CN104024438A - Snp loci set and usage method and application thereof - Google Patents

Snp loci set and usage method and application thereof Download PDF

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CN104024438A
CN104024438A CN201380004386.7A CN201380004386A CN104024438A CN 104024438 A CN104024438 A CN 104024438A CN 201380004386 A CN201380004386 A CN 201380004386A CN 104024438 A CN104024438 A CN 104024438A
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snp
rice
chip
site
snp site
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CN104024438B (en
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邓兴旺
周向阳
唐晓艳
陈浩东
何航
陈伟
王学林
陈竹锋
李永红
张文辉
侯红利
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Shenzhen Xingwang Biological Seed Industry Co., Ltd.
Shenzhen's agricultural science and technology promotes center
WEIMING XINGWANG SYSTEM CROP DESIGN FRONTIER LABORATORY (BEIJING) CO., LTD
Shenzhen Institute of Molecular Crop Design
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SHENZHEN AGRICULTURAL TECHNOLOGY PROMOTION CENTER
Shenzhen Xingwang Biological Seed Industry Co ltd
Xingwang Investment Co ltd
Weimingwang System Crop Design Frontier Laboratory Beijing Co ltd
Shenzhen Institute of Molecular Crop Design
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    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
    • C12Q1/6837—Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Abstract

The present invention discloses a chip for detecting rice genome SNP loci sets, wherein SNP loci are chosen from a nucleotide sequence as represented by SEQ ID NO: 1-9000. Said chip can be applied to the analysis of rice germplasm resource genetic fingerprinting, to the identification of genotypes of the offspring of rice hybrids, to the identification of rice varieties and to the selection of rice breeding materials.

Description

SNP LOCI SET AND USAGE METHOD AND APPLICATION THEREOF
SNP site set and its use trip and applied technical field
The present invention relates to a kind of set of highdensity SNP site and its application method and application, the specifically related to a kind of highdensity SNP site set of paddy rice and its application method and application, belong to the breeding of rice molecular mark, rice varieties fingerprint identification and functional gene research field.
Background technology
During breeding practice and related biological research, molecular labeling plays an important role.Its importance is embodied in：（1) position or clone the gene of the specific economical character of regulation and control；（2) new purpose character is incorporated into existing good rice varieties, while retain other ideal characters of the kind, such as is difficult to conventional character and transgene traits for observing etc.；(3) using the different rice varieties of DNA fingerprint precise Identification, the science of heredity affiliation of rice varieties is investigated.
Currently, in breeding at home and abroad, the use of molecular labeling is more and more extensive.Molecular labeling has many different species, including RFLP marks（Restriction Fragment Length Polymorphism, RFLP）, RAPD marks（Random Amplified Polymorphic DNA, DNA DNArandom amplified polymorphic DNAs）, SSR marks（Simple Sequence Repeat, microsatellite marker AFLP marks (Amplified Fragment Length Polymorphism, AFLP）, SNP marker（Single Nucleotide Polymorphism, SNP)
SNP is the molecular labeling of new generation that latest developments are got up, with the features such as abundance is high, detection easily realizes automation.The sequencing of different rice varieties full-length genomes and comparing shows that distributions of the SNP on rice genome is extremely enriched, much broader compared to the SSR marker commonly used in current rice breeding.Applications of the current SSR in the research such as molecular mark and the assignment of genes gene mapping is more, but because in genome there is unstability in SSR, the density of distribution is relatively low, the problems such as Genotyping automation relatively difficult to achieve, and its application has significant limitation.SNP mutation rate is low, and the SNP for being particularly in code area is highly stable, and its genetic stability is more much higher than genetic markers such as SSR, and the reappearance, accuracy when genetic analysis or gene diagnosis are better than SSR.Most SNP have dimorphism, either-or, unlike SSR marker often has the length possibility of a variety of DNA fragments, are conducive to the detection of automation.Although SNP only has the polymorphism information amount on two kinds of allelotypes, Single locus fewer than the information content of the multiple alleles type such as SSR, the high-frequency characteristics of SNP make the gross information content of its offer very high.SNP is easy to carry out the characteristic of high flux detection, has been doomed the genetic analysis that it is more suitable for complex character.
Detection SNP technology includes：Sequencing, PCR-RFLP, the specific hybrid of different equipotentials, Ligase chain reaction, TapMan, Single base extension etc..Genotyping company（Such as Illumina Affimetrix companies） The technology of SNP Genotypings can be quickly and efficiently carried out through developing, the current mankind (Murray SS, et al. A highly informative SNP linkage panel for human genetic studies. Nat Methods (2004) 1:113-117), corn (Mammadov JA, et al. Development of highly polymorphic SNP markers from the complexity reduced portion of maize [Zea mays L.] genome for use in marker-assisted breeding. Theor Appl Genet (2010) 121:577-588) etc. the high density paddy rice SNP chip of species has been widely used.But have to be developed suitable for the high density paddy rice SNP chip of breeding practice.
Because current conventional breeding methods depend on the selection of plant phenotype, it is largely dependent upon experience in breeding and opportunity, in the presence of very big blindness and unpredictability, molecular marker assisted selection breeding is increasingly widely used in breeding (Wang C in recent years, Chen S, Yu S. Functional markers developed from multiple loci in GS3 for fine marker-assisted selection of grain length in rice. Theor Appl Genet (2011) 122:905-913).The own assisted Selections of molecule mark i (marker assisted selection, MAS) are exactly in gene or QTL (quantitative trait locus, quantitative trait locus）On the basis of positioning, selected using the close linkage relation between target gene or QTL and molecular labeling, so as to realize breeding objective.Molecular marker assisted selection breeding can improve the accuracy rate and breeding efficiency of selection.But molecular marker assisted selection, dependent on the molecular labeling with gene or QTL site close linkage, highdensity molecular labeling can improve the accuracy rate of molecular marker assisted selection breeding.Thus highdensity SNP chip is significant in rice molecular marker assisted selection Breeding Application.
Whole-genome association（Genome wide association study, GWAS), be one kind with linkage disequilibrium (linkage disequilibrium, LD) based on, identify objective trait and genomic locus or the analysis method of candidate gene relation in a certain colony.Plant whole-genome association is one of focus of current International Plant genomics research, have a wide range of applications (Kump KL, et al. Genome-wide association study of quantitative resistance to southern leaf blight in the maize nested association mapping population. Nat Genet (2011) 43 in the thing standing grain such as corn:163-168; Riedelsheimer C, et al. Genome-wide association mapping of leaf metabolic profiles for dissecting complex traits in maize. Proc Natl Acad Sci U S A (2012) 109:8872-8877; Tian F, et al. Genome-wide association study of leaf architecture in the maize nested association mapping population. Nat Genet (2011) 43:159-162), it is supplement and raising to qtl analysis in molecular breeding.But in the research of paddy rice, due to not effective high density SNP site set, paddy rice association analysis is become economically viable one effective SNP site set and urgently develop.
Another important function of molecular labeling is the fingerprint identification to different cultivars in crop.With the continuous progress and the fast development of seed industry of Crops In China breeding research, kind quantity is dramatically increased.It there is that different degrees of kind is identical, similar phenomena in rice varieties, if can not identify well kind, it will regional testing, authorization in kind, The problem of important steps such as the producing and selling of seed produce serious, and finally influence the stabilization of agricultural production.In recent years, with the development of molecular biology, it is ripe to carry out DNA fingerprint identification using the molecular labeling of crop, and progressively develops, as a kind of quick, accurately and efficiently authentication method.Exemplified by paddy rice, in recent years in, the fingerprint judging technique using SSR as molecular labeling, which is widely popularized, to be used, 2007 Nian9Yue14 the Ministry of Agriculture issue agricultural industry criteria《NYT 1433-2007 rice varieties identification of dna fingerprint methods》In employ such a technology, and recommend the preferred SSR markers of 12 I classes and another 12 II class candidate SSR markers, totally 24 SSR markers.Specifically, this 24 SSR are evenly distributed on 12 chromosomes of paddy rice, every chromosome two, except 2 marks of No. 6 the short arm of a chromosome, 3rd, 7, each 2 marks of 10 and No. 12 chromosome long arms are outer, each 1 mark (Cheng Benyi etc., 2008) of remaining 7 long galianconism of chromosome.The accuracy and reliability of this set SSR marker technology are higher, also have good operational and repeatability in practice, are used as standard method in the identification to paddy rice region trial target kind.
But the DNA fingerprint that current SSR molecular marker is identified as rice varieties still has clearly disadvantageous and defect.Due to SSR marker negligible amounts, even if taking whole 24 SSR as the DNA fingerprint of kind, every chromosome also only has 2 SSR markers, and it can be found that there is mutant dna sequences more than 10 myriabit points on every chromosome of paddy rice in the large scale sequencing data of 520 kind paddy rice（Huang et, al, 2009), only taking two SSR markers to be can not the so many difference condition of representation DNA sequence, even if two rice varieties detect two identical SSR genotype on same chromosome, because not knowing about the genotype in most of region in addition to this 2 SSR, the whether shared identical chromosome of two rice varieties can not be also judged completely.If increasing SSR number to obtain more genotype informations, workload can be sharply increased.SSR detection process easily produces error simultaneously.
High-throughout SNP chip can solve these problems well, greatly improve the accuracy and detection efficiency of DNA fingerprint identification technology.First, high-throughout SNP chip includes a large amount of SNP sites being evenly distributed on rice chromosome, while detecting nearly ten thousand SNP genotype of rice varieties, obtains sufficient " fingerprint " information of kind；Second, in high-throughout SNP chips, chromosome distance is spaced small between SNP, and within lOOkb, therefore change small in rice varieties also can be detected；3rd, using high-throughout SNP chip, when carrying out cultivar identification to paddy rice, we can obtain more valuable information, such as new varieties and the affiliation of original rice varieties, and this kind to production estimation is significant safely.
In order to advantageously promote the progress and Research of Plant Genomics research of breeding practice, and improve DNA fingerprint identification technology means, the present invention have collected the set that SNP sites are uniformly distributed comprising SNP site and full-length genome inside all currently known paddy rice functional genes, design is prepared for high flux chip, and is widely used in the application and research of paddy DNA fingerprint identification, Molecular design breeding and correlation.
It is an advantage of the invention that:1st, the selected SNP site of inventor is based on large scale sequencing result, uniform on genome Preferable polymorphism is respectively provided between distribution, different rice varieties；2nd, SNP sites are picked in all known function gene internals；3rd, Illumina Infmium technology platforms have the features such as flux is high, accuracy is good, detection is easy in itself, and the site that we select is especially suitable for using on this platform.
The content of the invention
The present invention is intended to provide a kind of highdensity SNP set of paddy rice, the set includes 9000 SNP sites, high density is presented on rice genome and is uniformly distributed situation for these sites, it is highly applicable to the SEQ ID NO in the breeding of rice molecular mark, functional gene research and the research and development of paddy DNA fingerprint identification, 9000 SNP sites such as sequence table:Shown in 1 ~ 9000.
The present invention is in order to provide a kind of SNP site set being uniformly distributed between different rice varieties, 590 parts of rice germplasms are have selected to carry out resurveying sequence, 420 portions of long-grained nonglutinous rices and 170 portions of japonica rice are included in this 590 parts of rice materials, the title of specific selected materials is as shown in the subordinate list 1 in specification, by sequencing analysis, the present invention obtains the SNP marker site between substantial amounts of rice varieties from different rice materials, and totally 8,944,748 SNP sites.Further pick out highdensity covering full-length genome and equally distributed SNP site 7490 from these sites, specific selected site and its position on chromosome are as shown in specification subordinate list 2；The SNP site 1510 of known function gene internal is selected again, and specific selected site and its position on chromosome are as shown in specification subordinate list 2；Form the set in 9000 SNP marker sites altogether.
In order that the SNP site set obtained is applied to the analysis between paddy rice multi items, the present invention employs many indexes and algorithm and these SNP marker sites is screened when selecting SNP marker set.Including being uniformly distributed on genome, weak tendency gene frequency（Minor Allele Frequency, MAF), linkage disequilibrium（Linkage disequilibrium, LD), non-duplicate region, the index such as gene function area.
Specifically select flow as follows：
(one）The present invention have selected the SNP site for the high reliability that can detect in more than or equal to 3 parts of germplasm, totally 5,680,149 first from the SNP of 590 parts of rice germplasms.
(two）Filtered out from above-mentioned site at its upstream or remaining SNP or InDel site is not detected by the range of the 55bp genome sequences of downstream（Insertion-deletion, insertion and deletion mark）SNP site, can so exclude the SNP detection difficults caused by the variation of flanking sequence.
(three）On this basis, SNP site in the further non-duplicate region of Select gene group, by using blast softwares by the upstream of SNP site or downstream 55bp sequences and genome alignment, it is determined that being genome repeat region more than 0.7-cause property, rejected, finally obtain 1,376,113 SNP.
(four）In order to assess the probe face efficiency of selected 1,376,113 SNP sites, the success rate of the typing assay of SNP site is improved, the present invention is given a mark by Illumina infmium iSelect softwares to all SNP sites, and have chosen dozen Point>0.6 740,786 SNP.
(five）Rice genome is equally divided into 50kb interval, suitable SNP site is further screened by following steps and condition in each interval：
A) choose and meet the SNP site of following condition simultaneously, if having multiple, only retain a SNP for meeting condition:MAF in long-grained nonglutinous rice>0.2, MAF in japonica rice>0.2, long-grained nonglutinous rice japonica rice merges statistics MAF>0.2；
If b) a upper standard does not choose suitable SNP site, the SNP of condition are met in each interval selection 2 according to following standard:The SNP of MAF maximums in long-grained nonglutinous rice, and in japonica rice MAF maximums SNP.
By above-mentioned selection flow, 7490 SNP sites being evenly distributed on rice genome are obtained altogether.7490 SNP sites being evenly distributed on these rice genomes are in paddy rice reference gene group Nipponbare（MSU6.1 versions, http://rice.plantbiology.msu.edu/) on position be listed in subordinate list 2.In subordinate list 2, SNP titles the first two letter " Os " represents paddy rice, listed two numerals immediately following letter osbwk or osbwkg represent chromosome numbers in SNP titles, 8 bit digitals below are the particular location on the chromosome, the first two letter in last three letters represents two kinds of nucleotides possibilities in paddy rice in the SNP site, and last represents probe design in chromosome normal chain or minus strand.
(six）It is specifically selected to collect the SNP site being located on these gene extron subsequences as shown in table 2 according to 879 functional genes reported, and removal is not suitable for the site of chip probe design, obtains 8737 SNP sites.
(seven）By following steps in this 8737 SNP, 2 SNP are selected on each gene:
A) screening meets the nonsynonymous mutation site on 879 genes, and 2 maximum SNP of MAF values are chosen on these sites;
B) SNP, without nonsynonymous mutation site, chooses 2 maximum SNP of MAF values such as on fruit gene.
1510 SNP sites being located in functional gene are so obtained altogether.SNP site in these functional genes is in paddy rice reference gene group Nipponbare（MSU6.1 versions）On each 60bp in position and its both sides sequence as shown in subordinate list 2, the implication of SNP titles is with above-mentioned in the table（Five）Bar.
(eight）It is final to obtain the highdensity SNP marker set containing 9000 SNP sites, including equally distributed SNP site 7490, the SNP site inside functional gene 1510 on genome altogether as described in above-mentioned flow.
The disclosed 9000 SNP site set of the present invention is listed in subordinate list 2, and the average distance between adjacent sites is about 50Kb, and its distribution situation on genome is as shown in Figure 1.The distribution situation that the SNP of selection is integrated into effective SNP site number between the representative long-grained nonglutinous rice japonica rice Core Germplasms of any two is shown in Fig. 2（Two rice varieties arbitrarily are selected, effective SNP numbers between them are analyzed）.As a result show, this set SNP site set, number for effective SNP between any two long-grained nonglutinous rice is 2000 or so, and the SNP between any two japonica rice is 1500 or so, and the SNP between any one long-grained nonglutinous rice and a japonica rice is 5000 or so. Those skilled in the art should know; after the system of selection of above-mentioned SNP site and its physical location on chromosome are disclosed; anyone is analyzed by increasing or decreasing the sequencing result of the different rice materials in part; all SNP site set disclosed in this invention or the set of its subset are included so as to what is obtained, all within the scope of the present invention.
By different rice material sequencing results, flow is selected using same or like SNP, other SNP set is obtained, within the scope of the present invention.
After SNP site disclosed in this invention is obtained; by determine gene where the specific SNP site or with after the SNP site of the gene linkage; detected by PCR, probe hybridization, chip detection and the design such as kit and the method for application detection, all within the scope of the present invention.
By carrying out depth excavation to SNP site set disclosed in this invention; choose and a class economical character; such as one group of related SNP site of plant height, plant type, resistance; and applied to the designs such as PCR detections, probe hybridization, chip and kit and the method for application detection, all within the scope of the present invention.
The SNP site that the present invention is obtained, can apply to any experiment porch for being capable of detecting when SNP genotype, the Genotyping platform of the company such as Illumina, Affimetrix, Sequenom.The technological means of the parting platform of these companies is variant, but principle is similar.By the chip or kit of the oligonucleotide probe for synthesizing detection SNP marker, then Genotyping is carried out with corresponding SNP typing methods in corresponding Genotyping equipment.
SEQ ID NO of the present invention are included by what above-mentioned platform was developed:The chip of 1-9000 SNP detection site is also known as Rice9K chips, or Rice9K, or 9K chips in the present invention.
After the invention discloses all SNP sites physical location on chromosome, the flanking sequence of the site both sides can be obtained by public channel, and probe design is carried out by related software, so that effective probe of the SNP site can be detected by obtaining.
In an embodiment of the present invention, the Infmium technology platforms of the specifically chosen IUumina companies of inventor carry out SNP chip synthesis.The probe of IUumina Infmium Genotypings is designed and Cleaning Principle is：（1) to tetra- kinds of SNP polymorphisms of A/G, A/C, T/G and T/C, it is only necessary to design one bi-deoxyribose nucleotides of completion in the adjacent sites that a probe terminates in SNP site, detection process（DdNTP extension), base A and T dyeing is a kind of color, and base C and G dyeing are another color, it is possible thereby to distinguish SNP two kinds of allelism；（2) to two kinds of polymorphisms of A/T and C/G, it is necessary to design two probes, terminate at SNP site, difference is only that last nucleotides, correspondence SNP two kinds of possibilities.By detecting whether the two probes can extend two kinds of equipotential possibilities to distinguish SNP.
The principle designed according to above-mentioned probe, by experimental verification, we obtain the optimal probe for IUumina Infmium platforms, the probe sequence for detecting each SNP site is the nucleotide sequence close to the upstream 50bp of SNP site.
The beneficial effects of the invention are as follows the highdensity SNP site tag set for constructing paddy rice, these SNP sites have very High polymorphism and it is evenly distributed in whole gene group（High polymorphism ensure that the validity of SNP site in application process, and full-length genome is uniformly distributed the comprehensive and reliability that ensure that analysis result）.In addition, the SNP site on functional gene meets the demand of the related breeding of functional gene or research.
The important meaning of the present invention there is provided efficiently and cover the high density SNP marker site set of paddy rice full-length genome, it is adaptable to cultivar identification, hybrid analysis, molecular mark and the molecule genetics research of paddy rice.As described in the background art, in Crop Genetic Breeding, the accuracy rate and breeding efficiency of selection can be improved by carrying out assisted Selection using molecular labeling, but carry out very insufficient in China.Equally, Plant Genome association analysis is the research method of current international popular, but the SNP site set without effective paddy rice, only a laboratory carries out the association analysis of paddy rice by high-flux sequence at present, data analysis needs higher skill, and most of laboratory has no idea to realize.The present invention, which provides such a effective SNP sites set, makes paddy rice association analysis become economically feasible.Meanwhile, in the research such as the assignment of genes gene mapping, qtl analysis, SNP set can greatly improve the efficiency and flux of Genotyping.
In addition, during rice breeding, often there is uncontrollable product specific admixture in experiment in field, the selection of parent brings chaotic so as to cause the failure of breeding during this experimental design to breeding, and SNP collection of the invention is combined into cultivar identification and hybrid analysis provides economically feasible and efficient instrument.The pedigree analysis of core parental rice can verify the Main Agronomic Characters of paddy rice（Including yield, quality, resistance etc.）The relation with haplotype or gene delivery is developed, so that the improvement of parental rice is instructed, thus SNP set of the invention provides the marker site of the high-res of the change of research pedigree.
In crossbreeding work, the selection of parent is most important, the parent of usual affiliation farther out can significantly Ameliorative character, the nearer parent of affiliation being capable of the indivedual characters of quick improvement.But be only difficult accurately to judge the affiliation of parent from phenotype, thus need to be the parent of paddy rice the identification of genotype to analyze their affiliation, SNP set of the invention provides parent relationship and analyzes effective means.Quantitative trait locus is positioned（QTL mapping) it is widely used in the breeding process of paddy rice, more highdensity site-tag can obtain more accurate positioning, the SNP set of the present invention provides full-length genome and is uniformly distributed highdensity tag set, it can be positioned to paddy rice QTL more accurate, can further improve the efficiency that rice breeding is carried out using QTL site.
In a word, the high density SNP site set that the present invention is built not only makes it possible large-scale rice molecular marker-assisted breeding and whole-genome association, and for cultivar identification, hybrid analysis, pedigree analysis and QTL positioning analysises etc. provide economically feasible, efficiently and high-res instrument.The SNP site of the present invention can promote the development of rice breeding, be expected to make within a few years the Molecular design breeding field of China to have important breakthrough in many applications in rice breeding field.
The SNP site set obtained by the present invention, its embodiment is carried out by following steps：
A) prepared by probe, and the chip or kit of the oligonucleotide probe of 9000 SNP markers of detection, pin of the present invention are contained in Illumina, Af ymerix Sequenom or other Genotyping company customizations that can carry out oligonucleotide synthesis The oligonucleotide probe developed to Illumina chip technology platforms is sequence table SEQ ID NO:The nucleotides of the 11st to the 60th of nucleotide sequence listed by 1 9000, i.e., the upstream 50bp of adjacent each SNP pleomorphism sites is the probe sequence provided by the present invention for IUumina chip technology platforms.
B) sample DNA is extracted, the sample required for being collected according to designed rice breeding or other biological research related experiment.According to the requirement of customized gene typing chips or kit, the sample genomic dna of certain concentration is extracted and obtains, and preserve with felicity condition.
C) Genotyping in SNP marker site, according to the requirement of the chip or kit of customization, the genotype for obtaining SNP marker site is reacted by hybridization of sample genomic dna and the oligonucleotide probe of SNP marker etc. in corresponding genotyping system.
D) genotype data is analyzed, and the PRELIMINARY RESULTS to Genotyping carries out quality control, selects the site of high reliability.Then the genotype of SNP marker sites is combined to the experimental design related on rice breeding or other biological research, selects corresponding data analysing method, obtain corresponding result.Experimental design and data analysing method are referring to specific embodiment.
The present invention compared with prior art, with advantages below and effect：
1st, compared with conventional molecular mark is such as SSR, with flux height, the low advantage of single marking data cost.It is highly difficult in 200 polymorphism SSR markers of rice genome exploitation using conventional method, and Rice9K breedings chip is in detection any two kind, two rice varieties, two japonica rice varieties or a rice variety and a japonica rice variety, about 3 can be averagely obtained respectively, 100th, 2,400th, 1,800th, 4,400 high-quality polymorphic SNP sites.
2nd, compared with other genotyping systems based on genetic chip platform, with the advantage such as reproducible, flux is high, data analysis is simple.The technology repeatability of Rice9K breedings chip detection paddy rice sample can reach more than 99.99%, and this is that other current Genotyping platforms are extremely difficult to.
3rd, compared with the genotyping system based on second generation microarray dataset, simply there is the advantages such as comparativity between the different experiments number of chambers with data analysis.With the development of sequencing technologies, cost is constantly reduced, sequencing throughput is also improved constantly for sequencing.But, also more and more higher is, it is necessary to which the analysis software and bioinformatics professional person of specialty could be analyzed for the requirement of sequencing data analysis, and the data analysis of Rice9K breeding chip platforms is very simple；Sequencing has randomness, and the low cover degree sequencing data of different batches is difficult directly to be compared, and deep sequencing cost is still very high at present, and most of mark on Rice9K breeding chips is all fixed, and different batches data are very convenient by comparison.However, a large amount of sequencing datas that second generation sequencing system is produced provide important basic data for the design of genetic chip.So, second generation sequencing and Gene Chip system have complementarity. 4th, Rice9K chips of the invention have wider applicability compared with published Chinese invention patent 201210055775.X.The X of Chinese invention patent 201210055775. chip designs the identification and parting mainly for long-grained nonglutinous rice and japonica rice Liang Ge inter-subspecies hybrids colony, therefore SNP substantially stationary between two Xian round-grained rice rice subspecies in per lOOkb is chosen during design.This selection scheme has limited to the use scope of chip, be difficult to play inside good parting effect, actual test long-grained nonglutinous rice for the hybrid Population and close kind in the same subspecies that are mainly faced in actual breeding work and inside japonica rice average kind two-by-two detect pleomorphism site only has 1046 and 813.And the Rice9k chips design object of the present invention is genotype identification and typing chip generally applicable in paddy rice, it is preferential to be chosen at the SNP that long-grained nonglutinous rice is all higher than 0. 2 with MAF in japonica rice, aid in SNPs of the MAF more than 0. 2 in a subspecies, the SNP so chosen just has good applicability, inside long-grained nonglutinous rice and inside japonica rice average kind two-by-two detect pleomorphism site averagely reaches 2400 and 1800, the pleomorphism site to two subspecies is even more to reach 4400.In addition, the present invention devises 1510 SNP for 879 paddy rice knowns of collection, it may be directly applied in breeding, and only comprising 40 such probes in the X of Chinese invention patent 201210055775..To sum up, the X of Chinese invention patent 201210055775. is the chip that a application is limited to very much, and Rice9k chips of the invention have extensive universality, can be applied to various rice varieties and the actual breeding work of colony.Brief description of the drawings
Fig. 1 is distribution situation of 9000 SNP sites in paddy rice full-length genome.
Fig. 2 is that using 38 there is the representational Rice Core Germplasm of genetic diversity to detect the distribution situation of effective SNP site number between any two long-grained nonglutinous rice, two japonica rice, one japonica rice of a long-grained nonglutinous rice.Abscissa is effective SNP numbers, and ordinate is the paddy rice matched group number for corresponding SNP site number occur.
Fig. 3 is the result clustered in Illumina GenomeStudio using two methods to SNP site.The site of yellow is one group of parent and its heterozygote（Pl, P2, Fl), AA, AB, BB are the result of the three kinds of genotype clustered, respectively homozygosis, heterozygosis, homozygosis.
Fig. 4 is the SNP site parting comparison schematic diagram that 4 parts of yellow China account for sample.Grey is masked as accounting for consistent SNP site with the yellow China of standard in figure, and red marker is to account for inconsistent SNP site with the yellow China of standard.
Fig. 5 is the SNP site parting schematic diagram of Huang Huazhan and its EMS mutagenesis mutant.Grey is masked as accounting for consistent SNP site with the yellow China of wild type in figure, and red marker is to account for inconsistent SNP site with the yellow China of wild type.
Fig. 6 is the dendrogram for 65 Rice Core Germplasms analyzed based on SNP site.Figure Green is masked as the SNP site consistent with Nipponbare, and red marker is the SNP site inconsistent with Nipponbare, and yellow is heterozygous sites, and white is deletion segment. Fig. 7 is the haplotype analysis figure that Huang Hua accounts for pedigree.The left side is Huang Huazhan pedigree；Grey in the figure of the right represents that haplotype may be obtained from any parent, and other colors represent the kind in haplotype source, and first kind for certain color occur is the kind in the corresponding haplotype sources of yellow Hua Zhanzhong from the bottom up.
Fig. 8 is Huang Huazhan and its SNP site parting schematic diagram of derived varieties.Grey is masked as the SNP site consistent with Huang Huazhan in figure, and red marker is to account for inconsistent SNP site with yellow China.
Fig. 9 is the SNP site parting schematic diagram for 6 plants of BC5F1 that Gu Mei 4, R608 and its their hybridization are produced.Grey is masked as the SNP site with R608-cause in figure, and red marker is the site inconsistent with R608, and yellow is masked as the position that black designation in heterozygosis SNP site, Gu Mei 4 is rice blast resistance gene.
Figure 10 is the Manhattan figure for the amylose trait associations analysis that Huang Hua accounts for pedigree.
Figure 11 is 9311 and Nipponbare RIL Genotyping schematic diagram.Figure Green is masked as the SNP site consistent with Nipponbare, and red marker is the SNP site inconsistent with Nipponbare, and yellow is heterozygous sites, and white is deletion segment.Detailed description of the invention
The invention provides the SNP site of one group of covering paddy rice full-length genome, altogether comprising 9,000 marker site, the nucleic acid molecules of the separation related to these marker sites have such as SEQ ID NO in sequence table:Nucleotide sequence and feature shown in 1 9000.These SNP sites are found by the way that oryza sativa genomic dna is sequenced and polymorphism is determined by computer analysis.These SNP markers can be used for the application of several genes parting.The polymorphism paddy gene seat of the present invention includes at least 12 continuous nucleotides, and the nucleotide sequence includes or be adjacent to the pleomorphism site determined in the present invention（That is SNP site）, the nucleotide sequence SEQ ID NO in the corresponding gene code number of the SNP site and its SNP and the nucleotide sequence adjacent with the SNP site such as table 2 and sequence table:Shown in 1-9000.From table 2 it can be seen that SNP determined by the present invention has also specifically been navigated on rice chromosome.
The application in rice plants Genotyping based on above-mentioned SNP site is described more detail below.The nucleic acid of separation point ~ ~ because of seat, primer and probe
The paddy gene seat of the present invention includes a series of molecular labelings, and it includes at least 20 continuous nucleotides, and including or be adjacent to the one or more polymorphisms determined in table 2.These paddy genes seat nucleotide sequence and include or adjacent to polymorphism any chain of paddy DNA fragment in identical few nucleotide sequence have at least 90% sequence identity, more preferably at least 95%, or even more preferably it is at least 98% for some allele, at least 99% sequence identity in many cases.Can be in sequence table SEQ ID NO:The nucleotide sequence of a chain of such paddy DNA fragment is found in 1 9000 sequence.It is appreciated that according to the property of polymorphism at least some of allele, with disclosed polymorphism in itself without homogeneity.Therefore, it is right In the sequence in addition to disclosed polymorphic sequence, it may be determined that sequence identity.In other words, it is contemplated that there may be for other allele of polymorphism disclosed by the invention, easily it can be characterized by sequence measurement, and can be used for Genotyping.For example, it will be understood to those skilled in the art that for wherein disclose only two polymorphic residues (for example, " A'; or " G ") SNP can also include other polymorphic residues (for example, 〃 T', and/or " G ").
Polymorphism in the present invention in each locus is more specifically listed in Table 2.SNP is particularly useful as genetic marker, because they are more more stable than the polymorphism of other species, and is abundant in rice genome.SNP can be produced by insertion, missing and point mutation.In the present invention, SNP can represent an insertion that may be made up of one or more base-pairs and missing (indel) event, or SNP.There may be in the individual from common ancestor for the shared polymorphism of two or more individuals.It is this " source homogeneity ' '（IBD) characterize and carried by two or more individuals and all be from germanus two DNA locus/fragment." state homogeneity "（IBS two DNA locus/fragments for being carried by two or more individuals and there is detectable phase iso-allele in those locus) are characterized.When considering a group crop system greatly, and multiple tie up at marked locus when there is identical allele, it is necessary to determine the IBS at marked locus whether be IBD at chromosomal region around marked locus reliable prediction.The instruction that a large amount of marked locus in one fragment are enough to characterize the IBD of the fragment is that they can predict the allele existed in the fragment at other marked locus.In addition to the fact that they seldom independently occur, SNP stability and rich them are made to be determined for IBD.
As mark it is useful using the polymorphism from more than one locus for many genes parting application.Therefore, an aspect of of the present present invention provides the set of nucleic acid molecules, and it allows to carry out parting to the polymorphism of different genes seat.The number of locus in such set can be different, but will be limited numerical value, for example, as little as 2 or 5 or 10 or 25 locus or more, such as locus of for up to 40 or 75 or 100 or more.
Another aspect of the present invention provides nucleic acid molecules that can be with the separation of the polymorphism paddy gene seat hybridization of the present invention.In certain embodiments of the invention, for example there is provided in the embodiment of PCR primer, such molecule includes at least 15 nucleotide bases.Can be used as primer molecule can under high stringency conditions with the present invention polymorphic locus in DNA fragmentation a chain hybridize.Primer for DNA amplification is provided in pairs, i.e. forward primer and reverse primer.One chain complementation of one primer and the DNA in locus, and another chain complementation of another primer and the DNA in locus, that is primer sequence few nucleotide purpose sequence preferably at least 90% identical with a chain is identical, and more preferably at least 95% is identical.It is appreciated that such primer can be with remote polymorphism（For example, away from polymorphism at least 5,10,20,50,100,200,500 or at most about 1000 nucleotide bases）Locus in sequence hybridization.The design of the primer of the present invention depends on factor known in the art, for example, it is to avoid or repetitive sequence.
The another aspect of the nucleic acid molecules of the separation of the present invention is the hybridization probe design tested for polymorphism.In one aspect of the invention, such probe is the oligonucleotide sequence comprising at least 12 nucleotide bases or detectable mark.It is described to visit Pin sequence is under high high stringency conditions, and a chain of the DNA in nucleotide base fragment that can be with including or being adjacent to the target polymorphism in polymorphic locus amplification part hybridizes.The sequence of such oligonucleotide sequence few nucleotide purpose fragment identical with paddy DNA in polymorphic locus-bar chain preferably has at least 90% similitude, more preferably at least 95% similitude.The detectable mark can be radioactive element or dyestuff.It is highly preferred that hybridization probe can further comprise fluorescence labeling and quencher, for example, the hybridization probe experiment available for the type for being referred to as Taqman experiments that can be obtained from AB Biosystems.
The present invention separation nucleic acid molecules under certain condition can with including but not limited to oryza sativa genomic dna, clone oryza sativa genomic dna and amplification oryza sativa genomic dna other making nucleic acid molecular hybridizations.In the present invention, if two nucleic acid molecules can form antiparallel double-strandednucleic acid structure, then the two molecules are referred to as each other hybridizing.If two nucleic acid molecules are shown " complete complementary ", i.e. each nucleotides in a sequence and the base pairing nucleotide complementary in another sequence, then claim a nucleic acid molecules and another nucleic acid molecules " complementation ".If two molecules can hybridize mutually under conditions of at least conventional " low rigorous ", and with enough stability, so as to allow them to keep annealing with one another, then it is " bottom line is complementary " to claim the two molecules.Similarly, if two molecules can hybridize mutually under conditions of conventional " high rigorous ", and with enough stability, so as to allow them to keep annealing with one another, then it is " complementation " to claim the two molecules.For example, at least it is referred to as other nucleic acid molecules with the nucleic acid molecules of other making nucleic acid molecular hybridizations under low stringency condition " thing of the same clan can be hybridized,.Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Ed., Cold Spring Harbor Press, Cold Spring Harbor, New York (1989) standing grain mouthful Haymes et al., Nucleic Acid Hybridization, A Practical Approach, IRL Press, Washington, DC (1985) describes conventional high stringency conditions, and reference is used as present invention introduces document above.Thus deviateing complete complementary is allowed, as long as the ability of molecule formation duplex structure is not completely eliminated in this deviation.Therefore, in order that nucleic acid molecules are used as primer or probe, it is only necessary to fully complementary in sequence, so that stable duplex structure can be formed under the specific solvent and salinity used.
Identify polymorphism
SNP is the result of sequence variations, and new polymorphism can be detected by the way that random gene group or cDNA molecules are sequenced.On the one hand, the polymorphism in genome can be determined by comparing the cDNA sequence of not homology.Although detecting polymorphism relatively convenient by comparing cDNA sequence, the assessment of cDNA sequence can not obtain the information on the introne position in corresponding gene group DNA.In addition, the polymorphism during non-coding sequence can not be determined from cDNA.This is a shortcoming, for example, when using the polymorphism from cDNA as the mark for carrying out Genotyping to genomic DNA.If including those polymorphisms being present in non-coding unique sequences in the range of polymorphism, more effective genotypic assays can be designed.
Genomic dna sequence is more more useful than cDNA in terms of identifying and detecting polymorphism.The polymorphism in genome can be determined by comparing the genomic dna sequence of not homology.However, the genomic DNA of higher eucaryote-as comprising a high proportion of heavy Complex sequences and transposons.If being enriched with coding/differentiated part by subtracting or eliminating repetitive sequence, more effectively genomic DNA can be sequenced.
To the parting of the polymorphism in oryza sativa genomic dna sample
Polymorphism in DNA sequence dna can be detected by a variety of effective ways well known in the art or parting is carried out to it, and these methods include but is not limited to those in United States Patent (USP) 5,468 613 and 5,217,863,5,210,015th, 5,876,930,6,030,787,6,004,744th, 6,013,431,5,595,890,5,762,876th, 5,945,283,5,468,613,6,090,558th, 5,800,944 and 5, method disclosed in 616,464, the present invention completely introduces these patents as reference.However, the SNP marker of the present invention can be used in combination with any polymorphism classifying method, to carry out parting to the polymorphism in oryza sativa genomic dna sample.These oryza sativa genomic dna samples used include but is not limited to the oryza sativa genomic dna for the oryza sativa genomic dna, the oryza sativa genomic dna of clone or amplification directly isolated from rice plant.
For example, such as United States Patent (USP) 5,468,613 and 5, disclosed in 217,863, the polymorphism in DNA sequence dna can be detected by oligonucleotides (AS0) probe hybridization with allele-specific.United States Patent (USP) 5,468,613 disclose the hybridization of the oligonucleotides of allele-specific, wherein, sequence containing nucleotide diversity in this procedure, can be expanded to one or more of detection of nucleic acids nucleotide sequence nucleotide diversity by following procedure, point sample is handled with the sequence specific oligonucleotide probes of mark on film.
Can also be by United States Patent (USP) 5, Probe ligation methods disclosed in 800,944 detect target nucleotide sequences, wherein, target sequence is expanded, and it is hybridized with probe, then it is attached the part of the mark to detect the probe.
Microarray can also be used for polymorphic detection, wherein, oligonucleotide probe group is assembled in an overlapping manner to represent a kind of sequence, and so, target sequence can cause part probe hybridization in the difference of a point（Borevitz et al., Genome Res. 13: 513-523 (2003)；Cui et al., Bioinformatics 21: 3852-3858 (2005) ).On any one microarray, it is contemplated that have multiple target sequences.They can represent gene and/or noncoding region, wherein each target sequence is by a series of overlapping oligonucleotides, rather than as representated by a probe.The platform allows a variety of polymorphisms of high flux screening.Single feature polymorphism (SFP) is the polymorphism detected by the Single probe in oligonucleotide arrays, wherein, it is characterized in the probe in array.United States Patent (USP) 6,799,122,6,913,879 and 6,996,476 discloses the parting to target sequence by the method based on microarray.
United States Patent (USP) 5 can also be passed through, 616, Probe ligation methods disclosed in 464 detect target nucleic acid sequence, this method uses at least one pair of probe, the probe has the sequence homologous with the adjacent part of target nucleotide sequences and with side chain, the side chain in the probe and the target nucleic acid sequence base pairing Non-covalent binding to form stem.At least one side chain has the activable group of light, and the group can form covalent cross-linking with other side members of stem.
Detecting SNP other methods includes Single base extension（SBE) method.In a kind of method of preferred detection polymorphism, SNP and Indel can be detected by the method disclosed in United States Patent (USP) 5,210,015,5,876,930 and 6,030,787.A kind of useful experiment is the Taqman experiments that can be obtained from AB Biosystems.
^ B/ trait associations are set up using polymorphism
Polymorphism in the locus of the present invention can be used for marking/the identification of trait associations, and this association is inferred to from the statistical analysis of the genotype and phenotype of group member.These members can be single organism, such as paddy rice, closely related individual family, inbred strais, the closely related individual monoploid doubled or other colonies.Such Rice Population is referred to as " being ", represents origin system.Colony can be the single hybridization between (for example, colony of positioning) originating from two individuals or two, or, it can be made up of the individual with multiple origin systems.Each individual is to be characterised by single or average trait phenotypes and the genotype at one or more marked locus.
The association of mark/character can be inferred from phenotype/genotype data using the statistical analysis of several types, but a basic purpose is detection molecules mark, that is polymorphism, for polymorphism, alternative genotype has dramatically different average phenotypic.If for example, given marked locus A has 3 alternative genotype (AA, Aa and aa), and if that 3 class individual is with dramatically different phenotype, then may infer that locus A is related to the character.The conspicuousness of the difference of phenotype can be tested by the standard statistical tests of several types, as molecular marker gene type includes SAS Enterprise Miner (SAS Institute Inc. to the linear regression or variance analysis (ANOVA) of phenotype commonly used to carry out the commercially available statistical software of such analysis, Cary, NC) standing grain mouthful Splus (Insightful Corporation. Cambridge, MA).When testing many molecular labelings simultaneously, the adjustment corrected such as Bonferonni is carried out in the significance needed for announcing association.
In order to QTL map, including mark should be source characteristics, to be drawn an inference to subsequent colony.Molecular labeling based on SNP is preferable for mapping, because the possibility that specific SNP allele is derived from the separate source in the existing colony of particular species is extremely low.Therefore, SNP marker can be used for spike and assist QTL infiltration, particularly in the case of haplotype.
Generally, the target of association study is more than detecting mark/trait associations, can also assess the gene for directly affecting character（That is, QTL) relative to the position of mark position.In a simple method for realizing the target, the level for substituting difference size or the significance of difference between genotype is compared between marked locus.Infer character gene position in the mark closest to the genotypic difference with maximal correlation.The genetic linkage of other mark molecule can be set up by gene mapping model, the gene mapping model is such as, but not limited to, Lander et al.（Lander et al., Genetics, 121:185-199 (1989)) report flanking marker model, and Interval mapping（Interval mapping), it is performed based on wherein described maximum likelihood method, and with software kit MAPMAKER/QTL.Other software includes Qgene, Version 2. 23 (1996), Department of Plant Breeding and Biometry, 266 Emerson Hal l, Cornel l University, Ithaca, NY.The use of Qgene softwares is a kind of particularly preferred method. The structure of genetic map
In another aspect of this invention, the polymorphism in locus of the invention is positioned on rice genome, for example, as the genetic map of rice genome, it includes the Map Location of two or more polymorphisms as shown in table 2.This genetic map is as shown in Figure 1.Genetic map data can also be recorded on a computer-readable medium.The preferred embodiments of the invention can provide highdensity polymorphic genetic collection of illustrative plates (for example has at least 5000 kinds polymorphisms to be present in same subspecies, at least 20000 kinds polymorphisms are present between different subspecies on physical map of the rice genome）.The average distance that particularly useful genetic map is included in linkage group is no more than 0. 02 centimorgans（CM polymorphism).
Linkage disequilibrium is mapped and association study
Another method for determining character gene location is to analyze mark/trait associations in wherein individual character and marked locus all different colonies.In the colony, due to the genetic process of colony, such as unique origin of mutation, creator's event (founder events), random drift and group structure, some marker alleles may be associated with some trait locuses allele.This association is referred to as linkage disequilibrium.
In plant breeding colony, linkage disequilibrium (LD) is to leave the level of the random association in colony between two or more locus, and LD is frequently found on big chromosome segment.Although it is possible to pay close attention to the individual effect of each gene in the fragment, but for actual plant breeding, typically emphasize be when region is present in, in hybrid or mutation when to the average influence of objective trait.In linkage disequilibrium mapping, compare the individual character value at marked locus with different genotype.Generally, significant character difference shows between marked locus and one or more trait locuses closely.If mark density is suitably high, and linkage disequilibrium only closely occurs between chain site on chromosome, then the position of trait locuses can be very accurate.
The breeding of auxiliary and the selection of auxiliary
When quantitative trait locus (QTL) be located in molecular labeling it is neighbouring when, these marks can be selected for the character value for raising, without it is each selection circulation when carry out phenotypic analysis.In the breeding of mark auxiliary and the selection of mark auxiliary, analyzed by genetic mapping set up associating between QTL and mark first.During same, which molecular labeling allele and favourable QTL allele linkages determined.Then, the marker allele associated with favourable QTL allele is selected in colony.If having sufficiently tight chain between mark and QTL, this process will improve character value.Required linkage degree depends on the algebraically of selection, because in every generation, having an opportunity to break association by restructuring.
Association between specific markers allele and favourable QTL allele can be also used for predicting that the offspring of which type can separate from given hybridization.This prediction can allow the parent for selecting to be suitable for producing colony, and the Combination nova of favourable QTL allele is assembled from the colony to produce new inbred strais.For example, if to be A have at locus 1,20 and 31 in the past known to the marker allele associated with favourable QTL allele, and be B at locus 15,27 and 29 With the marker allele associated with favourable effect, then can be by hybridizing A X B and selecting at whole 6 QTL there is the offspring of favourable allele to develop new system.
SNP marker provided by the present invention can also be used to accelerate infiltration of the transgenosis into new genetic background（SP, into the germplasm of different range）.Simple gene transgression includes render transgenic system and excellent inbred line cross, then makes the hybrid and excellent (samsara）Parent is returned repeatedly, while the holding for transgenosis is selected.It is returned through excessive generation, by recombinating and separating, the genetic background of initial transgenosis system is gradually replaced by the genetic background of excellent inbred strais.By being selected according to the molecular labeling allele for being derived from backcross parent, this process can be accelerated.
In addition, the fingerprint of inbred strais is the combination of the allele at one group of two or more marked locus.High density fingerprint can be for setting up and following the trail of the identity of germplasm, and germplasm identity can be used for the database for setting up mark-trait associations, with beneficial to whole crop breeding program, and germplasm property protection.
Select parent for plant breeding, the trip of offspring's Huo Ce Township things
SNP polymorphisms set provided by the present invention can be also used for as plant breeding Selection parent, offspring or test plants.The ability of these plants is selected from the plant population that cannot be distinguished by phenotype can accelerate plant breeding and reduce because of expense caused by progress phenotypic character analysis.The method of the plant for breeding is selected to comprise the following steps:A) associating between a variety of polymorphisms determined in table 2 and a variety of characters at least the first and second paddy rice inbred strais is determined;B) allele status of one or more polymorphisms in parent, offspring or test plants is determined；And parent, offspring or the test plants that c) selection is combined with more favourable correlated traits.In some applications, parent, offspring or the test plants selected by this method are paddy rice inbred strais.In other embodiments, the advantageous combination of correlated traits provides improved hybrid vigour.
In one embodiment, determining the genotype of at least two polymorphisms contributes to parent of the selection for breeding cross.It is this to determine to provide the advantage for producing hybridization to breeder, wherein at least two preferred genome areas, to produce the offspring with least two preferred genome areas.On the other hand, determine that the genotype of at least two polymorphisms can determine to provide basis to make a choice in offspring, wherein, those offsprings comprising preferred genome area are selected in breeding plan.In still further aspect, the test system for assessing combination ability of the inbred strais in hybrid combination can be selected, is included based on presence or absence of in the inbreeding test plan of at least two genome areas, to ensure in different Germplasm Banks（I.e. different Heterotic Groups) between hybridized.
Hybrid is predicted
Hybridized between excellent inbred strais by belonging to different " Heterotic Groups " at two and produce the beautiful art seed of commodity.These groups are different enough on science of heredity so that the hybrid between them shows high-caliber hybrid vigour（That is, improved relative to parent system performance）.It is made up of the mark for analyzing superior hybrid, the allele group at the different genes seat in the male parent and female parent that good combination hybridizes advantage can be identified.Recognize these patterns and understand the mark composition of different inbred strais. The different levels to the hybrid vigour between strain can be predicted.These predictions can reduce the possibility of the performance for which the new inbred strais of strain test that should use opposite Heterotic Groups.
The invention provides the method for the hybrid vigour for improving hybrid rice.In these methods, associate being set up between the chain a variety of polymorphisms of polymorphic locus of the present invention and the character in more than two paddy rice inbred strais.The inbred strais that the complementary Heterotic Groups of hybrid vigour can be improved by having prediction as selection two is used for breeding.The method for improving hybrid vigour comprises the following steps：（A) associating between a variety of polymorphisms determined in table 2 and a variety of characters in more than two paddy rice inbred strais is determined；（B) two inbred strais of the inbred strais selected from step (a) are distributed to Heterotic Groups；（C) hybridized at least one times between at least two inbred strais of step (b), wherein, each inbred strais optimizes complementary Heterotic Groups from different and complementary Heterotic Groups, and wherein for improving the hereditary feature of hybrid vigour；With（(d) hybrid generation plant is obtained by the hybridization of step (c), wherein, relative to the offspring produced with unselected inbred line cross, the hybrid vigour that the hybrid generation plant display is improved.These methods can also include traditional single crosses in step (c)（That is, between two inbred strais, ideally from different Heterotic Groups）, three way cross（After single crosses, with the 3rd inbred line cross) and double cross (also referred to as the single crosses of quaternionic breeding, i.e., two advanced generation cross）.Can be by carrying out Hand Hybriding between educating parent in the male of selection or realizing by using male sterility crossing system to hybridize.In Bernardo, Breeding for Quantitative Traits in Plants, Stemma Press, Woodbury, MN, the exploitation and selection, these hybridization for being and the new excellent paddy rice hybrid of selection superior hybrid hybridization identification of excellent inbred strais are described in 2002.
Genetic origin homogeneity
A kind of theoretical prediction of hybrid vigour, the genetic origin homogeneity between the male parent hybridized and female parent（IBD) region can reduce hybrid vigour.The pattern inference genetic origin homogeneity of marker allele that can never in homology.If a series of a string of same tags at neighbouring locus can not possibly independently occur by accident, it is considered that they are that genetic origin is same.Mark fingerprint analysis in male parent and female parent can identify IBD regions.Knowledge to these regions helps to select Hybrid parent, because avoiding IBD to improve heterosis, hybrid vigor in hybrid.These information also contribute to formulate breeding plan, and to design, hybridization produces little or no IBD male parent and female parent is combined.
SNP for Genotyping gathers
The SNP site set that the present invention is provided can be used for the experiment related to rice germplasm improvement, including but not limited to it is cross-breeding using plant, further heredity or phenotype test to plant, plant is improved by autogamous, converted using plant or part thereof, and mutagenesis is carried out using plant or part thereof.Different group nucleic acid in SNP site set can be sampled, detected, or its any group, subgroup or combination are individually detected, to carry out parting to any oryza sativa genomic dna provided in table 2 of the present invention.In general, SNP site set includes at least two groups different nucleic acid molecules, wherein each in the different IPs acid molecule group allows to divide the corresponding oryza sativa genomic dna polymorphism determined in table 2 Type.
In one embodiment, it is allowed to which the different group nucleic acid molecules that parting is carried out to the corresponding oryza sativa genomic dna polymorphism determined in table 2 are distributed in each hole of microtiter plate.In certain embodiments, the nucleic acid molecules of parting can be carried out to the only a kind of paddy rice polymorphism determined in table 2 comprising one or more in each hole of microtiter plate.But, other embodiments are directed to, wherein, the nucleic acid molecules of parting can be carried out to more than one the paddy rice polymorphism determined in table 2 comprising one or more in each hole of microtiter plate.Microtiter plate can have as little as 8 holes, or up to 24,96,384,1536 or 3456 holes.Microtiter plate can be included but is not limited to, polystyrene, polypropylene or ring-olefin plastics by following material manufacture.Nucleic acid molecules in each hole in the solution or can be dried（That is, lyophilized form）.Generally, nucleic acid is assigned in the hole of microtiter plate so that the nucleic acid in the every hole of microtiter plate is known.But, in the nucleic acid molecules other embodiments associated with unique marker (as unique dyestuff or other unique identifications are marked), nucleic acid can be randomly assigned in the hole of microtiter plate.It can be clearly seen that being directed to the set for including distributing nucleic acid in micro titer plate well, being fixed on solid carrier (such as pearl) from this specification.
In other embodiments, it is allowed to the cDNA chip of parting is carried out to the rice genome polymorphism determined in table 2（S Jie, are covalently attached) on solid carrier.Solid carrier includes but is not limited to pearl, chip, array or filter.
Pearl as solid carrier can be magnetic bead, to help the purifying of hybridization complex.Or, pearl can include unique identification mark.Especially, with the pearl for the fluorescent dyeing that can be made a distinction according to its spectrophotometric or photoluminescent property, the nucleic acid molecules for carrying out parting to polymorphism can be coupled to.These are used to have been described (United States Patent (USP) 5,736,330) system based on pearl that polymorphism carries out parting.The pearl of dye marker, analytical reagent and also had been described for the device that parting is carried out to polymorphism（United States Patent (USP) 6,649,414,6,599,331 and 6,592,822), and can be obtained from Luminex Corporation (Austin, Texas, USA).As described above, it can also be that chip, array or filter can be also used for the nucleic acid molecules that the fixed polymorphism to table 2 carries out parting to be combined with the SNP nucleic acid molecules that pearl is connected.In certain embodiments, defined physical location will be fixed on array for carrying out the nucleic acid marking of parting to given polymorphism so that can produce and record the typing data from the position corresponding to given polymorphism, for subsequent analysis.Manufacture and included but is not limited to using the method for the array for being used to carry out polymorphism parting in United States Patent (USP) 5,858,659 (method based on hybridization) and (one base extension methods of United States Patent (USP) 6,294,3 36）Described in method.
DNA clone library is spouted to go using polymorphism analysis and mapped
The polymorphism and locus represented by the molecular labeling of the present invention can be used for identification and positioning and the chain QTL of molecular labeling and the DNA sequence dna of gene.It is, for example, possible to use BAC or yac clone storehouse are inquired about with the molecular labeling of the linkage of characters, to find the clone for including the specific QTL and gene related to character.For example, QTL and gene in a variety of (such as hundreds of or thousands of kinds) big polygenes sequence can by being identified with oligonucleotide probe hybridization, the oligonucleotide probe can with positioning and/or Chain molecular labeling hybridization, wherein it is possible to detect one or more molecular labelings.This screening by hybridization can be improved by providing cloned sequence in high density arrays.The screening technique is improved more preferably by using strategy is collected, to significantly reduce the hybridization number required for clone of the discriminating comprising molecular labeling.When being mapped to molecular labeling, screening effectively can map clone.
For example, in the case where thousands of clones are arranged in defined array for example in 96 orifice plates, these plates can be arranged arbitrarily, the stacking in the hole of three-dimensional arrangement is formed, each hole includes unique DNA clone.Hole in each stacking can be expressed as the individual elements in the cubical array of row, column and plate.In the one side of invention, the number for stacking plate in number and each stacking is roughly equal, so that test number (TN) minimize.The stacking of plate allows to build clone DNA ponds.
For the stacking of three-dimensional arrangement, clone DNA ponds can be created for following key element：（A) all key elements per a line,（B) all key elements of each row, and (c) every block of plate all key elements.To provide positive instruction for the pond in the pond of a row, the pond of row and one block of plate with the pond can be screened with the oligonucleotide probe hybridization hybridized for the unique molecular labeling of a clone, so as to indicate to include the hole unit (key element of target clone）.
In the case of multiple pileup, all clone DNA other ponds allow the stacking for indicating the plate coordinate of one row of row one with target clone in each stacking.For example, the group of 4608 clones can be arranged in 48 piece of 96 orifice plate.48 blocks of plates can be arranged in the stacking of 8 groups of each 6 blocks of plates includes 6 stackings of 8 rows and 12 row there is provided the key element of 6x12x8 cubical arrays, i.e. each stack.For whole clone's group, there are 36 ponds, the pond of SP6 stacking, the pond of 8 rows, the ponds of the pond of 12 row and 8 stackings.Accordingly, it would be desirable to which most 36 hybridization reactions are to find the clone included to the related or chain QTL of each mapping molecular labeling or gene.
Once identifying clone, the Oligonucleolide primers designed from the locus of molecular labeling may be used for the positional cloning of chain QTL and/or gene.
Spouted with unimolecule ^ the method for row Genotyping
The method for marking (for example, rice genome polymorphism) to carry out Genotyping with unimolecule can be used for the phenotypic character of rice plants is associated with genotype.DNA or mRNA in the tissue from least two rice plants with allelic dna is detected to determine whether there is the polymorphism that the present invention is provided as molecular labeling.Associating between identification molecular labeling and phenotypic character, wherein the mark is determined in table 2.On the other hand, have in the specific gene seat of chromosome in the rice plants segregating population of allelic dna, character is associated with genotype, and the locus has phenotypic effect to objective trait, and wherein Molecular mapping among the locus or near.
The method for marking (for example, rice genome polymorphism) to carry out Genotyping with unimolecule may also be used for mother plant, progeny plants or test plants of the selection for breeding.In this case, chromosomal region genetic linkage of the polymorphism with assigning one or more special shapes of preferably phenotype.Parent of the selection comprising the specific allele status associated with phenotypic character, offspring or test rice plants provide breeding accelerate and lower cost. It is expected that some rice genome polymorphisms of the present invention disclosed in table 2 can be directly related with given phenotypic character, because they include some regulation and control or allele status of coded sequence for changing and assigning sub- character or the gene for contributing to trait expression.These characters include yield, lodging, maturation, plant height, disease resistance and resistance, such as drought tolerance, cold resistance, heat resistance, nutritional deficiency, and qualitative character.When rice genome polymorphism by this way with character direct correlation when, it is highly useful in the works being intended to the rice breeding that the character is introduced in many different rice genetic backgrounds.
It can be marked by using multiple so that the chain resistance related to that may not provide the genome area of agronomy advantageous property minimizes, to accelerate the infiltration of the genome area related to this single mark.Can by using multiple marks located immediately at single label side Ji so that may the chain resistance related to closely related genome area minimize, come accelerate with this singly mark close association genome area infiltration.Therefore, it is located at the mark that list marks proximally and distally 10,5,2 or 1 cm using one group 2 of cluster, 5,10 or 20, the infiltration of the genome area related to single mark required for can providing, without the need for the infiltration of direct flank region minimize.Can also be by using the multiple marks being distributed in genome so as to may minimize with any chain resistance of the genome area close association on same distal chromosome region and on other chromosomes, to accelerate the infiltration that closely related genome area is singly marked with this.This, which organizes multiple marks, can include other 10 marks, and each chromosome arm has at least one mark.However, in preferred embodiments, mark density is at least about 10 marks of each chromosome arm, at least about 100 marks of more preferably each chromosome arm, to efficiently differentiate the genome area from donor and receptor parent.Thus, reclaim receptor parent using maximum in the filial generation that selection is can be provided in single mark directly multiple flanking markers that are chain or being distributed on genome.
Spouted the hero of row Genotyping with rice genome ■ polymorphism groups
The invention particularly relates to the methods of genotyping using the nucleic acid molecules group that can a variety of different polymorphisms be carried out with parting.In such method, at least two rice genome polymorphisms to limited quantity carry out parting.The rice genome polymorphism of this limited quantity of inquiry can include at least 2,5,10 or 20 kind of different genotype, and they are expressed as 2,5,10 or 20 kind of different nucleotide sequence in table 2.These methods of genotyping are necessarily required to use can carry out the nucleic acid molecules group of parting to rice genome polymorphism group.
In some applications, these methods of genotyping use the multiple molecular labelings concentrated in given chromosome interval（That is rice genome polymorphism）.High density fingerprint for setting up and following the trail of germplasm identity can be obtained by carrying out methods of genotyping, and methods described utilizes in specific chromosome interval and/or assigned multiple molecular labelings of concentration or cluster around some locus of some characters.High density finger print information can be used for assessing Germplasm Diversity, exercise Genetic quality assurance function, develop rare allele, assesses Exotic Germplasm storehouse and assesses genetic purity.These high density fingerprints can be beneficial to whole crop breeding plan for setting up mark-trait associations database.High density fingerprint may also be used for setting up and protect germplasm ownership.Mark of the selective aggregation around the chromosome interval or inhereditary feature of needs in the paddy rice polymorphism for the positioning that can be provided from table 2 Note group.
These methods for carrying out Genotyping with multiple molecular labelings can also be used for the phenotypic character of rice plants is associated with genotype.The DNA or mRNA in the tissue from least two rice plants with allelic dna are detected, to determine whether there is the polymorphism that the present invention is provided as one group of limiting serial of molecular labeling.Determine associating between this group of molecular labeling and this group of phenotypic character, wherein, this group of molecular labeling at least includes 2 kinds, at least five kinds of or at least ten kinds of and chain molecular labeling of polymorphic locus of the invention, for example, at least 10 kinds with position polymorphic sex-kink molecular labeling, for example, as determined in table 2.At a preferred aspect, have in the chromogene seat that phenotypic effect is assigned to objective trait in the rice plants segregating population of allelic dna, character is associated with genotype, and wherein the correlation degree between molecular labeling between polymorphism and character allows the linear precedence for determining polymorphism and trait locuses.In such method, at least five molecular labeling is with allowing the locus linkage of locus imbalance mapping.
In a specific embodiment, at least one kind of Molecular mapping used in the methods of genotyping using multiple marks is on each chromosome arm of all 12 rice chromosomes, it is therefore necessary to carry out parting at least 24 kinds oryza sativa genomic dna polymorphisms.But, other embodiments of this method are directed to, 24 kinds of oryza sativa genomic dna polymorphisms of wherein at least are positioned on each chromosome arm, it is therefore necessary to which parting is carried out at least 288 kinds oryza sativa genomic dna polymorphisms.Equally, other embodiments are directed to, it is necessary to which parting is carried out at least 24 kinds oryza sativa genomic dna polymorphisms on each chromosome arm（Parting must be carried out at least 576 kinds polymorphisms）, or parting is carried out at least 50 kinds oryza sativa genomic dna polymorphisms on each chromosome arm（Parting must be carried out at least 1200 kinds polymorphisms）.The mark group being distributed on rice genome can be selected from the paddy rice polymorphism of the positioning provided for the table 2 of these methods.
It can be used for a variety of applications using the methods of genotyping for the molecular labeling being distributed on rice genome.In one application, methods of genotyping is used to select mother plant, progeny plants or the test plants for breeding.It is related to a variety of applications of these methods of genotyping in rice breeding in the works.These methods of genotyping can be used for the insertion for promoting one or more characters, the infiltration of genomic locus and/or transgenosis from a genetic background into different genetic background.In general, inquiry comes from outbreeding（Out-crossed) the mark group selected in the progeny plants of colony, to identify and select the individual offspring for still including the allele of the different genetic backgrounds as much as possible from outbreeding comprising required character, genomic locus and/or transgenosis insertion.These methods can accelerate insertion of the required character, the infiltration of genomic locus and/or transgenosis into new genetic background by several generations.
Set of these methods also by inquiring after the molecular labeling such as SNP that averag density on rice genetic collection of illustrative plates is less than about 10 cM provides character screening.Can be in the range of one or more phenotypic characters, the presence or absence of the analysis molecular labeling chain with the polymorphic locus of table 2, to identify one or more specific molecule markers allele at the one or more genome areas related to one or more characters.In another aspect of this invention, using molecular markers for identification haplotype, The haplotype is the allele fragment of genomic DNA, it is characterized in that at least two polymorphisms in linkage disequilibrium, and the polymorphism is in the genomic window no more than 10 centimorgan length, for example, in window no more than about 8 centimorgans or smaller, for example, in the range of 1-5 centimorgans.In some embodiments of these methods, the group of such molecular labeling identifies a variety of haplotypes in a series of adjacent genomic windows in each rice chromosome, for example, the genome provided substantially completely with these windows is covered.Using sufficiently large and multifarious rice breeding colony, substantial amounts of haplotype can be identified in each window, so that allelic dna that can be related to one or more characters is provided, to allow the breeding of the mark auxiliary focused on.Therefore, the one side of paddy rice of the invention analysis further comprises the steps：One or more characters are characterized to the rice plants colony, and the character is associated with the allele SNP or Indel polymorphisms, tissue are preferably carried out with definition unit type.Such character includes yield, lodging, maturation, plant height, disease resistance and resistance.Specific embodiment
Below according to specific embodiment, the present invention will be described.It should be noted that these embodiments are intended to be merely illustrative of the present, and limitation of the present invention can not be construed in any way.In addition, unless stated otherwise, involved method is conventional method in the following embodiments, and involved material and preparation are also to be commercially available.
Unless otherwise specified, the conventional meanses that the technological means employed in embodiment is well known to those skilled in the art, are referred to《Molecular Cloning:A Laboratory guide》The third edition or Related product are carried out.Agents useful for same or the unreceipted production firm person of instrument, being can be by the conventional products of acquisition purchased in market.
The selection in 1. 9000 SNP sites of embodiment
In order to provide a kind of SNP site set being uniformly distributed between different rice varieties, the present invention have selected 590 parts of rice germplasms and carry out resurveying sequence, 420 portions of long-grained nonglutinous rices and 170 portions of japonica rice are included in this 590 parts of rice materials, the title of specific selected materials is as shown in the subordinate list 1 in specification, by sequencing analysis, the present invention obtains the SNP marker site between substantial amounts of rice varieties from different rice materials, and totally 8,944,748 SNP sites.Further pick out highdensity covering full-length genome and equally distributed SNP site 7490 from these sites, specific selected site and its position on chromosome are as shown in specification subordinate list 2；The SNP site 1510 of known function gene internal is selected again, and specific selected site and its position on chromosome are as shown in specification subordinate list 2；Form the set in 9000 SNP marker sites altogether.
In order that the SNP site set obtained is applied to the analysis between paddy rice multi items, the present invention employs many indexes and algorithm and these SNP marker sites is screened when selecting SNP marker set.Including being uniformly distributed on genome, weak tendency gene frequency（Minor Allele Frequency, MAF), linkage disequilibrium（Linkage disequilibrium, LD), non-duplicate region, the index such as gene function area.
Specifically select flow as follows：
(one）The present invention have selected and can detect in more than or equal to 3 parts germplasm first from the SNP of 590 parts of rice germplasms The SNP site of the high reliability arrived, totally 5,680,149.
(two）Filtered out from above-mentioned site at its upstream or remaining SNP or InDel site is not detected by the range of the 55bp genome sequences of downstream（Insertion-deletion, insertion and deletion mark）SNP site, can so exclude the SNP detection difficults caused by the variation of flanking sequence.
(three）On this basis, SNP site in the further non-duplicate region of Select gene group, by using blast softwares by the upstream of SNP site or downstream 55bp sequences and genome alignment, it is determined that being genome repeat region more than 0.7-cause property, rejected, finally obtain 1,376,113 SNP.
(four）In order to assess the probe face efficiency of selected 1,376,113 SNP sites, the success rate of the typing assay of SNP site is improved, the present invention is given a mark by Illumina infmium iSelect softwares to all SNP sites, and have chosen marking>0.6 740,786 SNP.
(five）Rice genome is equally divided into 50kb interval, suitable SNP site is further screened by following steps and condition in each interval：
A) choose and meet the SNP site of following condition simultaneously, if having multiple, only retain a SNP for meeting condition:MAF in long-grained nonglutinous rice>0.2, MAF in japonica rice>0.2, long-grained nonglutinous rice japonica rice merges statistics MAF>0.2；
If b) a upper standard does not choose suitable SNP site, the SNP of condition are met in each interval selection 2 according to following standard:The SNP of MAF maximums in long-grained nonglutinous rice, and in japonica rice MAF maximums SNP.
By above-mentioned selection flow, 7490 SNP sites being evenly distributed on rice genome are obtained altogether.7490 SNP sites being evenly distributed on these rice genomes are in paddy rice reference gene group Nipponbare（MSU6.1 versions, http://rice.plantbiology.msu.edu/) on position be listed in subordinate list 2.In subordinate list 2, SNP titles the first two letter " Os " represents paddy rice, listed two numerals immediately following letter osbak or osbakg represent chromosome numbers in SNP titles, 8 bit digitals below are the particular location on the chromosome, the first two letter in last three letters represents two kinds of nucleotides possibilities in paddy rice in the SNP site, and last represents probe design in chromosome normal chain or minus strand.
(six）It is specifically selected to collect the SNP site being located on these gene extron subsequences as shown in table 2 according to 879 functional genes reported, and removal is not suitable for the site of chip probe design, obtains 8737 SNP sites.
(seven）By following steps in this 8737 SNP, 2 SNP are selected on each gene:
A) screening meets the nonsynonymous mutation site on 879 genes, and 2 maximum SNP of MAF values are chosen on these sites;
B) SNP, without nonsynonymous mutation site, chooses 2 maximum SNP of MAF values such as on fruit gene.
1510 SNP sites being located in functional gene are so obtained altogether.SNP site in these functional genes is in paddy rice reference gene group Nipponbare（MSU6.1 versions）On each 60bp in position and its both sides sequence as shown in subordinate list 2, the table The implication of middle SNP titles is with above-mentioned（Five）Bar.
(eight）It is final to obtain the highdensity SNP marker set containing 9000 SNP sites, including equally distributed SNP site 7490, the SNP site inside functional gene 1510 on genome altogether as described in above-mentioned flow.The use in the Illumina platform SNP sites of embodiment 2.
1) prepared by probe.The infinium chips of the oligonucleotide probe containing 9000 SNP markers of detection are customized in Illumina companies, oligonucleotide probe described in the present embodiment is sequence table SEQ ID NO:Of nucleotide sequence listed by 1-9000
The nucleotides of 11 to the 60th, i.e., the probe sequence for Illumina chip technology platforms that the upstream 50bp of adjacent each SNP pleomorphism sites is provided by the present embodiment.
2) sample is collected and DNA is extracted.The leaf or seed of sample are collected, according to Qiagene kit application methods, genomic DNA is extracted.The need for being tested according to next step Genotyping, the DNA of extraction concentration should be in 50 η§/ μ more than 1
(Α260/Α280: 1. 8-1. 9； Α260/Α230:1. 5 3), and cumulative volume is in 50 μ more than 1.
3) Genotyping is tested.Using the genotyping system of Illumina companies, amplification oligonucleotide is carried out on Illumina HiScan chip platforms, probe hybridizes, chip scanning and signal analysis, finally obtains the genotype data in 7869 effective SNP markers of sample genomic dna.
What Genotyping was tested comprises the following steps that：
A. it is denatured the preparation of single stranded DNA：DNA sample is denatured into sodium hydroxide single-stranded, then neutralizes denaturant, add enzymatic amplification reaction solution.
B. whole genome amplification：The sample of upper step is placed on whole genome amplification in 37 degree of incubators, 37 degree are reacted 20-24 hours.
The genomic fragment of c amplifications：Fragment of the amplified production digestion into hundreds of base sizes.
D. DNA is precipitated:Product after digestion adds isopropanol, and 3000g centrifugations precipitate DNA in 20 minutes, drying at room temperature one hour.
E. dissolving DNA:Add hybridization solution, 48 degree 1 hour, vortex concussion makes DNA be substantially dissolved in hybridization solution.
F. chip, DNA and chip hybridization are put：The DNA of upper step is denatured 20 minutes at 95 degree, room temperature starting point chip is cooled to, notes avoiding cross pollution between different samples, the chip put is placed in 48 degree of hybrid heaters 16-24 hours, do not exceed 24 hours.
G. chip is cleaned：DNA of the non-hybridized or imperfect hybridization on chip is washed off, the DNA only matched completely with chip can be just retained on chip. H. Single base extension and dyeing：To hybridize to the DNA on chip as template progress Single base extension, the base of extension is being combined with dyestuff for advance modified, and different bases can have corresponding dye colour to determine.
I. chip, coating are cleaned, is fixed：Dyestuff unnecessary on chip is washed off, fixer is added and signal is fixed.
J. chip is scanned：The chip fixed is put into the chip slot of HiScan scanners and is scanned acquisition signal, the result of scanning further can carry out interpretation of result in the software that I l lumina companies provide.
4) genotypic results are analyzed.The result scanned using I l lumina GenomeStudio softwares to HiScan classification systems is analyzed.Clustered according to the result of dye colour during Single base extension, the genotype of material is divided into by 3 classes according to the result of cluster（AA, BB, AB).
Because I l lumina GenomeStudio softwares are when lacking heterozygosis cluster, it may appear that parting mistake, and paddy rice is self-pollinated plant, and site is based on homozygosis in stabilization of rice kind.In order that cluster preferably distinguishes homozygosis and heterozygous sites, we creatively pick 34 groups of parents and its hybridization first filial generation is used as the reference of cluster.This clustering method has higher accuracy than the method for the cluster of system default, can preferably distinguish 3 genoid types, and the SNP site parting shown in Fig. 3 improves accuracy after using this clustering method.As shown in Figure 3, acquiescence is clustered the heterozygous genotypes for being clustered into AB of part BB Genotyping errors, and the position of AB heterozygous genotypes can be preferably found in the new clustering method after increase control, so as to more accurately obtain genotypic results.We are according to new clustering method so as to obtain the genotypic results of 7869 effective SNP sites.
Embodiment 3. is spouted row cultivar identification using SNP sites
The present invention is analyzed the truth that yellow China accounts for kind.
Inventor have collected 3 kinds of separate sources and be referred to as the seed that yellow China accounts for（A, B, C), they cannot be distinguished by from phenotype, and also have collected the yellow China of standard from Huang Huazhan cultivations unit Guangdong academy of agricultural sciences in addition accounts for the control that material is used as cultivar identification.
The Genotyping of sample genomic dna：Method according to the probe preparation described in embodiment 1, the extraction that sample is touched and Genotyping obtains the genotype in 7869 effective SNP marker sites.
The genotypic results obtained to Infinium chips carry out quality control.The SNP site of GenTrain Score ＜ 0. 6 in the sample is removed, the poor site of genotyping result is not used to further data analysis due to the reliability of data.Huang Huazhan may be considered pure and mild parent by cultivation for many years, removes the site that Genotyping is heterozygosis, finally obtains 7566 SNP marker sites.
After screening-gene typing data, several kinds to Huang Huazhan carry out identification and analysis, and are visualized these sites according to method described in embodiment 2, as a result as shown in Figure 4.It can be seen that A, B, C are regarded as consistent with cultivating the yellow Hua Zhanjun of unit from result, rather than yellow China accounts for kind and will show the difference of more genotype. Embodiment 4. using SNP sites spout row variet complexity analyze
The mutant that the yellow China of present invention selection accounts for kind carries out Identification of The Origin.
Experimental subjects：The object of experiment material is as shown in Fig. 5.The yellow China of core rice germplasm that we collect accounts for and obtains 7 important mutant by EMS mutagenesis based on this.Cultivate yellow China of standard of unit Guangdong academy of agricultural sciences in addition, have collected Huang Hua and account for and account for material.
The Genotyping of sample genomic dna：Method according to the probe preparation described in embodiment 1, the extraction that sample is touched and Genotyping obtains the genotype in 7869 effective SNP marker sites.
The genotypic results obtained to Infinium chips carry out quality control.The SNP site of GenTrain Score ＜ 0. 6 in the sample is removed, the poor site of genotyping result is not used to further data analysis due to the reliability of data.Huang Huazhan may be considered pure and mild parent by cultivation for many years, removes the site that Genotyping is heterozygosis, finally obtains 7566 SNP marker sites.
After screening-gene typing data, identification and analysis is carried out to Huang Huazhan and its mutant.According to the SNP marker site obtained, program or can analyze and visual software analysis using oneself.Our analysis employs the pattern of oneself programming, and obtained result is as shown in Fig. 5.The genotype that we are used as the wild type Huang Hua Zhanyu Guangdong academy of agricultural sciences cultivation material of mutagenesis is completely the same, and material does not mix.Τ Χ Υ -1, Τ Χ Υ -2, Τ Χ Υ -3, Τ Χ Υ _ 4, Τ Χ Υ -5, Τ Χ Υ -6 and yellow China account for basically identical in mutant, it is believed that this 6 mutant are all derived from Huang Huazhan and do not mixed.But Τ Χ Υ -6 have 355 SNP to account for inconsistent with yellow China, it is believed that occur in that and mix.
Embodiment 5. is spouted this affiliation using SNP sites
The present invention have collected 65 core rice germplasm materials and will be as its affiliation of object analysis, the breeding to next step.
Method according to the probe preparation described in embodiment 1, the extraction of sample DNA and Genotyping obtains the genotype in 7869 effective SNP marker sites.
The genotypic results obtained to infinium chips carry out quality control.SNP site as removed GenTrain Score ＜ 0. 6 in the sample in embodiment 2.Finally give 7591 SNP marker sites.
The SNP marker site of 65 samples obtained using cluster softwares and treeview software analysis, as a result as shown in Fig. 6.According to cluster clustering, this 65 rice varieties can be divided into two groups, i.e. japonica rice and long-grained nonglutinous rice.It is japonica rice according to known Nipponbare, 26 kinds that most green background part above is obtained in figure are japonica rice, most 39 kinds of red background are long-grained nonglutinous rice in addition.The affiliation between rice varieties can be clearly judged in figure.
Embodiment 6. using SNP Wei Dian minute system's monotype
The present invention have collected Huang Huazhan and its pedigree totally 11 kinds.By the haplotype information for analyzing Huang Huazhan and its pedigree Obtain the situation of change that genome in breeding process is accounted in Huang Hua.
The Genotyping of sample genomic dna：Method according to the probe preparation described in embodiment 1, the extraction that sample is touched and Genotyping obtains the genotype in 7869 effective SNP marker sites.
The genotypic results obtained to infinium chips carry out quality control.SNP site as removed GenTrain Score ＜ 0. 6 in the sample in embodiment 2.And the site that the miss rate in this 11 kinds is more than 0. 2 is removed, deletion segment can not participate in further data analysis process due to the loss of data, finally give 7560 SNP marker sites
According to the SNP marker site obtained, the change of haplotype is calculated according to the algorithm of haplotype (Yamamoto et al., 2010), and according to these sites being visualized described in embodiment 2, obtain result as shown in Figure 7.The situation of change of genome in breeding process is accounted in Huang Hua by analyzing Huang Huazhan and its pedigree haplotype information acquisition.
Embodiment 7. uses SNP Locus Analysis in Shoots derived varieties
The present invention have collected Huang Huazhan and its derived varieties totally 11 kinds.Region and not conservative region that analysis Huang Huazhan is guarded during cultivating after which.Conservative region may be considered Huang Hua and account for metastable site in pedigree, it may be associated with the pedigree merit, mean that the region can optimize without conservative region, this analysis has important directive function as the rice breeding of background to accounting for pedigree material using Huang Hua.
The Genotyping of sample genomic dna：Method according to the probe preparation described in embodiment 1, the extraction that sample is touched and Genotyping obtains the genotype in 7869 effective SNP marker sites.
The genotypic results obtained to infinium chips carry out quality control.SNP site as removed GenTrain Score ＜ 0. 6 in the sample in embodiment 2.And the site that the miss rate in this 11 kinds is more than 0. 2 is removed, deletion segment can not participate in further data analysis process due to the loss of data, finally give 7552 SNP marker sites.
According to the SNP marker site obtained, these sites are visualized as described in example 2 above, result are obtained as shown in Fig. 8.We can clearly obtain the region and not conservative region guarded during Huang Huazhan is cultivated after which, so as to instruct with the rice breeding of yellow Hua Zhanwei backgrounds.
Embodiment 8. uses the auxiliary back cross breeding of SNP ^B sites
Improve Elite restorer line R288 resistance.Rice blast resistance gene site important in D3-10 is imported into R288, the preferable further back cross breeding of material of importing is selected in BC3F1 using SNP marker site.
The parent for collecting 6 plants of BC3F1 materials and 2 hybridization, the genotype in 7869 effective SNP marker sites is obtained according to the method for the probe preparation described in embodiment 1, the extraction of sample DNA and Genotyping.
The genotypic results obtained to infinium chips carry out quality control.SNP site as removed GenTrain Score ＜ 0. 6 in the sample in embodiment 2.And the site that the miss rate in this 8 kinds is more than 0. 2 is removed, deletion segment can not participate in further data analysis process due to the loss of data, finally give 7574 SNP marker sites. According to the SNP marker site obtained, these sites are visualized as described in example 2 above, result are obtained as shown in Fig. 9.The genomic fragment being imported into BC3F1 materials in D3-10 in R288 can be therefrom can see, important references are provided for the material selection of back cross breeding.
Embodiment 9. is spouted row association analysis using SNP sites
The present invention have collected Huang Huazhan and its pedigree totally 21 kinds, the change of genome and associating for character of analysis Huang Huazhan and its pedigree.
The Genotyping of sample genomic dna：Method according to the probe preparation described in embodiment 1, the extraction that sample is touched and Genotyping obtains the genotype in 7869 effective SNP marker sites.
The genotypic results obtained to infinium chips carry out quality control.SNP site as removed GenTrain Score ＜ 0. 6 in the sample in embodiment 2.And the site that the miss rate in this 11 kinds is more than 0. 2 is removed, deletion segment can not participate in further data analysis process due to the loss of data, finally give 7527 SNP marker sites.
The present invention have collected the amylose content of rice paddy seed, and according to the SNP marker site obtained, be associated analysis, obtain Manhattan figure as shown in Figure 10.There is site at two that there is significant relevance on chromosome by analysis, and No. 6 regions of the chromosome navigated to include known waxy genes.Analysis is thus associated by highdensity SNP chip, preferable result can be obtained.
Embodiment 10. uses the genotype in SNP Locus Analysis in Shoots hybrid Populations
The present invention have selected 9311 and be analyzed with the RIL data of Nipponbare.
The present invention have collected 9311 and wherein three materials of Nipponbare RIL and selection, and the genotype in 7869 effective SNP marker sites is obtained according to the method for the probe preparation described in embodiment 1, the extraction of sample DNA and Genotyping.
SNP site as removed GenTrain Score ＜ 0. 6 in the sample in embodiment 2.And the site that the miss rate in this 3 materials is more than 0. 2 is removed, deletion segment can not participate in further data analysis process due to the loss of data, finally give 7591 SNP marker sites.According to the SNP marker site obtained, these sites are visualized as described in example 2 above, are as shown in figure 11 its result.Recombinant inbred lines are carried out with Genotyping, next step regathers the phenotypic character of correlation, it is possible to QTL site positioning analysis.
Table 1:The kind list of file names of 590 parts of rice materials
Qiananshui Xishanba Dingzho Nantian Guang im Hongmidongz
〃02428" KT5 nuo i unuo zhan i-2 Jianan-9 han Wuke-4
Guangkehon Lengshu
lemont gmixianzha izi (Yeh Sanl icu Wulongbe Huangyad Youzha KT6 n Dal izhan e) n-1 i-3 ao Huangsinuo nnuo
Yangmianza Jinbaoyi Dazhang Mahuangn Zaokengj Baimihuangz Jiaozh
MH63 KT7 n Jinzhan u uo i han an
Yanghuoz Xiaohon Gaoganba
FH838 KT8
Sanshizi hong ggu Wangu Fengleng i Tongzinuo Dayegu
61
The Ζ OAV of l7Zll00/Cl0ZN3/X3d Z908 Please 0
i uo ndao ixian ongj iaoq oxuan nnuo
i
Ruanzhan Datouhu Miandian Xiaoj i
YB19
Xihuzao Banl ikenuo j iugu ang Mazinuo j iugu Jiegunuo Jiuyuenuogu ugu
Sanl icun Baimido Hongman Datiez
YB20
-6 Guihuanuo Taohuami ngzhan gnuo Zaodao Xiyeqing Chuangnuo-2 ao
Changl i j in Hongdou Taidong Hongjiao Changmaodao
KT1 YB21
Lizhidao gzhan Haonuobi nuo wuzhan Aizinuo wen -1-1
Dahongji Honghand Hangunu Shuihon Hongjiao
KT2 YB22 ao~3 Hunuo ao-2 gmi Wushizao zhan Erfuzao
Hongkenu Laohong Daguang Qianjinz
KT3 YB23
Anqinggu Handaozi gu-2 mazhan Zaonuo-2 ao Cungu
Chang j in Zunhuawenz Honggu- Gouyingd
The u Huanggu Yanong-1 tables 2 of KT4 YB24 gchi izui Haoxiang Youzhan 2:9,000 equally distributed SNP site and its corresponding gene numbered lists on genome（Note：Listed two numerals immediately following letter osbwk or osbwkg represent chromosome numbers in SNP titles, 8 bit digitals below are the particular location on the chromosome, the first two letter in last three letters represents two kinds of nucleotides possibilities in paddy rice in the SNP site, and last represents probe design in chromosome normal chain or minus strand）
Correspondence
Whether sequence nucleotide sequence is corresponded to
Whether being located in work(dyeing body surface in dyeing body surface
The SEQ of the positive minus strand SEQ ID energy positive minus strand of gene interval of SNP title SNP title functions
Gene NO: ID
It is interval
NO :
osbwk0100030071AGR 1 osbwk0714863477AGR 4501 osbwk0100071882CTF + 2 osbwk0714886545GAF + 4502 osbwk0100079943TCR 3 osbwk0714928798TCR 4503 osbwk0100149920TAF + 4 osbwk0714974452CTR 4504 osbwk0100194254GAF + 5 osbwk0714988971GTF + 4505 osbwk0100213775TGF + 6 osbwk0715025425TCF + 4506 osbwk0100287697CTF + 7 osbwk0715074908TCR 4507 osbwk0100333976TCR 8 osbwk0715205294GAF + 4508 osbwk0100400732TGR 9 osbwk0715210361GAF + 4509 osbwk0100479914TGF + 10 osbwk0715283320GAF + 4510 osbwk0100532873CTF + 11 osbwk0715336231TGR 4511 osbwk0100554916CTR 12 osbwk0715364585GTF + 4512 osbwk0100581591TCR 13 osbwk0715420950GAF + 4513 osbwk0100626145CGR 14 osbwk0715487838GAF + 4514 osbwk0100693597TCR 15 osbwk0715588736AGF + 4515 osbwk0100742720CAR 16 osbwk0715596678TGR 4516 osbwk0100803739AGR 17 osbwk0715653783GAR 4517 osbwk0100852739CTF + 18 osbwk0715664483CTR 4518 osbwk0100920661TCR 19 osbwk0715701842TCF + 4519 osbwk0100935862AGF + 20 osbwk0715716516GAF + 4520 osbwk0100961331GTF + 21 osbwk0715758580TCF + 4521 osbwk0101005881TCR 22 osbwk0715803349TCR 4522 osbwk0101045858GAR 23 osbwk0715846316GAF + 4523 osbwk0101092889GAR 24 osbwk0715885882AGR 4524
ε
+ + + + + + + + + + + + + + + +
+ + + + + + + + + + + + + + + + + + + + + +
o o o o o o o o o o
o o o o o o o o o o
+ 1 + 1 1 + 1 + 1 + 1 + 1 + + + 1 + 1 1 1 1 1 1 + 1 1 + 1 + + 1 + + + + + 1 1 + + 1 + 1 1 + 1 1 + +
^→ ^→ ^→
^→ ^ ^→
o o o o o o o o o o o o o o o
o n o o n o o n o o o n o o o
n o o o o n o o o o o o o o n o o o o o o o n o o o n o o o o o o o
o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o
o o o o o o o o o o
o o o o o o o o o
1 1 1 1 + 1 1 1 1 + + 1 + 1 1 1 1 1 + 1 + 1 + 1 1 + 1 1 + + 1 + 1 + + 1 + 1 1 1 1 + 1 + + 1 1 + 1 1
^
o o o o o o o o n
o o o o n o o oo o o o o n n o o o o o o
o o o o o n o o o n o o n o o o o o o o
o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o
+ + + + + + + + + + + + + + + + + + + + + + + + + + + + +
+ + + + + + + + + + + + + + + + +
o o o o o o o o o o
o o o o o o o o o
1 + + 1 1 1 + 1 1 1 + 1 1 1 1 1 1 + + 1 1 1 + 1 1 1 1 1 + + 1 1 1 + + 1 1 1 + + 1 + 1 1 + 1 1 + 1 +
o o o
o o o o o o o o o n o σ; o o o o o o n n o o o n o n o o
o o o o o o n o o o
o o o o o o o o o o
n o o o o o o o o o o o o o o o o o o o o o o o o o o o
o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o
o o o o o o o o o o
o o o o o o o o o o
1 1 1 1 + 1 + + 1 + + 1 1 1 1 + + 1 1 + + 1 1 1 + + + 1 1 1 + + + + 1 + 1 + + + 1 + 1 1 + 1 1 + + +
o o o o o o o o o o o o o o o o o o o o o o o
o o o o n o o n o o o
o o o o o o o o o o o o
o o o o o n o o o o o o o o o o o o o
o o o o o o o o o o o o o
o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o
+ + + + + + + + + + + + + + + + + + + + + + +
+ + + + + + + + + + + + + + + + + + + + + + + + + +
+ + + + + + + + + + + + + + + + + + + + + + + +
+ + + + + + + + + + + + + + + + + + + + + + + + + + + + + +
+ + + + + + + + + + + + + + + + + + + + + + + +
+ + + + + + + + + + + + + + + + + + bk0808010185TCF osw
bk0125262032AGR osw
+ + + + + + + + + + + + + + + +
bkbk0810041312AGR0126809955AGR osw osw
bkbk0810082969CTF0126844403TAF osw osw
bkbk0810130855ACF0126860884GAR osw osw
bkbk0810214081ACF0126885533GTF osw osw
bkbk0810277398GTF0126967808TCR osw osw
bkbk0810284615CTR0127046165TCR osw osw
bkbk0810341411CAR0127054406CTR osw osw
bkbk0810367314TGR0127097922AGR osw osw
bkbk0810398414TAR0127103550CTR osw osw
+ + + + + + + + + + + + + + + + + + + + bkbk0810436667AGF0127154833TGR osw osw
bkbk0810466537CAR0127177993TCR osw osw
bkbk0810507183CTF0127247593GAR osw osw
bkbk0810591120GAR0127326824AGF osw osw bkbk0810689011AGR0127389902GAF osw osw
bkbk17350TCF012740810767710GAR osw osw
bkbk0810754116GAR0127537931TCR osw osw
bkbk0810782265CAF0127543151GAF osw osw
bkbk0810836901CTR0127632121TCR osw osw
bkbk0810902739TGR0127681175GAR osw osw
bkbk0810946434TCR0127709050AGR osw osw
bkbk0811029927CTR0127739701GAF osw osw
bkbk0811107360CTF0127774070TCR osw osw
bkbk0811259293CTR0127816300CTF osw osw
bkbk0811278549TCR0127860557CAF osw osw
bkbk0811343103ACR0127906044TCR osw osw
bkbk0811368213CTR0128058408ACR osw osw
bkbk0811450554AGR0128125264GAF osw osw
bkbk0811481292TCR0128198151CAR osw osw
bkbk0811537309CTR0128266497GTR osw osw
bkbk0811591059GAF0128280959CTF osw osw
bkbk0811699885GAR0128349033GTF osw osw bkbk0811735054AGR0128425031TGR osw osw bkbk0811781404GAR0128433728ACF osw osw bkbk0811860060TCF0128465146TCR osw osw 3
s
+ + + + + + + + + + + + + + + + + + + + +
+ + + + + + + + + + + + + + + + + + + + + +
5
+ + + + + + + + + + + + + + + + + + + + + + + + +
+ + + + + + + + + + + + + + + + + + + + + +
+ + + + + + + + + + + + + + + + + + + + +
+ + + + + + + + + + + + + + + + + + + + + + + + + +
+ + + + + + + + + + + + + + + + + + +
+ + + + + + + + + + + + + + + + + + + + + + +
+ + + + + + + + + + + + + + + + + + + + + + + + + + +
+ + + + + + + + + + + + + + + + + + + + + + + +
+ + + + + + + + + + + + + + + + + + + + +
+ + + + + + + + + + + + + + + +
+ + + + + + + + + + + + + + + + + + + + +
+ + + + + + + + + + + + + + + +
o o o o o o o o o
o o o o o o o o o o
1 + 1 + + + 1 1 1 + + + 1 + + 1 1 1 1 + 1 + 1 1 + 1 + + + + 1 + + 1 1 1 1 1 1 1 + + 1 1 + 1 + 1 1 +
^→ ^→ ^→ ^
^→ ^→ ^→
o o o o o o n oo o o o o o o o o
o n o o o o o
o o o o o o
o o o n o o o o o
o o n o o o o n o oo o
n n
o o o o o o o o o o o o o o o o o o o o o o o o o o ο ο o o o o o o o o o o o o o o o o o o o o o
o o o o o o o o o o
o o o o o o o o o
+ 1 1 1 + 1 1 1 1 1 + 1 1 + + 1 + 1 1 + + 1 1 1 1 1 1 + 1 + + + 1 + + 1 1 + + 1 1 + 1 + 1 + 1 + + 1
^→ ^→ ^→
^ ^→
o o o o o o o n o
o o o o o o o o o o o
o o n o o n o o o o o o o o o o ο o o o
o n o o o o o o oo n o o o o ο o o
o o o o o o o o o o o
o O o o o o o o o o o o o o o o o o o o o o o o o o ο o o o o o o o o o o o o o o o o o o o o o o
+ + + + + + + + + + + + + + + + + + + + + + + +
+ + + + + + + + + + + + + + + + + + + + + + + + +
+ + + + + + + + + + + + + + + + + + + + + +
+ + + + + + + + + + + + + + + + + + + + + + + + +
n
o o
osw osw
+ + + + + + + + + + + + + + + + + + + + + + + + + + +
+ + + + + + + + + + + + + + + + + + + + + bkbkl003945619GAR0226106439TCF osw osw
2
+ + + + + + + + + + + + + + + + + + + + + +
bkbkl013042194CTF0235635047GAR osw osw
+ + + + + + + + + + + + + + + + + + + + + + bkbkl013476735GAF0300216106TGF osw osw
bkbkl013911428ATR0300855610TCR osw osw
5
+ + + + + + + + + + + + + + + + + + + + +
+ + + + + + + + + + + + + + + + + + + + + + + + +
+ + + + + + + + + + + + + + + + + + + + +
+ + + + + + + + + + + + + + + + + + + + + + + + + + + + + +
+ + + + + + + + + + + + + + + + + + + + + +
+ + + + + + + + + + + + + + + + + + + + +
u
+ + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +
+ + + + + + + + + + + + + + + + + + + + + +
+ + + + + + + + + + + + + + + + + + + + + + + +
+ + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +
+ + + + + + + + + + + + + + + + + + + + + + + + +
+ + + + + + + + + + + + + + + + + + + + +
+ + + + + + + + + + + + + + + + + + + + + + + + + + + +
+ + + + + + + + + + + + + + + + + + + + + +
69 H0V099 9SS0^0Wso
+ Z Z HID6 908SS0^0Wso
ZZ69 ZZ Z + dVD9S98TSS0^0Wso
TS69 HDI9 6 STTWS0 IZfZ HID89SS 0S0^0Wso
0S69 + dV086 90 STTWso OZ Z + dID8SS8S0S0^0Wso
6T69 6l Z HID80S^S0S0^0Wso
8T69 HIDTS69969STTWS0 Sl Z HID8 T8T0S0^0Wso T69 + dDI066^69STTWso Ll Z HV0^T^96T0^0Wso
9T69 HVO SS689STTWSO 9l Z HDI S6T0^0Wso
ST69 HDIS0^9889STTWSO + dV0SSS668T0^0Wso
W69 HI008TSS89STTWSO n z + d0VT8S9 8T0^0Wso
ST69 HOI 6^6S 9STTWSO HVD^S99^8T0^0Wso
ST69 + dID^906S 9STTWso Zl Z
Π69 HVOS SS899STTWSO U Z HDV6S6SS T0^0Wso
0Τ69 + dDI0^TT^99STTWso Ol Z HDVS00^ T0^0Wso
6069 + dOV9T9 T99STTWso 60 Z + dV0TS6 ^9T0^0Wso
8069 + 80 H0DSK 0^T0^0Wso 069 HIO 06^S9STTWSO LO Z + dV0^609SST0^0Wso
9069 HOV 9ST6^9STTWSO 90 + dDIS96SSST0^0Wso
S069 90 Z + d0V0ST^ST0^0Wso
W) 69+Getting S+dV088TS0ST0^0Wso
S069 + 0 Z HDV88 86TT0^0Wso
S069 ZO Z HDIS^ST9TT0^0Wso
Τ069 HV0T0 ^T9STTWso lO Z HIDST960TT0^0Wso
0069 HI00 609STTWso OO Z + dDIS90060T0^0Wso
6689 + H0VSS86T0T0^0Wso
8689 + H0I99 ST0T0^0Wso 689 HDI909S96SSTTWSO L6 Z + dOV9^S09600^0Wso
9689 + dDI 8TST600^0Wso
S689 + 6 Z HOITS888800^0WSO
^689 6 Z HVD^S0S^800^0Wso
S689 H0V0 8S6 SSTTWso H0V8T00½00^0Wso
S689 + Z6 Z + dI089 S9 00^0Wso
Τ689 HIO T 9SSTTWSO 16 Z + dDIS080 00^0Wso
0689 + dDI8TS0T9SSTTWso + dOVTSOS6900^0Wso
6889 + dOIS ^99900^0Wso
8889 + HIDSS080900^0WSO 889 + + dID00998S00^0Wso
9889 + dDVSSS6TS00^0Wso
S889 + + dID 6^66^00^0Wso 9 + dV00S09S^00^0Wso
S889 H0V 888S^00^0Wso
S889 + dDI69T½S00^0Wso
Τ889 + HV09SS S00^0Wso
0889 + H0VT0SSSS00^0Wso
6 89 6L Z + d0VSS9^TS00^0Wso
8 89 HDIT9T8S0SSTTWso + dDVS^T0TS00^0Wso 89 HDIS8S996^STTWS0 + dIDSTSS0S00^0Wso
9 89 HI099SS06^STTWSO 9L Z H0D9ST^ST00^0Wso
L Z HDISS9S6000^0WSOL7ZllOO/ClOZN3/X3d Z908 Please Ο Ζ OAV + + + + + + + + + + + + + + + + + + + + + + + + + +
+ + + + + + + + + + + + + + + + + + + + + + + + + +
bkbkl200556616CTF0404218344ACR osw osw bkl200750072TGR osw
osw
L7ZllOO/ClOZN3/X3d Z908 Please Ο Ζ OAV
.8
L7ZllOO/ClOZN3/X3d Z908 Please Ο Ζ OAV
1 1 1 1 1 + + 1
o
osw osw
osw osw
osw osw osw osw
osw osw
+ + + + + + + + + + + + + + + + + + + +
+ + + + + + + + + + + + + + + + + + + + + + + + + + + + + +
6
o o o o o o o o o
o o o o o o
1 + 1 1 + 1 1 + 1 1 1 1 1 1 1 1 1 + + + + + 1 + + + + 1 1 1 1 1 + + + 1 + 1 1 + 1 1 + 1 1 1 1 1 1 +
^→ ^→ ^o o o o n o
n o o o o o
o o o o n o o o o
o n o o o o
o n o o o o n o o
o o o o o o n n o o
o o o n n o o o o o o
O o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o
o o o o o o o o o o
o o o o o o o o o
+ 1 + + 1 + 1 1 1 + 1 + 1 + 1 + + 1 + + + + 1 1 + + + + + 1 1 + 1 + 1 1 1 + + + 1 1 1 + + 1 + 1 + 1 o o o o o o o o o o o o o
o o o o o o o o
o o o o o n
o o o o o o o o n o
o o o o o o o o o
o o o n o o o o n n o o
n n n o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o
o o o o o o o o
n n o o o o o o o o o o
1 1 + 1 + 1 1 1 1 1 1 + 1 + 1 + 1 + 1 + + 1 1 1 + 1 1 1 + 1 1 + + + + 1 + + 1 + 1 1 + 1 1 1 1 + 1 + o o o o o o o o o o o o o o o o o o o o o o o o o n o o o o o o n o o o
n n o o o o o o o o o o ο o o o
o o o o o o
o o o o o o o o o o o o o o o o o O O O o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o
o o o o o o o o o o
n n o o o o o o o o o o
1 1 + 1 + + + 1 1 1 1 + 1 1 + 1 1 1 + 1 1 1 + + + + 1 1 + 1 1 + + 1 1 + + 1 + 1 1 + + 1 1 + 1 1 1 +
o o o o
o o n o o o o o o o o o o o o o o σι o o n o o o
o n o o o o o o o o o n o o o o n o o o o
o o n n o o o o n o o σι σι n n n o o o o o o o o o o o o o o o o
o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o
+ + + + + + + + + + + + + + + + + + + + +
+ + + + + + + + + + + + + + + + + + + + + + + + + + +
+ + + + + + + + + + + + + + + + + + + + + +
+ + + + + + + + + + + + + + + +
i
L7ZllOO/ClOZN3/X3d Z908 Please Ο Ζ OAV + + + + + + + + + + + + + + + + + + + + + +
+ + + + + + + + + + + + + + + + + + + + + + + + + +
+ + + + + + + + + + + + + + + + + + + + + + + +
+ + + + + + + + + + + + + + + + + + + + + + + +
+ + + + + + + + + + + + + + + + + + + + + + + + +
+ + + + + + + + + + + + + + + + + + + + + + +
+ + + + + + + + + + + + + + + + + + + + + + + + + + +
+ + + + + + + + + + + + + + + + + + + + + +
o o o o o o o o
bk0617388801CTF osw o o o o o o o o
+ 1 + 1 1 1 1 + 1 1 + + 1 1 + + 1 1 + + + 1 1 + + + 1 1 1 1 1 + 1 1 1 1 1 1 + 1 + 1 + 1 + + + 1 + 1
^→ ^→ ^→
o o o o o o o o
o o o o o o
o o n o o n
o o o o o o
o o o o o o o o o
o o o o o o o o n
o o o o o n n o
o o o o o o o o o o o o o o o o o o o
o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o
o o o o o o
o o o o o o o o o
n n n n n n n n n n n n n n n n n n n n
+ + 1 + 1 + + 1 1 + + 1 1 + + + + + 1 1 1 + 1 1 1 1 1 + + + 1 + 1 + 1 1 1 + 1 1 1 1 1 1 + 1 + + 1 +
^→
o o o o o
n o o o o n n o
o n o o o o n n n o o n n o o n
o o o o o o o o o o o o n o o n o o
n n n n n n n n n
o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o ο o o o o ο
+ + + + + + + + + + + + + + + + + + + + + + + + +
+ + + + + + + + + + + + + + + + + + +
+ + + + + + + + + + + + + + + + + + + + + + +
+ + + + + + + + + + + + + + + + + + + + + + + + + + + +
+ + + + + + + + + + + + + + + + + + + +
+ + + + + + + + + + + + + + + + + + + + + + + +
+ + + + + + + + + + + + + + + + +
+ + + + + + + + + + + + + + + + + + + + + + + + +
+ + + + + + + + + + + + + + + + +
+ + + + + + + + + + + + + + + + + + + + + + +
o o o o
o o o o o o
+ + + + + + + + + + + + + + + + + + + + + + + + +
^→ ^→ ^→ ^→
o o o o o o n
o o o o n
o o o o n
o o n n
n n o n o
o o o n n o o o o
o o o o o o o
+ + + + + + + + + + + + + + + + + + + + + + + + + o o o
o n o n
o <iO
o o n o o n o o
n o n o
o o o o o o o o o o o o o
+ + + + + + + + + + + + + + + + + + + + +
+ + + + + + + + + + + + + + + + + + + + + + + + + + +

Claims (1)

1. a kind of SNP site set, including at least two different SNP sites, and parting can be carried out to corresponding oryza sativa genomic dna polymorphism, it is characterised in that the SNP site is selected from such as SEQ ID NO:Nucleotide sequence shown in wide 9000.
2. a kind of chip of SNP site set for described in test right requirement 1, it is characterised in that：Described chip can be used for the Genotyping of corresponding SNP site.
3. the chip described in claim 2, it is characterised in that nucleotide probe of the chip also containing the corresponding SNP site of a set of detection, the nucleotide probe is fixed at least one solid carrier or at least one microtiter plate.
4. the chip described in claim 2, it is characterised in that nucleotide probe of the chip also containing the corresponding SNP site of a set of detection, the nucleotide probe is located in the single or different hole of microtiter plate.
5. the chip described in claim 2, it is characterised in that nucleotide probe of the chip also containing the corresponding SNP site of a set of detection, the nucleotide probe is located at the different interrogation positions on the solid carrier.
6. claim 2-5 any described chip, wherein nucleotide sequence of the described nucleotide probe for the upstream and downstream 50bp in SNP sites or so, available for detecting corresponding SNP site.
7. the chip described in claim 2, wherein described Genotyping, its experimental procedure includes：A) Genotyping nucleotide probe and reagent are made；B) probe and sample are hybridized and scanned on genotyping system, genotype of the sample at the SNP site is analyzed in acquisition.
8. the chip described in claim 2 or 7, wherein described genotyping system is the Inf inium chip platforms of I l lumina companies.
9. the chip described in claim 8, it is characterised in that：Described chip refers to according to SEQ ID NO:L to SEQ ID NO:9000 DNA sequence dnas utilize the chip that Infmium Patent designs manufacturing technology makes.
10. application of the claim 2-9 any described chip in Rice Germplasm Resources genetic fingerprinting.
11. application of the claim 2-9 any described chip in Rice Progenies genotype identification.
12. application of the claim 2-9 any described chip in rice varieties identification.
13. application of the claim 2-9 any described chip in detection rice breed.
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