ABSTRACT. tropical and sub-tropical countries. Recent utility of turmeric by the pharmaceutical

Transcription

1 ABSTRACT Turmeric (Curcuma longa L; Zingiberaceae) is one of the most important herb in the tropical and sub-tropical countries. Recent utility of turmeric by the pharmaceutical industries as a source of antioxidant, hepatoprotectant, anti-inflammatory in addition to its use in cardiovascular and gastrointestinal disorders has categorized it as a major industrially important crop of high demand. The International Trade Centre, Geneva, has estimated an annual growth rate of 10% in the world demand for turmeric. However, turmeric yield is not adequate as per the demand in the recent times. Among several factors, diseases are the most important cause associated with low productivity. Crop losses upto 60% has been realized in the recent times mainly due to the infection by a necrotrophic oomycetic fungus Pythium aphanidermatum causing the rhizome rot disease. Conventional crop improvement methods are not suitable in turmeric because it is not only completely sterile but also propagate exclusively by vegetative means. Therefore, identification and development of new turmeric cultivars and genotypes resistant to P. aphanidermatum remains the most promising option to solve the economically important rhizome rot problem. Genetic improvement of turmeric for resistance to soft rot seriously suffers from lack of a suitable resistance source. Knowledge about the spatial distribution of pathogen resistance among populations of turmeric plant and the correspondence between this and the pattern of the genetic diversity of the species will provide a baseline for the efficient utilization and identification of valuable resistance source. These resistant turmeric sources can play a major role in the development of sequencetagged markers such as sequence characterized amplified regions (SCARs) and sequence tagged sites (STS) for identification and detection of genetic loci associated with disease resistance and their successful utilization in the marker assisted selection

2 programme. Further, cloning and characterization of resistance genes and their subsequent transformation can be another possibility in the development of disease resistance turmeric cultivars. However, owing to high stigmatic incompatibility and complete genetic sterility in Curcuma longa, the genetic variation for disease resistance is poor in the cultivated turmeric. Hence, the utilization of wild Curcuma species for isolation and characterization of resistance gene candidate(s) can be a possible alternative because they can evolve resistance specificities more efficiently than cultigens and can provide a lead towards retrieving resistance specificities suitable for the improvement of turmeric. Keeping all these factors into account, the present study was undertaken to (1) determine the population genetic structure of turmeric (Curcuma longa) and its response to Pythium aphanidermatum, (2) identify and evaluate sequence tagged diagnostic markers (SCAR/STS) linked to rhizome rot resistance in turmeric and (3) clone and characterize resistance related candidate gene sequences from wild Curcuma spp. for resistance against Pythium aphanidermatum. Eleven populations of Curcuma longa, including 62 individuals (5-7 individuals per population) were sampled from 11 localities across 10 districts of Odisha, India. Screening with two field isolate of P. aphanidermatum revealed large and significant variation in the degree of disease incidence in the overall distribution of the number of plants in all turmeric accessions within the 11 populations. All the six turmeric accessions from KR population had a disease index 10% and were considered resistant. Similarly, six out of seven accessions from MK population and five accessions from NP population exhibited the lowest disease index values. On the other hand, turmeric accessions from CTK and CTN population represented the highest DI values (58.35% to 76.2%). Turmeric accessions from KP, DK, KJ and MB populations also showed >20% DI and categorized as susceptible. The relative frequency of resistance

3 accessions was higher in the Southern region of Odisha. Molecular analysis turmeric accessions using 29 sets of combinational markers yielded 286 bands of which 186 (65.03%) were polymorphic. Nei s genetic diversity (He) of 0.051±0.033 and Shannon information index (I) of 0.078±0.043 revealed a very low genetic diversity in turmeric accessions as expected for a clonal species. Turmeric accessions were classified into two clusters according to the UPGMA dendrogram with low level of genetic differentiation, which is further confirmed by Bayesian model based STRUCTURE and PCA analysis. The genetic clustering of turmeric accessions was mostly according to their geographic location and no clear correspondence was observed between the clustering pattern of accessions and their response to rhizome rot pathogen. Nevertheless, the resistance turmeric accessions identified through this study could be used as potential donor for soft rot resistance for genetic improvement of turmeric. In our quest to develop rapid and reliable diagnostic molecular to track turmeric germplasm resistant against rhizome rot, eleven turmeric genotypes known to be either resistant or susceptible to Pythium aphanidermatum as identified through pathogen screening were used in this study. Bulk segregant analyses using pooled DNA from resistant and susceptible genotypes with 40 inter simple sequence markers resulted in the isolation of a putative resistance specific marker ClRSM. Fifty positive clones (10 each from the five resistant individuals) were selected and subjected to sequencing. The five resistant specific sequences have been deposited in the NCBI GenBank database (accession no KF to KF603885).The resistance specific fragment was converted into a sequence tagged marker ClSTS, which could successfully amplify a 720 bp fragment in resistance turmeric genotypes but not in the susceptible plants. The CISTS amplified product was cloned, sequenced and deposited in the NCBI database with accession number KF Southern blotting confirmed it as a single copy locus

4 found associated with the resistant genotypes. Further, the ClSTS marker precisely identified 10 resistant and five susceptible genotypes among the 15 turmeric germplasm with unknown disease response collected from different agroclimatic regions. Inoculation assessment of the 15 individuals with a virulent strain of P. aphanidermatum corroborated with the STS marker results. This suggest that ClSTS marker exhibited high stability and reproducibility in the identification of resistant turmeric genotypes collected from different agroclimatic zones, geographic regions and population structures and not influenced by environmental factors and management practices. Thus, the STS marker will not only help in fast proliferation of rot resistance genotypes, but also will facilitate better cultivation practices and improve breeding programmes through development of new turmeric cultivars resistant to rhizome rot. Further, the phenotypic evaluation of rhizome rot resistance in clonal population of turmeric is not only time consuming, the infected plant contaminated with the fungal inoculums cannot be used further for cultivation. Hence, scrutinizing the turmeric plantlets with the identified marker ClSTS could be rapid, safe and efficient method towards identification and elimination of susceptible genotypes at an early stage to decrease the effect of rhizome rot disease on the overall productivity of the important commercial crop. To fulfil the objective of cloning and characterization of resistance related gene sequences from wild turmeric genotypes, degenerative primers based on the NBS conserved motif of nucleotide binding site leucine rich repeats (NBS-LRR) class R- genes were used to isolate 21 NBS sequences from three wild turmeric genotypes- Curcuma aromatica, Curcuma angustifolia and Curcuma zedoaria and named as wild turmeric resistance gene candidates (RGCs) (NCBI accession number JN to JN426989). The phylogenetic tree based on percentage identity of the deduced amino

5 acid sequences of wild Curcuma RGCs identified their relatedness with each other as well with the known R-genes and classified them into four phenetic classes. The percentage identity of wild Curcuma RGCs to RGCs from other plant species ranged from 41% to 63% and RGCs from other Zingiberaceous species ranged from 85% to 99%. The 21 RGCs were characterized by the presence of a putative coiled-coil (CC) structure, the specific RNBS-A and RNBS-B domains and a tryptophan residue at the kinase-2 motif thereby classifying them with the non-toll/interleukin receptor (TIR) type NBS-LRR class R gene family. Southern hybridization showed a multi copy representation of RGCs in the wild turmeric genomes. Expression variability of wild turmeric RGCs were analyzed through reverse transcription PCR in root tissues of the three wild turmeric plants resistant or susceptible to Pythium aphanidermatum. Cap12 and Can12 showed a constitutive expression in both resistant and susceptible plants of Curcuma aromatica and Curcuma angustifolia respectively while Czp11 expression was realized only in Pythium aphanidermatum resistant lines of Curcuma zedoaria. The expression of Czp11 in resistant turmeric accession and non-expression in the susceptible lines further confirm their role in resistance development against the rhizome rot pathogen. Thus Czp11 can be used as a source material towards cloning and sequence characterization of full-length R-genes that can be subsequently transformed into susceptible turmeric plants to test its ability in conferring Pythium aphanidermatum disease resistance. Further, the potential role of the isolated wild turmeric RGCs in disease resistance could further be tested using other advanced methods such as RNAi and BIBAC (Binary bacterial artificial chromosome) technology to reveal new information on the organization, function and evolution of the NBS-LRR-encoding resistance genes in asexually reproducing plants.