hybrid F1 repeats all to be judged as the heterozygous genes type for twice; 775, to occupy and imitate 74.0% (3775/5102) of number of sites, gene type (Fig. 5) is carried out to 67 individual plants of F2 colony in these sites of usefulness.
Two parent's Barillas and Nanjing 11 have obvious phenotypes difference.Barilla is typical japonica rice variety, semi-short-stalked, compact, little, the seed oval of tassel of plant, and Nanjing 11 is typical rice varieties, high bar, plant are hung down loosely, tassel big, the seed ellipse.Utilize RICE6K SNP chip detection, two parent's correlation function gene genotype results conform to by (table 1) with phenotype, and in F2 colony, separation are arranged.For example: paddy rice Sd-1 gene (MSU:LOC_Os01g66100) SNP probe Os01g66100.1 detects 71 individual plants (2 parents+2 hybrid F1+67 hybrid F2) has 26 to be Barilla homozygous genotype (AA); 21 is Nanjing 11 homozygous genotypes (BB), and all the other 24 is that (Fig. 2 a) for heterozygous genes type (AB); INDEL probe I D01g00SD1.1 detected result has 50 individual plants for inserting genotype (Barilla homozygous genotype and heterozygous genes type), and all the other 21 is missing gene type (Nanjing 11 homozygous genotypes) (Fig. 2 b), and both results are consistent.
This result shows that RICE6K SNP chip has good gene type ability to the hybridization colony of long-grained nonglutinous rice and two subspecies of japonica rice, and important functional gene type is had good detectivity.
Table 1. Barilla and Nanjing 11 partial function gene test results
The application of embodiment 5:RICE6K paddy rice SNP chip in the genetic background that detects rice breed
For the effect of checking R ICE6K paddy rice SNP chip in breeding, the applicant has carried out the genetic background analysis to the back cross breeding intermediate materials.Two excellent genes of donor parents B (long-grained nonglutinous rice) import the defective of receptor parent A (japonica rice) with improvement A parent.Take turns to backcross obtaining BC4F1 through 4, carry out the target gene foreground selection with traditional molecule marker (SSR) in the backcross process, in backcross process, do not lose to guarantee excellent gene.With RICE6K SNP chip detection 29 parts of the BC4F1 materials that obtain through phenotypic screen and SSR molecular marker assisted selection, two representative family genotype results are as shown in Figure 6.Family shown in Fig. 6 a is except the target gene section, and other background genotypes are all consistent with receptor parent, and the family shown in Fig. 6 b has three sections to have the donor parents fragment at the 4th, 8 and 9 karyomit(e)s except that the target gene section.In order to keep A parent's good characteristic as far as possible, preferentially select the family shown in Fig. 6 a to carry out follow-up breeding work.
This result shows, RICE6K paddy rice SNP chip can the successful analysis long-grained nonglutinous rice and the genetic background of two subspecies back cross breedings of japonica rice colony, and guides breeding.Not only waste time and energy with traditional SSR equimolecular labeled analysis genetic background, and because limited amount can not cover whole genome.RICE6K paddy rice SNP chip can carry out full genome genetic background accurately, fast and efficiently and select; Preferentially be used for two inter-subspecies hybrid colonies of long-grained nonglutinous rice and japonica rice; Even be used for the hybridization colony in the subspecies, its mark density is also far above traditional SSR equimolecular mark.
The application of embodiment 6:RICE6K paddy rice SNP chip in detecting the rice paddy seed verity
It is same breed (State Standard of the People's Republic of China GB/T20396-2006) that China's series of three-series hybrid rice seed authenticity detection at present only uses 24 SSR sites to judge whether.Two parts of hybrid rice seeds verities that the applicant has used RICE6K paddy rice SNP chip detection, the 1st part of testing sample and standard model (special excellent 009) (seeing genetic resources table 3) detect 4,968 sites altogether; Wherein only 1 loci gene type is inconsistent; Consistence is that 99.98%, the 2 part of seed to be measured and standard model (Yue You 9264) (seeing genetic resources table 4) detect 4,876 sites altogether; All locus gene is consistent, and consistence is 100%.The 1st duplicate samples is in full accord through 24 SSR marker detection genotype of GB regulation.
The applicant has also used RICE6K paddy rice SNP chip detection 2 parts of conventional rice, the 1st part of testing sample and standard model (beautiful sesame oil accounts for) (seeing genetic resources table 5) detect 4,794 sites altogether; Wherein only 2 loci gene types are inconsistent; Consistence is that 99.96%, the 2 part of testing sample and standard model (R1303) (seeing genetic resources table 5) detect 4,696 sites altogether; Wherein have 4 loci gene types inconsistent, consistence is 99.91%.
The applicant with RICE6K paddy rice SNP chip detection 5 parts of testing samples (seeing genetic resources table 6,7) that are called as 93-11, deriving with the standard model (seeing genetic resources table 2) of 1 part of 93-11 and 93-11 is that to select No. 6 standard models (seeing genetic resources table 7) be contrast to peace.The result finds that wherein 2 parts of testing samples and 93-11 standard model genotype consistence reach more than 99.9%, only have 1-4 loci gene type inconsistent in more than 4,900 site; And select No. 6 standard model genotype consistence with peace is 99.3%, and 70 the SNP sites of having an appointment are variant.So the applicant has compared 93-11 and peace is selected the difference between No. 6, the result finds that about 70 difference sites all concentrate on the 8th a chromosomal interval (Fig. 7) between them.
Can find out that from above detected result RICE6K paddy rice SNP chip is suitable for carrying out the verity detection of series of three-series hybrid rice and conventional rice, its result has more cogency than SSR molecule marker.Reflect also simultaneously that the same sample technology repetition of RICE6K paddy rice SNP chip detection can reach more than 99.9%.
Sequence table
Claims (5)
1. the full genome SNP of paddy rice chip is characterized in that: said SNP chip is meant the chip that utilizes Infinium patent design and manufacturing technology to make according to sequence shown in SEQ No.001 ~ SEQ ID No.5636.
2. the application of the full genome SNP of the described a kind of paddy rice of claim 1 chip in the fingerprinting of rice germplasm resource molecule marker.
3. the application of the full genome SNP of the described a kind of paddy rice of claim 1 chip in detecting paddy rice cross breeding colony genotype.
4. the application of the full genome SNP of the described a kind of paddy rice of claim 1 chip in the genetic background that detects rice breed.
5. the application of the full genome SNP of the described a kind of paddy rice of claim 1 chip in detecting rice paddy seed.
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