CN101423870B - Method for rapid screening banana fusarium wilt resistance - Google Patents
Method for rapid screening banana fusarium wilt resistance Download PDFInfo
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- CN101423870B CN101423870B CN2008101984267A CN200810198426A CN101423870B CN 101423870 B CN101423870 B CN 101423870B CN 2008101984267 A CN2008101984267 A CN 2008101984267A CN 200810198426 A CN200810198426 A CN 200810198426A CN 101423870 B CN101423870 B CN 101423870B
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Abstract
The invention provides a method for quickly screening resistance of banana vasicular wilt, which respectively prepares two pairs of specific primers, and uses genetic group DNA of a banana plant to be tested as a template to quickly identify plants resisting the banana vasicular wilt and plants easily being infected by the vasicular wilt through a method of PCR amplification. The invention uses the banana genetic group as the template, and uses the two pairs of the specific primers to carry out the PCR amplification on the banana genetic group respectively, so that specific DNA segment can be amplified from a disease-resistant variety, while a target segment can not be amplified from an infected banana variety. The technology respectively carries out the PCR amplification on the two pairs of the specific primers through one experiment, amplification results can be identified with one another so as to effectively eliminate the phenomenon of PCR false positive, the test process is quick and simple, and the results are reliable, thereby the plants resisting the banana vasicular wilt and the plants being infected by the banana vasicular wilt can be quickly identified. Through a plurality of experiments, the results prove that the accuracy rate of the method for testing the resistance of the banana vasicular wilt reaches 100 percent, thereby providing reliable guarantee of selecting varieties resisting the banana vasicular wilt for banana peasants.
Description
Technical field
The invention belongs to a kind of method of rapid screening banana fusarium wilt resistance.
Background technology:
Banana is as one of important fruit in the world, and annual production reaches 10,400 ten thousand tons (FAO, 2004).The banana production development in recent years of China is swift and violent, reaches 26.96 ten thousand hm to national cultivated area in 2004
2Ultimate production reaches 6,420,000 tons, occupy the whole world the 5th, be the fruit of China's south output maximum, banana had become the mainstay industry in China's hot-zone agricultural already, but because banana cultivar resistance is poor at present, whole world banana industry is subjected to the blight harm of (claiming Panama disease again) deeply, its pathogenic bacteria is a Cuba point sickle spore bacterium (Fusarium oxysporum f.sp cuberse (E.F.Simth) Snyderethansen), it is the destructive quarantine venereal disease evil of a kind of tool, particularly this cause of disease germina number-four biological strain can be contaminated all banana species, is destructive disease.With Guangdong is example, and nineteen ninety-five reports that this disease is at the about 1.4hm of Guangdong harm banana cultivar area
2And in 2002, onset area increased sharply to 1.4 ten thousand hm
2, then be increased to 2.0 ten thousand hm in 2003
2Have at present that first annual morbidity is that 10%, the second annual morbidity is that the 20%～30%, the 3rd annual morbidity reaches more than 70% in the withered pathogenetic any of several broadleaf plants garden, suffer any of several broadleaf plants field of this harm can not continue to plant banana basically.After banana infects blight, currently can prevent and treat, so banana blight made ground part banana gardens such as Taiwan, Guangdong destroy, caused great financial loss without any medicament.Therefore the banana peasant has only the banana variety of selection anti-blight to plant, and just can prevent the generation of banana blight.Owing to also do not find a kind of chemical agent of effectively preventing banana blight at present, adopt the plantation disease-resistant variety, with aquatic crops crop rotation and biological control be relatively valid approach of current control banana blight, but the market management of banana test-tube plantlet is comparatively chaotic, many is not that disease-resistant variety is sold with disease-resistant variety by illegal merchant, causes banana peasant's financial loss at last.And in the area that does not have the paddy field, banana and aquatic crops crop rotation are impossible.
The method that the purpose of this invention is to provide a kind of rapid screening banana fusarium wilt resistance, screen respectively and prepare two pairs of Auele Specific Primers, genomic dna with banana plant to be detected is a template, method by pcr amplification can identify resisting banana vascular wilt plant and susceptible blight plant apace, show the rate of accuracy reached 100% of its detection through experimental result repeatedly.Plant effective assurance is provided for the banana peasant selects to plant the anti-blight banana kind.
The present invention includes following steps:
(1) extraction of banana genome DNA
Take by weighing the 0.5g banana tip of a root or blade, add 0.01-0.02 gram polyethylene pyrroles cyclic ketones, in liquid nitrogen, be ground into powder fast; Powder is changed over to (CTAB extracting solution: 2%CTAB, 100mmol L in the 4mL3%CTAB extracting solution of 65 ℃ of preheatings
-1Tris-Cl (pH8.0), 1.4mol L
-1NaCl, 20mmol L
-1EDTA (pH8.0), 2% beta-mercaptoethanol), add 20 μ L10mg mL simultaneously
-1Proteinase K, shake up back 65 ℃ of water-bath 60min, during constantly shake centrifuge tube gently; After the cooling, add 1/3 volume 5molL
-1KAc solution, ice bath 20min behind the mixing; Add isopyknic chloroform/primary isoamyl alcohol (24:1), behind the mixing, under 4 ℃ of conditions 12, the centrifugal 10min of 000rpm; Supernatant liquor is transferred in the new centrifuge tube, adds the Virahol of 2/3 times of volume precooling in supernatant liquor, abundant mixing, behind-20 ℃ of precipitation 1h, under 4 ℃ of conditions with 12, the centrifugal 15min of 000rpm; Abandon supernatant, add twice, 4 ℃ of 1mL75% washing with alcohol, 12, the centrifugal 5min of 000rpm; Abandon supernatant, air-dry throw out in stink cupboard adds 500 μ LTE damping fluids and (contains 10 μ g.ml
-1RNase) dissolving, 55 ℃ of water-bath 30min; Get upper phase, add the dehydrated alcohol deposit D NA of 2 times of volume precoolings, under 4 ℃ of conditions 12, the centrifugal 15min of 000rpm; Abandon supernatant, 75% washing with alcohol precipitation, under 4 ℃ of conditions 12, the centrifugal 15min of 000rpm abandons supernatant, puts to be inverted on the thieving paper in the Bechtop and dries, after the DNA drying, with the TE dissolution precipitation of 50-100 μ L; With the quality of 0.8% agarose gel electrophoresis Detection and Extraction DNA, use UV spectrophotometer measuring DNA concentration simultaneously, ℃ packing is preserved standby then-20;
(2) PCR detects the sequence of used two pairs of primers
After these two pairs of primer sequences design, give birth to worker biotech firm by Shanghai and synthesize, obtain primer after, be made into 10 times mother liquor with dd water, use on request during use;
(3) pcr amplification reaction condition
Utilize two couples of special primer OPU10F and OPU10R respectively, OPS09F and OPS09R enantiopathy kind ' William's Si bud mutation ' and susceptible variety ' William Si ' banana genome are carried out pcr amplification, and reaction system is: 2.5 μ L10 * buffer, 0.4 μ L25mmol.L
-1MgCl
2, 1.0 μ L2.5m mol.L
-1DNTP, 1U LATaq archaeal dna polymerase, 1.0 μ L10 μ molL
-1Primer, the 20ng template DNA adds 16 μ LddH at last
2O supplies the total reaction volume of 25 μ L; React on the PCR instrument then, the PCR response procedures is, 94 ℃ of pre-sex change 5min, and 94 ℃ of sex change 40S then, 38 ℃ of annealing 1min, 72 ℃ are extended 90S, circulate 72 ℃ of 10min 35 times; Get 10 μ LPCR reaction product and 2 μ L sample-loading buffer mixings, click and enter in 1.2% sepharose that contains EB, at 5V.cm
-1Behind the electrophoresis 1h, sepharose is transferred to FX automated imaging system (BIO-RAD company) take pictures under the condition, from the product of amplification, just can determine apace whether the strain to be measured of all detections can resisting banana vascular wilt.
The present invention is template with the banana genome, respectively its genome is carried out pcr amplification with two pairs of special primers, can both amplify specific DNA fragment from disease-resistant variety, and susceptible banana variety then can not amplify the purpose fragment.This technology is carried out pcr amplification respectively with two pairs of special primers simultaneously by once testing, amplification can be verified mutually, can eliminate the false-positive phenomenon of PCR effectively, testing process is simple fast, reliable results, thus can identify resisting banana vascular wilt plant and sense blight banana plant apace.Show that through experimental result repeatedly this method is to the rate of accuracy reached 100% of the detection of banana fusarium wilt resistance.For selecting the resisting banana vascular wilt kind, the banana peasant provides reliable assurance.
Description of drawings
Fig. 1 is the present invention with OPU10F and OPU10R primer to ' William Si ' and ' William's Si bud mutation ' genome amplification figure as a result;
Fig. 2 is the present invention with OPS09F and OPS09R primer to " William's Si bud mutation " and " William Si " genome amplification figure as a result;
Fig. 3 is that the present invention utilizes OPU10F and the amplification figure of OPU10R in " golden finger " and " William's Si bud mutation " and other four susceptible variety;
Fig. 4 is OPS09F of the present invention and the amplification figure of OPS09R in four susceptible variety, " William's Si bud mutation " and " golden finger ";
Fig. 5 is that the present invention utilizes OPU10F and OPU10R to " William's Si bud mutation " tissue cultured seedling detected result figure;
Fig. 6 is that the present invention utilizes OPS09F and OPS09R to " William's Si bud mutation " tissue cultured seedling detected result figure.
Embodiment
Use OPU10F and OPU10R, OPS09F and two pairs of primers of OPS09R from 4 plant of resisting banana vascular wilt kind " William's Si bud mutation ", to react respectively by PCR, amplify the dna fragmentation that is about 1800bp, in infecting 4 plant of sick kind " William Si ", then do not have this fragment and occur as depicted in figs. 1 and 2.Explanation is carried out pcr amplification by utilizing primer OPU10F and OPU10R, OPS09F and OPS09R to banana genome, can filter out the resisting banana vascular wilt kind exactly from banana plant, the appearance of this purpose band also can be used as vital signs of the anti-kind of banana fusarium wilt resistance.
Fig. 1. use OPU10F and OPU10R primer to ' William Si ' and ' William's Si bud mutation ' genome amplification result, 1,2,3,4th, " William's Si bud mutation " is disease-resistant variety; 5,6,7,8th, " William Si " is susceptible variety.
Fig. 2. use OPS09F and OPS09R primer to " William's Si bud mutation " and " William Si " genome amplification result, 1,2,3,4th, " William's Si bud mutation " is disease-resistant variety; 5,6,7,8th, " William Si " is susceptible variety.
With OPU10F and OPU10R, OPS09F and OPS09R primer are used for susceptible variety
(' Cavendish ', ' big honey is breathed out ', ' Brazilian any of several broadleaf plants ', ' wild any of several broadleaf plants ') and disease-resistant variety (' golden finger Goldfinger ') further verify.The result shows, amplify the identical purpose fragment of size with this a pair of primer of OPU10F and OPU10R is all stable in disease-resistant variety ' golden finger ' and " William's Si bud mutation ", and 4 susceptible kinds all can not amplify the purpose fragment as shown in Figure 3; Use OPS09F and OPS09R all to amplify the purpose fragment again at disease-resistant variety ' golden finger ' and " William's Si bud mutation ", but many swimming bands appear in when amplification in golden finger, this be possible be since during annealing temperature hang down the mismatching phenomenon that causes excessively and cause as shown in Figure 4.Experimental result shows, verifies mutually with two pairs of primers respectively to directly apply to the disease-resistant blight kind of banana rapid detection.
Fig. 3 utilizes OPU10F and the amplification of OPU10R in " golden finger " and " William's Si bud mutation " and other four susceptible variety.
Annotate: M is Marker2000; 1, " golden finger " is disease-resistant variety, 2, and " William's Si bud mutation " is disease-resistant variety; 3,4,5,6 are respectively susceptible variety (" Cavendish ", " big honey is breathed out ", " Brazilian any of several broadleaf plants ", " wild any of several broadleaf plants ")
Fig. 4 OPS09F and the OPS09R amplification in four susceptible variety, " William's Si bud mutation " and " golden finger ".
Annotate: M is Marker2000; 1,2,3,4 is four susceptible variety (" Cavendish ", " big honey is breathed out ", " Brazilian any of several broadleaf plants ", " wild any of several broadleaf plants "); 5,6th, " William's Si bud mutation " and " golden finger ".
This breadboard 40 strains " William's Si bud mutation " group training material of picked at random utilizes OPU10F and OPU10R respectively, and whether OPS09F and OPS09R primer also can the resisting banana vascular wilt situation be analyzed its tissue cultured seedling.Detect respectively 40 strains " William's Si bud mutation " seedling with two pairs of primers, the result shows has 39 young plants can both amplify the purpose fragment.In tissue cultured seedling, there is a strain that variation may take place, causes the segmental disappearance of purpose in the genome, so the purpose fragment that can not increase.
After simultaneously this 40 strain tissue cultured seedling being inoculated No. 4 physiological strain germs of blight, be planted in again in Southern Asia Institute of Tropical Crop, Chinese Academy of Tropical Agricultural Science's banana garden (Zhanjiang, Guangdong), between the whole growing of banana seedlings plantation back, do not have the generation of blight.Therefore from this experimental result, utilize these two pairs of primers to detect after plantation again, can guarantee that detected banana seedlings anti-blight plant does not infect blight, so this detection method fast is effective.
Fig. 5 utilizes OPU10F and OPU10R to " William's Si bud mutation " tissue cultured seedling detected result.Annotate: M is Marker2000; 1-40 is " William's Si bud mutation " tissue cultured seedling, and wherein variation has taken place the 6th plant, and PCR result is negative, and other 39 strains are all positive.
Fig. 6 utilizes OPS09F and OPS09R to " William's Si bud mutation " tissue cultured seedling detected result.Annotate: M is Marker2000; 1-40 is " William's Si bud mutation " tissue cultured seedling, and wherein variation has taken place the 6th plant, and PCR result is negative, and other 39 strains are all positive.
Needed instrument and medicine at home biological reagent company public offering is all arranged, can buy from the market at an easy rate.
Claims (3)
1. the method for a rapid screening banana fusarium wilt resistance is characterized in that comprising the steps:
(1) extraction of banana genome DNA
Take by weighing the 0.5g banana tip of a root or blade, add 0.01-0.02 gram polyethylene pyrroles cyclic ketones, in liquid nitrogen, be ground into powder fast; Powder is changed in the 4mL 3%CTAB extracting solution of 65 ℃ of preheatings, add 20 μ L10mg mL simultaneously
-1Proteinase K, shake up back 65 ℃ of water-bath 60min, during constantly shake centrifuge tube gently; After the cooling, add 1/3 volume 5molL
-1KAc solution, ice bath 20min behind the mixing; Add isopyknic chloroform/primary isoamyl alcohol, chloroform/primary isoamyl alcohol is 24: 1, behind the mixing, and under 4 ℃ of conditions 12, the centrifugal 10min of 000rpm; Supernatant liquor is transferred in the new centrifuge tube, adds the Virahol of 2/3 times of volume precooling in supernatant liquor, abundant mixing, behind-20 ℃ of precipitation 1h, under 4 ℃ of conditions with 12, the centrifugal 15min of 000rpm; Abandon supernatant, add twice, 4 ℃ of 1mL 75% washing with alcohol, 12, the centrifugal 5min of 000rpm; Abandon supernatant, air-dry throw out in stink cupboard adds the dissolving of 500 μ LTE damping fluids, 55 ℃ of water-bath 30min; Get upper phase, add the dehydrated alcohol deposit D NA of 2 times of volume precoolings, under 4 ℃ of conditions 12, the centrifugal 15min of 000rpm; Abandon supernatant, 75% washing with alcohol precipitation, under 4 ℃ of conditions 12, the centrifugal 15min of 000rpm abandons supernatant, puts to be inverted on the thieving paper in the Bechtop and dries, after the DNA drying, with the TE dissolution precipitation of 50-100 μ L; With the quality of 0.8% agarose gel electrophoresis Detection and Extraction DNA, use UV spectrophotometer measuring DNA concentration simultaneously, ℃ packing is preserved standby then-20;
(2) PCR detects the sequence of used two pairs of primers
OPU10F：5’-ACCTCGGCACTCGAAGACACAT-3’
OPU10R：5’-ACCTCGGCACTATTACCCATCAT-3’
OPS09F：5’-TCCTGGTCCCAGTACAAATAC-3’
OPS09R：5’-TCCTGGTCCCTCTGAATTTTC-3’
After these two pairs of primer sequences design, give birth to worker biotech firm by Shanghai and synthesize, obtain primer after, be made into 10 times mother liquor with dd water, use on request during use;
(3) pcr amplification reaction condition
Utilize two couples of special primer OPU10F and OPU10R respectively, OPS09F and OPS09R enantiopathy kind ' William's Si bud mutation ' and susceptible variety ' William Si ' banana genome are carried out pcr amplification, and reaction system is: 2.5 μ L, 10 * buffer, 0.4 μ L25mmolL
-1MgCl
2, 1.0 μ L 2.5mmolL
-1DNTP, 1U LA Taq archaeal dna polymerase, 1.0 μ L, 10 μ molL
-1Primer, the 20ng template DNA adds 16 μ L ddH at last
2O supplies the total reaction volume of 25 μ L; React on the PCR instrument then, the PCR response procedures is, 94 ℃ of pre-sex change 5min, and 94 ℃ of sex change 40S then, 38 ℃ of annealing 1min, 72 ℃ are extended 90S, circulate 72 ℃ of 10min 35 times; Get 10 μ L PCR reaction product and 2 μ L sample-loading buffer mixings, click and enter in 1.2% sepharose that contains EB, at 5V.cm
-1Behind the electrophoresis 1h, sepharose is transferred to FX automated imaging system take pictures under the condition, from the product of amplification, just can determine apace whether the strain to be measured of all detections can resisting banana vascular wilt.
2. the method for a kind of rapid screening banana blight of stating according to claim 1 is characterized in that the CTAB extracting solution is: 2%CTAB, 100mmolL
-1Tris-Cl, 1.4molL
-1NaCl, 20mmolL
-1EDTA, 2% beta-mercaptoethanol; The PH8.0 of described Tris-Cl, the PH8.0 of described EDTA.
3. according to the method for the described a kind of rapid screening banana blight of claim 1, it is characterized in that the TE damping fluid contains 10 μ g.ml
-1RNase.
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| CN101643772B (en) * | 2009-09-09 | 2012-07-18 | 广东省农业科学院果树研究所 | Method for quickly evaluating disease resistance of banana fusarium wilt |
| CN104232759B (en) * | 2014-08-18 | 2017-01-11 | 中国热带农业科学院热带生物技术研究所 | Gene marker for screening banana variety resistant to wilt |
| CN106119360B (en) * | 2016-06-29 | 2019-09-10 | 东莞市香蕉蔬菜研究所 | A kind of SCAR molecular labeling and its identification method for identifying banana blight resistance |
| CN107653335B (en) * | 2017-10-17 | 2021-04-13 | 东莞市香蕉蔬菜研究所 | Banana wilt resistance molecular marker and application thereof |
| CN107641641B (en) * | 2017-11-10 | 2021-06-01 | 福建农林大学 | Rapid and simple preliminary evaluation method for resistance of banana germplasm blight |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101113467A (en) * | 2006-07-03 | 2008-01-30 | 华南农业大学 | Detection primer for banana wilt germina number-four biological strain and method for detecting same |
| CN101205558A (en) * | 2006-12-22 | 2008-06-25 | 福建省农业科学院植物保护研究所 | Banana wilt bacterium molecule detecting genes and detecting method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101113467A (en) * | 2006-07-03 | 2008-01-30 | 华南农业大学 | Detection primer for banana wilt germina number-four biological strain and method for detecting same |
| CN101205558A (en) * | 2006-12-22 | 2008-06-25 | 福建省农业科学院植物保护研究所 | Banana wilt bacterium molecule detecting genes and detecting method thereof |
Non-Patent Citations (1)
| Title |
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| 王国芬等.香蕉枯萎病镰刀菌ITS序列的PCR扩增及其分子检测.《华南热带农业大学学报》.2007,第13卷(第3期),全文. * |
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