Contents lists available at ScienceDirect Gene journal homepage: www.elsevier.com/locate/gene Research paper Transcriptome profiling of the floral buds and discovery of genes related to sex-differentiation in the dioecious cucurbit Coccinia grandis (L.) Voigt Jatindra Nath Mohanty a , Sanghamitra Nayak a , Sumita Jha b , Raj Kumar Joshi a,⁎ a Functional Genomics Laboratory, Centre of Biotechnology, Siksha O Anusandhan University, Kalinga Nagar, Ghatikia, Bhubaneswar 751003, India b Centre of Advanced Study, Dept. of Botany, University of Calcutta, 35 Ballygunge Circular Road, Kolkata 700019, India ARTICLE INFO Keywords: Coccinia grandis Dioecious Sex determination RNA-seq Phytohormone signaling Transcription factors Methylatransferase ABSTRACT Dioecious species offer an inclusive structure to study the molecular basis of sexual dimorphism in angiosperms. Despite having a small genome and heteromorphic sex chromosomes, Coccinia grandis is a highly neglected dioecious species with little information available on its physical state, genetic orientation and key sex-defining elements. In the present study, we performed RNA-Seq and DGE analysis of male (MB) and female (FB) buds in C. grandis to gain insights into the molecular basis of sex determination in this plant. De novo assembly of 75 million clean reads resulted in 72,479 unigenes for male library and 63,308 unigenes for female library with a mean length of 736 bp. 61,458 (85.57%) unigenes displayed significant similarity with protein sequences from pub- licly available databases. Comparative transcriptome analyses revealed 1410 unigenes as differentially expressed (DEGs) between MB and FB samples. A consistent correlation between the expression levels of DEGs was ob- served for the RNA-Seq pattern and qRT-PCR validation. Functional annotation showed high enrichment of DEGs involved in phytohormone biosynthesis, hormone signaling and transduction, transcriptional regulation and methyltransferase activity. High induction of hormone responsive genes such as ARF6, ACC synthase1, SNRK2 and BRI1-associated receptor kinase 1 (BAK1) suggest that multiple phytohormones and their signaling crosstalk play crucial role in sex determination in this species. Beside, the transcription factors such as zinc fingers, homeodomain leucine zippers and MYBs were identified as major determinants of male specific expression. Moreover, the detection of multiple DEGs as the miRNA target site implies that a small RNA mediated gene silencing cascade may also be regulating gender differentiation in C. grandis. Overall, the present transcriptome resources provide us a large number of DEGs involved in sex expression and could form the groundwork for unravelling the molecular mechanism of sex determination in C. grandis. 1. Introduction Plants are characterized by multiple numbers of sexual systems duly regulated by ecological, developmental and genetical phenomenon (Ming et al., 2011). Hermaphroditism or coexistence of male and fe- male sex is the dominant form of sex expression, making up for 94% of plant species. While, a strict separation of the sexes (dioecy) and as- sociated sex determination system is distinct in animals, it is only re- flected in 6% of the 240,000 flowering plant species (Renner and Ricklefs, 1995). An inconsistent distribution of dioecy among the land plants suggests that separation of sexual function has evolved many times independently from a hermaphroditic ancestor (Tandurdzic and Banks, 2004). As such, the limited number of available dioecious plants can be effectively used as models for analyzing sex determination. Ex- tensive investigation of species like Silene latifolia, Carica papaya and Cucumis melo have led to the cloning and characterization of genes determining the male and female structures (Matsunaga, 2006; Yu et al., 2008; Martin et al., 2009). Nevertheless, sex determination is a complex phenomenon in higher plants due to the involvement of sev- eral genes that are differentially or specifically expressed at different developmental phases (Charlesworth, 2010). Under these circum- stances, the identification and characterization of a few sex-linked http://dx.doi.org/10.1016/j.gene.2017.05.058 Received 28 February 2017; Received in revised form 22 April 2017; Accepted 30 May 2017 ⁎ Corresponding author at: Functional Genomics Laboratory, Centre of Biotechnology, Siksha O Anusandhan University, Bhubaneswar 751003, Odisha, India. E-mail address: rajjoshi@soauniversity.ac.in (R.K. Joshi). Abbreviations: ACS, 1-aminocyclopropane-1-carboxylate synthase; AGL, Agamous like; ARFs, auxin response factors; BAK1, BRI1-associated receptor kinase 1; bHlH, basic loop helix loop; BRI1, brassinosteroid insensitive 1 protein; BSK, BR-signaling kinase; COG, cluster of orthologous group; DEG, Differentially expressed genes; EIN3, ethylene insensitive 3; FB, Female bud; GH3, Gretchen hagen3; GO, Genome annotation; HD-ZIP, homeodomain leucine zipper; KEGG, Kyoto encyclopedia of genes and genomes; MB, Male bud; mF, male fertility factor; MSE1, Male Specific Expression; MYB, Myloblastosis; PlantTFDB, Plant Transcription Factor Database; qRT-PCR, Quantitative reverse transcription PCR; RNA-Seq, RNA se- quencing; SAUR, small auxin up RNA; SNRK2, SNF1 related protein kinase 2; SPF, stamen promoting factor; SRK2, serine/threonine protein kinase 2; SuF, gynoecium suppression factor; TIR1, transport inhibitor response 1; ZF, Zinc finger Gene 626 (2017) 395–406 Available online 31 May 2017 0378-1119/ © 2017 Elsevier B.V. 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