Development of Monogenic and Digenic Rice Lines for Blast ...

M A P P I N G P O P U L AT I O N

Development of Monogenic and Digenic Rice Lines for Blast Resistance Genes Pi-ta, Pi-kh/Pi-ks Yulin Jia* and Karen Moldenhauer

ABSTRACT The major blast resistance (R) genes Pi-ta, Pi-ks , Pi-kh have been effectively deployed in rice (Oryza sativa L.) in the southern USA for preventing disease caused by the predominant U.S. races of Magnaporthe oryzae Cav. [=Magnaporthe grisea (Herbert) Barr.]. In the present study, the codominant single nucleotide length polymorphism DNA marker for the Pi-ta gene, and the simple sequence repeat markers, RM144 and RM224, that cosegregate with two alleles of the Pi-k gene, Pi-ks and Pi-kh, were used for R identification in a F10 recombinant inbred line population. This population (Reg. no. MP-3, NSL 452303) was derived from the cross RU9101001/‘Katy’. This population composed of 235 individual lines was jointly released on 24 Apr. 2009 by the USDA–ARS and the University of Arkansas Division of Agriculture Arkansas Agriculture Experiment Station. R gene containing RIL lines were verified with standard pathogenicity assays by means of a set of differential races of M. oryzae in the USA. One hundred eighty-two pure lines were identified from the population with the Pi-ta, Pi-ks , and Pi-kh genes. A total of 56 had Pi-ta and Pi-ks , 51 lines had Pi-ks , 27 had Pi-kh, and 48 lines had Pi-kh and Pi-ta. These monogenic and digenic rice lines with the major blast R genes are expected to be useful for studying effects of each R gene singly and in combination for their epistatic interaction with yield and for introducing blast resistance with marker assisted selection.

R

ICE, one of the most important food crops feeding half of the world’s population, is under extensive cultivation worldwide. Breeding for high yielding and improved resistance are the two most economically and environmentally benign methods of maintaining stable rice production. However, if resistance (R) genes are linked to genes that affect yield, this may affect the selection of resistant cultivars. Yield penalties for expressing R genes have been well documented in cereal crops except for rice (see review, Brown, 2002). Blast disease of rice caused by the filamentous fungus Magnaporthe oryzae has been one of the most damaging diseases of rice and remains one of the most difficult

Yulin Jia, USDA–ARS, Dale Bumpers National Rice Research Center, 2890 Hwy. 130 East, Stuttgart, AR 72160. Karen Moldenhauer, Univ. of Arkansas, Rice Research and Extension Center, 2900 Hwy. 130 East, Stuttgart, AR 72160. Registration by CSSA. Received 27 Apr. 2009. *Corresponding author ([email protected]). Abbreviations: DB NRRC, Dale Bumpers National Rice Research Center; RIL, Recombinant Inbred Line; UA RREC, University of Arkansas, Division of Agriculture Rice Research and Extension Center; USDA–ARS, US Department of Agriculture, Agriculture Research Service.

Published in the Journal of Plant Registrations 4:163–166 (2010). doi: 10.3198/jpr2009.04.0223crmp © Crop Science Society of America 5585 Guilford Rd., Madison, WI 53711 USA All rights reserved. No part of this periodical may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Permission for printing and for reprinting the material contained herein has been obtained by the publisher.

Journal of Plant Registrations, Vol. 4, No. 2, May 2010

crop diseases to manage (Khush and Jena, 2007). Genetic analysis demonstrated that each Pi (for Pyricularia) gene confers resistance to a race of M. oryzae that carries the corresponding avirulence (AVR) gene (Silué et al., 1992). Over 80 complete major resistance Pi genes have been described in rice germplasm worldwide (Ballini et al., 2008; McCouch et al., 1994). To date, Pi-ta, Pi2/Piz-t, Pi5, Pi9, Pikm, Pi-b, Pi36, Pi-37, Pi-d2, and Pit (Ashikawa et al., 2008; Bryan et al., 2000; Chen et al. 2006; Hayashi and Yoshida, 2009; Lin et al., 2007; Liu et al., 2007; Qu et al., 2006; Wang et al.,1999; Zhou et al., 2006) have been characterized molecularly and their gene structure and resistance functions have been extensively investigated. However, resistance responses to blast are often masked by different matched pairs of R and AVR genes (Silué et al., 1992). In addition, because R genes in germplasm and avirulence genes in the blast pathogen are often unknown, it is difficult to investigate the effects of individual R genes and their epistatic interactions. In the southern USA, the R genes Pi-ta and Pi-ks/kh have been used to prevent infections by the common races of M. oryzae IA, IB, IC, ID, IE, IG, and IH for over a decade, and they remain effective in commercial rice fields (Fjellstrom et al., 2004; Correll et al., 2000; Jia et al., 2009; Marchetti et al., 1987; Marchetti, 1994). Similar to most predicted blast R genes, Pi-ta and Pikm, alleles of Pi-ks and Pi-kh were predicted to encode proteins with nucleotide binding sites suggesting their putative roles as receptors for pathogen signaling molecules (Ashikawa et al., 2008; Bryan et al., 2000). Pi-ta has been studied extensively and information on its mode of action, distribution, and evolution has been documented (Jia and Valent, 2009). The Pi-kh/Pi-ks alleles

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have been extensively used in U.S. long-grain breeding programs. The physical locations of Pi-kh/Pi-ks have been delimited with two simple sequence repeat markers, RM144 and RM224 (Fjellstrom et al., 2004; Jia et al., 2009), but the individual and combined effects of Pi-ks/Pi-kh are unknown. Monogenic lines of rice each with different major blast R genes in genetic background of the susceptible cultivar Lijiangxintuanheigu (LTH) from Yunnan province in China were developed by means of a backcrossing breeding strategy (Tsunematsu et al., 2000). These lines are useful for determining the resistance spectra of individual R genes but are not suitable for studying epistatic interactions among different R genes. Monogenic and digenic recombinant inbred lines with Pi-ks, Pi-kh, Pi-ta and Pi-ks, and Pi-ta and Pi-kh are useful materials for studying individual effects of Pi-ks, Pi-kh, and the epistatic relationships between Pi-ta and Pi-ks and Pi-ta and Pi-kh as well as between the individual R genes and yield and yield components. The objective of this study was to identify RILs that contain Pi-ta and Pi-ks or Pi-ks or Pi-kh and Pi-ta and Pi-kh from a recombinant inbred line population (Reg. no. MP-3, NSL 452303) from a cross of an adapted breeding line and Katy, a U.S. tropical japonica cultivar.

Canada), and 100-seed weight. The seeds from each row were bulked to produce F9 families which were grown plots with two replications. In 2006, these families were grown in 0.46-m2 field plots with two replications at Stuttgart, AR. Data were again collected on the same characteristics. The 2006 bulk harvested seeds to be released as genetic stocks are being maintained at the GSOR collection, DB NRRC, Stuttgart, AR. DNA preparation and marker analysis was performed following the procedures by Jia et al. (2004a) for Pi-ta and Jia et al. (2009) for Pi-ks and Pi-kh. Seven to 10 seedlings (3–4 leaf stage) per RIL line were grown in the greenhouse for inoculation. The fungal isolates ZN61 (race IB49), ZN60 (race IC17) carrying AVR-Pita for Pi-ta, and isolates unnamed (race IB54) for Pi-ks and isolates unnamed (race IB45), ZN39 (race IG1), and isolate unnamed (race IH1) for Pi-kh were used for pathogenicity assays. The fungal isolate TM2 (race IE-1k) lacking AVR-Pita, AVR-Piks and AVR-Pikh was used to verify specificity of race reaction in both parents and RILs (Zhou et al., 2007). Pathogenicity assays were performed by procedures described in Valent et al. (1991). Disease infections were rated by a standard described in Valent (1997). Disease evaluations for each RIL line were repeated three times.

Materials and Methods

Results and Discussion

The monogenic and digenic blast resistance population of 235 individual lines originated from the cross RU9101001/Katy (cross no. 19930952) made at the University of Arkansas Division of Agriculture Rice Research and Extension Center (UA RREC), Stuttgart, AR, in 1993. The population was jointly developed by the Agricultural Research Service, U.S. Department of Agriculture Dale Bumpers National Rice Research Center (DB NRRC) and UA RREC at Stuttgart, AR, and officially released on 24 Apr. 2009. RU9101001 was developed by UA RREC from the cross ‘Bonnet73’/CI9837//PI 265116/5/ ‘Vegold’/CI9556//‘Dawn’/3/ ‘Starbonnet’/ ‘Taducan’/4/ ‘L-201’ (cross no. 19860135) made in 1986. RU9101001 originated from a bulk of F5 seed from the 1988 F4 panicle row Pll-076. RU9101001 has the gene Pi-kh and has been maintained in a stable form for many generations at the UA RREC, Stuttgart, AR (Jia et al., 2009). It is an extremely early maturing long-grain experimental line that has seedling cold tolerance. Katy (PI527707), also developed by UA RREC, Stuttgart, AR, is a mid-season, long-grain U.S. cultivar that possesses the major blast resistance genes Pi-ta and Pi-ks (Moldenhauer et al., 1990; Jia et al., 2004b). The F1 plant from the cross was grown in the greenhouse during the winter of 1993–1994 and transplanted to the field for further collection of F2 seed during the summer of 1994. The F2 population of 235 plants was grown during the summer of 2001. This population was advanced through single seed descent to the F8 generation in the greenhouse. In 2005, single F8 panicles rows 1 m in length were grown in the field at Stuttgart, AR. Data were collected from individual rows and plots on 50% heading (days from planting to 50% heading) plant height in centimeters (distance from soil surface to panicle tip before harvesting), length and width with Winseedle scanner and program (Windseedle, Regent Instruments Incorporated,

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Plant Characteristics Evaluation of the population in 2005 and 2006 demonstrated diversity for days to 50% heading (68–158 d), plant height (76–132 cm), seed length (0.82–1.087 cm), seed width (0.20–0.29 cm), seed length/width ratio (3.22–4.72), and 100-seed weight (1.7–2.76 g) as presented in (Table 1). In the present study, the RU9101001/Katy RIL population was created primarily to identify monogenic and digenic RILs that contain one or two major genes that provide resistance to the common U.S. blast races (Jia et al., 2004b, Jia et al., 2009). The codominant DNA markers for the Pi-ta gene and cosegregating simple sequence repeat (SSR) markers for Pi-ks and Pi-kh were first used for R gene identification. The presence of these R genes in identified lines was verified by means of differential races of M. oryzae.

Line Selection and Evaluation DNA markers developed previously were used to identify blast R genes (Jia et al., 2004a, Fjellstrom et al., 2004). The presence of a polymerase chain reaction (PCR) product of 181 bp indicates the presence of the Pi-ta allele. The presence of a PCR product of 182 bp indicates the absence of the Pi-ta allele (Jia et al., 2004a). The presence of a PCR 120-bp product determined by SSR marker RM224 and a 254-bp product determined by SSR RM144 indicates the presence of Pi-ks (Fjellstrom et al., 2004). The presence of a 139-bp product determined by RM224 and a 257-bp product determined by RM144 indicates the presence of Pi-kh. Pi-ta confers resistance to races IB49 and IC17 (Jia et al., 2004b). Pi-kh confers resistance to the races IB45, IG1, and IH1 (Fjellstrom et al., 2004). Race IB54 was recently identified to detect the presence of Pi-ks (Jia and Martin, 2008).


Table 1. Characteristics of recombinant inbred lines of the cross RU9101001/Katy.† Characteristics Heading days (d) Plant height (cm) Seed length (cm) Seed width (cm) Ratio Weight (g) /100 kernels †

Range

Recombinant inbred lines Average Standard deviation

68–158 76–132 0.82–1.09 0.20–0.29 3.22–4.72 1.7–2.76

83.6 99.7 0.97 0.24 4.04 2.17

9.9 10.05 0.039 0.014 0. 26 0.19

RU9101001

Katy

66 76 0.97 0.26 3.73 2.4

103 105 0.93 0.22 4.22 2.09

Phenotypic data were averaged over two noninoculated plots grown in Arkansas in 2005 and 2006. Data on individual lines were deposited at http://www.ars.usda.gov/ Main/docs.htm?docid=8318 (verified 5 Feb. 2010).

Table 2. Ranges of characteristics of mono and digenic lines containing different blast R genes.† Characteristics Heading days (d) Plant height (cm) Seed length (cm) Seed width (cm) Ratio Weight (g) /100 kernels †

Range of traits in mono and digenic lines containing different blast R genes Pi-ta + Pi-ks Pi-ks Pi-ta + Pi-kh Pi-kh 72–158 76–132 0.87–1.05 0.22–0.27 3.91–4.22 1.78–2.69

68–99 80–126 0.90–1.7 0.23–2.8 3.57–4.40 1.74–2.76

73–104 86–131 0.90–1.4 0.20–0.27 3.36–4.7 1.7–2.53

70–100 83–123 0.91–1.1 0.2–0.27 3.53–4.71 1.83–2.54

Mono and digenic lines containing different blast R genes can be accessed at http://www.ars.usda.gov/Main/docs.htm?docid=8318; accessed 5 Feb. 2010.

By means of these criteria, 51 RILs were identified which had only Pi-ks (Table 2). The presence of Pi-ks and the absence of Pi-ta and Pi-kh in these RILs were verified by the use of codominant markers and pathogenicity assays. Fiftyfive RILs were found to have Pi-ta and Pi-ks. The presence of Pi-ta and Pi-ks and the absence of Pi-kh in these RILs were verified by the use of codominant markers and pathogenicity assays with the races IB49, IC17, and IB54 (Table 2, Jia et al., 2004b). Twenty-seven RILs were identified with only the presence of Pi-kh. The presence of Pi-kh and the absence of both Pi-ta and Pi-ks were verified by the use of codominant markers and pathogenicity assays using the races IB45, IG1, and IH1 (Table 2). Finally, 48 RILs were identified with both the Pi-ta and Pi-kh alleles (Table 2). Pi-ta and Pi-kh and the absence of Pi-ks were verified by the use of codominant markers and the pathogenicity assays using IB45, IB49, IC17, IG1, and IH1 of M. oryzae. The remaining 53 RILs were still segregating, a mixture, or recombinants. In the past, possible epistatic interactions among different blast R genes and with R genes and other traits have been underexplored because of the lack of a monogenic and digenic homozygous rice population. Although evidence suggests that over expression of an R gene has a fitness cost in plants (Tian et al., 2003), investigations of epistatic interactions between R genes and other traits have not been studied as extensively as the characterization of structure and evolution of individual R genes in rice (Brown, 2002; Ballini et al., 2008). The original population has been useful in determining the spectra of resistance mediated by the Pi-ta and Pi-k genes in the USA (Jia et al., 2009). Although IE1k was able to defeat all R genes, its occurrence in southern U.S. rice fields is sporadic and insignificant (Zhou et al., 2007). Hence, the monogenic and digenic RIL lines developed in the present study are expected to be extremely useful for individual and combinational effects of Pi-ta, Pi-K,s and Pi-kh and their relationship with yield and other agroJournal of Plant Registrations, Vol. 4, No. 2, May 2010

nomic traits (Table 2). They will also be useful for mapping genes that are responsible for traits that are segregating in the population and as potential breeding lines with southern U.S. agronomic characteristics.

Availability The USDA–ARS Genetic Stocks-Oryza (GSOR) collection at Stuttgart, AR, will distribute these lines. Limited amounts of seed (about 10 seed of each line) may be obtained through the Dale Bumpers National Rice Research Center, GSOR, USDA–ARS, 2890 HWY, 130 East, Stuttgart, AR 72160 ([email protected]). Requests from outside the USA must be accompanied by a seed import permit. Seeds are available for research purposes, including hybridization to develop new cultivars. If these genetic stocks contribute to the advancement of science or development of new cultivars, it is requested that appropriate recognition be given to the source.

Acknowledgments The authors thank the Arkansas Rice Research and Promotion Board for their partial financial support, Michael Lin for population advancement and pathogenicity assays, Tony Beaty for population advancement in fields, Melissa H. Jia for all DNA marker analysis along with Jessica Harris, Kristen Pratt, Eli Eggerman, and Lorie Bernhardt for technical assistance.

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