WO2014048062A1 - Jeu de loci de snp et procédé d'utilisation et application de celui-ci - Google Patents

Jeu de loci de snp et procédé d'utilisation et application de celui-ci Download PDF

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WO2014048062A1
WO2014048062A1 PCT/CN2013/001124 CN2013001124W WO2014048062A1 WO 2014048062 A1 WO2014048062 A1 WO 2014048062A1 CN 2013001124 W CN2013001124 W CN 2013001124W WO 2014048062 A1 WO2014048062 A1 WO 2014048062A1
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rice
snp
chip
genotyping
marker
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PCT/CN2013/001124
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English (en)
Chinese (zh)
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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
候红利
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未名兴旺系统作物设计前沿实验室（北京）有限公司
深圳市农业科技促进中心
深圳市作物分子设计育种研究院
深圳兴旺生物种业有限公司
兴旺投资有限公司
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Priority to CN201380004386.7A priority Critical patent/CN104024438B/zh
Publication of WO2014048062A1 publication Critical patent/WO2014048062A1/fr

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    • 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
    • C12Q1/6876—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
    • C12Q1/6834—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
    • 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

Definitions

  • the invention relates to a collection of high-density SNP sites and a method and application thereof, in particular to a high-density SNP site collection of rice and a method and application thereof, belonging to rice molecular marker breeding, fingerprint identification of rice varieties and Functional gene research field.
  • molecular markers At present, in the breeding at home and abroad, the use of molecular markers is becoming more and more widespread.
  • molecular markers including RFLP markers (Restriction Fragment Length Polymorphism), RAPD markers (Random Amplified Polymorphic DNA), SSR markers (Simple Sequence Repeat, Amplified Fragment Length Polymorphism (Amplified Fragment Length Polymorphism), SNP molecular marker (Single Nucleotide Polymorphism)
  • SNP is a new generation of molecular markers recently developed, featuring high abundance and easy detection and automation. Sequence analysis and comparison of different genomes of different rice varieties indicated that the distribution of SNPs on rice genomes is extremely abundant, which is much more extensive than the SSR markers commonly used in rice breeding. At present, SSR is widely used in molecular marker-assisted breeding and gene mapping research. However, due to the instability of SSR in the genome, the density of distribution is relatively low, and the genotyping is difficult to achieve automation. Limitations. The SNP has a low mutation rate, especially the SNP in the coding region is highly stable, and its genetic stability is much higher than that of genetic markers such as SSR. The reproducibility and accuracy of genetic analysis or genetic diagnosis are superior to those of SSR.
  • SNPs are dimorphic, either or both, unlike SSR markers, which often have the potential for multiple DNA fragments, facilitating automated detection.
  • SNPs have only two allelic types, the amount of polymorphism information at a single locus is less than that of multiple alleles such as SSR, but the high frequency of SNPs provides a high amount of information.
  • the SNP is easy to carry out high-throughput detection and is destined to be more suitable for genetic analysis of complex traits.
  • Techniques for detecting SNPs include: sequencing, PCR-RFLP, specific allelic hybridization, Ligase chain reaction, TapMan, single base extension, and the like.
  • Genotyping companies such as Illumina Affimetrix
  • a technique for rapid and efficient SNP genotyping has been developed, currently available to humans (Murray SS, et al. A highly informative SNP linkage panel for human genetic studies. Nat Methods (2004) 1: 113-117), Maize (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.
  • Rice SNP chips have been widely used. However, high-density rice SNP chips suitable for breeding practice are yet to be developed.
  • Molecular marker-assisted selection breeding has been widely used in breeding in recent years. Wang C, 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 marker assisted selection (MAS) is based on the location of the gene or QTL (quantitative trait locus), using the target gene or a close linkage between the QTL and the molecular marker. Thereby achieving breeding goals.
  • MAS marker assisted selection
  • molecular marker-assisted selection relies on molecular markers that are tightly linked to genes or QTL sites, and high-density molecular markers can improve the accuracy of molecular marker-assisted selection breeding. Therefore, high-density SNP chips are of great significance in the application of rice molecular marker-assisted selection breeding.
  • Genome wide association study is an analytical method based on linkage disequilibrium (LD) to identify the relationship between target traits and genomic loci or candidate genes in a population.
  • Plant genome-wide association analysis is one of the hotspots of international plant genomics research. It has a wide range of applications in corn and other fields (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) 43:163-168; Riedelsheimer C, et al. Genome-wide association mapping of leaf metabolic profiles for dissecting complex traits in maize. Proc Natl Acad Sci USA (2012) 109:8872- 8877; Tian F, et al.
  • High-throughput SNP chips can solve these problems well, greatly improving the accuracy and efficiency of DNA fingerprinting technology.
  • the high-throughput SNP chip contains a large number of SNP loci distributed on the rice chromosome, and simultaneously detects nearly 10,000 SNP genotypes of rice varieties, and obtains sufficient "fingerprint" information.
  • Second, high-throughput SNPs In the chip the chromosomal distance between SNPs is small, within 100 kb, so small changes in rice varieties can also be detected.
  • the present invention collects a collection of SNP sites containing all currently known rice functional genes and a genome-wide uniformly distributed SNP site. High-throughput chips were designed and prepared, and they are widely used in rice DNA fingerprinting, molecular design breeding and related applications and research.
  • the advantages of the present invention are as follows: 1.
  • the SNP site selected by the inventors is based on large-scale sequencing results and is evenly distributed on the genome. Distribution, different rice varieties have good polymorphism; 2. SNP sites are selected within all known functional genes; 3. Illumina's Infmium technology platform itself has high throughput, good accuracy and easy detection. And so on, the sites we selected are very suitable for use on this platform.
  • the present invention aims to provide a high-density SNP collection of rice, which comprises 9,000 SNP sites, which exhibit high-density uniform distribution on rice genome, and is highly suitable for rice molecular marker breeding, functional gene research and rice.
  • the 9000 SNP sites are shown as SEQ ID NOs: 1 to 9000 in the sequence listing.
  • the present invention obtains a large number of SNP marker sites among rice varieties from different rice materials, and a total of 8,944,748 SNP sites.
  • the present invention uses a plurality of indicators and algorithms to screen these SNP marker sites when selecting SNP marker sets. Including genomically uniform distribution, weak allele frequency (MAF), linkage disequilibrium (LD), non-repetitive regions, gene functional regions and other indicators.
  • genomically uniform distribution weak allele frequency (MAF), linkage disequilibrium (LD), non-repetitive regions, gene functional regions and other indicators.
  • the present invention first selects 5,680,149 high-reliability SNP sites detectable from 3 or more germplasms from 590 SNPs of rice germplasm.
  • the rice genome is equally divided into 50 kb intervals, and appropriate SNP sites are further screened in each interval by the following steps and conditions:
  • the disclosed 9000 SNP site sets are listed in Schedule 2, and the average distance between adjacent sites is about 50 Kb, and its distribution on the genome is shown in FIG.
  • Figure 2 shows the distribution of effective SNP loci between the selected SNP sets in any two representative japonica rice japonica core collections (two rice varieties were randomly selected and the number of effective SNPs between them was analyzed). The results showed that the set of SNP loci was about 2000 for the number of effective SNPs between any two japonica rice, and about 1500 for any two japonica rice. The SNP between any japonica rice and one japonica rice was About 5,000.
  • a set of SNP loci associated with a class of agronomic traits such as plant height, plant type, stress resistance, etc.
  • PCR detection and probe hybridization The design and application detection methods of chips, kits, etc. are all within the scope of the present invention.
  • the SNP locus obtained by the present invention can be applied to any experimental platform capable of detecting the SNP genotype, such as a genotyping platform of companies such as Illumina, Affimetrix, and Sequenom.
  • a genotyping platform of companies such as Illumina, Affimetrix, and Sequenom.
  • the technical means of these companies' different types of platforms are different, but the principles are similar.
  • a chip or kit for detecting SNP-labeled oligonucleotide probes is synthesized and then genotyped using the corresponding SNP typing technique on the corresponding genotyping device.
  • the chip comprising the SEQ ID NO: 1 - 9000 SNP detection sites of the present invention developed by the above platform is also referred to as Rice9K chip, or Rice9K, or 9K chip, in the present invention.
  • the present invention discloses the physical position of all SNP loci on the chromosome, the flanking sequences on both sides of the locus can be obtained through a common channel, and the probe design is performed by the related software, thereby obtaining an effective detection of the SNP locus. Probe.
  • the inventors specifically selected IUumina's Infmium technology platform for SNP chip synthesis.
  • the probe design and detection principle of IUumina Infmium genotyping are as follows: (1) For SNP polymorphisms of A/G, A/C, T/G and T/C, only one probe needs to be designed to terminate at the SNP position. Adjacent sites of the point, an extension of a dideoxyribonucleotide (ddNTP) is completed during the assay, bases A and T are stained to one color, and bases C and G are dyed to another color.
  • ddNTP dideoxyribonucleotide
  • the probe sequence for each SNP site is the 50 bp nucleotide sequence immediately upstream of the SNP site.
  • the beneficial effects of the present invention are to construct a high density SNP site marker set of rice, which has very High polymorphism and uniform distribution throughout the genome (high polymorphisms ensure the validity of SNP loci during application, and uniform distribution of whole genomes ensures comprehensive and reliable analysis results).
  • SNP loci on functional genes meet the needs of functional gene-related breeding or research.
  • the important significance of the present invention is to provide a high-density SNP marker locus collection that is efficient and covers the whole genome of rice, and is suitable for rice variety identification, hybrid analysis, molecular marker-assisted breeding, and molecular genetics research.
  • the use of molecular markers for assisted selection can improve the accuracy of selection and breeding efficiency, but it is not fully developed in China.
  • plant genome association analysis is currently an internationally popular research method.
  • Currently, only one laboratory conducts high-throughput sequencing for rice correlation analysis. Data analysis requires high skill. Some laboratories have no way to achieve this.
  • the present invention provides such an efficient collection of SNP sites that makes rice correlation analysis economically viable.
  • the SNP collection can greatly improve the efficiency and throughput of genotyping.
  • the SNP collection of the present invention is the identification and mixing of the varieties. Analysis provides an economically viable and efficient tool.
  • the lineage analysis of the core rice parents can determine the relationship between the evolution of important agronomic traits (including yield, quality, resistance, etc.) and haplotype or gene transfer, thereby guiding the improvement of rice parents, and thus the SNP collection of the present invention provides High-resolution marker sites that study changes in lineages.
  • the SNP set of the present invention provides an effective means for parental affinity analysis. Quantitative trait locus mapping (QTL mapping) is widely used in the breeding process of rice. The higher density of site markers can be more accurately located.
  • QTL mapping Quantitative trait locus mapping
  • the SNP set of the present invention provides a genome-widely distributed high-density marker set, which can The rice QTL can be positioned more accurately, which can further improve the efficiency of rice breeding using QTL loci.
  • the high-density SNP site set constructed by the present invention not only enables large-scale rice molecular marker-assisted breeding and genome-wide association analysis, but also provides economic feasibility and efficiency for variety identification, hybrid analysis, lineage analysis, and QTL mapping analysis. And high-resolution tools.
  • the application of the SNP locus of the present invention in the field of rice breeding can promote the development of rice breeding, and it is expected that there will be an important breakthrough in the field of molecular design breeding in China within a few years.
  • oligonucleotide probes developed for the Illumina chip technology platform are nucleotides 11 to 60 of the nucleotide sequence set forth in SEQ ID NOs: 1 to 9000 of the sequence table, that is, next to each SNP.
  • the upstream 50 bp of the polymorphic site is the probe sequence provided by the present invention for the IUumina chip technology platform.
  • sample DNA extraction collect the required samples according to the designed rice breeding or other biological research related experiments. A specific concentration of sample genomic DNA is extracted and obtained according to the requirements of the customized genotyping chip or kit, and stored under appropriate conditions.
  • genotyping of SNP marker loci obtaining SNPs by hybridization of sample genomic DNA and SNP-labeled oligonucleotide probes in the corresponding genotyping system according to the requirements of a customized chip or kit The genotype of the marker locus.
  • Genotyping data analysis Quality control of preliminary results of genotyping, selection of sites with high reliability.
  • the genotype of the SNP marker locus is then combined with the experimental design related to rice breeding or other biological studies, and the corresponding data analysis method is selected to obtain the corresponding results.
  • the experimental design and data analysis methods are described in the specific examples.
  • the present invention has the following advantages and effects:
  • the Rice9K chip of the present invention has wider applicability than the disclosed Chinese invention patent 201210055775.X.
  • Chinese invention patent 201210055775. The chip design of X is mainly aimed at the identification and classification of the hybrids between the two subspecies of japonica and japonica rice. Therefore, the SNPs which are basically fixed between the two subspecies of rice in each lOOkb are selected. This option limits the scope of use of the chip, and it is difficult to achieve a good typing effect on the hybrid populations and similar varieties in the same subspecies that are mainly faced in the actual breeding work.
  • the actual test inside the japonica rice and the average inside the japonica rice are two or two.
  • the number of polymorphic sites detected by the breed was only 1046 and 813.
  • the SNP is more than 0. 2 in a subspecies, the MAF is greater than 0.2 in a subspecies.
  • the SNPs thus selected have good applicability.
  • the average number of polymorphic loci detected in the two indoor and in japonica rice varieties is 2400 and 1800, and the polymorphism between the two subspecies. The point is to reach 4,400.
  • the present invention has designed 1510 SNPs for the collected 879 known genes of rice, which can be directly applied to breeding, and the Chinese invention patent 201210055775. X contains only 40 such probes.
  • the Chinese invention patent 201210055775. X is a chip with a limited application range.
  • the Rice9k chip of the invention has wide universality and can be applied to the actual breeding work of various rice varieties and groups.
  • Figure 1 shows the distribution of 9000 SNPs in the whole genome of rice.
  • Figure 2 shows the distribution of effective SNP loci between any two indica rice, two indica rice, and one indica rice using one of the 38 core rice germplasms with genetic diversity.
  • the abscissa is the number of effective SNPs, and the ordinate is the number of rice pairings in which the number of corresponding SNP sites appears.
  • Figure 3 shows the results of clustering SNP loci using two methods in Illumina GenomeStudio.
  • the yellow loci are the results of a group of parents and their heterozygotes (Pl, P2, Fl), AA, AB, and BB are the three genotypes of the cluster, which are homozygous, heterozygous, and homozygous.
  • Fig. 4 is a schematic diagram showing the comparison of SNP loci typing of four samples of Huanghuazhan.
  • the grey mark in the figure is the SNP site that is consistent with the standard Huanghua, and the red mark is the SNP site that is inconsistent with the standard Huanghua.
  • Figure 5 is a schematic representation of the SNP locus typing of Huang Huazhan and its EMS mutagenized mutants.
  • the gray marker in the figure is the SNP locus consistent with the wild-type Huanghua, while the red marker is the SNP locus that is inconsistent with the wild-type Huanghua.
  • Figure 6 is a clustering diagram of 65 rice core collections based on SNP locus analysis.
  • the green mark is the SNP site consistent with Nipponbare
  • the red mark is the SNP site that is inconsistent with Nipponbare
  • yellow is the heterozygous site
  • white is the deletion site.
  • Figure 7 is a haplotype analysis diagram of the Huanghua pedigree. On the left is the pedigree of Huang Huazhan; the gray on the right shows that the haplotype may be obtained from any parent, and the other colors indicate the haplotype-derived variety.
  • the first variety that appears in the color from the bottom up is Huang Huazhanzhong. The corresponding haplotype-derived species.
  • FIG 8 is a schematic diagram of the SNP locus classification of Huang Huazhan and its derivatives.
  • the gray mark is the SNP site that is consistent with Huanghua
  • the red mark is the SNP site that is inconsistent with Huanghua.
  • Fig. 9 is a schematic diagram showing the SNP site typing of 6 strains of BC5F1 produced by the combination of Gumei 4, R608 and their hybrids.
  • the gray mark is the SNP site corresponding to R608
  • the red mark is the site inconsistent with R608
  • the yellow mark is the heterozygous SNP site
  • the black mark in Gumei 4 is the position of the rice blast resistance gene.
  • Figure 10 is a Manhattan view of the correlation analysis of the amylose traits of the Huanghua pedigree.
  • FIG 11 is a schematic diagram showing the genotyping of 9311 and Nipponbare recombinant inbred lines.
  • the green mark is the SNP site consistent with Nipponbare
  • the red mark is the SNP site that is inconsistent with Nipponbare
  • yellow is the heterozygous site
  • white is the deletion site.
  • the present invention provides a set of SNP sites covering the whole genome of rice, comprising a total of 9000 marker sites, and the isolated nucleic acid molecules associated with these marker sites are as shown in SEQ ID NOs: 1 to 9000 in the Sequence Listing. Nucleotide sequence and characteristics. These SNP loci were discovered by sequencing rice genomic DNA and determining polymorphism by computer analysis. These SNP markers can be used for a variety of genotyping applications.
  • the polymorphic rice locus of the present invention comprises at least 12 contiguous nucleotides comprising or adjacent to a polymorphic site (ie, a SNP site) identified in the present invention, said SNP locus
  • a polymorphic site ie, a SNP site
  • the corresponding gene coding number, its single nucleotide polymorphism, and the nucleotide sequence adjacent to the SNP site are shown in Table 2 and the nucleotide sequence in the sequence listing as shown in SEQ ID NO: 1-9000.
  • Table 2 the single nucleotide polymorphisms identified in the present invention have also been specifically mapped to rice chromosomes.
  • the rice locus of the invention comprises a series of molecular markers comprising at least 20 contiguous nucleotides and comprising or adjacent to one or more polymorphisms identified in Table 2.
  • the nucleic acid sequence of these rice loci is at least 90% sequence identical to the sequence of the same number of nucleotides in any strand of the rice DNA fragment comprising or adjacent to the polymorphism, more preferably at least 95%, or even more preferably for certain
  • the locus is at least 98%, and in many cases at least 99% sequence identity.
  • the nucleotide sequence of one strand of such a rice DNA fragment can be found in the sequence of SEQ ID NOs: 1 to 9000 of the Sequence Listing.
  • SNPs are especially useful as genetic markers because they are more stable than other types of polymorphisms and are abundant in the rice genome. SNPs can be produced by insertions, deletions, and point mutations.
  • a SNP may represent an insertion and deletion (indel) event, or a single nucleotide polymorphism, which may consist of one or more base pairs.
  • Polymorphisms shared by two or more individuals may result from individuals derived from a common ancestor. This "source identity" (IBD) characterizes two DNA loci/fragments carried by two or more individuals and all from the same ancestor.
  • IBD source identity
  • State identity is characterized by two or more individuals Two DNA loci/fragments carrying and having the same allele detectable at those loci.
  • IBS tate identity
  • a large number of marker loci in a fragment is sufficient to characterize the IBD of the fragment as an indication that they are capable of predicting within the fragment Alleles present at other marker loci.
  • the stability and richness of SNPs allows them to be used to determine IBD.
  • one aspect of the invention provides a collection of nucleic acid molecules that allow for the typing of polymorphisms at different loci.
  • the number of loci in such a collection may vary, but will be a finite number, for example, as few as 2 or 5 or 10 or 25 loci or more, for example up to 40 or 75 or 100 or more The locus.
  • Another aspect of the invention provides an isolated nucleic acid molecule capable of hybridizing to a polymorphic rice locus of the invention.
  • a PCR primer is provided
  • such a molecule comprises at least 15 nucleotide bases.
  • a molecule which can be used as a primer can hybridize under high stringency conditions to a strand of a DNA fragment in the polymorphic locus of the present invention.
  • Primers for amplifying DNA are provided in pairs, namely a forward primer and a reverse primer.
  • One primer is complementary to one strand of DNA in the locus, and the other primer is complementary to the other strand of DNA in the locus, ie, the sequence of the primer sequence is preferably at least 90% identical to the sequence of the same number of nucleotides in one strand, More preferably, at least 95% are the same. It will be appreciated that such primers may be associated with sequences in loci that are remote from polymorphisms (eg, at least 5, 10, 20, 50, 100, 200, 500 or up to about 1000 nucleotide bases from the polymorphism). Hybrid. The design of the primers of the invention depends on factors well known in the art, for example, avoidance or repetition of sequences.
  • a hybridization probe design for polymorphism assays is an oligonucleotide sequence comprising at least 12 nucleotide bases or a detectable label.
  • the needle sequence hybridizes under high stringency conditions to a strand of DNA in a nucleotide base fragment comprising or adjacent to a target polymorphism in the amplified portion of the polymorphic locus.
  • the sequence of such oligonucleotide sequences and fragments of the same number of nucleotides in the rice DNA-strand in the polymorphic locus preferably has at least 90% similarity, more preferably at least 95% similarity.
  • the detectable label can be a radioactive element or a dye. More preferably, the hybridization probe may further comprise a fluorescent label and a quencher, for example, a hybridization probe assay of the type known as the Taqman assay available from AB Biosystems.
  • the isolated nucleic acid molecules of the invention are capable of hybridizing under certain conditions to other nucleic acid molecules including, but not limited to, rice genomic DNA, cloned rice genomic DNA, and expanded rice genomic DNA.
  • other nucleic acid molecules including, but not limited to, rice genomic DNA, cloned rice genomic DNA, and expanded rice genomic DNA.
  • a nucleic acid molecule is said to be complementary to another nucleic acid molecule if it exhibits "complete complementarity", ie, each nucleotide in one sequence is complementary to a base-paired nucleotide in another sequence.
  • Two molecules are said to be “minimally complementary” if they hybridize to each other under at least conventional "low stringency” conditions and are sufficiently stable to allow them to remain annealed to each other. Similarly, two molecules are said to be “complementary” if they hybridize to one another under conventional "high stringency” conditions and are sufficiently stable to allow them to remain annealed to each other.
  • a nucleic acid molecule that hybridizes to other nucleic acid molecules at least under low stringency conditions is referred to as a "hybridizable homologue of the other nucleic acid molecule," Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Ed., Cold Spring Harbor Press , Cold Spring Harbor, New York (1989) and Haymes et al, Nucleic Acid Hybridization, A Practical Approach, IRL Press, Washington, DC (1985) describe conventional stringent conditions, the invention being incorporated by reference. Complete complementarity is permissible as long as such deviation does not completely eliminate the ability of the molecule to form a double-stranded structure. Therefore, in order for a nucleic acid molecule to be used as a primer or probe, it is only necessary to be sufficiently complementary in sequence to be The solvent and salt concentration can form a stable double-stranded structure.
  • SNPs are the result of sequence variation, and new polymorphisms can be detected by sequencing random genomes or cDNA molecules.
  • polymorphisms in the genome can be determined by comparing cDNA sequences from different lines.
  • the evaluation of cDNA sequences does not provide information on the position of introns in the corresponding genomic DNA.
  • polymorphisms in non-coding sequences cannot be determined from cDNA. This is a disadvantage, for example, when using a cDNA-derived polymorphism as a marker for genotyping genomic DNA. If the polymorphism includes polymorphisms that are present in non-coding unique sequences, a more efficient genotyping assay can be designed.
  • Genomic DNA sequences are more useful than cDNA in identifying and detecting polymorphisms. Polymorphisms in the genome can be determined by comparing genomic DNA sequences of different lines. However, genomic DNA in higher eukaryotes generally contains a high proportion of Complex sequences and transposons. If the coding/unique portion is enriched by subtracting or eliminating the repeat sequence, the genomic DNA can be sequenced more efficiently.
  • Polymorphisms in DNA sequences can be detected or typed by a variety of effective methods well known in the art, including, but not limited to, those in U.S. Patents 5,468,613 and 5,217,863, 5,210. 015, 5, 876, 930, 6, 030, 787, 6, 004, 744, 6, 013, 431, 5, 595, 890, 5, 762, 876, 5, 945, 283, 5, 468, 613, The methods disclosed in 6,090, 558, 5,800, 944, and 5, 616, 464, the entire disclosures of each of which are incorporated herein by reference.
  • the SNP markers of the present invention can be used in combination with any of the polymorphism methods to classify polymorphisms in rice genomic DNA samples.
  • rice genomic DNA samples used include, but are not limited to, rice genomic DNA isolated from rice plants, cloned rice genomic DNA or amplified rice genomic DNA.
  • polymorphisms in DNA sequences can be detected by hybridization to an allele-specific oligonucleotide (AS0) probe as disclosed in U.S. Patent Nos. 5,468,613 and 5,217,863.
  • AS0 allele-specific oligonucleotide
  • U.S. Patent No. 5,468,613 discloses the hybridization of allele-specific oligonucleotides in which one or more nucleotide variations in a nucleic acid sequence can be detected by a nucleic acid sequence, in which, Sequences containing nucleotide variations are added, spotted onto the membrane, and processed with labeled sequence-specific oligonucleotide probes.
  • the target nucleotide sequence can also be detected by the probe ligation method disclosed in U.S. Patent No. 5,800,944, wherein the target sequence is amplified and hybridized to the probe, followed by ligation to detect the labeled portion of the probe. .
  • Microarrays can also be used for polymorphism detection, in which an oligonucleotide probe set is assembled in an overlapping manner to represent a sequence such that a difference in the target sequence at one point results in partial probe hybridization (Borevitz et al., Genome Res. 13: 513-523 (2003); Cui et al, Bioinformatics 21: 3852-3858 (2005)).
  • multiple target sequences are expected. They may represent genes and/or non-coding regions, wherein each target sequence is represented by a series of overlapping oligonucleotides rather than by a single probe.
  • the platform allows for high throughput screening of multiple polymorphisms.
  • Single characteristic polymorphism is a polymorphism detected by a single probe in an oligonucleotide array, wherein the features are probes in the array.
  • SFP Single characteristic polymorphism
  • the target nucleic acid sequence can also be detected by the probe ligation method disclosed in U.S. Patent No. 5,616,464, which utilizes at least one pair of probes having sequences homologous to adjacent portions of the target nucleotide sequence and having a side chain that non-covalently binds to form a stem when the probe is base paired with the target nucleic acid sequence. At least one of the side chains has a photoactivatable group that can form covalent crosslinks with other side chain members of the stem.
  • SNPs and Indels can be detected by the methods disclosed in U.S. Patent Nos. 5,210,015, 5,876,930, and 6,030,787.
  • One useful test is the Taqman test available from AB Biosystems.
  • Polymorphisms in the loci of the invention can be used for the identification of marker/trait associations, which are inferred from statistical analysis of the genotype and phenotype of the population members.
  • These members may be a single organism, such as rice, a family of closely related individuals, inbred lines, doubled haploids of closely related individuals, or other groups.
  • Such a rice population is called a "line" and represents the origin system.
  • a population may originate from a single hybrid between two individuals or two lines (e. g., a localized population), or it may be composed of individuals having multiple origin families.
  • Each individual or line is characterized by a single or average trait phenotype and genotype at one or more marker loci.
  • the included markers should be characteristic of the source to make inferences for subsequent populations.
  • SNP-based molecular markers are ideal for mapping because the probability that a particular SNP allele is derived from an independent source in an existing population of a particular species is extremely low. Therefore, SNP markers can be used to trace and assist in the infiltration of QTLs, especially in the case of haplotypes.
  • the goal of association studies is not only to detect marker/trait associations, but also to assess the location of genes (ie, QTLs) that directly affect traits relative to marker locations.
  • the level of difference or the significance of the difference between the alternative genotypes is compared between marker loci.
  • the putative trait gene is located at the marker closest to the genotype difference with the greatest correlation.
  • Genetic linkage of additional marker molecules can be established by gene mapping models such as, but not limited to, flanking markers reported by Lander et al. (Lander et al., Genetics, 121: 185-199 (1989)).
  • the model, and interval mapping are based on the maximum likelihood method described therein and are executed with the software package MAPMAKER/QTL.
  • Additional 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 software is a particularly preferred method. Construction of genetic map
  • the polymorphism in the locus of the invention is localized to the rice genome, for example, as a genetic map of the rice genome, comprising two or more polymorphisms as shown in Table 2. Map location. This genetic map is shown in Figure 1. Genetic map data can also be recorded on a computer readable medium.
  • a preferred embodiment of the invention provides a high density polymorphic genetic map (eg, at least 5000 polymorphisms in the rice genome map are present in the same subspecies, and at least 20,000 polymorphisms are present between different subspecies) ).
  • a particularly useful genetic map includes polymorphisms with an average distance of no more than 0.02 centimeters (cM) on the linkage group.
  • Another way to determine the location of a trait gene is to analyze the marker/trait association in a population in which the individual's traits and marker loci are different.
  • certain marker alleles may be associated with certain trait locus alleles due to the genetic processes of the population, such as the unique origin of the mutation, founder events, random drift and population structure. . This association is known as linkage disequilibrium.
  • linkage disequilibrium is the level of random association between two or more loci in a population, and LD is often found on large chromosome segments. While it is possible to focus on the individual effects of each gene in the fragment, for actual plant breeding, the average effect on the target trait is generally emphasized when the region is present in a line, hybrid or variant.
  • linkage disequilibrium mapping the trait values of individuals with different genotypes at the marker locus are compared. In general, significant trait differences indicate that the marker locus is very close to one or more trait loci. If the marker density is suitably high and the linkage disequilibrium occurs only between sites that are very closely linked on the chromosome, the location of the trait locus can be very precise.
  • QTLs quantitative trait loci
  • markers can be used to select for improved trait values without the need for phenotypic analysis at each selection cycle.
  • marker-assisted breeding and marker-assisted selection the association between QTLs and markers was first established by genetic mapping analysis. In the same process, it is determined which molecular marker alleles are linked to a favorable QTL allele. Subsequently, a marker allele associated with a favorable QTL allele is selected in the population. If there is a tight enough linkage between the marker and the QTL, this process will increase the trait value. The degree of linkage required depends on the algebra chosen, because in each generation, there is an opportunity to break the association through reorganization.
  • the association between a particular marker allele and a favorable QTL allele can also be used to predict which types of progeny can be isolated from a given hybridization. This prediction may allow selection of a parent suitable for generating a population from which a new combination of favorable QTL alleles is assembled to generate a new inbred line. For example, if line A has a marker allele previously associated with a favorable QTL allele at loci 1, 20, and 31, and line B at loci 15, 27, and 29 With a marker allele associated with a favorable effect, the new line can then be developed by crossing AXB and selecting progeny with favorable alleles at all 6 QTLs.
  • the SNP molecular markers provided by the present invention can also be used to accelerate the infiltration of transgenes into new genetic backgrounds (SP, into different ranges of germplasm).
  • Simple introgression involves crossing the transgenic line with an elite inbred line and then repeatedly backcrossing the hybrid with the elite (recurrent) parent while selecting for maintenance of the transgene. After multiple generations of backcrossing, through genetic recombination and isolation, the genetic background of the initial transgenic lines was gradually replaced by the genetic background of elite inbred lines. This process can be accelerated by selection based on molecular marker alleles derived from the backcross parent.
  • the fingerprint of an inbred line is a combination of alleles at a set of two or more marker loci.
  • High-density fingerprints can be used to establish and track the identity of germplasm, which can be used to create a marker-trait correlation database that benefits the entire crop breeding program and the protection of germplasm ownership.
  • the SNP polymorphism set provided by the present invention can also be used to select a parent, progeny or test plant for plant breeding.
  • the ability to select these plants from phenotypically indistinguishable plant populations can accelerate plant breeding and reduce the cost of phenotypic trait analysis.
  • the method of selecting a plant for breeding comprises the steps of: a) determining the association between the plurality of polymorphisms identified in Table 2 and at least a plurality of traits in at least the first and second rice inbred lines; b) determining the parent , progeny or allelic status of one or more polymorphisms in the test plant; and c) selection of a parent, progeny or test plant having a more favorable combination of related traits.
  • the parent, progeny or test plant selected by this method is a rice inbred.
  • an advantageous combination of related traits provides improved heterosis.
  • determining the genotype of at least two polymorphisms facilitates selection of a parent for breeding crosses. This determination provides the breeder with the advantage of producing hybridization in which at least two preferred genomic regions are directed to produce progeny having at least two preferred genomic regions. In another aspect, determining the genotype of at least two polymorphisms can provide a basis for making selection decisions in the progeny, wherein those progeny that comprise the preferred genomic region are selected in the breeding program. In another aspect, a test line for assessing the combined ability of an inbred line in a hybrid combination can be selected for inclusion in an inbred test plan based on the presence or absence of at least two genomic regions to ensure a different genebank Hybridization is carried out between different heterotic groups.
  • the present invention provides a method for increasing the heterosis of hybrid rice.
  • a plurality of polymorphisms linked to the polymorphic locus of the present invention are associated with traits in two or more rice inbred lines. Two such inbred lines with complementary heterotic groups predicted to enhance heterosis were selected for breeding.
  • Methods for increasing heterosis include the steps of: (a) determining the association between multiple polymorphisms identified in Table 2 and multiple traits in more than two rice inbred lines; (b) being selected from the steps ( The two inbred lines of the inbred line a) are assigned to the heterotic group; (c) at least one cross between at least two inbred lines of step (b), wherein each inbred line is from a different And a complementary heterotic group, and wherein, the hybrid heterozygous group is optimized for genetic characteristics that enhance heterosis; and (d) the hybrid progeny plant is obtained by the hybridization of step (c), wherein, relative to and without selection Progeny produced by inbred crosses, the hybrid progeny plants exhibit increased heterosis.
  • These methods may also include conventional one-hybridization in step (c) (ie, between two inbred lines, ideally from different Heterosis group), ternary hybridization (after single hybridization, hybridization with the third inbred line) and two-hybrid (also known as quaternary hybridization, ie hybridization of two single-hybrid progeny).
  • Parenting Hybridization is carried out by hand or by using a male sterile hybrid system. The development and selection of elite inbred lines, hybridization and selection of these lines are described in Bernardo, Breeding for Quantitative Traits in Plants, Stemma Press, Woodbury, MN, 2002. Excellent hybrid hybrids identify new elite rice hybrids.
  • a theory of heterosis predicts that the genetic source identity (IBD) region between the male and female parents of the hybrid will reduce heterosis.
  • Genetic source identity can be inferred from patterns of marker alleles in different lines. If a string of identical markers at a series of adjacent loci cannot occur by chance independently, they can be considered to be of the same genetic origin. Marker fingerprinting in the parent and parent can identify IBD regions. Knowledge of these areas helps to select hybrid parents, as avoiding IBD in hybrids may increase the advantage of hybridization. This information also helps to develop a breeding program to design a cross between a male and a female parent with little or no IBD.
  • the set of SNP sites provided by the present invention can be used in experiments related to rice germplasm improvement, including but not limited to cross-breeding using plants, further genetic or phenotypic testing of plants, improvement of plants by self-fertilization, use of plants or Part of it is transformed and mutagenized using plants or parts thereof.
  • Different sets of nucleic acids in a set of SNP sites can be sampled, detected, or individually tested for any group, subgroup or combination thereof to classify any rice genomic DNA provided in Table 2 of the present invention.
  • a set of SNP sites comprises at least two different sets of nucleic acid molecules, wherein each of said different sets of nucleic acid molecules allows for the assignment of the corresponding rice genomic DNA polymorphisms identified in Table 2 Type.
  • each well of a microtiter plate contains one or more nucleic acid molecules that can classify only one rice polymorphism identified in Table 2.
  • each well of the microtiter plate contains one or more nucleic acid molecules that can classify more than one rice polymorphism identified in Table 2.
  • the microtiter plate can have as few as 8 wells, or as many as 24, 96, 384, 1536 or 3456 wells.
  • Microtiter plates can be made from materials including, but not limited to, polystyrene, polypropylene or cyclo-olefin plastics.
  • the nucleic acid molecules in each well can be either in solution or in a dry (ie, lyophilized form).
  • the nucleic acid is dispensed into the wells of a microtiter plate such that the nucleic acid in each well of the microtiter plate is known.
  • the nucleic acid molecule is associated with a unique identifier, such as a unique dye or other unique recognition signature
  • the nucleic acid can be randomly distributed into the wells of the microtiter plate. It will be apparent from this description that it also relates to a collection of nucleic acids immobilized on a solid support such as a bead, which is dispensed in the wells of a microtiter plate.
  • the nucleic acid that has been typed in the rice genomic polymorphism identified in Table 2 is allowed to be immobilized (i.e., covalently linked) to a solid support.
  • Solid carriers include, but are not limited to, beads, chips, arrays or filters.
  • the beads used as a solid support can be magnetic beads to aid in the purification of the hybridization complex.
  • the beads can contain unique identification marks.
  • beads dyed with a fluorescent dye that can be distinguished according to its spectrophotometric or fluorescent properties can be coupled to a nucleic acid molecule for typing a polymorphism.
  • a fluorescent dye that can be distinguished according to its spectrophotometric or fluorescent properties
  • Dye-labeled beads, analytical reagents, and devices for typing polymorphisms have also been described (U.S. Patents 6, 649, 414, 6, 599, 331 and 6, 592, 822) and are available from Luminex Corporation. (Austin, Texas, USA) obtained.
  • the binding of the SNP nucleic acid molecules linked to the beads can also be a nucleic acid molecule in which the chip, array or filter can also be used to immobilize the polymorphism of Table 2.
  • a nucleic acid marker for typing a given polymorphism will be immobilized to a defined physical location on the array such that a typing from a location corresponding to a given polymorphism can be generated and recorded. Data for subsequent analysis.
  • Methods of making and using arrays for typing polymorphisms include, but are not limited to, U.S. Patent 5,858,659 (hybridization based method) and U.S. Patent 6,294,336 (single base extension method) The method described in ).
  • Polymorphisms and loci can be used to identify and locate the DNA sequences of QTLs and genes linked to molecular markers.
  • a BAC or YAC clone library can be queried using a molecular tag linked to a trait to find a clone comprising a particular QTL and gene associated with the trait.
  • multiple (eg, hundreds or thousands) large QTLs and genes in a multi-gene sequence can be identified by hybridization to an oligonucleotide probe that is capable of localization with / or Linked molecular marker hybridization, wherein one or more molecular markers can be detected.
  • This hybridization screen can be improved by providing cloned sequences in high density arrays.
  • This screening method is more preferably improved by employing a pooling strategy to significantly reduce the number of hybridizations required to identify clones containing molecular markers. When mapping molecular markers, screening can effectively map clones.
  • the plates can be arbitrarily arranged to form a stack of three-dimensionally arranged wells, each well comprising a unique DNA clone.
  • the holes in each stack can be represented as separate features in a three-dimensional array of rows, columns, and plates.
  • the number of stacks and the number of plates in each stack are approximately equal to minimize the number of trials. The stacking of plates allows the construction of a pool of cloned DNA.
  • a cloned DNA pool for: (a) all features of each row, (b) all features of each column, and (c) all features of each panel. Screening the pool with an oligonucleotide probe that hybridizes to a unique molecular marker for a clone will provide a positive indication for a pool of columns, a pool of rows, and a pool of plates, indicating the well cells containing the target clone (Elements).
  • the other pools of all cloned DNA in each stack allow for a stack indicating the row-to-plate coordinates of the row with the target clone.
  • a group of 4,608 clones can be arranged in 48 96-well plates.
  • the 48 boards can be arranged in a stack of 8 groups of 6 boards, providing elements of a 6x12x8 3D array, ie 6 stacks of 8 rows and 12 columns per stack.
  • For the entire clone group there are 36 pools, SP6 stacked pools, 8 row pools, 12 column pools, and 8 stacked pools. Therefore, up to 36 hybridization reactions are required to find clones containing QTLs or genes associated or linked to each of the mapped molecular markers.
  • oligonucleotide primers designed from the molecularly tagged locus can be used for localized cloning of linked QTLs and/or genes.
  • Methods for genotyping with single molecule markers can also be used to correlate phenotypic traits of rice plants with genotypes.
  • DNA or mRNA in tissues from at least two rice plants having allele DNA is detected to determine the presence or absence of a polymorphism as a molecular marker provided by the present invention.
  • the association between the molecular marker and the phenotypic trait is identified, wherein the marker is determined in Table 2.
  • a population of rice plants having allele DNA in a particular locus of a chromosome associates a trait with a genotype, the locus having a phenotypic effect on the target trait, and wherein the molecular marker is localized In or near the locus.
  • Methods for genotyping with single molecule markers can also be used to select parental, progeny or test plants for breeding.
  • the polymorphism is genetically linked to a chromosomal region that confers one or more desirable phenotypic traits.
  • Selection of parental, progeny or test rice plants comprising a particular allelic state associated with a phenotypic trait provides accelerated and lower cost breeding.
  • certain rice genomic polymorphisms disclosed in Table 2 of the present invention may be directly related to a given phenotypic trait, as they include certain regulatory or coding sequences that alter the proton trait or contribute to the expression of the trait. Allelic status.
  • These traits include yield, lodging, maturation, plant height, disease resistance, and stress resistance, such as drought tolerance, cold tolerance, heat tolerance, nutrient deficiencies, and quality traits.
  • stress resistance such as drought tolerance, cold tolerance, heat tolerance, nutrient deficiencies, and quality traits.
  • Infiltration of genomic regions associated with this single marker can be accelerated by the use of multiple markers to minimize linkage resistance associated with genomic regions that may not provide agronomically superior properties.
  • Infiltration of genomic regions closely associated with this single marker can be accelerated by using multiple markers directly on the single marker side to minimize linkage resistance associated with closely related genomic regions.
  • using a cluster of 2, 5, 10 or 20 markers located at the proximal and distal ends of the single marker, 10, 5, 2 or 1 cm can provide the required infiltration of the genomic region associated with the single marker, At the same time, the infiltration of the direct flanking regions that are not required is minimized.
  • the plurality of markers can include an additional 10 markers, each chromosome arm having at least one marker.
  • the marker density is at least about 10 markers per chromosome arm, more preferably at least about 100 markers per chromosome arm to effectively distinguish genomic regions from donor and recipient parents.
  • the present invention relates to a genotyping method using a set of nucleic acid molecules that can classify a plurality of different polymorphisms.
  • a limited number of at least two rice genomic polymorphisms are typed.
  • This limited number of rice genomic polymorphisms can be queried to contain at least 2, 5, 10 or 20 different genotypes, which are represented in Table 2 as 2, 5, 10 or 20 different nucleotide sequences.
  • These genotyping methods necessarily require the use of a set of nucleic acid molecules that can be used to classify rice genomic polymorphisms.
  • these genotyping methods use multiple molecular markers (ie, rice genomic polymorphisms) that are concentrated in a given chromosome interval.
  • High-density fingerprints for establishing and tracking germplasm identities can be obtained by performing genotyping methods that utilize multiple molecules concentrated or clustered around specific loci and/or certain loci conferring certain traits. mark.
  • High-density fingerprint information can be used to assess germplasm diversity, perform genetic quality assurance functions, develop rare alleles, assess foreign genebanks, and assess genetic purity.
  • These high-density fingerprints can be used to build a marker-trait association database that benefits the entire crop breeding program.
  • High-density fingerprints can also be used to establish and protect germplasm ownership.
  • the markers that are clustered around the desired chromosomal interval or hereditary trait can be selected from the localized rice polymorphisms provided in Table 2. Group.
  • genotyping with multiple molecular markers can also be used to correlate phenotypic traits of rice plants with genotypes.
  • DNA or mRNA in tissues from at least two rice plants having allele DNA is detected to determine the presence or absence of a limited set of polymorphisms as molecular markers provided by the present invention. Determining the association between the set of molecular markers and the set of phenotypic traits, wherein the set of molecular markers comprises at least 2, at least 5, or at least 10 molecular markers linked to the polymorphic loci of the invention, eg At least 10 molecular markers linked to the located polymorphisms, for example, those identified in Table 2.
  • a rice plant isolated population having allele DNA in a chromosomal locus that confers a phenotypic effect on a target trait traits are associated with genotypes, wherein molecular markers and polymorphisms and traits The degree of association between them allows for the determination of the linear order of polymorphisms and trait loci.
  • at least 5 molecular markers are linked to loci that allow for locus imbalance mapping.
  • At least one of the molecular markers used in the genotyping method using multiple markers is localized on each chromosome arm of all 12 rice chromosomes, and thus must have at least 24 rice genomic DNAs The state is typed.
  • other embodiments of the method are also involved, wherein at least 24 rice genomic DNA polymorphisms are located on each chromosome arm, so at least 288 rice genomic DNA polymorphisms must be classified.
  • At least 24 rice genomic DNA polymorphisms on each chromosome arm must be typed (at least 576 polymorphisms must be typed), or on each chromosome arm At least 50 rice genomic DNA polymorphisms were typed (at least 1,200 polymorphisms must be typed).
  • the marker set distributed on the rice genome can be selected from the localized rice polymorphisms provided in Table 2 for these methods.
  • Genotyping methods using molecular markers distributed over the rice genome can be used in a variety of applications.
  • the genotyping method is used to select a parent plant, a progeny plant, or a test plant for breeding.
  • These genotyping methods can be used to facilitate the infiltration of one or more traits, genomic loci, and/or insertion of a transgene from a genetic background into a different genetic background.
  • a panel of markers selected from progeny plants from an out-crossed population is queried to identify and select for inclusion of the desired trait, genomic locus and/or transgene insertion, while still containing as much of the Individual progeny of alleles of different genetic backgrounds that are distant.
  • These methods can accelerate the insertion of desired traits, infiltration of genomic loci and/or transgenes into new genetic backgrounds over several generations.
  • These methods also provide for trait screening by interrogating a collection of molecular markers such as SNPs with an average density of less than about 10 cM on the rice genetic map.
  • the presence or absence of a molecular marker linked to the polymorphic locus of Table 2 can be analyzed within the scope of one or more phenotypic traits to identify one or more associated with one or more of the traits described One or more specific molecular marker alleles at a genomic region.
  • the molecular tag is used to identify the haplotype
  • the haplotype is an allelic fragment of genomic DNA characterized by at least two polymorphisms in linkage disequilibrium, and the polymorphism is in a genomic window of no more than 10 centimeters in length, for example, no more than In a window of 8 centimeters or less, for example, in the range of 1-5 centimeters.
  • such a set of molecular markers identifies a plurality of haplotypes in a series of adjacent genomic windows in each rice chromosome, for example, using these windows to provide substantially complete genomic coverage.
  • an aspect of the rice assay of the present invention further comprises the steps of: characterizing the rice plant population with one or more traits, and correlating the trait with the allele SNP or Indel polymorphism, preferably Organize to define haplotypes.
  • traits include yield, lodging, maturation, plant height, disease resistance and stress resistance.
  • the present invention selects 590 rice germplasms for resequencing, and the 590 rice materials include 420 indica rice and 170 indica rice, specifically selected materials.
  • the name is as shown in the attached table 1 in the specification.
  • the present invention obtains a large number of SNP marker sites among rice varieties from different rice materials, and a total of 8,944,748 SNP sites.
  • the present invention uses a plurality of indicators and algorithms to screen these SNP marker sites when selecting SNP marker sets. Including genomically uniform distribution, weak allele frequency (MAF), linkage disequilibrium (LD), non-repetitive regions, gene functional regions and other indicators.
  • genomically uniform distribution weak allele frequency (MAF), linkage disequilibrium (LD), non-repetitive regions, gene functional regions and other indicators.
  • the present invention first selects from among 590 rice germplasm SNPs that are detectable in three or more germplasms.
  • the high-reliability SNP sites reached 5,680,149.
  • the present invention scores all SNP sites by Illumina infmium iSelect software and selects the scores > 740,786 SNPs of 0.6.
  • the rice genome is equally divided into 50 kb intervals, and appropriate SNP sites are further screened in each interval by the following steps and conditions:
  • Probe preparation The infinium chip containing the 9000 SNP-labeled oligonucleotide probe was custom-made at Illumina, and the oligonucleotide probe described in this example is the nucleotide sequence set forth in SEQ ID NO: 1-9000 of the Sequence Listing.
  • the nucleotides from position 11 to position 60, i.e., 50 bp upstream of each SNP polymorphic site, are the probe sequences for the Illumina chip technology platform provided by this example.
  • a. Preparation of denatured single-stranded DNA The DNA sample is denatured into a single strand with sodium hydroxide, then the denaturant is neutralized, and the enzyme amplification reaction solution is added.
  • Amplified genomic fragmentation The amplified product is cleaved into fragments of several hundred bases in size.
  • Precipitation DNA The digested product was added with isopropanol, centrifuged at 3000 g for 20 minutes to precipitate DNA, and dried at room temperature for one hour.
  • Dissolved DNA Add the hybridization solution at 48 °C for 1 hour, and vortex the DNA to dissolve the DNA in the hybridization solution.
  • Point chip, DNA and chip hybridization The DNA of the previous step is denatured at 95 degrees for 20 minutes, cooled to room temperature to start the chip, pay attention to avoid cross-contamination between different samples, and place the chip in the 48-degree hybridization furnace. 16-24 hours, no more than 24 hours.
  • g. Cleaning the chip Wash the DNA that is not hybridized or incompletely hybridized on the chip, and only the DNA that exactly matches the chip can be retained on the chip.
  • h. Single base extension and staining Single base extension is carried out by using DNA hybridized to the chip as a template, and the extended base is pre-modified and can be combined with the dye, and different bases can be determined by corresponding dye colors. .
  • the fixed chip is placed in the chip slot of the HiScan scanner to scan for signals, and the result of the scanning can be further analyzed in the software provided by Illumina.
  • genotyping results The results of the HiScan typing system were analyzed using the Illumina GenomeStudio software. The clustering was performed according to the results of the dye color at the time of single base extension, and the genotypes of the materials were classified into three types according to the clustering results (AA, BB, AB).
  • the invention analyzes the real situation of Huanghuazhan variety.
  • the inventors collected three different sources, called Huanghuazhan seeds (A, B, C), which were indistinguishable from the phenotype.
  • the standard Huanghuazhan material was collected from the Huanghuazhan cultivation unit Guangdong Academy of Agricultural Sciences for the identification of varieties. .
  • Genotyping of sample genomic DNA The genotypes of 7869 effective SNP marker sites were obtained according to the probe preparation, sample collision extraction and genotyping methods described in Example 1.
  • the invention selects a mutant of Huanghuazhan variety for source identification.
  • Genotyping of sample genomic DNA The genotypes of 7869 effective SNP marker sites were obtained according to the probe preparation, sample collision extraction and genotyping methods described in Example 1.
  • Huang Huazhan and its mutants were identified and analyzed.
  • a software analysis that is self-programming or analyzable and visualizable can be used.
  • Our analysis uses our own programming model and the results are shown in Figure 5.
  • the wild type Huanghua which we used for mutagenesis, is identical to the genotype of the materials cultivated by the Guangdong Academy of Agricultural Sciences, and there is no mixture of materials.
  • ⁇ -1, ⁇ -2, ⁇ -3, ⁇ _4, ⁇ -5, ⁇ -6 and Huanghuazhan are basically the same. It can be considered that these six mutants are all derived from Huanghuazhan and are not mixed. However, there are 355 SNPs in the ⁇ -6 that are inconsistent with Huang Huazhan, and it can be considered that there is a mixture.
  • the present invention collects 65 core rice germplasm materials and analyzes their genetic relationship for the next breeding.
  • genotypes of 7869 effective SNP marker sites were obtained according to the probe preparation, sample DNA extraction and genotyping method described in Example 1.
  • the SNP marker sites of the 65 samples were analyzed using cluster software and treeview software. The results are shown in Figure 6.
  • these 65 rice varieties can be divided into two groups, namely japonica rice and japonica rice.
  • japonica rice According to the known Nipponbare as indica rice, 26 varieties of the above-mentioned mostly green background parts are indica, and most of the 39 varieties are indica. In the figure, the relationship between rice varieties can be clearly judged.
  • the invention collects 11 varieties of Huanghuazhan and its lineage. Analysis of haplotype information of Huang Huazhan and its lineage Obtain the changes in the genome during the breeding process of Huanghuazhan.
  • Genotyping of sample genomic DNA The genotypes of 7869 effective SNP marker sites were obtained according to the probe preparation, sample collision extraction and genotyping methods described in Example 1.
  • the haplotype changes were calculated according to the haplotype algorithm (Yamamoto et al., 2010), and these sites were visualized as described in Example 2, and the results are shown in Fig. 7. Show.
  • the genomic changes during the breeding process of Huanghuazhan were obtained by analyzing the haplotype information of Huanghuazhan and its lineage.
  • the invention collects 11 varieties of Huanghuazhan and its derivative varieties. Analyze the areas that Huang Hua holds in the process of its subsequent cultivation and the areas that are not conservative.
  • the conserved region can be considered as a relatively stable site in the Huanghua pedigree, which may be associated with the excellent traits of the pedigree.
  • the non-conservative region means that the region can be optimized. This analysis is important for rice breeding with the background of Huanghua pedigree. Guidance role.
  • Genotyping of sample genomic DNA The genotypes of 7869 effective SNP marker sites were obtained according to the probe preparation, sample collision extraction and genotyping methods described in Example 1.
  • Example 8 Use SNP ⁇ B site to assist backcross breeding
  • the SNP site of the GenTrain Score ⁇ 0.6 in the sample was removed as in Example 2.
  • the locus with a deletion rate greater than 0.2 in the 8 varieties was removed, and the missing locus could not participate in further data analysis process due to the loss of data, and finally 7574 SNP marker loci were obtained. Based on the obtained SNP marker sites, these sites were visualized as described in Example 2, and the results are shown in Fig. 9. From this, we can see the genomic fragments introduced into R288 in D3-10 of BC3F1 material, which provides an important reference for the material selection of backcross breeding.
  • the invention collects 21 varieties of Huanghuazhan and its lineage, and analyzes the relationship between the changes of the genome of Huanghuazhan and its lineage and the traits.
  • Genotyping of sample genomic DNA The genotypes of 7869 effective SNP marker sites were obtained according to the probe preparation, sample collision extraction and genotyping methods described in Example 1.
  • the present invention collects the amylose content of rice seeds, and performs correlation analysis according to the obtained SNP marker sites, and the Manhattan figure is shown in Fig. 10. After analysis, there are two sites on the chromosome that are significantly correlated, and the region of chromosome 6 that is located contains the known waxy gene. Therefore, correlation analysis can be performed by a high-density SNP chip to obtain ideal results.
  • Example 10 Analysis of genotypes in a hybrid population using SNP loci
  • the present invention selected the recombinant inbred line data of 9311 and Nipponbare for analysis.
  • the present invention collects 9311 and Nipponbare recombinant inbred lines and selects three of them, and obtains the genotype of 7869 effective SNP marker sites according to the probe preparation, sample DNA extraction and genotyping method described in Example 1. .
  • the SNP site of the GenTrain Score ⁇ 0.6 in the sample was removed as in Example 2.
  • the locus with a deletion rate greater than 0.2 in the three materials was removed, and the missing locus could not participate in further data analysis due to the loss of data, and finally 7591 SNP marker loci were obtained. These sites were visualized as described in Example 2, based on the obtained SNP marker sites, as shown in Figure 11.
  • the genotypes of the recombinant inbred lines were genotyped, and the relevant phenotypic traits were collected in the next step to analyze the QTL locus.
  • Table 1 List of 590 varieties of rice materials
  • Lemont gmixianzha izi (Yeh Sanl icu Wulongbe Huangyad Youzha KT6 n Dal izhan e) n-1 i-3 ao Huangsinuo nnuo

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Abstract

La présente invention concerne une puce pour la détection de jeux de loci de SNP du génome du riz, dans laquelle les loci de SNP sont choisis parmi une séquence nucléotidique comme représentée par SEQ ID NO:1-9000. Ladite puce peut être appliquée à l'analyse de l'empreinte génétique des ressources de plasma germinatif du riz, à l'identification de génotypes de la descendance d'hybrides du riz, à l'identification de variétés de riz et à la sélection de matériaux de culture du riz.
PCT/CN2013/001124 2012-09-28 2013-09-23 Jeu de loci de snp et procédé d'utilisation et application de celui-ci WO2014048062A1 (fr)

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