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Identification of a dominant bacterial blight resistance gene from Oryza nivara and its molecular mapping
 
P. NATARAJKUMAR#, K. SUJATHA#, G. S. LAHA, B. C. VIRAKTAMATH, C. S. REDDY, B. MISHRA, S. M. BALACHANDRAN, T. RAM, K. SRINIVASARAO, Y. HARI and R. M. SUNDARAM*

Directorate of Rice Research, Rajendranagar, Hyderabad 500030 India
# These authors contributed equally to the work
* Corresponding author (E-mail: rms_28@rediffmail.com)

Rice (Oryza sativa L.) is the most important crop of India and is grown all over the country in diverse ecosystems. The productivity of rice is being affected by a number of biotic and abiotic stresses. Among the biotic stresses, bacterial blight (BB) caused by Xanthomonas oryzae pv. oryzae (Xoo) is an important disease that results in significant yield reduction worldwide. The disease, in its severe form, is known to cause yield losses ranging from 74 to 81% in susceptible cultivars (Srinivasan and Gnanamanickam, 2005). Resistance breeding is considered as the most economical and environmentally safe approach for achieving yield stability. Over 30 BB resistance genes have been identified so far in rice (Khush and Angeles, 1999 Chu et al., 2006a & b) and a few of these like Xa4 and Xa7 have been incorporated widely into many high yielding varieties through conventional breeding (Khush et al., 1989). However, widespread cultivation of varieties with single BB resistance gene has led to predominance of Xoo races that can overcome the gene (Mew et al., 1992). The deployment of rice cultivars that have multiple BB resistance genes is expected to lead to more durable resistance. Through marker-assisted gene pyramiding, two or more resistance genes like Xa21, xa13, xa5 and Xa4 have been incorporated in the genetic background of elite varieties (Huang et al., 1997 Sanchez et al., 2000 Singh et al., 2001 Joseph et al., 2004 Sundaram et al., 2008). Even though gene pyramid combinations like Xa21 + xa13 + xa5 or Xa21 + xa5 or Xa21 + xa13 have been observed to possess high level of resistance against multiple isolates of Xoo, the durability of resistance in such gene pyramid lines has not been validated so far. In order to enhance the durability of resistance, it is desirable to identify and characterize new genes from wild relatives of rice so that they could be deployed along with Xa21 or xa13 or xa5 in elite rice varieties. Towards this objective, we have identified, characterized and genetically mapped a new BB resistance gene from an accession of Oryza nivara.

Fifty-five accessions belonging to O. nivara maintained at the Directorate of Rice Research, Hyderabad, were evaluated against five virulent strains of BB pathogen collected from different BB hot-spot locations of India as per the clip inoculation methodology of Kauffman et al. (1973). Six resistant accessions were reevaluated with three hyper virulent strains of the pathogen collected from Pantnagar, Aduthurai and Raipur, India. IRGC105710, an accession of O. nivara showed very high level of resistance against all the three pathogen strains. An interspecific cross was then made between O. nivara (IRGC105710) and TN1, a BB susceptible cultivar. The F1 plants were observed resistant when screened with a hyper virulent isolate of BB pathogen collected from Raipur, India (DX133), indicating the possible involvement of dominant gene(s) in governing BB resistance. The F1 plants were backcrossed to TN1 to generate 90 BC1F1 plants. Forty-two BC1F1 plants were observed BB resistant, while 48 were susceptible, indicating that a single dominant gene could be controlling BB resistance in IRGC105710 (χ2 = 0.40, P = 0.53). A single resistant BC1F1 plant was then selfed to generate a BC1F2 mapping population consisting of 435 plants. When the mapping population was screened with DX133 isolate, 317 BC1F2 plants were observed resistant, while 118 were susceptible. Their genotypes for BB resistance were further examined based on the reactions using BC1F3 plants. As a result, 101 BC1F2 plants were observed homozygous resistant and 216 were heterozygous resistant, and all the 118 susceptible BC1F2 plants produced susceptible progenies (1:2:1, χ2= 1.35, P = 0.51), demonstrating that a single dominant gene is governing BB resistance in IRGC105710. Allelism tests were carried out with different IRBB lines which carry single dominant BB resistance gene in the genetic background of IR24 (received from IRRI, Philippines) viz., IRBB1, IRBB4, IRBB7, IRBB21 and IRBB26 revealed that the dominant resistance gene present in IRGC105710 is non-allelic to the known dominant BB resistance genes Xa1, Xa4, Xa7, Xa21 and Xa26 respectively.

In order to map the dominant BB resistance gene in the O. nivara accession, DNA was extracted from the parental lines and individual BC1F2 plants using the procedure of Dellaporta et al. (1983). PCR analysis was performed using 210 SSR markers spread uniformly across the rice genome as per the conditions described by Panaud et al. (1996). Out of 210 SSR markersanalyzed, 85 displayed polymorphism among the parental lines and were used for bulked segregant analysis (Michelmore et al., 1991). In bulked segregant analysis, the SSR marker RM21330 located on chromosome 7 displayed co-segregation not only with the resistant and susceptible bulks but also with the individuals constituting the bulks with a few recombinants. RM21330 along with six other parental polymorphic SSR markers specific for chromosome 7 (RM20866, RM20948, RM21004, RM21177, RM21260 and RM21277) were then analyzed in all the 435 BC1F2 individuals constituting the mapping population for their co-segregation with BB resistance. Fig. 1 displays the segregation pattern of RM21330 in a set of BC1F2 plants. The markers RM21004 and RM21177 were observed to be closest to the BB resistance gene at a genetic distance of 2.0 cM and 4.5 cM, respectively and were flanking the gene on either side. RM20866 and RM20948 were observed to be located on one side of the gene while RM21260, RM21277 and RM21330 were located on the other side of the resistance gene. Fig. 2 displays the linkage map of chromosome 7 showing the genetic position of the linked SSR markers.

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Considering the fact that the O. nivara accession IRGC105710 is resistant to many virulent and hypervirulent isolates of Xoo and since no other BB resistance gene, particularly from O. nivara has been mapped on chromosome 7, it can be assumed that the dominant BB resistance gene present in IRGC105710 could be new. Hence, the gene has been tentatively named as Xa33t. Presently, fine mapping analysis and marker-assisted introgression of the gene into the genetic background of an elite fine grained rice variety Samba Mahsuri is in progress.

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