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Title: De novo transcriptome sequencing and customized abscission zone-specific microarray as a new molecular tool for analysis of tomato organ abscission

Author
item SUNDARESAN, SRIVIGNESH - Volcani Center (ARO)
item PHILOSOPH-HADAS, SONIA - Volcani Center (ARO)
item RIOV, JOSEPH - Hebrew University Of Jerusalem
item MUGASIMANGALAM, RAJA - Qtlomics Technologies Pvt Ltd
item KURAVADI, NAGESH - Qtlomics Technologies Pvt Ltd
item KOCHANEK, BETINA - Volcani Center (ARO)
item SALIM, SHOSHANA - Volcani Center (ARO)
item Tucker, Mark
item MEIR, SHIMON - Volcani Center (ARO)

Submitted to: Frontiers in Plant Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/24/2015
Publication Date: 1/14/2016
Citation: Sundaresan, S., Philosoph-Hadas, S., Riov, J., Mugasimangalam, R., Kuravadi, N.A., Kochanek, B., Salim, S., Tucker, M.L., Meir, S. 2016. De novo transcriptome sequencing and customized abscission zone-specific microarray as a new molecular tool for analysis of tomato organ abscission. Frontiers in Plant Science. 6(1258):1-29.

Interpretive Summary: Abscission, organ separation, is a highly regulated process occurring as a final stage of organ development. We used technological advancements in next generation sequencing (NGS) - RNA-Sequencing (RNA-Seq), to better understand changes in gene expression during tomato flower and leaf abscission. The sequence information obtained from this work was then used to design a custom abscission-specific microarray chip. The microarray chip is comprised of 180,880 short (60 nucleotide) sequences that enable a researcher to accurately quantify global changes in the expression of genes during tomato abscission under different environmental and growth conditions and in mutants with altered abscission responses. The customized abscission-specific microarray chip provides a cost-effective approach to analyze multiple samples in rapid succession. Results from this study will help researcher and industry identify genes essential for abscission that can be targeted for breeding or transgenic manipulation in tomato or other plant species.

Technical Abstract: Abscission, which is the process of organ separation, is a highly regulated process occurring as a final stage of organ development. In the tomato (Solanum lycopersicum) system, flower and leaf abscission was induced by flower removal or leaf deblading, leading to auxin depletion which results in increased sensitivity of the abscission zone (AZ) to ethylene. Transcriptome studies were conducted in various crops to identify the genes expressed in the AZ tissues. However, information on the molecular mechanisms that drive the acquisition of abscission competence and its modulation by auxin gradients is still lacking. In the present study we used the technological advancements in next generation sequencing (NGS) - RNA-Sequencing (RNA-Seq), to understand the complete transcriptome of tomato flower and leaf AZs (FAZ and LAZ, respectively). RNA-Seq was performed on a pool of cDNAs sampled during the abscission process from tomato FAZ and LAZ, generating 37.92 and 39.78 million high quality 73-bp paired end reads. AZ-specific transcript information and available tomato gene models were utilized to design a customized AZ-specific microarray chip. A 4×180K - 60 mer oligonucleotide microarray, comprised of 180,880 probes was designed using the transcripts derived from the pooled RNA-Seq analysis and known abscission-related transcripts, transcripts from the Solanaceae (Sol) genomics database, NCBI databases and Agilent control and random probe sets. The unique design of this chip allows us to quantify accurately global changes in the transcriptome of tomato AZs during the abscission process. Additionally, in this chip the probes were designed in both sense and antisense orientations, which will enable future analyses of expression profiles of naturally occurring antisense transcripts (NATs) in the AZs. This study is the first attempt to analyze the global gene expression in different AZs in tomato by combining the RNA-Seq technique with customized oligonucleotide microarrays. Our customized AZ-specific microarray chip provides a cost-effective approach for analysis of multiple samples in rapid succession. We are currently using this chip to quantify molecular shifts in gene expression in transgenic plants that display altered abscission phenotypes. Results from this study will help to identify target genes for further understanding and manipulating abscission, as well as markers for breeding.