CN102229986A - Method for assisted selection of wheat variety with stripe rust resistance and special PCR reagent used therein - Google Patents
Method for assisted selection of wheat variety with stripe rust resistance and special PCR reagent used therein Download PDFInfo
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Abstract
The invention discloses a method for screening a wheat variety with stripe rust resistance assisted by molecular markers and special primers used therein. Sequences of the primer pairs provided in the invention for assisted selection of a wheat variety with stripe rust resistance are described in a primer sequence table (Table 1). The method for assisted selection of a wheat variety with stripe rust resistance provided in the invention is carried out on the basis of conventional breeding and causes no interruption to the breeding process of a breeder except that in the generations of F2 and F6, after the selection of the breeder, candidate individual plants are subjected to molecular marker detection and only those individual plants containing two stripe rust resistant genes are reserved, thereby helping the breeder to more effectively select excellent strains containing a plurality of stripe rust resistant genes and enhancing endurance of stripe rust resistance of the strains. According to the invention, a novel stripe rust resistant gene Yrcaas and its special primers are discovered in the wheat variety Zhou 8425B; the gene is on wheat 1B chromosome and is located between SSR mark Xbarc8 and SSR mark Xgwm582, wherein the two marks can be used for assisted breeding of stripe rust resistant plants. The special primers of the stripe rust resistance genes in the invention are important to the breeding of stripe rust resistant wheat.
Description
Technical field
The present invention relates to biological technical field, relate in particular to a kind of method and special-purpose PCR reagent thereof of assist-breeding stripe rust resisting wheat kind.
Background technology
Stripe rust of wheat is a kind of gas biography property leaf diseases that is caused by wheat stripe rust (Puccinia striiformis f.sp.tritici), after taking place, this disease will influence the grouting of plant development and wheat, and then having a strong impact on wheat yield and quality, there are 4,300 ten thousand hm in the whole world
2Yi Faqu, account for the total cultivated area of wheat 19.4% (
Http:// www.cimmyt.org/Agricdb/fao/Default.aspx).China is the maximum in the world popular district of stripe rust of wheat, about 2,000 ten thousand hm
2(Stubbs, 1988; Wan etc., 2004,2007), cool and the high altitude localities particularly serious.Since the 1950's, China 8 stripe rust successively occur and is very popular, wherein be very popular for 1950,1964,1990 and 2002 four times the area maximum takes place, harm is the heaviest, preceding twice onset area reaches more than 200,000,000 mu, twice morbidity in back is more than 1.1 hundred million mu, lose 600,320,180 and 1,300,000 tons of wheats respectively, bring massive losses (Li Zhenqi etc., 1989 to Wheat Production; Li Shimo, 2001; Li etc., 2000; Wan etc., 2004).Recent years is owing to the reasons such as generally use of climate change, triadimefon, its relative importance descends to some extent, annual average about 0.63 hundred million mu of the area that takes place of 2004-09 China stripe rust, but also there are (Zhang Yuejin etc. about 100,000,000 mu in the maximum time, 2005-2009), and unusual along with global warming, climate change, stripe rust of wheat rises to once more probably and is main disease, thus the stable high yield of serious threat China wheat.
The wheat stripe rust resistance genes of definite designation in the world is totally 51 at present, is distributed in 48 chromosomal focis, i.e. Yr1-Yr48 (Mcintosh RA etc., 2010).These Stripe Rust Resistance Gene belong to the disease-resistant gene in seedling stage with microspecies specialization mostly, seedling stage, disease-resistant gene was because its Gao Kangshen to the stripe rust evil is liked by breeding man, the resistant gene of wheat population great majority are microspecies specialization resistance in recent decades, but the single kind of establishing in large scale disease-resistant gene, can quicken the orthoselection of pathogenic bacteria microspecies, the result causes disease-resistant variety " forfeiture " original resistance after the large-area applications several years on producing, and loses using value (village ingeniously living 1996).In history, because derive is " the Lip river class " and " numerous 6 " of establishing in large scale, causing newly causing a disease in the monoid bar, No. 32 (CYR32) physiological strains occur also rising to rapidly being dominant races among No. 31 (CYR31) and bar, thereby have caused that national stripe rust of wheat in 2002 is very popular.Recognize that after the potential hazard of single disease-resistant gene, breeding scholar and phytopathologist wish to prolong by methods such as multiple gene polymerization, gene layout and multiline varieties the resistance life-span of disease-resistant gene in seedling stage.If the disease-resistant gene of the different physiological strains of opposing can be aggregated in the kind, this kind just has the ability of the multiple physiological strain of opposing so, promptly have multiresistance, lose resistance with regard to being not easy because of the variation of the microspecies of causing a disease like this, thereby the resistance time length is prolonged.Realize this purpose, need abundant anti-source on the one hand, will use the more effective realization gene pyramiding of method easily on the other hand.At present China still keep the major gene of stripe rust resistance have Yr5, Yr10, Yr15, Yr24, Yr26 and YrZH84 etc. a few, therefore it is extremely urgent to excavate new anti-source, assist breeding for disease resistance by molecule marker simultaneously, thereby the anti-source of more efficient use is also important unusually.
Molecular marker assisted selection is by with objective trait close linkage or common isolating molecule marker offspring's strain system being carried out the screening of target gene or chromosome segment, and then in that early in generation, can obtain to contain the fine individual plant of target gene, improve breeding efficiency (Fang Xuanjun etc., 2001).Utilize the auxiliary breeding for disease resistance of molecule marker can deduct conventional disease-resistant evaluation work, and then overcome owing to falling ill insufficient or identifying the inaccurate selection mistake of bringing, simultaneously, this technology makes a plurality of disease-resistant gene importing work of detection become possibility, greatly improves the efficient of gene pyramiding.At present, molecular marker assisted selection has obtained some gratifying results in the breeding for disease resistance of world wheat main product state.Will be referred to the rust resistance gene at interior 42 proterties or gene as the western australia, it has been carried out breed breeding and germplasm improvement (Caki etc., 2008) by the molecular marker assisted selection technology; The Australia south utilizes molecule marker and DH technology, successfully changes rust-proofing in the wheat breed Annuello and high-quality gene among the susceptible strain Stylet (Kuche etc., 2008); The U.S. is under the support of " wheat cdna is applied to put into practice " project, by the auxiliary backcross breeding method of molecule marker, 27 disease-resistant, anti insect genes and 20 high-quality allelotrope have been changed effectively over to (Sorrell etc. in 180 strains of U.S.'s wheat belt, 2007), and the University of California that is arranged in the Davis utilizes the molecule marker ancillary technique successfully to change stripe rust resistance genes Yr17 and brown leaf rust-resistance gene Lr37 over to wheat breed Patwin (Hospita etc., 2009); International corn wheat improvement center (CIMMTY) utilizes molecular marker assisted selection and the conventional breeding method that combines, 25 different disease-resistant worms, high-quality and economical character excellent genes are carried out polymerization, make wheat breed be improved (Willia etc., 2007); Canada has obtained the molecule marker of proterties such as some rust, smut, head blight, high-quality and fringe germination, at present, pass through molecular marker assisted selection, existing 2 high-quality wheat varieties are promoted, be Lillian and Goodeve (DePauw etc., 2005,2009), and the disease molecule marker utilize work to be in to carry out.In sum, molecule marker (comprising RAPD, RFLP, SSR and RGAP) assisted Selection has been able to widespread use in global wheat breeding for disease resistance, wherein the SSR mark is fit to molecular mark owing to advantages such as its codominance, repeated height and technology are simple.
Though there are some correlative studys in China, the report of specifically using effect is less.Li Zaifeng etc. (2006) utilize the popular microspecies CYR32 of domestic stripe rust that all 8425B are carried out resistance evaluation in seedling stage, found that all 8425B carry dominance disease-resistant gene anti-in a pair of performance, and then utilize the SSR mark to be located on 7BL karyomit(e), name and be YrZH84, comprise with the closely linked molecule marker of this gene: Xgwm577, Xwmc276, Xwmc273, Xcfa2040, Xbarc32, Xbarc182 and Xwmc526, genetic distance is that 1.4cM is to 12.0cM, wherein be labeled as Xcfa2040-7B and Xbarc32-7B at the nearest SSR in YrZH84 both sides, genetic distance is respectively 1.4cM and 4.8cM.Yin Guihong etc. (2009) utilize the RGAP mark, by segregating population fractional analysis method (BSA) based on marker gene type and Phenotypic Selection, obtained and the chain RGA more closely of wheat stripe rust resisting ospc gene YrZH84 mark Xrga-I, with the linkage distance of YrZH84 be 0.8cM.Ren Yan etc. (2010) utilize the popular microspecies CYR32 of domestic stripe rust that all 8425B are carried out resistance evaluation in seedling stage recently, found that all 8425B carry a high anti-new dominance Stripe Rust Resistance Gene of performance, and then utilize the SSR mark to be located on 1B karyomit(e), tentative Yrcaas by name, comprise with the closely linked molecule marker of this gene: H20, Xbarc8, Xgwm582, Xgwm131 and Xwmc216, genetic distance is that 0.6cM is to 16.8cM, wherein be labeled as Xbarc8 and Xgwm582 at the nearest SSR in YrZH84 both sides, genetic distance is respectively 0.6cM and 0.7cM, as shown in Figure 1.
Summary of the invention
An object of the present invention is to provide the primer of a kind of assist-breeding stripe rust resisting plant.
Primer provided by the invention is following 1)-4) in any one:
1) primer shown in is made up of 5 (H20) 4 (Xgwm582) and primer 3 (Xbarc8), primer 2 (Xcfa2040), primer 1 (Xbarc32), primer primer, and primer sequence sees table 1 for details;
2) primer shown in by primer to 1, primer to 2 and following A-C in any two kinds form: A: primer to 3, B: primer is to 4 and C: primer is to 5;
3) primer shown in by primer to 1, primer forms 2;
4) primer shown in is formed by any two kinds among the following A-C: A: primer to 3, B: primer is to 4 and C: primer is to 5;
Described primer is classified sequence 1 in the sequence table as to the nucleotides sequence of a primer in 1, and the nucleotides sequence of another primer of described primer centering is classified the sequence 2 in the sequence table as;
Described primer is classified sequence 3 in the sequence table as to the nucleotides sequence of a primer in 2, and the nucleotides sequence of another primer of described primer centering is classified the sequence 4 in the sequence table as;
Described primer is classified sequence 5 in the sequence table as to the nucleotides sequence of a primer in 3, and the nucleotides sequence of another primer of described primer centering is classified the sequence 6 in the sequence table as;
Described primer is classified sequence 7 in the sequence table as to the nucleotides sequence of a primer in 4, and the nucleotides sequence of another primer of described primer centering is classified the sequence 8 in the sequence table as;
Described primer is classified sequence 9 in the sequence table as to the nucleotides sequence of a primer in 5, and the nucleotides sequence of another primer of described primer centering is classified the sequence 10 in the sequence table as.
Another object of the present invention provides the PCR reagent of assist-breeding stripe rust resisting plant.
Provided by the invention following 1) the PCR reagent of the assist-breeding stripe rust resisting plant shown in arbitrary-4):
1) reagent of PCR shown in is made up of PCR reagent 1, PCR reagent 2, PCR reagent 3, PCR reagent 4 and PCR reagent 5;
2) reagent of PCR shown in is formed by any two kinds among PCR reagent 1, PCR reagent 2 and the following A-C: A:PCR reagent 3, B:PCR reagent 4 and C:PCR reagent 5;
3) reagent of PCR shown in is made up of PCR reagent 1, PCR reagent 2;
4) reagent of PCR shown in is formed by any two kinds among the following A-C: A:PCR reagent 3, B:PCR reagent 4 and C:PCR reagent 5;
Described PCR reagent 1 by described primer to 1, dNTP, archaeal dna polymerase and pcr amplification damping fluid form;
Described PCR reagent 2 by described primer to 2, dNTP, archaeal dna polymerase and pcr amplification damping fluid form;
Described PCR reagent 3 by described primer to 3, dNTP, archaeal dna polymerase and pcr amplification damping fluid form;
Described PCR reagent 4 by described primer to 4, dNTP, archaeal dna polymerase and pcr amplification damping fluid form;
Described PCR reagent 5 by described primer to 5, dNTP, archaeal dna polymerase and pcr amplification damping fluid form;
The final concentration of all described each primers of primer centering in the PCR at its place reagent is 4pmol.
Each final concentration in the PCR at its place reagent described in described PCR reagent 1, described PCR reagent 2, described PCR reagent 3 and the described PCR reagent 4 among the dNTP is 0.2mM;
The final concentration of each among the dNTP in the described PCR reagent 5 is 0.15 μ M;
The final concentration of all described archaeal dna polymerases in the PCR at its place reagent is 0.067U/ μ L.
The pathogenic bacteria of described stripe rust is wheat stripe rust (Puccinia striifirmis f.sp.tritici), and described wheat stripe rust (Puccinia striiformis f.sp.tritici) is specially in wheat stripe rust (Puccinia striiformis f.sp.tritici) the microspecies bar No. 32.
The 3rd purpose of the present invention is the test kit of assist-breeding stripe rust resisting plant.
Provided by the invention following 1)-4) arbitrary shown in the test kit of assist-breeding stripe rust resisting plant:
1) test kit shown in is for containing described 1) shown in the test kit of PCR reagent;
2) test kit shown in is for containing described 2) shown in the test kit of PCR reagent;
3) test kit shown in is for containing described 3) shown in the test kit of PCR reagent;
4) test kit shown in is for containing described 4) shown in the test kit of PCR reagent.
Described primer or described PCR reagent or the application of described test kit in Resistant breeding also are the scope of protection of the invention;
Or described primer or described PCR reagent or the application of described test kit in assist-breeding stripe rust resisting plant also be the scope of protection of the invention, and described plant is specially wheat; The parent of described wheat is all wheats 22 and all wheats 24.
The pathogenic bacteria of described stripe rust is wheat stripe rust (Puccinia striiformis f.sp.tritici), and described wheat stripe rust (Puccinia striiformis f.sp.tritici) is specially No. 32 physiological strains in the bar.
The 4th purpose of the present invention provides a kind of the discriminating or the auxiliary method of differentiating stripe rust resisting wheat.
Method provided by the invention comprises the steps:
With described 1) shown in primer or described 1) shown in PCR reagent or described 1) shown in described primer in the test kit to 1, described primer to 2, described primer to 3, described primer to 4 and described primer respectively wheat to be measured is carried out pcr amplification to 5, detect pcr amplification product;
If the product of pcr amplification is following A)-in E) 5 kinds, then described wheat to be measured is or the candidate is a stripe rust resisting wheat;
If the product of pcr amplification is not following A)-in E) 5 kinds, then described wheat to be measured is or the candidate is non-stripe rust resisting wheat:
A) described primer obtains the PCR product of 200bp to 1 amplification;
B) described primer obtains the PCR product of 150bp to 2 amplifications;
C) described primer obtains the PCR product of 560bp to 3 amplifications;
D) described primer obtains the PCR product of 350bp to 4 amplifications;
E) described primer obtains the PCR product of 1598bp to 5 amplifications.
Described primer to 1, described primer to 2, described primer to 3 and the annealing temperature of described primer when carrying out pcr amplification to 4 be 55 ℃, annealing time is 1min;
Annealing temperature when described primer carries out pcr amplification to 5 is 60 ℃, and annealing time is 45S;
Agarose gel electrophoresis is adopted in described detection.
The 5th purpose of the present invention provides the method for a kind of assist-breeding high yield and/or stripe rust resisting wheat.
Method provided by the invention comprises the steps:
1) hybridizes with all wheats 24 and all wheats 22, obtain F
1Generation;
2) F that step 1) is obtained
1Carry out selfing for wheat, obtain F
2For wheat;
3) with described 1) shown in primer or described 1) shown in PCR reagent or 1) shown in described primer in the test kit to 1, described primer to 2, described primer to 3, described primer to 4 and described primer to 5 respectively to step 2) F that obtains
2Carry out pcr amplification for wheat, detect pcr amplification product;
If the product of pcr amplification is following A)-in E) 5 kinds, then described wheat to be measured is or the candidate is a stripe rust resisting wheat;
If the product of pcr amplification is not following A)-in E) 5 kinds, then described wheat to be measured is or the candidate is non-stripe rust resisting wheat:
A) described primer obtains the PCR product of 200bp to 1 amplification;
B) described primer obtains the PCR product of 150bp to 2 amplifications;
C) described primer obtains the PCR product of 560bp to 3 amplifications;
D) described primer obtains the PCR product of 350bp to 4 amplifications;
E) described primer obtains the PCR product of 1598bp to 5 amplifications.
After described step 3), also comprise the steps:
4) candidate that step 3) is obtained is stripe rust resisting F
2Carry out selfing for wheat and obtain F
3For wheat, with described F
3Obtain F for the wheat selfing
4For wheat, with described F
4Obtain F for the wheat selfing
5For wheat, with described F
5Obtain F for the wheat selfing
6For wheat;
5) with 1) shown in primer or 1) shown in PCR reagent or 1) shown in test kit F that step 4) is obtained
6Carry out pcr amplification for wheat, obtain the PCR product, detect pcr amplification product,
If the product of pcr amplification is following A)-E) shown in, then described F
2For wheat be or the candidate is stripe rust resisting F
6For wheat;
If the product of pcr amplification is not following A)-E) shown in, then described F
2For wheat be or the candidate is non-stripe rust resisting F
6For wheat:
A) described primer obtains the PCR product of 200bp to 1 amplification;
B) described primer obtains the PCR product of 150bp to 2 amplifications;
C) described primer obtains the PCR product of 560bp to 3 amplifications;
D) described primer obtains the PCR product of 350bp to 4 amplifications;
E) described primer obtains the PCR product of 1598bp to 5 amplifications;
6) be stripe rust resisting F with described candidate
6Carry out selfing for wheat, obtain F
7For wheat, screening output is higher than the F of all wheats 22
7For wheat, be high yield, stripe rust resisting wheat;
Described pcr amplification in the step 3) is all with F
2Genomic dna for wheat is a template;
Described pcr amplification in the step 5) is all with F
6Genomic dna for wheat is a template;
The pathogenic bacteria of described stripe rust is wheat stripe rust (Puccinia striiformis f.sp.tritici), and described wheat stripe rust (Puccinia striiformis f.sp.tritici) is specially in wheat stripe rust (Puccinia striiformis f.sp.tritici) the microspecies bar No. 32;
Agarose gel electrophoresis is all adopted in described detection.
The output of described high yield, stripe rust resisting wheat is compared with all wheats 22, and the ratio of raising is not less than 5%.
The molecule marker that is used for assistantly screening anti-stripe rust wheat that provides that experiment showed, of the present invention has 5, is respectively: Xcfa2040, Xbarc32, Xbarc8, Xgwm582 and H20.The invention provides the auxiliary resistant breeding method of a kind of molecule marker, this method is consistent with the process of conventional breeding, and different is after parents' apolegamy, at first to utilize above-mentioned primer that it is carried out Molecular Detection, and then determine which combination can utilize this method; To these combinations that is suitable for, at F
2The time, candidate's individual plant of breeding man being selected with described 2 SSR marks carries out pcr amplification respectively, detect amplified production, when the purpose fragment of above-mentioned all primer correspondences is arranged in the amplified production, this individual plant is the candidate's individual plant that contains above-mentioned two Stripe Rust Resistance Gene, only these individual plants is used for next step breeding work; At F
6The time, after the selection of breeding man, utilize described 5 pairs of primers that the strain system of isozygotying is substantially carried out Molecular Detection once more, thereby above-mentioned two Stripe Rust Resistance Gene whether still contained in roguing system of checking institute, keep the strain system of containing anti-bar rust gene, further by comprehensive agronomy proterties comparation and assessment, and then the height that obtains the good and high stripe rust resisting of economical character finally obtains kind for strain.
Description of drawings
Fig. 1 is the linkage map of 5 SSR marks and Yrcaas gene
Fig. 2 deposits the pedigree of wheat No. 7 for wheat breed
Fig. 3 is that the SSR primer is to F
2For plant with to F
6The amplification of strain system
Embodiment
Employed experimental technique is ordinary method if no special instructions among the following embodiment.
Used material, reagent etc. if no special instructions, all can obtain from commercial channels among the following embodiment.
The sequence of used primer is as shown in table 1 below among the following embodiment, is synthetic:
The discovery of a new anti-bar rust gene Yrcaas and the acquisition of SSR mark thereof among embodiment 1, the wheat lines week 8425B:
One, phenotypic acquisition and colony thereof show situation seedling stage
With all 8425B and Avocet S hybridization, obtain F
1Colony is with F
1Colony's selfing obtains F
2Colony, F
2Colony is 586 individual plants.With No. 32 physiological strains in the popular microspecies bar of the present stripe rust of China experiment material is inoculated evaluation, the result shows: all 8425B shows as high anti-(response type IT=0 seedling stage; ) to immunity (IT=0), AvocetS shows as high sense (IT=4), F seedling stage
1Colony all shows as high anti-(IT=0; ), F
2439 strains are disease-resistant individual plant in the colony, and 147 strains are susceptible individual plant, show the dominant gene that carries 1 pair of high stripe rust resisting among all 8425B.According to seedling stage qualification result select for use 10 extremely disease-resistant individual plants and 10 extremely susceptible individual plants respectively its DNA to be carried out balanced mix to form disease-resistant pond and susceptible pond.
Two, the right acquisition of primer special
Select for use 655 pairs of SSR primers that the parent is carried out the polymorphism screening, all primers are synthetic to be finished by Beijing AudioCodes biotech firm.The result shows at 3 mark H20, Xbarc8 on the 1B karyomit(e) and Xgwm582 all has polymorphism between parent and anti-sense pond.Show that tentatively these marks and Stripe Rust Resistance Gene (Yrcaas) are chain.
Three, the acquisition of linkage map and source thereof
With 3 mark H20, Xbarc8 on the 1B and Xgwm582 to 586 F
2Individual plant increases respectively, obtain colony's genotype, the gained data utilize mapping software MapMaker 3.0 and Map Manager QTX20 to carry out linkage analysis, the result shows that these 5 marks are all chain with Stripe Rust Resistance Gene Yrcaas, nearest Xbarc8 and the Xgwm582 of being labeled as wherein apart from the Yrcaas both sides, genetic distance is respectively 9.5cM and 9.6cM, sees shown in Figure 1.
The PCR reaction system of 2 SSR marks (being Xbarc8 and Xgwm582) is 15 μ l, that is: 8.8 μ l sterilized waters, 1.5 μ l10 * PCR buffer (remittance day east), 0.3 μ l 10mM dNTPs (remittance day east), every primer 4pmol, Taq archaeal dna polymerase 0.067U/ μ L (remittance day east), the 20ng template DNA; Response procedures is: 94 ℃ of pre-sex change 5 minutes, and each 94 ℃ of sex change 1min that circulate, 55 ℃ of annealing 1min, 72 ℃ are extended 1min, 35 circulations, last 72 ℃ are extended 10min; The PCR product is stored in 4 ℃; Every part of amplified production adds 3 μ l sex change load sample indicator [98% no ion methane amide, 10mM EDTA (pH 8.0), 0.1% dimethylbenzene green grass or young crops, 0.1% tetrabromophenol sulfonphthalein], 95 ℃ of sex change 10 minutes.Point sample 5 μ l carry out electrophoretic separation in 6% denaturing polyacrylamide gel.
The reaction system that silver dyes development STS mark H20 is 20 μ l, contains 10 * PCR buffer, 2 μ l, each 200 μ molL of dNTP (A, T, C, G)
-1, every primer 4pmol, Taq archaeal dna polymerase (TaKaRa) 0.067U/ μ L, template DNA 50ng; The PCR program is 94 ℃ of pre-sex change 5min; 94 ℃ of sex change 45s, 60 ℃ of annealing 45s, 72 ℃ are extended 1.5min, 38 circulations; 72 ℃ are extended 10min; Pcr amplification product is with 1.5% agarose gel electrophoresis separation detection, and buffering liquid is 1 * TAE solution, 220V voltage electrophoresis 10min, ethidium bromide staining.
3 are labeled as H20, Xbarc8 and Xgwm582, and the primer of above-mentioned 3 mark correspondences is to as shown in table 1.
Mark Xbarc32 and Xcfa2040 are for detecting the special marker of Stripe Rust Resistance Gene YrZH84.
Table 1 detection Stripe Rust Resistance Gene YrZH84 in seedling stage and the required primer sequence table of Yrcaas
Annotate: mark Xbarc32 and Xcfa2040 are the primer specials of screening Stripe Rust Resistance Gene YrZH84, and mark Xbarc8, Xgwm582 and H20 are the primer specials of screening Stripe Rust Resistance Gene Yrcaas.
Embodiment 2, deposit No. 7 breeding of wheat processes of wheat
Depositing wheat is for No. 7 semi-winterness, the middle dwarf wheat kind that forms with all wheat 24/ all wheat 22 selection cross, and its pedigree sees Fig. 2 for details.This kind is high anti-to the performance of stripe rust mixing fungus strain seedling stage, becomes not only high stripe rust resisting of strain phase, and high leaf rust resistance, has participated in the annual national the Yellow River and Huai He River sheet prerun of 2010-2011 at present.
Its seed selection process is as follows:
1, F
1Acquisition for wheat
With all wheat 24 (academy of agricultural sciences, Zhoukou City improved variety; obtained kind power protection (CNA002834E) in 2006; 2007 careful by state; all No. 24 features of wheat and cultivation technique; Wang Shigang plants the industry guide, 2010 the 05th phases; the 29th page, the public can deposit the acquisition of wheat improving technology institute from Institute of Crop Science, Chinese Academy of Agricultural Science and sky, Henan Province.) be maternal, all wheats 22 (improved variety of academy of agricultural sciences, Zhoukou City, all wheat 22 high-yield culture techniques, Gu Sumei plants the industry guide, 2011 the 1st phases, the 25-27 page or leaf, the public can deposit the acquisition of wheat improving technology institute from Institute of Crop Science, Chinese Academy of Agricultural Science and sky, Henan Province.) be male parent, hybridize, obtain F
1Generation, F
1In generation, do not eliminate, all F of results after the impurity elimination
1Plant;
2, F
2Acquisition and evaluation for wheat
With F
1Carry out selfing for wheat and obtain F
2For wheat, about program request 1200 strains, in its process of growth, breeding man by individual proterties (in snappiness of short stem, the stem stalk good, fringe big, thousand seed weight 48g) and the field show (fringe is many, filling speed is fast, the Huang that falls good) and select 54 strain F
2For individual plant.
Extract above-mentioned 54 strain F
2Genomic dna for individual plant, adopt Xbarc32-F in the table 1 of embodiment 1 and Xbarc32-R, Xcfa2040-F and these 2 pairs of primers of Xcfa2040-R to carry out pcr amplification respectively, primer Xbarc32 and Xcfa2040 are for detecting the primer special of Stripe Rust Resistance Gene YrZH84.With all 8425B (Molecular tagging of stripe rust resistance gene YrZH84 in Chinese wheat line Zhou 8425B, Li etc, Theor Appl Genet (2006) 112:1098-1103; The public can deposit the acquisition of wheat improving technology institute from Institute of Crop Science, Chinese Academy of Agricultural Science and sky, Henan Province; Above-mentioned studies show that, all 8425B carry Stripe Rust Resistance Gene YrZH84), (32 make the become rusty present situation in anti-source of its China's wheat bar, Yang Zuomin etc., Acta Agronomica Sinica, the 29th volume, the 2nd phase, in March, 2003,161-168 page or leaf to Avocet S in the back bar; The susceptible strain Avocet S of Australia does not contain Stripe Rust Resistance Gene YrZH84; The public can deposit the acquisition of wheat improving technology institute from Institute of Crop Science, Chinese Academy of Agricultural Science and sky, Henan Province; ) for contrasting.
The PCR reaction system is 15 μ l, 8.8 μ l sterilized waters, 1.5 μ l, 10 * PCR buffer (remittance day east), 0.3 μ l 10mM (final concentration is 0.2mM) dNTPs (remittance day east), Taq archaeal dna polymerase 0.067U/ μ L (remittance day east), 20ng template DNA, every primer 4pmol; The program of described pcr amplification is: 94 ℃ of pre-sex change 5 minutes; 94 ℃ of sex change 1min, 55 ℃ of annealing 1min, 72 ℃ are extended 1min, 35 circulations; Extend 10min at last.
Every part of pcr amplification product adds 3 μ l sex change load sample damping fluids, mixing, 95 ℃ of sex change 8 minutes.With 6% denaturing polyacrylamide gel electrophoresis separation detection, buffering liquid is: go up groove 0.3 * TBE, following groove 0.5 * TBE, and power 80W, electrophoresis 50-70min, silver dyes development.
The result is shown in Fig. 3 a and 3b, and wherein 3a is that SSR primer Xcfa2040 is to F
2The amplification of individual plant, Fig. 3 b are that SSR primer Xbarc32 is to F
2The amplification of individual plant, M is Marker, 1-30 is F
2For individual plant.
From Fig. 3 a as can be seen, with Xbarc32-F and Xbarc32-R is primer (primer sequence is respectively sequence 1 and sequence 2 in the sequence table), the 1-20 plant obtains the PCR fragment 1 (200bp) shown in the arrow, and the 21-30 plant obtains the PCR fragment 2 (170bp) shown in the arrow; With above-mentioned primer amplification, week, 8425B obtained PCR fragment 1, Avocet S obtains PCR fragment 2, and (specific fragment of disease-resistant plant is a PCR fragment 1, the specific fragment of disease plant is a PCR fragment 2, consistent with disease-resistant parent and susceptible parent respectively, thereby can distinguish disease-resistant plant and disease plant from clip size).
From Fig. 3 b as can be seen, be primer with Xcfa2040-F and Xcfa2040-R, the 1-20 plant obtains the PCR fragment 3 (150bp) shown in the arrow, and the 21-30 plant obtains the PCR fragment 4 (165bp) shown in the arrow; With above-mentioned primer amplification, all 8425B obtain PCR fragment 3, and Avocet S obtains PCR fragment 4.
Extract above-mentioned 54 strain F
2For the genomic dna of individual plant, adopt Xbarc8-F in the table 1 of embodiment 1 and Xbarc8-R, Xgwm582-F and Xgwm582-R, H20-F and these 3 pairs of primers of H20-R to carry out pcr amplification respectively.With all 8425B, Avocet S is contrast.
The result is as follows:
With Xbarc8-F and Xbarc8-R is primer amplification, 1-20 plant F
2Obtain the fragment of PCR fragment 5 (560bp) for individual plant, all 8425B also obtain the specific fragment of 560bp, and Avocet S does not obtain the as above fragment of size.
With Xgwm582-F and Xgwm582-R is primer amplification, 1-20 plant F
2Obtain the fragment of PCR fragment 6 (350bp) for individual plant, all 8425B also obtain the specific fragment of 350bp, and Avocet S does not obtain the as above fragment of size.
With H20-F and H20-R is primer amplification, 1-20 plant F
2Obtain the segmental fragment of PCR fragment 7 (1598bp) for individual plant, all 8425B also obtain the specific fragment of 1598bp, and Avocet S does not obtain the as above fragment of size.
From as can be seen above-mentioned, above-mentioned 1-20 plant obtains PCR fragment 1, PCR fragment 3, PCR fragment 5, PCR fragment 6, PCR fragment 7, this class F
2For the wheat individual plant is disease-resistant plant, obtains 30 stripe rust resisting F altogether
2For the wheat individual plant; 24 are stripe rust resisting F not
2For the wheat individual plant.
With 30 candidates is stripe rust resisting F
2Be stripe rust resisting F not for wheat and 24 candidates
2All carry out stripe rust resistance evaluation in seedling stage for wheat, method is as follows: material to be detected is planted in the polypots of 9*9*9,1 part of material to be identified of every alms bowl kind (that is candidate F,
2Seed for harvesting wheat, 20) and the susceptible contrast of 3 strains engrave virtuous 169 (Quantitative Trait Loci Mapping for Adult-Plant Resistance to Stripe Rust in Chinese Landrace Wheat Cultivar Pingyuan 50, Lan CX etc., Phytopathology (2010), 100:313-318; The public can deposit the acquisition of wheat improving technology institute from Institute of Crop Science, Chinese Academy of Agricultural Science and sky, Henan Province.Engrave and virtuous 169 all strip rust bacteria physiological strains are all shown as high sense in whole growing, think at present and do not contain any disease-resistant gene), when wheat seeding grows to wholeheartedly a leaf, with sweeping No. 32 ((Puccinaia striiformis f.sp.tritici in wiping manipulation inoculation wheat stripe rust (the Puccinaia striiformis f.sp.tritici) bar, Wan An people, Wu Li people, Jin Shelin, Yao Ge, Wang Baotong. No. 32 name and feature thereof in the Chinese puccinia striiformis bar. the plant protection journal, 2003,30 (4): 347-352., the public can deposit the acquisition of wheat improving technology institute from Institute of Crop Science, Chinese Academy of Agricultural Science and sky, Henan Province), inoculate back about 15 days, the documented response type infects the type grade scale and adopts 6 grade standards when treating that susceptible contrast is fully fallen ill, and promptly 0,0; , 1,2,3 and 4, and with+and-expression is strong and weak, wherein, 0～2+ type is disease-resistant, 3-～4 types are susceptible (Bariana and McIntosh, 1993).The result proves that 30 candidates are stripe rust resisting F
2Be disease-resistant plant for wheat, 24 candidates are stripe rust resisting F not
2For the equal disease plant of wheat.
The checking of proof present method is correct.
3, F
6Acquisition and evaluation for wheat
With above-mentioned 30 candidates is stripe rust resisting F
2Obtain F for the equal selfing of wheat
3For wheat, with described F
3Obtain F for the wheat selfing
4For wheat, with described F
4Obtain F for the wheat selfing
5For wheat, with described F
5Obtain F for the wheat selfing
6For wheat;
Extract 35 strain F respectively
6Genomic dna for individual plant, the 5 couples of primer Xbarc32-F that adopt respectively that embodiment 1 obtains and Xbarc32-R, Xcfa2040-F and Xcfa2040-R, Xbarc8-F and Xbarc8-R, Xgwm582-F and Xgwm582-R, H20-F and H20-R carry out pcr amplification, be respectively equipped with contrast, specifically see the result.
The PCR reaction system is the same, and annealing temperature sees Table 1.
The result is shown in Fig. 3 c-3g, and M is Marker, and 1-11 is F
6For individual plant, the red arrow indication is the pairing fragment of disease-resistant gene among the figure;
3c is the SSR primer to Xbarc32-F and Xbarc32-R to F
6The amplification of strain system; With all 8425B, AvocetS, all wheats 22, all wheats 24 is contrast, and the 1-7 plant obtains the PCR fragment 1 (200bp) of arrow indication; The 8-11 plant obtains the PCR fragment 2 (170bp) shown in the arrow; With above-mentioned primer amplification, all 8425B and all wheats 22 obtain PCR fragment 1, Avocet S and all wheats 24 and obtain PCR fragment 2.
Fig. 3 d is the SSR primer to Xcfa2040-F and Xcfa2040-R to F
6The amplification of strain system; With all 8425B, Avocet S, all wheats 22, all wheats 24 is contrast, and the 1-7 plant obtains the PCR fragment 3 (150bp) of arrow indication; The 8-11 plant obtains the PCR fragment 4 (165bp) shown in the arrow; With above-mentioned primer amplification, all 8425B and all wheats 22 obtain PCR fragment 3, Avocet S and all wheats 24 and obtain PCR fragment 4.
Fig. 3 e is the SSR primer to Xbarc8-F and Xbarc8-R to F
6The amplification of strain system; With all 8425B, Avocet S, all wheats 22, all wheats 24 is contrast, and the 1-5 plant obtains the PCR fragment 5 (560bp) of arrow indication; The 8-10 plant does not amplify above-mentioned specific fragment; With above-mentioned primer amplification, all 8425B and all wheats 22 obtain PCR fragment 5, and Avocet S and all wheats 24 do not amplify this fragment.
Fig. 3 f is the SSR primer to Xgwm582-F and Xgwm582-R to F
6The amplification of strain system; With all 8425B, Avocet S, all wheats 22, all wheats 24 is contrast, and the 1-7 plant obtains the PCR fragment 6 (350bp) of arrow indication; The 8-11 plant does not amplify above-mentioned specific fragment; With above-mentioned primer amplification, all 8425B and all wheats 22 obtain PCR fragment 6, Avocet S and all wheats 24 and do not amplify this fragment.
Fig. 3 g (: STS primer H20 is to F
6The amplification of strain system; With all 8425B, Avocet S, all wheats 22, all wheats 24 is contrast, and the 1-6 plant obtains the PCR fragment 6 (1598bp) of arrow indication; The 8-11 plant does not amplify above-mentioned specific fragment; With above-mentioned primer amplification, all 8425B and all wheats 22 obtain PCR fragment 6, and AvocetS and all wheats 24 do not amplify this fragment.
As can be seen, above-mentioned 1-7 plant can amplify specific PCR fragment 1,3,5,6 and 7, therefore thinks that it keeps above-mentioned 2 Stripe Rust Resistance Gene, is the stripe rust resisting plant, and its candidate is stripe rust resisting F
6For wheat, obtaining 11 candidates altogether is stripe rust resisting F
6For wheat; 24 candidates are stripe rust resisting F not
6For wheat.
With 11 candidates is stripe rust resisting F
6For wheat and 24 candidates stripe rust resisting F not
6All carry out stripe rust resistance evaluation in seedling stage for wheat, method is the same.
The result shows that 11 candidates are stripe rust resisting F
6Be the stripe rust resisting plant for wheat; 24 candidates are stripe rust resisting F not
6All feel the stripe rust plant for wheat.
The checking of proof present method is correct.
4, high yield, disease-resistant Screening and Identification of depositing wheat No. 7
With 11 candidates is stripe rust resisting F
6Obtain F for the equal selfing of wheat
7For wheat, with candidate F
7Carry out the strain evaluation for wheat strain system and (promptly under higher water and fertilizer condition, adopt randomized block design, three repetitions, every sub-district 6.67m
2, the amount of broadcasting is consistent with production level.Selecting local one or two best kinds or product is contrast, and the contrast of this research is all wheats 22.
Screening obtains the F that output is higher than contrast
7For the wheat family, its called after is deposited wheat No. 7.Wherein, contrast 22 super high-yielding of all wheats, the output three elements are coordinated (400,000 fringe/mu * 36 * 45.0 restrain/thousand), and yielding ability is good.
And the output three elements of depositing wheat No. 7 are/thousand of 410,000 fringe/mu * 36 * 46.5 grams.
Improved seeds are deposited wheat utilizes for No. 7 above-mentioned special primer to carry out Markers for Detection and stripe rust resisting evaluation in seedling stage, prove that this kind contains anti-bar rust gene YrZH84 and Yrcaas, pedigree analysis shows, all (Markers for Detection shows the resistant gene of this kind from all wheats 22, all wheats contain above-mentioned two disease-resistant genes for No. 22, therefore and all wheats 24 do not contain above-mentioned two disease-resistant genes, infer that the disease-resistant gene of depositing wheat No. 7 is from parent week wheat 22).
Claims (10)
1. the primer of an assist-breeding stripe rust resisting plant is following 1)-4) in any one:
1) primer shown in by primer to 1, primer to 2, primer to 3, primer to 4 and primer form 5;
2) primer shown in by primer to 1, primer to 2 and following A-C in any two kinds form: A: primer to 3, B: primer is to 4 and C: primer is to 5;
3) primer shown in by primer to 1, primer forms 2;
4) primer shown in is formed by any two kinds among the following A-C: A: primer to 3, B: primer is to 4 and C: primer is to 5;
Described primer is classified sequence 1 in the sequence table as to the nucleotides sequence of a primer in 1, and the nucleotides sequence of another primer of described primer centering is classified the sequence 2 in the sequence table as;
Described primer is classified sequence 3 in the sequence table as to the nucleotides sequence of a primer in 2, and the nucleotides sequence of another primer of described primer centering is classified the sequence 4 in the sequence table as;
Described primer is classified sequence 5 in the sequence table as to the nucleotides sequence of a primer in 3, and the nucleotides sequence of another primer of described primer centering is classified the sequence 6 in the sequence table as;
Described primer is classified sequence 7 in the sequence table as to the nucleotides sequence of a primer in 4, and the nucleotides sequence of another primer of described primer centering is classified the sequence 8 in the sequence table as;
Described primer is classified sequence 9 in the sequence table as to the nucleotides sequence of a primer in 5, and the nucleotides sequence of another primer of described primer centering is classified the sequence 10 in the sequence table as.
2. the PCR reagent of the assist-breeding stripe rust resisting plant shown in arbitrary following 1)-4):
1) reagent of PCR shown in is made up of PCR reagent 1, PCR reagent 2, PCR reagent 3, PCR reagent 4 and PCR reagent 5;
2) reagent of PCR shown in is formed by any two kinds among PCR reagent 1, PCR reagent 2 and the following A-C: A:PCR reagent 3, B:PCR reagent 4 and C:PCR reagent 5;
3) reagent of PCR shown in is made up of PCR reagent 1, PCR reagent 2;
4) reagent of PCR shown in is formed by any two kinds among the following A-C: A:PCR reagent 3, B:PCR reagent 4 and C:PCR reagent 5;
Described PCR reagent 1 by the described primer in the described primer of claim 1 to 1, dNTP, archaeal dna polymerase and pcr amplification damping fluid form;
Described PCR reagent 2 by the described primer in the described primer of claim 1 to 2, dNTP, archaeal dna polymerase and pcr amplification damping fluid form;
Described PCR reagent 3 by the described primer in the described primer of claim 1 to 3, dNTP, archaeal dna polymerase and pcr amplification damping fluid form;
Described PCR reagent 4 by the described primer in the described primer of claim 1 to 4, dNTP, archaeal dna polymerase and pcr amplification damping fluid form;
Described PCR reagent 5 by the described primer in the described primer of claim 1 to 5, dNTP, archaeal dna polymerase and pcr amplification damping fluid form;
The concentration of all described each primers of primer centering in the PCR at its place reagent is 4pmol.
3. PCR reagent according to claim 1 and 2 is characterized in that:
Each final concentration in the PCR at its place reagent described in described PCR reagent 1, described PCR reagent 2, described PCR reagent 3 and the described PCR reagent 4 among the dNTP is 0.2mM;
The final concentration of each among the dNTP in the described PCR reagent 5 is 0.15 μ M;
The final concentration of all described archaeal dna polymerases in the PCR at its place reagent is 0.067U/ μ L.
4. according to arbitrary described PCR reagent among the claim 2-3, it is characterized in that: the pathogenic bacteria of described stripe rust is wheat stripe rust (Puccinia striiformis f.sp.tritici), and described wheat stripe rust is specially No. 32 physiological strains in the bar.
5. following 1)-4) arbitrary shown in the test kit of assist-breeding stripe rust resisting plant:
1) test kit shown in is to contain described 1 in the described PCR reagent of claim 2-4) shown in the test kit of PCR reagent;
2) test kit shown in is to contain described 2 in the described PCR reagent of claim 2-4) shown in the test kit of PCR reagent;
3) test kit shown in is to contain described 3 in the described PCR reagent of claim 2-4) shown in the test kit of PCR reagent;
4) test kit shown in is to contain described 4 in the described PCR reagent of claim 2-4) shown in the test kit of PCR reagent.
6. arbitrary described PCR reagent or the application of the described test kit of claim 5 in Resistant breeding among described primer of claim 1 or the claim 2-4;
Or arbitrary described PCR reagent or the application of the described test kit of claim 5 in assist-breeding stripe rust resisting plant among described primer of claim 1 or the claim 2-4, described plant is specially wheat;
The pathogenic bacteria of described stripe rust is wheat stripe rust (Puccinia striiformis f.sp.tritici), and described wheat stripe rust (Puccinia striiformis f.sp.tritici) is specially in wheat stripe rust (Puccinia striiformis f.sp.tritici) the microspecies bar No. 32.
7. differentiate or the auxiliary method of differentiating stripe rust resisting wheat for one kind, comprise the steps:
With described 1 in the described primer of claim 1) shown in primer or claim 2-4 in described 1 in arbitrary described PCR reagent) shown in PCR reagent or the described test kit of claim 5 1) shown in described primer in the test kit to 1, described primer to 2, described primer to 3, described primer to 4 and described primer respectively wheat to be measured is carried out pcr amplification to 5, detect pcr amplification product;
If the product of pcr amplification is following A)-in E) 5 kinds, then described wheat to be measured is or the candidate is a stripe rust resisting wheat;
If the product of pcr amplification is not following A)-in E) 5 kinds, then described wheat to be measured is or the candidate is non-stripe rust resisting wheat:
A) described primer obtains the PCR product of 200bp to 1 amplification;
B) described primer obtains the PCR product of 150bp to 2 amplifications;
C) described primer obtains the PCR product of 560bp to 3 amplifications;
D) described primer obtains the PCR product of 350bp to 4 amplifications;
E) described primer obtains the PCR product of 1598bp to 5 amplifications.
8. according to arbitrary described method in the claim 7, it is characterized in that:
Described primer to 1, described primer to 2, described primer to 3 and the annealing temperature of described primer when carrying out pcr amplification to 4 be 55 ℃, annealing time is 1min;
Annealing temperature when described primer carries out pcr amplification to 5 is 60 ℃, and annealing time is 45S;
Agarose gel electrophoresis is adopted in described detection.
9. the method for a seed selection or assist-breeding stripe rust resisting and/or High-yield Wheat comprises the steps:
1) hybridizes with all wheat 24 wheats and all wheats 22, obtain F
1Generation;
2) F that step 1) is obtained
1Carry out selfing for wheat, obtain F
2For wheat;
3) with 1 in the described primer of claim 1) shown in primer or claim 2-4 in 1 in any described PCR reagent) shown in PCR reagent or the described test kit of claim 5 1) shown in described primer in the test kit to 1, described primer to 2, described primer to 3, described primer to 4 and described primer to 5 respectively to step 2) F that obtains
2Carry out pcr amplification for wheat, detect pcr amplification product;
If the product of pcr amplification is following A)-in E) 5 kinds, then described F
2For wheat be or the candidate is stripe rust resisting F
2For wheat;
If the product of pcr amplification is not following A)-in E) 5 kinds, then described F
2For wheat be or the candidate is non-stripe rust resisting F
2For wheat:
A) described primer obtains the PCR product of 200bp to 1 amplification;
B) described primer obtains the PCR product of 150bp to 2 amplifications;
C) described primer obtains the PCR product of 560bp to 3 amplifications;
D) described primer obtains the PCR product of 350bp to 4 amplifications;
E) described primer obtains the PCR product of 1598bp to 5 amplifications.
10. method according to claim 9 is characterized in that:
After described step 3), also comprise the steps:
4) candidate that step 3) is obtained is stripe rust resisting F
2Carry out selfing for wheat and obtain F
3For wheat, with described F
3Obtain F for the wheat selfing
4For wheat, with described F
4Obtain F for the wheat selfing
5For wheat, with described F
5Obtain F for the wheat selfing
6For wheat;
5) with 1) shown in primer or 1) shown in PCR reagent or 1) shown in test kit F that step 4) is obtained
6Carry out pcr amplification for wheat, obtain the PCR product, detect pcr amplification product;
If the product of pcr amplification is following A)-in E) 5 kinds, then described F
6For wheat be or the candidate is stripe rust resisting F
6For wheat;
If the product of pcr amplification is not following A)-in E) 5 kinds, then described F
6For wheat be or the candidate is non-stripe rust resisting F
6For wheat:
A) described primer obtains the PCR product of 200bp to 1 amplification;
B) described primer obtains the PCR product of 150bp to 2 amplifications;
C) described primer obtains the PCR product of 560bp to 3 amplifications;
D) described primer obtains the PCR product of 350bp to 4 amplifications;
E) described primer obtains the PCR product of 1598bp to 5 amplifications;
6) be stripe rust resisting F with described candidate
6Carry out selfing for wheat, obtain F
7For wheat, screening output is higher than the F of all wheats 22
7For wheat, be high yield, stripe rust resisting wheat;
Described pcr amplification in the step 3) is all with F
2Genomic dna for wheat is a template;
Described pcr amplification in the step 5) is all with F
6Genomic dna for wheat is a template;
The pathogenic bacteria of described stripe rust is wheat stripe rust (Puccinia striiformis f.sp.tritici), and described wheat stripe rust (Puccinia striiformis f.sp.tritici) is specially in wheat stripe rust (Puccinia striiformis f.sp.tritici) the microspecies bar No. 32;
Agarose gel electrophoresis is all adopted in described detection.
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| CN103789431A (en) * | 2014-01-26 | 2014-05-14 | 甘肃省农业科学院小麦研究所 | Special primers, reagent and kit for auxiliary screening and cultivation of stripe rust-resistant new wheat strains, and breeding method of stripe rust-resistant novel wheat strains |
| CN103789431B (en) * | 2014-01-26 | 2016-01-06 | 甘肃省农业科学院小麦研究所 | Assisting sifting and cultivate the primer special of stripe rust resisting wheat new lines, reagent and test kit and selection thereof |
| CN105200122A (en) * | 2014-11-17 | 2015-12-30 | 中国农业科学院植物保护研究所 | Quantitative detection kit for wheat stripe rust and application thereof |
| CN105200122B (en) * | 2014-11-17 | 2019-01-18 | 中国农业科学院植物保护研究所 | Quantitative detection kit for wheat stripe rust and application thereof |
| CN104774934A (en) * | 2015-04-01 | 2015-07-15 | 周口市农业科学院 | Method for identifying Zhoumai No.23 wheat variety |
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| CN104975015A (en) * | 2015-07-13 | 2015-10-14 | 中国农业科学院作物科学研究所 | Method for assisting in screening stripe-rust-resistant wheat and special primers thereof |
| CN110663542A (en) * | 2019-11-19 | 2020-01-10 | 中国科学院西北高原生物研究所 | Breeding method for long-acting resistance of wheat stem rust Ug99 |
| CN113832249A (en) * | 2021-09-29 | 2021-12-24 | 河南农业大学 | Molecular marker for detecting specific chromosome fragment of wheat backbone germplasm 8425B and application thereof |
| CN113832249B (en) * | 2021-09-29 | 2023-07-07 | 河南农业大学 | Molecular marker for detecting specific chromosome fragment of wheat diaphysis germplasm 8425B and application thereof |
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Application publication date: 20111102 Assignee: Henan Fengdekang Seed Co., Ltd. Assignor: Institute of crop science of Chinese Academy of Agricultural Sciences|Henan Institute of Wheat Improvement Technology Contract record no.: 2014410000110 Denomination of invention: Method for assisted selection of wheat variety with stripe rust resistance and special PCR reagent used therein Granted publication date: 20130417 License type: Exclusive License Record date: 20141110 |
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| EM01 | Change of recordation of patent licensing contract | ||
| EM01 | Change of recordation of patent licensing contract |
Change date: 20191029 Contract record no.: 2014410000110 Assignee after: Henan fengdekang Seed Industry Co., Ltd Assignee before: Henan Fengdekang Seed Co., Ltd. |