Gene expression atlas of fruit ripening and transcriptome assembly from RNA-seq data in octoploid strawberry (Fragaria × ananassa)

Abstract

RNA-seq has been used to perform global expression analysis of the achene and the receptacle at four stages of fruit ripening, and of the roots and leaves of strawberry (Fragaria × ananassa). About 967 million reads and 191 Gb of sequence were produced, using Illumina sequencing. Mapping the reads in the related genome of the wild diploid Fragaria vesca revealed differences between the achene and receptacle development program, and reinforced the role played by ethylene in the ripening receptacle. For the strawberry transcriptome assembly, a de novo strategy was followed, generating separate assemblies for each of the ten tissues and stages sampled. The Trinity program was used for these assemblies, resulting in over 1.4 M isoforms. Filtering by a threshold of 0.3 FPKM, and doing Blastx (E-value < 1 e-30) against the UniProt database of plants reduced the number to 472,476 isoforms. Their assembly with the MIRA program (90% homology) resulted in 26,087 contigs. From these, 91.34 percent showed high homology to Fragaria vesca genes and 87.30 percent Fragaria iinumae (BlastN E-value < 1 e-100). Mapping back the reads on the MIRA contigs identified polymorphisms at nucleotide level, using FREEBAYES, as well as estimate their relative abundance in each sample.

Characterizing the involvement of FaMADS9 in the regulation of strawberry fruit receptacle development

“…Previous studies have shown that ABA plays an important role in the regulation of strawberry fruit ripening (Chai et al , ; Jia et al , ; Li et al , ). Here, we found that three key genes involved in the synthesis of ABA, namely NCED1 ( FvH4_3g16740 ), NCED2 ( FvH4_3g05440 ) and NCED3 ( FvH4_3g16730 ), the expression of which increases during receptacle ripening (Sánchez‐Sevilla et al , ), were significantly down‐regulated (over 70%) in the red receptacles of FaMADS9 ‐silenced lines (Table S2). Then, the ABA level was measured in the receptacles of the transgenic fruits and the control.…”

“…At the white stage, a total of 2670 genes were significantly differentially expressed, while at the red stage, 1943 genes were differentially expressed (Table S2). Notably, only one of the 34 MADS genes expressed in fruits (Sánchez‐Sevilla et al , ) was down‐regulated in the silenced receptacles, namely FaMADS9 ( FvH4_6g46420 ), the expression of which decreased more than 90% in both the white and red stages (Figure f). This result was confirmed when the expression was evaluated by qRT‐PCR (Figure g).…”

“…Analysis of other fruit characteristics, such as firmness and total soluble solid content (Brix), showed that three of the silenced lines (L7, L23 and L27) exhibited a significantly reduced Brix content, whereas no significant changes were observed in fruit firmness (Figure b,c). Given that FaMADS9 is expressed in the early stage of achene development (Sánchez‐Sevilla et al , ), as well as in flowers (Seymour et al , ), we next evaluated the effect of FaMADS9 silencing on fruit abortion and fruit yield. The silenced lines exhibited no significant changes in these traits (Figure d,e).…”

“…Strawberry is a non‐climacteric fruit, and several studies have analysed the metabolic (Fait et al , ) and cell wall changes (Jiménez‐Bermúdez et al , ; Quesada et al , ) that occur during fruit ripening, as well as changes in gene expression associated with this process (Aharoni and O'Connell, ; Härtl et al , ; Pillet et al , ; Sánchez‐Sevilla et al , ). Recently, a number of reports have shed light on two key elements of the strawberry fruit developmental programme of strawberry fruit: the involvement of hormones and the participation of specific transcription factors.…”

“…In strawberry, silencing of the FaMADS9 gene, an orthologue of the tomato RIN , altered the normal development of the achene and receptacle, and transcriptomic analysis of the silenced fruits revealed pleiotropic effects on gene expression (Seymour et al , ). By analysis of the strawberry fruit transcriptome, we found that FaMADS9 presented weak expression in the achenes and high expression in the receptacles, showing two main peaks, one at the green developmental stage and the other at the red developmental stage (Sánchez‐Sevilla et al , ). In this work, we complement the preliminary study of this gene (Seymour et al , ) via the generation of four independent strawberry transgenic lines that were silenced in the FaMADS9 gene and present an in‐depth analysis.…”