CN1241630A

CN1241630A - Common wild-rice near isogenic line resisting bacterial leaf-blight and its breeding process

Info

Publication number: CN1241630A
Application number: CN 99111577
Authority: CN
Inventors: 章琦; 赵炳宇; 王春连; 杨文才; 阚更生; 邢全党; 周永力; 林世成
Original assignee: INST OF CROP BREEDING AND CULT
Current assignee: INST OF CROP BREEDING AND CULT
Priority date: 1999-08-18
Filing date: 1999-08-18
Publication date: 2000-01-19
Anticipated expiration: 2019-08-18
Also published as: CN1091151C

Abstract

The present invention relates to one rice Xanthomonas line PX099. One near isogenic line carrying common wild rice bacterial leaf-blight resisting new gene Xa23 is cultivated and one new technology system is established, which relates to biological technology, plant pathology, genetic breeding technology and molecular labelling and aims at obtaining near isogenic line Xa23 as one rare excellent highly disease resisting gene. The new gene has very wide resistance spectrum and very strong resistance entering effect, and may be used to effectively improve the disease resistance of hybrid rice and long-shaped rice.

Description

Common wild-rice near isogenic line resisting bacterial leaf-blight and method of cultivation thereof

The present invention relates to the genetically engineered field.Be specifically related to a near isogenic line from the new gene of the high anti-bacterial blight of rice of carrying of common wild-rice.

Bacterial blight of rice is 1 global important bacteria venereal disease evil.The rice varieties of China's establishing in large scale carries single disease-resistant gene Xa-4 since the eighties, causes the deleterious 5 type bacterium of Xa-4 are risen to dominant bacteria, the risk of potential kind forfeiture resistance.The sixties is thrown in the kind that carries gene Xa-4 in a large number by International Rice Research Institute (IRRI), in country in Southeast Asia's susceptibleization of big area already ^[1]So far, identify in 21 bacterial leaf spot resistant ospc genes with the IRRI cooperation by Japan, most narrower or be recessive gene for anti-spectrum, inconvenience is used for breeding, has only the new gene Xa-21 that is identified from long medicine wild-rice (O.longislaminata) by (1990) such as Khush in recent years ^[2], its resistance of wide spectrum is similar to the present invention, promptly all can resist 6 international bacterial leaf-blight physiological strains of the specialization fungus strain PXO99 of 1 wide virulence comprising at that time.But the Xa-21 gene has the following disadvantages: promptly (1) makes up when carrying the near isogenic line of this gene, only carries out backcrossing for 4 times and 1 selfing, from generation to generation; It is lower to be used for the breeding imported effect.(2) the gene line IRBB21 that carries Xa-21 is susceptible in seedling stage, and is just disease-resistant to the tillering phase.(3) may since when the representative fungus strain PXO99 of the not clear and definite physiological strain of fashion No. 6 be the crucial microspecies fungus strains of differentiating, and the genetics of resistance basis of adopting the representative fungus strain PXO86 of microspecies 2 to disclose Xa-21 is an appropriateness inadequately.

The object of the present invention is to provide 1 can differentiate the specific bacterial leaf-blight fungus strain PXO99 that extensively causes a disease, and made up one with this fungus strain and overlap the method that carries from the near isogenic line of common wild-rice specific gene Xa23.

Content of the present invention is:

1, in order to the yellow unit cell of 1 paddy rice of identifying special bacterial leaf spot resistant ospc gene cause a disease mutation (Xanthomonasoryzae pv.oryzae) fungus strain PXO99 on July 14th, 1999 by China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation.The numbering of registering on the books at this center is CGMCCNO.0406.

2, method of cultivation

Whether the special wide pathogenic fungus strain PXO99 that the whole programs carry special resistant gene Xa23 near isogenic line all must employing can be differentiated this gene is cultivated in success, can detect this specialization resistant gene and exist:

1) adopt comprise bacterial leaf-blight extensively cause a disease fungus strain PXO99 9 worlds, 3 Japan and totally 19 fungus strains 7 China to common wild-rice the inoculation of anti-source identify, obtains the anti-source of wide spectrum that resist whole fungus strains 1 time of infertility.

2) adopt the flower training haploid breeding technology resistance in this anti-source of common wild-rice of quickening to isozygoty.Plant hybridization by the open country, the disease resistance of this germplasm is imported 1 indica type cultivated rice JG30 that plant type is good, obtain the F1 plant, select the individual plant of the high antibiotic PXO99 of being to carry out anther culture acquisition H1, selfing 3 times, identify that by substituting the PXO99 fungus strain it is H4 that the disease-resistant resistance of acquisition stably express height in the time of infertility is isozygotied.

3) adopt PXO99 to disclose genetics of resistance basis and the resistance expression's type thereof of WBB1

In all resisting bacterial leaf-blight dominant genes of the cultivation seed rice of international definite designation so far, all can not resist the invasion and attack of PXO99, only have that to isozygoty from the Xa-21 of wild-rice and this be that H4 can resist PXO99, then analyzed the genetics of resistance pattern of H4, disclosed the resistant gene that H4 carries the high resistance characteristic of disease of PXO99 controlled by a pair of complete dominance disease-resistant gene PXO99.This gene be from seedling stage to becoming the strain phase all to express the resistance in the time of infertility of resistance.And Xa-21 could express resistance to the Sheng phase of tillering of PXO99.Allelomorphism is measured and is shown as non-allelic genes between the two, shows that WBB1 carries a pair of new gene, temporarily called after Xa23.

4) make up the near isogenic line that carries disease-resistant gene Xa-23

Is H4 hybridization with susceptible rice variety JG30 with disease-resistant the isozygotying of flower training, be again that recurrent parent is backcrossed with JG30, in per generation, identify with specialization resistance fungus strain PXO99 inoculation that respectively 14 strains of selecting and remain have the dominance resistance expression in seedling stage, show the individual plant of the proterties of recurrent parent, continue to be returned to BC with recurrent parent ₅F ₁, selfing is to BC again ₅F ₃, obtain a pair of genetic background and recurrent parent basically identical, the near isogenic line WBB1 that the target resistance is clear and stable.

5) the SSR molecule marker is selected

F with near isogenic line WBB1 that carries the Xa-23 resistant gene and susceptible variety JG30 hybridization ₁And F ₂Segregating population is selected and the chain SSR molecule marker of target disease-resistant gene.Earlier with fungus strain PXO99 inoculation F ₁And F ₂, obtain reaction parameter data anti-and sense, select 160 strain F ₂Carrying out the blade genome DNA extracts.160 pairs of SSR primers have been selected in the PCR reaction routinely, obtain 2 pairs of energy detect polymorphism between parent and anti-sense DNA pond primer, OSR6 and RM224 carry out coseparation analysis to 160 F2 individual plants selecting respectively with it, and adopt mapping software Mapmarker (3.0) to analyze genetic distance.Find OSRS and new gene Xa-23 close linkage, map distance is 5.3CM, and the genetic distance of RM224 and Xa-23 is 27.CM.

3, advantage of the present invention and positively effect

(1) new gene identification and the technical system that makes up near isogene have been designed

The principle and the common wild-rice disease resistance of the in store typical gene pairs interaction of genes relation of paddy rice and bacterial leaf-blight are in heterozygous state usually, and the core technology that the present invention adopts is: the 1. whole process using fungus strain PXO99 that extensively causes a disease; 2. adopt disease-resistant the isozygotying of the flower training new gene of haploid breeding technique construction to be; 3. in many generations, backcross by fungus strain detects disease resistance and economical character is selected for specially changing synchronously.Structure carries the disease-resistant near isogenic line of the Xa-23 of new gene.

(2) structure carries in the near isogenic line of Xa-23 gene, fully taking into account genetic element with recurrent parent JG30 returns to and carries in the target resistant gene system, thereby it is more with total algebraically of selfing to backcross, thereby it is consistent with the recurrent parent height and specially change clear, the stable near isogenic line of resistance to have built up genetic background, for gene clone is raised the efficiency.

(3) the new gene Xa-23 of the common wild-rice resisting bacterial leaf-blight that the near isogenic line WBB1 that breeds carries is the complete dominance gene that anti-spectrum is the widest in the international so far named disease-resistant gene, the resistance imported effect is extremely strong, rice at whole growth periods is all highly disease-resistant.It will utilize single tight at change China bacterial leaf spot resistant ospc gene

Play a positive role on high present situation, the improvement China paddy disease-resistant, and carrying out China's rice molecular mark

Play a driving role in the assisted selection.Fig. 1 set up the common wild-rice resisting bacterial leaf-blight isozygoty be and identify new gene map 2 cultivate carry the new gene of Xa23 the new gene Xa23 of disease-resistant near isogenic line WBB1 technical program Fig. 3 in the first-class position of paddy rice Sub_clause 11 karyomit(e) (other bacterial leaf spot resistant ospc gene and dna marker thereof draw from Causse etc. 1994) material disease-resistant resistance reaction resistance expression breeding time to 9 microspecies

Gene

P1 P2 P3 P4 P5 P6 P7 P8 P9WBB1 Xa-? R R R R R R R R R IRBB21 in time of infertility Xa21 R R R R R R R R R is tillered the Sheng phase IRBB3 Xa3 R R R R R S S S R one-tenth strain phase

) IRBB4 Xa4 R S S M R S R R S IRBB7 in time of infertility Xa7 MS R R S R S R R R (?) become strain phase IRBB10 Xa10 S R S S R S R S S IRBB14 in the time of infertility Xa14 S S S S R S S R S time of infertility

) JG30 (CK) S S S S S S S S S time of infertility

R=is anti-, anti-among the M=, S=sense table 1 reference (1) T.W.Mew 1987, Current status and Future Prospects ot research onBacterial Blight of Rice, Ann.Rev.Phytopathology, 1987,25:359 ~ 382 (2) (2) G.S.Khush et al, A new gene for resistance to bacterial blightFrom O.longistamanata, RCN, 1990.7:21 ~ 22

Claims

1, can in order to the yellow unit cell of 1 paddy rice of identifying special bacterial leaf spot resistant ospc gene cause a disease mutation (Xanthomonasoryzae pv.oryzae) fungus strain PXO99 on July 14th, 1999 by China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation, the numbering of registering on the books at this center is CGMCCNO.0406.;

2, a cover is characterized in that being made up of the following step from the method for cultivation of common wild-rice near isogenic line resisting bacterial leaf-blight, and all program is all necessary:

1) adopt comprise bacterial leaf-blight extensively cause a disease fungus strain PXO99 9 worlds, 3 Japan and totally 19 fungus strains 7 China to common wild-rice the inoculation of anti-source identify, obtains the anti-source of wide spectrum that resist whole fungus strains 1 time of infertility;

2) adopt the flower training haploid breeding technology resistance in this anti-source of common wild-rice of quickening to isozygoty, plant hybridization by the open country, the disease resistance of this germplasm is imported 1 Zhou type cultivated rice JG30 that plant type is good, obtain the F1 plant, select the individual plant of the high antibiotic PXO99 of being to carry out anther culture acquisition H1, selfing 3 times identifies that by substituting the PXO99 fungus strain it is H4 that the disease-resistant resistance of acquisition stably express height in the time of infertility is isozygotied;

Can not resist the invasion and attack of PXO99 at all resisting bacterial leaf-blight dominant genes middle parts of the cultivation seed rice of international definite designation so far, only have that to isozygoty from the Xa-21 of wild-rice and this be that H4 can resist PXO99, then analyzed the genetics of resistance pattern of H4 to PXO99, having disclosed the resistant gene that H4 carries is controlled by a pair of complete dominance disease-resistant gene the high resistance characteristic of disease of PXO99, this gene be from seedling stage to becoming the strain phase all to express the resistance in the time of infertility of resistance, and Xa-21 could express resistance to the Sheng phase of tillering of PXO99, allelomorphism is measured and is shown as non-allelic genes between the two, show that WBB1 carries a pair of new gene, temporarily called after Xa23;

4) make up the near isogenic line that carries disease-resistant gene Xa-23

Is H4 hybridization with susceptible rice variety JG30 with disease-resistant the isozygotying of flower training, be again that recurrent parent is backcrossed with JG30, in per generation, identify with specialization resistance fungus strain PXO99 inoculation that respectively 14 strains of selecting and remain have the dominance resistance expression in seedling stage, show the individual plant of the proterties of recurrent parent, continue to be returned to BC with recurrent parent ₅F ₁, selfing is to BC again ₅F ₃, obtain a pair of genetic background and recurrent parent basically identical, the near isogenic line WBB1 that the target resistance is clear and stable:

5) the SSR molecule marker is selected

F with near isogenic line WBB1 that carries the Xa-23 resistant gene and susceptible variety JG30 hybridization ₁And F ₂Segregating population is selected and the chain SSR molecule marker of target disease-resistant gene, earlier with fungus strain PXO99 inoculation F ₁And F ₂, obtain reaction parameter data anti-and sense, select 160 strain F ₂Carrying out the blade genome DNA extracts, 160 pairs of SSR primers have been selected in the PCR reaction routinely, obtain 2 pairs of energy detect polymorphism between parent and anti-sense DNA pond primer, OSR6 and RM224, respectively 160 F2 individual plants selecting are carried out coseparation analysis with it, and adopt mapping software Mapmarker3.0 to analyze genetic distance, and find OSRS and new gene Xa-23 close linkage, map distance is that the genetic distance of 5.3CM RM224 and Xa-23 is 27.CM.