Identification and expression analysis of genes associated with the early berry development in the seedless grapevine (Vitis vinifera L.) cultivar Sultanine

Abstract

Sultanine grapevine (Vitis vinifera L.) is one of the most important commercial seedless table-grape varieties and the main source of seedlessness for breeding programs around the world. Despite its commercial relevance, little is known about the genetic control of seedlessness in grapes, remaining unknown the molecular identity of genes responsible for such phenotype. Actually, studies concerning berry development in seedless grapes are scarce at the molecular level. We therefore developed a representational difference analysis (RDA) modified method named Bulk Representational Analysis of Transcripts (BRAT) in the attempt to identify genes specifically associated with each of the main developmental stages of Sultanine grapevine berries. A total of 2400 transcript-derived fragments (TDFs) were identified and cloned by RDA according to three specific developmental berry stages. After sequencing and in silico analysis, 1554 (64.75%) TDFs were validated according to our sequence quality cut-off. The assembly of these expressed sequence tags (ESTs) yielded 504 singletons and 77 clusters, with an overall EST redundancy of approximately 67%. Amongst all stage-specific cDNAs, nine candidate genes were selected and, along with two reference genes, submitted to a deeper analysis of their temporal expression profiles by reverse transcription-quantitative PCR. Seven out of nine genes proved to be in agreement with the stage-specific expression that allowed their isolation by RDA.

Transcriptome analyses of the Dof-like gene family in grapevine reveal its involvement in berry, flower and seed development

“…In order to identify putative Dof proteins in grapevine, a VviDofL sequence previously identified by the group 23 was used as query in BLAST searches, resulting in the identification of 25 genes possibly coding for Dof proteins. This result was further confirmed in a HMM search using the Dof-seed sequence available at the Pfam database (PF02701) against the grapevine genome database.…”

“…23 In this work, by exploring genome-wide analysis by microarray in combination with real-time PCR, specific VviDofL transcriptional patterns were found. Transcriptome data obtained from various grapevine samples showed different VviDofL expression signatures according to the tissue/organ tested (Figure 4).…”

“…A VviDofL sequence previously identified by the group 23 was used as query in a genome-wide search for putative Dofs using the BLASTP tool 38 implemented in the 12X Grape Genome Browser V1 (http://genomes.cribi.unipd.it/), 39,40 which was verified in the V2 version with the same 25 VviDofL. In order to confirm the results, the Dof-seed sequence (accession number PF02701) available in the Pfam database 41 was used to perform a HMM search using Hmmer v3.0 (http://hmmer.org/) to find predicted grapevine proteins in the 12X Grape Genome Browser V1.…”

“…1 Thereafter, Dof -like genes were identified in several other species such as grapevine, 23 Chlamydomonas reinhardtii , Jatropha curcas , maritime pine, pea, the moss Physcomitrella patens , sorghum, soybean and sweet potato. 21,22 In terms of family composition, the first genome-wide report for Dof genes was for Arabidopsis, which presented 36 Dof-encoding genes and one pseudogene.…”

“…23 Among the expressed sequence tags identified, a putative Dof gene was isolated in the developmental stage of four-weeks after fruit set. We therefore decided to further investigate the presence of other genes in the grapevine genome potentially encoding Dof proteins.…”