疣粒野生稻抗白叶枯病新基因xa32（t）的鉴定及其分子标记定位（英文）

虿北雉业学报 2008，1 7(6)：170 l74 Acta Agriculturae Borealioccidentalis Sinica Identifying and Mapping New Gene xa32( )for Resistance to Bacterial Blight(Xanthomonas oryzae pvoryzae， Xoo)from Oryza meyeriana L RUAN H uihui ，YAN Chengqi ，AN Derong ， LIU Ren_hu and CHEN Jianping (1College of Plant Protection，Northwest A&F UniversityYangling Shaanxi 712100，China；2Institute of Virology and Biotechnology，Zheiiang Academy of Agricultural Sciences Zhejiang Provincial Key Laboratory of Plant Virology，Key Laboratory of Animal and Plant Virology，Ministry ofgricultural，Hangzhou Zhejiang 310021，China) Abstract：A new bacterial blight resistance gene xa32(f)was identified from the descendant of somat ic hybridization between Oryza meyeriana I to 0sativa I sspjaponicaA F2 population including 132 plants was generated from a cross between the descendant of somatic hybridization and Osativa I sspjaponicaThe bacterial blight strain P6 was used to test the resistance or susceptible of this F2 population and the result is there were 32 resistance and 100 susceptible plantsA stochastic 412 SSR primer pairs covering 1 2 rice chromosomes were used for polymorphism survey of the parents and the F2 populationThere were 8 SSR markers found on rice chromosome 12Linkage analysis showed that the two markers RM8216 and RM20A which on the long arm chromosome 12 link with xa32()，loca ted on the same sideAnd the genetic distance was 69 cm and 17 cm Key words：Bacterial blight；Gene mapping；xa32( )；Descendant of somatic hybridization CLc number：S5119 Document code：A Article ID：10041389(2008)06-07005 疣粒野生稻抗白叶枯病新基因xa32(t)的鉴定 及其分子标记定位 阮辉辉 ，严成其 ，安德荣 ，刘仁虎 ，陈剑平 (1西北农林科技大学植物保护学院，陕西杨凌 712100；2浙江省农业科学院病毒与生物技术研究所 浙江省植物病毒学重点室，农业部动植物病毒学重点实验室，浙江杭州 310021) 摘 要：xa32( )是从疣粒野生稻与栽培稻经体细胞杂交途径获得的新种质Y76中鉴定出的水稻抗白叶枯病 新基因。将Y76与栽培稻“大粒香”回交构建F 群体，获得132个抗性稳定的株系。经致病菌株P6对该F 群体抗性鉴定，得到抗感株系数分别为32和100。随机选用覆盖水稻12条染色体的412对SSR引物对亲本 和Fz代群体进行多态性分析。筛选出8对多态性好的引物标记，连锁遗传分析发现位于z。32()同侧的 RM82l6和RM20A与其连锁，从而将xa32(f)定位在第12染色体长臂上，遗传距离为69cm和17cm。 关键词：水稻白叶枯病；分子标记定位；xa32( )；体细胞杂交后代 Received date：2008 04 1 4 Accepted date：20080608 Foundation item：Zhejiang Province Foundation Important Item(Z307451) Biography：Ruan Huihui(1982)，female，postgraduate，mainly engaged in plant virology and Molecular BiologyE-mail：ruan huihl63corn *Conrresp。nding author：An Derong Professor 6期 RUAN Huihui，et al：Identifying and Mapping New Gene xa32()for Resistance to Bacterial Blight(Xanthomonas oryzae pvoryzae，Xoo)from Oryza meyeriana L The bacterial blight of rice(BBR)which caused by(Xanthomonas oryzae pvoryzae， Xoo)is one of most important bacterial disease on the world。it take heavy lOSS to the world rice product For frequent pathogenic bacteria va riance，complex regularity of outbreak，large regional differences，the pharmacy control is poor timeconsuming and laborious，pollution of the environmentThus efficient use of broad spectrum resistance to genetic resources for ge netic improvement of rice varieties to control the disease has become the best way Research on resistance genes in bacteria1 blight of rice start from 1950sBetween 1960s to 1970sJapanese use their local bacterium identi fied three dominant genes，they are Xal，Xa2 and Xa3 International Rice Research Institute (IRRI)have also identified Xa4，xa5，Xa6，Xa7， Xa8，Xa9 and Xa l O，this seven resistance genes using Philippine race 1_3Japanese have cooper ated with IRRI，they apply near-isogenic lines founded an international BBR single gene identi fication system with uniform standard，and they have used this system to coordinate and identify the forepart 2 1 BBRresistance genesl_l1_In Chi na，some relevant institution have also coopera ted and made unified research programme into Iine with international standards，they identified and analysis of the local resources and varieties with resistance gene from 1991 As far as now，there are already 3 1 resistance genes have been identified and reported by international an thority of the publication 一 ，among them，the Xa1，Xa2，Xa3，Xa4，Xa5，Xa7，Xa1O，Xa12， Xal3，Xa14，Xa21，Xa22(t)，Xa23，Xa25，Xa25 (t)，Xa26，Xa27(t)，Xa29(t)and Xa30 have been fine mapped，with 6 genes cloned，they are Xa1，Xa5，Xa13，X21，Xn26，Xn27(t) However，the resistance genes of BBR often have race specificity，the variation of the physio logical race of the germ and new race will make the new cultivar lOSS resistance Wild rice re sources contains highyield，disease resistance ,and resilience。and other fine traits。it will pro vide valuable genes for breeding and expansion of cultivated varieties that Songetc al have longistaminata，Xa21 is Philippine race One typical sample is cloned Xa21 from O high resist to India and In China，the specialist begin to select R genes from wild rice just from 1980sPeng shao qiu，Wei Zisheng et al，used Hunan bacterial race inoculated to Orufipogon，00 ficinalis， 0meyeriana，and find that the Omeyeriana almost immune to the bacteriaI racec The 0 meyeriana growing in south Arial，southeast Arial and Yunnan，Hainan，Taiwan province in China In all the genus oryza species，the O meyeriana have many superior characters such as immunity to the BBR，high resistance to bac terial leaf streak in rice and rice brown planthop per，xeromorphic and shadetoleranceIt has great potential in genetic improving of cultivated riceNormally，hybridization could not hap pened between Omeyeriana(GG genome 2n一 24)and cultivated rice(AA genome) Using somatic hybridization technical to introduce the Omeyeriana useful genes into cultivated rice， and backcrossing with cultivated rice recurrent parents is a feasible method With this meth od，we obtained 1 30 somatic hybridization prog eny from the 0meyeriana and varieties of culti vated rice(0sativa Lsspjaponica)And we also culture 4 new germplasms with broad spec trum disease resistance from back across and selfcross to the eight generationThe chromo some numbers is similar to norma1 cultivated rice，and also have stable heredity and disease resistance We have used allele identification system from different countries and 30 strains to identification，they all have high resistance plants In order to identify this gene，this research used one Y76 line backcrossed and self crossed to obtain a BC4 F2 population，and make it as genetic location populationsFinally，it mapped this gene on the long arm of 1 2 chromo some，named xa32( )temporarily 1 Material and Method 11 Material 111 Rice material In this research，the broad spectrum and high disease resistance 西北农业学报 17卷 plants which used to construct location popula tion，is the offspring of somatic fusion and hy bridization between Omeyeriana(provided by professor Shen Zongtan，Zhejiang university， from Xishuangbanna，Yunnan)and Osativa I sspj aponica(provide by China rice institute) The backcross and selfcross process has contin ued for 8 generationWe at last got the resist ance parent Y76It has stable resistance genetic and chromosome numberThen we use Y76 crossed with Osativa L_sspjaponica，after got the plants，we let them self-cross and get the i solated population，which is our location for this research 112 Test strains The broad spectrum patho genic bacteria P6 of BB have been used in this re searchThe bacterial cultured 48h at 28on cul ture medium before inoculationThe bacterial SHS pension(310 cells per mL)in aseptic water 113 SSR(Simple Sequence Repeat)primer We use Bulked Segregant Analysis，BSA to screening the bacterial blight resistance genes which linked to SSR primer4 1 2 random SSR primers on the 1 2 chromosome of rice have been usedAnd there are 2 O SSR primer with poly morphism been screenedAl1 the SSR primer are synthesised by Genst Nanjing CompanyThe se quence of RM2OA and RM8216 in below table 1 Table 1 The sequence of SSR primer of RM20A and RM8216 12 Research method 121 Identification of the rice resistance Planted the F2 population breed of somatic fusion and hybridization between Omeyeriana and O sativa I sspj aponicaInclude the broadspec trum pathogenic bacteria P6 of BB in the booting stage in riceThe bacterial cultured 48h at 28。C on ATCC 470 culture medium before inocula tionThe bacterial suspension(31 0 cells per mI )in aseptic waterThe uppermost fully open leaves were inoculated by clipping methodBuilt the database of F2 population3 weeks after in clusion，we have an investigation on the retinop athy of prematurity，measure the length of the disease spot，statistics data 122 Extraction of rice DNA genome Using ( B method to extract rice DNA genome of Y76 sativa I sspjaponica and F2 population 123 PCR amplification and electrophoresis PCR amplification system：The total volume of PCR amplification is 20 uI ：10PCR buffer (200 mmolI TrisHCl， 100 mmolI (NH4)2SO1，100 mmolI KCl，1 Triton X一 100，20 mmolI MgC12，pH 88)，25 mmolI dNTP 02 uI ，10 Um primer，20ng DNA ge nome。06Utaq enzyme 、Process of PCR amplification：94_C prede tgeneration 30s，then have 35 cycles：94。C de generation 30s，56renaturation 1min，72。C ex tension 1 rainAt last，72。C holding 5 rain Electrophoresis and detection：The production of PCR amplification with 6loading buffer re solved on 60 denaturing polyacrylamide gels and visualized by 01 AgN03 silver staining，15 NaOH coloration，scan denaturing polyacrylamide gels，observe the scan picture 124 Result analysis We Used Mapmaker EXP 30 software to analysis the data including the resistance of every plant，the obtained mo lecular markers results for the genetic linkage of genes and molecular markers 1ocationWith Ko sambi map translate it to genetic distance(cm) 2 Result for resistance identifica- tion and genetic analysis 21 Genetic linkage map constructing for F2 Hybrided Y76 and sativa Lsspjaponica in 2005，and got their F1 populationBreed F1 popula tion in Ningbo，harvest and keep them from differ ent plant，then got F2 populationPlanted the F2 population in Hainan，obtained 132 plantsTest them with inoculating P6，find that there were 32 plants in resistance to BB，with 100 susceptible ones，the ratio of resistance and susceptible is al most 3：1。chisquare test find it is consistent with the role of one gene genetic mode1It showed that the resistance of Y76 was controlled by one recessive 6期 RUAN Huthui，el al：Identifying and Mapping New Gene xa32( )for Resistance to Bacterial Blight(Xanlh017101l15“ory “P pvoryzaeXoo)from Oryza meyeriana I l 73 genes(Fig1)，can be used for the molecular mark ers location 35 3O 25 2O 1 5 lO 5 0 75 95 115 135 155 17 195 215 235 255 Fig1 Frequency distribution of lesion length to philippines Xoo race 6 in BC4 F2 population Therefore，we extract rice DNA genome of this 1 32 ones to SSR analysis the resistance genes xa32(t)of Y76 22 Molecular markers linked with BB resist- ance genes in rice In order make polymorphic analysis for the Y7 6 and sativa I sspjaponica，there are 4 1 2 molec ular markers which covering the whole rice chromo some have been usedThe result is that 48 primers showed polymorphic，and 20 primers among them on the twelfth chromosome As to ensure the linkage connection with xa32(t)，we have applied this primers which showed polymorphic in the parents into analysis the F2 populationIt found that there are eight SSR primers on the twelfth chromosome electro phoresis bands accord with the consequence of inoculated test in the field(Fig2) 23 Linkage analysis between polymorphic SSR primers and xa32(t) We used those polymorphic SSR primers on the twelfth chromosome to detect the l 32 ones in F2 populationWith Mapmaker software，we a nalysis the consequence of evaluation of resist ance to bacteria1。and detected the molecular markerLinkage analysis showed that the two markers RM82 1 6 and RM20A which on the long arm chromosome 12 link with xa32(t)，1ocated on the same sideAnd the genetic distance was 69 em and 17cm(Fig3) P1Y73；P2Osativa cv Dalixiang；ARsistence plants；BSusceptible plant；Hrecombine Fig2 DNA bands of the parents and their progenies of BC4F2 on SSR molecular markers 69cm 17cm Fig3 The location of bacterial blight resistance gene xa32(f)on the molecular linkage map of chromosome 12 in F2 population 3 Discussion It is one of the most economical and envi ronment protection way to utilize the resistance gene to breed cuhivars in late blight resistance breeding to control the bacterial blight of rice For a long time，international experts are work ing on research in this area，and use of hybrid breeding techniques breed a series of rice varie ties which have resistance genesHowever，the variation and new appearance of physiological race has been a serious threat to the effectiveness of the existing resistance gene，which need peo pie to look for new diseaseresistant genes to ap- plied to breed resistance variety research Years of research showed that rice bacterial blight bears high host specificityThe rice cuhi var which resist to BB is controlled by a single gene(R)In the process of pathogen infecting pathogens carrying avirulence genes can be rec ognized by plant disease resistance genesthe recognition will then induce a series of defense responses and prevent the plant from being i1l As wild relalives of resources in high yield，stress tolerance and disease resistance，it can provide the breeding and genetic background of cuhivars with necessary germplasm re 西北农业学报 17卷 sourcesBreeding prove from home and abroad： AA genome of wild rice in rice played a tremen dous role in breedingAgricultural expert recog nize，if we want to get a breakthrough seed，the new germplasm resources must be foundThis shows that use of wild rice to be parents is im perativeHowever，though Osativa with AA genorne(2n一24)is easy to cross with Oni varait is difficult in hybridizing with no AA genome cuhivarIt always failed in sexual hy bridizationBut from many years experience in wild rice research，we found that wild rice with noAA genome have many superior characters In order to make full use of wild rice resource。it has great significance in developing useful wild rice germplasm research The Omeyeriana which we used come from Xishuanbanna Yunnan，it is immunity to bacte rial blightIt is an available way to apply somat ic hybridization technical to introduce the excel lent genes of Omeyeriana into cultured rice With adopting molecular marker technology SSR，we finally 1ocated the xa32(t)on the twelfth chromosome in riceIt is different from any reported genesMolecular marker assisted selection and clone and Molecular markerassis ted selection and its new resistance gene cloning and transformation provides us a new way in rapid development of new varieties of cropsThe discovery and location of the new resistance genes from the generation of somatic hybridiza tion and cultivated rice 0sativa cv Dalixiang hy brids，not only enriched rice bacterial blight re sistance gene pool，but also provide a foundation for rice bacterial blight resistance gene cloning Acknowledgment：Many thanks to doctor Wu Zhongchang in Zhejiang universityFrom whom we get the SSR primers，thank him from my heart! 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