CN101880710A - SCAR molecular marker interlocked with brown planthopper resistance genes - Google Patents
SCAR molecular marker interlocked with brown planthopper resistance genes Download PDFInfo
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Abstract
The invention discloses an SCAR molecular marker interlocked with brown planthopper resistance genes. Non-resistant pool DNA and resistant pool DNA in B3F4 segregation population of brown planthopper resistance near-isogenic lines, which are distantly hybridized by Oryza officinals Waii Ex Watt 1665 and cultivated rice Gui 99 by adopting 282 random primers, are subjected to specific RAPD marker screening, so an RAPD marker S229 with an obvious specific amplification band spectrum is screened out; and the S229 has the sequence length of 703bp, and has 51.86 percent of homology with a sequence (67114269100) of a BAC clone (No. OS JNBa0070011) of the rice No.4 chromosome which has been reported in a genbank. The RAPD marker is converted into a stable SCAR marker. The marker can be used for assisting the selective breeding of brown planthopper resistance rice, and lays a foundation for cloning new brown planthopper resistance genes.
Description
Technical field
The present invention relates to a kind of and the chain SCAR mark of brown planthopper resistant gene in rice, can be used for molecular mark, belong to paddy rice breeding for pest resistance and biology field.
Background technology
Since the seventies in 20th century, the various countries scientist is devoted to the development and utilization of the new gene of brown planthopper resistant.Up to the present, according to the different resistance reactions of resistant gene, 15 brown planthopper resistant genes (Athwal etc., 1971 have at least been identified to the different biotypes brown paddy plant hopper; Khush etc., 1985; Khush and Brar etc., 1991; Ishii etc., 1994; Liu Guoqing etc., 2001), wherein, Bph1 and Bph2 antibiont type 1 and 3; Bph3, Bph4, Bph8, Bph9 and Bph10 (t) antibiont type 1,2 and 3; Bph5, Bph6, Bph7 antibiont type 4.It is the basis that effectively utilizes brown planthopper resistant gene that the new gene of brown planthopper resistant is positioned.Now Bph1, bph2, Bph9, Bph10 have been positioned on No. 12 karyomit(e) of paddy rice, bph (t) and Bph11 are located on the 3rd karyomit(e), and Bph3, bph4, Bph (t) and bph12 are located in respectively on the 10th, 6,9, No. 4 karyomit(e) [16].Employing rflp analysis methods such as Ishii were positioned at Bph10 (t) on the position of No. 12 karyomit(e) distance R G4573.68cM in 1994; Employing qtl analysis methods such as Huang were positioned at a unnamed new resistance site between the 12nd chromosomal RG463 and the SdH-1 in 1997; Alam in 1998 etc. are the parent with IR64 and Azucena, made up a double haploid that contains 131 individual plants, use the resistant gene of QTL method research IR64, experimental result shows, the main resistant gene of imitating of IR64 is positioned on the 12nd karyomit(e), also has the resistance site simultaneously on the 1st, 2,3,4,6,8 karyomit(e)s; Utilize tight fringe wild-rice and cultivated rice kind 02428 distant hybrid progeny that a resistant gene is positioned on the 2nd karyomit(e) Liu National Day calendar year 2001 etc., between SSR mark RM140 and RM250, and called after Bph13 (t).In some rice varieties, also exist the suppressor gene of brown planthopper resistant gene, such as have resistant gene Bph1 and dominance suppressor gene Ibph1 at rice varieties TKM6, so TKM6 shows as the reaction of sense worm to brown paddy plant hopper, separate by TKM6 and IR24 being hybridized back two pairs of genes, in filial generation, can choose pest-resistant individual plant, cultivate and finally cultivate brown planthopper resistant new variety IR26.
SCAR (sequence characterized amplified region) sequence signature amplification region mark normally is transformed by the RAPD mark.The a certain RAPD that finds in order to improve is marked at the stability in the application, this RAPD labeled fragment can be reclaimed and clones and check order from gel, design a pair of Auele Specific Primer (18-24bp) according to its base sequence, also can check order by a end to this RAPD labeled fragment, increasing about 14 adjacent base on the basis of 10 original base primerses, becoming and the terminal paired Auele Specific Primer mutually of original RAPD labeled fragment.And genomic dna carried out pcr amplification, and just can amplify the specificity band spectrum with the original identical size of RAPD labeled fragment, thisly just become the SCAR mark through the specific DNA molecular mark that transforms.The SCAR mark generally shows as the whether existence of amplification band spectrum, is a kind of dominant marker.But also show as the length polymorphism mark sometimes, be codominant marker [38].Relative RAPD mark, because the used primer length of SCAR mark and and match fully with template, so under quite rigorous condition, we can obtain stability and the good result of repeatability, along with the continuous development of SCAR marker research and perfect, will obtain the SCAR mark of increasing Main Agronomic Characters, vital role in the performance in marker assisted selection in the future.
Develop rapidly along with molecular biological, the conversion of most farm crop has become possibility, the location with separate the gene relevant and become the important directions of research economical character growth course with Main Agronomic Characters, also be the key point of using gene engineering technique improvement plant genetic proterties.Development in recent years and the localization method of having used several genes.Utilize the molecular marker assisted selection breeding can effectively realize the fundamental shifts that breeding is selected to genotype from Phenotypic Selection.
Summary of the invention
The purpose of this invention is to provide a kind of and the chain SCAR mark of brown planthopper resistant gene in rice, this mark can be used in the assisted selection of paddy rice brown planthopper resistant, lays the foundation for cloning new brown planthopper resistant gene.
The present invention is achieved through the following technical solutions above-mentioned purpose: a kind of and chain SCAR mark of brown planthopper resistant gene in rice, the primer of being made up of SEQ ID NO.1 in the sequence table and SEQ ID NO.2 obtains pcr amplification.Described molecule marker SCAR
694, be to filter out S229 with the RAPD molecule marking method
703Molecule marker is again with S229
703Convert stable SCAR mark to, this mark adopts following method to obtain:
1, material therefor be oryza officinalis 1665 (Oryza officinals Waii Ex Wat t) with the B3F4 of cultivated rice osmanthus 99 distant hybirdization for the selfing segregating population.
2, extract rice total dna with the CTAB method, be dissolved among 1 * TE respectively, and standby in-20 ℃ of preservations, and the agarose gel electrophoresis through 1.0% detects the integrity of rice total dna, and calculates its concentration according to the EB staining power.
3, adopt the RAPD molecule marking method, to the screening of the molecule marker relevant with the paddy rice brown planthopper resistant.
4, select a RAPD molecule marker S229
703, identify through linksystem, find that this mark and brown planthopper resistant gene in rice are linked, with the genetic distance of resistant gene be 10.1CM.
5, with S229
703Behind the cloning and sequencing, according to check order row, design primer SEQ ID NO.1 and SEQ IDNO.2 are template with the rice total dna, and pcr amplification obtains SCAR mark, i.e. SCAR
694
Adopt the RAPD molecule marking method, carry out the molecule marker linked with brown planthopper resistant gene in rice, the specific practice of its screening is:
1) label screening:
The used material of this experiment be oryza officinalis 1665 (Oryza officinals Waii Ex Wat t) with the B3F4 of cultivated rice osmanthus 99 distant hybirdization for the selfing segregating population choose B3F4 for 10 strains in the selfing segregating population not the total DNA of brown planthopper resistant plant mix as not anti-pond DNA, the total DNA of 10 strain brown planthopper resistant plant mixes as anti-pond DNA, respectively not anti-pond DNA and anti-pond DNA are carried out RAPD PCR respectively with each bar RAPD primer, the pcr amplification reaction system is set up: 10 * buffer 2uL; 2.5mM dNTP2uL; 10pM primer 1uL; Total DNA (20～50ng/uL); 1uL Ex Taq (0.5u/uL) 2uL; Add ddH at last
2O complements to 20uL.Response procedures is 95 ℃ of pre-sex change 5min; 94 ℃ of 30s, 36 ℃ of 30s, 40 circulations of 72 ℃ of 1min; 72 ℃ are extended 10min (Yuan Yongfang etc., 2000) then.PCR reaction amplified production detects through EB dyeing then with 1.5% agarose gel electrophoresis, and writes down every amplification band spectrum position on sepharose, the random primer that screening has the brown planthopper resistant gene polymorphism.
2) mark S229
703Linksystem identify
The RAPD primer S229 that utilizes screening to obtain
703122 individual plants of near isogenic line colony are carried out pcr amplification.The result shows that have 66 strains to have special band in the 75 strain resistance individual plants, there is not special band in 9 strains; There is 1 strain to have special band in the not anti-individual plant of 47 strains, the 46 strains special band that do not increase.The result shows, specific fragment S229
703With the paddy rice brown planthopper resistant be closely linked, but in resistance individual plant and responsive individual plant, all have certain exchange.In conjunction with the phenotype resistance qualification result of near isogenic line colony, adopt MAPMAKER/EXP.Version 3.0 to analyze, software analysis shows S229
703And the genetic distance between brown planthopper resistant gene is 10.1cM.
3) RAPD mark sequencing analysis
Glue reclaims test kit available from Shanghai China Shun biotechnology company limited, and pGEM T-Easy vector kit is available from Promega company.The RAPD primer RAPD mark that increases in a large number with screening, PCR product 1.5% agarose gel electrophoresis, reclaim test kit with the centrifugal a small amount of glue of post again and reclaim RAPD specific marker fragment, and connection is cloned on the cloning vector pGEM T-Easy vector, schedule of operation according to BIORAD company electric impulser, adopt electrization Transformed E .coliDH5 α, select to select white colony on the substratum at the penbritin LA that contains X-gal and IPTG, and cut the evaluation recombinant plasmid with restriction enzyme EcoR I enzyme, send the order-checking of precious biotechnology Dalian company limited.Sequencing result shows that this RAPD specific marker clip size is 703bp.
4) conversion of SCAR mark
According to RAPD mark specific fragment dna sequencing result, design Auele Specific Primer SEQ ID NO.1 and SEQ ID NO.2.With Auele Specific Primer not anti-strain DNA of 15 strains and 15 strain resistance individual plant DNA are carried out PCR, system is: 10 * buffer 2uL, and 2.5mM dNTP 2uL, each 1uL of 10pM primer, (20～50ng/uL), 1uL Ex Taq (0.5u/uL) 2uL adds ddH to total DNA
2O complements to 20uL.Response procedures is: 95 ℃ of pre-sex change 5min, and 94 ℃ of 30s, 50 ℃ of 30s, 30 circulations of 72 ℃ of 1min, 72 ℃ are extended 10min then.PCR reaction amplified production detects through EB dyeing then with 1.0% agarose gel electrophoresis.The result has amplified the special band of 694bp in the resistance individual plant, and does not detect corresponding band in not anti-individual plant.
Outstanding effect of the present invention is:
This mark can be used in the assisted selection of paddy rice brown planthopper resistant, lays a good foundation for cloning new brown planthopper resistant gene.
Embodiment
Adopt the RAPD molecule marking method, obtain the molecule marker S229 relevant with the paddy rice brown planthopper resistant
703, the physical length of this molecule marker is 703bp, changes SCAR into the SCAR method
694Mark.
One, the preparation of rice material
With oryza officinalis 1665 and cultivated rice hybridization, cultivate the F1 generation that obtains through rataria and carry out distant hybirdization with the cultivated rice kind, continuous backcross, selfing and plant resistance are identified then, obtain the anti-sense of near isogenic line B3F4 colony, 122 strains altogether.The resistant phenotype qualification result sees Table 1
Table 1 near isogenic line B3F4 plant resistance qualification result
Two, rice total dna extracts
Get the fresh rice leaf of 1-50g, grind into powder in liquid nitrogen changes agar in the centrifuge tube of precooling, and the 2 * CTAB that adds 65 ℃ of preheatings of equal-volume (w/v) immediately extracts damping fluid, abundant mixing, 65 ℃ of insulations 10-20 minute, during shake frequently.Add isopyknic chloroform/primary isoamyl alcohol, the light and slow centrifuge tube mixing of putting upside down, under the room temperature, the centrifugal 10-20 of 12000r/mim minute.Supernatant is changed in another centrifuge tube, add the 10%CTAB of 1/10 volume, mixing adds isopyknic chloroform/primary isoamyl alcohol, puts upside down the centrifuge tube mixing, under the room temperature, and centrifugal 10 minutes of 12000r/min.Get supernatant liquor, repeat (4) operation 1 time.Supernatant liquor is changed over to new in the centrifuge tube that silanization is handled, add 1 * CTAB precipitation buffering liquid of 1-1.5 times of volume, mixing, placed 30 minutes under the room temperature, observe precipitation and generate, generate as no obvious sediment, prolong storage period, with prolonging storage period, precipitation capacity increases.The centrifugal 5-10 of 3500-4000r/min minute, remove supernatant, precipitation dries up, with 200uL 10 * TE dissolving.
Add 1-2.5uL RNAse (10g/L), 37 ℃ are incubated 1 hour.Add the 2-4uL Proteinase K, 55 ℃ are incubated 1 hour.Add 150uL H
2O and 360uL (24: 1) chloroform/primary isoamyl alcohol, light and slow shaking 6 minutes, mixing, centrifugal 10 minutes of room temperature 8000r/min.On change over to mutually in another new centrifuge tube, add the NaAC (3M) of 1/10 volume precooling, add the dehydrated alcohol of 2 times of volume precoolings again, deposit D NA places more than 30 minutes for-20 ℃, 12000r/min is centrifugal 10 minutes under the low temperature.DNA precipitates with 75% washing with alcohol twice, the centrifugal ethanol of outwelling.After air-dry, add 50uL1 * TE dissolving ,-20 ℃ of preservations, standby.
Three, the structure of near isogenic line (NIL) DNA mixing pit
Choose 10 the highest grades of strain resistance resistance individual plant near isogenic line, the sample DNA that thaws and diluted takes out 50uL respectively and mixes, and constitutes the anti-DNA mixing pit of near isogenic line.According to same principle, near isogenic line, choose the elementary resistance individual plant of 10 strain resistances, the sample DNA that thaws and diluted takes out 50uL respectively and mixes, and constitutes the not anti-DNA mixing pit of near isogenic line.
Four, amplification polymorphism DNA (RAPD) analyzes
Respectively not anti-pond DNA and anti-pond DNA are carried out RAPD PCR respectively with each bar RAPD primer, the pcr amplification reaction system is set up: 10 * buffer 2uL; 2.5mM dN TP 2uL; 10pM RAPDprimer 1uL; Total DNA (20～50ng/uL) 1uL; Ex Taq (0.5u/uL) 2uL; Add ddH2O and complement to 20uL.Response procedures is: 95 ℃ of pre-sex change 5min; 94 ℃ of 30s, 36 ℃ of 30s, 40 circulations of 72 ℃ of 1min, 72 ℃ are extended 10min then.PCR reaction amplified production detects through EB dyeing then with 1.5% agarose gel electrophoresis, and write down every amplification band spectrum position on sepharose, analyze the electrophoretogram of each RAPD primer at the amplified production in different DNA pond, searching is resisting, is feeling the RAPD primer that has polymorphism in the group, by 4 screenings of taking turns that reduce the scope gradually, random primer with the chain RAPD mark of brown planthopper resistant gene Bph tentatively obtains increasing.The random primer S229 that screening obtains having the brown planthopper resistant gene polymorphism
703
Six, mark S229
703Linksystem identify
The RAPD primer S229 that utilizes screening to obtain
703122 individual plants of near isogenic line colony are carried out pcr amplification.The result shows that have 66 strains to have special band in the 75 strain resistance individual plants, there is not special band in 9 strains; There is 1 strain to have special band in the not anti-individual plant of 47 strains, the 46 strains special band that do not increase.The result shows, specific fragment S229
703With the paddy rice brown planthopper resistant be closely linked, but in resistance individual plant and responsive individual plant, all have certain exchange.In conjunction with the phenotype resistance qualification result of near isogenic line colony, adopt MAPMAKER/EXP.Version 3.0 to analyze, software analysis shows S229
703And the genetic distance between brown planthopper resistant gene is 10.1cM.
Seven, RAPD mark sequencing analysis
Glue reclaims test kit available from Shanghai China Shun biotechnology company limited, and pGEM T-Easy vector kit is available from Promega company.The RAPD primer RAPD mark that increases in a large number with screening, PCR product 1.5% agarose gel electrophoresis, reclaim test kit with the centrifugal a small amount of glue of post again and reclaim RAPD specific marker fragment, and connection is cloned on the cloning vector pGEM T-Easy vector, schedule of operation according to BIORAD company electric impulser, adopt electrization Transformed E .coliTH5 α, select to select white colony on the substratum at the penbritin LA that contains X-gal and IPTG, and cut the evaluation recombinant plasmid with restriction enzyme EcoR I enzyme, send the order-checking of precious biotechnology Dalian company limited.Sequencing result shows that this RAPD specific marker clip size is 703bp.
Eight, the SCAR of molecule marker conversion
According to RAPD mark specific fragment dna sequencing result, design Auele Specific Primer SEQ ID NO.1 and SEQ ID NO.2.With Auele Specific Primer not anti-strain DNA of 15 strains and 15 strain resistance individual plant DNA are carried out PCR, system is: 10 * buffer 2uL, 2.5mM dNTP 2uL, each 1uL of 10pM primer, (20～50ng/uL) 1uL, Ex Taq (0.5u/uL) 2uL adds ddH20 and complements to 20uL total DNA.Response procedures is: 95 ℃ of pre-sex change 5min, and 94 ℃ of 30s, 50 ℃ of 30s, 30 circulations of 72 ℃ of 1min, 72 ℃ are extended 10min then.PCR reaction amplified production detects through EB dyeing then with 1.0% agarose gel electrophoresis.The result has amplified the special band of 703bp in the resistance individual plant, and does not detect corresponding band in not anti-individual plant.
Claims (4)
1. a SCAR molecule marker chain with brown planthopper resistant gene in rice is characterized in that, the primer of being made up of SEQ ID NO.1 in the sequence table and SEQ ID NO.2 obtains pcr amplification.
2. the described molecule marker of claim 1 is to filter out S229 with the RAPD molecule marking method
703Molecule marker is again with S229
703Convert stable SCAR mark to, this SCAR mark adopts following method steps to obtain:
(1) material therefor be oryza officinalis 1665 (Oryza officinals Waii Ex Wat t) with the B3F4 of cultivated rice osmanthus 99 distant hybirdization for the selfing segregating population,
(2) extract rice total dna with the CTAB method, it is standby to be dissolved among 1 * TE 220 ℃ of preservation respectively, the integrity of agarose gel electrophoresis detection rice total dna through 1.0%, and calculate its concentration according to the EB staining power,
(3) adopt the RAPD molecule marking method, to the screening of the molecule marker relevant with the paddy rice brown planthopper resistant,
(4) select a RAPD molecule marker S229
703, this mark and brown planthopper resistant gene in rice are linked, with the genetic distance of resistant gene be 10.1CM,
(5) with S229
703Behind the cloning and sequencing, according to check order row, design primer SEQ ID NO.1 and SEQID NO.2 are template with the rice total dna, and pcr amplification obtains SCAR mark, i.e. SCAR
694
According to claim 1 described with the chain SCAR molecule marker of brown planthopper resistant gene in rice, it is characterized in that, use the RAPD molecule marking method, carry out the molecule marker relevant with the paddy rice brown planthopper resistant, the specific practice of its screening is: choose B3F4 for 10 strains in the selfing segregating population not the total DNA of brown planthopper resistant plant mix as not anti-pond DNA, the total DNA of 10 strain brown planthopper resistant plant mixes as anti-pond DNA, respectively not anti-pond DNA and anti-pond DNA are carried out RAPD PCR respectively with each bar RAPD primer, the pcr amplification reaction system is set up: the total DNA of 10 * buffer 2uL 2.5mM dNTP 2uL 10pM RAPD primer 1uL (20～50ng/uL) 1uL ExTaq (0.5u/uL) 2uL, add ddH20 and complement to 20uL, 95 ℃ of pre-sex change 5min, 94 ℃ of 30s, 36 ℃ of 30s, 40 circulations of 72 ℃ of 1min, 72 ℃ are extended 10min then, and PCR reaction amplified production detects through EB dyeing then with 1.5% agarose gel electrophoresis.
4. the described and chain SCAR molecule marker of brown planthopper resistant gene in rice according to claim 2 is characterized in that, with S229
703Convert stable SCAR to
694Mark, specific practice is: at first with mark S229
703Be cloned on the cloning vector pGEM T-Easy vector, schedule of operation according to BIORAD company electric impulser, adopt electrization Transformed E .coliDH5 α, select to select white colony on the substratum at the penbritin LA that contains X-gal and IPTG, and cut the evaluation recombinant plasmid with restriction enzyme EcoRI enzyme, send the order-checking of precious biotechnology Dalian company limited, after the order-checking, according to this sequences Design 2 SCAR primers P1 and P2, carry out pcr amplification at B3F4 respectively in for the resistance individual plant of selfing segregating population, all can amplify the SCAR mark SCAR of expectation
694
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103509791A (en) * | 2013-07-31 | 2014-01-15 | 江西省农业科学院水稻研究所 | Gene marker of major gene Bph14 for resisting brown planthopper in rice and application thereof |
| CN106480052A (en) * | 2015-08-24 | 2017-03-08 | 中国种子集团有限公司 | Recombinant nucleic acid fragment RecCR020352 and its detection method |
-
2010
- 2010-01-26 CN CN2010101012120A patent/CN101880710A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103509791A (en) * | 2013-07-31 | 2014-01-15 | 江西省农业科学院水稻研究所 | Gene marker of major gene Bph14 for resisting brown planthopper in rice and application thereof |
| CN103509791B (en) * | 2013-07-31 | 2016-03-16 | 江西省农业科学院水稻研究所 | The genetic marker of Rice Resistance brown paddy plant hopper major gene Bph14 and application thereof |
| CN106480052A (en) * | 2015-08-24 | 2017-03-08 | 中国种子集团有限公司 | Recombinant nucleic acid fragment RecCR020352 and its detection method |
| CN106480052B (en) * | 2015-08-24 | 2020-06-19 | 中国种子集团有限公司 | Recombinant nucleic acid fragment RecCR020352 and detection method thereof |
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Application publication date: 20101110 |