Marker Assisted Screening of Nepalese Rice for Bacterial Leaf Blight (BLB) Resistance

Bacterial Leaf Blight (BLB) is the most important yield limiting factor in Nepalese rice. BLB resistance rice varieties are highly demanding in the country. Breeding efforts for developing disease resistant depends on availability and use of resistant gene donors. Nepalese rice landraces could be the source of resistant gene. Therefore, ninety six Nepalese rice accessions were screened using eight Simple Sequence Repeats (SSR) markers and one Sequence Tagged Sites (STS) marker for presence and absence of BLB resistance gene. We have detected BLB resistance gene Xa-10 on five accessions, Xa-13 on six accessions, Xa-7 on 23 accessions, Xa-3 and Xa-4 on 52 accessions, Xa-5 on 25 accessions, Xa-8 on 30 rice accessions. No any rice accessions tested have Xa-21. Similarly, 17 rice accessions showed three and more than three BLB resistance genes. Presence of Xa-13 on susceptible check variety CNTRL-85033 confirmed that this resistant gene is not working in Nepalese rice field. Therefore, Nepal need to pyramide the BLB resistant genes for durable resistance.

BLB resistance rice varieties are highly demanded worldwide.However, the continuous evolution of pathogenic races leading to the breakdown of resistance in many improved varieties [9].Thus, success of resistance breeding program depends on the availability of the resistant donors.Similarly, pyramiding different resistant genes in a single rice variety will increase the resistance.However, two or more resistance gene pyramiding in a single variety is easy through molecular marker assisted selection (MAS), and identification for the presence and absence of particular gene in a variety for MAS as donor and recipient parent through molecular marker assisted screening is very fast, reliable and cheaper.Therefore, this study was carried out to identify the accessions within Nepalese gene pool with the potential of BLB resistance genes.

Molecular Marker and check variety
Eight SSR markers and one STS marker (pTA248) for the presence and absence of BLB resistance gene.Molecular markers are selected based on their linkage with particular BLB resistance gene (Table 2).IR-64 and CNTRL-85033 were used as resistant and susceptible check respectively.

DNA extraction, PCR reaction and data analysis
Genomic DNA of rice accessions was prepared using modified CTAB method as described by Sul and Korban [11].Each PCR reaction was conducted with100ng of genomic DNA, 1 µM of each primer and 7.5 µl of 2x GoTaqGreen PCR Master Mix (Promega Corporation, Madison, WI, USA).PCR mixture was amplified in MJ Research PTC-100TM Programmable Thermal Controller (MJ Research, Inc, Watertown, MA, USA) with the following temperature regimes: initial denaturation for 2 min at 95oC followed by 33 cycles of 95oC for 30 sec, annealing as per primer for 1 min, extension at 72oC for 2 min and final extension at 72oC for 7 min followed by holding at 4oC as described on Table 2 and Gramene [12].Amplified PCR products were separated in 2% analytical grade agarose gel at 100V for 1H.Gels were stained with 0.1 µg/ml ethidium bromide (Promega Corporation, Madison, WI, USA) and then visualized under UV transilluminator gel documentation system (Wilber Lourmat, Marne-La-Valleen, France) using 1 µg guide size DNA ladder (Genetix, Biotech Asia Pvt.Ltd.).The presence and absence of particular band size was scored for screening disease resistance genes.

Results and Discussion
Different resistance genes were identified in Nepalese rice germplasm as defined by different molecular markers (Table 3).We identified BLB resistance gene Xa-10 on two accessions, Xa-13 on six accessions, Xa-7 on 23 accessions, Xa-10 on five accessions, Xa-3 and Xa-4 on 52 rice accessions (Figure 2), Xa-5 on 25 accessions, Xa-8 on 30 rice accessions.No any rice accessions have Xa-21 (Figure 1).Similarly, 17 rice accessions showed three and more than three BLB resistance genes (Table 4).Nepalese rice accessions lack the Xa-21 genes which is more important for controlling BLB epidemics throughout the world.Kameshwara Rao [16] reviewed and noted that Xa-21 gene is transferred from O. longistaminata and integrated into some IRRI developed rice varieties.Lacks of such varieties in our study may be the result for this.Similarly, our susceptible check 'CNTRL-85033' showed the presence of Xa-13 but Amgai [17] reported that it was heavily infected by BLB at Nepalese rice field.This may be due to the difference on BLB isolated found in Nepal which may break the resistance reaction developed by Xa-13 gene.Resistant check IR-64 showed the presence of Xa-5 indicating that it is effective for Nepalese BLB pathogen.

Conclusion
Nepalese rice landraces contains many marker alleles for different BLB resistant genes.The rice landraces with effective resistant gene can be used as donor parent for MAS.However, for the enhancement of resistance in Nepalese rice with absence and/or ineffective resistance gene can be done by transferring broad spectrum resistance gene like Xa-21.BLB resistance is also affected by multiple resistance genes and their interaction [17].Therefore, pyramiding BLB resistance gene on Nepalese rice variety is most important for durable resistance with BLB.