30. 
Identification and Tagging a new gene for resistance to bacterial blight
(Xanthomonas oryzae pv. oryzae) from 0. rufipogon
Q. ZFIANG’, S.C. Lin1 B.Y. ZHAo’, C.L. WANG’, W.C. YANG’, Y.L. ZH0U’,D.Y. Li2, C.B. CHEN2
and L.H. ZHU3
1) 
Institute of Crop Breeding and Cultivation, CAAS Beijing 100081, China
2) 
Institute of Germplasm Resources, GAAS, Naining 530007, China
3) 
Institute of Genetics, Academia Sinica, Beijing 100101, China
The gene Xa-4 for resistance to bacterial blight (BB) has been widely used in the development of almost all of the commercial hybrids and conventional cultivars of indica rice in the main rice growing regions of China which encompass more than 90% of the national rice area (Min 1992). Such narrow genetic base has increased the genetic vulnerability of hybrid rice and conventional indica rice to BB, since a strong selection on the pathogen population has increase the frequency of pathotype V (virulent to rice carrying Xa-4). This pathotype is the most virulent one among the 7 Chinese BB pathotypes (Zhang et a!. 1996). To overcome this problem, diversification of rice varieties with different resistance gene(s) is necessary.
Nine hundred seventy-one accessions of 14 wild species were evaluated with 3 strains of BB from China, the Philippines and Japan. Twenty-one resistant accessions were reevaluated with strains of 7 Chinese, 6 Philippine and 3 Japanese races at both seedling and adult stages. One accession (RBB16) of 0. rufipogon showed high level of resistance to all the strains used (Thang et a!. 1994).
An interspecific cross between RBB16 and Jiagang 30 (JG3O), a susceptible cultivar was made, the F, plants showed different levels of resistance to several strains of BB including PXO99. Since 0. rufipogon was heterozygous for resistance, the F, plants showing high resistance to BB were anther-cultured to obtain doubled haploids that would be homozygous for resistance. The anther-cultured progenies were inoculated with 3 strains: GD 1358 (Chinese pathotype V), PX099 (Philippine race 6), and T7133 (Japanese race 3). Lines showing high resistance and improved plant-type in H2 populations were selected and advanced to H4 which were collectively designated as WBB 1 (Zhang et al.1994).
Among all the known genes for resistance to BB, Xa21 is the only dominant gene from a wild species (0. longistaminata) resistant to all 6 races of BB in the Philippines (Khush eta!. 1989, 1990). Therefore, Xa21 was used as tester for identifying the resistance gene of 0. rufipogon.
Comparison of resistance reactions of WBB1 and 6 testers (with different major dominant resistance genes) using 9 Philippine races of BB at both seedling and adult stages
WBB1 showed highly resistant reaction to the 9 races at all growth stages; IRBB21 (Xa21) showed resistance to the 9 races at the tillering stage; and Xa3, Xa4, Xa7, Xa1O and Xa14 showed no resistance to race 6 (PX099), but showed resistance to some races. This suggests that the resistance reaction ofWBB1 to 9 Philippine races is different from Xa21 and the other testers (Table 1). 
 
Inheritance of BB resistance in WBB1 to the Philippine race 6 (PX099)
WBB1 was crossed with 2 susceptible Chinese cultivars, JG3O (indica) and 02428
(japonica), and IRBB21 (Xa21). The F,, F2 and B1F1 populations were inoculated with
PX099 at the seedling stage. As shown in Table 2, all the F,s were resistant. The F2
populations from the crosses of WBB 1 with susceptible parents segregated in a ratio of
3R: is. The backcross progenies segregated in a ratio of 1R:1S. The X2 values for the
1 5R: iS segregation expected for two independently segregating genes in reciprocal crosses of IRBB21 and WBB1 were significant indicating that the dominant gene of WBB1 may be linked to Xa21. The results suggest that a single dominant gene form 0. rufipogon confers resistance to PX099 in WBBI, and the gene is nonallelic but probably linked to Xa21.
PCR analysis of WBB1 with Xa21 specific primers
Since only Xa21 confers resistance to 9 races including race 6 (PX099) amongst all know dominant genes for resistance to BB, we checked the allelism of the gene from 0. rufipogon with Xa21 at the molecular level. Two Xa21 primers U1(U, 5’-CGA TCG GTATA CAG CAAACC-3’TA CAG CAAAAC-3’) and I1 (5’-ATA GCA ACT 5) were used to test the allelic relationship between Xa21 and WBB1, JG3O was used as check. Only IRBB21 (Xa21) showed the 1.4 Kb fragment which is specific to Xa21 locus, whereas WBB1 and JG3O showed 1.3 Kb fragments (Fig. 1). This again indicates that the resistance gene of WBB 1 is different from Xa21.
Tagging the new gene by using DNA markers
After screening 94 rice Microsatellite (SSR) primers, two markers linking with the resistance gene were identified. The linkage analysis was performed by Mapmaker 3.0 Via the SSR markers mapped, the resistance gene was located on rice chromosome 11(5.4 cM to OSR6 and 27.7 to RM224) (Fig. 2). Fine mapping is on going.
Thus, the gene for resistance to BB from 0. rufipogon appears to be distinct from all the known genes for resistance. We have designated the new gene as Xa23(t).

 
 
Table 1.
Comparison o
f reac
tion t
o9 Ph
ilippin
e races o
f BB
betwe
en WB
B! and
6 tester genes
Material
Resistance
gene
        
Race
        
Growth
moculated
1
2
3
4
5
6
7
8
9
WBB1
Xa-?
R
R
R
R
R
R
R
R
R
SS
IRBB21
Xa-21
R
R
R
R
R
R
R
R
R
TS*
IRBB3
Xa-3
R
R
K
R
R
S
S
S
R
ASS
IRBB4
Xa-4
R
S
S
MR
K
S
R
R
S
SS*
IRBB7
Xa-7
K
R
R
S
R
S
R
R
R
AS
IRBBIO
Xa-1O
S
K
S
S
R
S
R
S
S
SS
IRBB14
Xa-l4
S
S
S
S
K
S
R
S
R
SS
JG3O
None
S
S
S
S
S
S
S
S
S
  
SS. TS, AS = Resistant at seedling, tillering and adult stage, respectively.
 

 
 
Table 2. Reaction to PX099 ofF,, F2 and B,F, populations from the crosses of WBBI with susceptible cultivars and IRBB2I at seedling stage
Cross
Reaction
ofF1
F2 or
BC populations
X2
(expected ratio)
P Value
R
S
Total
JB30/WBB1
R
183
52
235
0.887 (3:1)
0.25-0.50
02428/WBB1
R
199
58
257
0.686 (3:1)
0.25-0.50
JG3OIWBBI//JG3O
  
35
32
67
0.06(1:1)
0.25-0.50
02428/WBBI//WBBI
  
24
30
54
0.463 (1:1)
0.25-0.50
IRBB21/WBBI
R
136
31
167
46.73 (15:1)
0.001
WBBI/IRBB2I
R
164
28
192
25.08 (15:1)
0.001
R = Resistant; S = Susceptible.


 
 


 
 
 

 
 
References
Khush, G.S., E. Bacalangco and T. Ogawa, 1990. A new gene for resistance to bacterial blight from 0. longistaminata, RGN 7: 121-122.
Khush, G.S., DJ. Mackill and G.S. Sidhu, 1989. Breeidng rice for resistance to BB. p. 207-217 In Bacterial Blight of Rice. International Rice Research Institute, P.O. Box 933, Manila, Philippines.
Min, S.K., 1992. An outline of rice breeding in China, P. 58-67 In Rice in China. China National Rice Research Institute, 310006, Hangzhou, China.
Zhang Qi, i.E. Leach and C.L. Wang, 1996. Genetic structure study of bacterial blight for rice breeding in China. Abstracts for East Asia Regional Meeting of the International Program on Rice Biotechnology,
26-27 
Hangzhou, China.
Zhang Qi, Shi-cheng Ling et al., 1994. Evaluation of resistance to bacterial blight (Xanthomonas oryzae pv. oryzae in Wild Rice Species, Scientia Agricultura Sinica 27(5): 1-9.