Versatile and efficient RNA extraction protocol for

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RNA extraction from grapevine tissues

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Versatile and efficient RNA extraction protocol for grapevine berry tissue, suited for next generation RNA sequencing M. RIENTH1,2, L. TORREGROSA2, M. ARDISSON3, R. DE MARCHI2 and C. ROMIEU2 1

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Fondation Jean Poupelain, 30 Rue Gâte Chien, 16100 Javrezac, France Unité Mixte de Recherche (UMR) Amélioration Génétique et Adaptation des Plantes méditerranéennes et tropicales) AGAP – équipe Diversité, Adaptation et Amélioration de la vigne (DAAV), Institut National de la Recherche Agronomique (INRA) Montpellier, 2 place Pierre Viala, 34060 Montpellier Cedex, France 3 UMR AGAP – équipe GE2POP, INRA Montpellier, 2 place Pierre Viala, 34060 Montpellier Cedex, France Corresponding author: Dr Charles Romieu, email [email protected] Abstract Background and Aims: New high-throughput tools for transcriptomic analysis such as RNA sequencing have been developed rapidly in recent years. These technologies provide new opportunities for biologists to improve the understanding of gene expression underlying important physiological processes. The sequencing of RNA by nextgeneration technologies is, in particular, dependent on the use of pure and un-degraded RNA. Many fruits from perennial crops share an elevated concentration of tannins and polysaccharides and a strong dilution of cytosolic content upon vacuole hypertrophy. The grapevine berry presents these characteristics together with high vacuole acidity, which has never been taken into account in previous protocols, and poses thereby a particular challenge for RNA extraction. Methods and Results: In order to counterbalance these limitations, we developed a new extraction protocol based on a tri-sodium-citrate extraction buffer that has a high buffer capacity and proved to be particularly suitable for low pH plant tissue. Conclusions: The proposed RNA extraction procedure produces consistently high-quality RNA (RNA integrity number > 8 up to 10; 260 nm/280 nm > 2; 260 nm/230 nm > 1.9) with an elevated yield [20 μg/g fresh mass (FM) for ripe berries up to 150 μg/FM for green berries]. The method was developed initially for berry tissues but proved also to be efficient for other grapevine tissues, such as nodes, roots, leaves, seeds, lignified shoots and flowers. Significance of the Study: The method is most suitable for modern gene expression analysis methods, such as RNA sequencing and microarray studies. Successful construction of cDNA libraries and high numbers of detected reads obtained by next-generation RNA sequencing underline the applicability of the protocol. Keywords: grapevine berry, microvine, next-generation sequencing, pH buffer capacity, RNA extraction

Introduction Next-generation sequencing technologies have considerably improved the throughput of transcriptomic studies (Wang et al. 2009, Haas and Zody 2010). Disregarding the higher computational needs, they constitute an economically viable alternative to whole genome microarrays, leading to the discontinuation of important platforms such as Nimblegen Vitis 12× (Roche Nimblegen 2013). In order to obtain optimum results from RNA sequencing, the sample needs to meet particularly high-quality standards [RNA integrity number (RIN) > 8; 260 nm/280 nm > 1.8; 260 nm/230 nm > 1.8] when compared with standard gene expression analysis methods such as real-time quantitative polymerase chain reaction (Gayral et al. 2011). Compared with other eukaryotic plant tissue or other fleshy fruits, the green grapevine berry possesses a low pH (2.7) (Romieu 2001), a high amount of RNA-interfering compounds, such as tannins that bind proteins and nucleic acids (Newbury and Possingham 1977), and polysaccharides (in particular during ripening), which can lead to co-precipitation with RNA (Lodhi et al. 1994, Richards et al. 2001). Many fleshy fruit species from other perennial crops, such as apple, citrus, plums and mangoes, share these characteristics. Additionally, rapid cell doi: 10.1111/ajgw.12077 © 2014 Australian Society of Viticulture and Oenology Inc.

expansion of ripening fruits (Ojeda et al. 1999, Coombe and McCarthy 2000) entails a dilution of RNA in the cells. Because of these features, commercial RNA extraction kits usually fail to yield reproducible results. Extraction problems are not only encountered with the grapevine berry but with other acidic fruits, such as citrus (Tao et al. 2004), apples (Gasic et al. 2004), papaya (Li and Chen 2008) and peaches (Meisel et al. 2005), where commercially available protocols fail completely or result in poor RNA yield and quality. Proposed alternative protocols rely mainly on empirical buffer formulations and complex prepurification/precipitation steps, increasing the hazard of RNA losses and RNAse contamination (Tesniere and Vayda 1991, Tattersall et al. 2005, Reid et al. 2006, Romieu 2010). The aim of the present study was to develop a new robust extraction protocol for RNA from tissue of all developmental stages of grapevine berries taking into account the dramatic changes in composition (Ollat et al. 2002, Conde et al. 2007). The proposed RNA extraction protocol, in particular, manages the high acidity of grapevine berries, using tri-sodium-citrate as a pH buffer solution. The method provides high reproducibility in quality and quantity on all extracted berry development stages; extraction tests on other grapevine organs demonstrated the high versatility of the protocol.

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Australian Journal of Grape and Wine Research 20, 247–254, 2014

Materials and methods Plant material The grapevine gai1 mutant – Dwarf Rapid Cycling and Flowering (DRCF) (Boss and Thomas 2002) – also named microvine, has recently been proposed as a new model for genetic (Chaïb et al. 2010) and transcriptomic research (Rienth et al. 2014). It has been shown that this genotype is suitable for experiments in small-scale climate chambers under precisely monitored environments (Rienth et al. 2012). For the present study, DRCF plants were grown in climate chambers under controlled conditions (day/night temperature, 30/20°C; photoperiod, 14 h; vapour pressure deficit, 1 kPa) during 3 months. To assess protocol efficiency, RNA was extracted from five stages of berry development classed by the adapted Eichorn and Lorenz (EL)

system (Coombe 1995), berry set (EL 27), green I (EL 31), green II (EL 33), maturation I (EL 35) and maturation II (EL 38), representing the diversity of fruit composition (Figure 1 and Table 1). The two green and the maturation I stages of berry growth were selected for cDNA library construction and subsequent RNA sequencing. Although the main objective was to design a protocol suitable for berry tissue, the extraction method developed was also assessed on other grapevine organs, such as winter nodes, roots, seeds, young/adult leaves, lignified shoots and flowers at flowering, where difficulties of extraction have been reported. To compare with commercially available protocols, the same samples have in addition been extracted with the Quiagen RNeasy Kit (Quiagen, Venlo, The Netherlands) as described in the manual.

RNA extraction protocol Preliminary steps. Used glassware, spatulas, mortar and pestle were wrapped in aluminium foil and heated over night at 230°C. All plastic ware, Eppendorfs and pipettes were RNAse free and designated for RNA use only. The pH electrode used for buffer pH adjustment was pre-incubated in autoclaved 0.1% SDS, 0.1 N NaOH for 1 h before use, and rinsed with diethylpyrocarbonate (DEPC)-treated water.

Extraction buffer. The buffer contained 6 mol guanidinhydrochloride, 0.15 mol tri-sodium-citrate, 20 mmol Na2EDTA (ethylenediaminetetraacetic acid) and 1.5% cetyltrimethylammonium bromide (CTAB). The buffer was adjusted to pH 7 with 37% HCl solution.

Figure 1. Dwarf Rapid Cycling and Flowering grapevine (microvine) plant line ML1 with leaves removed for illustration purposes. Stages sampled for RNA extraction are displayed within the picture.

Extraction. Up to 1 g (depending on organ, developmental stage and required RNA yield) of tissue, ground in liquid nitrogen and stored at −80°C, was weighed under liquid nitrogen, into pre-chilled 15 mL falcon tubes. Just prior to extraction, sample temperature was equilibrated at −20°C for ca. 20 min to reduce freezing of the extraction buffer in the following step. Thawing of the powder before buffer loading was strictly avoided. Five millilitres of room temperature extraction buffer supplemented with 1% β-mercapto-ethanol (β-MSH) were added to 1 g of powder which was immediately and vigorously shaken until the resulting mix had completely melted. Samples were kept on ice with occasional shaking until this first extraction step on all samples to be processed was completed. We recommend limiting the number of samples within one extraction batch to maximum of eight. Samples were then centrifuged at 5000 g, 5°C and 10 min to eliminate cell debris. The supernatant was transferred into new 15 mL tubes, chloroform was added (1 volume) and tubes were shaken immediately. This was followed by centrifugation at 5000 g, 7°C and 5 min. The

Table 1. Composition of five stages of development of grapevine berries selected for RNA extraction. Developmental stage

Berry mass (g)

Hexose (mol/kg FM)

Malic acid (meq/kg FM)

Tartaric acid (meq/kg FM)

Berry set Green I Green II Maturation I Maturation II

0.06 ± 0.10 0.22 ± 0.01 0.45 ± 0.20 1.18 ± 0.10 1.27 ± 0.10

nd nd nd 0.66 ± 0.03 0.73 ± 0.09

98 ± 34 167 ± 19 254 ± 22 140 ± 5 114 ± 7

219 ± 55 161 ± 15 145 ± 11 85 ± 3 91 ± 4

Values are the mean (± SE), n = 6. FM, fresh mass; nd = not detected. © 2014 Australian Society of Viticulture and Oenology Inc.

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Figure 2. Samples of extracted tissue in 15 mL tubes after elimination of cell debris and chloroform treatment. After centrifugation, an interphase is formed between the buffer (containing RNA) and the chloroform. Coloured phase on top consists of the buffer phase containing RNA. This phase is recovered for subsequent purification steps, and is coloured only when anthocyanin-containing samples are extracted.


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in which case the supernatant was removed carefully by pipetting. The pellet was re-suspended in 1 mL cold 75% ethanol and transferred to 2 mL Eppendorf tubes with a pipette. The RNA was re-precipitated by centrifugation (>10 000 g, 10 min, 15°C). From the Qiagen RNeasy Kit (Qiagen), RLC buffer (450 μL) previously supplemented with 1.5% CTAB and 1% β-MSH was then added. The pellet was suspended by pipetting and subsequently heated to 60°C for 2–5 min until completely dissolved. To reduce pectin and tannin residues, 600 μL chloroform were added, mixed and immediately centrifuged for 5 min at 8000 g. The supernatant was carefully racked into new Eppendorf tubes, without disturbing the interphase. Recovered volume ranged around 430 μL and was noted. Half volume of 100% ethanol was added and mixed carefully by pipetting. The mix was then transferred to spin columns from the Qiagen RNeasy Kit (Qiagen) and centrifuged at 10 000 g for 30 s. The flow through was recovered for a second passage on the same column for improved yield. The succeeding washing steps and the DNase treatment were performed according to the commercial kit. The final elution was undertaken with 40–100 μL DEPC water at 60°C and centrifuged at 13 000 g for 30 s (elution volume was adjusted according to expected yield and concentration required for further experiments).

Quality control of extracted RNA Absorbance at 260 and 280 nm of RNA was assessed with a NanoDrop 2000c Spectrophotometer (Thermo Fisher Scientific Inc., Waltham, MA, USA). Integrity of RNA (RIN) was evaluated using a 2100 Bioanalyser System (Agilent Technologies, Santa Clara, CA, USA) on Agilent RNA 6000 Pico chips. RNA was quantified by RiboGreen RNA assay (Invitrogen, Eugene, OR, USA).

Preparation of cDNA

Figure 3. Pellet, containing RNA, after isopropanol precipitation and centrifugation.

supernatant was carefully racked without disturbing the jelly, and pectin-like material accumulated at the interphase between buffer and chloroform (illustrated in Figure 2). It was then advisable to check which proportion of the supernatant was obtained for further calculation of the RNA yield. One volume of isopropanol was added and mixed for RNA precipitation at −20°C for at least 2 h. This precipitation period at −20°C could be extended up to 48 h without impacting the extraction result significantly. A small transparent pellet formed upon centrifugation of the samples for 30 min, 5000 g and 5°C (Figure 3). The sticky pellet usually allowed elimination of the supernatant upon pouring the tube and finally placing it top down on a filter paper for 1 min to remove residual liquid. This step was avoided if the pellet floated on traces of chloroform, © 2014 Australian Society of Viticulture and Oenology Inc.

The fragmented mRNA samples were subjected to cDNA synthesis using an Illumina TruSeq RNA sample preparation kit (low-throughput protocol) (Illumina, Inc., San Diego, CA, USA). Briefly, cDNA was synthesised from enriched and fragmented RNA using reverse transcriptase (Super-Script II, Illumina) and random primers. The cDNA was further converted into double-stranded DNA using the reagents supplied in the kit, and the resulting DNA was used for library preparation. Blunt ends of cDNAs were modified with an addition of a 3′adenine. The modified ends were then used to ligate an indexed adapter, which allowed different samples to be distinguished during flow-cell hybridisation and sequencing. The subsequent adapter-modified DNA fragments were then enriched using standard PCR.

Sequencing and data processing Cluster generation of cDNA libraries and hybridisations onto the flow-cell was performed with the Illumina cluster generation kit (Illumina). Paired-end sequencing was realised on a HiSeq 2000 Illumina sequencer using SBS (Sequence By Synthesis) technology (Illumina). Image analysis was carried out with Illumina HiSeq Control Software (Illumina) which identifies cluster position, intensity and background noise. Intensity was transformed into nucleotide bases by RTA software from Illumina.

Organic acid and sugar analysis Approximately 0.1 g of N2-ground powder was fivefold diluted and frozen at −20°C for sugar and organic acid analysis. Samples were heated (60°C for 30 min) and subsequently homogenised

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and diluted with 4.375 μmol acetate as internal standard. Potassium bitartrate precipitation was impeded by addition of 0.18 g of Sigma Amberlite IR-120 Plus (sodium form) (Sigma-Aldrich, St Louis, MO, USA) to 1 mL of sample before agitation in a rotary shaker for at least 10 h. Samples were centrifuged (15 493 g for 10 min) and the supernatant was transferred into high-performance liquid chromatography vials before injection onto an Aminex HPX87H column (Bio-Rad, Marnes-laCoquette, France) and eluted under isocratic conditions (0.05 mL/min, 60°C, H2SO4) (Schneider et al. 1987). Refractive index was measured with a Kontron 475 detector (Kontron Instruments, Rossdorf, Germany), and the concentration of fructose and glucose was calculated according to EyeggheBickong et al. (2012). Organic acids were detected at 210 nm with a Waters 2487 dual absorbance detector (Waters Corporation, Milford, MA, USA).

Results and discussion Commercially available protocols for RNA extraction often fail or result in low quality and quantity when applied to tissues rich in phenolic substances and polysaccharides, and need therefore to be adapted to the specific characteristics of the species (Tao et al. 2004, Li and Chen 2008, Rubio-Pina and Zapata-Perez 2011, Yu et al. 2012). Tissue from grapevine berry pulp, high in phenolic substances and polysaccharides combined with a low pH (Romieu 2001), represents a critical context for RNA extraction. The extraction of berry tissue undertaken with the Qiagen RNeasy Kit in this work yielded no RNA (data not shown), whereas the yield and purity of other extracted organs were poor (Supporting Information Table S1). Many protocols have been proposed for RNA extraction of tissue from grapevines and other species. The main differences in the available protocols are, in particular, found in the constitution of the first extraction buffer and can be divided into three groups: chaotrope, containing buffers based either on: (i) guanidine (Salzman et al. 1999, Gehrig et al. 2000, Ribaudo et al. 2001); (ii) sodium perchlorate (Rezaian and Krake 1987, Rathjen and Robbins 1992); or (iii) Tris with detergents [(Na2EDTA, sodium deoxycholate, CTAB, polyvinylpolypyrrolidone (PVPP)] (Tesniere and Vayda 1991, Bahloul and Burkard 1993, Chang et al. 1993, Franke et al. 1995, Gasic et al. 2004, Tao et al. 2004, Tattersall et al. 2005, Reid et al. 2006). Table 2 provides an overview of more recent protocols for RNA extraction from grapevine berry tissue with subsequent high-throughput transcriptomic studies (microarrays or RNA-Seq). Three main extraction methods are principally applied to grapevine berries, which are based on Tris buffers (Tesniere and Vayda 1991, Tattersall et al. 2005, Reid et al. 2006). All finalise RNA extraction with overnight lithium chloride precipitation whereas the most important difference consists in their constitution of extraction buffers. These contain Tris and detergents at several concentration values, whereas mainly the Tris concentration ranges between 100 and 300 mmol (Tesniere and Vayda 1991, Tattersall et al. 2005, Reid et al. 2006). All three protocols consist of several precipitation and cleaning steps, and are complex and time consuming (usually over 2 days), and thus prone to RNase contamination and degradation. To obtain RNA of sufficient purity, a subsequent clean-up step is often necessary following the protocol of Tesniere and Vayda (1991) (Chatelet et al. 2007) and of Tattersall et al. (2005) (Deluc et al. 2011). Furthermore, the latter has been tested only on poorly specified berry stages supposedly during ripening and requires a significant amount of berry tissue (up to 3 g in Tattersall et al. 2005) in order to obtain sufficient RNA. For other fleshy fruits, RNA yield is generally as


Table 2. Overview of recently used RNA extraction protocols in transcriptomic studies on grapevine berries using high throughput technologies. Extraction protocol Reid et al. (2006)

Tattersall et al. (2005)

Tesniere and Vayda (1991)

Moser et al. (2004) Rerie et al. (1991) Rezaian and Krake (1987) Adapted from Sigma Aldrich

Publication Carbonell-Bejerano et al. (2013) Lijavetzky et al. (2012) Pillet et al. (2012) Guillaumie et al. (2011) Ali et al. (2011) Peng et al. (2007) Tillett and Cushman (2011) Deluc et al. (2011) Deluc et al. (2009) Grimplet et al. (2007) Deluc et al. (2007) Fernandez et al. (2007) Ageorges et al. (2006) Terrier et al. (2005) Dal Santo et al. (2013) Pilati et al. (2007) Fortes et al. (2011) Zenoni et al. (2010) Fasoli et al. (2012)

Figure 4. Effect of stage of berry development on the yield of RNA extracted from berries and expressed as either content of a berry (■) or berry concentration (□). Vertical lines show the SD of six extracted replicates. low as for grapes, for example apple 13.4 μg/g fresh mass (FM) (Li and Chen 2008), blackcurrant 15–28 μg/g FM (Woodhead et al. 1997), peach ca. 28 μg/g FM (Tong et al. 2012) and citrus 50–70 μg/g FM (Tao et al. 2004). The proposed, chaotrope-based protocol compensates for the constraints listed above with a RNA yield for berries ranging from 16 μg/g FM at ripeness up to 161 μg/g FM at berry set (Figure 4). Such yields obtained on berry tissue are comparable with those reported in the literature for young berries but are significantly higher for ripe berries (Franke et al. 1995, Indolino et al. 2004, Tattersall et al. 2005, Romieu 2010). The yield calculated on a per-berry basis stays constant with the exception of berry set where the concentration is highest but the content per berry is lowest. These results are in accordance with the characteristics of berry development, where the volume of young © 2014 Australian Society of Viticulture and Oenology Inc.

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Table 3. Effect of stage of development of grapevine berries on the quality of extracted RNA. Stage Berry set Green I Green II Maturation I Maturation II

260 nm/280 nm

260 nm/230 nm

RIN

2.06 ± 0.05 2.12 ± 0.03 2.11 ± 0.02 2.10 ± 0.03 2.10 ± 0.02

1.9 ± 0.25 2.5 ± 0.42 2.1 ± 0.24 2.3 ± 0.13 2.2 ± 0.24

9.4 ± 0.27 9.2 ± 0.15 9.1 ± 0.51 8.2 ± 0.10 8.3 ± 0.35

Values are the mean (± SE), n = 6. RIN, RNA integrity number.

berries increases during the first growth phase because of both cell division and cell expansion, whereas during ripening berry growth occurs only by cell enlargement (Ojeda et al. 1999). Other proposed RNA extraction protocols observe such a decrease in yield with advanced berry development as well, some even fail completely for ripening berries (Franke et al. 1995). In addition to the high yield, one other important benefit of the new protocol is the consistent quality of extracted RNA. Table 3 shows the absorbance ratios for 260 nm/280 nm and 260 nm/230 nm with values >2 indicating a high purity of extracted RNA for all development stages which are superior to those obtained with other protocols where 260 nm/280 nm was lower than 1.8 (Tattersall et al. 2005). Such high purity is the result of the two chloroform-cleaning steps, which reduce tannin, polysaccharide and protein contamination. A RIN value consistently above 8 indicates low RNA degradation. The intactness of RNA is confirmed with Bioanalyser results as illustrated in Figure 5. The high quality of the extracted RNA makes the method particularly suitable for the construction of cDNA libraries and subsequent RNA sequencing. In the present study, the construction of cDNAs for Illumina sequencing was performed on the stages green I, green II and maturation I. The construction of cDNA libraries from extracted RNA was successful and is illustrated in Figure 6 for two synthesised cDNAs. The construction of such libraries with degraded or impure RNA results generally in low cDNA yield or complete failure of library construction. Berry development stages, green I, green II and maturation I, were subsequently sequenced on independent lanes with 12 replicates per lane. An average of 12.8 (±0.8) million reads for green I, 11.8 (±0.5) for green II and 17.6 (±2.9) for maturation I were obtained. Read quality with an average Phred score (Ewing et al. 1998) of 38 (Figure 7) emphasised the high initial quality of the extracted RNA. Quality scores per read position and average quality over all sequences are illustrated for one replicate of green II in Figure 6. On other extracted organs RNA purity was slightly decreased with a 260 nm/230 nm ratio of about 1.8, supposedly because of the high concentration of polysaccharides and tannins in these tissues (Table 4). A further chloroformcleaning step would possibly increase this ratio, but this level of purity can already be considered as acceptable, although other protocols developed for leaf tissue obtained a slightly higher purity level (Tattersall et al. 2005). Yield was heterogeneous according to different organs but showed a similar trend as other proposed protocols (Tattersall et al. 2005, Reid et al. 2006), where young leaves reached the highest and roots generally the lowest amount of RNA. For nodes, however, yield was slightly inferior (27 μg/g FM) to that obtained with other specifically adapted extraction methods (Acheampong et al. © 2014 Australian Society of Viticulture and Oenology Inc.

Figure 5. Assessment of RNA integrity number (RIN) with a Bioanalyser. One extracted replicate is shown for each stage of berry development: (a) berry set, RIN 10; (b) green I, RIN 9.2; (c) green II, RIN 9.3; (d) maturation I, RIN 8.2; and (e) maturation II, RIN 8.8.

2010). Low RNA degradation was consistently observed for all extracted organs. These results illustrate clearly the good performance of the protocol, particularly for acidic tissues, but as well on adult and

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Table 4. Yield and quality of RNA extracted from different grapevine organs with citrate buffer. Organ Node Root Young leaf Adult leaf Flower Lignified shoot Seed

260 nm/280 nm

260 nm/230 nm

RIN

μg/g FM

2.1 ± 0.1 2.1 ± 0.1 2.1 ± 0.1 2.1 ± 0.0 2.1 ± 0.1 2.2 ± 0.2 2.1 ± 0.1

1.7 ± 0.1 1.8 ± 0.5 2.4 ± 0.1 2.0 ± 0.1 1.8 ± 0.7 1.8 ± 0.2 2.0 ± 0.2

8.6 ± 0.5 9.0 ± 0.6 8.0 ± 1.0 7.5 ± 0.2 7.8 ± 0.2 7.9 ± 0.4 8.1 ± 0.1

27 ± 13 24 ± 14 435 ± 152 172 ± 32 58 ± 33 49 ± 14 79 ± 30

Values are the mean (±SE), n = 3. FM, fresh mass; RIN, RNA integrity number.

Figure 6. Quality control results of cDNA libraries that were migrated on an Agilent Technologies Bioanalyser System. Fluorescence absorption curves are shown on the left hand side, and pictures of the migration gel are shown on the right-hand side. cDNA was synthesised from enriched and fragmented RNA using reverse transcriptase (Super-Script II, Illumina) and random primers. The cDNA was further converted into double-stranded DNA, and the resulting DNA was used for library preparation. Blunt ends of cDNAs were ligated with indexed adapters, which allowed different samples to be distinguished during flow-cell hybridisation and sequencing.

lignified organs where RNA extraction problems are frequently encountered (Wang et al. 2000). Thus, it was demonstrated that the new proposed protocol for RNA extraction is versatile for many plant tissues. Obviously, these improvements can be associated with the increased pH-buffering capacity of the proposed extraction medium. Excessive tissue acidity has been overlooked until now in the large number of RNA extraction protocols reported for plant tissues which focus only on issues of RNase, tannin and polysaccharide interference (Schneiderbauer et al. 1991, Shirzadegan et al. 1991, Staub et al. 1995, Tattersall et al. 2005, Chan et al. 2007). The pH of the RNeasy RLC medium (Qiagen) dropped immediately to 3.2 ± 0.1 following extraction with powder prepared from young berries, whereas the buffer proposed here stabilised the pH around 5.6 ± 0.2 on the same sample. Titration of the commercial RNeasy RLC medium shows that a dramatically acidic pH can be expected if sample acidity exceeds 80 meq/kg FM (Figure 8), using 0.1 g of tissue in 450 μL buffer according to manufacturer instructions. Such tissue acidity is clearly exceeded, not only in berries, but also in other grapevine

Figure 7. Example of sequence quality of one sample. (a) Quality of sequences according to their position within the sequenced read assessed by the Phred score. For example, a Phred score of 40 indicates incorrect base call probability of 1 in 1000 (99.99% accuracy of base) and a Phred score of 20 indicates incorrect base call probability of 1 in 100 (99% accuracy). (b) Mean sequence qualityscore distribution over all sequences within the read.

organs. Indeed, we found that the concentration of total acidity (malic acid + tartaric acid) in fully expanded grapevine leaves ranged from 350 [Gora acidless mutant (Diakou et al. 2000)] up to 450 meq/kg FM (Sultanina) with a titratable acidity ranging between 170 and 350 meq/kg FM. These data are in accordance with previous reports obtained with the Cot and Négrette cultivars (Attia et al. 2005). We can suspect that excess of sample acidity may be encountered not only in young fleshy and acidic fruit, such as apples, plums, citrus and grapes, but also in vegetative organs, explaining the impairment of commercial RNA extraction kits. Consequently, the method © 2014 Australian Society of Viticulture and Oenology Inc.

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Figure 8. Titration curve of the commercial RNA easy buffer (▲) [y = –0.767ln(x) + 8.2971, R2 = 0.99924] and the developed citrate buffer (◆) (y = 0.0006x3 − 0.0335x2 + 5.5043, R2 + 0.98254).

presented in this study appears to be a good starting procedure for new or recalcitrant plant materials.

Acknowledgements Financial support was provided by the Fondation Jean Poupelain (Javrezac, Cognac), the Comité National des Interprofessions des Vins d’appellation d’origine (CNIV) and the French Research Agency (program DURAVITIS, ANR-2010GENM-004-01). We would like to thank as well Nathalie Luchaire, Anne Pellegrino, Rattaphon Chatbanyong, Gilbert Lopez, Agnès Agorges, Thérèse Marlin, Sandrine Vialet and Bertrand Muller for their help during climate chamber experiments, sampling and sample processing.

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Manuscript received: 16 October 2013 Revised manuscript received: 14 December 2013 Accepted: 8 January 2014 Supporting information Additional Supporting Information may be found in the online version of this article at the publisher’s web-site: http:// onlinelibrary.wiley.com/doi/10.1111/ajgw.12077/abstract Table S1. Yield and quality of RNA extracted from different grapevine organs with Qiagen RNeasy. © 2014 Australian Society of Viticulture and Oenology Inc.