Fruit transcriptional profiling of the contrasting genotypes for shelf life reveals the key candidate genes and molecular pathways regulating post-harvest biology in cucumber.

Cucumber fruits are perishable in nature and become unfit for market within 2-3 days of harvesting. A natural variant, DC-48 with exceptionally high shelf life was developed and used to dissect the genetic architecture and molecular mechanism for extended shelf life through RNA-seq for first time. A total of 1364 DEGs were identified and cell wall degradation, chlorophyll and ethylene metabolism related genes played key role. Polygalacturunase (PG), Expansin (EXP) and xyloglucan were down regulated determining fruit firmness and retention of fresh green colour was mainly attributed to the low expression level of the chlorophyll catalytic enzymes (CCEs). Gene regulatory networks revealed the hub genes and cross-talk associated with wide variety of the biological processes. Large number of SSRs (21524), SNPs (545173) and InDels (126252) identified will be instrumental in cucumber improvement. A web genomic resource, CsExSLDb developed will provide a platform for future investigation on cucumber post-harvest biology.

Strategies for utilization of crop wild relatives in plant breeding programs.

Crop wild relatives (CWRs) are weedy and wild relatives of the domesticated and cultivated crops, which usually occur and are maintained in natural forms in their centres of origin. These include the ancestors or progenitors of all cultivated species and comprise rich sources of diversity for many important traits useful in plant breeding. CWRs can play an important role in broadening genetic bases and introgression of economical traits into crops, but their direct use by breeders for varietal improvement program is usually not advantageous due to the presence of crossing or chromosome introgression barriers with cultivated species as well as their high frequencies of agronomically undesirable alleles. Linkage drag may subsequently result in unfavourable traits in the subsequent progeny when segments of the genome linked with quantitative trait loci (QTL), or a phenotype, are introgressed from wild germplasm. Here, we first present an overview in regards to the contribution that wild species have made to improve biotic, abiotic stress tolerances and yield-related traits in crop varieties, and secondly summarise the various challenges which are experienced in interspecific hybridization along with their probable solutions. We subsequently suggest techniques for readily harnessing these wild relatives for fast and effective introgression of exotic alleles in pre-breeding research programs.

Differential accumulation of ß-carotene and tissue specific expression of phytoene synthase (MaPsy) gene in banana (Musa sp) cultivars.

An experiment was conducted with twelve major Indian banana cultivars to investigate the molecular relationship between the differential accumulation of ß-carotene in peel and pulp of the banana fruit and carotenoid biosynthetic pathway genes. The high performance liquid chromatography showed that all banana cultivars accumulated two-three fold more ß-carotene in non-edible portion of the banana fruit. However, Nendran, a famous orange fleshed cultivar of South India, had high ß-carotene content (1362 µg/100 g) in edible pulp. The gene encoding Musa accuminata phytoene synthase (MaPsy) was successfully amplified using a pair of degenerate primers designed from Oncidium orchid. The deduced amino acid sequences shared a high level of identity to phytoene synthase gene from other plants. Gene expression analysis confirmed the presence of two isoforms (MaPsy1 and MaPsy2) of MaPsy gene in banana fruits. Presence of two isoforms of MaPsy gene in peel and one in pulp confirmed the differential accumulation of ß-carotene in banana fruits. However, Nendran accumulated more ß-carotene in edible pulp due to presence of both the isoforms of MaPsy gene. Thus, carotenoid accumulation is a tissue specific process strongly dependent on differential expression pattern of two isoforms of MaPsy gene in banana.

Genome wide identification of lncRNAs and circRNAs having regulatory role in fruit shelf life in health crop cucumber (Cucumis sativus L.).

Cucumber is an extremely perishable vegetable; however, under room conditions, the fruits become unfit for consumption 2-3 days after harvesting. One natural variant, DC-48 with an extended shelf-life was identified, fruits of which can be stored up to 10-15 days under room temperature. The genes involved in this economically important trait are regulated by non-coding RNAs. The study aims to identify the long non-coding RNAs (lncRNAs) and circular RNAs (circRNAs) by taking two contrasting genotypes, DC-48 and DC-83, at two different fruit developmental stages. The upper epidermis of the fruits was collected at 5 days and 10 days after pollination (DAP) for high throughput RNA sequencing. The differential expression analysis was performed to identify differentially expressed (DE) lncRNAs and circRNAs along with the network analysis of lncRNA, miRNA, circRNA, and mRNA interactions. A total of 97 DElncRNAs were identified where 18 were common under both the developmental stages (8 down regulated and 10 upregulated). Based on the back-spliced reads, 238 circRNAs were found to be distributed uniformly throughout the cucumber genomes with the highest numbers (71) in chromosome 4. The majority of the circRNAs (49%) were exonic in origin followed by inter-genic (47%) and intronic (4%) origin. The genes related to fruit firmness, namely, polygalacturonase, expansin, pectate lyase, and xyloglucan glycosyltransferase were present in the target sites and co-localized networks indicating the role of the lncRNA and circRNAs in their regulation. Genes related to fruit ripening, namely, trehalose-6-phosphate synthase, squamosa promoter binding protein, WRKY domain transcription factors, MADS box proteins, abscisic stress ripening inhibitors, and different classes of heat shock proteins (HSPs) were also found to be regulated by the identified lncRNA and circRNAs. Besides, ethylene biosynthesis and chlorophyll metabolisms were also found to be regulated by DElncRNAs and circRNAs. A total of 17 transcripts were also successfully validated through RT PCR data. These results would help the breeders to identify the complex molecular network and regulatory role of the lncRNAs and circRNAs in determining the shelf-life of cucumbers.

Elucidating Mitochondrial DNA Markers of Ogura-Based CMS Lines in Indian Cauliflowers (Brassica oleracea var. botrytis L.) and Their Floral Abnormalities Due to Diversity in Cytonuclear Interactions.

Mitochondrial markers can be used to differentiate diverse mitotypes as well as cytoplasms in angiosperms. In cauliflower, cultivation of hybrids is pivotal in remunerative agriculture and cytoplasmic male sterile lines constitute an important component of the hybrid breeding. In diversifying the source of male sterility, it is essential to appropriately differentiate among the available male sterile cytoplasms in cauliflower. PCR polymorphism at the key mitochondrial genes associated with male sterility will be instrumental in analyzing, molecular characterization, and development of mitotype-specific markers for differentiation of different cytoplasmic sources. Presence of auto- and alloplasmic cytonuclear combinations result in complex floral abnormalities. In this context, the present investigation highlighted the utility of organelle genome-based markers in distinguishing cytoplasm types in Indian cauliflowers and unveils the epistatic effects of the cytonuclear interactions influencing floral phenotypes. In PCR-based analysis using a set of primers targeted to orf-138, 76 Indian cauliflower lines depicted the presence of Ogura cytoplasm albeit the amplicons generated exhibited polymorphism within the ofr-138 sequence. The polymorphic fragments were found to be spanning over 200-280 bp and 410-470 bp genomic regions of BnTR4 and orf125, respectively. Sequence analysis revealed that such cytoplasmic genetic variations could be attributed to single nucleotide polymorphisms and insertion or deletions of 31/51 nucleotides. The cytoplasmic effects on varying nuclear-genetic backgrounds rendered an array of floral abnormalities like reduction in flower size, fused flowers, splitted style with the exposed ovule, absence of nonfunctional stamens, and petaloid stamens. These floral malformations caused dysplasia of flower structure affecting female fertility with inefficient nectar production. The finding provides an important reference to ameliorate understanding of mechanism of cytonuclear interactions in floral organ development in Brassicas. The study paves the way for unraveling developmental biology of CMS phenotypes in eukaryotic organisms and intergenomic conflict in plant speciation.

Identification, Validation and Utilization of Novel Nematode-Responsive Root-Specific Promoters in Arabidopsis for Inducing Host-Delivered RNAi Mediated Root-Knot Nematode Resistance.

The root-knot nematode (RKN), Meloidogyne incognita, is an obligate, sedentary endoparasite that infects a large number of crops and severely affects productivity. The commonly used nematode control strategies have their own limitations. Of late, RNA interference (RNAi) has become a popular approach for the development of nematode resistance in plants. Transgenic crops capable of expressing dsRNAs, specifically in roots for disrupting the parasitic process, offer an effective and efficient means of producing resistant crops. We identified nematode-responsive and root-specific (NRRS) promoters by using microarray data from the public domain and known conserved cis-elements. A set of 51 NRRS genes was identified which was narrowed down further on the basis of presence of cis-elements combined with minimal expression in the absence of nematode infection. The comparative analysis of promoters from the enriched NRRS set, along with earlier reported nematode-responsive genes, led to the identification of specific cis-elements. The promoters of two candidate genes were used to generate transgenic plants harboring promoter GUS constructs and tested in planta against nematodes. Both promoters showed preferential expression upon nematode infection, exclusively in the root in one and galls in the other. One of these NRRS promoters was used to drive the expression of splicing factor, a nematode-specific gene, for generating host-delivered RNAi-mediated nematode-resistant plants. Transgenic lines expressing dsRNA of splicing factor under the NRRS promoter exhibited upto a 32% reduction in number of galls compared to control plants.

Enhanced plant growth promoting role of phycomolecules coated zinc oxide nanoparticles with P supplementation in cotton (Gossypium hirsutum L.).

This report focuses on application of zinc oxide nanoparticles (ZnONPs) carrying phycomolecule ligands as a novel plant growth promoter aimed at increasing the crop productivity. The present investigation examined the effect of ZnONPs on plant growth characteristics, and associated biochemical changes in cotton (Gossypium hirsutum L.) following growth in a range of concentrations (25-200 mg L-l ZnONPs) in combination with 100 mM P in a hydroponic system. Treated plants registered an increase in growth and total biomass by 130.6% and 131%, respectively, over control. Results demonstrated a significant increase in the level of chlorophyll a (141.6%), b (134.7%), carotenoids (138.6%), and total soluble protein contents (179.4%); at the same time, a significant reduction (68%) in the level of malondialdehyde (MDA) in leaves with respect to control. Interestingly, a significant increase in superoxide dismutase (SOD, 264.2%), and peroxidase (POX, 182.8%) enzyme activities followed by a decrease in the catalase (CAT) activity, in response to above treatments. These results suggest that bioengineered ZnONPs interact with meristematic cells triggering biochemical pathways conducive to an accumulation of biomass. Further investigations will map out the mode of action involved in growth promotion.

Differential expression of salt overly sensitive pathway genes determines salinity stress tolerance in Brassica genotypes.

The objective of the present study was to examine the role of SOS pathway in salinity stress tolerance in Brassica spp. An experiment was conducted in pot culture with 4 Brassica genotypes, i.e., CS 52 and CS 54, Varuna and T 9 subjected to two levels of salinity treatments along with a control, viz., 1.65 (S(0)), 4.50 (S(1)) and 6.76 (S(2)) dS m(-1). Salinity treatment significantly decreased relative water content (RWC), membrane stability index (MSI) and chlorophyll (Chl) content in leaves and potassium (K) content in leaf, stem and root of all the genotypes. The decline in RWC, MSI, Chl and K content was significantly less in CS 52 and CS 54 as compared to Varuna and T 9. In contrast, the sodium (Na) content increased under salinity stress in all the plant parts in all the genotypes, however, the increase was less in CS 52 and CS 54, which also showed higher K/Na ratio, and thus more favourable cellular environment. Gene expression studies revealed the existence of a more efficient salt overly sensitive pathway composed of SOS1, SOS2, SOS3 and vacuolar Na(+)/H(+) antiporter in CS 52 and CS 54 compared to Varuna and T 9. Sequence analyses of partial cDNAs showed the conserved nature of these genes, and their intra and intergenic relatedness. It is thus concluded that existence of an efficient SOS pathway, resulting in higher K/Na ratio, could be one of the major factor determining salinity stress tolerance of Brassica juncea genotypes CS 52 and CS 54.

Transgenic Indian mustard (Brassica juncea) expressing tomato glucanase leads to arrested growth of Alternaria brassicae.

Brassica juncea is an important oilseed crop of the Indian sub-continent. Yield loss due to fungal disease alternaria leaf spot caused by Alternaria brassicae is a serious problem in cultivation of this crop. Nonavailability of resistance genes within crossable germplasms of Brassica necessitates use of genetic engineering strategies to develop genetic resistance against this pathogen. The pathogenesis related (PR) proteins are group of plant proteins that are toxic to invading fungal pathogens, but are present in plant in trace amount. Thus, overexpression of PR proteins leads to increased resistance to pathogenic fungi in several crops. The PR protein glucanase hydrolyzes a major cell-wall component, glucan, of pathogenic fungi and acts as a plant defense barrier. We report the expression of a class I basic glucanase gene, under the control of CaMV 35S promoter, in Indian mustard and its genetic resistance against alternaria leaf spot. Southern and Northern hybridization confirmed stable integration and expression of the glucanase gene in mustard transgenics. Several independent transgenics were screened in vitro and under poly house conditions for their resistance against Alternaria brassicae. In an in vitro antifungal assay, transgenics arrested hyphal growth of Alternaria brassicae by 15-54%. Under pathogen-challenged conditions in poly house, the transgenics showed restricted number, size and spread of lesions caused by Alternaria brassicae. Also, the onset of disease was delayed in transgenics compared to untransformed parent plants. The results demonstrate potentiality of a PR protein from a heterologous source in developing alternaria leaf spot resistance in Indian mustard.