CN1669410A

CN1669410A - Gibberellic disease resistant and breeding method for wheat

Info

Publication number: CN1669410A
Application number: CN 200510038978
Authority: CN
Inventors: 张旭; 陆维忠; 姚金保; 周淼平; 任丽娟
Original assignee: Jiangsu Academy of Agricultural Sciences
Current assignee: Jiangsu Academy of Agricultural Sciences
Priority date: 2005-04-20
Filing date: 2005-04-20
Publication date: 2005-09-21
Anticipated expiration: 2025-04-20
Also published as: CN1299568C

Abstract

The invention belongs to the field of wheat aneuploid biotechnics breeding. Adopt the integral series of 'Chinese spring' wheat and the wheat gibberellic disease resistant source 'numble 3 su-wheat' to cross and backcross, and microscopy every generation, then select the integral to backcross again. Until it backcrosses to the forth generation, cover the separated integral generation in the bag to cross, and select homozygosity disome to be the substitute material of monosome. Select the SSR primer whose colorant layer is located to do PCR augmentation to the monosome, and certificate the monosome according to the gained molecular label. The material is good for positioning and colonying the gibberellic disease resistant genes of the numble 3 su-wheat, lead the resistant genes into the wheat breeds which don't contains that gene, seclect the wheat breed with gibberellic disease resistance, and accelerate the disease-resistant and the breeding process, then the selecting efficiency is improved.

Description

A kind of wheat scab disease resistant and breeding method

One. technical field

The invention belongs to wheat aneuploid biotechnology breeding field.

Two. background technology

By Fusarium graminearum (Fusarium graminearum, Schw) wheat scab that causes is a worldwide disease, not only cause serious production loss, and the residual deoxynivalenol (deoxynivalenol of sick wheat, mycotoxin such as DON), serious threat people and animals' health.At present there is the Soviet Union wheat No. 3 of China in the anti-source of wheat scab of generally acknowledging in the world, and is peaceful 7840, Wangshuibai etc., but these wheat breeds are difficult to be applied directly in the agricultural production because economical character (as plant height, output etc.) is relatively poor.Therefore need the resistant gene of determining that these anti-sources are carried, and be transferred in the conventional production kind, the disease-resistant wheat kind that cultivation can commercial applications.

Present many research workers (Kolb, F.L., Crop Sci, 2001,41:611-619; Buerstmayr, H, Theor Appl Genet, 2003,107:503～508) utilize RIL or double haploid colony resistant gene research to be carried out in the anti-source of different wheat scabs from full genome angle, the result shows that the scab resistance of wheat is that genetic mechanism is comparatively complicated by the quantitative character of 2-3 to the common modification of minor gene that major gene resistance is controlled and quantity is not quite clear.Therefore the transformation work of resistant gene also be affected.

The application of aneuploid technology in genetic analysis is of long duration.Utilize monosome replacement technology between kind, can be at a certain chromosome, the location genes of interest.But double, drift about, reach reasons such as conversion owing to monosome automatically, in the initiative process of monosome substitution line, the substitution line of mistake can occur.The evaluation of traditional monosome substitution line is to utilize chromosomal cytological marker, but cytological marker quantity is few, and is difficult to obtain, and therefore, before molecular labeling occurred, the monosome substitution line of wheat was never identified.

Three. summary of the invention

The problem that the present invention need solve is: cover (21) monosomic substitution line that the anti-source of creation wheat scab Soviet Union wheat is No. 3, and carry out Molecular Identification, make it to become and to be used to revive the genetic stocks of chromosome mapping research of No. 3 scab resistance genes of wheat and other genes of interest, set up a kind of wheat scab disease resistant and breeding method.

Technical scheme of the present invention is: 1. the initiative of the monosomic substitution line of Soviet Union's No. 3 one covers of wheat (21).2 monosome substitution lines Molecular Identification.

1. the initiative of the monosomic substitution line of Soviet Union's No. 3 one covers of wheat (21).

A) wheat scab disease-resistant variety Soviet Union wheat carries out scab resistance inoculation evaluation for No. 3, selects the best individual plant of resistance;

B) cover (21) China spring series monomer difference microscopy tip of a root meristematic cell chromosome number, the plant of selection 2n=41;

C) be that female parent (♀) is hybridized with above-mentioned Chinese spring series monomer (♂) respectively with No. 3 resistance individual plants of above-mentioned Soviet Union wheat, obtain cover (21 a) hybrid F ₁

D) to hybrid F ₁Microscopy tip of a root meristematic cell chromosome number is selected 2n=41, and external form approaches the plant of China spring, as male parent (♂), with corresponding Chinese spring monomer (♀) hybridization, obtains cover (21 a) BC1 plant respectively;

E) to BC1 plant repeating step d) described operation, obtain cover (21 a) BC2 plant;

F) to BC2 plant repeating step d) described operation, obtain cover (21 a) BC3 plant;

G) to BC3 plant repeating step d) described operation, obtain cover (21 a) BC4 plant;

H) to BC4 plant microscopy tip of a root meristematic cell chromosome number, select 2n=41, external form approaches the plant of China spring, and the bagging selfing obtains cover (21 a) BC4F ₁

L) to BC4F ₁The plant microscopy, the homozygote plant of selection 2n=42, i.e. cover (21) China spring (Soviet Union wheat No. 3) monosome substitution line.

2 monosome substitution lines Molecular Identification.

A) clip one overlaps (21) China spring (Soviet Union wheat No. 3) monosome substitution line plant respectively, and the blade of parent's China spring and No. 3 plant of parent Su Mai extracts complete genome DNA;

B) choose simple repeated sequence mark (SSR) primer 2-4 that is positioned on each bar chromosome respectively;

C) to the DNA of monosome substitution line, use the SSR primer on the above-mentioned homologue, carry out pcr amplification, amplified production obtains the molecular labeling data after electrophoretic separation on the 6g/100ml polyacrylamide gel

D) contrast above-mentioned molecular labeling data, determine the accuracy of gained monosome substitution line.

Effect of the present invention is cover (21) China spring (Soviet Union wheat No. 3) monosome substitution line that the present invention formulates, with the individual chromosome transformation of scab resistance kind Soviet Union wheat No. 3 in Chinese spring.Compare with existing gene positioning method, utilize this cover material to carry out the resistant gene location, can overcome the shortcoming that to study full genome, only the wall scroll chromosome at Soviet Union wheat No. 3 carries out resistant gene research, not only can dwindle research range, reduce workload, the more important thing is to shield each intergenic mutual work and interference, effectively study all scab resistance genes.Compare with existing breeding method, after determining resistant gene, select the monosome substitution line at resistant gene place to hybridize combo as the resistance parent, in the offspring, select resistant strain system and enter traditional procedure of breeding, not only can avoid the parent to hybridize the blindness of combo, can also reduce the workload that offspring's resistant strain system is selected, quicken the seed selection process of new varieties.Can also be applied to the to revive genetic research of No. 3 other objective traits of wheat of this cover wheat lines, as characteristics at the height sense powdery mildew of Soviet Union wheat No. 3, utilize this cover wheat lines, powder mildew resistance inoculation appraising datum by to each monosome substitution line can carry out chromosome mapping to the wheat powdery mildew resistant gene.Can also be applied to the to revive genetic meticulous Position Research of No. 3 objective traits of wheat of this cover wheat lines.As determining the chromosome at scab resistance gene place, can utilize this monosome substitution line, make up large sample genetic group, carry out the meticulous location of gene.Along with the development of molecular marking technique, utilize molecular labeling can determine autoploidy between substitution line and the donor parents, identify the accuracy of monosome substitution line thus.And molecular marking technique is not subjected to the influence of plant tissue, developmental stage and environment, all can detect at any season, environment.Used molecular labeling numbers of poles is many, covers whole genome, and the performance codominance can be differentiated homozygote and heterozygote, compares with other technologies, has very strong technological merit.

Four. description of drawings:

Fig. 1: the electrophoresis pattern of China spring (Soviet Union wheat No. 3) 6D monosome substitution line Molecular Identification

1-19. China spring (Soviet Union wheat No. 3) 1A monosome substitution line--China spring (Soviet Union wheat No. 3) 5D monosome substitution line

20. China spring (Soviet Union wheat No. 3) 6D monosome substitution line

21. China spring (Soviet Union wheat No. 3) 7D monosome substitution line

22. China spring

23. Soviet Union wheat No. 3

Five. embodiment

1. to Chinese spring 6D chromosome monomer, microscopy tip of a root meristematic cell chromosome number is selected the plant of 2n=41.Wheat scab disease-resistant variety Soviet Union wheat is carried out the scab resistance inoculation for No. 3 identify that select the best individual plant of resistance, sick small ear rate is 6.85%.With No. 3 resistance individual plants of Soviet Union wheat is female parent (♀), with Chinese spring 6D chromosome monomer (♂) hybridization, obtains hybrid F1.F1 plant microscopy tip of a root meristematic cell chromosome number is selected 2n=41, and external form approaches the plant of China spring, as male parent (♂), with Chinese spring 6D chromosome monomer (♀) hybridization, obtains the BC1 plant again.Repeat microscopy, select and hybridization work, until obtaining the BC4 plant.BC4 plant microscopy tip of a root meristematic cell chromosome number is selected 2n=41, and external form approaches the plant of China spring, and the bagging selfing obtains BC4F ₁Plant.To BC4F ₁Microscopy, the homozygote plant of selection 2n=42 is China spring (Soviet Union wheat No. 3) 6D monosome substitution line.

2. the blade of clip China spring (Soviet Union wheat No. 3) 6D monosome substitution line plant, extract complete genome DNA, choose simple repeated sequence mark (SSR) the primer gwm325 and the gwm469 that are positioned on the 6D chromosome, carry out pcr amplification, amplified production is electrophoretic separation on the 6g/100ml polyacrylamide gel.Separating resulting (Fig. 1) as can be seen, No. 3 chromosomal pcr amplification products of 6D of chromosomal pcr amplification product of 6D and Soviet Union wheat of China spring (Soviet Union wheat No. 3) 6D monosome substitution line are identical, and it is identical with the chromosomal pcr amplification product of China spring 6D, illustrate that Soviet Union's wheat No. 3 6D chromosomes have replaced in the Chinese spring, resulting plant be China spring (revive wheat No. 3) 6D monosome substitution line really.

Claims

1. a wheat scab disease resistant and breeding method is characterized in that setting up No. 3 monosome substitution lines of a cover, and carries out Molecular Identification with it.

2. according to the described wheat scab disease resistant and breeding method of claim 1, No. 3 monosome replacement of wheat that it is characterized in that reviving are:

1) wheat scab disease-resistant variety Soviet Union wheat carries out scab resistance inoculation evaluation for No. 3, selects the best individual plant of resistance;

2) 21 China spring series monomers difference of cover microscopy tip of a root meristematic cell chromosome number, the plant of selection 2n=41;

3) be that maternal ♀ is hybridized with above-mentioned Chinese spring series monomer ♂ respectively with No. 3 resistance individual plants of above-mentioned Soviet Union wheat, obtain 21 hybrid F of a cover ₁

4) to hybrid F ₁Microscopy tip of a root meristematic cell chromosome number is selected 2n=41, and external form approaches the plant of China spring, as male parent ♂, with corresponding Chinese spring monomer ♀ hybridization, obtains 21 BC1 plant of a cover respectively;

5) to BC1 plant repeating step 4) described operation, obtain 21 BC2 plant of a cover;

6) to BC2 plant repeating step 4) described operation, obtain 21 BC3 plant of a cover;

7) to BC3 plant repeating step 4) described operation, obtain 21 BC4 plant of a cover;

8) to BC4 plant microscopy tip of a root meristematic cell chromosome number, select 2n=41, external form approaches the plant of China spring, and the bagging selfing obtains 21 BC4F of a cover ₁

9) to BC4F ₁The plant microscopy, the homozygote plant of selection 2n=42, i.e. No. 3 monosome substitution lines of 21 China spring Soviet Union wheats of a cover.

3. according to the described wheat scab disease resistant and breeding method of claim 1, it is characterized in that the method for identifying molecules of monosome substitution line is made of following several steps:

1) clip one overlaps No. 3 monosome substitution lines of 21 China spring Soviet Union wheats plant respectively, and the blade of parent's China spring and No. 3 plant of parent Su Mai extracts complete genome DNA;

2) choose the simple repeated sequence mark SSR primer 2-4 that is positioned on each bar chromosome respectively;

3) to the DNA of monosome substitution line, use the SSR primer on the above-mentioned homologue, carry out pcr amplification, amplified production obtains the molecular labeling data after electrophoretic separation on the 6g/100ml polyacrylamide gel;

4) contrast above-mentioned molecular labeling data, determine the accuracy of gained monosome substitution line.