Indian Journal of Biochemistry & Biophysics Vol. 42, August 2005, pp. 228-232

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Molecular and biochemical markers associated with leaffolder Phenol ( medinalis G.) resistance in ( L.) Scr as des Seema Sinha, R Balasaraswathi*, K Selvaraju and P Shanmugasundaram was n Department of Plant Molecular Biology and Biotechnology more *Department of Biochemistry, CPMB, Tamil Nadu Agricultural University, Coimbatore 641003 Received 27 October 2004; revised 31 May 2005 Bioche Firl Association of molecular markers namely isozymes and simple sequence repeats (SSRs) and various biochemical 60% markers to leaffolder (Cnaphalocrocis medinalis G., a predominant pest of rice) resistance were studied in rice sampl (Oryza sativa L.). Recombinant inbred lines (RILs) of Fs generation obtained by crossing IR36 (susceptible parent) and TNAULFR831311 (moderately resistant parent) were used in this study. Soluble protein content, protein profile, and plants peroxidase and phenylalanine ammonia lyase (PAL) activities were the various biochemical markers studied. Decrease in studie soluble leaf protein content was observed in all lines, due to insect infestation. Protein profiling revealed an enhanced methc expression of a high molecular mass (>97 kDa) protein in all the infested lines. Besides, there was an increased induction of insect a 38 kDa protein in infested resistant parent and resistant RILs. A significant increase if! peroxidase and PAL activities was health observed after infestation. In peroxidase isozyme analysis, carried out after infestation, "isoform I" was found to be more prominent in the susceptible lines and "isoforrn 2" in the resistant lines. Bulk segregant analysis (BSA) with twenty-five rice profil microsatellites (RM) resulted in identification of three polymorphic markers between bulks RM II and RM432 located on Perox chromosome 7 and RM271 on chromosome 10 of rice. These markers may be associated with leaffolder resistance in rice spectr and can be used in marker-assisted selection for leaffolder resistance in rice. Per leaffo Keywords: Rice, Oryza sativa L, simple sequence repeat, isozyme, leaffolder, Cnaphalocrocis medinalis G., peroxidase, phenylalanine ammonia lyase, SDS-PAGE, bulk segregant analysis, leaf protein profile, soluble protein. expre IPC Code: CI2NI5/65, C12N15/52 obtair insect Leaffolder (Cnaphalocrocis medinalis Guenee) is a population), along with parents IR36 and studie predominant insect pest in South India and causes TN AULFR831311 were selected as experimental paren significant loss in yield of rice, which is estimated to material. Different biochemical parameters, which play leaf. be around 4.8 kg/halo Yield loss is high when the flag important role in insect defense in plants, such as total leaf is damaged. The 5% damaged leaves is reported soluble protein content, and peroxidase and Molecl 2 Iso to reduce the yield by 200 kg/ha inIR50 • Among the phenylalanine ammonia lyase (PAL) activities were eight leaffolder species recorded in India, C medinalis studied. Besides, simple sequence repeats (SSRs) and polya is the most widespread, and can cause damage peroxidase isozymes markers were also used to (SSR: ranging from 18.3 to 58.4 per cent'. The losses due to determine their association with leaffolder resistance. suitat Reser insect pests can be reduced by developing insect- separ: resistant rice varieties. Identification of molecular and Materials and Methods biochemical markers associated with leaffolder by e resistance in rice would help in enhancing the Plant materials and raising of plants mIcro breeding efficiency. In this study, based on preliminary The rice variety IR36 was chosen as leaffolder On (Cnaphalocrocis medinalis Guenee) susceptible screening, thirty recombinant inbred lines (F; mapping the s parent. TNAULFR831311, a pre-release rice culture phenc of Tamil Nadu Agricultural University was selected were *Author for correspondence. as the donor parent for the trait of resistance to indivi Tel: 0422 -5511263 Fax: 0422 - 2431672 leaffolder. Thirty F, generation RILs developed from susce E-mail: [email protected] the cross of these two parents by single seed descent and ~ Abbreviations: RILs, recombinant inbred lines, RM, rice method were also used for the experiment. resist: microsatellite, SSR, simple sequence repeat; PAL, phenylalanine Seedlings were raised in pots in the screen house. five ammonia lyase; BSA, bulked segregant analysis; PCR, polymerase chain reaction;SDS, sodium dodecyl sulphate, PAGE, Leaf samples were collected for DNA extraction 15 Reser polyacrylamide gel electrophoresis. days after sowing. Eighteen days after sowing, the RM8' SINHA et al: MARKERS FOR LEAFFOLDER RESISTANCE IN RICE 229

remaining seedlings were transplanted in replications RM237, RM244, RM271, RM272, RM295, RM297, separately for phenotyping and biochemical analyses. RM304, RM320, RM333, RM428, RM432, RM472, Ten days after transplantation, five leaffolder larvae RM495, RM543, RM575 and RM580 were used in were released per plant. this study. These primer pairs are located on rice chromosomes 1, 7, 9 and 10 and were selected, based Phenotyping on our earlier study (communicated for publication). Screening of lines for leaffolder damage was done Bulked segregant analyses (BSA) were done as as described". Damage due to the larvae in the plants described 10. was recorded when susceptible parent (lR 36) showed more than 60% damage. Results and Discussion Phenotyping Biochemical analyses Twenty-eight days old plants of the parents and the Fifteen days after letting the larvae (when there was RILs were infested with second instar larvae of cal 60% damage in susceptible check IR36), the leaf C. medinalis G. The damage scores were calculated lce samples were collected from both healthy and infested ~ld and the results are shown in the Table 1. The damage plants and stored at -70DC for different biochemical nd scores were 9.0 and 5.0 for susceptible and resistant in studies. Protein estimation was done by Bradford parents, respectively and ranged from 4.0 to 9.0 for .ed methods. The changes in soluble leaf proteins due to different RILs. According to the phenotypic score, of insect were calculated from the estimated values of -as RILs were categorized into two groups viz., healthy and infected plant leaf samples. Protein ire 6 moderately resistant and susceptible. Seven RILs ice profiling of lines was done on 12% SDS-PAGE . having scores 5.0 or less were scored as moderately on Peroxidase and PAL activities were assayed resistant and the rest (damage score> 7) were scored ce spectrophotornetrically+'. as susceptible (Table 1). Peroxidase activity was assayed in healthy and leaffolder-infested RILs along with parents and was se, Biochemical studies expressed in units/mg protein/min. From the values obtained the change in peroxidase activity due to Total soluble leaf protein and leaf protein profile The soluble protein contents were 8.04 and 6.42 insect infestation was calculated. PAL activity was mg/g fresh leaf in healthy parents viz., IR36 and and studied in the leaves of healthy and leaffolder infested TNAULFR831311, respectively. This corresponds to 'imental parents and RILs, and expressed as mUIlOO mg fresh the earlier observations that the amount of total seed ch play leaf. as total Table 1-Phenotypic score of parents and RILs ~ and Molecular marker analyses .s were Isozyme analysis for peroxidase was carried out on Parents and RILs Phenotypic RILs Phenotypic score* score* b) and polyacrylamide gel? For simple sequence repeat sed to (SSR) analysis, DNA was extracted" and diluted Parents 5.0 245 8.8 tance. suitably for PCR analysis. PCR was carried out as per TNAULFR 83131 11(MRP) Research Genetics, USA protocol. PCR products were IR 36 (SP) 9.0 274 7.3 RILs 8.3 276 8.3 separated on 5% urea-polyacrylamide denaturing gel 16 by electrophoresis and silver stained to reveal 29 8.3 282 7.66 microsatellite bands". 40 8.3 289 7.6 iffolder On the basis of phenotypic data of RILs obtained in 43 8.3 302 8.3 .eptible the screen house, DNA of lines having extreme 45 8.6 327 8.6 66 5.0 348 7.3 culture phenotypes for leaffolder resistance and susceptibility 70 9.0 389 8.3 elected were bulked. For bulking, equal amount of DNA of 97 8.3 440 7.0 mce to individual RILs within moderately resistant or 108 7.0 528 4.8 d from susceptible group were pooled. Seven resistant lines 131 4.0 561 5.0 153 8.3 595 8.5 Iescent and seven susceptible lines were bulked to form 202 8.3 608 8.3 resistant and susceptible bulks respectively. Twenty- 218 4.3 616 4.6 house. five different SSR primer pairs received from 220 5.0 637 9.0 :ion 15 Research Genetics, USA. viz; RM5, RM11, RMI8, MRP, moderately resistant parent; SP, susceptible parent; RILs, ng, the RM84, RM151, RM180, RM212, RM216, RM229, recombinant inbred lines;*Mean of 3 replications 230 INDIAN J. BIOCHEM. BIOPHYS., VOL. 42, AUGUST 2005 proteins was less in rice varieties Co1, C027 and was absent in their healthy counterparts. However, no C032 resistant to angoumois grain (Silotraga such difference was noticed in the infested plants of signi cereallela) ". In case of healthy RILs, the protein either susceptible parent, IR36 or susceptible RIL 70. resis content ranged from 6.13 to 7.22 mg/g. After Though there was an overall decline in the soluble mere leaffolder infestation, there was a significant decrease leaf protein content due to leaffolder infestation, the cinru in protein content in all lines. However, no significant protein profile revealed increased levels of 97 and 38 rests difference was found in protein content between the kDa proteins. The 38 kDa protein may be a defense- The lines (Table 2). Similar results have been reported in related protein. Earlier, increase in expression of mere It corn leaf aphid resistant and susceptible varieties 12. defense-related protein after infestation 13 and The total soluble leaf protein profile of parents and induction of a specific protein (53 kDa), due to total RILs (70 and 218) on SDS-PAGE is shown in Fig. 1. leaffolder infestation in resistant/moderately resistant cont resis In both the RILs, there was enhanced expression of a rice varieties have been reported": high molecular mass protein (>97 kDa) after Mole Peroxidase and PAL activities infestation. Besides, in the infested resistant parent, Perm A significant increase 10 peroxidase activity was and infested moderately resistant RIL 218, there was In observed due to insect infestation in all lines as an increased induction of a 38 kDa protein, which evident from analysis of. variance. An increase in heal peroxidase activity has been reported in sugar beet RIL: Table 2-Analysis of variance for different biochemical after infestation by Lygus disponsi'". The increase in of 2 parameters foun peroxidase activity in plants may be associated with "iso: lignification of wound by polymerization of p-coumaryl Mean sum of square RIL: Total soluble Peroxidase Phenylalanine and coniferyl alcohols". Though there was an increased protein activity lyase activity activity of peroxidase due to leaffolder infestation, no pror indi: Treatments 0.4257 0.222 0.643 significant difference was found between susceptible and resistant RILs (Table 2). Earlier, little or no effect 10 ( Infestation (I) 4.19* 4.129* 6.957* nee Lines (L) 0.706 0.284 1.07 on insect resistance was reported, due to the increase perc IL 0.023 0.0321 0.038 in peroxidase activity 16-18. vari Error 1.005 0.997 1.01 A significant increase in PAL activity, due to pv *Significant at 5%)eve) leaffolder infestation was observed in all lines, but no

2 3 4 5 6 7 A A Br-"!~2_"3_ > 97 kDa >97 kDa

43 kDa B

43 kDa 38kDa 38 kDa

Fig. RIL: TN} infer RlL Fig. 1-(a); Total soluble protein profile of parents and RILs on SDS·PAGE [Lanel, marker proteins; lane 2, TNAULFR831311 healthy; pero lane 3, TNAULFR831311 leaffolder infested; lane 4, IR36 healthy; lane 5, IR36 leaffolder infested; lane 6, RIL 70 healthy; lane 7, RIL heal 70 infested];(b): Total soluble protein profile of RILs on SDS·PAGE [Lane 1, marker proteins; lane 2 RIL 218 healthy; and lane 3, RIL RIL 218 infested] 528 infe: 230 INDIAN J. BIOCHEM. BIOPHYS., VOL. 42, AUGUST 2005 proteins was less in rice varieties Co 1, C027 and was absent in their healthy counterparts. However, no C032 resistant to angoumois grain moth (Silotraga such difference was noticed in the infested plants of signifi. cereallela) 11. In case of healthy RILs, the protein either susceptible parent, IR36 or susceptible RIL 70. resisrai content ranged from 6.13 to 7.22 mg/g. After Though there was an overall decline in the soluble mcreas leaffolder infestation, there was a significant decrease leaf protein content due to leaffolder infestation, the cinnan. in protein content in all lines. However, no significant protein profile revealed increased levels of 97 and 38 resistar difference was found in protein content between the kDa proteins. The 38 kDa protein may be a defense- The cc lines (Table 2). Similar results have been reported in related protein. Earlier, increase in expression of mcreas It corn leaf aphid resistant and susceptible varieties 12. defense-related protein after infestation 13 and w, The total soluble leaf protein profile of parents and induction of a specific protein (53 kDa), due to total p RILs (70 and 218) on SDS-PAGE is shown in Fig. 1. leaffolder infestation in resistant/moderately resistant contrib resistan In both the RILs, there was enhanced expression of a rice varieties have been reported 14. high molecular mass protein (>97 kDa) after Molecul: Peroxidase and PAL activities infestation. Besides, in the infested resistant parent, Peroxida A significant increase III peroxidase activity was and infested moderately resistant RIL 218, there was observed due to insect infestation in all lines as In tl an increased induction of a 38 kDa protein, which evident from analysis of variance. An increase in healthy peroxidase activity has been reported in sugar beet RILs, t\ Table 2-Analysis of variance for different biochemical after infestation by Lygus disponsi'", The increase in of zym parameters peroxidase activity in plants may be associated with found tl "isoforr lignification of wound by polymerization of p-coumaryl Mean sum of square RILs ( Total soluble Peroxidase Phenylalanine and coniferyl alcohols". Though there was an increased protein activity lyase activity activity of peroxidase due to leaffolder infestation, no promine indicate Treatments 0.4257 0.222 0.643 significant difference was found between susceptible and resistant RILs (Table 2). Earlier, little or no effect in diffe Infestation (I) 4.19* 4.129* 6.957* rice line Lines (L) 0.706 0.284 1.07 on insect resistance was reported, due to the increase peroxide 1L 0.023 0.0321 0.Q38 in peroxidase activity'<". varieties Error 1.005 0.997 l.01 A significant increase in PAL activity, due to pv ory; *Significant at 5%level leaffolder infestation was observed in all lines, but no

A 2 3 4 5 6 7 2 3

> 97 kDa

43kDa

43 kDa 38kDa 38 kDa

Fig.2-(A RILs [Lane TNAULFR: infested; lar RIL 131 he: Fig. l-(a): Total soluble protein profile of parents and R1Ls on SDS-PAGE [Lanel, marker proteins; lane 2, TNAULFR831311 healthy; peroxidase lane 3, TNAULFR831311 leaffolder infested; lane 4, 1R36 healthy; lane 5, 1R36 leaffolder infested; lane 6, RIL 70 healthy; lane 7, RIL healthy; Ian 70 infested]; (b): Total soluble protein profile of R1Ls on SDS-PAGE [Lane 1, marker proteins; lane 2 R1L 218 healthy; and lane 3, RIL R1L 97 leaf 218 infested] 528 infester infested] 232 INDIAN 1. BIOCHEM. BIOPHYS., VOL. 42, AUGUST 2005

identified for brown planthopper (BPH) Nilaparvata 7 Sadasivam S & Manickam A (1996) Biochemical methods, 24 New Age International (P) Ltd. and Tamil Nadu Agricultural lugens resistance in rice • BSA was used to identifr RAPD markers associated for BPH resistance'". University 8 Dellaporta S L, Wood 1 & Hick 1 B (1983) Plant Mol Bio Molecular tagging of bacterial blight resistance gene Rep 1, 19-21 was also reported by BSA and one primer OPA 12 9 Panaud 0, Chen X & McCouch S R (1996) Mol Gen Genet was found linked to the trait"', 252,597-607 The present study revealed clear differences in the 10 Michelmore RW, Paran I & Kesseli R V (1991) Proc Natl protein profile, peroxidase isoforms and micro satellite Acad Sci (USA) 88, 9828-9832 11 Raghumoorthy K N & Gunathilagaraj K (1988) lnt Rice Res marker pattern between the moderately resistant and Newsl 13, 12 susceptible lines. These markers can further be 12 Beck D L, Dunn G M, Routley D 0 & Bowman 1S(1983) validated using larger set of RILs/germplasm lines for Crop Sci 23, 995-998 . use in marker-assisted selection for leaffolder 13 Edwards P 1& Wratten S D (1983) Oecologia 59, 88-93 resistance in rice. 14 Das A, Balasaraswathi R, Sadasivam S & Palanisamy S (1999) Indian J Agric Biochem 12, 1-4 15 Hori K (1973) Appl Ent Zool 8, 103-112 Acknowledgement 16 Dowd P F & Vega F E (1996) Nat Toxins 4, 85-91 Financial support from the Department of 17 Rahbe Y & Febvay G (1993) Entomol Exper Appl 67, 149· Biotechnology, Govt. of India and IeAR for the 160 18 Felton G W & Duffey S S (1991) J Chem Ecol 17, 1715· project and student fellowship respectively IS 1732 gratefully acknowledged. " 19 Cahill D M & Mc Comb 1 A (1992) Physiol Mol Plant PathoI46,315-332 References 20 Southerton S G & Deveral B 1(1990) Plant Physiol39, 223- 1 Herdt R W(1991) in Rice Biotechnology (G S Khush & G H 230 Toenniessen, eds), pp 320, CAB International and IRRl 21 Li C C, Chang S 1, Hsu FS & Tzeng DDS (1994) Plant 2 Pandi V (1997) M.Sc (Ag.) Thesis, Tamil Nadu Agricultural Pathol Bull 3, 107-118 University, Coirnbatore, India 22 Stavely 1R & Hanson E W (1967) Phytopathology 57, 482-485 3 Ramasamy C & laliecksono T (1996) Rice Research in : 23 Lagrimini L M & Rothstein S(1987) Plant Physiol84, 438-442 Progress and Priorities (Evanson R E & Hossain M, ed.), 24 Yang H, Rien X, Weng Q & Zhu L (2002) Hereditas 136, pp 418, CAB International and lRRl, 39-43 4 Heinrichs E A, Camanag E & Romena A(1985) J Econ 25 Renganayaki K,Fritz A K, Sadasivam S, Pammi S, Entomol78, 274-278 McCouch S R,Kumar SM & Reddy A S (2002) Crop Sci 5 Bradford M M (1976) Anal Biochem 72, 248-254 42,2112-2117 6 Laemmli U K (1970) Nature 227, 680-685 26 Rao K K (2003) Inti Rice Res News let 28, 1 L,i h) In: Tt en

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