Genetic diversity analysis and trait phenotyping for drought tolerance in Amaranth (Amaranthus spp) Germplasm

Drought is a major abiotic stress which causes severe crop loses worldwide. One way of enhancing food security in areas of limited or unpredictable rainfall is to exploit the wide genetic diversity of underutilised crop species with increased drought tolerance characteristics. This study aimed to develop a precise trait phenotyping strategy for drought tolerance in Amaranthus spp. (Amaranthus tricolor). This research provides a framework to identify the genetic basis of drought tolerance in amaranth germplasm through a panel of 188 amaranth mini core collections. A 188 amaranth mini core collection, derived from an original collection of 783 accessions, was made up of 131 World Vegetable Center Genebank (AVRDC) accessions, 52 United State Department of Agriculture (USDA) accessions, three commercial African varieties from East-West Seed (E-W) and two commercial local Malaysian varieties. It comprises of 18 species from diverse geographical origins and 120 out of 188 accessions belonging to A. tricolor. The choice of sampling strategy through stratification based on morphological database allows the core-set to retain more than 70% of the germplasm entire collection. The multivariate analysis using Jaccard’s similarity matrix based on 10 qualitative traits, including leaf, petiole and stem colours, growth habit, branching index, leaf shape and margin, and terminal inflorescence colour, shape and attitude revealed that morphological traits were less capable in demarcating plant-type, namely: grain, vegetable and weed in the 188 amaranth mini core collection. Structure-like population genetic analysis of a high density DArTseq SNPs was performed in two steps; all 188 amaranth accessions and only 120 A. tricolor accessions (3,898 SNP for 183 amaranth accessions and 4, 631 SNP for 118 A. tricolor accessions after SNP filering, respectively). Both structures produced three major sub-populations (K=3) and this DArTseq SNPs data generates consistent taxonomic classification of amaranth sub-genera (Amaranthus Amaranthus, Amaranthus Acnida and Amaranthus albersia), although the accessions were less likely demarcated by geographical origin and morphological traits. The genome-wide association study (GWAS) of 10 qualitative traits revealed that there was an association between specific phenotypes and genetic variants within a genome as 25 marker trait associations (MTAs) (P<0.01) associated with branching index, petiole pigmentation, inflorescence colour, and terminal inflorescence shape and attitude were found. To develop a precise trait phenotyping strategy for drought tolerance in A. tricolor, two pilot experiments were evaluated separately to evaluate the effect of drought on shoot and root traits; (1) Transpiration efficiency (TE) and (2) Root morphology, leaf gas exchange, cellular hydration and proline accumulation. In TE experiment, plants were subjected to either a gradual dry down or well-watered conditions. Results showed that TE was significantly higher (P<0.01) in water-deficient (WD) plants compared to water-sufficient (WS) plants. There was no significant difference in the fraction of transpirable soil water (FTSW) threshold decline between the amaranth genotypes. In second experiment, two contrasting amaranth varieties (red betalain and green acyanic) were subjected to gradual drought stresses. Genotypes that share similar morphological characteristics, specifically leaf colour may not necessarily have the same drought adaptive features. Green leaf amaranths rapidly reduced relative water content (RWC) as early as 10 days of water treatment (DAT, range: 70%-76%), while red leaf amaranth retained comparatively high RWC at 10 DAT and only began to decline at 15 DAT (range: 59%-61%). Green leaf amaranths showed no changes in proline content, while red leaf amaranths displayed variations in the adjustment of proline accumulation at each time point. Further, two drought tolerance screening trials were carried out on a sub-set of 44 A. tricolor accession to identify germplasms with potential drought-tolerant genotypes. Stress intensity was higher in Trial I (0.73) compared with Trial II (0.31) and low broad sense heritability was found for most of the growth traits (ranged: 0.12 to 0.31). Three drought tolerance indices, namely geometric mean productivity (GMP, P<0.01), mean productivity (MP, P<0.001) and stress tolerance index (STI, P<0.05) were consistent and stable predictors of highly drought tolerant genotypes regardless of different weather conditions. Ten tolerant genotypes and three susceptible genotypes were identified and had consistent drought tolerance performance across the two screening trials. This finding revealed that a change in stem biomass was probably the main mechanisms of drought tolerance in amaranth. Stem biomass was negatively correlated with PSII photochemistry (light-adapted quantum yield, Fv’/Fm’ and dark-adapted quantum yield, Fv/Fm) but positively correlated with RWC under drought stress, i.e. stem biomass improved yield performance by regulating osmotic adjustment and prevent photoinhibition to the plants. A total of 19 significant (P