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Introgression of Xa4, Xa7 and Xa21 for resistance

Euphytica

DOI 10.1007/s10681-008-9653-1

Introgression of Xa4, Xa7 and Xa21 for resistance

to bacterial blight in thermosensitive genetic male sterile rice (Oryza sativa L.) for the development of two-line hybrids Loida M. Perez · Edilberto D. Redo?a ·

Merlyn S. Mendioro · Casiana M. Vera Cruz ·

Hei Leung

Received: 7 February 2007 / Accepted: 23 January 2008

? Springer Science+Business Media B.V. 2008

Abstract Bacterial blight (BB) of rice caused by X.oryzae pv. oryzae is a major production constraint in commercial hybrid rice production in the Philippines because most of the parental lines used in hybrid production do not carry resistance genes against the pathogen. In this study, three bacterial blight resis-tance genes, Xa4, Xa7 and Xa21, were introgressed to a temperature-sensitive genetic male sterile (TGMS1) line. A three-way cross of AR32-19-3-3/TGMS1// IRBB4/7 (PR36944) was made to produce 1,364 F2 plants carrying various combinations of Xa4, Xa7 and Xa21. Individual plants were characterized for reaction to bacterial blight PXO61 (race 1), PXO86 (race 2), PXO99 (race 6) and pollen sterility. Of 144 F2 plants demonstrating resistance against PXO61, PXO86 and PXO99, 22 exhibited highly resistant phenotypes with mean lesion lengths ranging from 0.37–2.97cm. Analysis of disease reaction identi W ed 20 potential TGMS F2 plants containing Xa4, Xa7 and Xa21 while 78 plants with Xa4+Xa7. Phenotypic and polymerase chain reaction (PCR) analyses con W rmed PR36944-450, PR36944-473 and PR36944-700 as homozygous for Xa7 and Xa21 and highly resistant to all three Xoo races. Fertility of PR36944-450 and PR36944-700 was restored at permissive temperature in a growth chamber. BB-resistant TGMS lines should facilitate breeding two-line hybrids in the tropics.

Keywords Bacterial blight · Disease resistance · Hybrid rice · Marker-aided selection Abbreviations

BB Bacterial blight

CMS Cytoplasmic male sterile

PCR Polymerase chain reaction

STS Sequence tagged site

TGMS Thermo-sensitive genetic male sterile

Xoo Xanthomonas oryzae pv. oryzae Introduction

Bacterial blight (BB) caused by Xanthomonas oryzae pv. oryzae (Xoo) is considered the most serious bacte-rial disease in rice-growing countries worldwide (Leung et al. 2004). The disease causes signi W cant yield losses particularly in hybrid rice in China, Viet-nam, Indonesia, India, and the Philippines (Amar

L. M. Perez (&)

Plant Breeding and Biotechnology Division, Philippine Rice Research Institute, Maligaya, Science City of Munoz, Nueva Ecija 3119, Philippines

e-mail: lmperez@https://www.doczj.com/doc/df12576638.html,.ph;

lmoreno_perez@https://www.doczj.com/doc/df12576638.html,

E. D. Redo?a · C. M. Vera Cruz · H. Leung International Rice Research Institute, DAPO 7777, Metro Manila, Philippines

M. S. Mendioro

Institute of Biological Sciences, University of the Philippines Los Ba?os, Laguna 4031, Philippines

Euphytica

2004; Chen et al. 2000; Suwarno et al. 2003; Hoan and Nghia 2003; Singh et al. 2005). In Southern Korea, BB caused reductions in crop yields and dete-rioration of grain quality (Shin et al. 2004). It was also reported as a major constraint in the irrigated rice belt comprising Punjab and the adjoining Northwest-ern states of India (Goel et al. 1998). In the Philip-pines, commercial hybrid rice varieties su V er from signi W cant yield losses due to BB infection primarily in the wet season because most of the component lines of the commercially released hybrid rice varie-ties are susceptible to virulent and prevalent races of Xoo in the W eld (Obien, PhilRice, personal communi-cation). Furthermore, in the production of hybrid seeds, X ag leaf clipping of the male sterile line is used as a means to facilitate the transfer of pollen from the male parent to the female parent (male sterile). This practice creates wounds in the male sterile plant, generating an avenue for the entry of pathogens. Consequently, hybrid seed yield is reduced, increas-ing the cost of hybrid rice production. The situation necessitates the use of disease-resistant germplasm in developing hybrid rice varieties.

Breeding hybrid rice requires the development of elite parental lines in either a three-line or two-line system (Virmani et al. 1997). In a three-line hybrid system, a cytoplasmic male sterile (CMS) line is the female parent that is crossed to the restorer line (pol-len parent) to produce the hybrid. A CMS plant is multiplied by crossing it with its maintainer line either manually or by natural outcrossing in an iso-lated plot to produce large quantities of seed (Virmani et al. 1997). An alternative way of exploiting hybrid rice potential is the use of thermo-sensitive genetic male sterile (TGMS) lines (Mou et al. 2003) as female parents in developing two-line hybrids. Theo-retically, TGMS are superior to CMS lines in that they do not require the use of a B line to produce seeds. The line becomes fertile when grown at tem-peratures not exceeding 30°C. Thus, less materials, time, and labor are needed to produce the hybrid seeds. However, TGMS lines are generally suscepti-ble to major rice diseases like bacterial blight. Incor-poration of useful alleles of resistance genes in such lines would help in developing germplasm stocks in two-line hybrid breeding programs.

Use of molecular marker technology in rice breed-ing has been shown e V ective in tracking introgressed genes from resistant donor parents to susceptible but elite rice cultivars. Collard and Mackill (2007) pro-vided a comprehensive review of marker assisted selection (MAS) and its role in precision plant breed-ing. Jiang et al. (2007) discussed the prospects of molecular breeding including MAS and genetic engi-neering in the context of controlling allelic variation for all genes of agronomic signi W cance. Zhang (2007) also discussed the combination of MAS and trans-genic approach for developing disease resistant rice cultivars. In a computer simulation study conducted in self-fertilizing crops, MAS was found to improve genetic responses and dramatically increased the fre-quencies of superior genotypes (Liu et al. 2004).

Several major resistance genes (Xa) are su Y ciently W ne-mapped or cloned for MAS applications for the improvement of bacterial blight resistance (Yoshim-ura et al. 1995; Huang et al. 1997; Cheng et al. 2007). Resistance gene Xa4 was mapped to a region less than 1cM on chromosome 11 in a genetic mapping popu-lation involving IRBB4 and IR24 (Sun et al. 2003). The cloned Xa21 gene (Song et al. 1995) was used to improve resistance to bacterial blight disease in ‘Minghui 63’ and ‘6078’ in a recurrent backcrossing program (Chen et al. 2000, 2001). Both Xa21 and Xa4 showed complete dominance against the avirulent Xoo races and had large residual e V ects against the virulent races (Li et al. 2001). Their results further indicated that high level and durable resistance against Xoo should be more e Y ciently achieved by pyramiding di V erent types of R genes. On the other hand, Xa7 together with xa5 were e V ective against Z-173, a strongly virulent strain of Xoo in China (Wang et al. 2005). In Vietnam, high level of resis-tance was observed in plants with Xa4+Xa7+Xa21 combinations of Xoo R genes (Du and Loan 2007). In India, marker assisted selection was applied to detect Xa4 and Xa21 from a cross between IR64 and IET-14444 (Davierwala et al. 2001). Restorer lines and their derived hybrids developed with Xa7 and Xa21 showed a higher level of resistance to BB than the restorers and derived hybrids having only one of the resistance genes (Zhang et al. 2006).

The objective of this study was to produce TGMS lines containing Xa4, Xa7, and Xa21 by a combina-tion of phenotyping against Xoo diagnostic strains and marker-aided selection. The use of markers allowed us to combine resistance genes despite their epistatic interactions. We showed that these pyramid lines were resistant to the most virulent and prevalent

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races of Xoo in the Philippines. We recovered at least two BB-resistant plants with restored fertility under permissive temperatures, representing improved TGMS materials for two-line hybrid breeding.

Materials and methods Plant materials

Two sources of BB resistance genes, IRBB4/7 (for Xa4 and Xa7) and AR32-19-3-3 (for Xa21) were used as donors. The genes were introgressed to TGMS1(PhilRice Genebank Acc. No. PRT-1), a temperature-sensitive genetic male sterile rice susceptible to the disease via a three-way crossing method (Fig.1).TGMS1 is the background parent of numerous TGMS selections with stable pollen fertility/sterility expres-sion. It is currently being used as female parent in developing two-line hybrid rice varieties in the Phil-ippines. F 1 progenies of the crosses were advanced to F 2 by sel W ng. A segregating population of 1,364 indi-vidual F 2 plants was obtained for the characterization of resistance or susceptibility to BB and expression of pollen sterility/fertility.

Inoculation of F 2 progeny with Xoo isolates

Three races of Xoo pathogen including race 1 (PXO61),race 2 (PXO86) and race 6 (PXO99) were used to

inoculate individual F 2 plants. Tillers of each F 2 plant were divided into three groups, each consisted of 2–3tillers for inoculation with a speci W c Xoo isolate.Approximately 3–4 leaves were inoculated per isolate for each F 2 plant. BB inoculation was evaluated as described by Kau V man et al. (1973).PCR analysis for Xa4, Xa7 and Xa21

Selected resistant F 2 plants showing 0.27 to 4.67cm mean lesion length after inoculation of Xoo were assayed for the presence of Xa4, Xa7 and Xa21. Some F 2 plants that exhibited pollen sterility at harvest were also included in PCR W ngerprinting assays. Nuclear genomic DNA was isolated from 4-week-old seed-lings using a micro-scale extraction of DNA (Fulton et al. 1995).

Tightly linked DNA markers including MP1F/MP2R for Xa4, M5F/R for Xa7 (Porter et al. 2003)and Xa21F/R for Xa21 were used to con W rm gene pyramids based on the presence or absence of diag-nostic DNA bands or allele sizes. PCR reactions were carried out in 20 l containing 2.5ng/ l genomic DNA, 2.5ng/ l primer, 0.125mM dNTPs and 1£PCR bu V er with MgCl 2. Thermal cycling conditions for Xa21 followed an initial denaturation at 94°C for 4min followed by 35 cycles of denaturation at 94°C for 1min, annealing at 50°C for 1min and extension at 72°C for 1min and 50secs, and a W nal extension at 72°C for 7min. For Xa4 and Xa7, thermal cycles involved 94°C initial denaturation for 4min followed by 36 cycles of 94°C for 1min, 55°C for 1min and 72°C for 2min, and W nal extension for 5min at 72°C.Detection of ampli W ed fragments was done by horizontal gel electrophoresis (1.2% agarose) using 5 l of PCR products. Running time was approximately 2–3h at 85–96V with 15–20min gel staining in ethi-dium bromide solution.

Microsatellite W ngerprinting of PR36944-450, PR36944-473 and PR36944-700

Genetic background analysis was carried out using microsatellite markers. Genomic DNA was obtained from leaf tissues of PR36944-450, PR36944-473 and PR36944-700 as previously described (Fulton et al.1995). Sixty-W ve microsatellite loci covering 12 chro-mosomes were used in W ngerprinting. PCR reactions were carried out in 20 l volume containing 1£ PCR

Fig.1 A three-way crossing method used to incorporate bacte-rial blight resistance genes from AR32-19-3-3 (Xa21 donor) and IRBB4/7 (Xa4 and Xa7 donor) to the susceptible parent TGMS1

TGMS1AR32-19-3-3IRBB4/7

x F 1

x (3-way cross) F 1

F 2

(No X a g ene)

(X a 21)

(X a 4/X a 7)

Euphytica bu V er (10mM Tris–HCl, pH8, 50mM KCl, 0.001%

gelatin), 1.5mM MgCl2, 100 M of each nucleotides

(dATP, dCTP, dGTP, dTTP), 0.25 M each of for-

ward and reverse primers, 2units of Taq polymerase,

and 25ng template DNA. PCR cycling conditions

were as follows: 94°C for 5min, 35 cycles of 1min at

94°C, 1min at 55°C and 2min at 72°C, and 72°C for

7min. Reactions were run in a 96-well thin walled

polycarbonate V-bottom microtiter plate (Costar,

Corning Inc., New York, USA) on a programmable

thermal cycler (MJ Research model PTC-100, Water-

town, MA 02172, USA). PCR ampli W cation was

checked by electrophoresis of 5 l of the reaction

product in 2.5% agarose. Final electrophoresis was

carried out in 6% denaturing polyacrylamide gel as

described (Panaud et al. 1996).

Data analysis

Reaction of the F2 progeny to Xoo was evaluated by

computing the mean lesion length from lesion lengths

(cm) on individual leaves inoculated per plant. Analy-

sis of variance (ANOVA) was conducted using a ran-

domized complete block design (RCBD) with lines

and isolates (Xoo races) as factors.

DNA fragments generated in PAGE were scored as

1 and 0 for the presence or absence of the allele,

respectively. The data were analyzed using the

UPGMA (Unweighted Pair Group Arithmetic Mean)

clustering method of the Numerical Taxonomy Sys-

tem (NTSYS) program with Dice coe Y cient (Rohlf

2000). The resulting groups or clusters generated

from the dendrogram were tested for robustness by

bootstrapping analysis using the Winboot program

(Yap and Nelson 1996).

Results

Phenotypic analysis and gene combinations

The disease reactions of the 1,364 segregating F2 plants to PXO61, PXO86 and PXO99 are summarized in Fig.2. We obtained 157, 1,048 and 1,226 resistant (R) plants showing 0–5cm mean lesion lengths when plants were inoculated with PXO99, PXO86 and PXO61, respectively. Most of the plants did not have a consistent resistant phenotype in all 3BB pathogen race inoculated. For example, an individual F2 plant exhibited a resistant response to PXO61 but not to PXO86 and PXO99 while others exhibited vice versa. In total, 144 F2 plants demonstrated resistance against PXO61, PXO86 and PXO99.

In terms of potential TGMS materials, 111 sterile F2 plants (no seeds produced at harvest) were identi-W ed from the segregating population (1,364 F2 plants). The distribution of possible Xa gene/gene combinations present in these lines is summarized in Table1. Of these, 13 plants were resistant to Xoo

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races 1, 2 and 6 including PR36944-450, PR36944-473 and PR36944-700 (Table 2). Mean lesion length of PXO61-infected leaves ranged from 0.53–2.05cm while 0.60–3.53cm and 3.03–4.93cm for PXO86-and PXO99-infected leaves. After 14days of inocula-tion, PR36944-1150 was the most resistant to PXO61and PXO86 exhibiting 0.53cm and 0.60cm mean lesion length, respectively. With isolate PXO99,PR36944-396 was most resistant at 3.03cm mean lesion length. However, PR36944-450, PR36944-473and PR36944-700 were of special interest because of the presence of homozygous Xa7 and Xa21.

Phenotypic segregation based on disease reaction to diagnostic Xoo races among the sterile plants showed 78 plants (70%) containing Xa4+Xa7 (Table 1).Twenty plants (18%) had Xa21 or in combination with Xa4 and Xa7 while only one plant (0.9%) and 12 plants (11%) had Xa7 and Xa4, respectively.Molecular analysis of Xa4, Xa7 and Xa21

Seventy-three F 2 plants showing resistance to PXO61,PXO86 and PXO99 were tested for the presence of Xa7 and Xa21 using molecular markers. Results with the M5F/R sequence tagged site (STS) marker used to track Xa7 revealed that only 77% of the resistant lines yielded the 294bp fragment speci W c for Xa7, while the rest showed the 1,170bp band (Fig.3a). Mean lesion length of these lines exhibited a range of 0.27–1.75cm and 1.77–4.67cm in lines showing the 294bp and 1,170bp bands, respectively. Genotyping of the 73 resistant lines using the PCR STS marker

Xa21F/R demonstrated the presence of a 1,000bp fragment speci W c for Xa21 (Fig.3b). However, only 40 plants (55%) were homozygous for the allele while the rest (32 plants) showed the heterozygous bands.Altogether, of 73 plants identi W ed by phenotyping,nine plants were homozygous for Xa7 and Xa21 while two plants were heterozygous for the two genes and all of them showed high resistance to Xoo races 1, 2and 6 (Table 3). For example, PR36944-175 and PR36944-1147 were heterozygous for Xa21 showing mean lesion length of 0.8cm and 1.2cm to PXO99,respectively.

PCR analysis on 11 F 2 plants with variable reac-tion to PXO61, PXO86 and PXO99 showed the pres-ence of 294bp allele in lines carrying Xa7 alone or in combination with Xa4 and/or Xa21 (Fig.4). The Xa7

Table 1Distribution of Xa gene/gene combinations in 111potential TGMS F 2 plants showing pollen sterility under green-house condition and pollen fertility in ?25°C indoor growth chamber a a

Presence of Xa4, Xa7 and Xa21 were deduced from reactions of individual plants to diagnostic Xoo races 1 (PXO61), 2(PXO86) and 6 (PXO99)

Xa gene/gene combinations No. of plants (F 2)Mean lesion length (cm)PXO61PXO86PXO99

Xa4 alone 12 2.6511.4613.40Xa7 alone 110.80 3.0717.25Xa4/Xa778 1.16 1.5013.81Xa4/Xa7/Xa21, or Xa4/Xa21, or Xa7/Xa21, or Xa21 alone

20

1.27

1.65

4.72

Table 2Mean lesion length of sterile F 2 plants showing resis-tant (R) reaction to PXO61, PXO86 and PXO99 14days after inoculation a

Presence of Xa gene(s) were deduced from the genotypes of the individual plants using STS PCR primers M5F/R for Xa7and Xa21F/R for Xa21. No polymorphic allele(s) or band(s) was observed in PCR W ngerprinting assays using MP1F/MP2R STS PCR primer linked to Xa4 to di V erentiate the resistant from the susceptible phenotypes inoculated with PXO61b

PR36944-566 was not consistent for presence of Xa7 in 2PCR assays performed using M5F/R STS marker

The di V erent values indicated that means followed by the same letters are not signi W cantly di V erent at 5% level of signi W cance

Plant no.

Mean lesion length (cm)Xa gene(s) present a PXO61PXO86PXO99

PR36944-265

1.43 a

2.67 a 4.83 a Xa21(Aa)PR36944-3050.70 a 2.20 a

3.17 a Xa21(Aa)PR36944-375 1.53 a 3.53 a

4.90 a Xa21

PR36944-3960.97 a 0.77 a 3.03 a Xa7+Xa21(Aa)PR36944-4500.75 a 1.40 a 3.17 a Xa7+Xa21PR36944-473 2.05 a 1.10 a 4.50 a Xa7+Xa21PR36944-5240.83 a

0.77 a 4.93 a Xa7+Xa21(Aa)PR36944-566b

0.80 a 1.80 a 4.60 a Xa7+Xa21PR36944-633 1.03 a 1.67 a 4.40 a Xa7+Xa21(Aa)PR36944-6390.90 a 0.90 a 4.00 a Xa7+Xa21(Aa)PR36944-700 1.90 a 0.93 a 4.75 a Xa7+Xa21PR36944-11500.53 a 0.60 a 4.80 a Xa7+Xa21(Aa)PR36944-13520.83 a 0.63 a 4.83 a

Xa7+Xa21(Aa)

IRBB4 3.40 a 13.25 b 15.00 b Xa4

IRBB70.83 a 1.25 a 24.17 c Xa7IRBB21 2.80 a

2.40 a

4.17 a

Xa21

IR24

15.83 b 14.17 b 25.07 c No gene

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T I A M

M

1000 b p 1000 b p

)

Table 3Fertile F 2 plants

showing highly resistant to PXO61 (mean LL range 0.38–1.30cm), PXO86 (0.43–2.47cm), and PXO99 (0.80–2.97cm)

F 2 plant

Mean lesion length (cm)Xa gene(s) present PXO61

PXO86PXO99PR36944-960.73 1.00 1.50Xa7+Xa21PR36944-1310.550.45 2.37Xa7+Xa21PR36944-1580.570.57 2.17Xa7+Xa21PR36944-1690.450.53 1.55Xa7+Xa21PR36944-1750.450.430.8Xa7+Xa21(Aa)PR36944-176

0.530.53 2.77Xa7+Xa21PR36944-1900.400.50 2.97Xa7+Xa21PR36944-452 1.250.80 2.00Xa7+Xa21PR36944-470 1.30 2.47 2.35Xa21

PR36944-1147 1.300.50 1.20Xa7+Xa21(Aa)PR36944-1345

0.38

0.43

1.25

Xa7+Xa21

The plants can be useful ge-netic stocks for improve-ment of BB resistance and selection of potential TGMS materials for two-line hybrid breeding

M5F/R PCR evaluation of selected F 2 plants of rice, L. with variable reaction to Xanthomonas oryzae race 1 (PXO61), race 2 (PXO86) and race 6 plants resistant (R) to races 1, 2, and 6 contain susceptible (S) to race 1 but R to races 2 and 6 contain F 2 plants S to races 1, 2, and 6 have no to races 1 and 2 but S to race 6 have R to race 1 but S to races 2 and 6 have T I M

M

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fragment in these lines was the same as that of IRBB4/7, the donor of Xa7 in the 11 F 2 pyramided plants analyzed. TGMS1 (susceptible parent) and AR32-19-3-3 (Xa21 donor) showed the 1,170bp band.

Genotyping of the 63 sterile lines with strong or moderate resistance against PXO61, PXO86 and PXO99 showed that PR36944-450, PR36944-473 and PR36944-700were homozygous for Xa7 and Xa21(Fig.5, Table 1). The 294bp band was found in the three lines with the Xa7 donor-parent IRBB4/7 and IRBB7 control while the 1,000bp fragment was pres-ent in the same lines and Xa21 donor-parent AR32-19-3-3. PR36944-566 was resistant to the three Xoo races but PCR analysis of this line demonstrated an inconsistent genotype for the Xa7 allele (Table 2).MP1F/MP2R STS PCR primer linked to Xa4 did not di V erentiate the resistant from the susceptible phenotypes inoculated with PXO61 in PCR W nger-printing assays (data not shown).Genetic background analysis

Microsatellite W ngerprinting of PR36944-450,PR36944-473 and PR36944-700with their parents including TGMS1, AR32-19-3-3 and IRBB4/7revealed 121 allele types in 65 loci. An analysis of the genetic background of these lines showed 87%genetic similarity among the three F 2 progeny, while PR36944-473 was 92% similar to PR36944-700(Fig.6). Overall, the selected resistant lines were more similar to IRBB4/7 (83%) than to TGMS1 (48%).

Investigation of the F 3 progeny derived from PR36944-450 and PR36944-700 identi W ed 7 potential TGMS lines showing di V erent degrees of pollen fertility and sterility at 25°C and 27°C (Table 4).

Discussion

A growing number of rice-growing countries in the tropics have explored hybrid rice technology as a means to increase the yield potential of rice and to ensure rice self-su Y ciency. However the released

Fig.5Genotyping of PR36944-450, PR36944-473 and PR36944-700 and their parents (TGMS1, IRBB4/7 for Xa4 and Xa7, and AR32-19-3-3 for Xa21) using M5F/R for Xa7 (a ) and Xa21F/R for Xa21 (b ) STS PCR-based markers. Three homozygous lines of rice (O. sativa L.), IRBB7, IRBB4 and IR24 were used as control DNA. Marker is 1kb+ ladder

1

S M G T 7

/4B B R I 3

-3-91-23R A 7

B B R I 4

B B R I 4

2R I 0

54-44963R P 3

74-44963R P 0

07-44963R P M

294 bp

1170 bp

a )

b)

1000 bp

M

Fig.6UPGMA cluster analysis of PR36944-450, PR36944-473 and PR36944-700 with their parents at 65 microsatellite loci. The percentages in parentheses indicate % genetic similar-ity and numbers to the left side of the fork indicate the percent-age of times such group of lines occurred in 1,000 bootstrap iterations

PR36944-450

PR36944-473

PR36944-700

IRBB4/7

AR32-19-3-3

TGMS1

100.0

97.5

54.9(87%)(92%)

(83%)

(61%)

(48%)

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hybrid cultivars in the Philippines are seriously dam-aged by bacterial blight. Thus, improved disease resistance carried by the parents is essential to realize the potential of high-yielding hybrids. Furthermore, a greater degree of genetic diversity is needed in hybrid rice breeding program to reduce the vulnerability to disease. In this study, we sought to improve bacterial blight resistance in TGMS1 because it is genetically distant from other source of TGMS lines currently used in the Philippines as shown by microsatellite W ngerprinting analyses (data not shown). We showed that closely linked and diagnostic markers were e V ec-tive in tracking speci W c Xa genes, enabling early selection of desirable gene combinations in a large F 2breeding population. Although a small set of molecular markers did not reveal all the genes that contributed to the resistance phenotype, marker-aided analysis had enabled detection of potential new resistance genes in breeding lines.

Phenotypic characterization of the F 2 segregating plants inoculated with PXO61, PXO86 and PXO99suggested that epistatic interactions of genes involv-ing Xa4, Xa7 and Xa21 were responsible for the observed segregation of resistant phenotypes. This was evident in the frequency distribution of resistant and susceptible F 2 plants inoculated with PXO61.Earlier we argued that xa5 previously reported in AR32-19-3-3 (PhilRice 2002) may contribute to the observed resistance phenotype in concert with the

other Xa genes introgressed in the population, leading to a large number of plants expressing resistant response against PXO61. However, we were not suc-cessful in showing that xa5 was present in AR32-19-3-3 or the control IR24 (data not shown) using both STS markers used to select for xa5 in the early gener-ation and a PCR marker designed from the cloned xa5gene (Iyer and McCouch 2004; Vera Cruz et al. 2006,unpublished). Thus, with the current data, we cannot conclusively determine whether an additional resis-tance gene is involved.

In the case of Xa7, the use of M5 marker did not detect the 294bp diagnostic fragment in plants show-ing resistance to PXO86. Note that in addition to PXO86, these plants also exhibited resistance to both PXO61 and PXO99. Some of the plants might be carrying genes other than Xa7. Alternatively, the observed resistance could be due to epistatic e V ect of undetected genes. Marker M5 has been mapped using IRBB7 (resistant to PXO86) and IR24 (susceptible to PXO86) but did not showed any recombinant banding patterns on the 277 susceptible F 3 plants evaluated indicating that Xa7 is tightly linked (=0.16cM, one recombinant error factor) to M5 (Porter et al. 2003).This illustrates the problem that markers developed from a certain cross may not necessarily be linked to the target gene in di V erent genotypes. Also, during the breeding process, genetic recombination may uncouple linked markers from the target gene, leading to the selection of ‘false positives’. Thus, the use of linked markers alone in lieu of phenotypic traits must be practiced with caution. It further reinforces the bene W t of using diagnostic alleles of resistance genes that would essentially guarantee the selection of plants with appropriate phenotypes.

Introgression of Xa4, Xa7 and Xa21 genes in TGMS1 via conventional hybridization generated three potential TGMS lines (PR36944-450, PR36944-473 and PR36944-700) with resistance to PXO61,PXO86 and PXO99. Resistance to the three Xoo races observed in these plants could be attributed to various combinations of Xa4, Xa7 and Xa21. Genotyping using markers diagnostic to Xa7 and Xa21 was, there-fore, necessary to demonstrate that the three F 2pyramided plants were homozygous for the corre-sponding resistant alleles of the two genes. However,we were not able to ascertain the presence of Xa4based on markers because the MP1/MP2 primers linked to Xa4 could not di V erentiate the TGMS1

Table 4Selected F 3 progeny from PR36944 involving the cross AR32-19-3-3/TGMS1//IRBB4/7 showing pollen sterility (S), partial sterility (PS), partial fertility (PF), and complete sterility (CS) in 25°C and 27°C indoor growth chamber a

Seeds of the selected plants were used for seed production in the phytotron and further evaluation of critical pollen fertility/sterility points b

Pollen sterility (%) of plants considered as CS, S, PS and PF are as follows: CS (100), S (91–99), PS (71–90) and PF (31–70)

Line number a

Pollen fertility/sterility b 25°C

27°C PR36944-450-3PF CS PR36944-450-7PS CS PR36944-700-1PS CS PR36944-700-3PS S PR36944-700-4PS S PR36944-700-10PS S PR36944-700-11

PS

S

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parent (without the Xa4) and the IRBB4/7 donor line. Thus, only phenotypic detection of Xa4 was done on the F2 progeny.

The three selected plants did not produce seeds at maturity in the greenhouse (approximately 12h day and 12h at night). Potential TGMS lines usually exhibit sterility under greenhouse conditions because of high temperatures. Sterility/fertility variation in TGMS lines is controlled by temperature and male sterility in TGMS rice is associated with premature programmed cell death (PCD) of the tapetum, which plays a crucial role in pollen development (Ku et al. 2001, 2003). However, even at a relatively low tem-perature (approximately 25°C in the phytotron) favor-ing fertility reversion of the pollen, PR36944-450 and PR36944-700 produced only few seeds whereas PR36944-473 remained sterile. A sterile plant such as PR36944-473 is not useful for hybrid rice breeding as it cannot revert back to fertility at a permissive tem-perature. The partial fertility observed in PR36944-450 and PR36944-700 is characteristic of potential temperature-sensitive lines. The fact that complete fertility was not restored in these two plants at per-missive temperature suggested that multiple genetic factors may play a role in temperature-dependent male sterility in TGMS1. It is therefore essential to reconstitute these factors through additional selection.

Because it is important to restore TGMS character-istics for hybrid breeding programs, we examined the relationship between PR36944-450, PR36944-473 and PR36944-700 with the susceptible parent TGMS1 using 65 microsatellite loci (Fig.6). The dendrogram suggested that about 50% of the TGMS1 parent genome was retained in the selected plants. The method of crossing performed with TGMS1 and the bacterial blight gene donors in producing the three-way cross F1 seed has eliminated portions of the TGMS1 genome. The three F2 progeny that clustered with the two Xa gene donors at 61% genetic similarity, which was expected because of the three-way hybrid-ization of TGMS1 to the resistance gene donors. IRBB4/7 clustered with the three F2 progeny (83% similarity), while PR36944-473 and PR36944-700 were highly similar (92%). PR36944-700 could be used instead of PR36944-473 because the former was able to revert back to partial fertility in the phytotron.

The results of this study showed that marker aided selection is not only for increasing breeding e Y ciency but also essential for pyramiding genes with epistatic e V ect. The identi W cation of PR36944-450 and PR36944-700 from the large F2 segregating popula-tion is possible only through marker-aided evaluation. These lines, together with additional F2 sterile lines showing moderate resistance against bacterial blight provide the needed germplasm towards developing two-line hybrids.

Acknowledgements This work was conducted as part of the MS Thesis Research Scholarship (LM Perez) provided by the International Rice Research Institute (IRRI) and the Philippine Rice Research Institute (PhilRice).

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