CN1904063A - Method of selecting and breeding wheat seed capable of resisting powdery mildow stripe rust and yellow dwarf disease - Google Patents
Method of selecting and breeding wheat seed capable of resisting powdery mildow stripe rust and yellow dwarf disease Download PDFInfo
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Abstract
The present invention discloses a method for breeding multigene polymerized wheat germplasm capable of resisting powdery mildew, stripe rust and verticillium wilt. Said method includes the following steps: utilizing STS marker of Pm4 to detect that the wheat to be detected has the gene Pm4a for resisting powdery mildew or not; utilizing SCAR marker of Pm13 to detect that the wheat to be detected has the gene Pm13 capable of resisting powdery mildew or not; utilizing SCAR marker of Pm21 to detect that the wheat to be detected has the gene PmV capable of resisting powdery mildew or not; utilizing SCAR marker of Bdv2 to detect that the wheat to be detected has the gene Bdv2 for resisting verticillium wilt or not; and utilizing SSR marker of YrX to detect that the wheat to be detected has the gene YrX for resisting stripe rust or not. Said invention provides a new method for breeding new wheat variety capable of resisting powdery mildew, stripe rust and verticillium wilt.
Description
Technical field
The present invention relates to the polygenic double mildew-resistance of a kind of seed selection polymerization, the method for stripe rust and yellow dwart wheat germplasm.
Background technology
In recent years, owing to reasons such as the change of cropping system, fertilizer and water condition and weather condition and wheat breed anti-source simplification, a Mai Qu often suffers the invasion of disease and pest more than 2 kinds.Powdery Mildew and stripe rust have become the main disease of China's wheat stable and high yields.Yellow dwart is owing to propagate the wheat aphid of its cause of disease (barly yellow dwarf virus) and seriously take place, and has from Gansu, master such as Shanxi, Shaanxi send out the trend that spreads diffusion to Yangze river and Huai river and southwestern Mai Qu of distinguishing.Therefore, the double anti-white powder of creation, bar rust and yellow dwart new wheat germplasm are significant for the high and stable yields and the sustainable development of wheat.
Ceng Xuanyu such as old filial piety the new wheat germplasm YW243 (Xie Hao of the anti-white powder of holding concurrently, yellow dwarf and three rust, old filial piety, Zhang Zengyan, Xin Zhiyong, Lin Zhishan, Du Lipu, horse is strong-willed, Xu Huijun. the seed selection of wheat with resistance to yellow dwarf new lines YW243 and cellular elements biological assay. Acta Agronomica Sinica, 2000,26 (6): 687-691).YW243 is the hybridization F by Y90136 and RW1685
2Obtain for the material anther culture, wherein Y90136's is combined as PP9-1/ Shan 7859//rich anti-No. 8; RW1685 is combined as the rich anti-13/Khapli of 3*, and PP9-1 is combined as CSph * 2/L1//CSN5BT5D/3/Zhong 7902/4/Shan7859.Infer from pedigree, powder mildew resistance is by the mildew-resistance gene Pm4a control that is derived from Khapli, yellow dwarf resistance is by the control of the resistance to yellow dwarf gene Bdv2 on the middle couchgrass 7X karyomit(e) that comes from transposition (Zhang Z Y, Xu J S, Xu Q J, Larkin Philip, Xin Z Y.Development of novel PCR markers linked to the BYDV resistance gene Bdv2 usefulin wheat for marker-assisted selection.Theoretical and Applied Genetics, 2004,109:433-439).(Xie Hao such as Xie Hao, ox Yongchun, old filial piety, horse is strong-willed, Wu Liren, Lin Zhishan. anti-bar rust property evaluation of new strain of wheat YW243 and genetic analysis. and Plant Pathology, 2003,33 (3): 234-247) carry out that gene is derived and with known be that the result of test cross shows that YW243 is that disease-resistant spectrum is different to stripe rust resistance and known, may be the new resistant gene or the assortment of genes.It shows as the dominance monogenic inheritance to the resistance of No. 31 microspecies in the bar.Liu Chaohui etc. are positioned at YW243 on the 2BL to the resistant gene (called after YrX temporarily) of No. 31 microspecies in the bar, filter out (Liu Chaohui, Lin Zhishan, the old filial piety such as SSR mark Xgwm501 chain with it, horse is strong-willed, Xu Shichang, Zhang Zengyan, Xin Zhiyong, Wang Hui. the chromosomal localization of new wheat germplasm YW243 stripe rust resisting, the wheat crops journal, 2005,25 (1): 13-16).
Because wheat powdery mildew (Blumeria gramini f.sp.tritici) and puccinia striiformis (Puccinia striiformis f.sp.tritici) colony are big, the physiological strain variation soon, easily causes former resistant variety forfeiture disease resistance.If a kind polymerization a plurality of disease-resistant genes, not only can discern a plurality of different pathogenic bacteria physiological strains, enlarged disease-resistant spectrum, and a plurality of nontoxic genes of pathogenic bacteria must all undergo mutation (probability is very low), just can cause resistant lose (the Singh S of kind, Sidhu JS, Huang N, Vikal Y, Li Z, Brar D S.Pyramiding Three Bacterial Blight Resistance Genes[xa5, Xa13, Xa21] UsingMarker-assisted Selection into indica Rice Cultivar PR106.Theoretical andApplied Genetics, 2001,102:1011-1015; Li Z K, Sanche Z A, Angeles E, SinghS, Domingo J, Huang N.Are the Dominant and Recessive Plant Disease ResistanceGene Similar? A Case Study of Rice R Genes and Xanthomonas oryza pv.Oryzaeraces.Genetics, 2001,159:757-765).Therefore, the disease-resistant gene polymerization is the Critical policies that improves the varietal resistance persistence, enlarges disease-resistant spectrum and regional adaptability.By on purpose hybridizing, reestablishing diplomatic relations, a plurality of disease-resistant genes can be aggregated in the same individuality, but these genes to be transmitted in the offspring, breed stable height for strain or kind, must have fast accurate, practicable identification of means to per generation material follow the tracks of detection.For the different disease-resistant gene of phenotype, can be distinguished by the inoculation and the phenotypic evaluation of various pathogenic bacteria.For the different genes polymer and the offspring thereof of same disease, traditional phenotypic evaluation is difficult to accurately differentiate the wherein number and the concrete ownership of disease-resistant gene.Divide the microspecies artificial inoculation to identify, can be according to the composition of the contained disease-resistant gene of anti-spectrum derivation.But cause of disease divides microspecies inoculations to identify that desired condition and experience are all quite high, and segregating generation also is subjected to same individual plant to be difficult to simultaneously restriction with a plurality of microspecies inoculation evaluations in early days.
Because the variation of white powder germ physiological strain, mildew-resistance gene Pm4a gene pairs powder mildew resistance day by day weakens, and better to present popular white powder germ microspecies resistance in seedling stage, strain-forming period resistance is gradually lost.And be derived from tall and big goatweed karyomit(e) 3S long-armed, drawn and be seeped into chromosome of wheat 3D, the 3B mildew-resistance gene Pm13 on long-armed, high anti-the time of infertility to present popular white powder germ microspecies; Be derived from mildew-resistance gene PmV and the Pm21 of cluster hair wheat karyomit(e) 6VS, wide to the anti-spectrum of white powder germ, immunity in the time of infertility.PmV and Pm21 derive from the Soviet Union and cluster hair wheat Britain respectively.
Wheat/cluster hair wheat 6DL/6VS translocation line the Pm97033 that contains PmV, Pm97034, pm97035 forms (Li H by seed selections such as the old filial piety of crop science institute of the Chinese Academy of Agricultural Sciences, Chen X, Xin Z Y, Ma Y Z, Chen X Y, JiaX.Development and Identification of wheat-Haynaldia villosa T6DL.6VSChromosome Translocation Lines Conferring Resistance to Powdery mildew.PlantBreeding, 2005,124:203-205), wheat/cluster hair wheat 6AL/6AS translocation line the 92R137 that contains Pm21,92R149,92R178 is taught by the Chen Peidu of Agricultural University Of Nanjing, seed selections such as the big an ancient unit of weight academician of Liu form (Chen P D, Qi L L, Zhou B, Zhang S Z, Liu D J.Development and molecular cytogeneticanalysis of wheat-Haynaldia villosa 6VS/6AL translocation lines specifyingresistance to powdery mildew.Theoretical and Applied Genetics, 1995,91 (6): 1125-1128).PmV and Pm21 all are positioned on the cluster hair wheat karyomit(e) 6V galianconism, because 6VS and wheat homeologous chromosome are difficult to reorganization, exchange, so the PCR mark of Pm21 also can be used for detecting PmV.
Summary of the invention
Technical problem to be solved by this invention provides a kind of seed selection mildew-resistance of holding concurrently, the method for stripe rust and yellow dwart wheat germplasm.
The seed selection provided by the present invention mildew-resistance of holding concurrently, the method of stripe rust and yellow dwart wheat germplasm, be to utilize in the STS marker detection wheat to be measured of Pm4a to have or not mildew-resistance gene Pm4a, utilize in the SCAR marker detection wheat to be measured of Pm13 and have or not mildew-resistance gene Pm13, utilize in the SCAR marker detection wheat to be measured of Pm21 and have or not mildew-resistance gene PmV, utilize in the SCAR marker detection wheat to be measured of Bdv2 to have or not resistance to yellow dwarf gene Bdv2, utilize in the SSR marker detection wheat to be measured of YrX to have or not Stripe Rust Resistance Gene YrX.
Wherein, have or not the method for mildew-resistance gene Pm4a as follows in the STS marker detection wheat to be measured of the described Pm4a of utilization: the genomic dna with wheat to be measured is a template, use a pair of primer of forming by the nucleotide sequence of the nucleotide sequence of sequence in the sequence table 1 and sequence 2 to carry out pcr amplification, obtain the dna fragmentation of 470bp as wheat to be measured, then contain mildew-resistance gene Pm4a in this wheat to be measured.
Have or not the method for mildew-resistance gene Pm13 as follows in the SCAR marker detection wheat to be measured of the described Pm13 of utilization: the genomic dna with wheat to be measured is a template, use a pair of primer of forming by the nucleotide sequence of the nucleotide sequence of sequence in the sequence table 3 and sequence 4 to carry out pcr amplification, obtain the dna fragmentation of 564bp as wheat to be measured, then contain mildew-resistance gene Pm13 in this wheat to be measured.
Have or not the method for mildew-resistance gene PmV as follows in the SCAR marker detection wheat to be measured of the described Pm21 of utilization: the genomic dna with wheat to be measured is a template, use a pair of primer of forming by the nucleotide sequence of the nucleotide sequence of sequence in the sequence table 5 and sequence 6 to carry out pcr amplification, obtain the dna fragmentation of 1400bp as wheat to be measured, then contain mildew-resistance gene PmV in this wheat to be measured.
The SCAR of the described Bdv2 of utilization detects and has or not the method for resistance to yellow dwarf gene Bdv2 as follows in the wheat to be measured: the genomic dna with wheat to be measured is a template, use a pair of primer of forming by the nucleotide sequence of the nucleotide sequence of sequence in the sequence table 7 and sequence 8 to carry out pcr amplification, obtain the dna fragmentation of 450bp as wheat to be measured, then contain resistance to yellow dwarf gene Bdv2 in this wheat to be measured.
The described YrX-SSR of utilization detects and has or not the method for Stripe Rust Resistance Gene YrX as follows in the wheat to be measured: the genomic dna with wheat to be measured is a template, use a pair of primer of forming by the nucleotide sequence of the nucleotide sequence of sequence in the sequence table 9 and sequence 10 to carry out pcr amplification, as the increase dna fragmentation of 160-180bp of wheat to be measured, then contain Stripe Rust Resistance Gene YrX in this wheat to be measured.
The present invention utilizes mildew-resistance gene Pm4a, Pm13, PmV, the PCR mark that Stripe Rust Resistance Gene YrX, resistance to yellow dwarf gene Bdv2 are special, detected the polymer of mildew-resistance, stripe rust resisting, resistance to yellow dwarf 5 genes, select double anti-white powder, bar rust, 1 strain of yellow dwart winter habit material of five disease-resistant genes of polymerization Pm4a+Pm13+PmV+YrX+Bdv2, winter habit wheat 6 strains of four disease-resistant genes of Pm4a+PmV+YrX+Bdv2, spring habit BC
2F
3Material 18 strains, winter habit material 2 strains of the anti-above-mentioned three kinds of diseases of holding concurrently of four disease-resistant genes of Pm4a+Pm13+YrX+Bdv2, spring habit BC
2F
3Material 3 strains, tool Pm13+YrX+Bdv2, PmV+YrX+Bdv2 hold concurrently and resist the spring habit BC of three kinds of diseases
2F
3Individual plant is respectively 7 strains and 46 strains, can be cultivate lasting, resistance of wide spectrum kind provide hereditary basis clear and definite enrich anti-source.Method of the present invention can be carried out Molecular Detection and selection fast in arbitrary breeding time of the envrionment conditions of not having morbidity, plant, particularly detects at a plurality of disease-resistant gene polymers, shortens breeding cycle, accelerates more to embody its superiority on the breeding speed.Therefore, the molecule marker quick breeding of the present invention mildew-resistance of holding concurrently, the method for stripe rust and yellow dwart wheat germplasm is seed selection both mildew-resistance, the stripe rust wheat germplasm of a plurality of gene pyramidings of resistance to yellow dwarf again provides a kind of system of selection efficiently.
Description of drawings
Fig. 1 is YrX-SSR primer gwm501 amplification BC
2F
2Per plant
Fig. 2 is Pm4a-STS marker detection BC
2F
3The part per plant
Fig. 3 is Pm13-SCAR marker detection BC
2F
3The part per plant
Fig. 4 is PmV-SCAR marker detection BC
2F
3The part per plant
Fig. 5 is Bdv2-SCAR marker detection BC
2F
3The part per plant
Fig. 6 is YrX-SSR primer gwm501 amplification BC
2F
3Per plant
Embodiment
Experimental technique among the following embodiment if no special instructions, is ordinary method.
One, the acquisition of test material
1, the field spring habit wheat germplasm 04P161～04P170 (BC of mildew-resistance, stripe rust, yellow dwart that holds concurrently
2F
2) and self-mating system BC
2F
3Obtain
04P161～04P170 is 3 * M269/GP22-15, BC
2F
2Material is to be female parent with spring habit wheat with resistance to yellow dwarf strain M269, and the GP22-15 (carrying Pm4a, Pm13, PmV, YrX) of hold concurrently mildew-resistance, stripe rust is paternal hybrid acquisition F
1, be male parent and F again with M269
1Backcross and obtain BC
1Generation is male parent and BC with M269 (carrying Bdv2) again
1Backcross and obtain BC
2For plant, selfing promptly obtains BC 1 time
2F
2Material 04P161～04P170, selfing promptly obtains BC once more
2F
3Material.The selfing individual plant of 04P161 is designated as 04P161-1～04P161-10, and the rest may be inferred, and disease resistance was identified and therefrom obtained disease-resistant 04P161-1～04P170-10 totally 95 strains in seedling stage seedling stage, as table 3.And M269 is 2 * Ning Chun 12/PP9-1, F
6In generation, PP9-1 is the wheat with resistance to yellow dwarf material that carries Bdv2, is combined as among CSph * 2/L1//CSN5BT5D/3/ in 7902/4/ 8601.GP22-15 be YW243/70283 (carrying Pm13) //Pm97033 (carrying PmV), F
3High mildew-resistance, stripe rust wheat lines.YW243 obtains F by Y90136 (doing female parent) with RW1685 (doing male parent) hybridization
2The germplasm of the anti-yellow dwarf that must hold concurrently, Powdery Mildew, 3 rust for the anther cultural progeny selection of material, wherein Y90136 is combined as PP9-1/ Shan 7859//rich resisting No. 8, and promptly PP9-1 (doing female parent) obtains cross-fertilize seed F with Shan 7859 (doing male parent)
1Obtain with rich anti-No. 8 (doing male parent) hybridization again; RW1685 is combined as 3 * rich anti-13/Khapli (containing Pm4a), does female parent with rich anti-13, and the Khapli that carries Pm4a is a male parent, and hybridization obtains F
1, backcrossing with rich anti-13 (doing male parent) obtains for 2 times again.
Wherein, CSph be the wheat breed China spring the ph cryptic mutant, can not suppress homologous chromosomes pairing (Britain Camb plant breeding institute); L1 is wheat with resistance to yellow dwarf-middle couchgrass disomic addition line (Cauderon Y, Saigne B, Dauge is resistance to wheat rusts of Agropyron INtermediumand its use in wheat improvement.In:Proc.4th Int.Wheat Genet.Symp. M.1973.The, pp.401-407.University of Missouri, Columbia, Mo); CSN5BT5D (Britain Camb plant breeding institute) for the 5B nullisomic 5D limbs of wheat breed China spring, can not suppress homologous chromosomes pairing; In 7902 with in 8601 (the Chinese Academy of Agricultural Sciences crop breeding cultivation institutes); 70283 is the powdery-mildew-resistance wheat material, is combined as VPM/ hundred farmings 3217 * 3; Pm97033 is the powdery-mildew-resistance wheat-cluster hair wheat translocation line that carries PmV, breed (Li H by crop breeding cultivation institute of the Chinese Academy of Agricultural Sciences, Chen X, Xin Z Y, Ma Y Z, Chen X Y, Jia X.Development and Identification of wheat-Haynaldia villosa T6DL.6VSChromosome Translocation Lines Conferring Resistance to Powdery mildew.PlantBreeding, 2005,124:203-205); Spring wheat variety, high yield, wide suitable that the peaceful spring 12 is bred for Yinchuan City, the Ningxia State Scientific and Technological Commission; Shan 7859 (academy of agricultural sciences, Shaanxi food crop institute) is the stripe rust resisting wheat kind; Rich anti-No. 8 (crop breeding cultivation institute of the Chinese Academy of Agricultural Sciences) has been combined as No. 10, the red No. 7/Luo Fulin of awns, is the high yield wheat breed of stripe rust resisting; Rich anti-13 (crop breeding cultivation institutes of the Chinese Academy of Agricultural Sciences) are combined as that Beijing 14/ is anti-draws 655, are the high yield wheat breeds of stripe rust resisting; Khapli is the powdery-mildew-resistance wheat germplasm (Britain Camb plant breeding institute) that carries Pm4a).
2, the field acquisition of winter habit wheat germplasm 04S003-1～04S003-10 of mildew-resistance, stripe rust, yellow dwart of holding concurrently
04S003-1～04S003-10 is 2 * M269/GP22-13//CA9722 * 4 (BC
3F
1), be to be female parent with spring habit wheat with resistance to yellow dwarf strain M269, the GP22-13 of hold concurrently mildew-resistance, stripe rust is that paternal hybrid obtains F
1, be male parent and F again with M269
1Backcross and obtain BC
1In generation, is again with winter wheat CA9722 (doing male parent) hybridization 1 time, 3 times the material of backcrossing.Wherein, GP22-13 be combined as YW243/70283 (carrying Pm13) //Pm97033 (carrying PmV), F
3CA9722 (crop breeding cultivation institute of the Chinese Academy of Agricultural Sciences) is anti-bar rust, leaf rust wheat breed.
3, the Khapli that contains Pm4a, contain Pm4a, YW243 (the Xie Hao of the new gene YrX of stripe rust resisting, old filial piety, Zhang Zengyan, Xin Zhiyong, Lin Zhishan, Du Lipu, horse is strong-willed, Xu Huijun. the seed selection of wheat with resistance to yellow dwarf new lines YW243 and cellular elements biological assay. Acta Agronomica Sinica, 2000,26 (6): 687-691), 70823 (being Pm776-1) of containing Pm13, Pm97033 (the Li H that contains PmV, Chen X, Xin Z Y, Ma Y Z, Chen X Y, JiaX.Development and Identification of wheat-Haynaldia villosa T6DL.6VSChromosome Translocation Lines Conferring Resistance to Powdery mildew.PlantBreeding, 2005,124:203-205), HW642 (the Zhang Z Y that contains Bdv2, Xu J S, Xu QJ, Larkin Philip, Xin Z Y.Development of novel PCR markers linked to theBYDV resistance gene Bdv2 useful in wheat for marker-assisted selection.Theoretical and Applied Genetics, 2004,109:433-439)) respectively as the positive control of each gene test, wheat breed China spring (CS), in 8601, CA9722 is as negative control.
Two, disease-resistant evaluation
Powdery mildew disease-resistant evaluation: under field and greenhouse experiment, to No. 15 physiological strains of the artificial earlier inoculation white powder germ of above-mentioned test material seedling, inoculate the mixing microspecies of Powdery Mildew natural occurrence after 14 days, 2-3 is after week, 8601 fully during morbidity in the susceptible contrast, investigation 04P161～04P170 (BC
2F
2) and (BC of selfing strain system
2F
3) with 04S003-1～04S003-10 seedling stage with become strain phase disease resistance.It is disease-resistant that this research does not have being decided to be of white powder germ spore powder heap with the inoculation blade, and blade has being decided to be of white powder germ spore powder heap susceptible more than 5%.
The yellow dwart disease resistance is identified: carry barly yellow dwarf virus aphid (GAV strain system) in field inoculation in seedling stage, observe incidence after 40 days.Plant with no yellow dwarf's illness blade is disease-resistant, and the plant that yellow dwarf's illness blade is arranged is susceptible.
The stripe rust disease resistance is identified: in No. 30 popular microspecies of a leaf one heart stage inoculation stripe rust bacterium, treat that check variety engraves fully back (about 15 days) investigation disease resistances of virtuous 169 morbidities with rubbing manipulation.The plant that does not have the obvious sorus of stripe rust bacterium with blade is disease-resistant.
In the field, the spring habit material of 2 combinations of transformation and winter habit material are carried out resistance identify that the result shows, spring habit 04P161～04P170 (BC
2F
2) with each 10 equal mildew-resistance of individual plant of winter habit 04S003-1～04S003-10, stripe rust, yellow dwart.Above-mentioned materials is carried out Markers for Detection according to the method for step 3, the results are shown in Table 1, table 2.And to 04P161～04P170 selfing (BC
2F
3) strain is the Molecular Detection that 04P161-1～04P170-21 carries out disease-resistant evaluation and resistant gene thereof.
Under greenhouse experiment, to spring habit material 04P161～(BC of 04P170 selfing strain system
2F
3) 04P161-1～04P170-21 inoculation white powder germ, identify its seedling stage, become the powder mildew resistance of strain phase, pull out the disease plant in seedling stage, preserve disease-resistant plant, proceed into the disease-resistant evaluation of strain phase, extract 04P161-1～04P170-10 totally 95 individual plant DNA, carry out the Molecular Detection of 5 disease-resistant genes, the results are shown in Table 3.
Three, Markers for Detection
1, utilize molecule marker Pm4-STS to detect mildew-resistance gene Pm4a
The pcr amplification of Pm4-STS: with reference to (Ma Z.Q such as Ma, Wei J.B, Cheng S.H.2004, PCR-basedmarkers for the powdery mildew resistance gene Pm4a in wheat.Theoretical andApplied Genetics, 109:140-145) method is carried out pcr amplification with the Pm4F, Pm4R sequence synthesized primer thing Pm4F, the Pm4R that deliver.
(1) Pm4a-STS special primer sequence
Pm4F:5 '-TCCAGTGACCCCATCTGCTCATAC-3 ' (sequence 1)
Pm4R:5 '-GTGGTGTATCAAA TGTCATCAGTACTAC-3 ' (sequence 2)
(2) reaction system
10 * PCR damping fluid, 2.5 μ l
25mM?MgCl
2 2.0μl
10mM?dNTPs 0.5μl
TaqE(5u/ul) 0.2μl
Pm4F(60ng/L) 1.0μl
Pm4R(60ng/L) 1.0μl
Genomic dna (50ng/ul) 2.0 μ l
ddH
2O 15.8μl
Amount to 25 μ l
(3) reaction conditions: 96 ℃ of pre-sex change 1min of elder generation; 94 ℃ of 1min then, 60 ℃ of 1.5min, 72 ℃ of 1.5min totally 35 circulations; 72 ℃ are extended 10min again.
The PCR product that obtains is carried out electrophoretic analysis at 1.2% sepharose, as obtain the special band of the Pm4a-STS of the 470bp identical with tool Pm4a positive control Khapli, wheat plant then to be measured contains mildew-resistance gene Pm4a.
2, utilize molecule marker Pm13-SCAR to detect mildew-resistance gene Pm13
The pcr amplification of Pm13-SCAR: with reference to (Ceici A such as Cenci, D ' Ovidio R, TanzareLLa 0A, Ceoloni C, Porceddu E.Identification of molecular markers linked to Pm13, an Aegilops Longissiuma gene conferring resistance to powdery mildew in wheat, Theor.Appl.Genet., 1999,98:448-454) method and the P13F that delivers and P13R sequence synthesized primer thing carry out pcr amplification.
(1) Pm13-SCAR special primer sequence
P13F:5 '-CGCCAGCCAATTATCTCCATGA-3 ' (sequence 3)
P13R:5 '-AGCCATGCGCGGTGTCATGTGAA-3 ' (sequence 4)
(2) reaction system
10 * PCR damping fluid, 2.5 μ l
25mM?MgCl
2 1.5μl
10mM?dNTPs 0.5μl
TaqE(5u/ul) 0.2μl
P13F(50ng/L) 1.0μl
P13R(50ng/L) 1.0μl
Genomic dna (50ng/ul) 2.0 μ l
ddH
2O 16.3μl
Amount to 25 μ l
(3) reaction conditions: 94 ℃ of pre-sex change 5min of elder generation; 94 ℃ of 1min then, 55 ℃ of 1min, 72 ℃ of 2min, totally 40 circulations; 72 ℃ are extended 10min again.
The PCR product that obtains is carried out electrophoretic analysis at 1.2% sepharose, if obtain the special band of Pm13-SCAR of the 564bp identical with tool Pm13 positive control PM776-1, wheat plant then to be measured contains mildew-resistance gene Pm13.
3, utilize molecule marker Pm21-SCAR to detect mildew-resistance gene PmV
PMV and Pm21 all are positioned on the cluster hair wheat karyomit(e) 6V galianconism, because 6VS and wheat homeologous chromosome are difficult to reorganization, exchange, so the PCR mark of Pm21 can be used for detecting PmV.
The pcr amplification of Pm21-SCAR: utilize Pm21D and method (Liu Z Y such as Pm21C primer and Liu, SunQ X, Ni Z F.Development of SCAR markers linked to the Pm21 gene conferringresistance to the powdery mildew in common wheat.Plant Breeding, 1999,118:215-219) carry out.
(1) Pm21-SCAR special primer sequence
Pm21C:5 '-CACTCTCCTCAAACCTTGCAAG-3 ' (sequence 5)
Pm21D:5 '-CACTCTCCTCCACTAACAGAGG-3 ' (sequence 6)
(2) reaction system same Pm13 except that primer.
(3) reaction conditions: 94 ℃ of pre-sex change 5min of elder generation; 94 ℃ of 1min then, 55 ℃ of 1min, 72 ℃ of 2min, totally 35 circulations; Last 72 ℃ of 10min.
The PCR product that obtains is carried out electrophoretic analysis at 1.2% sepharose, if obtain Pm21 (V)-SCAR specific band of the 1400bp identical with tool PmV positive control Pm97033, wheat plant then to be measured contains mildew-resistance gene PmV.
4, utilize molecule marker Bdv2-SCAR to detect resistance to yellow dwarf gene Bdv2
The pcr amplification of Bdv2-SCAR: adopt primer SC-W37U and methods such as SC-W37L and Zhang Zengyan (Zhang Zengyan, Xin Zhiyong, old filial piety, Wang Xiaoping, Liu Jingfang, Du Lipu. come from the specific PCR mark of wheat resistance to yellow dwarf gene of L1 and the research of assistant breeding. Acta Agronomica Sinica, 2000,28 (4): 486-491) carry out.
(1) Bdv2-SCAR special primer sequence
SC-W37U:5 '-ACGAATTCCCAGCTAAACGCCCTC-3 (sequence 7)
SC-W37L:5 '-TCATTGTTGATCTTGCATGGTCGTAG-3 ' (sequence 8)
(2) Bdv2 reaction system
10 * PCR damping fluid, 2.5 μ l
25mM?MgCl
2 2.5μl
2.5mM?dNTPs 2.0μl
TaqE(5u/ul) 0.2μl
SC-W37U(5umol/L) 1.0μl
SC-W37U(5umol/L) 1.0μl
Genomic dna (25ng/ul) 2.0 μ l
ddH
2O 13.8μl
Amount to 25 μ l
(3) amplification condition: 96 ℃ of pre-sex change 1min of elder generation; 94 ℃ of 1min then, 64 ℃ of 1min, 72 ℃ of 2min, totally 35 circulations; Last 72 ℃ of 10min.
The PCR product that obtains is carried out electrophoretic analysis at 1.2% sepharose, if obtain the special band of Bdv2-SCAR of the 450bp identical with tool Bdv2 positive control HW642, wheat plant then to be measured contains resistance to yellow dwarf gene Bdv2.
5, utilize molecule marker YrX-SSR to detect Stripe Rust Resistance Gene YrX
The pcr amplification of YrX-SSR: utilize methods such as SSR primer gwm501 and Liu Chaohui (Liu Chaohui, Lin Zhishan, old filial piety, horse is strong-willed, Xu Shichang, Zhang Zengyan, Xin Zhiyong, Wang Hui. the chromosomal localization of new wheat germplasm YW243 stripe rust resisting. wheat crops journal, 2005,25 (1): 13-16) carry out.
(1) YrX-SSR primer gwm501 sequence
Gwm501-F 5 '-GGCTATCTCTGGCGCTAAAA-3 ' (sequence 9)
Gwm501-R 5 '-TCCACAAACAAGTAGCGCC-3 ' (sequence 10)
(2) YrX reaction system
10 * PCR damping fluid, 2.5 μ l
25mM?MgCl
2 0.8μl
2.5mM?dNTPs 1.6μl
TaqE(5u/ul) 0.2μl
gwm501-F(4umol/L) 1.5μl
gwm501-R(4umol/L) 1.5μl
Genomic dna (40ng/ul) 2.0 μ l
ddH
2O 14.9μl
Amount to 25 μ l
(3) amplification condition: 94 ℃ of pre-sex change 5min of elder generation; 94 ℃ of 1min then, 60 ℃ of 1min, 72 ℃ of 2min, totally 45 circulations; Last 72 ℃ are extended 7min.
Pcr amplification product carries out electrophoresis at 6% polyacrylamide gel, Silver Nitrate silver dyes analysis, if obtain the special band of YrX-SSR of the 160-180bp identical with tool YrX positive control YW243, wheat plant then to be measured contains Stripe Rust Resistance Gene YrX.
To the spring habit 04P161～04P170 (BC that identifies through the field resistance of step 2
2F
2) utilize molecule marker to carry out mildew-resistance gene, resistance to yellow dwarf gene and Stripe Rust Resistance Gene detection according to the method described above with each 10 individual plant of winter habit 04S003-1～04S003-10.
The result is as shown in table 1 for the spring habit material tests, from spring habit material 04P161～04P170 (BC of field mildew-resistance, stripe rust, yellow dwart
2F
2) 10 individual plants, all detect Stripe Rust Resistance Gene YrX (Fig. 1), at least one mildew-resistance gene Pm4a, Pm13, PmV, do not detect in 04P164 the resistance to yellow dwarf gene Bdv2, other 9 individual plants all detect resistance to yellow dwarf gene Bdv2, and molecule marker result and field resistance be basically identical as a result.
Table 1 spring habit material B C
2F
2The Markers for Detection of individual plant
| Strain number | Bdv2 | Pm4a | Pm13 | PmV | YrX |
| 04P161 04P162 04P163 04P164 04P165 04P166 ? 04P167 04P168 04P169 04P170 | + + + - + + ? + + + + | + + - + + + ? + + + + | - - - - - - ? - + - - | + + + + + + ? + - + + | + + + + + + ? + + + + |
Annotate: "+" expression detects this gene, and "-" expression does not detect this gene.
Wherein, utilize SSR primer Xgwm501PCR amplification spring habit 04P161-170 (BC
2F
2) 10 individual plants YrX-SSR result as shown in Figure 1,10 individual plants all can amplify the special band of YrX-SSR (arrow shows the purpose band) of 160-180bp; M:pBR322 DNA/Msp I marker; 1.YW243; 2. China spring (CS); 3.04P161; 4.04P162; 5.04P163; 6.04P164; 7.04P165; 8.04P166; 9.04P167; 10.04P168; 11.04P169; 12.04P170.
Mildew-resistance, stripe rust resisting, resistance to yellow dwarf gene to the winter habit material 04S003-1～04S003-10 of the mildew-resistance of holding concurrently, stripe rust, yellow dwart carry out Molecular Detection, result such as table 2.Table 2 shows, 10 winter habit individual plants that detected, all are 2～5 disease-resistant gene polymers, one of them individual plant 04S003-4 polymerization five disease-resistant genes of Pm4a+Pm+PmV+YrX+Bdv2,04S003-1～04S003-2, six individual plant polymerizations of 04S003-7～04S003-10 four disease-resistant genes of Pm4a+PmV+YrX+Bdv2,04S003-3, two individual plant polymerizations of 04S003-6 four disease-resistant genes of Pm4a+Pm13+YrX+Bdv2, molecule marker result and field resistance be basically identical as a result.
Table 2 winter habit wheat germplasm BC
3F
1The individual plant molecule marker
| Strain number | Bdv2 | Pm4a | Pm13 | PmV | YrX |
| 04S003-1 04S003-2 04S003-3 04S003-4 | + + + + | + + + + | - - + + | + + - + | + + + + |
| 04S003-5 04S003-6 04S003-7 04S003-8 04S003-9 04S003-10 | - + + + + + | - + + + + + | + + - - - - | - - + + + + | + + + + + + |
Learn 10 spring habit wheat BC from table 1
2F
2The individual plant major part has 3-4 disease-resistant gene.If want in the offspring to keep a plurality of resistant genes, just require each gene in the per generation material is all followed the tracks of detection, select stable resistant gene polymer as how anti-parent.
To spring habit wheat BC
2F
2(the BC of selfing strain system of material
2F
3) 95 individual plants molecule marker of having carried out Pm4a, Pm13, PmV, Bdv2, five resistant genes of YrX follows the tracks of and detect, the selfing individual plant of 04P161 is designated as 04P161-1～04P161-10, and the rest may be inferred, and the result is shown in table 3 and Fig. 2～6.
Table 3 spring habit BC
2F
3For the selfing strain is Markers for Detection result (becoming the strain phase)
| 04P161-1 | -2 | -3 | -4 | -5 | -6 | -7 | -8 | -9 | -10 | 04P162-1 | -2 | -3 | -4 | -5 | -6 | -7 | -8 | -9 | -10 | |
| Pm4a Pm13 PmV Bdv2 YrX powdery mildew | ? - ? - + + + ? R 04P163-1 | ? - ? - + + + ? R -2 | ? - ? - + + + ? R -3 | ? - ? - + + + ? R -4 | ? - ? - + + + ? R -5 | ? - ? - + + + ? R -6 | ? + ? - + + + ? R -7 | ? - ? - + + + ? R -8 | ? - ? - + + + ? R -9 | ? + ? - + + + ? R -10 | ? - ? - + + + ? R 04P164-1 | ? - ? - + + + ? R -2 | ? + ? - + + + ? R -3 | ? - ? - + + + ? R -4 | ? - ? - + + + ? R -5 | ? + ? - + + + ? R -6 | ? + ? - + + + ? R -7 | ? - ? - + + + ? R -8 | ? - ? - + + + ? R -9 | ? + ? - + + + ? R -10 |
| Pm4a Pm13 PmV Bdv2 YrX Powdery Mildew | - - + + ? + R 04P165-1 | - - + + ? + R -2 | - - + + ? + R -3 | - - + + ? + R -4 | - - + + ? + R -5 | - - + + ? + R -6 | - - + + ? + R -7 | - - + + ? + R -8 | - - + + ? + R -9 | - - + + ? + R -10 | - - + + ? + S 04P166-1 | - - + - ? + R -2 | - - + - ? + R -3 | - - + - ? + R -4 | - - + - ? + R -5 | - - + - ? + R -6 | - - + - ? + R -7 | - - + - ? + R -8 | - - + - ? + R -9 | + - + - ? + R -10 |
| Pm4a Pm13 PmV Bdv2 YrX Powdery Mildew | + ? - + + + R 04P167-2 | - ? - + + + R -3 | + ? - + + + R -4 | - ? - + + + R -5 | - ? - + + + R -6 | - ? - + + + R / | - ? - + + + R / | + ? - + + + R / | - ? - + + + R / | - ? - + + + R / | - ? - + + + R 04P168-1 | + ? - - + - S -2 | + ? - + - + R -3 | - ? - - + + S -4 | - ? - - - - S -5 | - ? - + + + R -6 | - ? - - + + S -7 | - ? - + + + R -8 | - ? - + + + R -9 | - ? - + + + R -10 |
| Pm4a Pm13 PmV Bdv2 YrX powdery mildew | + - - ? - ? - S 04P169-1 | + - - ? - ? + S -2 | - - + ? + ? + R -3 | + - - ? - ? + S -4 | + - - ? - ? + S -5 | / / / ? / ? / / -6 | / / / ? / ? / / -7 | / / / ? / ? / / -8 | / / / ? / ? / / -9 | / / / ? / ? / / -10 | - + - ? + ? + R 04P170-1 | - + - ? + ? + R -2 | - + - ? + ? + R -3 | - + - ? + ? + R -4 | - + - ? + ? + R -5 | - + - ? + ? + R -6 | + + - ? + ? + R -7 | + + - ? + ? + R -8 | - + - ? + ? + R -9 | + + - ? + ? + R -10 |
| Pm4a Pm13 PmV Bdv2 YrX powdery mildew | - ? - ? + + + R | - ? - ? + + + R | + ? - ? - + + S | - ? - ? + + + R | - ? - ? - + + S | - ? - ? + + + R | - ? - ? + + + R | + ? - ? + + + R | - ? - ? + + + R | - ? - ? + + + R | - ? - ? + + + R | + ? - ? + + + R | + ? - ? + + + R | + ? - ? + + + R | - ? - ? + + + R | + ? - ? + + + R | + ? - ? + + + R | + ? - ? + + + R | + ? - ? + + + R | + ? - ? + + + R |
Annotate: R shows mildew-resistance; S shows the sense Powdery Mildew; + show to detect this gene;-show not detect this gene)
Being learnt by table 3, is BC 95 selfing strains
2F
3In the individual plant, choosing the hold concurrently individual plant of mildew-resistance, stripe rust and 3 kinds of diseases of yellow dwart of four genes of polymerization Pm4a+PmV+YrX+Bdv2, Pm4a+Pm13+YrX+Bdv2 has 18 and 3 respectively, three resistant genes of polymerization PmV+Bdv2+YrX, Pm13+Bdv2+YrX hold concurrently mildew-resistance, stripe rust and 3 kinds of diseases of yellow dwart 46 and 7 individual plants are arranged respectively.The above results confirms, selects resistant gene polymer self progeny by molecule marker, and obtaining 3-4 the polymeric number of disease-resistant gene increases.From BC
2F
2Individual plant and selfing strain thereof are BC
2F
3The disease-resistant gene molecule detected result of individual plant learns, disease-resistant gene particularly heterozygous genes type and external source disease-resistant gene is being backcrossed, can separated in the selfing process, thus utilize molecule marker to per generation material select very necessity.
Among Fig. 2, arrow shows the special band of the Pm4a-STS of 470bp; M.PCR markers (available from Huamei Bio-Engrg Co.); 1.Khapli; 2CS; 3.04P169-7; 4.0469-8; 5.4P169-9; 6.04P169-10; 7.04P170-1; 8.04P170-2; 9.04P170-3; 10.04P170-4; 11.04P 170-5; 12.04P170-6; 13.04P 170-7; 14.04P170-8; 15.04P170-9; 16.04P170-10.Among Fig. 3, arrow shows the special band of the Pm13-SCAR of 564bp; M.100bp ladders; 1.Pm776-1; 2.CS; 3.04P168-3; 4.04P168-4; 5.04P168-5; 6.04P168-6; 7.04P168-7; 8.04P168-8; 9.04P168-9; 10.04P168-10; 11.04P165-1; 12.04P165-2; 13.04P165-3; 14.04P165-4; 15.04P165-5; 16.04P165-6.Among Fig. 4, arrow shows the special band of the PmV-SCAR of 1400bp; M.100bpladders; 1.CS; 2.Pm97033; 3.04P166-1; 4.04P166-2; 5.04P166-3; 6.04P166-4; 7.04P 166-5; 8.04P 166-6; 9.04P 166-7; 10.04P 166-8; 11.04P 166-9; 12.04P166-10; 13.04P167-2; 14.04P167-3; 15.04P167-4; 16.04P167-5.Among Fig. 5, arrow shows the special band of the Bdv2-SCAR of 450bp; M.100bp ladders; 1.HW642; 2.CS; 3.04P166-1; 4.04P166-2; 5.04P166-3; 6.04P166-4; 7.04P166-5; 8.04P166-6; 9.04P166-7; 10.04P166-8; 11.04P166-9; 12.04P166-10; 13.04P167-2; 14.04P167-3; 15.04P167-4; 16.04P167-5.Among Fig. 6, arrow shows the special band of the YrX of 160-180bp, M:pBR322DNA/Msp I markers; 1.YW243; 2. China spring; 3.04P165-1; 4.04P166-2; 5.04P161-1; 6.04P161-2; 7.04P161-3; 8.04P161-4; 9.04P161-5; 10.04P161-6; 11.04P161-7; 12.04P161-8; 13.04P161-9; 14.04P161-10; 15.04P162-1; 16.04P162-2.
Sequence table
<160>10
<210>1
<211>24
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>1
tccagtgacc?ccatctgctc?atac 24
<210>2
<211>28
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>2
gtggtgtatc?aaatgtcatc?agtactac 28
<210>3
<211>22
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>3
cgccagccaa?ttatctccat?ga 22
<210>4
<211>23
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>4
agccatgcgc?ggtgtcatgt?gaa 23
<210>5
<211>22
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>5
cactctcctc?aaaccttgca?ag 22
<210>6
<211>22
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>6
cactctcctc?cactaacaga?gg 22
<210>7
<211>24
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>7
acgaattccc?agctaaacgc?cctc 24
<210>8
<211>26
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>8
tcattgttga?tcttgcatgg?tcgtag 26
<210>9
<211>20
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>9
ggctatctct?ggcgctaaaa 20
<210>10
<211>19
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>10
tccacaaaca?agtagcgcc 19
Claims (6)
1, a kind of assist-breeding mildew-resistance of holding concurrently, the method of stripe rust and yellow dwart wheat germplasm, be to utilize in the STS marker detection wheat to be measured of Pm4a to have or not mildew-resistance gene Pm4a, utilize in the SCAR marker detection wheat to be measured of Pm13 and have or not mildew-resistance gene Pm13, utilize in the SCAR marker detection wheat to be measured of Pm21 and have or not mildew-resistance gene PmV, utilize in the SCAR marker detection wheat to be measured of Bdv2 to have or not resistance to yellow dwarf gene Bdv2, utilize in the SSR marker detection wheat to be measured of YrX to have or not Stripe Rust Resistance Gene YrX.
2, method according to claim 1, it is characterized in that: have or not the method for mildew-resistance gene Pm4a as follows in the STS marker detection wheat to be measured of the described Pm4a of utilization: the genomic dna with wheat to be measured is a template, use a pair of primer of forming by the nucleotide sequence of the nucleotide sequence of sequence in the sequence table 1 and sequence 2 to carry out pcr amplification, obtain the dna fragmentation of 470bp as wheat to be measured, then contain mildew-resistance gene pm4a in this wheat to be measured.
3, method according to claim 1, it is characterized in that: have or not the method for mildew-resistance gene Pm13 as follows in the SCAR marker detection wheat to be measured of the described Pm13 of utilization: the genomic dna with wheat to be measured is a template, use a pair of primer of forming by the nucleotide sequence of the nucleotide sequence of sequence in the sequence table 3 and sequence 4 to carry out pcr amplification, obtain the dna fragmentation of 564bp as wheat to be measured, then contain mildew-resistance gene Pm13 in this wheat to be measured.
4, method according to claim 1, it is characterized in that: have or not the method for mildew-resistance gene PmV as follows in the SCAR marker detection wheat to be measured of the described Pm21 of utilization: the genomic dna with wheat to be measured is a template, use a pair of primer of forming by the nucleotide sequence of the nucleotide sequence of sequence in the sequence table 5 and sequence 6 to carry out pcr amplification, obtain the dna fragmentation of 1400bp as wheat to be measured, then contain mildew-resistance gene PmV in this wheat to be measured.
5, method according to claim 1, it is characterized in that: the SCAR of the described Bdv2 of utilization detects and has or not the method for resistance to yellow dwarf gene Bdv2 as follows in the wheat to be measured: the genomic dna with wheat to be measured is a template, use a pair of primer of forming by the nucleotide sequence of the nucleotide sequence of sequence in the sequence table 7 and sequence 8 to carry out pcr amplification, obtain the dna fragmentation of 450bp as wheat to be measured, then contain resistance to yellow dwarf gene Bdv2 in this wheat to be measured.
6, method according to claim 1, it is characterized in that: the described YrX-SSR of utilization detects and has or not the method for Stripe Rust Resistance Gene YrX as follows in the wheat to be measured: the genomic dna with wheat to be measured is a template, use a pair of primer of forming by the nucleotide sequence of the nucleotide sequence of sequence in the sequence table 9 and sequence 10 to carry out pcr amplification, as the increase dna fragmentation of 160-180bp of wheat to be measured, then contain Stripe Rust Resistance Gene YrX in this wheat to be measured.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102220430A (en) * | 2011-05-17 | 2011-10-19 | 中国农业科学院作物科学研究所 | Auxiliary screening method for stripe rust-resistance wheat and its special primers |
| CN106258937A (en) * | 2016-08-12 | 2017-01-04 | 西北农林科技大学 | A kind of quickly breeding is disease-resistant, the method for good quality wheat new lines |
| CN110607388A (en) * | 2019-09-29 | 2019-12-24 | 四川农业大学 | Wheat stripe rust resistant gene related SNP molecular marker in adult stage, primer and application 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 |
-
2005
- 2005-07-27 CN CN 200510087141 patent/CN1904063A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102220430A (en) * | 2011-05-17 | 2011-10-19 | 中国农业科学院作物科学研究所 | Auxiliary screening method for stripe rust-resistance wheat and its special primers |
| CN106258937A (en) * | 2016-08-12 | 2017-01-04 | 西北农林科技大学 | A kind of quickly breeding is disease-resistant, the method for good quality wheat new lines |
| CN106258937B (en) * | 2016-08-12 | 2018-03-13 | 西北农林科技大学 | A kind of quickly breeding is disease-resistant, the method for good quality wheat strain |
| CN110607388A (en) * | 2019-09-29 | 2019-12-24 | 四川农业大学 | Wheat stripe rust resistant gene related SNP molecular marker in adult stage, primer and application 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 |
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