a new blast resistance gene identified in the indian native rice cultivar aus373 through allelism...

A new blast resistance gene identified in the Indian nativerice cultivar Aus373 through allelism and linkage tests

Q. H. Pan*†, L. Wang† and T. TanisakaFaculty of Agriculture, Kyoto University, Kyoto 606-8502, Japan

Blast, caused by Pyricularia grisea, is a major constraint on rice production. To widen genetic diversity for diseaseresistance, the Indian native rice cultivar Aus373 was screened by F2 segregation analyses to investigate the geneticbasis of its high resistance. Aus373 was crossed with a series of Japanese differential cultivars (JDCs) and the Chinesesusceptible cultivar Lijiangxintuanheigu (LTH). The resistance ratios of subsequent F2 progenies were used todetermine the number of blast-resistance loci present as well as allelic relationships with known loci. Resistance ofAus373 was governed by dominant alleles at two loci, one at the Pi-k locus and the second apparently at a new locuslinked to an isozyme gene Amp-1 with a recombination fraction of 37·9 6 3·0% on chromosome 2. This putative newlocus and allele were designated Pi16(t).

Keywords: allelism, genetic linkage, isozyme marker, Japanese differential cultivar, resistance genes, rice blast

Introduction

Rice blast is widespread throughout the world and thereare hundreds of genotypically distinct isolates of thecausal fungus, Pyricularia grisea (P. oryzae) (Ou, 1979;Bonman et al., 1986). The pathogen causes lesions on allaerial parts of the plant, including the leaf (blade, sheathand ligule), stem (section), panicle (rachilla and neck)and grain (hull), and limits yield potential in environ-ments favouring the disease (Shindo, 1980). Control isachieved largely by the use of resistant cultivars (Hiranoet al., 1980; Pan, 1990; Bonman et al., 1992). To breedeffectively for blast resistance, genetic information aboutresistance genes is required.

Genetic studies on blast resistance have been con-ducted extensively in Japan. Sasaki (1922) carried outgenetic analysis by inoculation with unidentified fungusstrains, and showed that the Japanese cultivar Tsurugihas a single dominant gene for resistance. Geneticstudies were advanced when Goto established a primaryset of differential cultivars for races of P. grisea in Japanin the early 1960s (Goto et al., 1961, 1964). Yamasaki &Kiyosawa (1966) then selected seven representativefungus strains (subsequently called Japanese differentialraces) from the 16 races of the pathogen found by Gotoet al. (1961, 1964). Using these races, they tested

resistance of many Japanese cultivars and classifiedthem into several groups, based on reaction patterns.They then selected representative cultivars from eachgroup and made crosses between cultivars in the sameand different groups. Segregation analyses wereconducted in F2 populations or F3 lines of these crosses.From these data, Kiyosawa and colleagues identified 13resistance alleles at seven loci: Pi-a, Pi-i, Pi-k (alleles:Pi-k, Pi-ks, Pi-km, Pi-kh and Pi-kp), Pi-z (Pi-z and Pi-zt),Pi-ta (Pi-ta and Pi-ta2), Pi-b, and Pi-t (Kiyosawa &Ando, 1990). Based on this work, Kiyosawa establishedthe term Japanese differential cultivars (JDC), each JDCcarrying a single gene at the above seven loci forresistance to specific Japanese races of blast (Kiyosawa,1979). These genes, and a new one, Pi-sh, which waslater found by Imbe & Matsumoto (1985), give a total of14 resistance genes identified in Japan so far.

Genetic studies in other countries have lagged becauseof a lack of continuity (Kiyosawa & Ando, 1990),caused by the extremely changeable virulence of thepathogen P. grisea (Bonman et al., 1986; Ling et al.,1989), and by the presence of many resistance genes intropical rice cultivars (Mackill et al., 1985; Yu et al.,1987). A major breakthrough came in the 1990s withdevelopment of breeding lines, including near-isogeniclines (NILs), recombinant inbred lines (RILs), anddoubled-haploid lines (DHLs), that revealed thecomplex genetic constitution of tropical cultivars. Atthe same time, a number of different types of DNAmarkers became available (Tanksley et al., 1989). At theInternational Rice Research Institute (IRRI) in thePhilippines, a set of NILs for blast resistance genes wasdeveloped, and Pi-1, Pi-2 (later designated as Pi-z5),

Plant Pathology (1999) 48, 288–293

Q 1999 BSPP288

*To whom correspondence should be addressed.

†Present address: BIOTEC, National Science and TechnologyDevelopment Agency, 73–1 Rama VI Road, Rajdhevee, Bang-kok 10400, Thailand.

Accepted 23 November 1998.

Pi-3, Pi-4a(t) (later Pi-ta), and Pi-4b(t) were identified(Yu et al., 1991; Mackill & Bonman, 1992; Inukai et al.,1994). Wang et al. (1994) identified Pi-5(t) and Pi-7(t)through RFLP analysis using RILs of ‘CO39/Morobere-kan’; and Zhu et al. (1993) identified Pi-zh(t) (laterdesignated as Pi11) through RAPD analysis using DHLs.

Rice cultivars native to Yunnan Province insouth-west China and to the Indian subcontinent wererecently examined as new sources of blast resistance. It iswell known that these regions contain plentiful geneticresources for rice (Nakagahra, 1985; Wang, 1993).Preliminary screening identified several cultivars thatconfer high resistance to all the Japanese blastdifferential races and even to some races that are virulenton cultivars carrying Pi-zt, Pi-ta2 and Pi-b genes,introduced from indica cultivars and expressing highresistance (Ling et al., 1990; Q. H. Pan, unpublisheddata). From this work, several new resistance genes werefound, including Pi8 in the Indian cultivar Kasalath (Panet al., 1996), Pi13(t) and Pi14(t) in the Chinese cultivarMaowangu (Pan et al., 1998a), and Pi-kg(t) and Pi15(t)in the Chinese cultivars GA20, and GA25, respectively(Pan et al., 1998b).

In the present study, Aus373, a cultivar from Indiawith high resistance to many blast races, was examined.The aims were to determine the inheritance of theresistance in Aus373, to identify any allelic relationshipsof its genes with the known genes in the JDCs and todefine any new resistance genes by using monogenicsegregating populations and isozyme markers.

Materials and methods

Plant materials

Nine of the JDCs and the Chinese susceptible cultivar

Lijiangxintuanheigu (LTH) (Table 1) were crossed toAus373. F1 plants were grown in a field to obtain F2

seeds. Seeds were pre-germinated by soaking in water at358C for 48 h and sown (36 files 18 rows) in paper pots(Japan Sugar Beet Ltd, Japan) filled with granulated soilused in the commercial production of rice seedlings(Kureha Chemistry Ltd, Japan). To confirm the successof inoculation and the pathogenicity of tested races,Aus373, the 12 JDCs and the susceptible check LTHwere sown at both ends of each F2 population in a tray.Seedlings were grown in a glasshouse at 20–358C forabout 3 weeks before inoculation.

Fungus races, inoculation and disease evaluations

Four P. grisea races (00.7, 031.1, 031.5, and 433.5) wereused. Their virulence on the 12 JDCs, LTH and Aus373is given in Table 1. The former two races are Japanesedifferentials that were used with the other five differ-ential races in Japan to identity the 13 known resistancegenes (Kiyosawa, 1974). The latter two races were usedto distinguish the Pi-z, Pi-zt and Pi-t genes, because thedifferential races used are avirulent on plants with thesethree genes (Table 1).

Inoculum was prepared as described previously (Panet al., 1996). When seedlings reached the four- to six-leafstage, the inoculum suspension (10–50 ×104 conidia permL) was sprayed onto leaves in an inoculation incubatorequipped with a temperature controller and a humidifier.After inoculation, the seedlings were kept in theinoculation incubator at 258C and above 95% relativehumidity for 24–36 h and then transferred to a moistvinyl tunnel at 25–358C. Disease reactions wererecorded about 10 days after inoculation, when typicallesions developed on leaves of susceptible plants. Therecording system described by Pan et al. (1996) was

A new gene for rice blast resistance 289

Q 1999 BSPP Plant Pathology (1999) 48, 288–293

Table 1 Reaction pattern of Indica rice cultivar Aus 373, the Chinese susceptible cultivar Lijiangxintuanheigu (LTH) andJapanese differential cultivars to two Japanese differential races and three additional races of Pyricularia grisea

P. grisea strain (race number)a

Resistance CultivarCultivar gene codea Hoko1 (007.0) Ina72 (031.1) TH67-22 (031.5) Ai75-61 (433.5)

Shin 2 Pi-ks 1 Sb S S SAichiasahi Pi-a 2 S R R SFujisaka 5 Pi-i 4 S R R RKusabue Pi-k 10 R S S STsuyuakec Pi-km 20 R S S SFukunishiki Pi-z 40 R R R RK1c Pi-ta 100 R R R RPiNo.4 Pi-ta2 200 R R R RToride 1 Pi-zt 400 R R R SK60c Pi-kp 0.1 R S S SBL1 Pi-b 0.2 R R R RK59 Pi-t 0.4 R R S SLTH þ S S S SAus373 ? R R R R

aThe race number of each fungus strain is the sum of the cultivar codes for cultivars susceptible to the race.bS, susceptible; R, resistant.cNot used in the crosses in this study.

used: 0 ¼ no lesions; 1 ¼ small brown spots of pinheadsize; 2 ¼ brown spots with a long axis less than 2 mm;3 ¼ brown spots with a long axis more than 2 mm;4 ¼ uncoloured or purple spots with a long axis less than2 mm; and 5 ¼ uncoloured or purple spots with a longaxis 2 mm or more. For data analysis, seedlings of classes0–3 were regarded as resistant, and those of classes 4and 5 as susceptible.

Isozyme assay

Enzyme extraction, gel electrophoresis, and enzymeactive staining were performed as described previously(Pan et al., 1996). The following 10 isozymes of nineenzymes were examined: alcohol dehydrogenase (Adh,EC 1.11.1.6), catalase (Cat, EC 1.11.1.6), esterase (Est,EC 3.1.1.1), aminopeptidase (Amp (Amp-1 and Amp-3),EC 3.4.11.1), malate dehydrogenase (Mal, EC 1.1.1.40),peroxidase (Pox, EC 1.11.1.7), phosphogluconatedehydrogenase (Pgd, EC 1.1.1.44), phosphoglucoseisomerase (Pgi, EC 5.3.1.9) and shikimate dehydrogen-ase (Sdh, EC 1.1.1.25).

Data analysis

The number of resistance genes in Aus373 was estimatedfrom segregation ratios of the F2 populations inoculatedwith the races virulent on the JDC and avirulent onAus373. Allelism of the gene(s) in Aus373 to the knownresistance genes in the JDCs was then tested in F2

populations inoculated with races avirulent on bothparents. Segregation in the F2 progenies from the crosses

was tested by Chi-square analysis for goodness of fit to3R : 1S, 15R : 1S and 63R : 1S (resistance : susceptible)ratios. If the F2 progeny of a cross did not includesusceptible plants, the parents were assumed to carryallelic resistance genes. The chromosomal location ofany new resistance gene was determined by analysis ofits linkage with isozyme marker genes, and therecombination fraction was estimated by the maximumlikelihood method (Allard, 1956).

Results

Inheritance

Aus373 exhibited high resistance to all four races tested(Table 1). When races virulent on the JDCs were used, allF1 plants were resistant, indicating that resistance inAus373 was dominant.

For F2 segregation analysis, 10 F2 populations weretested with one or two races of P. grisea (Table 2). Toestimate the number of resistance genes in Aus373, theF2 population from the cross between LTH and Aus373was inoculated with races 433·5 and 007·0. The crosssegregated in a 3R : 1S ratio with 433·5 and a 15R : 1Sratio with 007·0 (Tables 1 and 2, tests 1–2), indicatingthat Aus373 carries two genes, one of which probablyconfers resistance to both races, the other only to race007·0. Similarly, when the cross with Fujisaka 5 wastested with race 007·0 (test 6) to which Fujisaka 5 issusceptible and Aus373 is resistant, the segregation ratiofitted a 15R : 1S ratio. The two resistance genes weretemporarily designated G1 and G2.

Q. H. Pan et al.290


Table 2 Reactions to four races of Pyricularia grisea of F2 populations from crosses between rice cultivar Aus 373 and the susceptible cultivarLijiangxintuanheigu (LTH) and nine Japanese differential cultivars

Reaction of parent Reaction of F2 plantsTest Differential Known Test Expected P valueb

no. cultivara gene race Femail Aus373 Rc Sc ratio

1 LTH þ 433.5 S R 255 104 3:1 0·05–0·102 LTH þ 007.0 S R 401 21 15:1 0·30–0·403 Aichiasahi Pi-a 031.5 R R 146 8 15:1 0·70–0·804 Aichiasahi Pi-a 031.1 R R 364 25 15:1 >0·955 Fujisaka 5 Pi-i 031.5 R R 404 24 15:1 0·60–0·706 Fujisaka 5 Pi-i 007.0 S R 361 29 15:1 0·30–0·407 Shin 2 Pi-ks 031.5 S R 250 72 3:1 0·30–0·408 Kusabue Pi-k 031.5 S R 299 91 3:1 0·40–0·509 Kusabue Pi-k 007.0 R R 278 0

10 Kusabue Pi-k 007.0 R R 436 011 Fukunishiki Pi-z 007.0 R R 413 6 63:1 >0·9512 Toride 1 Pi-zt 007.0 R R 409 7 63:1 >0·9513 Pi No.4 Pi-ta2 007.0 R R 407 8 63:1 0·60–0·7014 BL1 Pi-b 007.0 R R 331 8 63:1 0·30–0·4015 BL1 Pi-b 031.5 R R 365 29 15:1 0·40–0·5016 K59 Pi-t 007.0 R R 411 5 63:1 0·60–0·7017 K59 Pi-t 031.0 R R 358 19 15:1 0·30–0·40

aJapanese differential cultivars and Lijiangxintuanheigu (LTH) were used as the female parent in each cross.bProbabilities of Chi-square test for the resistant:susceptible ratios.cR, resistant; S, susceptible.

Allelism tests

To determine if either of the two resistance genes fromAus373 is an allele of gene Pi-k, the F2 progeny from thecross of Kusabue with Aus373 was inoculated with race007·0, which is avirulent to both parents. No susceptibleplants were observed in this cross (tests 9–10), indicat-ing that one of the two resistance genes of Aus373 isallelic to Pi-k. The gene is arbitrarily designated G1.When the F2 populations from the crosses of Kusabueand Shin 2 with Aus373 were inoculated with race031·5, virulent to the other known resistance alleles atthe Pi-k locus (Pi-k, Pi-ks, Pi-km, and Pi-kp (Kiyosawa,1974)), a segregation ratio of 3R : 1S was observed (tests7–8). These results supported the hypothesis thatAus373 has two resistance genes, G1, an allele at thePi-k locus that does not confer resistance to race 031·5as do other known resistance alleles, plus a second gene,G2.

Possible allelic relationships between G2 and six otherknown loci (Pi-a, Pi-i, Pi-z, Pi-ta, Pi-b, and Pi-t) wereexamined using races avirulent on both parents (tests 3–5, and 11–17). The segregation ratios in these tests fellinto two distinct classes. Ratios of 15R : 1S wereobtained when races 031·1 or 031·5 were used (tests3–5, 15 and 17), both races being virulent to G1 andavirulent to G2 and the known genes. Ratios of 63R : 1Swere obtained with race 007·0 (tests 11–14 and 16),which was avirulent to G1, G2 and the known genes.

These results indicated that G2 was not allelic to any ofthe resistance genes at the above six loci.

Linkage tests

To determine the chromosomal location of the newlydetected gene carried by Aus373, the F2 progeny fromthe cross with LTH were tested with race 433·5 (test 1).After surveying the disease reaction, the F2 plants weretransplanted into the field and at the maximum tilleringstage fresh tillers from each plant were sampled forisozyme analysis. In all, 359 viable F2 plants were tested.G2 is on chromosome 2, linked to Amp-1 with arecombination fraction of 37·9 6 3·0% (Table 3). Inconformity with the new rules of gene designation forrice blast resistance (Kinoshita, 1993), this resistancegene has been designated Pi16(t) (Iwata, 1996).

Discussion

Chromosomal locations of resistance genes

The present study showed that the Indian native cultivarAus373 carries an allele at the Pi-k locus and a newresistance gene, Pi16(t), which confers high resistance toraces 007·0, 031·1, 031·5 and 433·5. Linkage wasdetected between Pi16(t) and Amp-1 on chromosome 2with a recombination fraction of 37·9 6 3·0%. In a



Table 3 Linkage analysis of a rice blast resistance gene with isozyme markers in the F2 population of a cross between rice cultivarsLijiangxintuanheigu (LTH) and Aus373 using race 433·5 of Pyricularia grisea

Locia Locus Bb X 2,c

RecombinationA B Locus Ab BB B- Bb b- bb Locus A Locus B Linkage fraction (%)

Chromosome 2 G2 Amp-1 A- 82 128 45 2·8 3·2 16·3***d 37·963·0aa 15 54 35

Chromosome 4 G2 Mal-1 A- 59 127 69 2·8 0·7 1·0aa 24 56 24

Chromosome 6 G2 Amp-3 A- 85 124 46 2·8 18·8*** 2·1aa 33 57 14

G2 Pgi-2 A- 77 126 52 2·8 7·8* 3·3aa 27 61 16

G2 Cat-1 A- 86 121 48 2·8 11·4** 2·5aa 31 45 28

Chromosome 7 G2 Est-9 A- 53 133 69 2·8 3·5 0·4aa 22 52 30

Chromosome 11 G2 Pgd-1 A- 74 110 71 2·8 5·2 1·1aa 25 48 31

G2 Adh-1 A- 75 133 47 2·8 5·5 1·4aa 25 55 24

Chromosome 12 G2 Pox-2 A- 191 64 2·8 0·5 1·9aa 72 32

G2 Sdh-1 A- 68 149 38 2·8 14·2*** 1·2aa 29 54 21

aSymbols A and B represent a tentative resistance gene G2 of Aus373, or isozyme genes, respectively.bSymbols A-, B- and b- are used when the heterozygous genotypes cannot be identified because of segregation of a dominant allele.cX 2

A, X 2B and X 2

L represent the calculated Chi-square values for loci A, B and linkage, respectively.d*, ** and *** represent significance at P ¼ 0·05, P ¼ 0·01 and P ¼ 0·001 levels, respectively, for a X 2.

previous study, the authors identified a new resistancegene, Pi14(t), which was also linked to Amp-1 with arecombination fraction of 34·2 6 3·2% on chromosome2 (Pan et al., 1998a), and expressed resistance to theabove three races. Further work is required to establishwhether theses genes are alleles or tightly linked.

It is of interest that many disease resistance genes aretightly clustered on particular chromosomes (Islam &Shepherd, 1991; Sudupak et al., 1993; Inukai et al.,1994), but the origin and/or the geographic distributionof blast resistance genes bear no relation to theirchromosomal locations. For the Pi-k cluster on chromo-some 11, Pi-ks was found in American, Chinese andJapanese cultivars, Pi-k and Pi-km in Chinese and Indiancultivars, Pi-kp in a Pakistan cultivar, Pi-kh in Indian andVietnamese cultivars (Kiyosawa & Ando, 1990), Pi-kg(t)in a Chinese cultivar (Pan et al., 1998b), Pi1 inVietnamese and African cultivars (Inukai et al., 1994),Pi7 and Pi12(t) in African cultivars (Wang et al., 1994;Inukai et al., 1996) and Pi18(t) in a Korean cultivar (Ahnet al., 1996). On chromosome 2, Pi-b was found inIndonesian and Malaysian cultivars (Kiyosawa & Ando,1990), Pi14(t) in a Chinese cultivar (Pan et al., 1998a),and Pi16(t) in a Indian cultivar.

Further studies with more races are needed to identifythe Pi-k allele carried by Aus373, and DNA markers areneeded for a precise map location of Pi14(t) and Pi16(t).

Strategies for gene analysis of resistance in complexcultivars

In the early 1970s, Kiyosawa suggested that analysis ofblast resistance genes was almost complete for Japanesecultivars with Japanese races, and that additionalresistance genes may be present in other cultivars,particularly indica (Kiyosawa, 1974). However, theimplementation of gene analysis of these cultivars hasbeen hindered because they are genetically more com-plex than Japanese cultivars (Mackill et al., 1985; Yu etal., 1987). Kiyosawa et al. (1986) proposed a scheme forthe systematic analysis of resistance genes in the world’srice cultivars through international cooperation, butclassified them only on reaction patterns to blast races.Early in the 1990s, some workers identified several newresistance genes through linkage analysis using NILs,RILs and DHLs in combination with DNA markers.Despite these successes, however, production of breedinglines is inefficient, especially as new resistance gene(s)are urgently needed.

For the rapid provision of useful information on newresistances, simultaneous allelism tests with JDCs andlinkage tests with markers, should make it possible toidentify directly at least the minimum number of majorresistance genes present in cultivars. With the help of theJDCs, the procedure provides a technology for unravel-ling allelic relationships with known resistance genes inJDCs, i.e. if a known resistance gene is present it wouldbe directly identified. With the help of a diverse set ofpathogen races that are virulent to known resistances,

populations segregating for single unknown resistancescould be obtained and used. With this approach, theauthors have identified several new resistance genes asdescribed above.

Acknowledgements

The authors thank H. Yamagata and S. Kiyosawa fortheir kind and helpful support; M. Inoue, T. Higashi, andH. Saito for providing the rice blast fungus races.

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a new blast resistance gene identified in the indian native rice cultivar aus373 through allelism...

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