GENETIC IMPROVEMENT OF SUGARCANE BY CONVENTIONAL AND MOLECULAR APPROACHES
Location: Sugarcane Research Unit
Title: Isolation and characterization of resistant gene analogs from RSD resistant and susceptible sugarcane clones: A progress report
Submitted to: American Society of Sugar Cane Technologists
Publication Type: Abstract Only
Publication Acceptance Date: June 2, 2010
Publication Date: June 16, 2010
Citation: Hameed, U., Pan, Y.-B., Grisham, M.P., Iqbal, J. 2010. Isolation and characterization of resistant gene analogs from RSD resistant and susceptible sugarcane clones: A progress report [abstract]. Journal of the American Society of Sugar Cane Technologists. 30:153.
Ratoon stunting disease (RSD) can decrease sugarcane yield by 5 to 15% if not properly managed. Currently, the disease is primarily managed by two approaches: breeding for RSD resistance and through sanitation, i.e., cleaning equipment and a clean seed program. Understanding the molecular basis of disease response would provide critical information for breeders and pathologists to efficiently develop cultivars resistant to RSD. Resistant gene analogs (RGAs) have been isolated from many crops and are found to be associated with disease resistance gene(s). The aims of this study were to isolate and characterize RGAs from the sugarcane cultivar LCP 85-384 and to identify single nucleotide polymorphism (SNP) DNA marker(s) among the RGAs that appear to be associated with RSD response. Based on real-time PCR and tissue-blot immune binding assays, we identified two distinctive groups of clones, one which was RSD resistant and the other RSD susceptible, from a mapping population that was produced by self-pollination of LCP 85-384. From these two groups, we selected five resistant and five susceptible clones that shared similar data for several other phenotypic traits, such as plant height, stalk number, sugar and fiber content, etc. A total of 25 primer pairs were used, 19 of which were taken directly from RGA publications on four other crops, namely, rice, maize, soybean, and cotton. The remaining six were designed based on reported sugarcane RGAs. All primer pairs were used to amplify RGAs following a standard PCR procedure on genomic DNA samples of LCP 85-384 along with the five resistant and the five susceptible clones. Amplified RGA products, along with DNA size standards, were separated in 1.5% agarose gels by electrophoresis, stained with ethidium bromide, visualized under UV lights, and photographically documented. Discrete bands within the size range of 350 to 600 base pairs were gel purified and cloned into the Invitrogen’s pCR4-TOPO vector, and the recombinant plasmids were sequenced. To date, we have isolated 10 RGAs from one resistant self progeny of LCP 85-384 and found that the DNA sequences of these RGAs were homologous to reported RGAs. Cloning of RGAs from LCP 85-384 and the rest of resistant and susceptible clones will allow us to determine the mode of transmission of RGA and to identify any sequence difference between RSD resistant and susceptible clones.