水稻条纹病毒和介体灰飞虱抗性的QTL分析_英文_

Vol 32,No 6pp 805-810 Jun.,2006作 物 学 报ACTA AGRONOMICA SINICA第32卷第6期2006年6月 805~810页Detection of QTLs for Resistance to Rice Stripe Virus and Small Brown Planthopperin Rice (O ryza sativa L.)SUN Dai Zhen 1,JI ANG Ling 1,LIU Shi Jia 1,Z HANG Ying Xin 1,HUANG Pei Hong 1C HENG Xia Nian 1,Z HAI Hu Qu 2and WAN Jian Min1,2,*(1S tate Key Lab oratory for Crop Genetics an d Germpl asm Enhance men t,Nanjin g Agricultu ral University/J iangsu Research Center of Plan t Gene En gi neerin g,Nan jing 210095,Jian gsu;2Insti tu te of Crop Sciences,Chin ese Academy of Agricu ltural Sciences,Beijin g 100081,Chin a)Abstract :An indica va riety,Kasalath,is highly resistant to rice stripe virus and small brown planthopper.In order to dissect the relationship between rice stripe virus and small brown planthopper resistance genes,the QTLs were detected using Nipponbase/Kasalath//Nipponbare backcross inbred lines.One putative QTL (qSTV 11)for the ratio of disease rating index was mapped be tween the marker S2260and G257on c hromosome 11,which e xplained 35.79%of the total phenotypic variation with LO D score of 9.2.The positive re sistant effec t ca me from Kasala th.The two QTLs (q SBPH3 a and qSBP H3 b )for non prefe rence index we re detected on c hromosome 3,and their contributions to the total varia t ion were 11.69%and 11.36%with LO D scores of 3.12and 2.96,respec tively.This suggests that the rice stripe virus and small brown planthoppe r resistance are c ontrolled by differe nt genes and this view is te stified by no significant correlation between RSV and SBP H resistance.Then,two pairs of epista t ic QTLs for ra t io of disease rating inde x and non preference index were respectively detected,showing the rice stripe virus and small brown planthopper resistance were influenc ed by main effec t QTLs and epistatic QTLs.Additionally,fur ther study found that SSR marker BJ11 8was tightly linked to qSTV 11.This facilitate s the breeding process for re sistance to rice stripe disease by marker assisted selec tion.Key words :Rice stripe virus;Laodelpha x striatellus Fallen;Backcross inbred lines (BILs);Q TL a nalysis;Epistasis水稻条纹病毒和介体灰飞虱抗性的QTL 分析孙黛珍1 江 玲1 刘世家1 张迎信1 黄培鸿1 程遐年1 翟虎渠2 万建民1,2,*(1南京农业大学作物遗传与种质创新国家重点实验室/江苏省植物基因工程技术研究中心,江苏南京210095;2中国农业科学院作物科学研究所,北京100081)摘 要:水稻品种Kasalath 高抗条纹病毒和介体灰飞虱。

为剖析不同抗性类型基因之间的关系,利用回交重组自交系群体Nipponbare/Kasalath//Nipponbare 分析了对条纹病毒和介体灰飞虱抗性的数量性状基因座。

结果在第11染色体S2260~G257标记区间检测到1个与条纹病毒抗性相关的QTL(qSTV 11),LOD 值为9 2,贡献率为35 79%;在第3染色体R1618~C595和R2170~C1135标记区间各检测到1个与介体灰飞虱抗性相关的QTL (qSBP H3 a,qSBPH3 b),LOD 值和贡献率分别为3 12和2 96,11 69%和11 36%,表明条纹病毒和介体灰飞虱抗性由不同基因所控制,而且两者之间不相关。

此外,还分别检测到两对与条纹病毒和介体灰飞虱抗性相关的上位性QTL,暗示水稻对条纹病毒和介体灰飞虱的抗性受主效和上位性QTL 的共同影响。

进一步分析发现SSR 标记BJ11 8与qST V 11紧密连锁,为分子标记辅助选择高抗条纹叶枯病水稻品种提供了基础。

关键词:水稻条纹叶枯病;介体灰飞虱;回交重组自交系;QTL 分析;上位性中图分类号:S511Foun dation item:Supported by National Tackle Key Problem Foundation(2004BA525B02 04),Jiangs u Hi gh Technol ogy Project (BG2004303)and Scienceand Technology Program of Educati on Mini stry (105090)and PCSIRT.B iography:SUN Dai Zhen (1964),present addres s:A gronomy College of Shanxi Agricultural University,Taigu 030801*Corres pondi ng author:WAN Jian Min.E mail:wanjm@,Tel/Fax:025 ********Received:2005 07 05;Accepted:2005 11 08.Rice stripe is caused by rice stripe virus(RSV), which mainly transmitted by the small bro wn planthopper (Laodelphax striatellus Fallen,SB PH).Resistance to rice stripe disease can be divided into resistance to virus or to vector,or to both.Furthermore,resistance to virus can be classified into resistance to virus infection and tolerance to virus[1].Nemoto et al.[1]studied the mechanism of resistance to rice stripe in variety IR50and found that IR50was moderately resistant to virus infection and tolerance to vector,variety TN1was moderately resistant to virus infection and susceptible to vector, variety Musashikogane and Minamihatamochi were highly resistant to virus infection and susceptible to vector. Ishika wa et al.[2]reported that RSV resistance c ould be classified into the Musashikogane type or the TN1 type depending on their RSV incidence and symptoms. Varieties belonging to the Musashikogane type showed a lower percentage of RSV incidences but symptoms were serious,while TN1 type sho wed moderate values of percentage of RSV incidence but symptoms were not serious.We found that an indica variety,Kasalath is highly resistant to rice stripe virus and small bro wn planthopper.Ding et al.[3]mapped the QTLs associated with rice stripe resistance on chromosomes11and7,but it has not been clear that the relationship between those QTLs and SBPH resistance.Here,QTLs for RSV and SBPH resistance were detected using Nipponbare/ Kasalath//Nipponbare backcross inbred lines(BILs),in order to verify expression stability of QTL associated with RSV resistance,clarify the relationship between genetic factors associated with the detec ted QTLs and resistance, screen the SSR marker whic h was tightly linked to QTL associated with RSV resistance so as to be used in marker assisted selection breeding progra m.1 Materials and methods1.1 Plant materialsNinety eight backcross inbred lines(BI Ls)were developed from a backcross of Nipponbare(japonica)/ Kasalath(indica)//Nipponbare by the method of single seed descent(SSD),provided by Yano at Rice Genome Research Program,National Institute of Agrobiological Resources,Tsukuba,Japan.Ninety eight BILs and their parents were planted at the farm of Jiangsu Acade my of Agricultural Sciences and BC1F11seeds were harvested for evaluation of RSV resistance.IR36and W uyujing3were used as control cultivar highly resistant and susceptible to RSV,respectively.1.2 Evaluation for RSV resistanceMass inoculation test was employed to evaluate RSV resistance.Fifteen seeds of each line were sown in rows in a70cm 45c m 17cm plastic tray which was placed in a cage of100c m 160c m 120cm.When the first leaf emerged,the weak seedling were pulled out and ten health seedlings of each line were kept.SB PH nymphs with second to third instars were released into the cage at the rate of about8SB PH per seedlings,when the seedlings were at one and a half leaf stage.The rate of viruliferous SB PH was estimated to be35%by ELISA method[4].Three days later,all SBPH nymphs were removed and seedlings were transferred to a greenhouse, where they were cultivated for about a month until symptoms were observed.The e xperiments were performed with two replications.Resistances to RSV in plants were classified into6 classes(A,B,Bt,Cr,C and D)30d after inoculation based on their symptoms.The disease rating index was calculated acc ording to the severity of symptom as follows[5]:Disease rating index=100A+80B+60B t+40Cr+20C+5DNumber of seedlings exa minedIn the above formula,A,B,Bt,Cr,C and D are plant numbers of each class,respectively.The ratio for the disease rating index of each line to that of Wuyujing3was used as phenotype value.1.3 Evaluation for SBPH resistanceInoculation method was the same as mass inoculation test.During inoculation,the number of SB PH that settled on each line was counted every day.Total number of attached SB PH for three days was utilized as the index of the non preference of each line.1.4 QTL and epistasis analysisMolecular data in the BI L population were provided by Yano[6].The whole linkage map included245RFLP markers,covering1179.9c M of rice genome with a4.8 cM avera ge distance between markers.QTL analysis was performed using composite interval mapping with QTL Cartographer version2.0computer program[7],with a model specifying five cofactors to control for genetic806 作 物 学 报第32卷background,and a windo w size of10cM that blocked out a region of5c M on each side of the markers flanking the test site.The specific cofactors used were obtained by forward backward stepwise regression with F in=0.1and F out=0.1using the sa me software.The significance thresholds were calculated based on 1000permutations at an experiment wise significant level P<0.05[8].A13 34mean of the likelihood ratios corresponding to an LOD score of2.9was used as the criteria to detect the significant association between marker loci and resistance loci.The effects and contributions of QTLs to whole variation were analyzed by using mixed genetic model.For the designation of QTLs, we follo wed the recommendation by McC ouch et al.[9] The significant test of interac tion between all putative non allelic loci was performed and digenic epistatic QTLs were analyzed using QTL Mapper2.0[10].1.5 Analysis of SSR marker tightly linked to QTLDNA samples of98lines and two parents were e xtracted from fresh leaves of each plant using the method described by Dellaporta et al.[11]with minor modifications.Fourteen pairs of SSR markers,named by BJ11 1BJ11 14,located on chromosome11between C1172and G202were developed using primer 5.0 software.Eight pairs of SSR markers were selected in the Gramene database(/microsat)and RGP(http://rgp.dna.affrc.go.jp)[12 13]. Polymorphisms of the22pairs of SSR marker were analyzed following the procedure of Chen et al.[14]with minor modifications.Linka ge map of chromosome11was reconstruc ted using MAPMARKE R/EXP3 0,according to molecular data resulted from polymorphic SSR primers and RFLP markers provided by Yano.QTL analysis was again performed by QTL Cartographer version2 0 computer program[7].2 Results and Analysis2.1 QTL analysis for RSV resistanceSignificant difference for the ratio of disease rating inde x between the parents was observed(Table1). Meanwhile,the continuous distribution of the ratio of disease rating index suggests genetic characteristic of quantitative resistance to RSV in BIL population (Fig.1).By the QTL Cartographer version2 0computer program,one major QTL(qSTV11)for the ratio of disease rating index was detected at the region of RM229 G257on chromosome11with LOD score9 2and contribution of35 79%to the total variation.The positive resistant effect at the locus was contributed by Kasalath alleles(Table2).T able1Rea ctio n to RSV a nd SB PH o f two pa rents and BIL popula tio n seedling sTrai tParent BIL populati onNi pponbare Kasal ath Range Mean SDRati o of disease rating inde x(%)106 2928 636 86165.2086 6337 58 Non preference index2 981 731 036 202 50 78 T able2Pu ta tiv e QT Ls a sso cia ted with RS V a nd S BPH resistanceTrait Loc us Chr Markerinterval LODscoreVarianceexplainedAdditiveeffectRatio of dis eas e rating index(%)q STV1111S2260G2579 235 79-17 23 Non preference index qSBPH3 a3R1618C5953 1211 693 63qSBPH3 b3R2170C11352 9611 36-2 732 2 QTL analysis for SBPH resistanceSBPH resistance in BI Ls and their parents was showed in Table1 Significant difference between two parents for non preference index was observed The continuous distribution of non preference inde x suggests a polygenic control of the resistance to vec tor,small bro wn planthopper in BIL population(Fig 1)Using QTL Cartographer2 0,two QTLs for non preference index were detected at the regions of R1618 C959and R2170C1135on chromosome3,their LOD scores and variations e xplained were3 12,2 96and 11 69%,11 36%,respectively The positive resistant effect at the qSB P H3 a was contributed by Nipponbare, and the other QTL qSBP H3 b was from Kasalath alleles (Table2)807第6期S UN Dai Zhen et a l.:Detection of QTLs for Resistance to Rice Stripe Virus and Small Brown Planthopper...Fig 1Frequ ency distribution of the ratio of diseas e rating index and non preference index in Nipponbare/Kasalath//Nipponbare B IL population2 3 Digenic epistatic resistance QTLsTwo pairs of epistatic loci,which increased RSV resistance,were detected and mapped on chromosomes 5and 11(Table 3),of which interaction between marker regionC82G376and S2137G320onchromosome11washighly significant,explained 11 11%of total variation Additionally,main effect QTL,qSTV 11,was involved in interaction This indicates that many marker intervals on chromosome 11are associated with RSV resistanceTwo pairs of epistatic loci associated with SBPHTa ble 3Putative epis ta tic Q TLs for RSV a nd SBPH resis ta nce in BI L po pulatio n TraitChr M arker interval Chr Marker i nterval L O D score AAij PVE(%)R atio of di seas e 5R1436R228911S2260G 257*16 5-3 461 28rating index 11C82G37611S2137G3204 98-10 3311 11Non preference2R26C4998R617R33754 60 358 46inde x 3C595C944*5R1436R22898 44-0 264 67Notes:*The bold markers mean the position of main effect Q TLs.resistance were also detec ted and mapped on chromosome2,3,5and 8(Table 3) And these two epistatic loci could explain 8 46%and 4 67%of the total trait variation when non preference index used as phenotype value,respectively SB PH resistance was decreased by the interac tion between marker interval R26C499on chromosome 2and R617R3375on chrom osome 8,while increa sed by that between marker interval C595C944on chromosome 3and R1436R2289on chromosome 82 4 SSR marker tightly linked to the QTL,qSTV 11,for RSV resistanceThe QTL,qSTV 11,detected in this research by mass inoculation test,corresponded to the QTL detectedby individual inoculation test and field test [3],suggesting that q STV11is non environment specific and would be very useful in marker assisted selection breeding program Because it is very difficult to utilize RFLP markers in marker assisted selection,qSTV11map position was again detected using the linkage map of chromosome 11re constructed The result showed that qSTV11was located at the region of BJ11 8G257with a distance of 1 9c M (Fig 2)A classified analysis for the ratio of disea se rating inde x in BI L popula tion wa s perfor med acc ording toB J11 8and G257marker genotypes and their combination.For the twomarker,N/-,-/N and N/NgenotypesmatchedT able 4Distributio n o f ra tio o f d is ea se rating index in BIL po pulation cla ss ified by marker g eno type a nd their co mbina tio nsMarker genotype and their combinations Number of BILs in different resi stance levelsBJ11 8G257029%30%39%40%69%70%Total b Mean t value cN a0113546898 19K a 661032551 87 N 00135265101 54 K 66832348 03N N 00135262100 92KK 66832044 4624 7728 014 26Notes:a N denoted Ni pponbare genotype,K denoted Kas al ath genotype;bMolecular data of 4lines for BJ11 8and 7lines for G257were mis sed;cShowedsignificant di fference between means of two genotypes at 0.1%probability level(t 0.001=5.959,12.924,3.46,respec tively).808 作 物 学 报第32卷Fig 2Chromosome location of pu tative Q TLs for RS V and its vector resistance in BILs detec ted Q TLs for SBP H resi stance;detected QTLs for RSV resistance;Chr.11 A denotes linkage map of chromosome 11wi th RFLP markers;Chr.11 B denotes linkage map of chromoso me 11with R FLP and SSR markers.with higher ratio of disease rating index,while K/-,-/K and K/K genotypes did with lower ratio of disease rating index,and significant difference (P =0.001)was found between genotypes N/-and K/-,-/N and -/K,N/N and K/K (Table 4).These results further verified that qSTV 11was closely linked to the two markers.2.5 Correlation analysis between resistance to RSV and SBPHFig 3Correlation analysis between the ratio of diseas e rating index and non p reference indexFig.3sho wed that the correlation between the ratio of disease rating index and non preference index in BI Ls was not significant (r =0.11,P <0.05),namely,resistance to RSV and SBP H was not related.This result supports above the mapping of QTLs for RSV and SB PH.3 DiscussionRecently,rice stripe became more and more serious in our country and had become a devastating virus disease in Jiangsu province.One of the reasons is that the number of SB PHs rapidly increased.Based on statistical data,3 105SBP Hs per ha in wheat field were counted on the average in 2005and were 1to 3times of that in 2004(;;ne ).Many kinds of insecticides were used to kill vector pest in rice produc tion,but little effect could be seen.Therefore,it is very important to study mechanism of resistance to rice stripe for polymerizing genes resistant to RSV and SB PH and breeding rice varieties with high resistance to rice stripe.So far,there have not been any reports about location of QTL for SB PH resistance.In this study,QTLs associated with specific mechanism of resistance to SBPH were firstly de tected to be located on chromosome 3.By comparing Ding !s [3]report with the result in this study,the QTL,qSTV 11,was associated with RSV resistance,not SBP H resistance,indicating that resistances to RSV and SBPH are controlled by different genes.This view coincides with no significant correlation (P <0.05)809 第6期S UN Dai Zhen et a l .:Detection of QTLs for Resistance to Rice Stripe Virus and Small Brown Planthopper...between resistances to RSV and SBPH.Classic quantitative genetics assumes that the genetic basis of trait correlations is the result of pleiotropic effects or the tight linkage of genes.Previous researches also found that QTLs for related traits of grain quality,grain yield and drought resistance in plant were mapped in QTL clusters [15 17],which illustrated genetic base of trait correlations in molecular level.Therefore,no significant correlation between two traits controlled by different genes in this study is reasonable.On the other hand,epistatic QTLs resistant to RSV and SBPH were firstly detected using mixed linear model.Altogether,four pairs of non allelic interaction for RSV and SBPH resistance were found,of which interaction effect between marker interval C82G376and S2137G320was significant,c ontributed to 11.11%of the total variation.Furthermore,main effec t QTLs,qSTV 11and qSBP H3 b ,also involved in interactions for RSV and SBPH resistance,respectively.This suggests that genetic controlling mechanism of rice stripe resistance is very comple x.Based on integrated linkage map,q SBP H3 bdetected in this research and the QTL [18]for whitebacked planthopper resistance mapped by Yamasaki using IR24/Asominori RI L were all near the sa me marker C1135on chromosome 3.This indicates the region near the marker C1135is related to distribution of genes for piercing sucking insects resistance.Further study is necessary to elucidate the relationship between these QTLs.By comparing with the previous study,it is found that qSTV11detected in this study and the QTLs detected by Hayano Saito et al .[19],Ding et al .[20],Maeda et al .[21 22]using different populations were all loca ted at the vicinity of G257.It is speculated that those QTLs are common.Furthermore,qSTV11detec ted in this study under mass inoculation test was also detected by Ding et al .[3]using the sa me 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