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Improvement of bacterial blight resistance of hybrid rice in China using the Xa23gene 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 ChinabWin-All Hi-Tech Seed Company Limited, 3 Tian-Zhi Road, Hefei 230012, PR Chinac International Rice Research Institute, DAPO Box 7777, Metro Manila, Philippines

a r t i c l e i n f o

Article history:Received 28 July 2010Received in revised form30 November 2010Accepted 4 December 2010

Keywords:Bacterial blightXa23 geneHybrid riceMarker-assisted backcrossResistance improvement

a b s t r a c t

A novel bacterial blight (BB) resistance gene, Xa23, identified from Oryza rufipogon was introgressed intothree popular restorer lines (Minghui63, YR293 and Y1671) for wild abortive cytoplasmic male sterilityby marker-assisted backcross breeding approach in combination with artificial inoculation and stringentphenotypic selections. The three derived BB resistant restorer lines (Minghui63eXa23, YR293eXa23 andY1671eXa23) and their hybrid combinations with Zhenshan97A (Shanyou63eXa23), NongfengA (Fen-gyou293eXa23) and Zhong9A (Zhongyou1671eXa23) demonstrated similar BB resistance spectrum asthe donor parent, CBB23 (B). The newly developed BB resistant restorers and their derived hybrids wereidentical 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 significantlyhigher grain weight and spikelet fertility as compared to the respective original restorer lines thusfurther resulting in BB resistant hybrids with significantly higher grain yields than their respectivepopular original hybrids. The results indicated that the Xa23 gene could completely express its dominantbroad spectrum resistance in different backgrounds of both restorer and male sterile lines acrossdifferent growth stages, suggesting its immense breeding value in BB resistance improvement for hybridrice. Moreover, a reasonable utilization and deployment of Xa23 gene for efficient control of BB disease inhybrid rice production was recommended.

� 2010 Elsevier Ltd. All rights reserved.

1. Introduction

Exploitation of hybrid vigor in rice is the most viable option toenhance the rice production levels across Asia. Hybrid rice area issteadily expanding across Asia with potential yield advantagesranging from 15 to 20% over the high yielding semi-dwarf varieties.Hybrid rice is commercially grown in many countries across theglobe particularly in China, where it accounts for half of the riceacreage and contributes 60% of the total rice yield of the country(Cheng et al., 2004). Despite the impressive yield gain throughheterosis breeding the problem of hybrid combination being

susceptible to many diseases due to narrow genetic base stillpersists. Amongst the major prevalent diseases, bacterial blight(BB) disease happens to be one of the major barriers to high andsustainable hybrid rice production (Virmani, 1996).

BB disease of rice (Oryza sativa), caused by Xanthomonas oryzaepv. oryzae (Xoo), is one of the most widely distributed and devas-tating rice diseases for both inbred and hybrid rice in Asia. BBdamage on this crop was reported to range from 20 to 30% andcould sometimes get as high as 50% (Mew, 1987; Ou, 1985). The BBdisease became prominent in the 1960s, when new high yieldingvarieties were first developed and introduced (Mew,1987). Varietalresistance is the main strategy for controlling this disease.

As many as 31 resistance genes (22 dominant and 9 recessive) toBB have been identified and registered (Chu et al., 2006; NiÑo-Liuet al., 2006; Wang et al., 2009). Among them, Xa4 has beenwidely used in developing almost all the commercial hybrids andconventional cultivars of indica rice in the main rice growing

q Yong-Li Zhou, Veronica NE Uzokwe and Cong-He Zhang contributed equally tothis work.* Corresponding author. Tel.: þ86 10 82105854; fax: þ86 10 82108559.

E-mail addresses: xujlcaas@yahoo.com.cn, xujl@caas.net.cn (J.-L. Xu).

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Crop Protection 30 (2011) 637e644

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regions of China (Zhang, 1991). This narrow genetic basis forresistance to BB in Chinese rice cultivars has imposed strongselection on the pathogen population, resulting in a dramaticincrease in frequency of race V that is virulent to both Xa4 andXa3 during the past decade in China (Zhang et al., 1996). Currentlymost of the parental lines of the popular hybrid rice in China aresusceptible to most Chinese and Philippines races (Xia et al., 2009).

Another widely used Xa21 gene in rice breeding programs wasidentified from the wild rice O. longistaminata (Khush et al., 1990;Song et al., 1995). This gene was recently broken down by newvirulent strains in Southern and Yangtze River Valley in China (Zenget al., 2002; Zhang, 2009).

Amongst all the known resistance genes to BB, Xa23, a singlecompletely dominant resistance gene effective at all growth stages,identified fromwild rice species of Oryza rufipogon emerged as themost promising one (Zhang et al., 1998, 2001). The Xa23 gene wasfound to be highly resistant to all the 20 strains of BB consisting of10 Philippine races (P1eP10), 7 Chinese pathotypes (C1eC7) and3 Japanese races (T1eT3) at the maximum tillering stage. It wasindicated that Xa23 gene possessed a broad resistance spectrumamong all known R genes (Zhang et al., 2001). In this study, weevaluated the practical utility of the Xa23 gene in improvement ofBB resistance for hybrid rice through introgression of the gene intothree popular restorer lines to develop new BB resistant restorerlines and their respective hybrid rice combinations.

2. Materials and methods

2.1. Parental materials

In this study, CBB23(B) carrying Xa23 gene, derived from BC4F5progeny of the cross between an O. rufipogon accession (RBB16, theXa23 donor) and a recipient indica BB susceptible variety IR24(Zhang, 2005), was used as the donor of resistance to BB. Threepopular restorer lines i.e. Minghui63, YR293 and Y1671 for wildabortive cytoplasmic male sterility (WAeCMS), also widely used forhybrid rice seed production in China but highly susceptible to BBwere used as recurrent parents. These three restorers were derivedfrom the following respective crosses IR30/Gui630, 9311/Min-ghui63 and YR293/02428 through pedigree breeding approach. Theseeds of the three restorer lines were provided by the Win-AllHi-Tech Seed Company Limited, Anhui province, China. The IR24variety 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 transferXa23 gene into elite restorer parental lines. The three restorer lineswere used as female to cross with CBB23(B) to produce three F1sand then backcrossed with the F1s to get three BC1F1 populations.A mini-scale DNA isolation for PCR analysis was carried out onthreeeleaf old seedlings in the seedbed for the parents and BC1F1plants following the procedure of Zheng et al. (1995). Foregroundselection against Xa23 gene was used to select plants carrying Xa23gene prior to transplanting through marker-assisted selection(MAS) using a closely linked SSR marker (RM206) of 1.9 cm apartfrom the Xa23 locus (Pan et al., 2003).

The 25 days old seedlings with Xa23 heterozygous gene selectedfrom BC1F1 were transplanted in the screenhouse. At early tillering(3 weeks after transplanting) and booting (7 weeks after trans-planting) stages, three to six uppermost leaves of each plant wereinoculated with a highly virulent Chinese Xoo strain, GD1358 usingthe leaf-clipping method (Kauffman et al., 1973). Resistant BC1F1plants that were determined by MAS and inoculation were furtherbackcrossed to the respective recurrent parents to produce their

BC2F1 populations. Consecutive backcrosses were carried out inthe same way to produce BC4F1 populations.

Resistant plants carrying homozygous Xa23 gene and identicalphenotype as that of the recurrent parents were identified by MASand inoculation in BC4F2. These phenotypically identical BB resis-tant plants to their respective restorer lines were then crossed withthe three respective WAeCMS lines (Zhenshan97A, NongfengA andZhong9A), to test their restoration and specific combining ability.All the BC4F3 lines were confirmed for the presence of Xa23 geneand studied for their phenotypic similarities to their respectiveoriginal restorer lines. The F1 progenies of the test-crosses involvingthe selected BB restorer lines in BC4F2 with their respective CMSlines were evaluated for their spikelet fertility and level of heter-osis. Based on the performance of BB resistance and morphologicalcharacters of BC4F3 lines and the level of heterosis of theirrespective test-crosses, only one line that was phenotypicallyidentical to the recipients with homozygous Xa23 gene was finallyselected from the progeny of each recipient, and it was designatedas Minghui63eXa23, YR293eXa23 and Y1671eXa23 for furtherevaluation.

2.3. Evaluation of BB resistance and agronomic traits

The original restorer lines Minghui63, YR293 and Y1671 andtheir derived BB resistant restorer lines Minghui63eXa23,YR293eXa23 and Y1671eXa23 were crossed with three WAeCMSlines, Zhenshan97A, NongfengA and Zhong9A to produce hybrid F1for evaluation of BB resistance and agronomic traits. BB resistanceevaluation of these materials were carried out by sowing them on20th of April, 2008 and 30 days old seedlings were transplantedinto a nine-row plot of six plants per row with a plant to rowspacing of 17 � 20 cm and three replications in the screenhouse ofthe Institute of Crop Sciences, Chinese Academy of AgriculturalSciences, Beijing, China. Three plants were artificially inoculated bypartial clipping of 3e6 uppermost leaves separately (Kauffmanet al., 1973) for each of the 7 different Chinese Xoo races (C1eC7)(Fang et al., 1990) and 10 Philippine Xoo races (P1eP10) at theearly tillering (3 weeks after transplanting) and booting (7 weeksafter transplanting) stages. Lesion length (LL) was measured on allpartially clipping inoculated leaves after 2e3 weeks of inoculation.

BB field evaluation and agro-morphological characterization ofthe newly developed restorer lines (Minghui63eXa23, YR293eXa23and Y1671eXa23) and their derived hybrid combinations i.e. Sha-nyou63eXa23 (Zhenshan97/Minghui63e Xa23); Fengyou293eXa23(NongfengA/YR293eXa23) and Zhongyou1671eXa23 (Zhong9A/Y1671eXa23), were compared to their original restorer lines (Min-ghui63, YR293 and Y1671) and their respective hybrid combinationsi.e. Shanyou63 (Zhenshan97/Minghui63); Fengyou293 (NongfengA/YR293) and Zhongyou1671 (Zhong9A/Y1671) by conducting fieldtrials both under disease and disease free environments in arandomized complete block design (RCBD) with 3 replications inHainan breeding station during 2009 winter season. Testing of allthe materials under severe diseased condition was carried out byplanting each line in three rows plot with twelve plants per rowand a spacing of 17 � 20 cm in a RCBD with three replications. Themiddle row in each plot was inoculated with Xoo race C5. However,for testingunderdisease free condition, thesamesetofmaterialswasplanted in the field without artificial BB inoculation and any changein plot size and layout.

Measurements were taken on ten plants from the middle rowin each plot for different agro-morphological traits, such as plantheight (PH), heading date (HD), panicle length (PL), panicle numberper 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 atboth early tillering and booting stages to all 7 Chinese (C1eC7) and10 Philippine (P1eP10) Xoo races, while the three sterile lines,Zhenshan97A, NongfengA and Zhong9A showed complete sus-ceptibility (Table 1, Fig. 1). The three restorer lines Minghui63,YR293 and Y1671 were found to be susceptible to most of the 17races, especially to the Chinese race 5 (GD1358) and the Philippinerace 6 (PXO99), that shows strongest pathogenicity in China andPhilippines at both early tillering and booting stages with similarLL to that of the susceptible variety IR24 (Table 2, Fig. 1).

The F1 plants derived from the crosses between the threerestorer lines and the CBB23(B) were each divided into two groupsand inoculated with Chinese race 5 (GD1358) and Philippine race 6(PXO99). All the F1 plants exhibited resistance to Xoo race PXO99 atearly tillering stage with the average LL ranging from 1.1 to 1.5 cmbut were highly resistant to GD1358with the LL ranging from 0.2 to0.9 cm, respectively (Fig. 2). At booting stage, all F1 plants showedenhanced resistance to PXO99 and GD1358 compared with thoseat 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 indifferent backgrounds and developmental stages by comparable LLto that of CBB23(B) thereby clearly indicating that the Xa23 couldcompletely express its dominant resistance in different geneticbackgrounds and varied growth stages.

Table 1Resistant 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) atearly tillering (TS) and booting (BS) stages.

Growth stage Xoo Resistant donor and the 3 parental CMS lines

Racea Strain IR24 (CK) CBB23(B) Zhenshan97A NongfengA Zhong9A

TS C1 JS97-2 9.5 � 2.3 0.3 � 0.2 7.5 � 2.8 7.5 � 0.5 8.7 � 1.6C2 KS6-6 10.3 � 1.8 0.5 � 0.1 8.8 � 1.3 7.7 � 0.8 10.2 � 1.0C3 JS158-2 13.5 � 1.1 0.3 � 0.1 7.2 � 1.6 13.0 � 2.7 8.2 � 1.1C4 Z173 10.3 � 1.7 0.4 � 0.2 10.0 � 3.4 15.3 � 2.3 12.9 � 1.9C5 GD1358 14.8 � 2.6 0.4 � 0.1 12.8 � 2.1 16.1 � 1.8 15.1 � 1.7C6 OS198 9.9 � 1.0 0.3 � 0.1 12.4 � 1.7 9.3 � 2.8 9.7 � 2.1C7 JS49-6 9.3 � 1.3 0.3 � 0.0 11.2 � 2.3 10.5 � 1.9 7.7 � 1.0P1 PXO61 11.5 � 2.1 0.5 � 0.1 9.5 � 1.2 6.2 � 1.3 6.6 � 1.3P2 PXO86 12.8 � 2.9 0.6 � 0.2 12.7 � 1.5 13.3 � 1.3 10.8 � 2.9P3 PXO340 12.5 � 2.4 0.3 � 0.0 9.8 � 2.9 10.7 � 1.8 12.8 � 2.0P4 PXO71 10.4 � 1.0 0.7 � 0.2 11.7 � 3.1 8.5 � 1.5 14.7 � 1.0P5 PXO112 11.5 � 2.0 0.5 � 0.0 10.2 � 1.3 7.0 � 0.5 7.8 � 1.3P6 PXO99 14.2 � 1.7 0.5 � 0.1 14.9 � 2.3 18.7 � 1.1 16.2 � 1.8P7 PXO145 13.5 � 0.9 0.2 � 0.1 9.7 � 0.3 6.8 � 1.0 9.8 � 1.3P8 PXO280 8.8 � 1.7 0.3 � 0.0 7.9 � 0.9 12.3 � 1.9 13.5 � 1.3P9 PXO339 10.2 � 0.5 0.1 � 0.1 12.6 � 1.4 16.5 � 3.8 12.8 � 1.3P10 PXO341 13.2 � 1.8 0.4 � 0.5 12.0 � 2.7 11.5 � 4.0 18.5 � 2.3

BS C1 JS97-2 11.9 � 2.3 0.2 � 0.1 8.8 � 0.3 7.7 � 0.7 7.4 � 1.2C2 KS6-6 12.4 � 0.7 0.5 � 0.0 10.9 � 0.7 6.4 � 1.2 8.8 � 0.6C3 JS158-2 15.3 � 1.5 0.2 � 0.1 11.8 � 1.0 12.3 � 0.8 9.8 � 2.5C4 Z173 13.6 � 1.3 0.4 � 0.3 14.1 � 1.0 13.2 � 1.7 7.8 � 1.2C5 GD1358 16.2 � 1.1 0.3 � 0.1 16.5 � 1.3 17.9 � 1.3 14.6 � 1.5C6 OS198 15.1 � 0.8 0.5 � 0.3 8.9 � 0.6 7.2 � 0.3 8.9 � 0.8C7 JS49-6 12.8 � 1.6 0.2 � 0.1 9.7 � 0.7 6.9 � 0.7 9.8 � 0.8P1 PXO61 16.1 � 2.3 0.3 � 0.1 7.8 � 0.6 8.6 � 1.2 7.8 � 1.9P2 PXO86 15.2 � 1.4 0.4 � 0.2 7.4 � 0.5 10.2 � 1.9 10.8 � 1.7P3 PXO340 13.9 � 0.5 0.5 � 0.3 6.2 � 1.1 13.0 � 0.7 10.6 � 2.0P4 PXO71 13.6 � 1.2 0.6 � 0.2 4.7 � 0.7 12. 2 � 0.8 11.6 � 1.7P5 PXO112 12.7 � 2.0 0.6 � 0.1 10.9 � 0.5 8.2 � 1.3 6.5 � 1.3P6 PXO99 18.2 � 3.1 0.4 � 0.1 20.8 � 1.4 18.5 � 2.1 17.6 � 1.7P7 PXO145 14.1 � 0.2 0.3 � 0.2 13.8 � 0.5 8.3 � 1.5 7.0 � 1.4P8 PXO280 11.9 � 1.3 0.5 � 0.2 14.0 � 1.1 10.6 � 1. 6 9.0 � 2.3P9 PXO339 13.2 � 0.7 0.2 � 0.1 13.2 � 0.6 6.6 � 1.5 9.5 � 0.9P10 PXO341 12.8 � 1.2 0.3 � 0.1 13.7 � 0.6 11.7 � 3.3 10.8 � 3.5

a C1eC7 and P1eP10 represent 7 Chinese and 10 Philippines races of Xoo, respectively.

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 earlytillering stage (3 weeks after transplanting), B: Effects at the booting stage (7 weeksafter transplanting). Leaves 1e7 represent the effects on CBB23(B), Minghui63, YR293,Y1671, Zhenshan97A, NongfengA and Zhong9A.

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Table 2Resistance 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 Xanthomonasoryzae pv. oryzae at early tillering (TS) and booting (BS) stages.

Growth stage Race Minghui63 Minghui63eXa23 YR293 YR293eXa23 Y1671 Y1671eXa23

TS C1 2.5 � 0.3 0.2 � 0.1 10.7 � 1.5 0.3 � 0.2 2.3 � 0.5 0.1 � 0.0C2 15.7 � 4.0 0.2 � 0.0 9.2 � 0.8 0.3 � 0.2 5.5 � 1.3 0.1 � 0.0C3 5.5 � 0.9 0.3 � 0.0 8.0 � 1.5 0.1 � 0.1 9.4 � 2.4 0.1 � 0.1C4 13.5 � 2.1 0.8 � 0.1 10.7 � 3.5 0.2 � 0.1 10.1 � 3.0 0.4 � 0.2C5 12.3 � 1.9 0.3 � 0.2 10.7 � 1.8 0.2 � 0.1 10.3 � 1.5 0.1 � 0.0C6 6.7 � 0.8 0.4 � 0.3 12.2 � 1.6 0.1 � 0.0 11.7 � 2.5 0.4 � 0.6C7 12.7 � 1.1 0.3 � 0.0 9.5 � 1.3 0.2 � 0.0 9.5 � 2.0 0.2 � 0.1P1 14.8 � 1.4 0.4 � 0.2 9.8 � 1.9 0.3 � 0.2 11.7 � 2.0 0.1 � 0.1P2 13.3 � 4.3 0.7 � 0.2 6.5 � 1.3 0.2 � 0.0 9.1 � 1.5 0.2 � 0.0P3 11.7 � 2.8 0.3 � 0.2 7.4 � 1.2 0.3 � 0.2 12.7 � 1.5 0.3 � 0.2P4 12.0 � 1.3 0.3 � 0.2 12.8 � 1.2 0.2 � 0.0 9.3 � 1.5 0.2 � 0.0P5 10.0 � 2.0 0.2 � 0.1 3.1 � 0.4 0.3 � 0.1 9.8 � 2.5 0.1 � 0.0P6 14.8 � 2.0 0.4 � 0.2 12.5 � 2.0 0.8 � 0.2 12.3 � 2.9 0.3 � 0.1P7 4.3 � 1.9 0.3 � 0.1 13.7 � 2.5 0.2 � 0.1 6.3 � 1.2 0.1 � 0.0P8 12.0 � 2.7 0.2 � 0.1 12.7 � 3.1 0.2 � 0.1 9.2 � 1.6 0.1 � 0.0P9 11.8 � 1.9 0.1 � 0.0 9.5 � 1.3 0.2 � 0.2 8.0 � 2.1 0.1 � 0.0P10 12.1 � 1.5 0.2 � 0.1 8.2 � 1.6 0.2 � 0.1 9.5 � 4.1 0.1 � 0.0

BS C1 1.2 � 0.1 0.2 � 0.1 1.3 � 0.1 0.2 � 0.0 1.7 � 0.3 0.1 � 0.0C2 1.5 � 0.2 0.2 � 0.0 1.2 � 0.2 0.1 � 0.1 3.5 � 0.5 0.2 � 0.1C3 2.9 � 0.7 0.1 � 0.0 11.9 � 4.9 0.1 � 0.0 2.9 � 0.5 0.1 � 0.0C4 13.6 � 2.3 0.7 � 0.1 2.2 � 0.4 0.2 � 0.1 6.0 � 1.5 0.2 � 0.1C5 13.2 � 0.3 0.2 � 0.0 15.1 � 1.9 0.1 � 0.0 14.7 � 2.6 0.3 � 0.1C6 6.8 � 1.3 0.5 � 0.2 8.5 � 4.2 0.1 � 0.1 2.7 � 1.4 0.1 � 0.0C7 10.4 � 1.3 0.1 � 0.0 12.3 � 2.3 0.1 � 0.0 6.3 � 1.6 0.1 � 0.0P1 7.5 � 0.6 0.3 � 0.2 4.5 � 1.7 0.3 � 0.1 5.4 � 2.4 0.3 � 0.2P2 8.4 � 1.7 0.6 � 0.4 1.6 � 0.31 0.2 � 0.0 6.9 � 1.6 0.3 � 0.1P3 4.3 � 0.8 0.8 � 0.3 1.8 � 0.75 0.2 � 0.0 6.7 � 1.0 1.0 � 0.3P4 4.6 � 0.6 1.0 � 0.4 15.2 � 4.3 0.6 � 0.3 5.6 � 0.8 0.2 � 0.1P5 4.9 � 0.9 0.3 � 0.2 1.2 � 0.0 0.1 � 0.1 3.5 � 0.8 0.2 � 0.1P6 17.2 � 1.4 0.4 � 0.1 16.5 � 2.9 0.4 � 0.1 19.5 � 3.1 0.5 � 0.1P7 2.3 � 0.4 0.3 � 0.1 5.7 � 2.3 0.2 � 0.1 5.3 � 0.9 0.1 � 0.0P8 3.3 � 0.1 0.3 � 0.2 5.2 � 0.5 0.1 � 0.0 8.5 � 0.5 0.1 � 0.0P9 2.4 � 0.5 0.1 � 0.0 6.1 � 2.5 0.1 � 0.0 9.3 � 2.6 0.1 � 0.1P10 3.50 � 0.2 0.2 � 0.1 1.4 � 0.1 0.1 � 0.0 8.14 � 1.7 0.1 � 0.0

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 F1plants 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 plantsinfected with the Philippine race 6 at the booting stage.

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3.2. Evaluation of BB resistance of the newly-bred restorer lines andtheir hybrids

The BB resistance of the newly developed restorer lines (Min-ghui63eXa23, YR293eXa23 and Y1671eXa23) is presented inTable 2 by comparing the average LL with their original versionscaused by the 10 Philippine and 7 Chinese Xoo races at the earlytillering and booting stages. All improved restorer lines carryingXa23 showed a high level of resistance to all Xoo races includingthe highly virulent Chinese race 5, GD1358 and the Philippinerace 6, PXO99 with LL < 1.0 cm even at the early tillering stage,thus indicating that Xa23 gene possessed a broad spectrum of BB

resistance to over 17 races tested. In addition, no significantdifferences existed for BB resistance for Xa23 gene in differentrestorer line backgrounds.

The hybrids derived from the newly developed BB resistantrestorer lines were highly significant for their resistance to PXO99and GD1358, with the average LL ranging from 1.0 to 1.5 cm and0.4e1.2 cm at early tillering stage; and from 0.9 to 1.4 cm and0.5e0.7 cm at booting stage, respectively, as compared to theiroriginal hybrid combinations with the three different sterile linesZhenzhan97A, NongfengA and Zhong9A, respectively (Table 3 andFig. 3). No significant differences were observed for resistance toPXO99 and GD1358 for the hybrids derived from each BB resistantrestorer line and different sterile lines in both the two stages.This further indicated that the expression of BB resistance of Xa23gene was independent of genetic background of the male sterilelines.

3.3. Agronomic characterization and grain yield performance of thenewly developed restorer lines and their hybrid combinations

The three newly developed BB resistant restorers and their cor-responding original versions consisting of Minghui63eXa23 andMinghui63; YR293eXa23 and YR293; Y1671eXa23 and Y1671 werefound to be identical for all the agronomic traits studied underdisease free field conditions. Likewise, we found the same fortheir hybrid combinations i.e. Shanyou63eXa23 and Shanyou63;Fengyou293eXa23 and Fengyou293; Zhongyou1671eXa23 andZhongyou1671 (Table 4). However, Minghui63eXa23 showedsignificantly higher 1000-grain weight and spikelet fertility thanMinghui63 under severe diseased conditions. Grain yield of Sha-nyou63eXa23 was significantly higher than that of Shanyou63 onaccount of pronounced differences in 1000-grain weight andspikelet fertility (Table 4). A similar pattern was observed for theother two BB resistant restorers and their derived hybrids. TheYR293eXa23 and Y1671eXa23 lines had significantly higher 1000-grain weight, spikelet fertility and grain yield than YR293 andY1671. Fengyou293eXa23 and Zhongyou1671eXa23 had signifi-cantly higher grain yield than Fengyou293 and Zhongyou1671(Table 4). Grain yield and its related traits such as spikelet fertility

Table 3Resistance levels of F1 rice hybrids with and without Xa23 gene against the virulentChinese race 5 (C5) and the Philippine race 6 (P6) of Xanthomonas oryzae pv. oryzaeboth at early tillering and booting stages.

F1 combination Early tillering stage Booting stage

P6 C5 P6 C5

NongfengA/Minghui63 22.0 � 5.0 21.3 � 3.6 18.9 � 4.3 7.9 � 1.0Zhenshan97A/Minghui63

(Shanyou63)20.8 � 3.5 19.6 � 3.2 16.3 � 2.9 8.2 � 1.1

Zhong9A/Minghui63 21.3 � 3.7 20.0 � 4.3 19.7 � 3.8 7.7 � 1.4NongfengA/Minghui63eXa23 1.2 � 0.5 1.0 � 0.7 1.4 � 0.3 0.5 � 0.2Zhenshan97A/Minghui63eXa23

(Shanyou63eXa23)1.5 � 0.6 1.2 � 0.4 1.4 � 0.5 0.6 � 0.4

Zhong9A/Minghui63eXa23 1.1 � 0.1 1.0 � 0.4 1.3 � 0.4 0.4 � 0.3

NongfengA/YR293 (Fengyou293) 21.9 � 4.9 15.9 � 2.8 20.4 � 5.7 11.3 � 2.5Zhenshan97A/YR293 22.5 � 3.8 16.0 � 4.2 20.0 � 5.0 10.8 � 2.3Zhong9A/YR293 22.1 � 3.1 16.8 � 2.9 17.3 � 2.4 10.6 � 1.5NongfengA/YR293eXa23

(Fengyou293eXa23)1.5 � 0.3 0.4 � 0.3 1.2 � 0.4 0.5 � 0.2

Zhenshan97A/YR293eXa23 1.4 � 0.2 0.6 � 0.3 1.4 � 0.2 0.6 � 0.2Zhong9A/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 � 4.4Zhenshan97A/Y1671 20.7 � 2.5 20.8 � 2.9 18.6 � 3.5 17.8 � 3.8Zhong9A/Y1671 (Zhongyou1671) 22.2 � 3.9 20.9 � 2.9 19.6 � 4.7 19.0 � 3.5NongfengA/Y1671eXa23 1.0 � 0.5 0.7 � 0.6 1.4 � 0.4 0.7 � 0.4Zhenshan97A/Y1671eXa23 1.2 � 0.2 0.6 � 0.5 1.4 � 0.4 0.5 � 0.5Zhong9A/Y1671eXa23

(Zhongyou1671eXa23)1.0 � 0.6 0.8 � 0.6 0.9 � 0.4 0.5 � 0.4

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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and 1000-grain weight were higher for Shanyou63eXa23, Fen-gyou293eXa23 and Zhongyou1671eXa23 than Shanyou63, Fen-gyou293 and Zhongyou1671, respectively.

4. Discussion

Prevalence of BB disease in China is jeopardizing the expansionof the popular hybrids as a result of their susceptibility. The threedifferent restorer lines i.e. Minghui63, YR293 and Y1671 have beenwidely utilized in hybrid rice production in China. Incorporation ofa broad spectrum resistance gene into these restorer lines willgreatly improve BB resistance of hybrid rice in China. Carefulanalysis of the literature showed CBB23(B) with Xa23 gene to be aneffective donor against all races collected from China, Philippinesand Japan (Zhang, 2005). In this study we incorporated a broadspectrum BB resistance Xa23 gene into the three commerciallypopular restorer lines using MAS-based backcrossing and stringentphenotypic selections within a short period. Minghui63eXa23,YR293eXa23, Y1671eXa23 and their hybrid combinations, Sha-nyou63eXa23, Fengyou293eXa23 and Zhongyou1671eXa23exhibited a significant yield advantage over their original coun-terparts with the same level and spectrum of resistance as that of

the resistant donor, CBB23(B). This study clearly indicated thatXa23 gene is valuable for the improvement of BB resistance inhybrid rice production. Improvement of restorer lines for BBresistance through MAS-based backcross schemes using Xa23 genewith broad spectrum resistance raised certain key issues in ricebreeding for increasing production.

Firstly, the BB resistance improvement of parental lines ofhybrids is much different from that of conventional varieties. Inthe backcross progenies of restorer parental lines, selections wereperformed not only for similarity to the recurrent parents, but alsofor their fertility restoring gene(s) and specific combining abilityto the CMS lines. The expected frequencies for individuals homo-zygous genotype of the recurrent parent on a single locus basis is0.5, 0.75, 0.875, and 0.9375 in BC1F1, BC2F1, BC3F1 and BC4F1,respectively. All individuals in each backcross generation carryeither the homozygous or heterozygous genes from the recurrentparent and the majority of them are homozygous genotype.However, the qualitatively inherited fertility restoring gene(s) ofthe recurrent parent most probably maintained in the subsequentbackcross progenies. Specific combining ability being quantita-tively inherited (Zha et al., 2008) from the recurrent parents willbe gradually recovered through backcrossing in different individ-uals to a varying extent. In each backcross generation in this study,MAS for Xa23 gene before transplanting combined with resistanceconfirmation by inoculating twice at early tillering and bootingstages and very strict phenotypic selections against the recurrentparents at maturity were performed over a relatively larger back-cross population. Eighty BC4F2 plants, identical to each recurrentparent derived from the progenies of each restorer line wereselected to make test-crosses with the respective male sterile lines.Normal spikelet fertility was observed in most of the test-crossesbut their grain yields varied (data not shown). Some test-crossesshowed high spikelet fertility and good specific combining ability(high grain yields), implying that their corresponding BB resistantlines were identical to their recurrent parents. Thus, it is clear fromthis study that a minimum of four backcrosses in conjunctionwith stringent phenotypic selection for the recurrent parent in eachBC progenies and combining ability testing on a relatively largescale, guarantees the recovery of recurrent parental characteristicseven without MAS against the background of the recurrent parentsas 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 breedingprogram for BB resistance was observed in this study. It wasreported that the level of hybrid rice resistant to Xoo was deter-mined by the restorer line when CMS was susceptible, whereasthe resistance level of F1 was controlled by the interaction betweenCMS and restorer line when CMS was resistant (Lu and Shen, 1984;Wang et al., 2000). Majority of the male sterility sources widelyused in China such as wild abortive (WA), dwarf abortive (DA),Gang, D, Yinshui and Honglian types were found to be susceptibleto BB (Zhang, 2009). As many as 31 resistance genes to BB havebeen reported (NiÑo-Liu et al., 2006; Wang et al., 2009), but mostof these genes have not been used in breeding programs due toeither their lower resistance level or narrow resistance spectrum.Hitherto, only Xa4 and Xa21 have been widely used in hybrid ricebreeding in China. However, these two genes do not confer theideal kind of resistance for BB in breeding programs. The level ofresistance of F1 rice hybrids carrying Xa4 gene to BB is low becauseof its incomplete dominance nature. While Xa21 gene with a broadresistance spectrum is a completely dominant, but it generallyexpresses resistance from the late tillering stage onwards and itsresistance level is affected by the genetic background (Zhai et al.,2001). Conversely, Xa23 gene has a broader resistance spectrumto different BB races collected from the Philippines, Japan and

Table 4Agronomic performance of restorer lines with and without Xa23 gene and theirrespective commercial hybrids tested under both severe disease (artificial inocula-tion) and disease free field conditions during 2009 winter season in Hainan.

Restorer lines/Hybrids PH(cm)

HD(d)

PL(cm)

PN SNP SF (%) GWT(g)

GY(g/plant)

Without inoculationMinghui63 95.5 105.5 21.5 7.6 114.5 85.6 27.5 17.3Minghui63eXa23 96.1 104.7 20.1 7.9 112.3 85.7 27.1 16.9Shanyou63 101.4 96.3 23.2 8.3 131.4 82.7 27.6 22.2Shanyou63eXa23 100.3 95.5 22.5 8.5 129.5 83.3 28.1 21.5LSD(0.05)a 2.5 0.9 1.6 0.5 5.1 2.3 1.1 1.7LSD(0.01)b 4.8 1.7 3.1 1.1 9.4 4.3 1.8 3.2YR293 99.8 110.7 22.3 7.3 122.3 85.6 29.2 17.1YR293eXa23 98.7 109.9 21.8 7.5 121.5 86.9 29.1 17.5Fengyou293 105.5 100.7 24.4 8.1 140.2 84.7 28.8 23.1Fengyou293eXa23 104.1 101.5 23.2 8.3 139.2 85.1 28.5 24.7LSD(0.05)a 1.9 2 1.4 0.4 3.1 2.1 1.5 1.9LSD(0.01)b 3.4 3.7 2.5 0.7 5.8 3.5 2.8 3.7Y1671 103.5 109.3 23.1 6.2 124.2 84.6 28.8 16.1Y1671eXa23 104.2 109.6 23.6 6.5 125.8 86.5 27.9 17.1Zhongyou1671 110.5 101.4 24.1 7.8 135.4 83.6 28.1 22.8Zhongyou1671eXa23 109.2 102.1 24.8 8.7 136.7 85.5 28.1 24.9LSD(0.05)a 1.4 2.3 0.9 1.1 4.1 2.5 1.2 2.4LSD(0.01)b 2 4.4 1.6 1.9 7.8 4.6 2.3 3.1

Inoculated by Xoo. C5 at the booting stageMinghui63 95.1 105.1 21.3 7.6 113.5 80.9 25.6 13.5Minghui63eXa23 96.3 104.5 21.2 7.8 112.1 84.5 27.3 15.1Shanyou63 102.7 97.5 23.1 8.4 127.5 75.5 25.2 17.5Shanyou63eXa23 100.6 96.8 22.2 8.6 128.1 83.6 27.9 21.3LSD(0.05)a 2.5 0.9 1.6 0.5 5.1 2.3 1.3 2.8LSD(0.01)b 4.8 1.7 3.1 1.1 9.4 4.3 2.4 5.2YR293 98.9 109.9 22.1 7.7 120.6 77.2 26.1 14.9YR293eXa23 98.3 110.2 22.3 7.6 122.1 85.5 28.9 17.6Fengyou293 105.1 101.8 24.2 8 138.6 75.7 25.7 17.9Fengyou293eXa23 103.8 102.3 23.1 8.4 140.1 83.1 28.1 23.5LSD(0.05)a 2.1 2.2 1.3 0.6 3.3 3.5 1.3 2.6LSD(0.01)b 3.8 4 2.4 1.1 6.1 6.7 2.1 5Y1671 103.2 109.5 22.9 6.4 123.3 77.6 25.7 14.5Y1671eXa23 104.5 110.3 23.3 6.8 125.2 82.3 27.2 17.6Zhongyou1671 109.1 100.8 24.2 7.7 131.3 77.7 26.3 18.1Zhongyou1671eXa23 108.6 102.3 24.7 7.9 133.9 83.9 28.3 22.5LSD(0.05)a 1.9 3 1.1 1.3 3.3 3.5 1.3 2.2LSD(0.01)b 3.5 5.8 2 2.4 6 6.7 2.4 4

PHeplant height, HDeheading date, PLepanicle length, PNepanicle number perplant, SNPespikelet number per panicle, SFespikelet fertility, GWTe1000-grainweight, GYegrain yield per plant.

a Least significant differences at 0.05 probability level.b Least significant differences at and 0.01 probability level.

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China, and shows very high resistance level during all the growthstages besides its strong effectiveness to inheritance of resistance(Zhang et al., 1998, 2001).

Recently, Xa23 has been introduced into several conventionalcultivars and restorer lines (Luo et al., 2005; Li et al., 2006; Qin et al.,2007; Zhou et al., 2009). However, in those studies the resistancelevel of F1 hybrid carrying Xa23 gene in different genetic back-grounds was not evaluated. We observed that the three differentrestorer lines with Xa23 and their derived hybrid combinationswith the three different sterile lines were highly resistant to all 17races tested at both seedling and booting stages with no significantdifferences for LL. This indicated that there was no genetic back-ground effect on the expression of this gene, thus strongly sug-gesting that Xa23 is of great value in breeding rice hybrids with BBresistance.

Thirdly, proper utilization and deployment of Xa23 gene in thecontrol of BB disease need to be outlined for protecting or delayingthe break down of this valuable gene. A sharp percentage declinefrom 35.6 to 10.7% in the release of BB resistant threeeline indicahybrids in the period (1996e2005) as compared to the duration(1973e1986) in China was primarily due to the ineffectiveness ofXa4 gene to some virulent strains of Xoo (Zhang, 2009). Thisresulted in the introduction of Xa21 gene into several rice varietiesand 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 inChina and other Asian countries led to the break down of BBresistance of Xa21 gene (Lee et al., 1999; Marella et al., 2001; Zenget al., 2002). As indicated in this study, the three hybrid parentscarrying Xa23 did not only express high resistance with broadspectrum during the entire growth stages, but also exhibitedgood agronomic traits, that could serve as immediate sources of BBresistance for hybrid rice breeding and its direct use in hybrid riceseed production. This step towards the development of BB resistantrestorer lines will be playing a crucial role in controlling BB inChina and other Asian countries.

It is expected that overtime the new virulent Xoo races willcertainly overcome the Xa23 gene once it is widely used in breedingprograms. In order to delay the loss of another broad spectrumresistance gene such as Xa23, it is highly encouraged to use differentsources of resistance genes for BB in varietal development andeffective deployment strategies. Chen et al. (2000) and Zhai et al.(2001) developed a BB resistant restorer line, Minghui63eXa21 bytransgenic means. Therefore, BB resistant Shanyou63 producedfrom the two resistant restorer lines, Minghui63eXa23 and Min-ghui63eXa21 could be rotated for hybrid rice production. Anotherstrategy to achieve enhanced and durable resistance against plantdiseases is to pyramid Xa23 with other resistant genes therebyincreasing resistance level and spectrum as reported in all previousgeneepyramiding 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 geneticbackground with different resistance genes could be co-cultivated(Ayliffe et al., 2008; Zhu et al., 2000). BB resistant genes Xa23 andXa21 as reported above have been derived from wild rice and arenow being considered as of great value in hybrid rice breedingprograms among all reported genes (NiÑo-Liu et al., 2006). Carefulutilization of the broad spectrum BB resistant genes is urgentlyrequired to efficiently control BB and to delay the speed of devel-opment of new virulent Xoo strains by the way of mixed plantingof the hybrid rice combinations derived from the same geneticbackground restorer lines with Xa23 and Xa21, respectively. Simul-taneous search for new resistant sources for BB from other wildrice varieties needs to be carried out for diversifying varieties pos-sessing different sets of BB resistant genes.

Acknowledgements

The authors are grateful to the Ministry of Science and Tech-nology and the Ministry of Agriculture, the People’s Republic ofChina for their financial support under the scientific programs-‘863’ (2007AA10Z191, 2006AA10Z158), ‘948’ (2006-G51).

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