CN102154470A - Molecular marker method for anti-rice blast gene Pi-hk1 (t) of paddy - Google Patents
Molecular marker method for anti-rice blast gene Pi-hk1 (t) of paddy Download PDFInfo
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Abstract
The invention belongs to the field of molecular genetics, and discloses a molecular marker method for anti-rice blast gene Pi-hk1 (t) of paddy. The method comprises the following steps of: (1) selecting a paddy sample, and extracting genome deoxyribonucleic acid (DNA) of the paddy sample; and (2) performing polymerase chain reaction (PCR) amplification on the genome DNA of the paddy sample by utilizing any one, two or three pairs of molecular marker primers in s01, in5 and in3, performing electrophoretic detection on a PCR amplification product, and if corresponding DNA fragments of molecular markers are amplified, indicating that the Pi-hk1 (t) gene exists. In the method, the selection efficiency of a single marker is over 99.92 percent, and the selection efficiency of any double markers is 100 percent. The resistance level of the gene on rice blast can be forecasted by detecting whether the paddy contains the gene or not by the molecular marker primers provided by the invention, so the selection efficiency of the rice blast resistance of the paddy is improved greatly, and the process for breeding for disease resistance is accelerated.
Description
Technical field
The invention belongs to the molecular genetics field, the molecule marking method that relates to rice blast resistant gene Pi-hk1 (t), be specifically related to the molecule marking method of Heikezijing blast resistant gene Pi-hk1 (t), be used for the utilization of molecular marker assisted selection breeding of rice anti-rice blast kind and germ plasm resource.
Background technology
Paddy rice is the important food crop of China.By the fungus-caused rice blast of rice blast, be that Rice Production is threatened one of maximum disease, it all has generation in the Rice Production area.According to estimates, the whole world is annual causes about billions dollars loss (Jeon et al, 2003) because of rice blast endangers.China loses the several hundred million kilograms of paddy because of rice blast harm every year.In the popular time of rice blast, the production loss that causes, generally at 10-20%, serious reaches more than 50%, local field piece even No kernels or seeds are gathered, as in a year of scarcity, and cause rice quality to descend.Cultivate disease-resistant variety and be that the control rice blast is the safest, effective, one of abridged edition, eco-friendly measure.
Genetic research shows that paddy rice is controlled by the dominance major gene loci mainly to rice blast resistance, has located about more than 50 disease-resistant sites at present in different resistant materials, is distributed in the karyomit(e) different positions, and the cluster that has distributes.Modern varieties be introduced or be aggregated to these disease-resistant genes can, selects kind high anti-, wide spectrum.But traditional breeding way is time-consuming, effort, phenotypic evaluation difficulty, breeding efficiency are low, because disease-resistant gene mostly is and often exists epistasis to do mutually between dominance, gene, the disease-resistant gene polymerization is more difficult.Can effectively address this problem by molecular mark.
Heikezijing is Taihu Lake basin japonica rice local variety, has the feature of the high blast resisting of wide spectrum, and preliminary positioning result shows, its main effect disease-resistant gene Pi-hk1 (t) is a new disease-resistant gene (Li Peifu etc., the rice in China science, 2007,21:579～584).On this basis, further screen and develop closely linked molecule marker, be molecular marker assisted selection breeding and the service of clone Pi-hk1 (t) gene.
Summary of the invention
The objective of the invention is: the molecule marking method that paddy rice Heikezijing blast resistant gene Pi-hk1 (t) is provided.By detecting and the closely linked molecule marker of disease-resistant gene Pi-hk1 (t), can define no disease-resistant gene Pi-hk1 (t), and the prediction rice plant rice blast resistance, the quickening anti-rice blast rice the selection progress.
Purpose of the present invention can be achieved through the following technical solutions:
The molecule marking method of paddy rice Heikezijing blast resistant gene Pi-hk1 (t) is characterized in that operation steps is as follows:
(1) water intaking rice sample extracts paddy rice sample gene group DNA;
(2) utilize in the table 1 any 1 pair, 2 pairs or the molecule marker primer of 3 couples of paddy rice Heikezijing blast resistant gene Pi-hk1 (t) that described paddy rice sample gene group DNA is carried out pcr amplification, pcr amplification product carries out electrophoresis detection on 8% non-denaturing polyacrylamide gel, if amplify the molecular marker DNA fragment of corresponding size, indicate the existence of Pi-hk1 (t) gene:
Table 1
Wherein, the reaction system of described pcr amplification is: 10 * buffer (contains Mg
2+) 1.0 microlitres, the InDel of 4pmol/ microlitre or SSR primer be to 1 microlitre, 2.5mM dNTPs 0.2 microlitre, and Taq enzyme 0.1 microlitre of 5 units/microlitre, paddy rice sample gene group template DNA 1 microlitre of 10 nanograms/microlitre adds water to 10 microlitres; Response procedures is: behind the DNA94 ℃ of pre-sex change 5min; 94 ℃ of sex change 30sec, 55 ℃ of annealing 30sec, 72 ℃ are extended exhibition 1min, circulate 35 times; Last 72 ℃ are extended 10min.
Beneficial effect
The molecule marking method of paddy rice Heikezijing blast resistant gene provided by the present invention has the following advantages:
(1) utilize the molecule marker primer of any 1 the paddy rice Heikezijing blast resistant gene Pi-hk1 of the present invention (t) to carry out the discriminating of blast resistant gene Pi-hk1 (t), select accuracy rate all to reach more than 99.92%, utilize any 2 or 3 molecule marker selection of primers efficient to reach 100%.
(2) accurate by the localized gene locus of molecule marker of the present invention, it is convenient to identify.Because the recombination fraction very low (≤0.08%) of these marks and disease-resistant gene Pi-hk1 (t), by detecting these and the closely linked molecule marker of disease-resistant gene Pi-hk1 (t), the existence that can determine blast resistant gene Pi-hk1 (t) whether, the rice blast resistance of prediction rice plant, thereby quick breeding for disease resistance process.
(3) the assistant breeding select target is clear and definite, saves cost.In traditional disease resistant and breeding method, the rice blast resistance of breeding material is carried out phenotypic evaluation, generally at seedling stage or heading stage, it is bigger to be inoculated environmental influence, and the result reliability of phenotypic evaluation is low.Therefore breeding for disease resistance is not only time-consuming, and difficulty is big, the cost height.By detecting blast resistant gene Pi-hk1 (t), can take a sample in seedling stage, extract DNA, utilize above-mentioned mark just can identify the individual plant of blast resisting, eliminate other plant, not only save production cost, control the breeding population scale, and improve the efficiency of selection of blast resisting individuality greatly.
Description of drawings
Fig. 1. 8% native polyacrylamide gel electrophoresis figure of rice blast resistant gene Pi-hk1 (t) close linkage InDel mark.
Wherein A is the 8% native polyacrylamide gel electrophoresis figure of in3 and in5;
B is the 8% native polyacrylamide gel electrophoresis figure of in2 and in7;
C is the 8% native polyacrylamide gel electrophoresis figure of in15;
M: molecular weight Marker, H: Heikezijing, S: Suyunuo, F: heterozygous.
Fig. 2. the 8% native polyacrylamide gel electrophoresis figure of rice blast resistant gene Pi-hk1 (t) close linkage SSR mark s01.
Wherein: M: molecular weight Marker, H: Heikezijing, S: Suyunuo, F: heterozygous.
Embodiment
Embodiment 1
(1) materials and methods:
1. Li Peifu etc. utilizes anti-rice blast rice local variety Heikezijing (♀) and susceptible variety Suyunuo (♂) hybridization to obtain F
1, selfing obtains F
2Segregating population is used for the location, and with Pi-hk1 (t) assignment of genes gene mapping (rice in China science, 2007,21:579～584) between SSR mark RM7654 and RM27381.The present invention therefrom selects to contain segmental 3 the homozygous disease-resistant individual plants of target dna and plants on this basis, backcrosses with Suyunuo respectively, selfing, forms BC
2F
2Separate in the large group, be used for Fine Mapping.
2. spawn culture and inoculation identification method are with reference to (rice in China science, 2007,21:579～584) such as Li Peifu.
3.DNA extracting method: with the DNA of each individual plant of SDS method extraction separation colony.
4. the exploitation of new mark: between first localized target area RM7654-RM27381, design new mark.
(1) SSR indicia designs: the genomic sequence (http://rgp.dna.affrc.go.jp) of downloading target gene place BAC, utilize biosoftware Bioxm (Huang Ji etc., 2004) carry out the search of SSR site, the base repetition number is greater than 2, select the bigger forward and reverse primer of zone design, PCR product size designs the SSR mark for 100-300bp.
(2) InDel indicia designs: the genome sequence of downloading target gene place BAC, then genome sequence is carried out predictive genes GENSCAN, RiceHMM, FGENESH, the length of choosing those introns between 200-500bp sequence as the purpose fragment of amplification, site with primer is located at exon region simultaneously, and design is the InDel mark based on the intron length multiformity mark ILP of EST.
(3) design of primers of mark: with Primer Premier 5.0 (http://www.premierbiosoft.com) design primer, the primer length of design is about 20bp, general requirement GC content is between 45%～60%, the Tm value is between 55～65 ℃, no mispairing, dimer and hairpin structure, 3 ' end is G or C preferably.After finding the candidate's primer sequence that meets above condition, with it with international biotechnology (the National Center for Biotechnology Information of information center, NCBI) the primer-blast software on the website carries out primer specificity comparison, selects the higher sequence of specificity in the genome right as the primer of this mark.Design 4 pairs of marking primer 2s altogether.
(4) mark polymorphism screening: the DNA with parent's Heikezijing and Suyunuo is a template, and is right with the labeled primer of exploitation, through PCR reaction carrying out polymorphism analysis.
5.PCR reaction system: volume is 10 microlitres, and wherein 10 * buffer, 1.0 microlitres (contain Mg
2+), InDel or SSR primer be to (4pmol/ microlitre) 1 microlitre, 2.5mM dNTPs 0.2 microlitre, and Taq enzyme (5 units/microlitre) 0.1 microlitre, template DNA (10 nanograms/microlitre) 1 microlitre adds water to 10 microlitres.Response procedures is: behind the DNA94 ℃ of pre-sex change 5min, and (72 ℃ are extended exhibition 1min for 94 ℃ of sex change 30sec, 55 ℃ of annealing 30sec) circulation 35 times, last 72 ℃ are extended 10min.In the enterprising performing PCR amplification of biometre amplification instrument, amplified production carries out electrophoretic separation on 8% non-denaturing polyacrylamide gel (containing 7.6 gram acrylamides and 0.4 gram methylene diacrylamide in the 100ml polyacrylamide solution), on ultraviolet transilluminator, take a picture the record result then.In 24 marks of exploitation 5 InDel mark: in2 (in2L:SEQ ID NO.7 is arranged, in2R:SEQ ID NO.8), in3, in5, in7 (in7L:SEQ ID NO.9, in7R:SEQ ID NO.10), in15 (in15L:SEQ ID NO.11, in15R:SEQ ID NO.12) and 1 SSR mark s01 between the parent, exist Percentage of polymorphic (Fig. 1, Fig. 2).
6. population analysis: polymorphic being marked in 915 susceptible BC2F2 individualities analyzed, and obtains the marking type data.1600 BC have been analyzed simultaneously
2F
2The individual mark type, at the individuality of RM7654-RM27381 generation exchange, the offspring carries out resistance and identifies, determines previous generation's genotype.According to chain polymorphism mark and corresponding disease-resistant phenotype, evaluation of markers and intergenic recombination frequency calculate the antagonistic selection accuracy rate of mark.
(2) result and analysis:
Between mark RM7654-RM27381, the polymorphic molecular marker of exploitation is analyzed above-mentioned 2515 individualities (i.e. 5030 gametes).Result's (table 2) is as follows:
Occur 1 crossover-type gamete between SSR mark s01 and disease-resistant gene Pi-hk1 (t), recombination fraction only is 0.02%, and the antagonistic efficiency of selection of this mark reaches 99.98%;
Occur 3 crossover-type gametes between in5 and disease-resistant gene Pi-hk1 (t), recombination fraction only is 0.06%, and the antagonistic efficiency of selection of this mark reaches 99.94%;
Occur 4 crossover-type gametes between in3 and disease-resistant gene Pi-hk1 (t), recombination fraction only is 0.08%, and the antagonistic efficiency of selection of this mark reaches 99.92%.
Other marks drop on outside the RM7654-RM27381, and efficiency of selection is not as good as above-mentioned 3 marks.
Can identify efficiently that by above-mentioned 3 molecule markers whether blast resistant gene Pi-hk1 (t) exists, single mark efficiency of selection all reaches more than 99.92%, and the efficiency of selection of double-tagging combination reaches 100% arbitrarily.Can effectively improve the breeding process of paddy disease-resistant kind, control breeding population scale.
Table 2.
| Mark | Upstream primer sequence L | Downstream primer sequence R | Heikezijing fragment (bp) | Suyunuo fragment (bp) | Select accuracy rate (%) |
| in5 | SEQ?ID?NO.1 | SEQ?ID?NO.2 | 871+713 | 780 | 99.92 |
| in3 | SEQ?ID?NO.3 | SEQ?ID?NO.4 | 360 | 432 | 99.94 |
| S01 | SEQ?ID?NO.5 | SEQ?ID?NO.6 | 96 | 109 | 99.98 |
| in2 | SEQ?ID?NO.7 | SEQ?ID?NO.8 | 380+285 | 396 | 96.00 |
| in7 | SEQ?ID?NO.9 | SEQ?ID?NO.10 | 390 | 465 | 99.20 |
| in15 | SEQ?ID?NO.11 | SEQ?ID?NO.12 | 480 | 515 | 97.20 |
Annotate: primer design template and expection expanding fragment length are with reference to the fine genome sequence of Japan.
Claims (2)
1. the molecule marking method of paddy rice Heikezijing blast resistant gene Pi-hk1 (t) is characterized in that comprising the steps:
(1) water intaking rice sample extracts paddy rice sample gene group DNA;
(2) utilize in the table 1 any 1 pair, 2 pairs or the molecule marker primer of 3 couples of paddy rice Heikezijing blast resistant gene Pi-hk1 (t) that described paddy rice sample gene group DNA is carried out pcr amplification, pcr amplification product carries out electrophoresis detection on 8% non-denaturing polyacrylamide gel, if amplify the molecule marker fragment of corresponding size, indicate the existence of Pi-hk1 (t) gene
Table 1
2. the molecule marking method of paddy rice Heikezijing blast resistant gene Pi-hk1 according to claim 1 (t), the reaction system that it is characterized in that described pcr amplification is: 10 * buffer, 1.0 microlitres, the InDel of 4pmol/ microlitre or SSR primer are to 1 microlitre, 2.5mM dNTPs 0.2 microlitre, Taq enzyme 0.1 microlitre of 5 units/microlitre, paddy rice sample gene group template DNA 1 microlitre of 10 nanograms/microlitre adds water to 10 microlitres; Response procedures is: behind 94 ℃ of pre-sex change 5min of DNA; 94 ℃ of sex change 30sec, 55 ℃ of annealing 30sec, 72 ℃ are extended exhibition 1min, circulate 35 times; Last 72 ℃ are extended 10min.
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Cited By (11)
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| CN102703443A (en) * | 2012-05-23 | 2012-10-03 | 华南农业大学 | Functional specific molecular marker of rice blast resistance gene Pia and method and application thereof |
| CN103205506A (en) * | 2013-05-08 | 2013-07-17 | 辽宁省农业科学院 | Method for detecting rice blast resistant gene pi5 by using coseparation mark pi5-2-4 |
| CN103215370A (en) * | 2013-05-08 | 2013-07-24 | 辽宁省农业科学院 | Method for detecting rice blast-resistant gene pi5 |
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| CN102703443B (en) * | 2012-05-23 | 2014-09-24 | 华南农业大学 | Functional specific molecular marker of rice blast resistance gene Pia and method and application thereof |
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| CN105713983B (en) * | 2016-04-21 | 2019-03-05 | 南京农业大学 | A kind of molecular labeling and its application with rice Jiangnan evening neck blast resistance gene close linkage |
| CN106636335A (en) * | 2016-10-09 | 2017-05-10 | 南京农业大学 | Molecular marker method of rice panicle blast resistance gene |
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| CN106755471B (en) * | 2017-01-08 | 2017-12-08 | 王依然 | Rice blast resistant gene Pilk (t) linked marker and its primer special |
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