Identification of genetic stock for drought tolerance and candidate gene analysis in cocoa(Theobroma cacao L.)

Abstract

Water deficit stress is considered as one of the most limiting factor for production in cocoa. Preliminary efforts have been made in Kerala Agricultural University to identify drought tolerant cocoa genotypes. The present study was an attempt made to identify the cocoa genotypes and hybrids that can tolerate drought condition. The study was conducted at Department of Plantation crops and spices, College of Agriculture (CoA) and Cocoa Research Centre and College of Agriculture, Vellanikkara, Thrissur. Thirty exotic genotypes and six pre-released hybrids were taken as materials for the study. They were budded on 6 month old rootstock of seedling raised from polyclonal gardens. GIV 18.5 (progeny of pods from Nileshwar) identified susceptible genotype was used as check in the experiment. Five month old budded plants of thirty exotic cocoa genotypes and six hybrids along with check variety were subjected to drought screening by following gravimetric method. Control plants were maintained at fully irrigated condition under 100 per cent field capacity. Field capacity was maintained at 40 per cent for stress condition. Based on the percentage of retained leaves genotypes and hybrids were categorised as highly tolerant (more than 70% leaves retained), tolerant (40-70% leaves retained), susceptible (10-40% leaves retained) and highly susceptible (less than 10% leaves retained). Various physiological and biochemical parameters related to drought were analyzed. The physiological parameters, relative water content, chlorophyll stability index, membrane stability, photosynthetic rate and chlorophyll content were recorded high in tolerant and highly tolerant genotypes and hybrids whereas these parameters were comparatively low in susceptible and check variety. The control plants at 100 per cent field capacity recorded high value for all these parameters. However in terms of transpiration rate a reverse trend was observed among genotypes and hybrids. Low transpiration rate was recorded in highly tolerant and tolerant hybrids whereas ii susceptible genotypes and hybrids had high transpiration rate under stress condition. The control plants under fully irrigated condition of 100 per cent field capacity also had high transpirational rate. This indicated the ability of tolerant genotypes and hybrids to conserve water during stress. The leaf temperature did not shown any significant variation among the genotypes and hybrids studied under both the field capacity level. In case of biochemical parameters, proline content, glycine betaine content and superoxide dismutase activity were found to be high in tolerant and highly tolerant genotypes and hybrids and was low in susceptible and check variety. The control plants kept under 100 per cent filed capacity level shown less values for all these parameters. However, all the plants showed reduced nitrate reductase activity under drought stress. The control plants recorded high nitrate reductase activity at 100 per cent field capacity level. Correlation studies showed that all physiological and biochemical parameters except transpiration rate and leaf temperature have positive correlation with percentage of leaves retained. Effect of these parameters on percentage of leaves retained was estimated using path analysis. Parameters having direct effect on leaf retention were proline, nitrate reductase activity, SOD, glycine betaine, cell membrane stability and relative water content. These characters were analyzed for their genetic parameters and it was found that all these characters are having high heritability and genetic gain. D 2 statistics analysis revealed that the 30 genotypes and 6 hybrids along with check were grouped into 6 clusters indicating the presence of diversity for different traits. Four different isolation methods were compared in this experiment to identify the best method that could produce high-quality total RNA free from contaminants and genomic DNA. RNA obtained through the SDS- tertiary butanol method (Gesteira et al., 2003) has high quantity and quality, followed by the modified SDSphenol method (Deepa et al., 2014) in both fresh and frozen leaves. iii The transcriptome of highly drought tolerant exotic genotype (T85/799) and highly drought sensitive check variety CCRP 5 was analysed using mRNA sequencing under control (100% FC) and drought stress condition (40% FC). Drought has up-regulated 1540 and 1148 DEGs and downregulated 1502 and 1250 DEGs respectively in tolerant and sensitive libraries. The PCA scatter plot shows that replicates of the well watered samples (control) of highly drought tolerant genotype and drought susceptible check variety CCRP 5 form distinct groups highlighting differences between the two genotypes. Volcano plot depicted the significance of differentially expressed genes in control and drought stress treated samples of highly tolerant and susceptible genotype. In both the tolerant and susceptible genotypes, the majority of the genes within the biological process category were linked to GO terms that fall under the subcategories of "metabolic process," "cellular process," and "single organism process." “Enzyme catalytic activity”, “water channel activity”, “binding” and “transporter activity” were the most abundant terms in molecular function category in both the genotypes under stress. There were 17 pathways significantly enriched (FDR-adjusted p-value < 0.05) in tolerant genotype for upregulated genes and the most enriched pathways including metabolic pathways (178 genes) followed by biosynthesis of secondary metabolites (121 genes), phenyl propanoid biosynthesis (29 genes), carbon metabolism (29 genes), glyoxylate and decarboxylate metabolism (16 genes), MAPK signalling pathway plant (14 genes) and starch and sucrose metabolism etc. Most of the Transcription Factors (TFs) identified families were enriched in, B3, bHLH, WRKY, ARF, ARR B, FAR, LBD, MADS and ERF. These TFs could be grouped into 10 families. The major upregulated genes families in tolerant genotype includes protein kinase, protein phosphatase type 2c, zing finger proteins, laccase gene, cytokinin dehydrogenase, cytochrome p450, galactinol synthase, NAC Domain protein, peroxidase, protein ubiquitin E3 ligase, mannitol dehydrogenase, fatty acid desaturase, UDP glycosyl transferase, Aquaporin PIP, Glutathione dependent genes, iv cellulose synthase and heat shock proteins etc.The major upregulated genes families in susceptible genotype includes LEA protein, PSII protein, peroxisomal membrane proteins, ABC transporter, ABA hydrolyse, phenyl alanine n-monoxygenase, tyrosine carboxylase, plastocyanin, trehalose phosphate phosphatase and expansin etc. RNA-Seq analysis showed that highly drought tolerant exotic genotype (T85/799) activated more number of drought responsive genes than drought sensitive check variety CCRP 5. Several drought-responsive genes were upregulated in the tolerant genotype such as those encoding TFs, cytochrome 450, and membrane transporters, and those associated with carbohydrate metabolism and flavonoid biosynthesis. These genes might confer drought tolerance in this cocoa genotype at the molecular level. Identified drought- responsive genes and metabolic pathways were targets for future studies in order to understand the molecular mechanism of drought tolerance in cocoa. Physiological and biochemical parameters also indicated the better performance of the tolerant genotype over the susceptible genotype under drought stress conditions. These results were a first step to understanding the molecular mechanisms of drought tolerance in cocoa and lay a foundation for its genetic improvement.