Draft of Zucchini ( Cucurbita pepo L.) Proteome: A Resource for Genetic and Genomic Studies

摘要

Introduction The Cucurbitaceae family is the second most large horticultural family in terms of economic importance after Solanaceae. It includes several important crops, such as melon ( Cucumis melo ), watermelon ( Citrullus lanatus ), cucumber ( Cucumis sativus ) and many Cucurbita species with edible fruits (Jeffrey, 1980 ). The genus Cucurbita (2x = 2n = 40), originated in the Americas, encompasses three economically important crop species such as Cucurbita pepo, Cucurbita moschata , and Cucurbita maxima , cultivated throughout temperate, sub-tropical, and tropical regions (Wang et al., 2011 ). Cucurbita pepo includes a wide assortment of varieties and cultivars, known for their unique fruit shape and color and appreciated for their culinary properties. Among different species of this genus, Cucurbita pepo have the greatest monetary value (Paris, 2008 ). Botanical classification based on allozyme variation recognized three subspecies in this species including: pepo, ovifera (syn. texana ), and fraterna . Paris ( 1986 ) classified edible-fruited C. pepo into eight cultivar-groups: Acorn, Crookneck, Scallop, and Straightneck that belong to subsp. ovifera and Pumpkin, Zucchini, Cocozelle, and Vegetable Marrow that belong to subsp. pepo (Paris, 2010 ). The genome size of Cucurbita spp. is approximately 500 Mb (Arumuganathan and Earle, 1991 ). Recently, a high-quality draft of C. pepo (subsp. pepo cultivar-group Zucchini) genome with a sequences length of about 265 million base pairs (Mbp) was made available on CucurbiGene database as well as several C. pepo transcriptomes have been explored (Blanca et al., 2011 ; Wyatt et al., 2015 ; Vitiello et al., 2016 ; Xanthopoulou et al., 2016 , 2017 ; Montero-Pau et al., 2017 ). However, still little is known about the genetic diversity of this noteworthy crop and even less has been done to explore its proteome. High-throughput sequencing of transcriptomes has opened the way to study the genetic and functional information stored within any organism at an unprecedented scale and speed.Transcriptome generation through RNA sequencing (RNA-seq) is a technology that can be used in the high resolution and broad dynamic range gene expression studies and in the simultaneous understanding of the genes function (Wang et al., 2009 ). Basically, the protein-coding genes function is inferred by the analysis of structure, function and evolution of the proteins they encode (Guo, 2013 ). For the characterization of unannotated proteins, can result particularly useful to undertake orthology analysis. Proteome data are important resources for having an overall genome vision but at the same time achieving a high level of accuracy in comparative studies (Andolfo et al., 2014a ). To this end, we sequenced and assembled the first transcriptome of zucchini cultivar “True French,” founder of important pathogen resistant commercial varieties and to harness the full potential of such data we performed also an high-quality proteome annotation. A total of 33,966 protein sequences were predicted, functionally annotated and compared to cucumber, melon, watermelon and Arabidopsis proteomes. In addition, disease resistance ( R ) gene family was finely characterized and several specie-specific R -genes expansion was detected in C. pepo . Value of the data The transcriptome obtained can be used as reference for gene expression analysis. Genetic and breeding studies will be enhanced by tools and insights developed from this resource. The transcriptome sequence data were assembled and annotated to create a C. pepo reference proteome for future genomic works in this species. Zucchini is an important crop that lack of molecular genetics information. The transcriptome and proteome released will drive new discovery to understand complex agronomic traits and to identify novel resistance gene loci. The predicted proteome and comparative dataset provided will facilitate the understanding of evolutionary mechanisms of expansion/contraction of important gene families, such as resistance genes, in Cucurbita spp. Experimental design, materials and methods Plant material, total RNA extraction and quality control, library preparation and RNA-seq Plants of Cucurbita pepo subsp. pepo cultivar-group Zucchini, variety True French, were grown in greenhouse facility at Department of Agricultural Science of University of Naples “Federico II” using standard horticultural practices. C. pepo cv. True French tissue samples were collected from young plants of about 10 cm high. Total RNA was isolated from ground, frozen leaf tissues using the SpectrumTM Plant Total RNA Kit (Sigma-Aldirch). A complete removal of traces of DNA was performed using On-Column DNase I Digest Set (Sigma-Aldirch). Quantity and integrity of the extracted total RNA were determined using NanoDrop ND-1000 Spectrophotometer (Thermo Fisher Scientific Inc., USA), on a denaturing formaldehyde gel and Agilent 2100 bioanalyzer (Agilent Technologies, USA) respec