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Improvement of bacterial blight resistance of hybrid rice in China using the Xa23 gene derived from wild rice (Oryza rufipogon)
Crop Protection 30 (2011) 637e644
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Improvement of bacterial blight resistance of hybrid rice in China using the Xa23 gene derived from wild rice (Oryza rufipogon)q Yong-Li Zhou a, Veronica NE Uzokwe a, Cong-He Zhang b, Li-Rui Cheng a, Lei Wang a, Kai Chen a, Xiao-Qing Gao a, Yong Sun a, Jin-Jie Chen b, Ling-Hua Zhu a, Qi Zhang a, Jauhar Ali c, Jian-Long Xu a, *, Zhi-Kang Li a, c a
Institute of Crop Sciences/ National Key Facility for Crop Gene Resources and Genetic Improvement, Chinese Academy of Agricultural Sciences, 12 South Zhong-Guan-Cun Street, Beijing 100081, PR China Win-All Hi-Tech Seed Company Limited, 3 Tian-Zhi Road, Hefei 230012, PR China c International Rice Research Institute, DAPO Box 7777, Metro Manila, Philippines b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 28 July 2010 Received in revised form 30 November 2010 Accepted 4 December 2010
A novel bacterial blight (BB) resistance gene, Xa23, identified from Oryza rufipogon was introgressed into three popular restorer lines (Minghui63, YR293 and Y1671) for wild abortive cytoplasmic male sterility by marker-assisted backcross breeding approach in combination with artificial inoculation and stringent phenotypic selections. The three derived BB resistant restorer lines (Minghui63eXa23, YR293eXa23 and Y1671eXa23) and their hybrid combinations with Zhenshan97A (Shanyou63eXa23), NongfengA (Fengyou293eXa23) and Zhong9A (Zhongyou1671eXa23) demonstrated similar BB resistance spectrum as the donor parent, CBB23 (B). The newly developed BB resistant restorers and their derived hybrids were identical to their respective original versions for agronomic traits especially under disease free condition. However, under severe disease condition, the three BB resistant restorer lines exhibited significantly higher grain weight and spikelet fertility as compared to the respective original restorer lines thus further resulting in BB resistant hybrids with significantly higher grain yields than their respective popular original hybrids. The results indicated that the Xa23 gene could completely express its dominant broad spectrum resistance in different backgrounds of both restorer and male sterile lines across different growth stages, suggesting its immense breeding value in BB resistance improvement for hybrid rice. Moreover, a reasonable utilization and deployment of Xa23 gene for efficient control of BB disease in hybrid rice production was recommended. Ó 2010 Elsevier Ltd. All rights reserved.
Keywords: Bacterial blight Xa23 gene Hybrid rice Marker-assisted backcross Resistance improvement
1. Introduction Exploitation of hybrid vigor in rice is the most viable option to enhance the rice production levels across Asia. Hybrid rice area is steadily expanding across Asia with potential yield advantages ranging from 15 to 20% over the high yielding semi-dwarf varieties. Hybrid rice is commercially grown in many countries across the globe particularly in China, where it accounts for half of the rice acreage and contributes 60% of the total rice yield of the country (Cheng et al., 2004). Despite the impressive yield gain through heterosis breeding the problem of hybrid combination being
q Yong-Li Zhou, Veronica NE Uzokwe and Cong-He Zhang contributed equally to this work. * Corresponding author. Tel.: þ86 10 82105854; fax: þ86 10 82108559. E-mail addresses: [email protected], [email protected] (J.-L. Xu). 0261-2194/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.cropro.2010.12.002
susceptible to many diseases due to narrow genetic base still persists. Amongst the major prevalent diseases, bacterial blight (BB) disease happens to be one of the major barriers to high and sustainable hybrid rice production (Virmani, 1996). BB disease of rice (Oryza sativa), caused by Xanthomonas oryzae pv. oryzae (Xoo), is one of the most widely distributed and devastating rice diseases for both inbred and hybrid rice in Asia. BB damage on this crop was reported to range from 20 to 30% and could sometimes get as high as 50% (Mew, 1987; Ou, 1985). The BB disease became prominent in the 1960s, when new high yielding varieties were first developed and introduced (Mew, 1987). Varietal resistance is the main strategy for controlling this disease. As many as 31 resistance genes (22 dominant and 9 recessive) to BB have been identified and registered (Chu et al., 2006; NiÑo-Liu et al., 2006; Wang et al., 2009). Among them, Xa4 has been widely used in developing almost all the commercial hybrids and conventional cultivars of indica rice in the main rice growing
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regions of China (Zhang, 1991). This narrow genetic basis for resistance to BB in Chinese rice cultivars has imposed strong selection on the pathogen population, resulting in a dramatic increase in frequency of race V that is virulent to both Xa4 and Xa3 during the past decade in China (Zhang et al., 1996). Currently most of the parental lines of the popular hybrid rice in China are susceptible to most Chinese and Philippines races (Xia et al., 2009). Another widely used Xa21 gene in rice breeding programs was identified from the wild rice O. longistaminata (Khush et al., 1990; Song et al., 1995). This gene was recently broken down by new virulent strains in Southern and Yangtze River Valley in China (Zeng et al., 2002; Zhang, 2009). Amongst all the known resistance genes to BB, Xa23, a single completely dominant resistance gene effective at all growth stages, identified from wild rice species of Oryza rufipogon emerged as the most promising one (Zhang et al., 1998, 2001). The Xa23 gene was found to be highly resistant to all the 20 strains of BB consisting of 10 Philippine races (P1eP10), 7 Chinese pathotypes (C1eC7) and 3 Japanese races (T1eT3) at the maximum tillering stage. It was indicated that Xa23 gene possessed a broad resistance spectrum among all known R genes (Zhang et al., 2001). In this study, we evaluated the practical utility of the Xa23 gene in improvement of BB resistance for hybrid rice through introgression of the gene into three popular restorer lines to develop new BB resistant restorer lines and their respective hybrid rice combinations. 2. Materials and methods 2.1. Parental materials In this study, CBB23(B) carrying Xa23 gene, derived from BC4F5 progeny of the cross between an O. rufipogon accession (RBB16, the Xa23 donor) and a recipient indica BB susceptible variety IR24 (Zhang, 2005), was used as the donor of resistance to BB. Three popular restorer lines i.e. Minghui63, YR293 and Y1671 for wild abortive cytoplasmic male sterility (WAeCMS), also widely used for hybrid rice seed production in China but highly susceptible to BB were used as recurrent parents. These three restorers were derived from the following respective crosses IR30/Gui630, 9311/Minghui63 and YR293/02428 through pedigree breeding approach. The seeds of the three restorer lines were provided by the Win-All Hi-Tech Seed Company Limited, Anhui province, China. The IR24 variety was used as a susceptible control for BB inoculation. 2.2. Development of BB resistant restorer lines with Xa23 gene Marker-assisted backcrossing procedure was applied to transfer Xa23 gene into elite restorer parental lines. The three restorer lines were used as female to cross with CBB23(B) to produce three F1s and then backcrossed with the F1s to get three BC1F1 populations. A mini-scale DNA isolation for PCR analysis was carried out on threeeleaf old seedlings in the seedbed for the parents and BC1F1 plants following the procedure of Zheng et al. (1995). Foreground selection against Xa23 gene was used to select plants carrying Xa23 gene prior to transplanting through marker-assisted selection (MAS) using a closely linked SSR marker (RM206) of 1.9 cm apart from the Xa23 locus (Pan et al., 2003). The 25 days old seedlings with Xa23 heterozygous gene selected from BC1F1 were transplanted in the screenhouse. At early tillering (3 weeks after transplanting) and booting (7 weeks after transplanting) stages, three to six uppermost leaves of each plant were inoculated with a highly virulent Chinese Xoo strain, GD1358 using the leaf-clipping method (Kauffman et al., 1973). Resistant BC1F1 plants that were determined by MAS and inoculation were further backcrossed to the respective recurrent parents to produce their
BC2F1 populations. Consecutive backcrosses were carried out in the same way to produce BC4F1 populations. Resistant plants carrying homozygous Xa23 gene and identical phenotype as that of the recurrent parents were identified by MAS and inoculation in BC4F2. These phenotypically identical BB resistant plants to their respective restorer lines were then crossed with the three respective WAeCMS lines (Zhenshan97A, NongfengA and Zhong9A), to test their restoration and specific combining ability. All the BC4F3 lines were confirmed for the presence of Xa23 gene and studied for their phenotypic similarities to their respective original restorer lines. The F1 progenies of the test-crosses involving the selected BB restorer lines in BC4F2 with their respective CMS lines were evaluated for their spikelet fertility and level of heterosis. Based on the performance of BB resistance and morphological characters of BC4F3 lines and the level of heterosis of their respective test-crosses, only one line that was phenotypically identical to the recipients with homozygous Xa23 gene was finally selected from the progeny of each recipient, and it was designated as Minghui63eXa23, YR293eXa23 and Y1671eXa23 for further evaluation. 2.3. Evaluation of BB resistance and agronomic traits The original restorer lines Minghui63, YR293 and Y1671 and their derived BB resistant restorer lines Minghui63eXa23, YR293eXa23 and Y1671eXa23 were crossed with three WAeCMS lines, Zhenshan97A, NongfengA and Zhong9A to produce hybrid F1 for evaluation of BB resistance and agronomic traits. BB resistance evaluation of these materials were carried out by sowing them on 20th of April, 2008 and 30 days old seedlings were transplanted into a nine-row plot of six plants per row with a plant to row spacing of 17 20 cm and three replications in the screenhouse of the Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing, China. Three plants were artificially inoculated by partial clipping of 3e6 uppermost leaves separately (Kauffman et al., 1973) for each of the 7 different Chinese Xoo races (C1eC7) (Fang et al., 1990) and 10 Philippine Xoo races (P1eP10) at the early tillering (3 weeks after transplanting) and booting (7 weeks after transplanting) stages. Lesion length (LL) was measured on all partially clipping inoculated leaves after 2e3 weeks of inoculation. BB field evaluation and agro-morphological characterization of the newly developed restorer lines (Minghui63eXa23, YR293eXa23 and Y1671eXa23) and their derived hybrid combinations i.e. Shanyou63eXa23 (Zhenshan97/Minghui63e Xa23); Fengyou293eXa23 (NongfengA/YR293eXa23) and Zhongyou1671eXa23 (Zhong9A/ Y1671eXa23), were compared to their original restorer lines (Minghui63, YR293 and Y1671) and their respective hybrid combinations i.e. Shanyou63 (Zhenshan97/Minghui63); Fengyou293 (NongfengA/ YR293) and Zhongyou1671 (Zhong9A/Y1671) by conducting field trials both under disease and disease free environments in a randomized complete block design (RCBD) with 3 replications in Hainan breeding station during 2009 winter season. Testing of all the materials under severe diseased condition was carried out by planting each line in three rows plot with twelve plants per row and a spacing of 17 20 cm in a RCBD with three replications. The middle row in each plot was inoculated with Xoo race C5. However, for testing under disease free condition, the same set of materials was planted in the field without artificial BB inoculation and any change in plot size and layout. Measurements were taken on ten plants from the middle row in each plot for different agro-morphological traits, such as plant height (PH), heading date (HD), panicle length (PL), panicle number per plant (PN), spikelet number per panicle (SNP), spikelet fertility (SF), 1000-grain weight (GWT) and grain yield per plant (GY) according to the standard evaluation system of rice (IRRI, 1996).
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3. Results 3.1. BB resistance levels of parental lines and F1 hybrids The Xa23 donor, CBB23(B) showed highly resistant reactions at both early tillering and booting stages to all 7 Chinese (C1eC7) and 10 Philippine (P1eP10) Xoo races, while the three sterile lines, Zhenshan97A, NongfengA and Zhong9A showed complete susceptibility (Table 1, Fig. 1). The three restorer lines Minghui63, YR293 and Y1671 were found to be susceptible to most of the 17 races, especially to the Chinese race 5 (GD1358) and the Philippine race 6 (PXO99), that shows strongest pathogenicity in China and Philippines at both early tillering and booting stages with similar LL to that of the susceptible variety IR24 (Table 2, Fig. 1). The F1 plants derived from the crosses between the three restorer lines and the CBB23(B) were each divided into two groups and inoculated with Chinese race 5 (GD1358) and Philippine race 6 (PXO99). All the F1 plants exhibited resistance to Xoo race PXO99 at early tillering stage with the average LL ranging from 1.1 to 1.5 cm but were highly resistant to GD1358 with the LL ranging from 0.2 to 0.9 cm, respectively (Fig. 2). At booting stage, all F1 plants showed enhanced resistance to PXO99 and GD1358 compared with those at early tillering stage, with LL of 0.1e0.6 cm and 0.8e1.4 cm, respectively. The Xa23 gene showed the same level of resistance in different backgrounds and developmental stages by comparable LL to that of CBB23(B) thereby clearly indicating that the Xa23 could completely express its dominant resistance in different genetic backgrounds and varied growth stages.
Fig. 1. Leaves showing the effects of Xanthomonas oyzae pv. oryzae Philippines race 6 (2e3 weeks post-inoculation) on rice restorer and sterile lines. A: Effects at the early tillering stage (3 weeks after transplanting), B: Effects at the booting stage (7 weeks after transplanting). Leaves 1e7 represent the effects on CBB23(B), Minghui63, YR293, Y1671, Zhenshan97A, NongfengA and Zhong9A.
Table 1 Resistant reactions of CBB23(B) and the three parental cytoplasmic male sterile (CMS) lines to the 7 Chinese and 10 Philippine races of Xanthomonas oryzae pv. oryzae (Xoo) at early tillering (TS) and booting (BS) stages. Growth stage
TS
BS
a
Xoo
Resistant donor and the 3 parental CMS lines
Racea
Strain
IR24 (CK)
CBB23(B)
Zhenshan97A
NongfengA
Zhong9A
C1 C2 C3 C4 C5 C6 C7 P1 P2 P3 P4 P5 P6 P7 P8 P9 P10 C1 C2 C3 C4 C5 C6 C7 P1 P2 P3 P4 P5 P6 P7 P8 P9 P10
JS97-2 KS6-6 JS158-2 Z173 GD1358 OS198 JS49-6 PXO61 PXO86 PXO340 PXO71 PXO112 PXO99 PXO145 PXO280 PXO339 PXO341 JS97-2 KS6-6 JS158-2 Z173 GD1358 OS198 JS49-6 PXO61 PXO86 PXO340 PXO71 PXO112 PXO99 PXO145 PXO280 PXO339 PXO341
9.5 2.3 10.3 1.8 13.5 1.1 10.3 1.7 14.8 2.6 9.9 1.0 9.3 1.3 11.5 2.1 12.8 2.9 12.5 2.4 10.4 1.0 11.5 2.0 14.2 1.7 13.5 0.9 8.8 1.7 10.2 0.5 13.2 1.8 11.9 2.3 12.4 0.7 15.3 1.5 13.6 1.3 16.2 1.1 15.1 0.8 12.8 1.6 16.1 2.3 15.2 1.4 13.9 0.5 13.6 1.2 12.7 2.0 18.2 3.1 14.1 0.2 11.9 1.3 13.2 0.7 12.8 1.2
0.3 0.2 0.5 0.1 0.3 0.1 0.4 0.2 0.4 0.1 0.3 0.1 0.3 0.0 0.5 0.1 0.6 0.2 0.3 0.0 0.7 0.2 0.5 0.0 0.5 0.1 0.2 0.1 0.3 0.0 0.1 0.1 0.4 0.5 0.2 0.1 0.5 0.0 0.2 0.1 0.4 0.3 0.3 0.1 0.5 0.3 0.2 0.1 0.3 0.1 0.4 0.2 0.5 0.3 0.6 0.2 0.6 0.1 0.4 0.1 0.3 0.2 0.5 0.2 0.2 0.1 0.3 0.1
7.5 2.8 8.8 1.3 7.2 1.6 10.0 3.4 12.8 2.1 12.4 1.7 11.2 2.3 9.5 1.2 12.7 1.5 9.8 2.9 11.7 3.1 10.2 1.3 14.9 2.3 9.7 0.3 7.9 0.9 12.6 1.4 12.0 2.7 8.8 0.3 10.9 0.7 11.8 1.0 14.1 1.0 16.5 1.3 8.9 0.6 9.7 0.7 7.8 0.6 7.4 0.5 6.2 1.1 4.7 0.7 10.9 0.5 20.8 1.4 13.8 0.5 14.0 1.1 13.2 0.6 13.7 0.6
7.5 0.5 7.7 0.8 13.0 2.7 15.3 2.3 16.1 1.8 9.3 2.8 10.5 1.9 6.2 1.3 13.3 1.3 10.7 1.8 8.5 1.5 7.0 0.5 18.7 1.1 6.8 1.0 12.3 1.9 16.5 3.8 11.5 4.0 7.7 0.7 6.4 1.2 12.3 0.8 13.2 1.7 17.9 1.3 7.2 0.3 6.9 0.7 8.6 1.2 10.2 1.9 13.0 0.7 12. 2 0.8 8.2 1.3 18.5 2.1 8.3 1.5 10.6 1. 6 6.6 1.5 11.7 3.3
8.7 1.6 10.2 1.0 8.2 1.1 12.9 1.9 15.1 1.7 9.7 2.1 7.7 1.0 6.6 1.3 10.8 2.9 12.8 2.0 14.7 1.0 7.8 1.3 16.2 1.8 9.8 1.3 13.5 1.3 12.8 1.3 18.5 2.3 7.4 1.2 8.8 0.6 9.8 2.5 7.8 1.2 14.6 1.5 8.9 0.8 9.8 0.8 7.8 1.9 10.8 1.7 10.6 2.0 11.6 1.7 6.5 1.3 17.6 1.7 7.0 1.4 9.0 2.3 9.5 0.9 10.8 3.5
C1eC7 and P1eP10 represent 7 Chinese and 10 Philippines races of Xoo, respectively.
Table 2 Resistance levels on the basis of lesion length (LL) of restorer lines carrying Xa23 and their original versions against the 7 Chinese and 10 Philippines races of Xanthomonas oryzae pv. oryzae at early tillering (TS) and booting (BS) stages. Growth stage
Race
Minghui63
Minghui63eXa23
YR293
YR293eXa23
Y1671
Y1671eXa23
TS
C1 C2 C3 C4 C5 C6 C7 P1 P2 P3 P4 P5 P6 P7 P8 P9 P10 C1 C2 C3 C4 C5 C6 C7 P1 P2 P3 P4 P5 P6 P7 P8 P9 P10
2.5 0.3 15.7 4.0 5.5 0.9 13.5 2.1 12.3 1.9 6.7 0.8 12.7 1.1 14.8 1.4 13.3 4.3 11.7 2.8 12.0 1.3 10.0 2.0 14.8 2.0 4.3 1.9 12.0 2.7 11.8 1.9 12.1 1.5 1.2 0.1 1.5 0.2 2.9 0.7 13.6 2.3 13.2 0.3 6.8 1.3 10.4 1.3 7.5 0.6 8.4 1.7 4.3 0.8 4.6 0.6 4.9 0.9 17.2 1.4 2.3 0.4 3.3 0.1 2.4 0.5 3.50 0.2
0.2 0.1 0.2 0.0 0.3 0.0 0.8 0.1 0.3 0.2 0.4 0.3 0.3 0.0 0.4 0.2 0.7 0.2 0.3 0.2 0.3 0.2 0.2 0.1 0.4 0.2 0.3 0.1 0.2 0.1 0.1 0.0 0.2 0.1 0.2 0.1 0.2 0.0 0.1 0.0 0.7 0.1 0.2 0.0 0.5 0.2 0.1 0.0 0.3 0.2 0.6 0.4 0.8 0.3 1.0 0.4 0.3 0.2 0.4 0.1 0.3 0.1 0.3 0.2 0.1 0.0 0.2 0.1
10.7 1.5 9.2 0.8 8.0 1.5 10.7 3.5 10.7 1.8 12.2 1.6 9.5 1.3 9.8 1.9 6.5 1.3 7.4 1.2 12.8 1.2 3.1 0.4 12.5 2.0 13.7 2.5 12.7 3.1 9.5 1.3 8.2 1.6 1.3 0.1 1.2 0.2 11.9 4.9 2.2 0.4 15.1 1.9 8.5 4.2 12.3 2.3 4.5 1.7 1.6 0.31 1.8 0.75 15.2 4.3 1.2 0.0 16.5 2.9 5.7 2.3 5.2 0.5 6.1 2.5 1.4 0.1
0.3 0.2 0.3 0.2 0.1 0.1 0.2 0.1 0.2 0.1 0.1 0.0 0.2 0.0 0.3 0.2 0.2 0.0 0.3 0.2 0.2 0.0 0.3 0.1 0.8 0.2 0.2 0.1 0.2 0.1 0.2 0.2 0.2 0.1 0.2 0.0 0.1 0.1 0.1 0.0 0.2 0.1 0.1 0.0 0.1 0.1 0.1 0.0 0.3 0.1 0.2 0.0 0.2 0.0 0.6 0.3 0.1 0.1 0.4 0.1 0.2 0.1 0.1 0.0 0.1 0.0 0.1 0.0
2.3 0.5 5.5 1.3 9.4 2.4 10.1 3.0 10.3 1.5 11.7 2.5 9.5 2.0 11.7 2.0 9.1 1.5 12.7 1.5 9.3 1.5 9.8 2.5 12.3 2.9 6.3 1.2 9.2 1.6 8.0 2.1 9.5 4.1 1.7 0.3 3.5 0.5 2.9 0.5 6.0 1.5 14.7 2.6 2.7 1.4 6.3 1.6 5.4 2.4 6.9 1.6 6.7 1.0 5.6 0.8 3.5 0.8 19.5 3.1 5.3 0.9 8.5 0.5 9.3 2.6 8.14 1.7
0.1 0.0 0.1 0.0 0.1 0.1 0.4 0.2 0.1 0.0 0.4 0.6 0.2 0.1 0.1 0.1 0.2 0.0 0.3 0.2 0.2 0.0 0.1 0.0 0.3 0.1 0.1 0.0 0.1 0.0 0.1 0.0 0.1 0.0 0.1 0.0 0.2 0.1 0.1 0.0 0.2 0.1 0.3 0.1 0.1 0.0 0.1 0.0 0.3 0.2 0.3 0.1 1.0 0.3 0.2 0.1 0.2 0.1 0.5 0.1 0.1 0.0 0.1 0.0 0.1 0.1 0.1 0.0
BS
Fig. 2. Resistant F1 plants of the crosses between the restorer lines and CBB23(B) to Xanthomonas oyzae pv. oryzae Chinese race 5 and Philippine race 6. A: The lesion length of F1 plants at the early tillering stage (3 weeks after transplanting); B: The lesion length of F1 plants at the booting stage (7 weeks after transplanting); C: The lesion length of F1 plants infected with the Philippine race 6 at the booting stage.
Y.-L. Zhou et al. / Crop Protection 30 (2011) 637e644 Table 3 Resistance levels of F1 rice hybrids with and without Xa23 gene against the virulent Chinese race 5 (C5) and the Philippine race 6 (P6) of Xanthomonas oryzae pv. oryzae both at early tillering and booting stages. F1 combination
Early tillering stage
Booting stage
P6
P6
NongfengA/Minghui63 Zhenshan97A/Minghui63 (Shanyou63) Zhong9A/Minghui63 NongfengA/Minghui63eXa23 Zhenshan97A/Minghui63eXa23 (Shanyou63eXa23) Zhong9A/Minghui63eXa23
22.0 5.0 21.3 3.6 18.9 4.3 20.8 3.5 19.6 3.2 16.3 2.9
7.9 1.0 8.2 1.1
21.3 3.7 20.0 4.3 19.7 3.8 1.2 0.5 1.0 0.7 1.4 0.3 1.5 0.6 1.2 0.4 1.4 0.5
7.7 1.4 0.5 0.2 0.6 0.4
1.1 0.1
C5
1.0 0.4
1.3 0.4
C5
0.4 0.3
NongfengA/YR293 (Fengyou293) 21.9 4.9 15.9 2.8 20.4 5.7 11.3 2.5 Zhenshan97A/YR293 22.5 3.8 16.0 4.2 20.0 5.0 10.8 2.3 Zhong9A/YR293 22.1 3.1 16.8 2.9 17.3 2.4 10.6 1.5 NongfengA/YR293eXa23 1.5 0.3 0.4 0.3 1.2 0.4 0.5 0.2 (Fengyou293eXa23) Zhenshan97A/YR293eXa23 1.4 0.2 0.6 0.3 1.4 0.2 0.6 0.2 Zhong9A/YR293eXa23 1.4 0.3 0.8 0.5 1.3 0.3 0.4 0.2 NongfengA/Y1671 22.0 3.5 21.5 3.8 15.3 2.5 18.7 4.4 Zhenshan97A/Y1671 20.7 2.5 20.8 2.9 18.6 3.5 17.8 3.8 Zhong9A/Y1671 (Zhongyou1671) 22.2 3.9 20.9 2.9 19.6 4.7 19.0 3.5 NongfengA/Y1671eXa23 1.0 0.5 0.7 0.6 1.4 0.4 0.7 0.4 Zhenshan97A/Y1671eXa23 1.2 0.2 0.6 0.5 1.4 0.4 0.5 0.5 Zhong9A/Y1671eXa23 1.0 0.6 0.8 0.6 0.9 0.4 0.5 0.4 (Zhongyou1671eXa23)
3.2. Evaluation of BB resistance of the newly-bred restorer lines and their hybrids The BB resistance of the newly developed restorer lines (Minghui63eXa23, YR293eXa23 and Y1671eXa23) is presented in Table 2 by comparing the average LL with their original versions caused by the 10 Philippine and 7 Chinese Xoo races at the early tillering and booting stages. All improved restorer lines carrying Xa23 showed a high level of resistance to all Xoo races including the highly virulent Chinese race 5, GD1358 and the Philippine race 6, PXO99 with LL < 1.0 cm even at the early tillering stage, thus indicating that Xa23 gene possessed a broad spectrum of BB
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resistance to over 17 races tested. In addition, no significant differences existed for BB resistance for Xa23 gene in different restorer line backgrounds. The hybrids derived from the newly developed BB resistant restorer lines were highly significant for their resistance to PXO99 and GD1358, with the average LL ranging from 1.0 to 1.5 cm and 0.4e1.2 cm at early tillering stage; and from 0.9 to 1.4 cm and 0.5e0.7 cm at booting stage, respectively, as compared to their original hybrid combinations with the three different sterile lines Zhenzhan97A, NongfengA and Zhong9A, respectively (Table 3 and Fig. 3). No significant differences were observed for resistance to PXO99 and GD1358 for the hybrids derived from each BB resistant restorer line and different sterile lines in both the two stages. This further indicated that the expression of BB resistance of Xa23 gene was independent of genetic background of the male sterile lines. 3.3. Agronomic characterization and grain yield performance of the newly developed restorer lines and their hybrid combinations The three newly developed BB resistant restorers and their corresponding original versions consisting of Minghui63eXa23 and Minghui63; YR293eXa23 and YR293; Y1671eXa23 and Y1671 were found to be identical for all the agronomic traits studied under disease free field conditions. Likewise, we found the same for their hybrid combinations i.e. Shanyou63eXa23 and Shanyou63; Fengyou293eXa23 and Fengyou293; Zhongyou1671eXa23 and Zhongyou1671 (Table 4). However, Minghui63eXa23 showed significantly higher 1000-grain weight and spikelet fertility than Minghui63 under severe diseased conditions. Grain yield of Shanyou63eXa23 was significantly higher than that of Shanyou63 on account of pronounced differences in 1000-grain weight and spikelet fertility (Table 4). A similar pattern was observed for the other two BB resistant restorers and their derived hybrids. The YR293eXa23 and Y1671eXa23 lines had significantly higher 1000grain weight, spikelet fertility and grain yield than YR293 and Y1671. Fengyou293eXa23 and Zhongyou1671eXa23 had significantly higher grain yield than Fengyou293 and Zhongyou1671 (Table 4). Grain yield and its related traits such as spikelet fertility
Fig. 3. Restorer lines and F1 combinations resistance to Xanthomonas oryzae pv. oryzae Chinese race 5 at the booting stage (7 weeks after transplanting).
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Table 4 Agronomic performance of restorer lines with and without Xa23 gene and their respective commercial hybrids tested under both severe disease (artificial inoculation) and disease free field conditions during 2009 winter season in Hainan. Restorer lines/Hybrids PH (cm)
HD (d)
PL PN SNP (cm)
SF (%) GWT GY (g) (g/plant)
Without inoculation Minghui63 Minghui63eXa23 Shanyou63 Shanyou63eXa23 LSD(0.05)a LSD(0.01)b YR293 YR293eXa23 Fengyou293 Fengyou293eXa23 LSD(0.05)a LSD(0.01)b Y1671 Y1671eXa23 Zhongyou1671 Zhongyou1671eXa23 LSD(0.05)a LSD(0.01)b
105.5 104.7 96.3 95.5 0.9 1.7 110.7 109.9 100.7 101.5 2 3.7 109.3 109.6 101.4 102.1 2.3 4.4
21.5 20.1 23.2 22.5 1.6 3.1 22.3 21.8 24.4 23.2 1.4 2.5 23.1 23.6 24.1 24.8 0.9 1.6
7.6 7.9 8.3 8.5 0.5 1.1 7.3 7.5 8.1 8.3 0.4 0.7 6.2 6.5 7.8 8.7 1.1 1.9
114.5 112.3 131.4 129.5 5.1 9.4 122.3 121.5 140.2 139.2 3.1 5.8 124.2 125.8 135.4 136.7 4.1 7.8
85.6 85.7 82.7 83.3 2.3 4.3 85.6 86.9 84.7 85.1 2.1 3.5 84.6 86.5 83.6 85.5 2.5 4.6
27.5 27.1 27.6 28.1 1.1 1.8 29.2 29.1 28.8 28.5 1.5 2.8 28.8 27.9 28.1 28.1 1.2 2.3
17.3 16.9 22.2 21.5 1.7 3.2 17.1 17.5 23.1 24.7 1.9 3.7 16.1 17.1 22.8 24.9 2.4 3.1
at the booting 95.1 105.1 96.3 104.5 102.7 97.5 100.6 96.8 2.5 0.9 4.8 1.7 98.9 109.9 98.3 110.2 105.1 101.8 103.8 102.3 2.1 2.2 3.8 4 103.2 109.5 104.5 110.3 109.1 100.8 108.6 102.3 1.9 3 3.5 5.8
stage 21.3 21.2 23.1 22.2 1.6 3.1 22.1 22.3 24.2 23.1 1.3 2.4 22.9 23.3 24.2 24.7 1.1 2
7.6 7.8 8.4 8.6 0.5 1.1 7.7 7.6 8 8.4 0.6 1.1 6.4 6.8 7.7 7.9 1.3 2.4
113.5 112.1 127.5 128.1 5.1 9.4 120.6 122.1 138.6 140.1 3.3 6.1 123.3 125.2 131.3 133.9 3.3 6
80.9 84.5 75.5 83.6 2.3 4.3 77.2 85.5 75.7 83.1 3.5 6.7 77.6 82.3 77.7 83.9 3.5 6.7
25.6 27.3 25.2 27.9 1.3 2.4 26.1 28.9 25.7 28.1 1.3 2.1 25.7 27.2 26.3 28.3 1.3 2.4
13.5 15.1 17.5 21.3 2.8 5.2 14.9 17.6 17.9 23.5 2.6 5 14.5 17.6 18.1 22.5 2.2 4
Inoculated by Xoo. C5 Minghui63 Minghui63eXa23 Shanyou63 Shanyou63eXa23 LSD(0.05)a LSD(0.01)b YR293 YR293eXa23 Fengyou293 Fengyou293eXa23 LSD(0.05)a LSD(0.01)b Y1671 Y1671eXa23 Zhongyou1671 Zhongyou1671eXa23 LSD(0.05)a LSD(0.01)b
95.5 96.1 101.4 100.3 2.5 4.8 99.8 98.7 105.5 104.1 1.9 3.4 103.5 104.2 110.5 109.2 1.4 2
PHeplant height, HDeheading date, PLepanicle length, PNepanicle number per plant, SNPespikelet number per panicle, SFespikelet fertility, GWTe1000-grain weight, GYegrain yield per plant. a Least significant differences at 0.05 probability level. b Least significant differences at and 0.01 probability level.
and 1000-grain weight were higher for Shanyou63eXa23, Fengyou293eXa23 and Zhongyou1671eXa23 than Shanyou63, Fengyou293 and Zhongyou1671, respectively. 4. Discussion Prevalence of BB disease in China is jeopardizing the expansion of the popular hybrids as a result of their susceptibility. The three different restorer lines i.e. Minghui63, YR293 and Y1671 have been widely utilized in hybrid rice production in China. Incorporation of a broad spectrum resistance gene into these restorer lines will greatly improve BB resistance of hybrid rice in China. Careful analysis of the literature showed CBB23(B) with Xa23 gene to be an effective donor against all races collected from China, Philippines and Japan (Zhang, 2005). In this study we incorporated a broad spectrum BB resistance Xa23 gene into the three commercially popular restorer lines using MAS-based backcrossing and stringent phenotypic selections within a short period. Minghui63eXa23, YR293eXa23, Y1671eXa23 and their hybrid combinations, Shanyou63eXa23, Fengyou293eXa23 and Zhongyou1671eXa23 exhibited a significant yield advantage over their original counterparts with the same level and spectrum of resistance as that of
the resistant donor, CBB23(B). This study clearly indicated that Xa23 gene is valuable for the improvement of BB resistance in hybrid rice production. Improvement of restorer lines for BB resistance through MAS-based backcross schemes using Xa23 gene with broad spectrum resistance raised certain key issues in rice breeding for increasing production. Firstly, the BB resistance improvement of parental lines of hybrids is much different from that of conventional varieties. In the backcross progenies of restorer parental lines, selections were performed not only for similarity to the recurrent parents, but also for their fertility restoring gene(s) and specific combining ability to the CMS lines. The expected frequencies for individuals homozygous genotype of the recurrent parent on a single locus basis is 0.5, 0.75, 0.875, and 0.9375 in BC1F1, BC2F1, BC3F1 and BC4F1, respectively. All individuals in each backcross generation carry either the homozygous or heterozygous genes from the recurrent parent and the majority of them are homozygous genotype. However, the qualitatively inherited fertility restoring gene(s) of the recurrent parent most probably maintained in the subsequent backcross progenies. Specific combining ability being quantitatively inherited (Zha et al., 2008) from the recurrent parents will be gradually recovered through backcrossing in different individuals to a varying extent. In each backcross generation in this study, MAS for Xa23 gene before transplanting combined with resistance confirmation by inoculating twice at early tillering and booting stages and very strict phenotypic selections against the recurrent parents at maturity were performed over a relatively larger backcross population. Eighty BC4F2 plants, identical to each recurrent parent derived from the progenies of each restorer line were selected to make test-crosses with the respective male sterile lines. Normal spikelet fertility was observed in most of the test-crosses but their grain yields varied (data not shown). Some test-crosses showed high spikelet fertility and good specific combining ability (high grain yields), implying that their corresponding BB resistant lines were identical to their recurrent parents. Thus, it is clear from this study that a minimum of four backcrosses in conjunction with stringent phenotypic selection for the recurrent parent in each BC progenies and combining ability testing on a relatively large scale, guarantees the recovery of recurrent parental characteristics even without MAS against the background of the recurrent parents as indicated in the previous studies (Chen et al., 2000; Joseph et al., 2004; Sundaram et al., 2008). Secondly, the effectiveness of Xa23 gene in backcross breeding program for BB resistance was observed in this study. It was reported that the level of hybrid rice resistant to Xoo was determined by the restorer line when CMS was susceptible, whereas the resistance level of F1 was controlled by the interaction between CMS and restorer line when CMS was resistant (Lu and Shen, 1984; Wang et al., 2000). Majority of the male sterility sources widely used in China such as wild abortive (WA), dwarf abortive (DA), Gang, D, Yinshui and Honglian types were found to be susceptible to BB (Zhang, 2009). As many as 31 resistance genes to BB have been reported (NiÑo-Liu et al., 2006; Wang et al., 2009), but most of these genes have not been used in breeding programs due to either their lower resistance level or narrow resistance spectrum. Hitherto, only Xa4 and Xa21 have been widely used in hybrid rice breeding in China. However, these two genes do not confer the ideal kind of resistance for BB in breeding programs. The level of resistance of F1 rice hybrids carrying Xa4 gene to BB is low because of its incomplete dominance nature. While Xa21 gene with a broad resistance spectrum is a completely dominant, but it generally expresses resistance from the late tillering stage onwards and its resistance level is affected by the genetic background (Zhai et al., 2001). Conversely, Xa23 gene has a broader resistance spectrum to different BB races collected from the Philippines, Japan and
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China, and shows very high resistance level during all the growth stages besides its strong effectiveness to inheritance of resistance (Zhang et al., 1998, 2001). Recently, Xa23 has been introduced into several conventional cultivars and restorer lines (Luo et al., 2005; Li et al., 2006; Qin et al., 2007; Zhou et al., 2009). However, in those studies the resistance level of F1 hybrid carrying Xa23 gene in different genetic backgrounds was not evaluated. We observed that the three different restorer lines with Xa23 and their derived hybrid combinations with the three different sterile lines were highly resistant to all 17 races tested at both seedling and booting stages with no significant differences for LL. This indicated that there was no genetic background effect on the expression of this gene, thus strongly suggesting that Xa23 is of great value in breeding rice hybrids with BB resistance. Thirdly, proper utilization and deployment of Xa23 gene in the control of BB disease need to be outlined for protecting or delaying the break down of this valuable gene. A sharp percentage decline from 35.6 to 10.7% in the release of BB resistant threeeline indica hybrids in the period (1996e2005) as compared to the duration (1973e1986) in China was primarily due to the ineffectiveness of Xa4 gene to some virulent strains of Xoo (Zhang, 2009). This resulted in the introduction of Xa21 gene into several rice varieties and hybrids in view of its wide BB resistance spectrum (Tu et al., 1998; Wang et al., 1996; Xu, 2007; Zhang et al., 1998; Zhai et al., 2001). However, recent emergence of new virulent strains in China and other Asian countries led to the break down of BB resistance of Xa21 gene (Lee et al., 1999; Marella et al., 2001; Zeng et al., 2002). As indicated in this study, the three hybrid parents carrying Xa23 did not only express high resistance with broad spectrum during the entire growth stages, but also exhibited good agronomic traits, that could serve as immediate sources of BB resistance for hybrid rice breeding and its direct use in hybrid rice seed production. This step towards the development of BB resistant restorer lines will be playing a crucial role in controlling BB in China and other Asian countries. It is expected that overtime the new virulent Xoo races will certainly overcome the Xa23 gene once it is widely used in breeding programs. In order to delay the loss of another broad spectrum resistance gene such as Xa23, it is highly encouraged to use different sources of resistance genes for BB in varietal development and effective deployment strategies. Chen et al. (2000) and Zhai et al. (2001) developed a BB resistant restorer line, Minghui63eXa21 by transgenic means. Therefore, BB resistant Shanyou63 produced from the two resistant restorer lines, Minghui63eXa23 and Minghui63eXa21 could be rotated for hybrid rice production. Another strategy to achieve enhanced and durable resistance against plant diseases is to pyramid Xa23 with other resistant genes thereby increasing resistance level and spectrum as reported in all previous geneepyramiding programs on BB resistance (Huang et al., 1997; Sanchez et al., 2000; Singh et al., 2001; Yoshimura et al., 1995). Alternatively, the mixture of the varieties in the same genetic background with different resistance genes could be co-cultivated (Ayliffe et al., 2008; Zhu et al., 2000). BB resistant genes Xa23 and Xa21 as reported above have been derived from wild rice and are now being considered as of great value in hybrid rice breeding programs among all reported genes (NiÑo-Liu et al., 2006). Careful utilization of the broad spectrum BB resistant genes is urgently required to efficiently control BB and to delay the speed of development of new virulent Xoo strains by the way of mixed planting of the hybrid rice combinations derived from the same genetic background restorer lines with Xa23 and Xa21, respectively. Simultaneous search for new resistant sources for BB from other wild rice varieties needs to be carried out for diversifying varieties possessing different sets of BB resistant genes.
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Improvement of bacterial blight resistance of hybrid rice in China using the Xa23 gene derived from wild rice (Oryza rufipogon)q Yong-Li Zhou a, Veronica NE Uzokwe a, Cong-He Zhang b, Li-Rui Cheng a, Lei Wang a, Kai Chen a, Xiao-Qing Gao a, Yong Sun a, Jin-Jie Chen b, Ling-Hua Zhu a, Qi Zhang a, Jauhar Ali c, Jian-Long Xu a, *, Zhi-Kang Li a, c a
Institute of Crop Sciences/ National Key Facility for Crop Gene Resources and Genetic Improvement, Chinese Academy of Agricultural Sciences, 12 South Zhong-Guan-Cun Street, Beijing 100081, PR China Win-All Hi-Tech Seed Company Limited, 3 Tian-Zhi Road, Hefei 230012, PR China c International Rice Research Institute, DAPO Box 7777, Metro Manila, Philippines b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 28 July 2010 Received in revised form 30 November 2010 Accepted 4 December 2010
A novel bacterial blight (BB) resistance gene, Xa23, identified from Oryza rufipogon was introgressed into three popular restorer lines (Minghui63, YR293 and Y1671) for wild abortive cytoplasmic male sterility by marker-assisted backcross breeding approach in combination with artificial inoculation and stringent phenotypic selections. The three derived BB resistant restorer lines (Minghui63eXa23, YR293eXa23 and Y1671eXa23) and their hybrid combinations with Zhenshan97A (Shanyou63eXa23), NongfengA (Fengyou293eXa23) and Zhong9A (Zhongyou1671eXa23) demonstrated similar BB resistance spectrum as the donor parent, CBB23 (B). The newly developed BB resistant restorers and their derived hybrids were identical to their respective original versions for agronomic traits especially under disease free condition. However, under severe disease condition, the three BB resistant restorer lines exhibited significantly higher grain weight and spikelet fertility as compared to the respective original restorer lines thus further resulting in BB resistant hybrids with significantly higher grain yields than their respective popular original hybrids. The results indicated that the Xa23 gene could completely express its dominant broad spectrum resistance in different backgrounds of both restorer and male sterile lines across different growth stages, suggesting its immense breeding value in BB resistance improvement for hybrid rice. Moreover, a reasonable utilization and deployment of Xa23 gene for efficient control of BB disease in hybrid rice production was recommended. Ó 2010 Elsevier Ltd. All rights reserved.
Keywords: Bacterial blight Xa23 gene Hybrid rice Marker-assisted backcross Resistance improvement
1. Introduction Exploitation of hybrid vigor in rice is the most viable option to enhance the rice production levels across Asia. Hybrid rice area is steadily expanding across Asia with potential yield advantages ranging from 15 to 20% over the high yielding semi-dwarf varieties. Hybrid rice is commercially grown in many countries across the globe particularly in China, where it accounts for half of the rice acreage and contributes 60% of the total rice yield of the country (Cheng et al., 2004). Despite the impressive yield gain through heterosis breeding the problem of hybrid combination being
q Yong-Li Zhou, Veronica NE Uzokwe and Cong-He Zhang contributed equally to this work. * Corresponding author. Tel.: þ86 10 82105854; fax: þ86 10 82108559. E-mail addresses: [email protected], [email protected] (J.-L. Xu). 0261-2194/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.cropro.2010.12.002
susceptible to many diseases due to narrow genetic base still persists. Amongst the major prevalent diseases, bacterial blight (BB) disease happens to be one of the major barriers to high and sustainable hybrid rice production (Virmani, 1996). BB disease of rice (Oryza sativa), caused by Xanthomonas oryzae pv. oryzae (Xoo), is one of the most widely distributed and devastating rice diseases for both inbred and hybrid rice in Asia. BB damage on this crop was reported to range from 20 to 30% and could sometimes get as high as 50% (Mew, 1987; Ou, 1985). The BB disease became prominent in the 1960s, when new high yielding varieties were first developed and introduced (Mew, 1987). Varietal resistance is the main strategy for controlling this disease. As many as 31 resistance genes (22 dominant and 9 recessive) to BB have been identified and registered (Chu et al., 2006; NiÑo-Liu et al., 2006; Wang et al., 2009). Among them, Xa4 has been widely used in developing almost all the commercial hybrids and conventional cultivars of indica rice in the main rice growing
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regions of China (Zhang, 1991). This narrow genetic basis for resistance to BB in Chinese rice cultivars has imposed strong selection on the pathogen population, resulting in a dramatic increase in frequency of race V that is virulent to both Xa4 and Xa3 during the past decade in China (Zhang et al., 1996). Currently most of the parental lines of the popular hybrid rice in China are susceptible to most Chinese and Philippines races (Xia et al., 2009). Another widely used Xa21 gene in rice breeding programs was identified from the wild rice O. longistaminata (Khush et al., 1990; Song et al., 1995). This gene was recently broken down by new virulent strains in Southern and Yangtze River Valley in China (Zeng et al., 2002; Zhang, 2009). Amongst all the known resistance genes to BB, Xa23, a single completely dominant resistance gene effective at all growth stages, identified from wild rice species of Oryza rufipogon emerged as the most promising one (Zhang et al., 1998, 2001). The Xa23 gene was found to be highly resistant to all the 20 strains of BB consisting of 10 Philippine races (P1eP10), 7 Chinese pathotypes (C1eC7) and 3 Japanese races (T1eT3) at the maximum tillering stage. It was indicated that Xa23 gene possessed a broad resistance spectrum among all known R genes (Zhang et al., 2001). In this study, we evaluated the practical utility of the Xa23 gene in improvement of BB resistance for hybrid rice through introgression of the gene into three popular restorer lines to develop new BB resistant restorer lines and their respective hybrid rice combinations. 2. Materials and methods 2.1. Parental materials In this study, CBB23(B) carrying Xa23 gene, derived from BC4F5 progeny of the cross between an O. rufipogon accession (RBB16, the Xa23 donor) and a recipient indica BB susceptible variety IR24 (Zhang, 2005), was used as the donor of resistance to BB. Three popular restorer lines i.e. Minghui63, YR293 and Y1671 for wild abortive cytoplasmic male sterility (WAeCMS), also widely used for hybrid rice seed production in China but highly susceptible to BB were used as recurrent parents. These three restorers were derived from the following respective crosses IR30/Gui630, 9311/Minghui63 and YR293/02428 through pedigree breeding approach. The seeds of the three restorer lines were provided by the Win-All Hi-Tech Seed Company Limited, Anhui province, China. The IR24 variety was used as a susceptible control for BB inoculation. 2.2. Development of BB resistant restorer lines with Xa23 gene Marker-assisted backcrossing procedure was applied to transfer Xa23 gene into elite restorer parental lines. The three restorer lines were used as female to cross with CBB23(B) to produce three F1s and then backcrossed with the F1s to get three BC1F1 populations. A mini-scale DNA isolation for PCR analysis was carried out on threeeleaf old seedlings in the seedbed for the parents and BC1F1 plants following the procedure of Zheng et al. (1995). Foreground selection against Xa23 gene was used to select plants carrying Xa23 gene prior to transplanting through marker-assisted selection (MAS) using a closely linked SSR marker (RM206) of 1.9 cm apart from the Xa23 locus (Pan et al., 2003). The 25 days old seedlings with Xa23 heterozygous gene selected from BC1F1 were transplanted in the screenhouse. At early tillering (3 weeks after transplanting) and booting (7 weeks after transplanting) stages, three to six uppermost leaves of each plant were inoculated with a highly virulent Chinese Xoo strain, GD1358 using the leaf-clipping method (Kauffman et al., 1973). Resistant BC1F1 plants that were determined by MAS and inoculation were further backcrossed to the respective recurrent parents to produce their
BC2F1 populations. Consecutive backcrosses were carried out in the same way to produce BC4F1 populations. Resistant plants carrying homozygous Xa23 gene and identical phenotype as that of the recurrent parents were identified by MAS and inoculation in BC4F2. These phenotypically identical BB resistant plants to their respective restorer lines were then crossed with the three respective WAeCMS lines (Zhenshan97A, NongfengA and Zhong9A), to test their restoration and specific combining ability. All the BC4F3 lines were confirmed for the presence of Xa23 gene and studied for their phenotypic similarities to their respective original restorer lines. The F1 progenies of the test-crosses involving the selected BB restorer lines in BC4F2 with their respective CMS lines were evaluated for their spikelet fertility and level of heterosis. Based on the performance of BB resistance and morphological characters of BC4F3 lines and the level of heterosis of their respective test-crosses, only one line that was phenotypically identical to the recipients with homozygous Xa23 gene was finally selected from the progeny of each recipient, and it was designated as Minghui63eXa23, YR293eXa23 and Y1671eXa23 for further evaluation. 2.3. Evaluation of BB resistance and agronomic traits The original restorer lines Minghui63, YR293 and Y1671 and their derived BB resistant restorer lines Minghui63eXa23, YR293eXa23 and Y1671eXa23 were crossed with three WAeCMS lines, Zhenshan97A, NongfengA and Zhong9A to produce hybrid F1 for evaluation of BB resistance and agronomic traits. BB resistance evaluation of these materials were carried out by sowing them on 20th of April, 2008 and 30 days old seedlings were transplanted into a nine-row plot of six plants per row with a plant to row spacing of 17 20 cm and three replications in the screenhouse of the Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing, China. Three plants were artificially inoculated by partial clipping of 3e6 uppermost leaves separately (Kauffman et al., 1973) for each of the 7 different Chinese Xoo races (C1eC7) (Fang et al., 1990) and 10 Philippine Xoo races (P1eP10) at the early tillering (3 weeks after transplanting) and booting (7 weeks after transplanting) stages. Lesion length (LL) was measured on all partially clipping inoculated leaves after 2e3 weeks of inoculation. BB field evaluation and agro-morphological characterization of the newly developed restorer lines (Minghui63eXa23, YR293eXa23 and Y1671eXa23) and their derived hybrid combinations i.e. Shanyou63eXa23 (Zhenshan97/Minghui63e Xa23); Fengyou293eXa23 (NongfengA/YR293eXa23) and Zhongyou1671eXa23 (Zhong9A/ Y1671eXa23), were compared to their original restorer lines (Minghui63, YR293 and Y1671) and their respective hybrid combinations i.e. Shanyou63 (Zhenshan97/Minghui63); Fengyou293 (NongfengA/ YR293) and Zhongyou1671 (Zhong9A/Y1671) by conducting field trials both under disease and disease free environments in a randomized complete block design (RCBD) with 3 replications in Hainan breeding station during 2009 winter season. Testing of all the materials under severe diseased condition was carried out by planting each line in three rows plot with twelve plants per row and a spacing of 17 20 cm in a RCBD with three replications. The middle row in each plot was inoculated with Xoo race C5. However, for testing under disease free condition, the same set of materials was planted in the field without artificial BB inoculation and any change in plot size and layout. Measurements were taken on ten plants from the middle row in each plot for different agro-morphological traits, such as plant height (PH), heading date (HD), panicle length (PL), panicle number per plant (PN), spikelet number per panicle (SNP), spikelet fertility (SF), 1000-grain weight (GWT) and grain yield per plant (GY) according to the standard evaluation system of rice (IRRI, 1996).
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3. Results 3.1. BB resistance levels of parental lines and F1 hybrids The Xa23 donor, CBB23(B) showed highly resistant reactions at both early tillering and booting stages to all 7 Chinese (C1eC7) and 10 Philippine (P1eP10) Xoo races, while the three sterile lines, Zhenshan97A, NongfengA and Zhong9A showed complete susceptibility (Table 1, Fig. 1). The three restorer lines Minghui63, YR293 and Y1671 were found to be susceptible to most of the 17 races, especially to the Chinese race 5 (GD1358) and the Philippine race 6 (PXO99), that shows strongest pathogenicity in China and Philippines at both early tillering and booting stages with similar LL to that of the susceptible variety IR24 (Table 2, Fig. 1). The F1 plants derived from the crosses between the three restorer lines and the CBB23(B) were each divided into two groups and inoculated with Chinese race 5 (GD1358) and Philippine race 6 (PXO99). All the F1 plants exhibited resistance to Xoo race PXO99 at early tillering stage with the average LL ranging from 1.1 to 1.5 cm but were highly resistant to GD1358 with the LL ranging from 0.2 to 0.9 cm, respectively (Fig. 2). At booting stage, all F1 plants showed enhanced resistance to PXO99 and GD1358 compared with those at early tillering stage, with LL of 0.1e0.6 cm and 0.8e1.4 cm, respectively. The Xa23 gene showed the same level of resistance in different backgrounds and developmental stages by comparable LL to that of CBB23(B) thereby clearly indicating that the Xa23 could completely express its dominant resistance in different genetic backgrounds and varied growth stages.
Fig. 1. Leaves showing the effects of Xanthomonas oyzae pv. oryzae Philippines race 6 (2e3 weeks post-inoculation) on rice restorer and sterile lines. A: Effects at the early tillering stage (3 weeks after transplanting), B: Effects at the booting stage (7 weeks after transplanting). Leaves 1e7 represent the effects on CBB23(B), Minghui63, YR293, Y1671, Zhenshan97A, NongfengA and Zhong9A.
Table 1 Resistant reactions of CBB23(B) and the three parental cytoplasmic male sterile (CMS) lines to the 7 Chinese and 10 Philippine races of Xanthomonas oryzae pv. oryzae (Xoo) at early tillering (TS) and booting (BS) stages. Growth stage
TS
BS
a
Xoo
Resistant donor and the 3 parental CMS lines
Racea
Strain
IR24 (CK)
CBB23(B)
Zhenshan97A
NongfengA
Zhong9A
C1 C2 C3 C4 C5 C6 C7 P1 P2 P3 P4 P5 P6 P7 P8 P9 P10 C1 C2 C3 C4 C5 C6 C7 P1 P2 P3 P4 P5 P6 P7 P8 P9 P10
JS97-2 KS6-6 JS158-2 Z173 GD1358 OS198 JS49-6 PXO61 PXO86 PXO340 PXO71 PXO112 PXO99 PXO145 PXO280 PXO339 PXO341 JS97-2 KS6-6 JS158-2 Z173 GD1358 OS198 JS49-6 PXO61 PXO86 PXO340 PXO71 PXO112 PXO99 PXO145 PXO280 PXO339 PXO341
9.5 2.3 10.3 1.8 13.5 1.1 10.3 1.7 14.8 2.6 9.9 1.0 9.3 1.3 11.5 2.1 12.8 2.9 12.5 2.4 10.4 1.0 11.5 2.0 14.2 1.7 13.5 0.9 8.8 1.7 10.2 0.5 13.2 1.8 11.9 2.3 12.4 0.7 15.3 1.5 13.6 1.3 16.2 1.1 15.1 0.8 12.8 1.6 16.1 2.3 15.2 1.4 13.9 0.5 13.6 1.2 12.7 2.0 18.2 3.1 14.1 0.2 11.9 1.3 13.2 0.7 12.8 1.2
0.3 0.2 0.5 0.1 0.3 0.1 0.4 0.2 0.4 0.1 0.3 0.1 0.3 0.0 0.5 0.1 0.6 0.2 0.3 0.0 0.7 0.2 0.5 0.0 0.5 0.1 0.2 0.1 0.3 0.0 0.1 0.1 0.4 0.5 0.2 0.1 0.5 0.0 0.2 0.1 0.4 0.3 0.3 0.1 0.5 0.3 0.2 0.1 0.3 0.1 0.4 0.2 0.5 0.3 0.6 0.2 0.6 0.1 0.4 0.1 0.3 0.2 0.5 0.2 0.2 0.1 0.3 0.1
7.5 2.8 8.8 1.3 7.2 1.6 10.0 3.4 12.8 2.1 12.4 1.7 11.2 2.3 9.5 1.2 12.7 1.5 9.8 2.9 11.7 3.1 10.2 1.3 14.9 2.3 9.7 0.3 7.9 0.9 12.6 1.4 12.0 2.7 8.8 0.3 10.9 0.7 11.8 1.0 14.1 1.0 16.5 1.3 8.9 0.6 9.7 0.7 7.8 0.6 7.4 0.5 6.2 1.1 4.7 0.7 10.9 0.5 20.8 1.4 13.8 0.5 14.0 1.1 13.2 0.6 13.7 0.6
7.5 0.5 7.7 0.8 13.0 2.7 15.3 2.3 16.1 1.8 9.3 2.8 10.5 1.9 6.2 1.3 13.3 1.3 10.7 1.8 8.5 1.5 7.0 0.5 18.7 1.1 6.8 1.0 12.3 1.9 16.5 3.8 11.5 4.0 7.7 0.7 6.4 1.2 12.3 0.8 13.2 1.7 17.9 1.3 7.2 0.3 6.9 0.7 8.6 1.2 10.2 1.9 13.0 0.7 12. 2 0.8 8.2 1.3 18.5 2.1 8.3 1.5 10.6 1. 6 6.6 1.5 11.7 3.3
8.7 1.6 10.2 1.0 8.2 1.1 12.9 1.9 15.1 1.7 9.7 2.1 7.7 1.0 6.6 1.3 10.8 2.9 12.8 2.0 14.7 1.0 7.8 1.3 16.2 1.8 9.8 1.3 13.5 1.3 12.8 1.3 18.5 2.3 7.4 1.2 8.8 0.6 9.8 2.5 7.8 1.2 14.6 1.5 8.9 0.8 9.8 0.8 7.8 1.9 10.8 1.7 10.6 2.0 11.6 1.7 6.5 1.3 17.6 1.7 7.0 1.4 9.0 2.3 9.5 0.9 10.8 3.5
C1eC7 and P1eP10 represent 7 Chinese and 10 Philippines races of Xoo, respectively.
Table 2 Resistance levels on the basis of lesion length (LL) of restorer lines carrying Xa23 and their original versions against the 7 Chinese and 10 Philippines races of Xanthomonas oryzae pv. oryzae at early tillering (TS) and booting (BS) stages. Growth stage
Race
Minghui63
Minghui63eXa23
YR293
YR293eXa23
Y1671
Y1671eXa23
TS
C1 C2 C3 C4 C5 C6 C7 P1 P2 P3 P4 P5 P6 P7 P8 P9 P10 C1 C2 C3 C4 C5 C6 C7 P1 P2 P3 P4 P5 P6 P7 P8 P9 P10
2.5 0.3 15.7 4.0 5.5 0.9 13.5 2.1 12.3 1.9 6.7 0.8 12.7 1.1 14.8 1.4 13.3 4.3 11.7 2.8 12.0 1.3 10.0 2.0 14.8 2.0 4.3 1.9 12.0 2.7 11.8 1.9 12.1 1.5 1.2 0.1 1.5 0.2 2.9 0.7 13.6 2.3 13.2 0.3 6.8 1.3 10.4 1.3 7.5 0.6 8.4 1.7 4.3 0.8 4.6 0.6 4.9 0.9 17.2 1.4 2.3 0.4 3.3 0.1 2.4 0.5 3.50 0.2
0.2 0.1 0.2 0.0 0.3 0.0 0.8 0.1 0.3 0.2 0.4 0.3 0.3 0.0 0.4 0.2 0.7 0.2 0.3 0.2 0.3 0.2 0.2 0.1 0.4 0.2 0.3 0.1 0.2 0.1 0.1 0.0 0.2 0.1 0.2 0.1 0.2 0.0 0.1 0.0 0.7 0.1 0.2 0.0 0.5 0.2 0.1 0.0 0.3 0.2 0.6 0.4 0.8 0.3 1.0 0.4 0.3 0.2 0.4 0.1 0.3 0.1 0.3 0.2 0.1 0.0 0.2 0.1
10.7 1.5 9.2 0.8 8.0 1.5 10.7 3.5 10.7 1.8 12.2 1.6 9.5 1.3 9.8 1.9 6.5 1.3 7.4 1.2 12.8 1.2 3.1 0.4 12.5 2.0 13.7 2.5 12.7 3.1 9.5 1.3 8.2 1.6 1.3 0.1 1.2 0.2 11.9 4.9 2.2 0.4 15.1 1.9 8.5 4.2 12.3 2.3 4.5 1.7 1.6 0.31 1.8 0.75 15.2 4.3 1.2 0.0 16.5 2.9 5.7 2.3 5.2 0.5 6.1 2.5 1.4 0.1
0.3 0.2 0.3 0.2 0.1 0.1 0.2 0.1 0.2 0.1 0.1 0.0 0.2 0.0 0.3 0.2 0.2 0.0 0.3 0.2 0.2 0.0 0.3 0.1 0.8 0.2 0.2 0.1 0.2 0.1 0.2 0.2 0.2 0.1 0.2 0.0 0.1 0.1 0.1 0.0 0.2 0.1 0.1 0.0 0.1 0.1 0.1 0.0 0.3 0.1 0.2 0.0 0.2 0.0 0.6 0.3 0.1 0.1 0.4 0.1 0.2 0.1 0.1 0.0 0.1 0.0 0.1 0.0
2.3 0.5 5.5 1.3 9.4 2.4 10.1 3.0 10.3 1.5 11.7 2.5 9.5 2.0 11.7 2.0 9.1 1.5 12.7 1.5 9.3 1.5 9.8 2.5 12.3 2.9 6.3 1.2 9.2 1.6 8.0 2.1 9.5 4.1 1.7 0.3 3.5 0.5 2.9 0.5 6.0 1.5 14.7 2.6 2.7 1.4 6.3 1.6 5.4 2.4 6.9 1.6 6.7 1.0 5.6 0.8 3.5 0.8 19.5 3.1 5.3 0.9 8.5 0.5 9.3 2.6 8.14 1.7
0.1 0.0 0.1 0.0 0.1 0.1 0.4 0.2 0.1 0.0 0.4 0.6 0.2 0.1 0.1 0.1 0.2 0.0 0.3 0.2 0.2 0.0 0.1 0.0 0.3 0.1 0.1 0.0 0.1 0.0 0.1 0.0 0.1 0.0 0.1 0.0 0.2 0.1 0.1 0.0 0.2 0.1 0.3 0.1 0.1 0.0 0.1 0.0 0.3 0.2 0.3 0.1 1.0 0.3 0.2 0.1 0.2 0.1 0.5 0.1 0.1 0.0 0.1 0.0 0.1 0.1 0.1 0.0
BS
Fig. 2. Resistant F1 plants of the crosses between the restorer lines and CBB23(B) to Xanthomonas oyzae pv. oryzae Chinese race 5 and Philippine race 6. A: The lesion length of F1 plants at the early tillering stage (3 weeks after transplanting); B: The lesion length of F1 plants at the booting stage (7 weeks after transplanting); C: The lesion length of F1 plants infected with the Philippine race 6 at the booting stage.
Y.-L. Zhou et al. / Crop Protection 30 (2011) 637e644 Table 3 Resistance levels of F1 rice hybrids with and without Xa23 gene against the virulent Chinese race 5 (C5) and the Philippine race 6 (P6) of Xanthomonas oryzae pv. oryzae both at early tillering and booting stages. F1 combination
Early tillering stage
Booting stage
P6
P6
NongfengA/Minghui63 Zhenshan97A/Minghui63 (Shanyou63) Zhong9A/Minghui63 NongfengA/Minghui63eXa23 Zhenshan97A/Minghui63eXa23 (Shanyou63eXa23) Zhong9A/Minghui63eXa23
22.0 5.0 21.3 3.6 18.9 4.3 20.8 3.5 19.6 3.2 16.3 2.9
7.9 1.0 8.2 1.1
21.3 3.7 20.0 4.3 19.7 3.8 1.2 0.5 1.0 0.7 1.4 0.3 1.5 0.6 1.2 0.4 1.4 0.5
7.7 1.4 0.5 0.2 0.6 0.4
1.1 0.1
C5
1.0 0.4
1.3 0.4
C5
0.4 0.3
NongfengA/YR293 (Fengyou293) 21.9 4.9 15.9 2.8 20.4 5.7 11.3 2.5 Zhenshan97A/YR293 22.5 3.8 16.0 4.2 20.0 5.0 10.8 2.3 Zhong9A/YR293 22.1 3.1 16.8 2.9 17.3 2.4 10.6 1.5 NongfengA/YR293eXa23 1.5 0.3 0.4 0.3 1.2 0.4 0.5 0.2 (Fengyou293eXa23) Zhenshan97A/YR293eXa23 1.4 0.2 0.6 0.3 1.4 0.2 0.6 0.2 Zhong9A/YR293eXa23 1.4 0.3 0.8 0.5 1.3 0.3 0.4 0.2 NongfengA/Y1671 22.0 3.5 21.5 3.8 15.3 2.5 18.7 4.4 Zhenshan97A/Y1671 20.7 2.5 20.8 2.9 18.6 3.5 17.8 3.8 Zhong9A/Y1671 (Zhongyou1671) 22.2 3.9 20.9 2.9 19.6 4.7 19.0 3.5 NongfengA/Y1671eXa23 1.0 0.5 0.7 0.6 1.4 0.4 0.7 0.4 Zhenshan97A/Y1671eXa23 1.2 0.2 0.6 0.5 1.4 0.4 0.5 0.5 Zhong9A/Y1671eXa23 1.0 0.6 0.8 0.6 0.9 0.4 0.5 0.4 (Zhongyou1671eXa23)
3.2. Evaluation of BB resistance of the newly-bred restorer lines and their hybrids The BB resistance of the newly developed restorer lines (Minghui63eXa23, YR293eXa23 and Y1671eXa23) is presented in Table 2 by comparing the average LL with their original versions caused by the 10 Philippine and 7 Chinese Xoo races at the early tillering and booting stages. All improved restorer lines carrying Xa23 showed a high level of resistance to all Xoo races including the highly virulent Chinese race 5, GD1358 and the Philippine race 6, PXO99 with LL < 1.0 cm even at the early tillering stage, thus indicating that Xa23 gene possessed a broad spectrum of BB
641
resistance to over 17 races tested. In addition, no significant differences existed for BB resistance for Xa23 gene in different restorer line backgrounds. The hybrids derived from the newly developed BB resistant restorer lines were highly significant for their resistance to PXO99 and GD1358, with the average LL ranging from 1.0 to 1.5 cm and 0.4e1.2 cm at early tillering stage; and from 0.9 to 1.4 cm and 0.5e0.7 cm at booting stage, respectively, as compared to their original hybrid combinations with the three different sterile lines Zhenzhan97A, NongfengA and Zhong9A, respectively (Table 3 and Fig. 3). No significant differences were observed for resistance to PXO99 and GD1358 for the hybrids derived from each BB resistant restorer line and different sterile lines in both the two stages. This further indicated that the expression of BB resistance of Xa23 gene was independent of genetic background of the male sterile lines. 3.3. Agronomic characterization and grain yield performance of the newly developed restorer lines and their hybrid combinations The three newly developed BB resistant restorers and their corresponding original versions consisting of Minghui63eXa23 and Minghui63; YR293eXa23 and YR293; Y1671eXa23 and Y1671 were found to be identical for all the agronomic traits studied under disease free field conditions. Likewise, we found the same for their hybrid combinations i.e. Shanyou63eXa23 and Shanyou63; Fengyou293eXa23 and Fengyou293; Zhongyou1671eXa23 and Zhongyou1671 (Table 4). However, Minghui63eXa23 showed significantly higher 1000-grain weight and spikelet fertility than Minghui63 under severe diseased conditions. Grain yield of Shanyou63eXa23 was significantly higher than that of Shanyou63 on account of pronounced differences in 1000-grain weight and spikelet fertility (Table 4). A similar pattern was observed for the other two BB resistant restorers and their derived hybrids. The YR293eXa23 and Y1671eXa23 lines had significantly higher 1000grain weight, spikelet fertility and grain yield than YR293 and Y1671. Fengyou293eXa23 and Zhongyou1671eXa23 had significantly higher grain yield than Fengyou293 and Zhongyou1671 (Table 4). Grain yield and its related traits such as spikelet fertility
Fig. 3. Restorer lines and F1 combinations resistance to Xanthomonas oryzae pv. oryzae Chinese race 5 at the booting stage (7 weeks after transplanting).
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Table 4 Agronomic performance of restorer lines with and without Xa23 gene and their respective commercial hybrids tested under both severe disease (artificial inoculation) and disease free field conditions during 2009 winter season in Hainan. Restorer lines/Hybrids PH (cm)
HD (d)
PL PN SNP (cm)
SF (%) GWT GY (g) (g/plant)
Without inoculation Minghui63 Minghui63eXa23 Shanyou63 Shanyou63eXa23 LSD(0.05)a LSD(0.01)b YR293 YR293eXa23 Fengyou293 Fengyou293eXa23 LSD(0.05)a LSD(0.01)b Y1671 Y1671eXa23 Zhongyou1671 Zhongyou1671eXa23 LSD(0.05)a LSD(0.01)b
105.5 104.7 96.3 95.5 0.9 1.7 110.7 109.9 100.7 101.5 2 3.7 109.3 109.6 101.4 102.1 2.3 4.4
21.5 20.1 23.2 22.5 1.6 3.1 22.3 21.8 24.4 23.2 1.4 2.5 23.1 23.6 24.1 24.8 0.9 1.6
7.6 7.9 8.3 8.5 0.5 1.1 7.3 7.5 8.1 8.3 0.4 0.7 6.2 6.5 7.8 8.7 1.1 1.9
114.5 112.3 131.4 129.5 5.1 9.4 122.3 121.5 140.2 139.2 3.1 5.8 124.2 125.8 135.4 136.7 4.1 7.8
85.6 85.7 82.7 83.3 2.3 4.3 85.6 86.9 84.7 85.1 2.1 3.5 84.6 86.5 83.6 85.5 2.5 4.6
27.5 27.1 27.6 28.1 1.1 1.8 29.2 29.1 28.8 28.5 1.5 2.8 28.8 27.9 28.1 28.1 1.2 2.3
17.3 16.9 22.2 21.5 1.7 3.2 17.1 17.5 23.1 24.7 1.9 3.7 16.1 17.1 22.8 24.9 2.4 3.1
at the booting 95.1 105.1 96.3 104.5 102.7 97.5 100.6 96.8 2.5 0.9 4.8 1.7 98.9 109.9 98.3 110.2 105.1 101.8 103.8 102.3 2.1 2.2 3.8 4 103.2 109.5 104.5 110.3 109.1 100.8 108.6 102.3 1.9 3 3.5 5.8
stage 21.3 21.2 23.1 22.2 1.6 3.1 22.1 22.3 24.2 23.1 1.3 2.4 22.9 23.3 24.2 24.7 1.1 2
7.6 7.8 8.4 8.6 0.5 1.1 7.7 7.6 8 8.4 0.6 1.1 6.4 6.8 7.7 7.9 1.3 2.4
113.5 112.1 127.5 128.1 5.1 9.4 120.6 122.1 138.6 140.1 3.3 6.1 123.3 125.2 131.3 133.9 3.3 6
80.9 84.5 75.5 83.6 2.3 4.3 77.2 85.5 75.7 83.1 3.5 6.7 77.6 82.3 77.7 83.9 3.5 6.7
25.6 27.3 25.2 27.9 1.3 2.4 26.1 28.9 25.7 28.1 1.3 2.1 25.7 27.2 26.3 28.3 1.3 2.4
13.5 15.1 17.5 21.3 2.8 5.2 14.9 17.6 17.9 23.5 2.6 5 14.5 17.6 18.1 22.5 2.2 4
Inoculated by Xoo. C5 Minghui63 Minghui63eXa23 Shanyou63 Shanyou63eXa23 LSD(0.05)a LSD(0.01)b YR293 YR293eXa23 Fengyou293 Fengyou293eXa23 LSD(0.05)a LSD(0.01)b Y1671 Y1671eXa23 Zhongyou1671 Zhongyou1671eXa23 LSD(0.05)a LSD(0.01)b
95.5 96.1 101.4 100.3 2.5 4.8 99.8 98.7 105.5 104.1 1.9 3.4 103.5 104.2 110.5 109.2 1.4 2
PHeplant height, HDeheading date, PLepanicle length, PNepanicle number per plant, SNPespikelet number per panicle, SFespikelet fertility, GWTe1000-grain weight, GYegrain yield per plant. a Least significant differences at 0.05 probability level. b Least significant differences at and 0.01 probability level.
and 1000-grain weight were higher for Shanyou63eXa23, Fengyou293eXa23 and Zhongyou1671eXa23 than Shanyou63, Fengyou293 and Zhongyou1671, respectively. 4. Discussion Prevalence of BB disease in China is jeopardizing the expansion of the popular hybrids as a result of their susceptibility. The three different restorer lines i.e. Minghui63, YR293 and Y1671 have been widely utilized in hybrid rice production in China. Incorporation of a broad spectrum resistance gene into these restorer lines will greatly improve BB resistance of hybrid rice in China. Careful analysis of the literature showed CBB23(B) with Xa23 gene to be an effective donor against all races collected from China, Philippines and Japan (Zhang, 2005). In this study we incorporated a broad spectrum BB resistance Xa23 gene into the three commercially popular restorer lines using MAS-based backcrossing and stringent phenotypic selections within a short period. Minghui63eXa23, YR293eXa23, Y1671eXa23 and their hybrid combinations, Shanyou63eXa23, Fengyou293eXa23 and Zhongyou1671eXa23 exhibited a significant yield advantage over their original counterparts with the same level and spectrum of resistance as that of
the resistant donor, CBB23(B). This study clearly indicated that Xa23 gene is valuable for the improvement of BB resistance in hybrid rice production. Improvement of restorer lines for BB resistance through MAS-based backcross schemes using Xa23 gene with broad spectrum resistance raised certain key issues in rice breeding for increasing production. Firstly, the BB resistance improvement of parental lines of hybrids is much different from that of conventional varieties. In the backcross progenies of restorer parental lines, selections were performed not only for similarity to the recurrent parents, but also for their fertility restoring gene(s) and specific combining ability to the CMS lines. The expected frequencies for individuals homozygous genotype of the recurrent parent on a single locus basis is 0.5, 0.75, 0.875, and 0.9375 in BC1F1, BC2F1, BC3F1 and BC4F1, respectively. All individuals in each backcross generation carry either the homozygous or heterozygous genes from the recurrent parent and the majority of them are homozygous genotype. However, the qualitatively inherited fertility restoring gene(s) of the recurrent parent most probably maintained in the subsequent backcross progenies. Specific combining ability being quantitatively inherited (Zha et al., 2008) from the recurrent parents will be gradually recovered through backcrossing in different individuals to a varying extent. In each backcross generation in this study, MAS for Xa23 gene before transplanting combined with resistance confirmation by inoculating twice at early tillering and booting stages and very strict phenotypic selections against the recurrent parents at maturity were performed over a relatively larger backcross population. Eighty BC4F2 plants, identical to each recurrent parent derived from the progenies of each restorer line were selected to make test-crosses with the respective male sterile lines. Normal spikelet fertility was observed in most of the test-crosses but their grain yields varied (data not shown). Some test-crosses showed high spikelet fertility and good specific combining ability (high grain yields), implying that their corresponding BB resistant lines were identical to their recurrent parents. Thus, it is clear from this study that a minimum of four backcrosses in conjunction with stringent phenotypic selection for the recurrent parent in each BC progenies and combining ability testing on a relatively large scale, guarantees the recovery of recurrent parental characteristics even without MAS against the background of the recurrent parents as indicated in the previous studies (Chen et al., 2000; Joseph et al., 2004; Sundaram et al., 2008). Secondly, the effectiveness of Xa23 gene in backcross breeding program for BB resistance was observed in this study. It was reported that the level of hybrid rice resistant to Xoo was determined by the restorer line when CMS was susceptible, whereas the resistance level of F1 was controlled by the interaction between CMS and restorer line when CMS was resistant (Lu and Shen, 1984; Wang et al., 2000). Majority of the male sterility sources widely used in China such as wild abortive (WA), dwarf abortive (DA), Gang, D, Yinshui and Honglian types were found to be susceptible to BB (Zhang, 2009). As many as 31 resistance genes to BB have been reported (NiÑo-Liu et al., 2006; Wang et al., 2009), but most of these genes have not been used in breeding programs due to either their lower resistance level or narrow resistance spectrum. Hitherto, only Xa4 and Xa21 have been widely used in hybrid rice breeding in China. However, these two genes do not confer the ideal kind of resistance for BB in breeding programs. The level of resistance of F1 rice hybrids carrying Xa4 gene to BB is low because of its incomplete dominance nature. While Xa21 gene with a broad resistance spectrum is a completely dominant, but it generally expresses resistance from the late tillering stage onwards and its resistance level is affected by the genetic background (Zhai et al., 2001). Conversely, Xa23 gene has a broader resistance spectrum to different BB races collected from the Philippines, Japan and
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China, and shows very high resistance level during all the growth stages besides its strong effectiveness to inheritance of resistance (Zhang et al., 1998, 2001). Recently, Xa23 has been introduced into several conventional cultivars and restorer lines (Luo et al., 2005; Li et al., 2006; Qin et al., 2007; Zhou et al., 2009). However, in those studies the resistance level of F1 hybrid carrying Xa23 gene in different genetic backgrounds was not evaluated. We observed that the three different restorer lines with Xa23 and their derived hybrid combinations with the three different sterile lines were highly resistant to all 17 races tested at both seedling and booting stages with no significant differences for LL. This indicated that there was no genetic background effect on the expression of this gene, thus strongly suggesting that Xa23 is of great value in breeding rice hybrids with BB resistance. Thirdly, proper utilization and deployment of Xa23 gene in the control of BB disease need to be outlined for protecting or delaying the break down of this valuable gene. A sharp percentage decline from 35.6 to 10.7% in the release of BB resistant threeeline indica hybrids in the period (1996e2005) as compared to the duration (1973e1986) in China was primarily due to the ineffectiveness of Xa4 gene to some virulent strains of Xoo (Zhang, 2009). This resulted in the introduction of Xa21 gene into several rice varieties and hybrids in view of its wide BB resistance spectrum (Tu et al., 1998; Wang et al., 1996; Xu, 2007; Zhang et al., 1998; Zhai et al., 2001). However, recent emergence of new virulent strains in China and other Asian countries led to the break down of BB resistance of Xa21 gene (Lee et al., 1999; Marella et al., 2001; Zeng et al., 2002). As indicated in this study, the three hybrid parents carrying Xa23 did not only express high resistance with broad spectrum during the entire growth stages, but also exhibited good agronomic traits, that could serve as immediate sources of BB resistance for hybrid rice breeding and its direct use in hybrid rice seed production. This step towards the development of BB resistant restorer lines will be playing a crucial role in controlling BB in China and other Asian countries. It is expected that overtime the new virulent Xoo races will certainly overcome the Xa23 gene once it is widely used in breeding programs. In order to delay the loss of another broad spectrum resistance gene such as Xa23, it is highly encouraged to use different sources of resistance genes for BB in varietal development and effective deployment strategies. Chen et al. (2000) and Zhai et al. (2001) developed a BB resistant restorer line, Minghui63eXa21 by transgenic means. Therefore, BB resistant Shanyou63 produced from the two resistant restorer lines, Minghui63eXa23 and Minghui63eXa21 could be rotated for hybrid rice production. Another strategy to achieve enhanced and durable resistance against plant diseases is to pyramid Xa23 with other resistant genes thereby increasing resistance level and spectrum as reported in all previous geneepyramiding programs on BB resistance (Huang et al., 1997; Sanchez et al., 2000; Singh et al., 2001; Yoshimura et al., 1995). Alternatively, the mixture of the varieties in the same genetic background with different resistance genes could be co-cultivated (Ayliffe et al., 2008; Zhu et al., 2000). BB resistant genes Xa23 and Xa21 as reported above have been derived from wild rice and are now being considered as of great value in hybrid rice breeding programs among all reported genes (NiÑo-Liu et al., 2006). Careful utilization of the broad spectrum BB resistant genes is urgently required to efficiently control BB and to delay the speed of development of new virulent Xoo strains by the way of mixed planting of the hybrid rice combinations derived from the same genetic background restorer lines with Xa23 and Xa21, respectively. Simultaneous search for new resistant sources for BB from other wild rice varieties needs to be carried out for diversifying varieties possessing different sets of BB resistant genes.
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