CN101294161A - Numerator mark concatenated with whitebacked planthopper resistance genes of rice, and developing method thereof - Google Patents
Numerator mark concatenated with whitebacked planthopper resistance genes of rice, and developing method thereof Download PDFInfo
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- CN101294161A CN101294161A CNA2008100613306A CN200810061330A CN101294161A CN 101294161 A CN101294161 A CN 101294161A CN A2008100613306 A CNA2008100613306 A CN A2008100613306A CN 200810061330 A CN200810061330 A CN 200810061330A CN 101294161 A CN101294161 A CN 101294161A
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Abstract
The invention discloses a molecular mark linked with rice gene resistant to Whitebacked Planthopper. Rice is selected, and a molecular mark primer is selected from following primer pairs, wherein the nucleotide sequence is 5' arrow 3'; RM6917 forward: CTGGTTGTTTTCTGAACTCCGT, reverse: GCATTAACCATAGCTGTCCACTC; and AP4741 forward: AATCATGCTAGGTTATTACTCG, reverse: GTGCAGAGATCATGCTTTTG. The invention also provides the development method of the molecular mark. The molecular mark is tightly linked with the gene resistant to Whitebacked Planthopper, and can be used for auxiliary selection and breeding of rice resistant to Whitebacked Planthopper.
Description
Technical field
The invention belongs to agricultural biotechnology engineering, particularly linked molecule marker and preparation method thereof with paddy rice white backed planthopper resistant gene.
Background technology
Paddy rice white backed planthopper (Sogatella furcifera, Horv á th) is one of the primary pest in China and paddy rice producing region, South East Asia, since the eighties, along with the popularization of China's high-yield hybrid rice technology and plant physiology factor that may be relevant with hybrid vigour, white backed planthopper has become the paddy rice primary pest.According to the record of Chinese agriculture yearbook, retransmit the time planthopper, injured area surpasses 50% of China's paddy rice total area.1991, the planthopper on a large scale that China takes place was caused harm and causes the loss of nearly 1,600,000,000 kilograms of paddy, injured area to reach 2,933 ten thousand hm
2Planthopper great outburst again in 2005, in addition with the synergistic effect of Cnaphalocrocis medinali(rice leaf roller), planthopper is caused harm and reaches more than 2.6 hundred million mu times.Therefore, the control of white backed planthopper has been become the important content of Guarantee Grain Production.
The main rice varieties of establishing in large scale is not with resistant gene, is the immanent cause of white backed planthopper outburst, adds using in a large number of chemical fertilizer, and the sense worm paddy rice of flourishing provides competent food source for the quick breeding of white backed planthopper.For a long time, the control of white backed planthopper mainly is to rely on to use chemical insecticide.Yet the abuse of agricultural chemical insecticide has killed the rice planthopper natural enemy in the rice field, brings out the rampant again of white backed planthopper on the contrary.In addition, white backed planthopper belongs to migratory pest on a large scale, and in the paddy growth season in every year, white backed planthopper is pursued for China rice district of moving into district by district toward north by south with the spring and summer warm moist air.The white backed planthopper early period of origination, the polypide of nymph is little, and is disguised strong, is difficult for being found by the peasant, prevents and treats untimelyly to make it be able to a large amount of breedings, and insect pest just is able to popular; The ripe filling stage of the paddy rice of insect pest outburst, rice strain growing way is vigorous, and the operation of sterilant being executed rice strain base portion is very difficult, adds that adult has good movability and stronger resistance, and the result works the mischief and the underproduction repeatedly.
Paddy disease-resistant worm breeding practice proves that utilizing anti-white backed planthopper gene to cultivate new variety is the most cost-effective methods in the integrated control of paddy rice white backed planthopper.Even the rice varieties of plantation only has the resistant gene of medium level, also be enough to the colony of white backed planthopper is controlled at work the mischief below horizontal, can not cause white backed planthopper great outburst or actual harm and production loss.
On the other hand, along with development of molecular biology, make be difficult to identify, disease and insect resistance shape easily affected by environment adopts marker assisted selection to become possibility, this technology not only can be carried out accurate, stable selection in generation morning, and can overcome the apparent recessiveness and the pure heterozygosis problem of separating individual plant, thereby the acceleration breeding process improves breeding efficiency, but the basis of this technology is to have good target gene and closely linked with it molecule marker.
For realizing above-mentioned task, excavate new resistant gene, develop the key that closely linked new mark becomes the anti-white backed planthopper molecular breeding of paddy rice.That the earliest paddy rice white backed planthopper resistance is carried out the molecule Position Research is McCouch, at first using TN1 (No. 1, this locality in the platform)/IR36 near isogenic line navigates to Wbphl between two RFLP mark RG146 and the RG445, but, and be difficult to as selective marker because this RFLP mark is the multiple copied mark; After this, Wbph2 and Wbph6 (t) also successively are positioned, but their genetic distance is respectively 25.6cM and 21.2cM, and practical application still has very big difficulty.
Summary of the invention
The technical problem to be solved in the present invention provides a kind of and linked molecule marker and the development approach thereof of the anti-white backed planthopper gene of paddy rice, the molecule marker of gained of the present invention and white backed planthopper resistant gene close linkage can be used for paddy rice white backed planthopper resistance assisted selection.
In order to solve the problems of the technologies described above, the invention provides a kind of and the linked molecule marker of the anti-white backed planthopper gene of paddy rice, as species, it is right that this molecule marker primer is selected from following primer with paddy rice, and nucleotides sequence wherein classifies 5 ' → 3 as ',
RM6917 forward: CTGGTTGTTTTCTGAACTCCGT
Oppositely: GCATTAACCATAGCTGTCCACTC;
AP4741 forward: AATCATGCTAGGTTATTACTCG
Oppositely: GTGCAGAGATCATGCTTTTG.
Improvement as molecule marker of the present invention: RM6917 is positioned on paddy rice the 6th karyomit(e); AP4741 also is positioned on paddy rice the 6th karyomit(e).
The present invention also provides the development approach of above-mentioned molecule marker, may further comprise the steps:
1), with japonica rice variety spring river 06 as the anti insect gene donor parents with as hybridizing for local No. 1 in the platform of sense worm kind, thereby obtain pest-resistant individual plant as filial generation;
2), extract parental rice seedling and filial generation seedling genomic dna with CTAB (cetyltriethylammonium bromide, Hexadecyl trimethyl ammonium Bromide) method;
3), adopt SSR (simple repeated sequence, simple sequence repeat) and/or STS (sequence tagged site sequence-tagged site) molecule marking method to carry out the screening of paddy rice white backed planthopper resistance molecule marker;
4), filter out a SSR molecule marker RM6917 and a STS molecule marker AP4741.
With linked molecule marker RM6917 and the AP4741 of the anti-white backed planthopper gene of paddy rice, specifically obtain with following method:
1), the anti insect gene donor parents is from the japonica rice variety spring river 06 (CJ06) of China Paddy Rice Inst, hybridize with local No. 1 (TN1) in the common rice variety platform of sense worm kind, anti-, sense worm individual plant is by hybridization, backcrosses and selfing, and anti-, the sense worm near isogenic line of acquisition are used for the combo and the assignment of genes gene mapping.
2), extract parental rice seedling and filial generation seedling genomic dna with the CTAB method.
3), adopt SSR and STS molecule marking method to carry out the screening of paddy rice white backed planthopper resistance molecule marker;
4), filter out a SSR molecule marker RM6917 and a STS molecule marker AP4741, through linkage analysis, find that these two marks and the anti-white backed planthopper gene of paddy rice are linked.
Adopt SSR and STS molecule marker screen with the linked molecular marker method of the anti-white backed planthopper gene of paddy rice specifically:
(1), SSR, STS primer are resisting, are feeling dna polymorphism analysis between parent and their filial generation:
Screen with the SSR labeling technique, employing is distributed in SSR primer on 12 karyomit(e)s of paddy rice according to what Temnykh delivered in 2000, primer entrusts the Shen, Shanghai betting office to synthesize, in the enterprising performing PCR amplification of PTC-225 PCR instrument, the PCR reaction system is: 20ng/ul oryza sativa genomic dna 1ul, 10 * PCR Buffer 2.0ul, 25mM MgCl
22.0ul, 2mM dNTP 2.0ul, 10uM primer 2 .0ul, 5U/ul Taq archaeal dna polymerase 0.2ul, ddH
2O 11.8ul, total system 20ul.Response procedures: 95 ℃ of sex change 5 minutes; 94 sex change 1 minute, 55 annealing 1 minute, 72 extended 40 circulations 1 minute; 72 polishings 10 minutes; Product detects: you are electrophoresis at 4.0% the agarose that contains 0.5%ug/ul EB, and ultraviolet lamp is observed also photographic recording result down.
Screen with the STS labeling technique, at first genome has been finished Japanese fine (the http://rgp.dna.affrc.go.jp) of order-checking and the sequence of 93-11 (http://www.rise.genomics.org.cn) is carried out sequence alignment, be used for the polymorphism screening of CJ06 and TN1 according to the suitable primer of consequence devised of the warm and fine 93-11 sequence alignment of Japan, primer entrusts the Shen, Shanghai betting office to synthesize, in the enterprising performing PCR amplification of PTC-225PCR instrument, the PCR reaction system is: 20ng/ul oryza sativa genomic dna 1ul, 10 * PCR Buffer 2.0ul, 25mM MgCl
22.0ul, 2mM dNTP 2.0ul, 10uM primer 2 .0ul, 5U/ul Taq archaeal dna polymerase 0.2ul, ddH
2O 11.8ul, total system 20ul.Response procedures: 95 ℃ of sex change 5 minutes; 94 ℃ of sex change 1 minute, 52 ℃ of annealing 1 minute, 72 ℃ were extended 40 circulations 1 minute; 72 ℃ 10 minutes; Product detects: containing 4.0% the agarose gel electrophoresis of 0.5%ug/ulEB, ultraviolet lamp is observed down and the photographic recording result.
(2), the linkage analysis of molecule marker
CJ06 and TN1 parents and offspring's thereof white backed planthopper resistance evaluation is asked in the field and is carried out, under the spontaneous induction condition of field, from the borer population of moving into, aphid evil aspects such as density and killed degree take place respectively in early days in tillering parents and offspring's segregating population are carried out the insect-resistance evaluation; Extract total DNA pest-resistant, sense worm individual plant respectively, with reaction system, the polymorphism primer of screening acquisition and the pcr amplification that program carry out individual plant same with the front, statistics resists, feels the frequency of occurrences and the switch type of individual plant indicia band and calculates crossover value, with the genetic distance between MapnakerEXP3.0b calculating mark and the anti insect gene.
The present invention is to be material with molecular biology method with the anti-white backed planthopper rice varieties of height CJ06, screening and seek new and stable existence and closely linked molecule marker of white backed planthopper resistant gene and method thereof is used for paddy rice white backed planthopper resistance assisted selection; Because the material that institute uses is to the high anti-characteristic of white backed planthopper performance, its white backed planthopper to China rice district has general resistance.In addition, the clone who also helps the new gene of the anti-white backed planthopper of paddy rice according to the gained molecule marker.
White backed planthopper is the main insect pest that hazard rice is produced.The present invention utilizes molecule marking method to obtain two new and the closely linked molecule markers of white backed planthopper resistant gene.Utilize this method, shortcomings such as conventional breeding method required time cycle length have not only been overcome, can targetedly anti insect gene be selected to obtain and on purpose carry out the polymerization of a plurality of resistant genes in the laboratory, thereby cultivate new rice variety with stable resistance.Simultaneously, also can utilize this two white backed planthopper resistant gene molecule markers, deeply carry out the clone of anti-white backed planthopper gene and it is carried out the 26S Proteasome Structure and Function analysis, this molecular genetics mechanism for the anti-white backed planthopper of further understanding paddy rice has positive meaning.Therefore, result of the present invention is significant in rice breeding practice and pest-resistant theoretical investigation.Its advantage specifically is summarized as follows:
(1) the of the present invention and closely linked molecule marker of white backed planthopper resistant gene, it is the new mark that in the stable filial generation individual plant of the CJ06 of the high anti-white backed planthopper to paddy rice and resistance thereof, obtains, the paddy rice white backed planthopper had very strong resistance, and stable existence, can be used for the evaluation of paddy rice white backed planthopper resistant variety and the pest-resistant offspring's of paddy rice assisted selection.
(2) the used high anti-paddy rice white backed planthopper of material of the present invention also has stronger brown planthopper resistant ability, has general pest-resistant characteristic.Therefore, the molecule marker according to gained is expected to be cloned into new white backed planthopper resistant gene.
(3) for cloning paddy rice white backed planthopper resistant gene, gene sequencing and changeing anti-white backed planthopper trans-genetic hybrid rice research and established good basis.
Description of drawings
Below in conjunction with accompanying drawing the specific embodiment of the present invention is described in further detail.
Fig. 1 is the SSR molecule marker RM6917 screening electrophoretic band figure of two rice varieties and offspring's genomic dna thereof;
Fig. 2 is the STS molecule marker AP4741 screening electrophoretic band figure of two rice varieties and offspring's genomic dna thereof;
Fig. 3 is the electrophoretic band figure of detection molecules mark RM6917 and pest-resistant sex-linked molecule marker;
Fig. 4 is the electrophoretic band figure of detection molecules mark AP4741 and pest-resistant sex-linked molecule marker;
Fig. 5 is the genetic distance synoptic diagram between SSR molecule marker RM6917 and STS molecule marker AP4741 and anti insect gene WBPH;
Symbol in above-mentioned Fig. 1～5 is explained as follows respectively:
1 representative: pest-resistant parent spring river 06;
2 representatives: sense worm parent TN1;
3 representatives: the F1 plant of spring river 06/TN1;
4,5 all representatives: the pest-resistant individual plant of spring river 06 and TN1 filial generation, this pest-resistant individual plant are by the selfing of hybridizing, backcrossing, and obtain from the stable advanced lines strain of anti-white backed planthopper is; Promptly 4 and 5 is the anti insect gene in spring river 06 to be imported TN1 by the marker assisted selection technology and the pest-resistant material that obtains;
R representative: with the pest-resistant individual plant of the filial generation of anti-white backed planthopper gene;
S representative: not with the sense worm individual plant of the filial generation of anti-white backed planthopper gene.
Embodiment
Specific practice is: the anti insect gene donor parents is from the japonica rice variety spring river 06 (CJ06) of China Paddy Rice Inst, hybridize with local No. 1 (TN1) in the common rice variety platform of sense worm kind, pest-resistant individual plant is by hybridization, backcrosses and selfing, anti-, the sense worm near isogenic line that obtain are used for the combo and the assignment of genes gene mapping, identify that from the offspring of this colony obtaining the recessive individuality of 76 strains is used for the linkage relationship analysis.
Following institute in steps in, all employings deionized water of not informing in detail is as solution.
One, extracts DNA
1), preparation DNA extraction damping fluid:
DNA extraction solution (the 0.35M sorbitol that adds 1 volume in order successively; 0.1M Tris, pH8.2; 0.005MEDTA; ), karyorhexis liquid (0.2M Tris, the pH7.5 of 1 volume; 0.05M EDTA; 2M NaCl; 0.055M CTAB; ) and the 5%sarkosyl of 0.4 volume; Add sodium bisulfite at last, making it final concentration is 0.02M.
2), above-mentioned CJ06, TN1 and the pest-resistant recessive individual rice leaf of 76 strains are carried out following processing respectively:
1., take by weighing the rice leaf liquid nitrogen grinding powdering of 0.1g, add the DNA extraction damping fluid of the above-mentioned preparation of 700 μ l then, 65 ℃ of water-baths 40 minutes.The chloroform that adds 700 μ l again: primary isoamyl alcohol (24: 1 volume ratios), and mixing.10, centrifugal 5 minutes of 000rpm transfers to supernatant liquor in the new centrifuge tube.
2., in the supernatant liquor of the 1. centrifugal back of above-mentioned steps gained, add the Virahol of 2/3～1 times of volume precooling, mixing precipitates to DNA gently.13, centrifugal 8 minutes of 000rpm pours out supernatant liquor.
3., again with the 2. DNA throw out of gained of 70% hexanol washing above-mentioned steps.
4., with the DNA airing after the above-mentioned washing and be dissolved in 100 μ l TE damping fluids or the pure water.
5., ultraviolet spectrophotometry detects the 4. concentration of the DNA sample of gained of above-mentioned steps, the integrity of 0.7% agarose gel electrophoresis detection DNA.
Two, ssr analysis:
Be distributed in SSR primer sequence on 12 karyomit(e)s of paddy rice according to what Temnykh delivered in 2000, entrust the Shen, Shanghai can betting office's synthetic primer 90 pairs, specifically as shown in table 1:
Table 1,90 pairs of SSR primer sequences of synthetic
| The primer title | Left wing's primer sequence | The right flank primer sequence |
| RM428 | AACAGATGGCATCGTCTTCC | CGCTGCATCCACTACTGTTG |
| RM151 | GGCTGCTCATCAGCTGCATGCG | TCGGCAGTGGTAGAGTTTGATCTGC |
| RM259 | TGGAGTTTGAGAGGAGGG | CTTGTTGCATGGTGCCATGT |
| RM493 | TAGCTCCAACAGGATCGACC | GTACGTAAACGCGGAAGGTG |
| RM9 | GGTGCCATTGTCGTCCTC | ACGGCCCTCATCACCTTC |
| RM488 | CAGCTAGGGTTTTGAGGCTG | TAGCAACAACCAGCGTATGC |
| RM128 | AGCTTGGGTGATTTCTTGGAAGCG | ACGACGAGGAGTCGCCGTGCAG |
| RM486 | CCCCCCTCTCTCTCTCTCTC | TAGCCACATCAACAGCTTGC |
| RM472 | CCATGGCCTGAGAGAGAGAG | AGCTAAATGGCCATACGGTG |
| RM14 | CCGAGGAGAGGAGTTCGAC | GTGCCAATTTCCTCGAAAAA |
| RM233 | CCAAATGAACCTACATGTTG | GCATTGCAGACAGCTATTGA |
| RM53 | ACGTCTCGACGCATCAATGG | CACAAGAACTTCCTCGGTAC |
| RM145 | CCGGTAGGCGCCCTGCAGTTTC | CAAGGACCCCATCCTCGGCGTC |
| RM324 | CTGATTCCACACACTTGTGC | GATTCCACGTCAGGATCTTC |
| RM341 | CAAGAAACCTCAATCCGAGC | CTCCTCCCGATCCCAATC |
| RM475 | CCTCACGATTTTCCTCCAAC | ACGGTGGGATTAGACTGTGC |
| RM263 | CCCAGGCTAGCTCATGAACC | GCTACGTTTGAGCTACCACG |
| RM240 | CCTTAATGGGTAGTGTGCAC | TGTAACCATTCCTTCCATCC |
| RM250 | GGTTCAAACCAAGCTGATCA | GATGAAGGCCTTCCACGCAG |
| RM535 | ACTACATACACGGCCCTTGC | CTACGTGGACACCGTCACAC |
| RM22 | GGTTTGGGAGCCCATAATCT | CTGGGCTTCTTTCACTCGTC |
| RM545 | CAATGGCAGAGACCCAAAAG | CTGGCATGTAACGACAGTGG |
| OSR13 | CATTTGTGCGTCACGGAGTA | AGCCACAGCGCCCATCTCTC |
| RM251 | GAATGGCAATGGCGCTAG | ATGCGGTTCAAGATTCGATC |
| RM473D | TATCCTCGTCTCCATCGCTC | AAGGATGTGGCGGTAGAATG |
| RM16 | CGCTAGGGCAGCATCTAAA | AACACAGCAGGTACGCGC |
| RM426 | ATGAGATGAGTTCAAGGCCC | AACTCTGTACCTCCATCGCC |
| RM55 | CCGTCGCCGTAGTAGAGAAG | TCCCGGTTATTTTAAGGCG |
| RM520 | AGGAGCAAGAAAAGTTCCCC | GCCAATGTGTGACGCAATAG |
| RM422 | TTCAACCTGCATCCGCTC | CCATCCAAATCAGCAACAGC |
| RM570 | GTTCTTCAACTCCCAGTGCG | TGACGATGTGGAAGAGCAAG |
| RM335 | GTACACACCCACATCGAGAAG | GCTCTATGCGAGTATCCATGG |
| RM456B | TTGTAGTCCGGGTCGTAACC | GATAGAATAGGGAGGGGGGG |
| RM471 | ACGCACAAGCAGATGATGAG | GGGAGAAGACGAATGTTTGC |
| RM564 | CATGGCCTTGTGTATGCATC | ATGCAGAGGATTGGCTTGAG |
| RM252 | TTCGCTGACGTGATAGGTTG | ATGACTTGATCCCGAGAACG |
| RM241 | GAGCCAAATAAGATCGCTGA | TGCAAGCAGCAGATTTAGTG |
| RM255 | TGTTGCGTGTGGAGATGTG | CGAAACCGCTCAGTTCAAC |
| RM280 | ACACGATCCACTTTGCGC | TGTGTCTTGAGCAGCCAGG |
| RM413 | GGCGATTCTTGGATGAAGAG | TCCCCACCAATCTTGTCTTC |
| RM437 | ACACCAACCAGATCAGGGAG | TGCTCGTCAATGGTGAGTTC |
| RM169 | TGGCTGGCTCCGTGGGTAGCTG | TCCCGTTGCCGTTCATCCCTCC |
| RM430 | AAACAACGACGTCCCTGATC | GTGCCTCCGTGGTTATGAAC |
| RM440 | CATGCAACAACGTCACCTTC | ATGGTTGGTAGGCACCAAAG |
| RM31 | GATCACGATCCACTGGAGCT | AAGTCCATTACTCTCCTCCC |
| RM334 | GTTCAGTGTTCAGTGCCACC | GACTTTGATCTTTGGTGGACG |
| RM587 | ACGCGAACAAATTAACAGCC | CTTTGCTACCAGTAGATCCAGC |
| RM6917 | CTGGTTGTTTTCTGAACTCCGT | GCATTAACCATAGCTGTCCACTC |
| RM253 | TCCTTCAAGAGTGCAAAACC | GCATTGTCATGTCGAAGCC |
| RM539 | GAGCGTCCTTGTTAAAACCG | AGTAGGGTATCACGCATCCG |
| RM3 | ACACTGTAGCGGCCACTG | CCTCCACTGCTCCACATCTT |
| RM454 | CTCAAGCTTAGCTGCTGCTG | GTGATCAGTGCACCATAGCG |
| RM162 | GCCAGCAAAACCAGGGATCCGG | CAAGGTCTTGTGCGGCTTGCGG |
| RM528 | GGCATCCAATTTTACCCCTC | AAATGGAGCATGGAGGTCAC |
| RM494 | GGGAGGGGATCGAGATAGAC | TTTAACCTTCCTTCCGCTCC |
| RM481 | TAGCTAGCCGATTGAATGGC | CTCCACCTCCTATGTTGTTG |
| RM125 | ATCAGCAGCCATGGCAGCGACC | AGGGGATCATGTGCCGAAGGCC |
| RM418 | TCGCGTATCGTCATGCATAG | GAGCACATATGCCACGTACG |
| RM70 | GTGGACTTCATTTCAACTCG | GATGTATAAGATAGTCCC |
| RM18 | TTCCCTCTCATGAGCTCCAT | GAGTGCCTGGCGCTGTAC |
| RM248 | TCCTTGTGAAATCTGGTCCC | GTAGCCTAGCATGGTGCATG |
| RM38 | ACGAGCTCTCGATCAGCCTA | TCGGTCTCCATGTCCCAC |
| RM25 | GGAAAGAATGATCTTTTCATGG | CTACCATCAAAACCAATGTTC |
| RM331 | GAACCAGAGGACAAAAATGC | CATCATACATTTGCAGCCAG |
| RM210 | TCACATTCGGTGGCATTG | CGAGGATGGTTGTTCACTTG |
| RM80 | TTGAAGGCGCTGAAGGAG | CATCAACCTCGTCTTCACCG |
| RM264 | GTTGCGTCCTACTGCTACTTC | GATCCGTGTCGATGATTAGC |
| RM219 | CGTCGGATGATGTAAAGCCT | CATATCGGCATTCGCCTG |
| RM105 | GTCGTCGACCCATCGGAGCCAC | TGGTCGAGGTGGGGATCGGGTC |
| RM434 | GCCTCATCCCTCTAACCCTC | CAAGAAAGATCAGTGCGTGG |
| RM242 | GGCCAACGTGTGTATGTCTC | TATATGCCAAGACGGATGGG |
| RM215 | CAAAATGGAGCAGCAAGAGC | TGAGCACCTCCTTCTCTGTAG |
| RM205 | CTGGTTCTGTATGGGAGCAG | CTGGCCCTTCACGTTTCAGTG |
| RM311 | TGGTAGTATAGGTACTAAACAT | TCCTATACACATACAAACATAC |
| RM467 | GGTCTCTCTCTCTCTCTCTCTCTC | CTCCTGACAATTCAACTGCG |
| RM184 | ATCCCATTCGCCAAAACCGGCC | TGACACTTGGAGAGCGGTGTGG |
| RM304 | TCAAACCGGCACATATAAGAC | GATAGGGAGCTGAAGGAGATG |
| RM484 | TCTCCCTCCTCACCATTGTC | TGCTGCCCTCTCTCTCTCTC |
| RM333 | GTACGACTACGAGTGTCACCAA | GTCTTCGCGATCACTCGC |
| RM332 | GCGAAGGCGAAGGTGAAG | CATGAGTGATCTCACTCACCC |
| RM202 | CAGATTGGAGATGAAGTCCTCC | CCAGCAAGCATGTCAATGTA |
| RM209 | ATATGAGTTGCTGTCGTGCG | CAACTTGCATCCTCCCCTCC |
| RM473E | TATCCTCGTCTCCATCGCTC | AAGGATGTGGCGGTAGAATG |
| RM254 | AGCCCCGAATAAATCCACCT | CTGGAGGAGCATTTGGTAGC |
| RM224 | ATCGATCGATCTTCACGAGG | TGCTATAAAAGGCATTCGGG |
| RM19 | CAAAAACAGAGCAGATGAC | CTCAAGATGGACGCCAAGA |
| RM101 | GTGAATGGTCAAGTGACTTAGGTGGC | ACACAACATGTTCCCTCCCATGC |
| RM519 | AGAGAGCCCCTAAATTTCCG | AGGTACGCTCACCTGTGGAC |
| RM270 | GGCCGTTGGTTCTAAAATC | TGCGCAGTATCATCGGCGAG |
| RM12 | TGCCCTGTTATTTTCTTCTCTC | GGTGATCCTTTCCCATTTCA |
1, pcr amplification
(1), reaction system is:
Oryza sativa genomic dna 20ng/ul 1ul, 10 * PCR Buffer 2.0ul, 25mM MgCl
22.0ul, 2mM dNTP2.0ul, 10uM primer 2 .0ul, 5U/ul Taq archaeal dna polymerase 0.2ul, ddH
2O 10.8ul, total system 20ul.
(2), response procedures:
95 ℃ of sex change 5 minutes; 94 sex change 1 minute, 55 annealing 1 minute, 72 extended 40 circulations 1 minute; 72 polishings 10 minutes.
2, electrophoresis detection
Get above-mentioned amplified production 20ul, the sepharose with 4.0% (containing 0.5%ug/ul EB) electrophoresis, ultraviolet lamp are observed down and the photographic recording result.
We at first utilize 90 pairs of SSR primers that CJ06 and TN1 are carried out polymorphism analysis, wherein have 52 pairs of primers to show polymorphism between parents.Further the filial generation individual plant of CJ06, TN1 and its 76 strain is carried out ssr analysis, find that the same banding pattern of pest-resistant parent CJ06 all appears in SSR primer RM6917 in all pest-resistant filial generation individual plants with these 52 pairs of primers.Illustrating that the anti-white backed planthopper characteristic of SSR primer RM6917 and paddy rice exists gets in touch.
The RM6917 primer sequence is: left wing 5 '-CTG GTT GTT TTC TGA ACT CCG T ' (SEQ ID NO:1), right flank 5 '-GCA TTA ACC ATA GCT GTC CAC TC-3 ' (SEQ ID NO:2) is positioned on paddy rice the 6th karyomit(e).
Three, the linksystem of SSR molecule marker RM6917 is identified
CJ06 and TN1 parents and offspring's thereof white backed planthopper resistance is identified and is carried out in the field, under the spontaneous induction condition of field, carry out the insect-resistance evaluation from aspects such as the borer population of moving into, honeydew secretory volume, insect pest generation density and killed degree to parents and offspring's segregating population respectively in early days in tillering; Extract total DNA of recessive pest-resistant individual plant, with reaction system and the program same with the front, carry out the pcr amplification of individual plant with primer RM6917, statistics resists, feels the frequency of occurrences and the switch type of individual plant indicia band and calculates crossover value, with the genetic distance between MapnakerEXP3.0b calculating mark and the anti insect gene.With RM6917 76 individual plants of filial generation colony are carried out pcr amplification, amplification condition is the same.The result shows to have the banding pattern of 72 individual plants the same with pest-resistant parent CJ06 in 76 pest-resistant individual plants, has the banding pattern of 4 strains to have parents' banding pattern simultaneously, discovery and feels the same individual plant of worm parent's TN1 banding pattern.This experimental result shows: the white backed planthopper resistance of SSR molecule marker RM6917 and paddy rice is closely linked, but still has certain exchange in pest-resistant individual plant, and as shown in Figure 3, the individual plant that parents' banding pattern occurs shows exchange has taken place.As calculated, its recombination fraction is 2.6%, and the genetic distance between SSR molecule marker and anti insect gene is 2.6cM.
Embodiment 2: use the linked molecule marker AP4741 vegetable material of acquisition of STS molecule marker and the anti-white backed planthopper gene of paddy rice with embodiment 1, specific practice is as follows:
One, extracts DNA
1), at first prepare the DNA extraction damping fluid:
With embodiment 1.
2), every kind of rice leaf is carried out following processing respectively:
With embodiment 1.
Two, STS analyzes:
The STS design of primers is used for the polymorphism screening of CJ06 and TN1 according to the suitable primer of consequence devised of the warm and fine 93-11 sequence alignment of Japan, entrusts 3 pairs of the STS primers that the Shen, Shanghai can the synthetic design of betting office, and is specifically as shown in table 2:
Table 2,3 pairs of STS primer sequences of synthetic
| The primer title | Left wing's primer sequence | The right flank primer sequence |
| AP4687 | AGCTTCCCCACGACTCCC | GAGCCACACCGCCTCCAC |
| AP4741 | AATCATGCTAGGTTATTACTCG | GTGCAGAGATCATGCTTTTG |
| AP4754 | TGACACTGACACGTTTAGCTG | CTTCAATGGTCGTATCTTCTCC |
1, pcr amplification
(1), reaction system is:
Oryza sativa genomic dna 20ng/ul 1ul, 10 * PCR Buffer 2.0ul, 25mM MgCl
22.0ul, 2mM dNTP2.0ul, 10uM primer 2 .0ul, 5U/ul Taq archaeal dna polymerase 0.2ul, ddH
2O 10.8ul, total system 20ul.
(2), response procedures:
95 ℃ of sex change 5 minutes; 94 sex change 1 minute, 55 annealing 1 minute, 72 extended 40 circulations 1 minute; 72 polishings 10 minutes.
2, electrophoresis detection
Get above-mentioned amplified production 20ul, the sepharose with 4.0% (containing 0.5%ug/ul EB) electrophoresis, ultraviolet lamp are observed down and the photographic recording result.
We utilize 3 pairs of STS primers that CJ06 and TN1 are carried out polymorphism analysis, wherein have 1 pair of primer to show polymorphism between parents.Further primer is carried out the STS analysis to CJ06, TN1 and its filial generation individual plant, find that STS primer AP4741 all has the same banding pattern of pest-resistant parent CJ06 in all pest-resistant filial generation individual plants with this.Illustrating that the anti-white backed planthopper characteristic of STS primer AP4741 and paddy rice exists gets in touch.
The AP4741 primer sequence is: left wing 5 '-AAT CAT GCT AGG TTA TTA CTC G-3 ' (SEQ ID NO:3), right flank 5 '-GTG CAG AGA TCA TGC TTT TG-3 ' (SEQ ID NO:4) is positioned on paddy rice the 6th karyomit(e).
Three, the linksystem of STS molecule marker AP4741 is identified
CJ06 and TN1 parents and offspring's thereof white backed planthopper resistance is identified and is carried out in the field, under the spontaneous induction condition of field, carry out the insect-resistance evaluation from aspects such as the borer population of moving into, honeydew secretory volume, insect pest generation density and killed degree to parents and offspring's segregating population respectively in early days in tillering; Extract total DNA of recessive pest-resistant individual plant, with reaction system and the program same with the front, carry out the pcr amplification of individual plant with primer AP4741, statistics resists, feels the frequency of occurrences and the switch type of individual plant indicia band and calculates crossover value, with the genetic distance between MapnakerEXP3.0b calculating mark and the anti insect gene.With AP4741 76 individual plants of filial generation colony are carried out pcr amplification, amplification condition is the same.The result shows to have the banding pattern of 71 individual plants the same with pest-resistant parent CJ06 in 76 pest-resistant individual plants, has the banding pattern of 5 individual plants to have parents' banding pattern simultaneously, discovery and feels the same individual plant of worm parent's TN1 banding pattern.This experimental result shows: the white backed planthopper resistance of STS molecule marker AP4741 and paddy rice is closely linked, but still has certain exchange in pest-resistant individual plant, shows as Fig. 3, and the individual plant that parents' banding pattern occurs shows exchange has taken place.As calculated, its recombination fraction is 3.3%, and the genetic distance between STS molecule marker and anti insect gene is 3.3cM.
At last, it is also to be noted that what more than enumerate only is several specific embodiments of the present invention.Obviously, the invention is not restricted to above embodiment, many distortion can also be arranged.All distortion that those of ordinary skill in the art can directly derive or associate from content disclosed by the invention all should be thought protection scope of the present invention.
Sequence table
SEQ ID NO：1
ctggttgttt tctgaactcc gt 22
SEQ ID NO：2
gcattaacca tagctgtcca ctc 23
SEQ ID NO：3
aatcatgcta ggttattact cg 22
SEQ ID NO：4
gtgcagagat catgcttttg 20
Claims (3)
1, the linked molecule marker of the anti-white backed planthopper gene of a kind of and paddy rice,, it is characterized in that as species with paddy rice: it is right that described molecule marker primer is selected from following primer, and nucleotides sequence wherein classifies 5 ' → 3 as ',
RM6917 forward: CTGGTTGTTTTCTGAACTCCGT
Oppositely: GCATTAACCATAGCTGTCCACTC;
AP4741 forward: AATCATGCTAGGTTATTACTCG
Oppositely: GTGCAGAGATCATGCTTTTG.
2, the linked molecule marker of the anti-white backed planthopper gene of according to claim 1 and paddy rice is characterized in that: described RM6917 is positioned on paddy rice the 6th karyomit(e); Described AP4741 also is positioned on paddy rice the 6th karyomit(e).
3, the development approach of molecule marker as claimed in claim 1 or 2 is characterized in that may further comprise the steps:
1), with japonica rice variety spring river 06 as the anti insect gene donor parents with as hybridizing for local No. 1 in the platform of sense worm kind, thereby obtain pest-resistant individual plant as filial generation;
2), extract parental rice seedling and filial generation seedling genomic dna with the CTAB method;
3), adopt SSR and/or STS molecule marking method to carry out the screening of paddy rice white backed planthopper resistance molecule marker;
4), filter out a SSR molecule marker RM6917 and a STS molecule marker AP4741.
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