Molecular Characterization of Resistant and Susceptible Sugarcane (Saccharum Hybrids L.) Cultivars to Smut Disease

Abstract

Sugarcane (Saccharum hybrids L.) is a highly treasured perennial crop and happens to be the economic backbone of many countries including Pakistan. Apart from abiotic factors, diseases inflicted by fungal, bacterial or viral pathogens have the most damaging effect over the growth of sugarcane. Whip smut of sugarcane is one such disease caused by fungus Sporisorium scitamineum which posses a major threat to cane yield. The most effectual remedy to tackle the problem is to develop resistant cultivars that stand a far greater chance of surviving the disease outbreak. The advent of molecular marker techniques has massively enhanced the process of selection for disease resistance in sugarcane employed in breeding programs. The current work was a small contribution towards the same goal and exploited the potential of molecular markers to distinguish between smut resistant and susceptible cultivars of sugarcane. Moreover, identification of Resistance Gene Analogues (RGAs) was another strategy to understand the defense mechanism utilized by the crop. The initial screening experiments to differentiate between two completely resistant and two completely susceptible Pakistani cultivars involved detection of DNA polymorphism among the respective samples using 200 RAPD primers. Forty four of these primers turned out to be highly polymorphic, 20 of which produced trait specific loci in the four sugarcane genotypes. Furthermore, it was discovered that decamers A-20, E-05 and OPAV-10 produced 4 loci linked with resistant cultivars, while 4 loci were generated by primers B-17, OPAD-01, OPAD-13 and OPAX-14 specific to susceptible cultivars. However, primer A-09 amplified 2 markers of 1200 bp and 500 bp from resistant and susceptible cultivars, respectively. The disparity between resistant and susceptible cultivars was further highlighted when cluster analysis separated the two genotypes into two discrete groups. In order to detect RAPD markers associated with smut resistance phenotype in sugarcane, six pooled bulks of DNA from Pakistani cultivars (i.e. comprising 2 completely resistant, 2 completely susceptible, 4 moderately resistant and 4 moderately susceptible genotypes) and USA cultivar (i.e. containing 5 resistant and 5 susceptible clones of LCP85-84 F2 mapping population) were prepared. The screening of the six DNA bulks with 500 arbitrary decamers eventually revealed two RAPD markers (B-17 and I-20) linked with smut responses in Pakistani and USA cultivars, respectively. The marker B-17 produced a reproducible polymorphic fragment which appeared to cosegregate in repulsion with sugarcane smut resistance in Pakistani cultivars. On the other hand, RAPD decamer I-20 was tightly linked with resistance in smut resistant clones of USA cultivar LCP85-384 F2 population. Two Sequence Characterized Amplified Region (SCAR) markers were designed from the sequences of B-17 and I-20 products and were found to be specific to resistant cultivars of Pakistan and resistant clones of LCP85-384 F2 population, respectively. The SCAR marker developed from B-17 sequence was used to screen seven additional sugarcane cultivars of Pakistan with SCAR marker, verified the earlier findings by showing essentially similar results. In order to conduct further study, ten sets of oligonucleotides were designed to tag the nucleotide-binding site attached to leucine-rich repeat (NBS-LRR) domain of RGAs (resistance gene analogues) and other related sequences reported in the members of family Poaceae (maize, rice, sorghum and foxtail millet). The PCR amplifications of Pakistani and USA cultivars using these 10 primers resulted in the identification of three RGAs (MRGA3, MRGA5 and MRGA2) showing discriminating products with respect to smut resistance trait in sugarcane. The sequences of three isolated RGAs were analyzed and compared with that of documented R genes. The results of NCBI blast, multiple sequence alignment and phylogenetic analysis showed sequence homology of the three RGAs with reported R genes and RGA-like sequences. The final study was based on targeting sequences in sugarcane genome which may have a part to play in the resistance mechanism of plants against fungal attack. These sequences included a reported SCAR marker linked with covered smut resistance in barley and a canecystatin partial mRNA sequence. In a nut-shell, our study provided molecular basis to distinguish between smut resistant and smut susceptible sugarcane cultivars of both Pakistan and USA.