CN106498027A - A kind of anti-rough dwarf disease authentication method of Semen Maydiss - Google Patents
A kind of anti-rough dwarf disease authentication method of Semen Maydiss Download PDFInfo
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Abstract
The invention discloses a kind of anti-rough dwarf disease authentication method of Semen Maydiss, the method step is as follows：1）Select identification scale；2）Hypoxia inducible Semen Maydiss to be measured and the plumule of identification scale；3）Light culture after the malicious small brown rice planthopper of plumule inoculation band；4）Quantitative fluorescent PCR obtains the RBSDV viruses S7 of each point corn variety cycle time and the relative expression quantity of reference gene（RQ values）；5）Draw RBSDV proliferation index Trendline；6）Resistance level and resistance class of the corn variety to be measured to MRDV are evaluated according to RBSDV proliferation index Trendline formula.The present invention evaluates corn variety to rough dwarf disease resistance level by plumule intrathecal RBSDV increment trend difference, the resistance level and resistance class of the anti-rough dwarf disease of corn variety can be determined in a short time from molecular level, qualification cycle is short and reliability is high, is worth with larger Breeding Application.
Description
Technical field
The present invention relates to agricultural high-tech field, the anti-rough dwarf disease authentication method of the Semen Maydiss that specifically a kind of qualification cycle is short, reliability is high.
Background technology
Semen Maydiss belong to grass family Semen Maydiss race Zea, and its domestication origin can be traced back to before modern 7000-10000, be the big crop of current the first in the world.China is the second largest Maize Production state for being only second to the U.S. in the world, and Semen Maydiss also become the first big crop of China in recent years, are important grain ration, feed grain, the raw material of industry and energy source raw material, have very important status aborning.In recent years, with the industrial structure adjustment and population be continuously increased, the demand of yield, quality for Semen Maydiss also more and more higher, maize sown area also increase year by year.
MRDV is a kind of virosiss of worldwide serious harm Maize Production.The disease, was subsequently also found in other European countries and south American countries first in Italy's discovery in succession in 1949.China found in southern Xinjiang and West Part of Gansu first in 1954, subsequently spread the whole nation.MRDV causes large area to ruin kind or have no harvest in some areas in China North China, northwest and northeast since the seventies in last century, becomes the important disease for threatening Maize Production.According to incompletely statistics, China's MRDV morbidity field in 1996 is up to 2,330,000 hectares, wherein has no harvest more than 40,000 hectares.After 2004, MRDV is broken out in areas such as Jiangsu, Anhui, Jiangsu, Zhejiang, Shanghai again.2007 are only in Shandong province occurring area just up to 3,400,000 mu, further expand to more than 1,110 ten thousand mu in Shandong within 2008, wherein cause to have no harvest 250,000 mu, and the MRDV of the province such as Anhui, Henan, Hebei is also than more serious.From 2008 to 2014, MRDV is above 3,000,000 hectares in the annual onset area of China, general diseased plant rate is between 2% to 10%, serious field is up to more than 80%, had even has no harvest, the outburst of the disease and prevalence have a strong impact on and constrain the Maize Production of China, and cause huge economic loss.In recent years, with the change and the adjustment of pattern of farming of weather, the generation of MRDV starts in rising trend again in China, especially even more serious in the generation of the area such as Hebei, Shanxi, Shandong, Jiangsu, Zhejiang, Shanghai, the harm for controlling MRDV is of great significance to the stable high yield tool for ensureing Semen Maydiss.
The initial symptoms of MRDV are that have transparent dotted line point between the thready pulse of spire middle arteries both sides, and transparent point gradually increases later, produce the wax projection that thickness differs on the vein of leaf back, and handss touch with obvious harsh feeling.Diseased plant plant stunts, shortened internodes；Leaf color is dark green；Blade is short wide plump, stiff stand upright, often have gauffer；Female tassel dysplasia, severe patient can not be solid.
MRDV, MRCV and RBSDV are the three kinds of cause of diseases that can cause MRDV of hitherto reported, and wherein MRDV and MRCV is the cause of disease of Europe and South America MRDV respectively, and the cause of disease of China's MRDV is RBSDV.These three viruses belong to second group of Reoviridae Fijivirus category, and three is closely similar at the aspect such as viral particle morphology, host range, vector, serology and genome electrophoresis and sequence.
It is intracellular that Chinese maize rough dwarf disease virus RBSDV is primarily present in the disease leaf cell for swelling and the fat-body for protecting kissing bug, salivary gland, digestive tract, muscle, trachea etc..In milpa, virion has focused largely on formation viroplast at cell wall, and Maize rough dwarf virus are only limitted to phloem and its neighbouring cell.Can only be by entomochory, infection insect is mainly ash and flies, and small brown rice planthopper once obtains poison, lifelong band poison, still breeds in vivo after casting off a skin, and category persistency passes poison.
Small brown rice planthopper is to propagate the MRDV topmost vector of virus, inhales juice without poisonous insect and obtain poison on the plant for infected RBSDV, obtains the malicious time and is generally 24-48h, most short lh, poison is passed without poisonous insect all the life can after band poison, not transovarial passage, poisonous insect is most to pass poison for short-term interval.In " Wheat Maize " growing area, the 1st generation small brown rice planthopper occurred on Semen Tritici aestivi is the main source of infection for causing MRDV popular with poisonous insect.Spring first generation striatellus imago take food on overwintering host malicious after migrate on Semen Maydiss, formed in wheat harvest and migrated peak, there is MRDV onset peak in about 21 days behind peak in migration.Migrate wheatland and field side weeds of 2nd, 3,4 generation small brown rice planthoppers pass poison and more summer.After autumn winter wheat emerges, migrate wheatland and weeds upload poison and survive the winter small brown rice planthopper, form then annual Disease Cycle.
The prophylactico-therapeutic measuress such as chemical pesticide control and adjustment sowing time, the generation and propagation of MRDV can be controlled to a certain extent, but wasted time and energy, bring pollution by pesticides environmental problem, effect might not be obvious, and it is preventing and treating rough dwarf disease most efficient method to plant disease-resistant corn variety.Cultivate and identify that excellent disease-resistant maize is the matter of utmost importance of breeding resistant variety.Local main breed and China's major corn varieties are carried out with rough dwarf disease Resistance Identification and evaluation, high anti-kind and germplasm is screened, is most direct foundation and the guidance for carrying out anti-MRDV Genetic improvement and breeding research.
At present for the research of MRDV is main or the method for relying on field natural occurrence, the identification consuming cycle is long, and qualification result fluctuation is big, poor repeatability, and this brings difficulty for the rough dwarf disease resistance and genetic analyses for identifying milpa.Therefore, set up one and stablize effective rough dwarf disease method of resistance identification for identifying Resistant gerplasm resource, the disease resistance mechanisms of research plant, identify and clone disease-resistant gene and breeding for disease resistance all has very important significance.Therefore, it is a very necessary job to improve MRDV identification technology, and accurately and reliably rough dwarf disease Resistance Identification technology can ensure the cause of disease of MRDV and the seriality and repeatability of Disease Resistance Identification work.
We have discovered that and RBSDV is inoculated with to milpa, in either disease-resistant or susceptible milpa body, RBSDV virus is detected, after but milpa seedling stage has infected RBSDV viruses, symptom is not highlighted, its disease resistance is not can determine that, needs further field production its disease resistance could be investigated to aobvious disease.Stem-winding it is, we have discovered that the RBSDV growth rates after Semen Maydiss inoculation RBSDV viruses in early stage seedling body are directly related with corn variety resistance, the growth rate for being inoculated with restrovirus particle by inquiry can intuitively reflect resistance level of the corn germplasm to rough dwarf disease.Our research finds to provide new approaches to the identification of MRDV disease resistance, the present invention is exactly detected using RBSDV in early stage corn seedling body after fluorescence quantitative RT-RCR interface differential technique kind virus accordingly, the accurate measurement for connecing RBSDV virus multiplications speed in early stage corn seedling after poison is achieved by making RBSDV propagation trendgrams, so as to quickly and accurately identify resistance level and resistance class of the Semen Maydiss to rough dwarf disease.
Content of the invention
The purpose of the present invention is that have the shortcomings that length qualification cycle, qualification result are inaccurate for the anti-rough dwarf disease authentication method of current Semen Maydiss, there is provided a kind of anti-rough dwarf disease authentication method of qualification cycle is short, reliability is high Semen Maydiss.
The purpose of the present invention is solved by the following technical programs：
A kind of anti-rough dwarf disease authentication method of Semen Maydiss, it is characterised in that：The authentication method is comprised the following steps：
1）Select identification scale：High anti-, three identification scales of middle resistant, susceptible are arranged to MRDV performance according to different cultivars in poison identification is manually connect, and three corresponding corn varieties are selected as identification scale；
2）Hypoxia inducible Semen Maydiss to be measured and the plumule of identification scale：Each kind takes 10-16 grain seeds, is sterilized with 0.6% liquor natrii hypochloritises immersion 15min, and ultrapure water is clean, is then placed into hypoxia inducible culture 3 days in the IAA solution of suitable concentration under the conditions of 25 DEG C, induces plumule；
3）Light culture after the malicious small brown rice planthopper of plumule inoculation band：Plumule is cleaned with ultra-pure water, it is put into and is covered with the test tube of absorbent cotton, one seed of each test tube, quantitative inoculation band poison small brown rice planthopper, with horizontal after breathable gauze closed test tube, pass poison 24h, will pass malicious after plumule ultrapure water, light culture on the absorbent cotton of the ABA solution for being then placed under the conditions of 25 DEG C having infiltrated suitable concentration；
4）Quantitative fluorescent PCR obtains the RBSDV viruses S7 of each point corn variety cycle time and the relative expression quantity of reference gene（RQ values）：According to the plumule 40-60g that point cycle time gathers Semen Maydiss to be measured and identification scale ad-hoc location, each kind each time point of drawing materials is drawn materials 2-3 repetitions, -70 DEG C of preservations, extract RNA purification, take the above-mentioned viral RNA extracting solution of 1 μ L, quantitative fluorescent PCR reaction system is added, using primer RBSDV-F：5’-AGA
GCT CTT CTA GTT ATT GCG-3 ' and RBSDV-R：5 '-TCA GCA AAA GGT AAA GGA ACG-3 ' expand RBSDV virus S7 fragments according to fluorescent quantitative PCR program, amplification is finished, enter interpretation of result interface, with GAPDH as internal reference, compared with matched group, the relative expression quantity of RBSDV viruses S7 that each kind each sampling time point repeats and reference gene is obtained（RQ values）；
5）Draw RBSDV proliferation index Trendline：Using, the RQ value averages of each sampling time point after poison raised by each kind to be measured of the RQ Data-Statistics for repeating and identification scale more, with RQ value averages as vertical coordinate, after raising poison, cycle sampling time point is as abscissa, data are carried out with statistical analysiss, RBSDV proliferation index Trendline y=Ae is obtainedkx；
6）Resistance level and resistance class of the corn variety to be measured to MRDV are evaluated according to RBSDV proliferation index Trendline formula：According to each kind RBSDV proliferation index Trendline formula y=AekxEvaluate the resistance level of corn variety to be measured, i.e. in formula, the big kind of the k value kind less than k value is low to RBSDV resistances, kind k value to be measured is high anti-kind less than or equal to high anti-scale, kind k value to be measured more than high anti-scale and in being less than or equal to anti-scale be in anti-kind, kind k value to be measured be more than in anti-scale and be susceptible variety less than or equal to susceptible scale, kind k value to be measured is telegraphy kind more than susceptible scale.
The step 1）With 6）In high anti-identify that scale is connect for interior and disease-resistant during poison is identified show in local corn variety resource that ranking is 9%-11%；In anti-identify scale ranking be 23%-27%；Susceptible identify scale ranking be 46%-54%.
The step 2）In suitable concentration IAA solution concentrations be 2.0-3.0 μm of ol/L；Described hypoxia inducible method is placed under liquid level at 5-10cm for seed and cultivates.
The step 3）In band poison small brown rice planthopper obtaining step be：
3.1）RBSDV poison source obtains：From the Semen Maydiss tender diseased plant of children that the collection of MRDV severe disease area field shows as MRDV symptom, -70 DEG C of preservations；
3.2）Pass virus mediator successive propagation：Land for growing field crops collection purification small brown rice planthopper advanced age nymph, are raised with corn seedling and are passed on；
3.3）Amboceptor raises poison：MRDV diseased plant is taken out when raising poison from -70 DEG C of refrigerators, room temperature 2-3h makes diseased plant launch naturally, then closely wrapped up with plastic bag after wrapping up diseased plant root with water suction cotton, guarantee that water will not flow out, then move it in crisper, the small brown rice planthopper that indoors artificial is raised is proceeded on the RBSDV poison source Semen Maydiss in crisper, after 25 DEG C of feeding poison 48h, remove malicious source, small brown rice planthopper is transferred on corn seedling to raise and spends the malicious small brown rice planthopper of the phase of walking around to as band.
The step 3）In the ABA solution concentrations of suitable concentration be controlled to 35-45 μm of ol/L；The step 3）In quantitative inoculation band poison small brown rice planthopper be controlled to a 10-15 worms/plumule.
The step 4）In periodic sampling time point be that RBSDV passes after poison 3-4 time point equally distributed every 2.5-3.5 days in 4-18 days；Middle position of the plumule of ad-hoc location for plumule total length.
The step 4）In the method for extraction RNA be：Take appropriate blade liquid feeding nitrogen to be ground in mortar rapidly, it is proportionally added in TRI-Reagent to 1.5ml centrifuge tubes, sample volume is not to be exceeded TRI-Reagent volumes 10%, 4 DEG C of 12000 × g low-temperature centrifugation 1.5min, then carefully transfer supernatant in new centrifuge tube.
The step 4）In the method for purifying RNA be：a）Directly add a volume（95%-100%）To being dissolved in the supernatant of TRI-Reagent, whirlpool is mixed ethanol；b）It is transferred in adsorption column and filters（Adsorption column is placed in collecting pipe）, then 12000 × g centrifugations 1min, is transferred to adsorption column in new collecting pipe afterwards；c）Plus 400 μ l RNA Wash
To in centrifugal column, 12000 × g is centrifuged 1min to Buffer；d）The DNAase I cocktail that 80 μ l are prepared
Buffer is applied directly in centrifugal column, 25-37 DEG C of heating in water bath centrifugal column 15min, and 12000 × g is centrifuged 30s；e）Add 400 μ l RNA Prewash in adsorption column, 12000 × g is centrifuged 1min, collect the liquid for filtering, repeat this step；f）Add 700 μ l RNA Wash
To in adsorption column, 12000 × g is centrifuged 1min to Buffer, then from collecting pipe in the new RNase-free 1.5ml centrifuge tubes of careful transfer adsorption column to；g）At least add 25 μ l DNase/RNase-Free Water in centrifuge tube substrate, maximum velocity centrifugation 1min, eluted RNA can directly using or be stored in -70 DEG C of ultra cold storage freezers.
The step 4）In the every 20 μ L of fluorescent quantitation reaction system in contain：2 × qPCR master mix (Promega) of 10 μ L, the cDNA templates of 1.0 μ L, the ddH of 8.2 μ L210 M the primer RBSDV-R and RP of O and 0.8 μ L；Real-time fluorescence quantitative PCR amplification program is：95 DEG C of denaturations 2min；95 DEG C of degeneration 15s, 60 DEG C of annealing 15s, 72 DEG C of extension 20s, carry out 40 circulations altogether；95 DEG C of degeneration 15s, 60 DEG C of annealing 1min, 95 DEG C of degeneration 15s.
The step 3.3）In RBSDV poison source corn sources in step 3.1）In the Semen Maydiss tender diseased plant of children for showing as MRDV symptom.
Description of the drawings
Accompanying drawing is drawn materials by the cycle after identification scale plumule biography poison selected to embodiment and is detected that RBSDV concentration change obtains RBSDV proliferation index trendgrams.
Specific embodiment
With reference to embodiment, the present invention is further illustrated.
A kind of method for identifying molecules of MRDV, it is characterised in that：The authentication method is comprised the following steps：
1）Select identification scale：High anti-, three identification scales of middle resistant, susceptible are arranged to MRDV performance according to different cultivars in poison identification is manually connect, three corresponding corn varieties are selected as identification scale, wherein, high anti-identify that scale is connect for interior and disease-resistant during poison is identified show that ranking is 9%-11% in local corn variety resource, in anti-identify scale ranking for 23%-27%, susceptible identify that scale ranking is 46%-54%；
2）Hypoxia inducible Semen Maydiss to be measured and the plumule of identification scale kind：Each kind takes 10-16 grain seeds, is sterilized with 0.6% liquor natrii hypochloritises immersion 15min, and ultrapure water is clean, is then placed under the IAA liquid level of solution of 2.0-3.0 μm of ol/L under the conditions of 25 DEG C hypoxia inducible culture 3 days at 5-10cm, induces plumule；
3）Light culture after the malicious small brown rice planthopper of plumule inoculation band：
3.1）RBSDV poison source obtains：From the Semen Maydiss tender diseased plant of children that the collection of MRDV severe disease area field shows as MRDV symptom, -70 DEG C of preservations；
3.2）Pass virus mediator successive propagation：Land for growing field crops collection purification small brown rice planthopper advanced age nymph, are raised with corn seedling and are passed on；
3.3）Amboceptor raises poison：MRDV diseased plant is taken out when raising poison from -70 DEG C of refrigerators, room temperature 2-3h makes diseased plant launch naturally, then closely wrapped up with plastic bag after wrapping up diseased plant root with water suction cotton, guarantee that water will not flow out, then move it in crisper, the 1.5-2.5 age small brown rice planthopper nymphs that indoors artificial is raised are proceeded on the RBSDV poison source Semen Maydiss in crisper, the RBSDV poison source Semen Maydiss are 3.1）In the tillering regularity Semen Maydiss diseased plant for showing as MRDV symptom, 25 DEG C raise poison 48h after remove malicious source, by small brown rice planthopper be transferred on corn seedling raise spend the phase of walking around to be band poison small brown rice planthopper；
3.4）The malicious small brown rice planthopper of plumule inoculation band：Plumule is cleaned with ultra-pure water, is put into and is covered with the test tube of absorbent cotton, one seed of each test tube is quantitatively inoculated with the malicious small brown rice planthopper of band according to the ratio of a 10-15 worms/plumule, with horizontal after breathable gauze closed test tube, passes poison 24h；
3.5）Plumule connects light culture after poison：By plumule ultrapure water, light culture on the absorbent cotton of the ABA solution for being then placed under the conditions of 25 DEG C having infiltrated 35-45 μm of ol/L；
4）Quantitative fluorescent PCR obtains the RBSDV viruses S7 of each point corn variety cycle time and the relative expression quantity of reference gene（RQ values）：Small brown rice planthopper passes the middle position 40-60g that poison terminates to gather Semen Maydiss to be measured and identification scale coleoptile length in latter 4-18 days in the equally distributed 3-4 time point every 2.5-3.0 days, each kind each time point of drawing materials is drawn materials 2-3 repetitions, -70 DEG C of preservations, extract RNA purification（RNA extraction method is：Take appropriate blade liquid feeding nitrogen to be ground in mortar rapidly, it is proportionally added in TRI-Reagent to 1.5ml centrifuge tubes, sample volume is not to be exceeded TRI-Reagent volumes 10%, 4 DEG C of 12000 × g low-temperature centrifugation 1.5min, then carefully transfer supernatant in new centrifuge tube；RNA purification process is：a）Directly add a volume（95%-100%）To being dissolved in the supernatant of TRI-Reagent, whirlpool is mixed ethanol；b）It is transferred in adsorption column and filters（Adsorption column is placed in collecting pipe）, then 12000 × g centrifugations 1min, is transferred to adsorption column in new collecting pipe afterwards；c）Plus 400 μ l RNA Wash
To in centrifugal column, 12000 × g is centrifuged 1min to Buffer；d）The DNAase I cocktail that 80 μ l are prepared
Buffer is applied directly in centrifugal column, 25-37 DEG C of heating in water bath centrifugal column 15min, and 12000 × g is centrifuged 30s；e）Add 400 μ l RNA Prewash in adsorption column, 12000 × g is centrifuged 1min, collect the liquid for filtering, repeat this step；f）Add 700 μ l RNA Wash
To in adsorption column, 12000 × g is centrifuged 1min to Buffer, then from collecting pipe in the new RNase-free 1.5ml centrifuge tubes of careful transfer adsorption column to；g）At least add 25 μ l DNase/RNase-Free Water in centrifuge tube substrate, maximum velocity centrifugation 1min, eluted RNA can directly using or be stored in -70 DEG C of ultra cold storage freezers.）The above-mentioned viral RNA extracting solution of 1 μ L is taken, quantitative fluorescent PCR reaction system is added, using primer RBSDV-F：5’-AGA
GCT CTT CTA GTT ATT GCG-3 ' and RBSDV-R：5 '-TCA GCA AAA GGT AAA GGA ACG-3 ' expand RBSDV virus S7 fragments according to fluorescent quantitative PCR program, amplification is finished, enter interpretation of result interface, with GAPDH as internal reference, compared with matched group, the relative expression quantity of RBSDV viruses S7 that each kind each sampling time point repeats and reference gene is obtained（RQ values）；
5）Draw RBSDV proliferation index Trendline：Using, the RQ value averages of each sampling time point after poison raised by each kind to be measured of the RQ Data-Statistics for repeating and identification scale more, with RQ value averages as vertical coordinate, after raising poison, cycle sampling time point is as abscissa, data are carried out with statistical analysiss, RBSDV proliferation index Trendline y=Ae is obtainedkx；
6）Resistance level and resistance class of the corn variety to be measured to MRDV are evaluated according to RBSDV proliferation index Trendline formula：
6.1）Kind resistance level to be measured is evaluated：According to each kind RBSDV proliferation index Trendline formula y=AekxEvaluate the big kind of the k value kind less than k value in the resistance level of corn variety to be measured, i.e. formula low to RBSDV resistances；
6.2）Kind resistance class to be measured is divided：The resistance class of kind to be measured is evaluated according to kind to be measured and identification scale k value size, kind k value i.e. to be measured is high anti-kind less than or equal to high anti-scale, kind k value to be measured more than high anti-scale and in being less than or equal to anti-scale be in anti-kind, kind k value to be measured be more than in anti-scale and less than or equal to susceptible scale be susceptible variety, kind k value to be measured more than susceptible scale be telegraphy kind.
Embodiment
2014, rough dwarf disease Resistance Identification has been carried out to the part corn variety resource that collection is obtained using the authentication method of the invention, it is respectively provided with red 340, pearl is sweet No. 1, single No. 7 three corn varieties that help identify scale as high anti-, middle resistant, susceptible, wherein, red 340 connect in 2012-2013 interiors and disease-resistant in poison identification show ranking the 7th in 68 corn variety sources groups, sweet No. 1 ranking 17 of pearl, single No. 7 rankings 34 of Ji；The IAA solution concentrations of process are chosen as 2.5 μm of ol/L；ABA solution concentrations are chosen as 40 μm of ol/L；Seed is placed under liquid level culture induction plumule at 8cm；Quantitative inoculation band poison small brown rice planthopper is controlled to a 12 worms/plumule；Plumule acquisition time is set to connect the 5th, 8,11,14 days four time points after the completion of poison.
Real-time fluorescence quantitative PCR obtains the RQ values of each kind periodic sampling time point, as shown in table 1：
Then RBSDV proliferation index Trendline is drawn：With RQ value averages as vertical coordinate, after raising poison, sampling time point is abscissa, and data are carried out with statistical analysiss, obtains RBSDV proliferation index Trendline, as shown in drawings.
The rough dwarf disease resistance level and resistance class of corn variety to be measured are evaluated according to the difference of each kind k value of RBSDV proliferation index trendgrams.In this experiment, 340 k value of high anti-scale pellet are calculated as 0.6368；In sweet No. 1 k value of anti-scale pearl be calculated as 1.4271；Susceptible scale Ji No. 7 k value of list are calculated as 1.9715, as shown in drawings.Kind k value to be measured compares the corresponding resistance class of acquisition with identification scale, and we identify germplasm rough dwarf disease resistance to following 8 corn varieties using the authentication method of the present invention, as a result such as table 2.
By the comparison of result that the qualification result of table 2 and field multiple years rough dwarf disease are fallen ill, we have found that the qualification result is coincide with land for growing field crops morbidity performance very much, and the present invention just can be accurately detected to germplasm rough dwarf disease resistance in the short time after biography virus mediator connects malicious maize coleoptile, so as to substantially reduce qualification time.
By embodiment it is found that the new Identification Method of the present invention compared with the method for anti-perception is evaluated using illness is investigated plumule inner virus growth rate to evaluate MRDV varietal resistance after connecing malicious maize coleoptile, substantially reduces qualification cycle；And by detecting that plumule inner virus growth rate determines resistance level and resistance class of the corn germplasm to MRDV from molecular level, reliability is higher, thus there is the present invention larger Breeding Application to be worth.
Above example technological thought only to illustrate the invention, it is impossible to which protection scope of the present invention is limited with this, every according to technological thought proposed by the present invention, any change that is done on the basis of technical scheme, each fall within the scope of the present invention；The technology that the present invention is not directed to can be realized by prior art.
Claims (10)
1. the anti-rough dwarf disease authentication method of a kind of Semen Maydiss, it is characterised in that：The authentication method is comprised the following steps：
1）Select identification scale：High anti-, three identification scales of middle resistant, susceptible are arranged to MRDV performance according to different cultivars in poison identification is manually connect, and three corresponding corn varieties are selected as identification scale；
2）Hypoxia inducible Semen Maydiss to be measured and the plumule of identification scale：Each kind takes 10-16 grain seeds, is sterilized with 0.6% liquor natrii hypochloritises immersion 15min, and ultrapure water is clean, is then placed into hypoxia inducible culture 3 days in the IAA solution of suitable concentration under the conditions of 25 DEG C, induces plumule；
3）Light culture after the malicious small brown rice planthopper of plumule inoculation band：Plumule is cleaned with ultra-pure water, it is put into and is covered with the test tube of absorbent cotton, one seed of each test tube, quantitative inoculation band poison small brown rice planthopper, with horizontal after breathable gauze closed test tube, pass poison 24h, will pass malicious after plumule ultrapure water, light culture on the absorbent cotton of the ABA solution for being then placed under the conditions of 25 DEG C having infiltrated suitable concentration；
4）Quantitative fluorescent PCR obtains the RBSDV viruses S7 of each point corn variety cycle time and the relative expression quantity of reference gene（RQ values）：According to the plumule 40-60g that point cycle time gathers Semen Maydiss to be measured and identification scale ad-hoc location, each kind each time point of drawing materials is drawn materials 2-3 repetitions, -70 DEG C of preservations, extract RNA purification, take the above-mentioned viral RNA extracting solution of 1 μ L, quantitative fluorescent PCR reaction system is added, using primer RBSDV-F：5 '-AGA GCT CTT CTA GTT ATT GCG-3 ' and RBSDV-R：5’-TCA GCA AAA
GGT AAA GGA ACG-3 ' expand RBSDV virus S7 fragments according to fluorescent quantitative PCR program, amplification is finished, enter interpretation of result interface, with GAPDH as internal reference, compared with matched group, the relative expression quantity of RBSDV viruses S7 that each kind each sampling time point repeats and reference gene is obtained（RQ values）；
5）Draw RBSDV proliferation index Trendline：Using, the RQ value averages of each sampling time point after poison raised by each kind to be measured of the RQ Data-Statistics for repeating and identification scale more, with RQ value averages as vertical coordinate, after raising poison, cycle sampling time point is as abscissa, data are carried out with statistical analysiss, RBSDV proliferation index Trendline y=Ae is obtainedkx；
6）Resistance level and resistance class of the corn variety to be measured to MRDV are evaluated according to RBSDV proliferation index Trendline formula：According to each kind RBSDV proliferation index Trendline formula y=AekxEvaluate the resistance level of corn variety to be measured, i.e. in formula, the big kind of the k value kind less than k value is low to RBSDV resistances, kind k value to be measured is high anti-kind less than or equal to high anti-scale, kind k value to be measured more than high anti-scale and in being less than or equal to anti-scale be in anti-kind, kind k value to be measured be more than in anti-scale and be susceptible variety less than or equal to susceptible scale, kind k value to be measured is telegraphy kind more than susceptible scale.
2. the anti-rough dwarf disease authentication method of Semen Maydiss according to claim 1, it is characterised in that：The step 1）With 6）In high anti-identify that scale is connect for interior and disease-resistant during poison is identified show in local corn variety resource that ranking is 9%-11%；In anti-identify scale ranking be 23%-27%；Susceptible identify scale ranking be 46%-54%.
3. the anti-rough dwarf disease authentication method of Semen Maydiss according to claim 1, it is characterised in that：The step 2）In suitable concentration IAA solution concentrations be 2.0-3.0 μm of ol/L；Described hypoxia inducible method is placed under liquid level at 5-10cm for seed and cultivates.
4. the anti-rough dwarf disease authentication method of Semen Maydiss according to claim 1, it is characterised in that：The step 3）In band poison small brown rice planthopper obtaining step be：
3.1）RBSDV poison source obtains：From the Semen Maydiss tender diseased plant of children that the collection of MRDV severe disease area field shows as MRDV symptom, -70 DEG C of preservations；
3.2）Pass virus mediator successive propagation：Land for growing field crops collection purification small brown rice planthopper advanced age nymph, are raised with corn seedling and are passed on；
3.3）Amboceptor raises poison：MRDV diseased plant is taken out when raising poison from -70 DEG C of refrigerators, room temperature 2-3h makes diseased plant launch naturally, then closely wrapped up with plastic bag after wrapping up diseased plant root with water suction cotton, guarantee that water will not flow out, then move it in crisper, the small brown rice planthopper that indoors artificial is raised is proceeded on the RBSDV poison source Semen Maydiss in crisper, after 25 DEG C of feeding poison 48h, remove malicious source, small brown rice planthopper is transferred on corn seedling to raise and spends the malicious small brown rice planthopper of the phase of walking around to as band.
5. the anti-rough dwarf disease authentication method of Semen Maydiss according to claim 1, it is characterised in that：The step 3）In the ABA solution concentrations of suitable concentration be controlled to 35-45 μm of ol/L；The step 3）In quantitative inoculation band poison small brown rice planthopper be controlled to a 10-15 worms/plumule.
6. the anti-rough dwarf disease authentication method of Semen Maydiss according to claim 1, it is characterised in that：The step 4）In periodic sampling time point be that RBSDV passes after poison 3-4 time point equally distributed every 2.5-3.5 days in 4-18 days；Middle position of the plumule of ad-hoc location for plumule total length.
7. the anti-rough dwarf disease authentication method of Semen Maydiss according to claim 1, it is characterised in that：The step 4）In the method for extraction RNA be：Take appropriate blade liquid feeding nitrogen to be ground in mortar rapidly, it is proportionally added in TRI-Reagent to 1.5ml centrifuge tubes, sample volume is not to be exceeded TRI-Reagent volumes 10%, 4 DEG C of 12000 × g low-temperature centrifugation 1.5min, then carefully transfer supernatant in new centrifuge tube.
8. the anti-rough dwarf disease authentication method of Semen Maydiss according to claim 1, it is characterised in that：The step 4）In the method for purifying RNA be：a）Directly add a volume（95%-100%）To being dissolved in the supernatant of TRI-Reagent, whirlpool is mixed ethanol；b）It is transferred in adsorption column and filters（Adsorption column is placed in collecting pipe）, then 12000 × g centrifugations 1min, is transferred to adsorption column in new collecting pipe afterwards；c）Plus 400 μ l RNA Wash Buffer in centrifugal column, 12000 × g be centrifuged 1min；d）The DNAase I cocktail buffer that 80 μ l are prepared are applied directly in centrifugal column, 25-37 DEG C of heating in water bath centrifugal column 15min, and 12000 × g is centrifuged 30s；e）Add 400 μ l RNA Prewash in adsorption column, 12000 × g is centrifuged 1min, collect the liquid for filtering, repeat this step；f）Add 700 μ l RNA Wash Buffer in adsorption column, 12000 × g is centrifuged 1min, then from collecting pipe in the new RNase-free 1.5ml centrifuge tubes of careful transfer adsorption column to；g）At least add 25 μ l DNase/RNase-Free Water in centrifuge tube substrate, maximum velocity centrifugation 1min, eluted RNA can directly using or be stored in -70 DEG C of ultra cold storage freezers.
9. the anti-rough dwarf disease authentication method of Semen Maydiss according to claim 1, it is characterised in that：The step 4）In the every 20 μ L of fluorescent quantitation reaction system in contain：2 × qPCR master mix (Promega) of 10 μ L, the cDNA templates of 1.0 μ L, the ddH of 8.2 μ L210 M the primer RBSDV-R and RP of O and 0.8 μ L；Real-time fluorescence quantitative PCR amplification program is：95 DEG C of denaturations 2min；95 DEG C of degeneration 15s, 60 DEG C of annealing 15s, 72 DEG C of extension 20s, carry out 40 circulations altogether；95 DEG C of degeneration 15s, 60 DEG C of annealing 1min, 95 DEG C of degeneration 15s.
10. the anti-rough dwarf disease authentication method of Semen Maydiss according to claim 4, it is characterised in that：The step 3.3）In RBSDV poison source corn sources in step 3.1）In the Semen Maydiss tender diseased plant of children for showing as MRDV symptom.
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| CN104789703A (en) * | 2015-05-20 | 2015-07-22 | 王开 | Method for rapid evaluation of variety resistance to bushy stunt of wheat by detecting proliferation rate of virus in coleoptiles |
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