Development and characterization of genic SSR-FDM for stem gall disease resistance in coriander (Coriandrum sativum L.) and its cross species transferability.

Coriander (Coriandrum sativum L.) is well known vegetable and spice crop grown globally for its leaves and seeds. Stem gall (Protomyces macrosporus L.) is a fungal disease affecting its quality and yield. However, no information is available on SSR markers linked to disease resistance in coriander. Hence, development of co-dominant genetic markers is prerequisite for disease investigations in coriander. In-house stem gall resistance and susceptible cultivars transcriptome data were utilized. Totally, 59,933 and 56,861 transcripts were examined, 9141 and 8346 Simple Sequence Repeats (SSR) were identified and the most abundant type was the tri, followed by di, tetra, penta and hexa nucleotide repeats. A total of ten selected SSR-Functional Domain Markers (FDM) were developed based on functional annotation terms associated with pathogen response and validated among ten coriander cultivars and their transferability was examined in five fennel (Foeniculum vulgare L.) cultivars. Nine primer pairs resulted from amplified bands. Marker ACorSGD-1 shown monomorphic bands among coriander genotypes except Acr-1 showed heterologouse and multiple bands in fennel cultivars. Markers ACorSGD-4, 5, 7 and 9 shown presence in resistant cultivars and absence of bands among susceptible cultivars of coriander and thus, considered to be the candidate markers for disease screening. Marker ACorSGD-6 shown monomorphic bands among coriander. Markers ACorSGD-1, 2, 3, and 5 shown transferability among fennel cultivars. A total of 136 alleles in coriander and fennel were produced. Using UPGMA clustering method a dendrogram was generated and cultivars were grouped into two separate clusters with coriander and fennel. Identified and developed SSR-FDM markers are useful for linkage mapping for disease resistant in coriander.

Identification and expression analysis of candidate genes associated with stem gall disease in Coriander (Coriandrum sativum L.) cultivars.

Coriander (Coriandrum sativum L.) is a well-known spice and aromatic crop cultivated globally. Stem gall disease is one of the major constraints for its leaf and seed quality used for consumption and also affecting the yield. The identification of resistance genes and further characterization of such genes could help to understand the molecular basis of resistance and lay a solid ground for cloning of stem gall resistance genes in coriander. To evaluate the genetic expression of disease resistance-relevant genes in popularly grown coriander cultivars in India such as Pant Haritma, Hisar Sugandh, Hisar Surabhi, Hisar Anand, Rajendra Swathi, ACr-1, ACr-2, AgCr-1, CO-2 and CS-6 were used for LRR, GDSL, USP, ANK and PDR gene expression using Real Time PCR along with 18S housekeeping gene as internal control for the normalization. Result revealed the different expression pattern of genes among the cultivars tested. Highest expression was shown in cultivar AgCr-1 followed by Pant Haritma, Hisar Sugandh and ACr-1, and least expression in Hisar Anand, ACr-2, CO-2, Rajendra Swathi and CS-6. Domain analysis revealed the conserved domain relevance of the genes. This is the first report on stem gall resistance gene expression in coriander. The identified genes have a potential role in coriander and further utilize in crop improvement program. We hypothesize that contrasting cultivars can be a good source for candidate gene evaluation and further to use them as potential markers and used in hybridization program focus on incorporating and develop durable disease-resistance into the adapted cultivars of the region.

Transcriptome Analysis Reveals Candidate Genes for Petroselinic Acid Biosynthesis in Fruits of Coriandrum sativum L.

Petroselinic acid (18:1Δ6), a monounsaturated cis Δ-6 fatty acid, has many prospective applications in functional foods and for the nutraceutical and pharmaceutical industries. Up to 80% of petroselinic acid has been found in the oil from fruits of coriander (Coriandrum sativum L.), which make it an ideal source for investigating the biosynthesis of petroselinic acid. A coriander acyl-acyl carrier protein desaturase was identified to be involved in its biosynthesis more than two decades ago, but since then little further progress in this area has been reported. In this study, the fatty acid profiles of coriander fruits at six developmental stages were analyzed. Fruit samples from three developmental stages with rapid accumulation of petroselinic acid were used for RNA sequencing using the Illumina Hiseq4000 platform. The transcriptome analysis presented 93 323 nonredundant unigenes and 8545 differentially expressed genes. Functional annotation and combined gene expression data revealed candidate genes potentially involved in petroselinic acid biosynthesis and its incorporation into triacylglycerols. Tissue-specific examination of q-PCR validation further suggested that ACPD1/3, KAS I-1, FATB-1/3, and DGAT2 may be highly involved. Bioinformatic analysis of CsFATB and CsDGAT2 identified their putative key amino acids or functional motifs. These results provide a molecular foundation for petroselinic acid biosynthesis in coriander fruit and facilitate its genetic engineering in other hosts.

Transcriptome landscaping for gene mining and SSR marker development in Coriander (Coriandrum sativum L.).

Coriander (Coriandrum sativum L.) is an aromatic herb, widely used as a spice and is of great pharmaceutical interest. Despite high medicinal and economic value, there is a dearth of genomic information about profiling as well as the expressed sequence-based genic markers. In this study, transcriptome was sequenced from seeds, leaves, and flower for gene mining and identification of SSR markers. A total of 9746 SSR containing loci were identified, the most abundant type of SSR identified were the di-nucleotide repeat motifs (45.5%), followed by tri- (34.6%), tetra- (4.5%), penta- (1.5%) and hexanucleotide repeats (1%). A total of 3795 primers were designed, out of which 120 randomly selected were validated in 14 accessions of coriander cultivated in India. The current study provides useful information about preliminary transcriptome sketch and genic markers, which can be useful in breeding and genetic diversity estimation of coriander.

Transcriptome profiling of coriander: a dual purpose crop unravels stem gall resistance genes.

Stem gall (Protomyces macrosporus Unger), a serious disease that affects leaves, petioles, stems and fruits of coriander (Coriandrum sativum L.) causing heavy loss in yield. Genetic improvement of coriander for stem gall disease is indispensable. Coriander cultivars of stem gall resistance (ACr-1) and susceptible (CS-6) leaf samples were utilized and transcriptome sequenced using Illumina NextSeq500 platform. After trimming low-quality reads and adapter sequences, a total of 49,163,108 and 43,746,120 high-quality reads were retained and further assembly resulted validated transcripts of 59,933 and 56,861. We have predicted 52,506 and 48,858 coding sequences (CDS) ofwhich 50,506 and 46,945 were annotated using NCBInr database. Gene ontology analysis annotated 19,099 and 17,625 terms; pathway analysis obtained 24 different functional pathway categories; signal transduction, transport, catabolism, translation and carbohydrate metabolism pathways etc. were dominated. Differentially expressed genes analysis predicted 13,123 CDS commonly expressed of which 431 and 400 genes were significantly upregulated and downregulated, respectively, in which Rgenes, stress inducible transcription factors such as ERF, NAC, bZIP, MYB, DREB and WRKY and antifungal related genes were predicted. The real-time PCR analysis of HSP20 gene expression in resistance showed upregulation by 10-fold over susceptible sample and 18s used as a housekeeping gene for normalization. The present results provide an insights into various aspects underlying the development of resistance to stem gall in coriander.

Effects of methyl jasmonate and carotenogenic inhibitors on gene expression and carotenoid accumulation in coriander (Coriandrum sativum L.) foliage.

Coriander (Coriandrum sativum L.), a commonly used annual herb that accumulates carotenoids upon methyl jasmonate (MeJA) treatment, provides an excellent model to investigate carotenogenesis and gene regulation. To explore key mechanisms involved in enhancing carotenoids, transcriptional expression profile of ten carotenogenic genes in the presence of MeJA and various gene specific inhibitors were investigated. Foliar application of MeJA (10 µM) increased expression levels of CsPDS (phytoene desaturase), CsZDS (ς-carotene desaturase), CsCHYE (carotene ε - hydroxylase) and CsLCYE (lycopene ß-cyclase) genes, and their transcript levels were strongly associated with carotenoid content, where, three days after treatment, 3.9 & 6.1 fold increase was observed for ß-carotene and lutein respectively. The regulatory effect of key genes, CsPDS, CsZDS, CsLCYE and LCYB were further confirmed by using gene-specific inhibitors fosmidomycin, norflurazon and amitrol. Norflurazon- the phytoene desaturation inhibitor leads to a decrease in ß-carotene and lutein content correlated with CsPDS, CsZDS gene induction. Our results clearly demonstrate that MeJA induced-signalling network evokes carotenogenic genes, leading to the accumulation of carotenoids. This knowledge may help to develop precise strategies for remodelling carotenoid pathway so that desired levels of a particular carotenoid in leafy vegetables is achievable.

Genetic combining ability of coriander genotypes for agronomic and phytochemical traits in response to contrasting irrigation regimes.

Knowledge of genetic combining ability and gene action would help breeders to choose suitable parents and devise an appropriate breeding strategy for coriander. In the present study, six diverse genotypes of coriander, their 15 F1s and 15 F2s were evaluated through randomized complete block design with three replications to study genetic combining ability for agronomic and phytochemical traits in coriander. Plants were subjected to well-watered (WW), mild water-deficit stress (MWDS) and severe water-deficit stress (SWDS) irrigation regimes. The results indicate that water-deficit stress decreased all of the measured traits in both the F1 and F2 generations. General combining ability and specific combining ability effects were highly significant for all of the traits in both the F1 and F2 generations. Additive gene action was predominant for phonology and fruit yield component traits in all irrigation regimes in both the F1 and F2 generations. For fatty acid content and total lipid yield, non-additive gene action was predominant in the F1 generation while additive gene action was predominant in the F2 generation under MWDS and SWDS conditions. The P4 parent had the highest general combining ability for fruit yield components in both the F1 and F2 generations. The P6 parent had the highest general combining ability for phenological and phytochemical traits. The P4 and P6 parents are promising material to develop early flowering and early maturing genotypes coupled with high total lipids in advanced generations of segregation.

Evaluation of folate-binding proteins and stability of folates in plant foliages.

The present study reports the presence of folate binding proteins (FBPs) in the plants, coriander and Arabidopsis, and their contributions toward folate enhancement. After observing that salicylic acid (SA) enhanced the accumulation of folates in coriander, a study was conducted in Arabidopsis, where twofold increase in folates occurred in foliage upon SA treatment. For obtaining insights into genes involved in SA-induced folate accumulation, microarray data of responsive genes in Arabidopsis were screened. Based on the expression profiles, 19 genes were further analysed by qPCR. The results revealed that folate biosynthetic genes were largely down-regulated, whereas a gene of a putative folate-binding protein (FPB) was up-regulated, which correlated with a significant increase of FPBs in foliage. This new information on a plant FBP appears useful for metabolic engineering of a wide range of crops to enhance the content and stability of the folates during post-harvest storage.

UV-B antagonises shade avoidance and increases levels of the flavonoid quercetin in coriander (Coriandrum sativum).

Despite controlling a diverse array of regulatory processes in plants, UV-B wavelengths (280-315 nm) are attenuated by common greenhouse materials such as glass and polycarbonate and are therefore depleted in many commercial growing environments. In this study, we analysed the architecture, pigment accumulation and antioxidant capacity of coriander (Coriandrum sativum, also known as cilantro) plants grown with and without supplementary UV-B (1.5 µmol m-2 s-1). We demonstrate that UV-B limits stem elongation responses to neighbour proximity perception (shade avoidance), promoting a more compact plant architecture. In addition, UV-B increased leaf quercetin content and total antioxidant capacity. Arabidopsis thaliana mutants deficient in flavonoid biosynthesis were not impaired in shade avoidance inhibition, suggesting that UV-B-induced flavonoid synthesis is not a component of this response. Our results indicate that UV-B supplementation may provide a method to manipulate the architecture, flavour and nutritional content of potted herbs whilst reducing the deleterious impacts of dense planting on product quality.

Plant regeneration through somatic embryogenesis and genome size analysis of Coriandrum sativum L.

In the present study, an improved plant regeneration protocol via primary and secondary somatic embryogenesis was established in two Co-1 and Rajendra Swathi (RS) varieties of Coriandrum sativum L. Callus was induced from root explants on 2, 4-D (0.5-2.0 mg/l) supplemented MS. The addition of BA (0.2 mg/l) improved callus induction and proliferation response significantly. The maximum callus induction frequency was on 1.0 mg/l 2, 4-D and 0.2 mg/l BA added MS medium (77.5 % in Co-1 and 72.3 % in RS). The callus transformed into embryogenic callus on 2, 4-D added MS with maximum embryogenic frequency was on 1.0 mg/l. The granular embryogenic callus differentiated into globular embryos on induction medium, which later progressed to heart-, torpedo- and cotyledonary embryos on medium amended with 0.5 mg/l NAA and 0.2 mg/l BA. On an average, 2-3 secondary somatic embryos (SEs) were developed on mature primary SEs, which increased the total embryo numbers in culture. Histology and scanning electron microscopy (SEM) studies are presented for the origin, development of primary and secondary embryos in coriander. Later, these induced embryos converted into plantlets on 1.0 mg/l BA and 0.2 mg/l NAA-amended medium. The regenerated plantlets were cultured on 0.5 mg/l IBA added ½ MS for promotion of roots. The well-rooted plantlets were acclimatized and transferred to soil. The genetic stability of embryo-regenerated plant was analyzed by flow cytometry with optimized Pongamia pinnata as standard. The 2C DNA content of RS coriander variety was estimated to 5.1 pg; the primary and secondary somatic embryo-derived plants had 5.26 and 5.44 pg 2C DNA content, respectively. The regenerated plants were genetically stable, genome size similar to seed-germinated coriander plants.

Studies on genetic divergence among Indian varieties of a spice herb, Coriandrum sativum.

Coriander (Coriandrum sativum L.) is an annual spice herb that belongs to umbel family Apiaceae with diversified uses. We investigated the extent of variability among 22 Indian varieties of coriander using phenotypic and genetic markers. Multilocus genotyping by nine RAPD primers detected an average of intraspecific variations amounting to 66.18% polymorphism in banding patterns. Analysis of molecular variance indicated that a greater proportion of total genetic variation exists within population (98%) rather than among populations (2%). Higher values of Nei's gene diversity (h) and Shannon Information Index (i) and genetic distance analysis validate wider genetic diversity among Indian coriander varieties. Besides total internal transcribed spacer (ITS) length variations and single nucleotide polymorphisms, insertions/deletions (INDELS) were detected at seven sites in ITS-1 region. Multiple sequence alignment of 12 sequenced varieties revealed cent per cent identities of 5.8S gene region (162 bp) that validates its conserved nature. Multiple sequence alignment of ITS-1 region may be of phylogenetic significance in distinguishing and cataloguing of coriander germplasm. The representative sequences of each subgroup and all distinct varieties of RAPD clusters have been submitted to NCBI database and assigned Gen Accession numbers HQ 377194-377205. The measures of relative genetic distances among the varieties of coriander did not completely correlate the geographical places of their development. Eventually, the knowledge of their genetic relationships and DNA bar coding will be of significance.

Genomic structures and characterization of the 5'-flanking regions of acyl carrier protein and Delta4-palmitoyl-ACP desaturase genes from Coriandrum sativum.

The seed-specific or seed-predominant promoters of acyl carrier protein (Cs-ACP1) and Delta4-palmitoyl-acyl carrier protein desaturase (Cs-4PAD) genes, which are involved in the biosynthesis of petroselinic acid, were isolated from coriander (Coriandrum sativum) and analyzed in coriander endosperms and transgenic Arabidopsis. The expression of Cs-ACP1 and Cs-4PAD genes was coordinately regulated during seed development.

Heterologous expression of Arabidopsis ERS1 causes delayed senescence in coriander.

The phytohormone ethylene is involved in many developmental processes, including leaf and flower senescence. Ethylene is perceived by plants through receptors that trigger the downstream signal transduction pathway. The mutated ethylene receptor ERS1 (ethylene response sensor) from Arabidopsis is of a dominant negative nature and confers ethylene insensitivity in Arabidopsis. To investigate if the altered ERS1 gene can affect the tissue senescence in heterologous plants, we introduced it into coriander by Agrobacterium-mediated transformation. Transgenic plants were regenerated by cocultivating hypocotyl segments with A. tumefaciens harboring binary vector pCGN1547 that carried the ERS1 gene. The presence and expression of the transgene were confirmed by genomic Southern blot and reverse transcriptase-PCR analyses. Leaf and flower senescence were delayed significantly in the transgenic plants. The ability of the mutated ERS1 gene to confer the ethylene-insensitive phenotype can be exploited for extending the shelf-life of leafy vegetables.

Coriander essential oil composition from two genotypes grown in different environmental conditions.

The objective was to study the essential oil composition of coriander fruits in plants growing in environments differing in soil conditions and weediness level. Factorial field experiments were conducted in two locations from the Rolling Pampas, Argentina, and two coriander landraces (European and Argentinean) were tested under two levels of nitrogen fertilization and weediness. Data were evaluated with uni- and multivariate techniques. The variation in the oil composition was related to the relative proportion of the constituents and not to the presence/absence of a particular component. Weather conditions in 1997 favored linalool and camphor in both landraces. Location, fertilization, and weediness also affected the chemical profile. The European landrace showed a more stable concentration of the major components than the Argentinean landrace. These results, which show the relationships between some environmental conditions and the essential oil composition, are useful in the development of innovative strategies aimed to improve oil composition and to manage crop pests.

An unusual seed-specific 3-ketoacyl-ACP synthase associated with the biosynthesis of petroselinic acid in coriander.

Petroselinic acid (18:1 delta6) is the major component of the seed oil of Umbelliferae species such as coriander (Coriandrum sativum) as well as Araliaceae and Garryaceae species. This unusual fatty acid is synthesized in plastids by the delta4 desaturation of palmitoyl-acyl carrier protein (16:0-ACP) and subsequent elongation of delta4-hexadecenoyl (16:1 delta4)-ACP. To characterize the enzymatic nature of the elongation reaction, an in vitro assay was developed with 16:1 delta4-ACP and 16:0-ACP as substrates. Extracts from developing coriander seeds elongated 16:1 delta4-ACP in a competitive assay at rates ten-fold greater than that with 16:0-ACP. In contrast, extracts from castor seeds, which do not synthesize petroselinic acid, displayed a strong preference for the elongation of 16:0-ACP rather than 16:1 A4-ACP. In addition, the elongation of 16:1 A4-ACP and 16:0-ACP by coriander seed extracts was strongly inhibited by cerulenin at concentrations as low as 10 microM. This finding suggested that the elongation of 16:1 A4-ACP and 16:0-ACP in coriander seed is catalyzed by a 3-ketoacyl-ACP synthase (KAS) 1-type enzyme(s), rather than a KAS II-type activity that is typically associated with 16:0-ACP elongation. Consistent with this, a cDNA for a diverged form of KAS I was isolated from a cDNA library prepared from developing coriander seed. Using a variety of heterologous probing techniques, no KAS II-type cDNAs could be identified in this library. Multiple alignment of KAS amino acid sequences indicated that, although the polypeptide corresponding to the coriander cDNA is more closely related to KAS I. its active site motif deviates from those found in both KAS I and KAS II enzymes. Also suggestive of a possible role in petroselinic acid synthesis, antibodies raised to the recombinant protein recognize an abundant 45 kDa polypeptide in coriander endosperm that is not detected in coriander leaves. These antibodies also recognize a major band of similar size in developing seeds of English ivy (Hedera helix), an Araliaceae species that also accumulates petroselinic acid in a seed-specific manner.