31. Tagging of bacterial blight resistance gene in rice using RAPD markers

Y. VIKAL, J.S. SIDHU, S. SINGH, M. SODHI and H.S. DHALIWAL
Biotechnology Center, PAU, Ludhiana. India

Bacterial blight (BB) caused by Xanthomonas oryzae pv. oryzae is one of the most devastating disease of rice throughout the world. Genetic resistance is the most effective and economical control for bacterial blight. Nineteen genes conferring resistance to specific races of Xoo have been identified by classical genetic analysis (Kinoshita 1989: Khush et al. 1990). It is important to tag these genes using molecular markers to facilitate breeding efforts aimed at developing more durable forms of resistance to BB. Since, the development of PCR based RAPD markers, in which random fragment of DNA are amplified from DNA samples using short, arbitrary primers, several resistance genes have been tagged with RAPD markers (Ronald et al. 1992; Yoshimura et al. 1995).

The F2 population of 74 individuals from the cross of TJ1 (japonica) x IR24 (indica) was used for mapping BB resistance gene. TJ1 is resistant to pathotypes 2 and 3 of BB (prevalent in Punjab, India) whereas IR24 is susceptible. Disease reaction was determined based on lesion length following inoculation with pathotypes 2 and 3 using the clipping method (Kauffman et al. 1973).

Sixty five primers of arbitrary nucleotide sequence were used to amplify DNA fragments from the genomic DNA of IR24 and TJ1 genotypes. Twenty five of these primers detected polymorphism between the parents. A total of 231 DNA fragments were amplified and 60 (26%) of these showed polymorphism. Fig. 1 shows the amplification products of different primers. The number of different amplification products for each primer depends upon the primer sequence, the genome sequence and the genome size. The size of the amplified products ranged from 0.2 to 3.7kb. The total number of loci as detected in IR24, TJ1 and number of polymorphic loci with each primer are listed in Table 1. Eight primers, OPA-18, OPC-03, OPC-12, OPC-19, OPD-05, OPD-09, OPK-09 and OPV-06 generated polymorphic loci that were present in IR24 and not in TJ1. Nine of the primers viz., OPB-19, OPC-16, OPD-14, OPE-11, OPE-17, OPJ-12, OPS-12, OPV-16 and OPX-11 produced polymorphic products in both the parents. Polymorphic products exhibited by TJ1 were generated by OPA-15, OPB-15, OPB-16, OPC-20, OPD-07, OPE-15, OPK-18 and OPX-07.

Twenty two polymorphic RAPD markers were surveyed on F2 population. With primer OPB-16, two of the RAPD markers OPB-163 and OPB-166 cosegregated with disease resistance. OPB-163 amplified fragment was of about 1.3kb whereas OPB-166 fragment was approximately 320bp. Both markers had an amplified fragment in resistant individuals and absent in susceptible individuals. The resistance locus was flanked by OPB-163 at a distance of 5.1cM. Bulked segregant analysis of F3 progenies showed that the RAPD marker (OPB-163) of 1.3kb cosegregated in F3 resistant bulk but was absent in the susceptible bulk confirming that this marker was closely linked to the BB resistance gene (Fig. 2). OPB-166 marker also cosegregated with resistance in F2 individuals but was not able to distinguish the resultant F3 bulks suggesting that it may not be closely linked to the resistant gene.

The results of this study indicate that bulked segregant analysis using RAPD markers on F3 progenies provides a highly efficient strategy to tag target genes. RAPD and bulked segregant analysis could be used as the initial method for tagging the genes of interest and then polymorphic RAPD products obtained would be mapped relative to the molecular markers (RFLP) with known map position.


Fig. 1. Polymorphism survey among IR24 (I) and TJ1 (T) with different RAPD primers. M represents Kb ladder molecular marker.

Table 1. Polymorphic loci between TJ1 and IR24 as detected through RAPD analysis
Primer
Total number of loci
Number of polymorphic loci
@
IR24
TJ1
IR24
TJ1
OPA-15
5
6
0
1
OPA-18
4
2
2
0
OPB-15
9
10
0
1
OPB-16
2
6
0
4
OPB-19
9
10
1
2
OPC-03
5
3
2
0
OPC-12
4
2
2
0
OPC-16
6
5
3
2
OPC-19
6
5
1
0
OPC-20
2
3
0
1
OPD-05
7
3
4
0
OPD-07
4
5
0
1
OPD-09
6
4
2
0
OPD-14
4
4
1
1
OPE- 11
5
4
2
1
OPE -15
4
7
0
3
OPE-17
3
5
2
4
OPJ-12
2
2
1
1
OPK-09
2
1
1
0
OPK.-18
1
2
0
1
OPS-12
4
4
2
7
OPV-06
5
3
2
0
OPV-16
5
4
2
1
OPX-07
3
4
0
1
OPX-11
4
5
1
2

Fig. 2. Bulked segregant analysis with primer, OPB-16 for detection of linkage. Lane 1: TJ1: Lane 2: IR24; Lane 3: Resistant bulk and Lane 4: Susceptible bulk. M represents molecular marker (Kb ladder).

Acknowledgments

The authors are grateful to Dr. D.S. Brar and Dr. T.S. Bharaj for providing the parents and their F2 seeds for this study.

References

Kauffman, H., A.P.K. Reddy, S.P.Y. Hsieh and S.D. Merca, 1973. An improved technique for evaluating resistance of rice varieties to Xanthomonas oryzae. Plant Dis. Report 57: 537-541.

Khush, G.S., E. Bacalangco and T. Ogawa, 1990. A new gene for resistance to bacterial blight from O.longistaminata. RGN 7:121-122.

Kinoshita, T., 1989. Report of the Committee on gene symbolization, nomenclature and linkage groups. RGN 6:2-29.

Ronald, P.C., B. Albano, R. Labien, L. Abenes, K. Wu, S. McCouch and S.D. Tanksley, 1992. Genetic and physical analysis of the rice bacterial blight disease resistance locus, Xa21. Mol. Gen. Genet. 236: 113-120.

Yoshimura, S., A. Yoshimura, R.J. Nelson, T.W. Mew and N. Iwata, 1995. Tagging Xa-1, the bacterial blight resistance gene in rice by using RAPD markers. Breed. Sci. 45: 81-85.