CN101828521B - Method for quick transformation of major QTL with resistance to fusarium head blight through combination of embryo culture of wheat and sign selection - Google Patents
Method for quick transformation of major QTL with resistance to fusarium head blight through combination of embryo culture of wheat and sign selection Download PDFInfo
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- CN101828521B CN101828521B CN2010101751508A CN201010175150A CN101828521B CN 101828521 B CN101828521 B CN 101828521B CN 2010101751508 A CN2010101751508 A CN 2010101751508A CN 201010175150 A CN201010175150 A CN 201010175150A CN 101828521 B CN101828521 B CN 101828521B
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Abstract
The invention relates to a method for quick transformation of major QTL with resistance to fusarium head blight through the combination of embryo culture of wheat and sign selection, which is characterized in that: the method for the quick transformation of major QTL with resistance to fusarium head blight combines the backcross transformation of fusarium head blight resistance, strong seedling culture of embryos, the auxiliary selection of signs and quick seedling technology; wheat varieties or strains which have excellent economical characters but are susceptible to the fusarium head blightare taken as a receptor parent, and wheat varieties or strains with resistance to the fusarium head blight are taken as an antigen male parent; the receptor parent is subjected to castration at the heading stage and is hybridized with the antigen male parent, and then the embryos are picked off; the embryos are cultured into seedlings, and DNA of lamina is extracted; SSR primers Xgwm493 and Xgwm533 are taken as the signs to perform PCR detection on the DNA samples, plants containing the signs are left to continue growing, the embryos are picked off again, and the backcross is performed for multiple times by the same method; and finally the selfing is performed to obtain new germplasm of the transformation major QTL with the resistance to the fusarium head blight. The method has the advantages of short culture period and high seed selection efficiency of the new germplasm.
Description
Technical field
The present invention relates to a kind of wheat immature embryo and cultivate the method that incorporation of markings is selected rapid transfer anti gibberellic disease main effect QTL, the plant rataria is cultivated with molecular labeling be combined in the application in the wheat anti gibberellic disease breeding.
Technical background
Little head blight is one of main pathogenic bacteria of harm improving yield of wheat, stable yields.Since the later stage eighties; Because the influence of Global climate change and cropping system; Wheat scab frequently takes place on Europe, the U.S., Canada, China, South America and other places; Wheat yield and quality have been caused serious loss, and people have carried out research extensively and profoundly to wheat scab for this reason, and have obtained significant achievement.China is the maximum country of global wheat scab injured area, and the middle and lower reach of Yangtze River and south China winter wheat district and east, northeast spring wheat district are particularly serious, and the generation area of annual wheat scab has surpassed 7,000,000 hm
2, account for 1/4 of the national wheat gross area.Therefore, the germplasm of middle and lower reach of Yangtze River Mai Qu initiative anti gibberellic disease, cultivation anti-disease wheat new varieties are the fundamental ways that overcome head blight harm.
But because Fusarium graminearum (Fusarium graminearum) is infected the wheat head at flowering stage of wheat; Infecting and expanding of it depends on that locality, weather condition at that time are weather conditions such as temperature and humidity (Lu Weizhong etc. 2000), and this relies on phenotype to resist the selection of red property to bring difficulty to breeding man.In addition, wheat scab is by the quantitative character of controlled by multiple genes (Zhang Yong etc., 2005), and the resistance qualification process is prone to affected by environment and wastes time and energy.Molecular marker assisted selection (MAS; Marker-assisted selection) brand-new approach is provided for the selection that makes a variation in the breeding material; Molecular labeling be based upon on the DNA basis a kind of genetics mark it have not affected by environment; The advantage that in any developmental stage of crop and any organ, tissue, can detect so just can realize indirectly objective trait is carried out identifying seedling stage with early for selection, thereby the qualification result that makes genes of interest more accurately and reliably.Not only improve the accuracy that proterties is selected, and accelerated the speed of character test, thereby reached purpose (Dudley etc., 1993 of improving breeding efficiency; Lee etc., 1995).
Except the application of molecular marker assisted selection in breeding, problem such as traditional breeding technology still exists breeding cycle long partially, and breeding efficiency is not ideal enough.Although the strange land adds generation and can realize 1 year 2-3 generation, testing expenses are higher, therefore explore the cultivation research that breeding cycle is shortened, quickens new wheat germplasm just seem very necessary and urgent (Wang Haibo etc., 2003).Accelerate the speed of wheat breeding; At first must accelerate the rate of development of wheat whole growth phase; Yet existing wheat immature embryo culture technique is mainly used in still that the distant hybridization embryo is saved or be the research (Wang Haibo etc. of explant induction callus plant regeneration and transgenosis aspect with the rataria; 2006), to how combining with the MAS back cross breeding of anti gibberellic disease breeding through the rataria cultivation under the many wet conditions of southern summer high temperature, the research of realization anti gibberellic disease gene rapid transfer but seldom.
Research shows, on the 3BS of wheat-resistance to scab kind or strain chromosome, has the disease-resistant QTL of main effect (all vast equality, 2003; Zhang etc. 2004), Xgwm493, Xgwm533 are SSR molecular labeling closely linked with it (Bai etc., 2002; 2003; Lu Weizhong, 2000).We utilize molecular marker assisted selection in the scab resistance improvement, to obtain first-stage success in recent years, have bred a plurality of anti-red property new varieties preferably in succession.But must could obtain the background resistant material consistent with recurrent parent owing to backcross at advanced lines in the strategy, needs employing proper method shortening cycle of backcrossing is good and carry the disease-resistant germplasm of high yield of resistance QTL chromosome segment to obtain yielding ability as early as possible.Therefore; How the wheat immature embryo cultivation is expected to shorten the back cross breeding time limit with the molecular labeling application in conjunction in the back cross breeding strategy; Set up the efficient anti gibberellic disease gene transformation procedure of breeding, obtain to have the material of backcrossing of genes of interest (QTL) as early as possible, be the focus that this area is paid close attention to always.
At present, cultivate the technology that combines with molecular labeling through wheat immature embryo, anti gibberellic disease main effect QTL transformation in wheat-resistance to scab kind or the strain is good but feel the method in head blight wheat breed or the strain to economical character, do not appear in the newspapers.
Summary of the invention
The objective of the invention is to: the transformation of anti gibberellic disease main effect QTL is poor to anti gibberellic disease to present applied molecular labeling technique; But the long practical problem of breeding cycle in the good kind of other comprehensive proterties provides a kind of new wheat immature embryo to cultivate the method that incorporation of markings is selected rapid transfer anti gibberellic disease main effect QTL.
The objective of the invention is to realize like this: a kind of wheat immature embryo is cultivated the method that incorporation of markings is selected rapid transfer anti gibberellic disease main effect QTL; It is characterized in that: comprise the rapid transfer anti gibberellic disease main effect QTL that anti gibberellic disease backcross transformation, rataria strong seedling culture, marker assisted selection, fast rapid-result seedling technology combine, concrete steps are following:
A) good but sense head blight wheat breed or strain are receptor parent with economical character, be anti-source male parent with wheat-resistance to scab kind or strain, receptor parent castrate heading stage with anti-source paternal hybrid after strip rataria;
B) transfer to 25 ℃/15 ℃ illumination cultivation Cheng Miao at daytime/night after 4 ℃ of following spring flowers are cultivated 7d; After becoming seedling 5d; Be transplanted to and grow in 28 ℃/15 ℃ greenhouses daytime/night; Getting half blade simultaneously and extract DNA, is that mark carries out the PCR detection to the DNA sample with SSR primer Xgwm493, Xgwm533, leaves and takes to contain underlined plant continued growth;
C) plant castration at heading stage is hybridized with backcross parent, and described backcross parent is sense head blight wheat breed receptor parent;
D) pollination back 12d strips rataria under the sterile working of laboratory;
E) repeat 4～5 times according to the samsara of b to d step, last samsara repeats to finish the back in the b step and directly gets into f;
F) blooming stage selfing, seed selection have anti gibberellic disease main effect QTL and the good new germ plasm of economical character.
In the present invention: the described rataria that strips is meant: the young grain of spending back 12d is through 75% alcohol disinfecting 30s and 0.1% mercuric chloride sterilization, 10～15min, aseptic washing 3 times; Under aseptic condition, rataria is stripped out; Described vernalization is cultivated and is meant: with the rataria pointed end in the 1/2MS medium that adds 0.3% malt extract 4 ℃ cultivated 7 days.
In the present invention: after becoming seedling 5d; Being transplanted to grows in 28 ℃/15 ℃ greenhouses daytime/night is meant: after becoming seedling 5d; In nutrition soil, the 2～3d that shades cultivates in 28 ℃/18 ℃ greenhouses of diurnal temperature behind the slow seedling with the rataria transplantation of seedlings; Shooting stage blade face spray is every at a distance from one time 0.3% potassium dihydrogen phosphate of 5d spray, sprays secondary continuously; Described nutrition soil is mixed by land for growing field crops soil, peat soil and the perlite volume ratio according to 6: 3: 1.
In the present invention: described economical character good but sense head blight wheat breed or strain are for raising wheat No. 15 or raising wheat 13 or Huaihe River wheat 18; Wheat-resistance to scab kind or strain are Soviet Union wheat No. 3 or peaceful 894037 or peaceful 7840.
The invention has the advantages that: combine with molecular labeling through wheat immature embryo is cultivated; Set up a kind of anti gibberellic disease transformation, samsara and repeated that rataria strong seedling culture, seedling replanting are educated soon, molecular marker assisted selection; Rapid transfer anti gibberellic disease main effect QTL in perceptual head blight wheat breed or strain; Shorten cultivation period greatly, improved new wheat germplasm seed selection efficient effectively.
Embodiment
Embodiment 1
1, receptor parent and recurrent parent: the weak muscle wheat of high-quality is raised wheat 15
2, anti-source male parent: wheat-resistance to scab kind Soviet Union wheat No. 3
3, medium: adopt and add 0.3% malt extract (malt extract) in 1/2MS (pH 5.8) solid culture medium.
4, nutrition soil: land for growing field crops soil, peat soil, perlite mix with 6: 3: 1 volume ratio.
5, primer design and synthetic:
Qfhs.ndsu.3 BS primer Xgwm493, Xgwm533 sequence are delivered referring to (1998) such as
, give birth to worker company by Shanghai and synthesize.
6, leaf DNA is extracted:
Half blade of each rataria seedling of clip put into and clayed into power after mortar adds liquid nitrogen, divides to install in the 1.5mlenpperdof pipe, adds the CTAB extract of 65 ℃ of preheatings of 0.6ml, 65 ℃ of water-bath 60min.Sample places room temperature after taking out, and adds isopyknic chloroform: isoamyl alcohol (24: 1) mixed liquor, and after the careful upset, the centrifugal 10min of 10000rpm (25 ℃).Centrifugal back supernatant changes in the enpperdof pipe of isoamyl alcohol of another precooling that contains 0.6 volume, shakes gently to the DNA deposition and separates out, and leaves standstill the back hook and goes out the DNA deposition.Add the 0.7ml70% washing with alcohol 3 times, the supernatant that inclines dries DNA is adherent.Add an amount of TE buffer solution, the dissolving DNA deposition.Add 3-5ulRNase (5mg/ml), 37 ℃ of water-bath 60min.Add isopyknic chloroform: isoamyl alcohol (24: 1), jog, the centrifugal 10min of 12000rpm.Supernatant changes in the enpperdof pipe of 3M NaAc of another absolute ethyl alcohol that contains 2 times of volumes (800ul) and 1/10 volume (40ul), slowly upset, mixing;-20 ℃ leave standstill 2h after; Hook goes out DNA deposition, adds 0.7ml 70% washing with alcohol 3-5 time, dries DNA is adherent.Add an amount of TE buffer solution, the dissolving DNA deposition.DNA is carried out 0.8% agarose electrophoresis, confirm DNA quality and concentration.
7, agarose electrophoresis detects
Behind the DNA extraction, adopt 0.8% agarose electrophoresis, confirm quality and the concentration of DNA.
8, molecular labeling electrophoresis detection
PCR is reflected at PTC-100TM Programmable Thermal Controller, and (MJ Research carries out on INC.), and reaction volume is 20 μ l.Reaction mixture comprises 1 * buffer, 1.5mmol/L MgCl2,2.0mmol/L dNTPs, 250 μ mol/L primers, 50-100ng template DNA, 1U Taq enzyme.Response procedures is: 94 ℃, and 5min; 94 ℃ of 45sec, 55-60 ℃, 45sec, 72 ℃, 45sec, 40 circulations; 72 ℃, 10min.4 ℃ of preservations.Amplified production is through 6% polyacrylamide gel electrophoresis, and silver dyes the colour developing imaging.
Detailed process:
Castrate and anti-source paternal hybrid with receptor parent (female parent) heading stage in the field, field pollination on April 10th, 2007, and strip rataria and cultivate April 22 (spending back 12d).The method of getting rataria is: the young grain of heading just is through 75% alcohol disinfecting 30s and 0.1% mercuric chloride sterilization, 10～15min, aseptic washing 3 times; Under aseptic condition, rataria is stripped out (get under the method for rataria with).
The first generation: got rataria on April 22 and cultivate, April 26 vernalization (with the rataria pointed end in the 1/2MS medium that adds 0.3% malt extract 4 ℃ cultivated 7 days, down with); After becoming seedling 5d, in nutrition soil, 2～3d shades with the rataria transplantation of seedlings; In 28 ℃/18 ℃ greenhouses of diurnal temperature, cultivate behind the slow seedling, May 10 transplanted, and got half blade and extract DNA; With SSR primer Xgwm493, Xgwm533 is that mark carries out the PCR detection to the DNA sample, leaves and takes to contain underlined plant continued growth, and shooting stage blade face spray is every at a distance from one time 0.3% potassium dihydrogen phosphate of 5d spray; Spray secondary continuously, flowering on June 15 was got the 12d rataria and is cultivated on June 28.(67d breeding time) (second generation to the five generations part process is identical, only is the clue summary with time)
The second generation: embryo culture on June 28 in 2007, July 1, vernalization was transplanted July 16 (Markers for Detection), and September, flowering on the 1st castration at heading stage was backcrossed with backcross parent, got rataria (78d breeding time) on September 14.
The third generation: embryo culture on September 14 in 2007, vernalization on September 18 was transplanted October 2 in booth (Markers for Detection), and November, flowering on the 8th castration at heading stage was backcrossed with backcross parent, and can get rataria on November 23 and cultivate (71d breeding time),
The 4th generation: embryo culture on November 23 in 2007, vernalization on November 27, intermediate house on December 12 (Markers for Detection), February in 2008, flowering on the 24th castration at heading stage was backcrossed with backcross parent, got 12d rataria (103d breeding time) on March 8.Condition is relatively poor because winter greenhouse is heated, and prolong breeding time.
The 5th generation: embryo culture on March 8 in 2008, vernalization on March 11 was transplanted March 25 in booth nutritive cube (Markers for Detection), flowering selfing on April 27, June 1 was gathered in the crops seed (84d breeding time), and planted in fall screens, identifies to the land for growing field crops.
On April 25th, 2009 offspring with mark of being transplanted to the field is carried out scab resistance and identify, at 87 BC
4F
2Detecting the strain that has from resistance parent Xgwm493 loci in offspring's strain system is 21, resistance marker recall rate 24.1%; Strain with resistance parent Xgwm533 mark is 16, resistance marker recall rate 18.4%; The strain that has two kinds of marks simultaneously is 12, and mark recall rate 13.8% is carried out the head blight inoculation and identified and show detecting the strain system with mark, contains simultaneously anti-ly in the strain system of Xgwm493, Xgwm533 mark to reach 75% to high anti gibberellic disease plant ratio; Contain that anti gibberellic disease strain system accounts for 68.7% in the strain system of Xgwm533 mark, secondly be contain have in the strain system of Xgwm493 14 anti gibberellic diseases reach in anti-above level, the efficient of label screening reaches 66.7%; It is thus clear that in the transformation of wheat scab resistance main effect QTL, adopting molecular marker assisted selection to combine rataria to cultivate is efficiently.
Above embodiment is not to concrete restriction of the present invention, and those of ordinary skill in the art combines the open method according to the present invention, selects different receptor parents, recurrent parent and anti-source male parent, all falls into protection scope of the present invention.
Claims (4)
1. a wheat immature embryo is cultivated the method that incorporation of markings is selected rapid transfer anti gibberellic disease main effect QTL; It is characterized in that: comprise the rapid transfer anti gibberellic disease main effect QTL that anti gibberellic disease backcross transformation, rataria strong seedling culture, marker assisted selection, fast rapid-result seedling technology combine, concrete steps are following:
A) good but sense head blight wheat breed or strain are receptor parent with economical character, be anti-source male parent with wheat-resistance to scab kind or strain, receptor parent castrate heading stage with anti-source paternal hybrid after strip rataria;
B) transfer to 25 ℃/15 ℃ illumination cultivation Cheng Miao at daytime/night after 7d is cultivated in 4 ℃ of following vernalization; After becoming seedling 5d; Be transplanted to and grow in 28 ℃/15 ℃ greenhouses daytime/night; Getting half blade simultaneously and extract DNA, is that mark carries out the PCR detection to the DNA sample with SSR primer Xgwm493, Xgwm533, leaves and takes to contain underlined plant continued growth;
C) plant castration at heading stage is hybridized with backcross parent, and described backcross parent is sense head blight wheat breed receptor parent;
D) pollination back 12d strips rataria under the sterile working of laboratory;
E) repeat 4～5 times according to the samsara of b to d step, last samsara repeats to finish the back in the b step and directly gets into f;
F) blooming stage selfing, seed selection have anti gibberellic disease main effect QTL and the good new germ plasm of economical character.
2. wheat immature embryo according to claim 1 is cultivated the method that incorporation of markings is selected rapid transfer anti gibberellic disease main effect QTL; It is characterized in that: the described rataria that strips is meant: the seed of pollination back 12d is through 75% alcohol disinfecting 30s and 0.1% mercuric chloride sterilization, 10～15min, aseptic washing 3 times; Under aseptic condition, rataria is stripped out; Described vernalization is cultivated and is meant: with the rataria pointed end in the 1/2MS medium that adds 0.3% malt extract 4 ℃ cultivated 7 days.
3. wheat immature embryo according to claim 2 is cultivated the method that incorporation of markings is selected rapid transfer anti gibberellic disease main effect QTL; It is characterized in that: after becoming seedling 5d, being transplanted to grow in 28 ℃/15 ℃ greenhouses daytime/night is meant: behind the one-tenth seedling 5d, with the rataria transplantation of seedlings in nutrition soil; 2～3d shades; In 28 ℃/18 ℃ greenhouses of diurnal temperature, cultivate behind the slow seedling, the shooting stage blade face is every at a distance from one time 0.3% potassium dihydrogen phosphate of 5d spray, sprays secondary continuously; Described nutrition soil is mixed by land for growing field crops soil, peat soil and the perlite volume ratio according to 6: 3: 1.
4. cultivate the method that incorporation of markings is selected rapid transfer anti gibberellic disease main effect QTL according to the described wheat immature embryo of one of claim 1～3, it is characterized in that: described economical character good but sense head blight wheat breed or strain are for raising wheat No. 15 or raising wheat 13 or Huaihe River wheat 18; Wheat-resistance to scab kind or strain are Soviet Union wheat No. 3 or peaceful 894037 or peaceful 7840.
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| Application Number | Priority Date | Filing Date | Title |
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| CN2010101751508A CN101828521B (en) | 2010-05-18 | 2010-05-18 | Method for quick transformation of major QTL with resistance to fusarium head blight through combination of embryo culture of wheat and sign selection |
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| CN101828521B true CN101828521B (en) | 2012-05-23 |
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| WO2014011859A2 (en) * | 2012-07-13 | 2014-01-16 | Pioneer Hi-Bred International, Inc. | Molecular markers for various traits in wheat and methods of use |
| CN107760767B (en) * | 2017-09-27 | 2021-04-20 | 山东省农业科学院生物技术研究中心 | Wheat scab-resistant multiple-fluorescence SSR (simple sequence repeat) marker detection method |
| CN111631130A (en) * | 2020-06-09 | 2020-09-08 | 石河子大学 | Method for accelerating strong winter wheat hybridization breeding process by embryo culture technology for 4 generations in 1 year |
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