CN103725675A - Molecular tagging method for paddy rice - Google Patents

Abstract

Paddy rice is one of the most important crops, and is also an important monocotyledonous model plant. Research based on the origination mode, differentiation process, formation mechanism, genetic diversity analysis, identification of indica rice-japonica rice introgression lines and the like of two subspecies of cultivated rice, i.e. indica rice-japonica rice, is the scientific problems concerning domestic and foreign scholars all the time. Option of excellent combination in genetic breeding of paddy rice, utilization of indica rice-japonica rice heterosis and other practical problems also have important use value and research significance. Development of molecular tagging based on indica rice-japonica rice differences can be a powerful tool for solving the scientific problems.

Description

The molecule marking method of paddy rice

Technical field

The molecule marking method of paddy rice belongs to biology field.

Background technology

Paddy rice is important in the world food crop, and world's population more than half is as life.China is the plantation big country of long-grained nonglutinous rice and hybridisation rice, the gramineous crops such as paddy rice and wheat, corn have colinearity in sequence in the gene, become at present the model plant of genetics and genome research, therefore carry out the research of rice molecular marking method, significant with cultivation new variety to promoting China's crop breeding level, and will help the whole world to solve food problems.

Paddy rice is not only one of topmost food crop, but also be one of important model plant, the researchs such as its relevant heredity, growth, evolution and breeding thereof enjoy scientist to pay attention to always, research contents relates to the basis of paddy rice and all respects of applied research thereof, and the molecule marker based on DNA polymorphism becomes one of powerful solving these problem in science because of its advantage having.

Its superiority is embodied in: 1. performance is stable, and polymorphism is directly with DNA form performance, and inorganization organ, developmental stage specificity, are not affected by envrionment conditions, interaction of genes; 2. quantity is many, spreads all in theory whole genome; 3. polymorphism is high, and nature exists many allelic variations, and without the special artificial particular inheritance material of creating, this has created condition for the closely linked label screening of a large amount of important character genes; 4. objective trait is expressed and had no adverse effects, chain without certainty with bad proterties; 5. part mark mode of inheritance is codominance, can differentiate homozygote and heterozygote; 6. cost is low, and common laboratory all can be carried out.For particular probe or primer can be introduced or synthetic voluntarily according to the particular sequence of delivering.Based on these features, molecule marker has now been widely used in paddy rice about many aspects researchs such as the molecular marker assisted selection of the drafting of functional gene mark, gene clone, genetic map and physical map, economical character, organic evolution, genetic diversity Journal of Sex Research.

Summary of the invention

Paddy rice is one of topmost food crop, is also important individual character leaf model plant.The research of the aspects such as the evaluation of origin mode, atomization, formation mechanism, analysis of genetic diversity and long-grained nonglutinous rice-japonica rice introgression line based on two subspecies long-grained nonglutinous rice-japonica rice of cultivated rice is the problem in science that Chinese scholars is paid close attention to always, for practical problemss such as the apolegamy of fine combination in the genetic breeding of paddy rice, the heterotic utilizations of long-grained nonglutinous rice-japonica rice, also has important utility value and Research Significance.The molecule marker of exploitation based on long-grained nonglutinous rice-japonica rice difference is one of powerful solving these problem in science.

 

1, the molecule marker based on hybridization

Typical Representative is DNA restriction fragment length polymorphism (restriction fragment length polymorphism, RFLP), and this technology is the method for the molecule marker based on Southern hybridization, is first-generation molecule marker.Its ultimate principle is with after digestion with restriction enzyme by Different Individual genomic dna, with with labelled with radioisotope or nonisotopically labelled DNA molecular probe (RFLP mark), carry out molecular hybridization, by technology such as radioactive automatic developings, show the size of endonuclease bamhi, thereby detect the allelic variation (polymorphism) of different genetic locuses.RFLP mark is codominance, and mark is accurate, does not affect phenotype, and number is also more.But the DNA that it needs amount is larger, routine analyzer complexity, technical difficulty is large, needs isotopic labeling probe time-consuming, and cost is higher.

2, based on pCRmolecule marker

The common RAPD (Random amplified polymorphic DNA) that comprises, AFLP (Amplified fragment length polymorphism), SSR (simple sequence repeats, claims again microsatellite), SCAR (sequence-characterized amplified region), STS (sequence-tagged site), CAPS (cleaved amplified polymorphiic sequence) etc.Be embodied in:

(1) random amplification fragment length polymorphism DNA(Random Amplified Polymorphic DNA)

With random primer, carry out the molecule marker of pcr amplification.Randomly amplified polymorphic DNA (Random amplified polymorphic DNA, RAPD) is the technology being grown up in nineteen ninety by Williams and Welsh simultaneously.This technology, is carried out pcr amplification and is produced polymorphism studied genomic dna as primer with short (being generally 10 bases) stochastic sequence DNA.Compared with other technology, RAPD technology has some outstanding advantages: 1. easy and simple to handle, experimental period is short, can screen a large amount of samples within a short period of time; 2. needn't understand in advance goal gene and fragment sequence; 3. required sample size is few, and once amplification only needs the template DNA of 20-100 ng; 4. primer tool general applicability, commercialization primer can be applicable to different biological research; 5. be adapted to automated operation and analysis.Owing to having above advantage, RAPD mark has been widely used in genetic evolution relation between belonging to of goal gene mark and location, research sibling species, has built in the researchs such as genetic map.But there are two shortcomings in RAPD technology, the one, the mark performance in most sites is dominant, and complete information can not be provided, and is stability and poor repeatability on the other hand.RAPD is as a kind of method of preliminary screening, once find needed DNA cloning product, then RAPD mark is converted into SCAR mark by the method for distinguished sequence amplification.

(2) restriction enzyme digestion combine with pcr amplification produce molecule marker.AFLP (amplified fragment length polymorphism) is amplified fragment length polymorphism, it is a kind of method of the restriction fragment that optionally increases, this method first design for the universal joint of certain restriction enzyme and can with the primer special of the sequence pairing of joint sequence and restriction enzyme site, with above-mentioned restriction endonuclease digested genomic dna, universal joint is connected with restriction fragment two ends, again by primer special amplification, finally electrophoresis showed result.Obvious this technology has the advantage of RFLP and two kinds of methods of RAPD concurrently, and its joint and primer are applicable to different biotypes, and AFLP can provide than the polymorphism information of RFLP and the more specific gene of RAPD.The polymorphism of AFLP mark is strong, utilizes radio-labeling 100-150 amplified production on the polyacrylamine gel of sex change, can be detected, is applicable to very much drawing the DNA fingerprinting of kind and carrying out sort research.

(3) molecule marker of specific primer PCR amplification.Micro-satellite (microsatellites) or simple sequence repeat (simple sequence repeats, SSRs) claim again microsatellite DNA (Microsatellite DNA), STR (Short seqence repeats, STR), it is the tandem repetitive sequence being formed for repeating unit's bunch collection by minority nucleic acid (general 1-6 bp), total length is tens to a hundreds of bp, is randomly dispersed on the different positions of whole rice genome.SSR mark is take PCR as basic molecule marker, has simple to operate, the feature such as polymorphism is high, stability is strong, codominance.Especially along with the completing of international paddy rice genome sequencing plan, this mark has obtained again development fast.2002, McCouch etc. developed 2240 pairs of new paddy rice SSR molecule markers.2005, international Sequencing of Rice Genome planning studies thought, rice genome contains 18828 SSR sites altogether.This mark is widely used in the research of the aspects such as location, gene map based cloning and comparative genomics and the molecular marker assisted selection breeding of the quantitative trait locus (QTL) of paddy rice, is to be applied at present one of class mark maximum on paddy rice.

(4) the specific primer PCR amplification molecule marker of generation that combines with restriction enzyme digestion.CAPS (c1eaved amplified polymorphic sequences, CAPS) mark, enzyme is cut amplification polymorphism sequence, is that specific primer PCR combines with restriction enzyme digestion and a kind of DNA marker of producing.Its ultimate principle is to remove to design specific PCR primer according to specific DNA sequence dna, then applies these primers and carries out pcr amplification; Then with narrow spectrum restriction enzyme, amplified production is carried out to enzyme and cut, finally by agarose or polyacrylamide gel electrophoresis, detect its polymorphism.DCAPS (derived c1eaved amplified polymorphic sequences, dCAPS) be that Neff etc. develops on CAPS basis, by introduce base mismatch in amplimer, to introduce new restriction enzyme action site on the basis of CAPS mark, the polymorphism of generation and CAPS designate similar after restriction enzyme digestion and electrophoresis.CAPS mark can be extended by the multiple mark such as SCAR, STS, EST, SNP, and can apply the various software such as Blast Digester (http://bbc. botany. utoronto. ca/ntools/cgi-bin/ntools blast_digester.cgi), SNP2CAPS (http: // pgrc. ipk-gatersleben. de/snp2 caps /), dCAPS Finder 2.0 (http: // helix. wustl.edu/dcaps/dcaps. html) and develop.At present, CAPS mark has been used in the aspects such as the development, molecular marker assisted selection of paddy gene location and clone, functional label.This label codominant inheritance, the advantage such as stability is high, high specificity, but detection technique relative complex, first to amplified production carry out enzyme cut after row polymorphic detection again.

3, based on dNAthe molecule marker of sequence and chip

Mainly comprise Expressed sequence tags (expressed sequence tags, EST), diversity sequence chip technology (Diversity A rrays Technology, DArT), mononucleotide polymorphic sequence (single nucleotide polymorphism, SNP), insertion and deletion length polymorphism (insertion deletion length polymorphism, InDel) etc.

(1) EST mark.EST is the expressed sequence fragment that is about 300-400bp.EST technology is extensive random choose cDNA clone the cDNA library from deriving from different tissues and organ, to its 5 ' or 3 ' end check order, the expressed sequence that is about 300-400bp that obtains.The length of est sequence is enough to the essential information that comprises its representative gene, therefore can use the specific marker gene of EST.In this sequence and gene database, known array compares, thereby obtain the technology to a series of physiological and biochemical procedures such as biology growing, growth, metabolism, breeding, decline death understanding, it is DNA sequence dna is checked order as one of representative of the New molecular marker of core.When genome sequencing not yet completes, EST identified gene, find new gene, gene mapping structure, utilize EST to carry out the aspects such as large scale analysis gene expression dose to play an important role.The quantity of EST was by the end of on December 13rd, 2010, and the EST quantity that discharges different plant species in public database NCBI has reached 52,858,766, and wherein the EST quantity of paddy rice has 25,019,080.

The weak point of EST approach is that by random sequencing, being sometimes difficult to obtain low abundance expresses the gene of coercing lower abduction delivering with those at special environment condition.Make up these deficiencies, must carry out genome sequencing.By analyzing gene group sequence, obtain the complete information of gene structure, as gene putting in order on karyomit(e), intergenic spacer structure, the structure of promotor and the distribution of intron etc.

(2) DArT mark.DArT mark is in a kind of New molecular marker technology of invention in 2001 ^[17], it is the method that dependence and chip hybridization are distinguished polymorphism between different genes group.The feature of DArT mark is to have high-throughput low cost, and an array can detect the hundreds of pleomorphism site being distributed in genome simultaneously; The discovery of polymorphism DArT mark is parallel carrying out with detection, and new mark is found and mark evaluation is to carry out on same chip, without the further appraisement system of development; In the discovery of mark with in detecting, do not need to know in advance DNA sequence dna information, this just makes this method can be applicable to that DNA sequence dna information has or all species of nothing, for the isolated germplasm materials of sibship rareness, has more suitability; Genome mutation can comprise the Cultivar of specific region, also can cover the heritable variation that comprises wild relatives in this kind, the diversity being disclosed by DArT can constantly be expanded on basis in the past, and therefore user can carry out to determine the scope of DArT genetic analysis as requested; This technology repeatability is stable, and experiment is reliable, is subject to the impact of karyomit(e) ploidy and Genome Size very little; DArT mark has dominant and codominant feature, can clone and carries out sequential analysis and develop into new mark etc.

(3) SNP molecule marker.SNP is called as third generation DNA genetic marker.It refers to by the conversion of single base in genomic level, transversion, and the DNA sequence polymorphism that causes such as the insertion of single base or disappearance.It extensively appears in single-copy DNA sequence, has higher frequency in most of genomes, can find SNP, and some SNP marks is immediate causes of phenotypic variation in transcriptional domain and the nontranscribed domain of DNA.SNP quantity is abundant, and genetic stability is good, and the extensive automatization being especially based upon on DNA chip technology detects, and makes it have application prospect more widely.The draft genome sequence of Indica in 2002, two subspecies of round-grained rice is completed, and this research that is SNP provides a great convenience.Shen etc. utilize bioinformatics method, by the difference of the whole genome sequence of the warm and fine 93-11 of rice varieties Japan relatively, built Japanese warm and fine 93-11 complete genome DNA sequence polymorphism database, this database comprises 1703176 SNP and 479406 InDel fragments, average every 286 bases just have a SNP, and every 953 bases just have an InDel.

Delivered again rice genome sequence full figure in August, 2005, so far more than 95% examining order of the paddy rice whole genome sequence that has completed total length 389Mb, the rice genome sequence full figure of delivering shows, nearly 80127, SNPs site between Xian, japonica rice, just there is a SNP in average every 154 bases, its frequency is 20 times of SNP frequency between two environmental Columbia of Arabidopis thaliana and Landsberg.At present this mark is used to the many aspects such as drafting, gene clone, molecular marker assisted selection of the genetic map of paddy rice.

Generally speaking; the research emphasis of SNP is also the discovery of SNPs at present; although there is a large amount of SNPs in indica rice genome sequence; but these SNPs just obtain based on two that represent two subspecies concrete genotype; lack representativeness widely, such SNP frequency usually can be over-evaluated.Between kind, SNPs quantity may be than the much less more of the SNPs in database in subspecies for Jin etc., in actual genetic breeding, we may be just for the improvement of indivedual objective traits, the material utilizing is usually the very near breeding material of sibship, SNP frequency will be more low, finds with the possibility of the closely linked SNPs of objective trait just less.Therefore to accurately evaluate in actual applications the frequency of SNP in paddy rice, will increase the genotype quantity of experiment material, to obtain the data of more DNA sequence dna, do relatively comprehensive.

At present to be that SNP detection technique need perfect for another factor of restriction SNPs application, although realized the increasingly automated and high-level efficiency of detection technique, experimental cost is high, has greatly limited the widespread use of SNP labeling technique.Although SNP mark can be converted into PCR or CAPS mark, its workload fails to reduce than other molecule markers, can not bring into play the superiority of SNP labeling technique completely.Therefore we also need to continue to improve level of automation and the detection efficiency of SNP detection technique, reduce testing cost, and a large amount of SNPs that are associated with paddy rice important economical trait or physiological character that excavate play a great role SNPs in Genetic and breeding in rice simultaneously.

(4) Indel mark.Indel mark is insertion and deletion length polymorphism.Insertion/deletion, refer to the difference of two kinds of parents in full genome, another parent relatively, in one of them parent's genome, there is the Nucleotide of some amount to insert, according to insertion/deletion site in genome, design the PCR primer in some these insertion/deletion sites of increasing, the difference that produces thus PCR product length scale be InDel mark.The same with SNP mark, InDel mark is also the New molecular marker of a class based in rice genome sequence basis.Along with the development of large scale sequencing technology and the completing of the kind genome sequence such as short No. 4 of the rice subspecies such as Japan is fine, 93-11 and Guangdong, development and the application of I in Rice nDel mark have been promoted widely.

Compared with SSR mark, the advantage such as InDel mark has in genome that distribution density is high, amplification banding pattern is few, good separating effect, result are stable; Compared with SNP mark, InDel mark has the advantages such as detection technique is simple, stability is high, economic and practical.The same with other molecule markers, InDel mark can be used to outside the aspects such as the Fine Mapping of paddy rice genes involved and mapping, gene clone, molecular marker assisted selection breeding, as also having following application advantage from the mark of two subspecies genome sequences exploitations of paddy rice: the evaluation of paddy rice long-grained nonglutinous rice-japonica rice and the rational deployment of kind; Indica-round-grained rice genetic variation and genetic differentiation and hybrid vigour Study on Molecular Mechanism; The identification and analysis of breeding population structure and long-grained nonglutinous rice-japonica rice introgression line.

Claims (1)

1. compared with SSR mark, the advantage such as InDel mark has that distribution density is high in genome, amplification banding pattern is few, good separating effect, result are stable; Compared with SNP mark, InDel mark has the advantages such as detection technique is simple, stability is high, economic and practical, the same with other molecule markers, InDel mark can be used to the Fine Mapping of paddy rice genes involved and mapping, gene clone, molecular marker assisted selection breeding etc.