Rice bacterial blight resistance Screening of mutant and breeding for recombinant inbred line

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1 Rice bacterial blight resistance Screening of mutant and breeding for recombinant inbred line Wang, A. Z. 1, T. H. Tseng 2, L. J. Hsieh 3, and C. S. Wang 1 * 1 Department of Agronomy, National Chung-Hsing University, 2 Biological Technology Division, and 3 Plant Pathology Division, Agricultural Research Institute, Council of Agriculture, Taiwan * wangchansen@nchu.edu.tw Abstract Rice bacterial blight (BB) resistance is caused by Xanthomonas oryzae pv. oryzae (Xoo), and leads over 50% yield loss while severe damage. To explore the BB resistance genes for rice improvement, we chose three typical Xoo isolates (XM1, XF89b and XM42) collected in Taiwan to evaluate the BB resistance in rice lines including: 38 sodium azide induced mutants (SA lines) of TNG67, 26 IRBB lines with known Xa resistance genes, and 22 cultivars (lines). When separately challenge with three Xoo isolates, the IRBB lines showed differential responses to isolates indicated that various resistant genes may involve. Only IRBB5 (xa5) and IRBB7 (Xa7) carrying a single Xa gene were resistant (responsive score 1) or even very resistant (0) to three isolates. Rice IRBB lines carry more than three Xa genes were resistant to three isolates that was consistent with previous reports. However, the well known BB resistance line IRBB21 (Xa21) was susceptible (7) or moderate susceptible (5) to three isolates suggesting that Xoo in Taiwan were more virulent than other resources. The responses of 22 cultivars (lines) to three Xoo isolates were very diverse ranging from resistant (1) to very susceptible (9). Among those, 93-11, IR28 and TK8 were with moderate resistance (3) but TN1, TCS10 and IR24 were very susceptible (9), and TNG67 was susceptible (7) to three Xoo isolates. The responses of 38 SA lines to the three Xoo isolates were highly diverse with resistant (1) to very susceptible (9) indicated that sodium azide induced mutants of TNG67 mutation pool contain a wide diversity in BB resistance. Though, no remarkable segregation was found in the F2 population of TNG67/SA0419 in respond to isolate XM1. Using pedigree method and continuously screening by isolate XM1, recombinant inbred lines (RILs) containing resistant (1) to very susceptible (9) were successfully bred 1

2 from TNG67/SA0419 F2 population. Various BB resistances can be pyramided by crossing to develop a variety with the broad-spectrum resistance. Our system demonstrated that sodium azide mutagenesis can not only generate diverse resistances but also provide an efficient method for breeding disease resistant variety. Keywords: Rice bacterial blight resistance, Mutant, Recombinant inbred line (RILs), Resistance gene, Xa 2

3 Introduction Plant disease management has three approachs: agriculture management, protectant application, and resistant cultivar cultivation. Disease resistant cultivar cultivation is not only the core of plant disease management but also one kind of vigorous plant disease control, and consequently can eliminate the amount of chemical protectant application. Particularly, more consumers demand foods without chemical protectant reveals the importance of disease resistant cultivar cultivation. Therefore, disease resistance breeding is the priority of plant disease control. Rice bacterial blight (BB) caused by Xanthomonas oryzae pv oryzae (Xoo) is one of the most destructive diseases of rice throughout the world, and it can reduce crop yield by up to 50% and grain quality. The most effective approach to struggle with BB is the cultivation of resistant varieties (Khush et al. 1989). So far, 30 resistance genes have been identified (collected from website Oryzabase, )(Kurata and Yamazaki 2006), and some of these have been transmit to modern rice varieties. The exploitation of gene Xa4 resulted in the development of many BB-resistant rice varieties that have played an important role in protecting rice from Xoo (Khush et al. 1989). However, the large-scale and long-term cultivation of varieties carrying Xa4 has resulted in significant shifts in the race frequency of Xoo (Mew et al. 1992). In many areas of Indonesia, India, China and Phillippines, rice varieties with only Xa4 for defense against Xoo have become susceptible to the pathogen. One way to delay such a breakdown of BB resistance is to pyramid multiple resistance genes into rice varieties in order to develop the broad-spectrum resistance. This approach can however be very difficult or impossible by using conventional breeding methods due to epistatic effect of genes, for example Xa21, and unimaginable works of inoculation assay with all known Xoo isolates. However, if molecular markers were available for each resistance gene, the identification of plants with multiple genes would become easy. Several BB resistance genes have been mapped with restriction fragment length polymorphism (RFLP) markers (Ronald et al. 1992; Yoshimura et al. 1995; Zhang et al. 1996). Yoshimura et al. (1995) combined resistance genes in pairs (Xa4/xa5, and xa5/xa10) and showed that plants with two genes can have a higher level of resistance to Xoo than would be expected from the sum of the parental levels. Zhang et al. (1996) identified xa13 via linkage to RFLP markers in three F4 populations in which the xa13 gene would otherwise have been masked by Xa21. Furthermore, 3

4 Huang et al. (1997) use marker-assisted selection (MAS) to pyramid four BB resistance genes, Xa4, xa5, xa13 and Xa21. The effect of resistance genes can be useful integrated and inherit steadily to next generation. The gene pyramid varieties with higher resistant are successfully bred means that more resistance genes pyramided in one individual is equal to more resistance. Finally, Li et al. (2001) discovered that to transmit one high resistance gene and to collocate with 2 to 3 other resistance genes can facilitate the broad-spectrum and durable resistance. In this paper, we report the inoculation assay of our SA mutants and 26 IRBB lines with Xa genes pyramid by using three typical Xoo isolates, XM1, XF89b and XM42, collected in Taiwan to explore the BB resistance genes for rice improvement. Materials and Methods Rice materials A rice mutation pool was derived from cultivar Tainung No. 67 (TNG67), which was developed by sodium azide mutagenesis. After more than 10 generations of self-crossing, selection, and purification by pedigree method, over 3,000 mutants, named SA mutant, on the same genetic background of TNG67 variety were maintained (Wang et al. 2002). 38 SA mutant lines with morphological genetic analysis, 26 IRBB lines with known Xa resistance genes introduced from International rice research institute (IRRI) (Table 1), and 22 cultivars (lines) with diverse genetic background were chosen for this study. All cultivars and lines were planted more than 30 plantlets and field grown according to traditional managements. TNG67/SA0419 RILs were bred from TNG67/SA0419 hybrid based on challenge to isolate XM1 and consequently the resistant and susceptible lines were bred following pedigree procedure. All parents and hybrids were planted in the field simultaneously. The levels of resistance are converted from the median of lesion length investigation after inoculation respectively with isolate XM1, XF89b, XM42. The mean is the average of the levels of 3 isolates; the smaller number the more resistant. Xanthomonas oryzae pv. oryzae (Xoo) inoculums preparation and inoculation Xanthomonas oryzae pv. oryzae (Xoo) isolate XM1, XF89b and XM42 were chosen for this study according to experience of Dr. Chih-Sheng Sheu. The inoculums were grown on Wakimoto solid medium (potato 300 g, sucrose 20 g, Na2HPO4 12H2O 2 g, Ca(NO3)2 4H2O 0.5 g, agar 25 g, H2O 1L) at 25 for 72 hours (Ou 4

5 1985), and then preserved at 4 (Chien and Shieh 1989; Hsieh et al. 2005). Single colony was subcultured in Wakimoto liquid medium with agitation at room temperature for 72 hours, and then suspended with distilled water into cells/ml. The leaf-clipping inoculation method was performed to evaluate rice bacterial blight resistance, which is soaked the clip in Xoo inoculums, cut leaf tip, and then investigated the disease response at 14-day after inoculation (Kauffman et al. 1973; Chien and Shieh 1989; Hsieh et al. 2005). The disease response was measured at 21-day after inoculation by lesion length of three plantlets, and scored as follows: very resistant (0, HR, < 3 cm), resistant (1, R, 3-5 cm), moderate resistant (3, MR, 5-8 cm), moderate susceptible (5, MS, 8-13 cm), susceptible (7, S, cm), and very susceptible (9, HS, >22 cm). The levels of resistance are converted from the median of lesion length investigation after inoculation respectively with isolate XM1, XF89b, XM42. The mean is the average of the levels of 3 isolates; the smaller number the more resistant. Results and Discussions To realize the pathogenicity of three chosen isolates, we used 26 IRBB lines with known Xa genes and resistance to challenge three isolates. The inoculation assay is resulted that IRBB5 (xa5) and IRBB7 (Xa7) were resistant and IRBB11 (Xa1) and IRBB13 (xa13) wre susceptible to these three Xoo isolates in Taiwan. However, the well known BB resistance line IRBB21 (Xa21) was susceptible. The more than 3 genes pyramid lines were very resistant, which demonstrates that 3 genes pyramid lines are very resistant. This result is as same as Huang et al. (1997) and Li et al. (2001). Each Xa gene has an individual response to different isolates. Finally, Xoo isolates in Taiwan have high virulence of pathogenicity than other resources (Fig. 1). To understand the inoculation assay ability and representative of these three isolates, we used 22 varieties with diverse genetic background to challenge theses three isolates. IR24 and TCS10 were very susceptible to three isolates and Nipponbare and TP309 were however very susceptible to isolates XF89b. This result is consistent with previous reports. TK8, and IR28 were resistant to three isolates according average resistance. TNG67, the wild type of SA mutant, was susceptible to three isolates. Rice varieties had a diverse resistance to three isolates means that the average resistance derived from different virulent isolates has steady 5

6 resistant result. The pathogenicity of three examined isolates is sequentially as follow: XF89b > XM1 > XM42. (Fig. 2). To demonstrate that the mutant can be implemented the disease resistance breeding, we explored the resistance of 38 SA mutants derived from susceptible TNG67. We found that 7 SA mutants were resistant to these three isolates. According to Xa gene pyramid lines, those 7 SA mutants maybe have more than 3 resistance genes. SA0422 was very susceptible to these three isolates. SA mutants had diverse resistance, from resistant to very susceptible, to these three isolates. It means that TNG67 mutant pool has a widespread variation. After compared the breeding cost of IRBB lines and SA mutants, self-cross more than 10 generations SA mutant are more efficient. We know that mutagenesis can provide very diverse resistance genes and is one high efficient disease resistance breeding (Fig. 3). To proof that the resistance obtained from mutants can inherit and recombine, we crossed TNG67 and SA0419 and selected progenies by isolate XM1 and pedigree method. The F2 population of TNG67/SA0419 had no remarkable segregation to isolate XM1 (Fig.4). Following continuously screening by isolate XM1, recombinant inbred lines (RILs) containing resistant (1) to very susceptible (9) were successfully bred from TNG67/SA0419 F2 population. It indicates that BB resistances derived from mutant can be inherited and recombined significantly. Unfortunately, RILs 23-1 and 23-2 even with resistant (1) was not dominant, according to TNG67/RILs F1 resistance assay result (Fig. 5). Nevertheless, various BB resistances maybe pyramided by crossing these RILs to obtain a broad-spectrum resistance. Our system demonstrated that sodium azide mutagenesis not only generates diverse resistant genes but also provides an efficient disease resistance breeding method. 6

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