Application of molecular marker ... 51Jurnal Bioteknologi Pertanian, Vol. 10, No. 2, 2005, pp. 51-54

ABSTRAK

Blas merupakan salah satu penyakit yang sangat merugikanpada padi sawah maupun padi gogo. Menanam varietas tahanyang mengandung lebih dari satu gen major merupakan carapenanggulangan penyakit yang efisien dan diharapkan ke-tahanannya dapat berlangsung lebih lama. Galur-galur pe-muliaan yang membawa piramida gen Pi-1 dan Pi-2 diseleksidengan markah DNA yang terpaut dengan gen tahan blasmelalui analisis RFLP and PCR. Penelitian ini bertujuan untukmengkaji penggunaan markah DNA guna mendukung programpiramida gen tahan blas pada varietas padi Indonesia. Galur-galur padi yang membawa piramida gen diseleksi berdasarkanada atau tidaknya alel Pi-1 pada analisis RFLP dengan markahRZ536 dan Pi-2 pada analisis PCR dengan primer STS-RG64.Hasil penelitian menunjukkan bahwa pemasukan gen Pi-1 danPi-2 pada galur-galur yang dikembangkan dari dua set persi-langan dengan varietas populer Indonesia seperti Way Rarem,Jambu, dan Cabacu telah berhasil dilakukan. Enam daridelapan galur piramida gen Pi- yang diuji, terdeteksi telahmembawa gen Pi-1 dan Pi-2 yang diinginkan. Galur ini siapuntuk dipelajari lebih lanjut baik sifat-sifat agronomisnyamaupun ketahanannya terhadap penyakit blas di lapangan.

[Kata kunci: Oryza sativa, seleksi, markah molekuler, ketahananterhadap penyakit, Pyricularia oryzae]

ABSTRACT

Blast is one of the most devastating diseases of rice thatappear both on upland and rainfed lowland rice-growing area.Growing rice carrying more than one major gene for rice blastresistance is an efficient approach to obtain a variety withmore durable. Marker-assisted selection (MAS) using RFLPand PCR markers flanking to resistant genes were done toassort breeding lines carrying pyramided blast resistance genesPi-1 and Pi-2. This experiment aimed to study the applicationof DNA markers in the program of pyramiding blastresistance genes in Indonesian rice varieties. The lines weredifferentiated based on the presence or absence of resistancealleles in RFLP and PCR analysis using RZ536 and STS-RG64,respectively. The results showed that two additional blastresistance genes Pi-1 and Pi-2 had been incorporated intoIndonesian popular cultivars such as Way Rarem, Cabacu, andJambu. They are ready for further agronomic and phenotypicanalyses.[Keywords: Oryza sativa, selection, molecular markers, diseaseresistance, Pyricularia oryzae]

INTRODUCTION

Growing resistant varieties is the best way to sustainrice productivity from crop losses due to abiotic andbiotic stresses such as diseases. Blast is one of themost damaging diseases of rice that occurs both onupland and rainfed lowland rice-growing area. Becauseof high genetic variability of Pyricularia oryzae Cav,rice varieties with single major resistance gene have atendency to become susceptible to the pathogenwithin a few year of varieties released. Consequently,to obtain a variety with more durable resistance,incorporation of more than one blast resistant geneinto a variety has become a priority in rice impro-vement (Wang et al. 1994; Mackill and Bonman 1992;Hittalmani et al. 1995b).

Combining several genes and monitoring their pres-ence are difficult by conventional breeding because oftheir epistatic effects and masking of one gene byanother (Hittalmani et al. 2000). The development ofmolecular markers enhances the efficiency of breedingselection. Target genes in a segregating populationcan be identified with assistance of molecular markers,subsequently propose to hasten the transfer ofdesirable genes among varieties (Zheng et al. 1995).Molecular markers have a capacity to differentiatehomozygote and heterozygote plants in population.Moreover, molecular markers may select severalcharacters at the same time and allow selecting morethan one gene with the same phenotypic expression ina variety (Hittalmani et al. 1995 ab; Hittalmani et al.2000). The availability of saturated molecular map ofthe rice genome facilitated mapping blast resistancegene and locating closely linked markers. DevelopingRFLP markers into PCR markers has increased thepowerful of molecular markers as marker assistedselection (MAS) in confirmation of the presence ofdesirable gene in varieties (Paterson et al. 1991; Huanget al. 1997).

Several reports showed that some major blastresistance genes were identified, designated and

Application of molecular marker for the assessment of blast resistancegenes, Pi-1 and Pi-2, incorporated into Indonesian rice cultivars

Masdiar Bustamam1, Reflinur1, Dita Agisimanto2, Erwina Lubis3, and Suwarno3

1Indonesian Center for Agricultural Biotechnology and Genetic Resources Research and Development,Jalan Tentara Pelajar No. 3A. Bogor 16111, Indonesia

2Citrus and Subtropic Horticulture Research Station, Tlekung PO Box 22 Batu 65301, Malang, Indonesia3Indonesian Institute for Rice Research, Jalan Raya 9, Sukamandi PO Box 11, Cikampek, Subang 41255, Indonesia

52 Masdiar Bustamam et al.

located as Pi-1, Pi-2, Pi-4, Pi-5, Pi-7, and Pi-zh (Yu etal. 1991; Zhu et al. 1993; Wang et al. 1994). Some nearisogenic lines carrying one gene are available andfrequently used as a donor in crosses. Hittalmani et al.(2000) reported that three major genes (Pi-1, Pi-2, andPi-ta) were incorporated in individual rice using RFLPmarkers in MAS. The objective of present experimentwas to detect the presence of Pi-1 and Pi-2 genes inpyramiding blast resistance genes in Indonesianpopular rice cultivars using DNA markers.

MATERIALS AND METHODS

Plant materials

Three popular rice cultivars, namely Way Rarem,Cabacu, and Jambu as recipient parents and C101LAC(carrying Pi-1) and C104A51 (carrying Pi-2) as donorparent were used. To incorporate blast resistancegenes from NILs into popular cultivars, three doublecrosses were performed, namely Way Rarem/C101LAC//Cabacu/C104A51 (cross #1), Jambu/C104A51// Cabacu/C101LAC (cross #2), and B8503E/C101LAC//Cabacu/C104A51 (cross #3). Five progenies (three from cross#1, one each from cross #2 and #3) of F5 were used todevelop F6 progenies. Six lines of the F6 generation(four from cross #1 and two from cross #2) were chosenbased on previous confirmation of Pi-1 and Pi-2 geneson F5 generation. All breeding lines together with theirrespective parents were planted at Pusakanegaraexperiment station, West Java, during the wet seasonof 2000 for field test.

Molecular analysis

Leaf sample of parents and progenies were collectedand genomic DNA was isolated using modifiedmethod of Delaporta et al. (1983). Quality and quantityof DNA were tested based on Sambrook et al. (1989).To confirm the incorporation of Pi-1, RFLP probeRZ536 located at 7.9 cM away from Pi-1 was used,while the incorporation of Pi-2 was detected usingSTS-RG64 located at 2.1 cM from Pi-2 (Fig. 1).

Genomic DNA of the test plant in RFLP analysiswere digested with HindIII and then probed withRZ536 using a nonradioactive, dig-11-dUTP, labelingmethod according to Roche instruction. Confirmationof the Pi-2 gene was completed using PCR markerSTS- RG64. The PCR reaction was consisted of 2.5 mlbuffer PCR (10 x), 3.2 ml 15 mM MgCl2, 0.25 ml dNTPseach, 2.5 ml primer (forward and reverse), 1 ml Taqpoly-merase and 2 ml sample DNA (50 ng/ml) in a 25 ml

total reaction. The conditions of PCR amplificationwere consisted of 5 minutes at 94oC for the initial dena-turation followed by 40 cycles 1 minute denaturationat 94oC, 1 minute annealing at 60oC, and 1 minuteextension at 72oC, and completed with additionalprimer extension for 5 minutes at 72oC on MJ ResearchThermocycler. The PCR products were cut with HaeIIIand run on a 2% agarose gel.

RESULTS AND DISCUSSION

The potential application of DNA markers for assistingselection of breeding materials in pyramiding rice blastresistance genes have been well documented (Hittal-mani et al. 1995; Marchetti 1999; Hittalmani et al. 2000;Khush and Brar 2002; Yuqing et al. 2004). Results ofexperiment indicated that hybridization of genomicDNA of donor parent and the breeding lines withRFLP marker RZ536 specify a distinct fragment at thelevel of 750 (data not shown). The occurrence of 750bp fragment on C104 A51 revealed the presence of Pi-1 allele as reported earlier by Hittalmani et al. (1995;2000). Consequently DNA hybridization of RFLPprobe RZ536 to 20 progenies of F5 and respectiveparents successfully selected five lines containing Pi-1 gene that is Bio 530 A-5-14-2-2-8, Bio 528 B-12-1-1,Bio 530 A-14-2-3-4, Bio 530 A-39-3-6, and Bio 532 B-46-1-2, while another progenies were undetected.

Amplification of genomic DNA of respected donorparents and selected breeding lines with STS-RG64revealed a 700 bp fragment. The occurrence of 700 bpon C101 LAC suggested the presence of allele Pi-2 asreported by Hittalmani et al. (1995; 2000). Therefore,selection of those progenies using STS-RG64 ex-plained that the entire five progenies mentionedabove also have Pi-2 gene. This present observation

6 .3

5 .1

7 .2

2 .1

RG123

RG456

RG612

Pi-2

RG64

7 .9

3 .5

10.0

RZ536

Pi-1

NpB181

RG303

Chromosome 6

Fig. 1. Map location of molecular markers used in detectionof the two blast resistance genes, Pi-1 and Pi-2 (Hittalmaniet al. 2000).

Chromosome 11

Application of molecular marker ... 53

indicated that five progenies of F5 lines have bothPi-1 and Pi-2 gene based on RZ536 and STS-RG64selection (Table 1).

Based on MAS on F5 lines, six breeding lines of F6containing four lines from cross #1 (indicated by Bio-530) and two entities from cross # 2 (indicated by Bio-528), were analyzed. They were consisted of twoplants from each line except Bio 530 A-14-2-3-4 andBio 532 B-46-1-2 that were not included due to poorseed viability in the field experiment. RFLP and PCRanalyses to confirm the presence of Pi-1 and Pi-2genes indicated that all F6 progenies tested showedsimilar band position like of the donor parents. Theresults developing from this F6 progeny are shown inFig. 2. Results of this study correspond with thepreceding analysis on F5 generation. In other word theincorporation of Pi-1 and Pi-2 genes detected on F5generation is confirmed at the F6 generation. Thisstudy imply that the incorporation of two new genes(Pi-1and Pi-2) in new lines developed in presentbreeding program were stable.

These results also indicated the importance ofRFLP and PCR analyses in MAS. One could differenti-ate the presence of two different genes in single plant

at one time analysis. As stated by Paleman and van derVoort (2003), DNA markers are extremely dependableselection utensils as they are stable, relativelystraightforward to score in a qualified laboratory, andnot affected by ecological condition. This study alsodemonstrated that MAS could enhance the efficiencyof breeding material selection. The use of DNA

Table 1. Breeding lines used for confirmation of Pi-1 and Pi-2 incorporation intoIndonesian popular rice cultivars.

Previous study Present study

F5 lines Remarks F6 lines Remarks

Bio 530 A-5-14-2-2-8 Pi-1 & Pi-2 Bio 530 A-5-14-2-2-8 Pi-1 & Pi-2Bio 530 A-5-14-2-2-8* Pi-1 & Pi-2

Bio 528 B-12-1-1 Pi-1 & Pi-3 Bio 528 B-12-1-1 Pi-1 & Pi-2Bio 528 B-12-1-1* Pi-1 & Pi-2

Bio 530 A-14-2-3-4 Pi-1 & Pi-2 Bio 530 A-14-2-3-4 Not testedBio 530 A-39-3-6 Pi-1 & Pi-3 Bio 530 A-39-3-6 Pi-1 & Pi-2

Bio 530 A-39-3-6* Pi-1 & Pi-2Bio 532 B-46-1-2 Pi-1 & Pi-2 Bio 532 B-46-1-2 Not tested**Bio 530 A-4-1-2-1 UndetectedBio 530 A-5-7-2-9 UndetectedBio 530 A-5-6-5-3 UndetectedBio 530 A-5-10-2-2 UndetectedBio 528 B-12-3-1 UndetectedBio 528 B-12-4-4 UndetectedBio 528 B-12-5-1 UndetectedBio 528 B-96-2-1 UndetectedBio 532 B-45-9-1 UndetectedBio 532 B-46-3-4 UndetectedBio 530 A-5-10-5-6 UndetectedBio 530 A-5-10-5-8 Undetected

Notes:*Sister lines. **Not tested because of bad seed viabilityBio 530 = Cross #1 (Way Rarem/C101LAC//Cabacu/C104A51)Bio 528 = Cross #2 (Jambu/C104A51//Cabacu/C101LAC)Bio 532 = Cross #3 (B8503E/C101LAC//Cabacu/C104A51)

Fig. 2. DNA profile of Pi- gene pyramiding lines (F6) and oftwo donor parents C101 LAC (bearing Pi-1) and C101 A51(bearing Pi-2); a = assortment of six individual F6 having Pi-1in RFLP analysis using RZ536; b = Pi-2 loci in PCR analysisusing STS-RG64.

54 Masdiar Bustamam et al.

markers permits the selection of plants with more thanone gene at a time. However, before those lines areused to overcome blast disease incidence, phenotypicconfirmation both in the greenhouse and in the blastnursery are requested.

CONCLUSION

This study suggested that RZ536 and STS-RG64 canbe used as marker assisted selection of the incorpora-tion of Pi-1 and Pi-2 in breeding lines of pyramidingrice blast resistance gene program in Indonesia. Theselected breeding lines could be developed into moreadvanced generation to produce elite lines, and testedfor further phenotypic performance such as resistanceto blast pathogen in the greenhouse and in thescreening sites as well as their performance to otheragronomic characters.

ACKNOWLEDGMENT

The great appreciations are expressed to Drs. RebeccaNelson, Hei Leung, Casiana Vera Cruz, and Ms.Marichu Bernardo for their kind guidance in settingand facilitating the ARBN program. Our sinceregratitude is also addressed to Mr. Mahrup, FajarSuryawan, and Ms. Suzie Christyanty for their kindhelp in laboratory activities. This study was madepossible by the research grant 977001#554 from theRockefeller Foundation and complimentary project ofARBN Phase II to our Institute. The breeding activitieswere funded by the Government of Indonesia.

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