Volatile components were identified by comparing a private library spectra, built with chemical standards, and the spectral library (NIST 98 /EPA/MSDC 49 K Mass Spectral Database, Hewlett–Packard Co., Palo Alto, CA, USA). When available, MS identifications were confirmed by comparing GC retention times with pure standards. Total RNA was extracted according to manufacturer’s instructions (Pure Link, Invitrogen®). For RT- PCR, DNase-treated RNA (2 μg) was reverse transcribed in NVP-BGJ398 in vivo a total volume of 20 μl using Omniscript Reverse Transcription Kit (Qiagen, Valencia, CA, USA) and then PCR was performed using 2 μl of cDNA in a 25 μl reaction
volume using SYBR GREEN PCR Master Mix (PE-Applied Biosystems, Foster City, CA, USA) on an ABI PRISM 7500 sequence-detection system. Primer Express software (Applied Biosystems) was used to design gene-specific primers ( Table 1). Fourteen genes were chosen based on putative roles in strawberry quality traits, such as cell wall disassembling (Exp2 from Civello, Powell, Sabehat, & Bennett, 1999; Exp5 from Harrison, McQueen-Mason, and Manning, 2001; PLa, PLb and PLc from Benítez-Burraco et al., selleck chemicals llc 2003;
PME from Castillejo, Fluente, Iannetta, Botella, & Valpuesta, 2004; PG from Redondo-Nevado et al., 2001; and β-Gal from Trainotti et al., 2001), phenolic and anthocyanin compounds synthesis (PAL from Usami, Kantou, & Amemiya, 2007; and ANS from Almeida et al., 2007), ascorbic acid synthesis (LGalDH from Gatzek, Wheeler, & Smirnoff, 2002; and GLDH from Pineau, Layoune, Danon, & De Paepe, 2008) and esters synthesis (ADH from Longhurst et al., 1990; AAT
from Aharoni et al., 2000). Optimal primer Clomifene concentration was 50 nM. Real time-PCR conditions were as follows: 50 °C for 2 min, 95 °C for 10 min, followed by 40 cycles of 95 °C for 30 s, 60 °C for 1 min, 72 °C for 1 min, and one cycle 72 °C for 5 min. Samples were run in triplicate on a 96-well plate. For each sample, a Ct (threshold cycle) value was calculated from the amplification curves by selecting the optimal ΔRn (emission of reporter dye over starting background fluorescence) in the exponential portion of the amplification plot. Relative quantitation (RQ) was calculated based on the comparative Ct method ( Livak & Schmittgen, 2001), using β-actin ( Almeida et al., 2007) as an internal standard. All experiments were done in triplicate. Data was analysed using analysis of variance (ANOVA) and means comparison using Tukey’s test at P ⩽ 0.05 using SAS. Transcript accumulation of Exp2 and Exp5, and of genes encoding enzymes acting in cell wall disassembly (PLa, PLb, PLc, PME, PG and β-Gal) was monitored in order to understand the role of these putative genes during the development of strawberry. Firmness decreased over time during fruit development; descending from 26.5 N at stage 1 (green, 3.0 g ± 0.9) to 2.7 N at stage 5 (red, 16.2 g ± 1.2) ( Fig. 1A).