Plant materials and ABA treatments
Four years old rabbiteye blueberry ‘Brightwell’ were grown in experimental base of Baima district , Nanjing city , China. Blueberries of same size and growing condition were selected to do the treatment. Each treatment was arranged in a randomised complete block design with 6 replications and each block replication contains 6 shrubs.
The (+)-Abscisic Acid ( Purity 95%, Coolaber company, China ) was dissolved in double distilled H2O containing 5% (v/v) ethanol and 0.1% Tween 80. When most of blueberries fruits were in a growing stage of ‘green mature’ and the top of the fruit at the top of the branch has just begun to turn red, 0, 500 and 1000 mg L−1 ABA solutions were sprayed on fruit clusters. Tiny sprayer were used to spray the ABA solutions on the peels untill the peels are wetted. The leaves and branches were carefully avoided.
In different development stage of fruits, 30 fruits were immediately frozen in liquid nitrogen and stored at -80℃ for later experiments.
Physiological characterization
The color of fruit peel
Fruit peel color was measured by colorimeter ( Ci64, X-Rite, US ) and shown by the International Commission on Illumination a∗ and b∗ colour space co-ordinates [29]. The a* value is negative for green and positive for red an the b* value is negative for blue and positive for yellow, both of the values range from -100 to 100. Due to the coloration of rabbiteye fruits starts from top to bottom, the top, side and bottom of fruit peel were separately measured in the same stage.
The total anthocyanins content
The total anthocyanins content were determined by the double pH differential method[30]: absorbance of the extract was measured at 510 and 700 nm in buffers at pH 1.0 (hydrochloric acid–potassium chloride, 0.2 M) and 4.5 (acetate acid–sodium acetate, 0.2 M). Total anthocyanins content was calculated using a molar extinction coefficient of 29,600 (cyanidin-3-glucoside) and absorbance of A = [(A510 - A700)pH 1.0 -(A510 - A700)pH 4.5].
Transcriptome analysis
RNA preparation and sequencing
Total RNA was extracted from whole fruits using Trizol reagent (Invitrogen, Carlsbad, CA). The RNA integrity was analyzed on agarose gel. RNA concentration and integrity were measured by Qubit® RNA Assay Kit in Qubit® 2.0 Flurometer (Life Technologies, CA, USA) and RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system respectively. Sequencing libraries were generated using NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer’s recommendations. Then, the libraries were sequenced on an Illumina Hiseq platform and 150 bp paired-end reads were generated.
Reads mapping to the reference genome
Clean reads were obtained by in-house perl scripts that remove reads containing adapter, reads containing ploy-N and low quality reads from raw reads. Reference genome and gene model annotation files of Vaccinium corymbosum were downloaded from Giga science(http://gigadb.org/dataset/100537). Paired-end clean reads were aligned to the reference genome using STAR (v2.5.1b) by the method of Maximal Mappable Prefix.The function of the transcript was annotated by UniProt database(https://www.uniprot.org/).
Differential expression analysis
HTSeq v0.6.0 was used to count the reads numbers mapped to each gene and FPKM(fragments per kilobase per million reads) was calculated based on the length of the gene and reads count mapped to this gene.Then, differential expression analysis of two groups was performed using the DESeq2 R package (1.10.1). Genes with P-value using Benjamini and Hochberg’s approach were assigned as differentially expressed. Enrichment analysis of differentially expressed genes of physiological processes was carried out by KEGG(http://www.genome.jp/kegg/) and GO( clusterProfiler R package).
Construction of phylogenetic trees
The maximum likelihood phylogenetic trees was constructed by by IQ-tree [31].The optimal alternative model is selected after calculation. The bootstrap value is 1000. The following sequence was downloaded from GenBank accessions:
MYB:AtMYB75(NC_003070.9), AtMYB113(NC_003070.9),AtMYB90(NC_003070.9),AtMYB114(NC_003070.9),PhAN2(AAF66727.1),PhPHZ(ADQ00388.1),PhDPL(ADQ00393.1),
VvMYBA1(BAD18977),VvMYBA2(BAD18978),MdMYB10(ACQ45201),MdMYBA(NC_041797.1),VcMYBA(MH105054),AcMYB10(PSS35990),AcMYB110(AHY00342),
AtMYB4(At4g38620),PhMYB4(ADX33331.1),PhMYB27(AHX24372),VvMYBC2-L2(ACX50288.2),VuMYBC2(AKR80571),VvMYBC2 L1(AFX64995.1),AtMYB12(ABB03913),SlMYB12(ACB46530.1),VvMYBF1(ACV81697)),AtMYB123(CAC40021),VvMYBPA2(ACK56131.1),VcMYB17(ALP43798.1),
MdMYB11(NC_041797.1),MdMYB9(NC_041796.1),VuMYBPA1(AKC94840.1),VvMYBPA1(CAJ90831.1),AtMYB5(NP_187963.1),
VvMYB5b(AAX51291),AtMYB56(AT5G17800),MdMYB1(NC_041789.1).
bHLH:AtEGL3(AT1G63650),AtGL3(AT1G11130),AmDELILA(Uniprot:Q38736),PhJAF13(Uniprot:O64908),
AtTT8(AT4G09820),MdbHLH3(ADL36597.1),PhAN1(FJ227329.1),VvbHLH1(Uniprot:A0A438KI27),MdBHLH33(EI011581.1).
WD40:AtTTG1(AT5G24520),MdTTG1(GU173814.1),PhAN11(Uniprot:O24514),VvWDR1(Uniprot:Q19N39),VvWDR2(Uniprot:Q19N38).
Quantitative real-time PCR (qRT-PCR) analysis
Eleven transcription factors with altered expression levels were selected for qRT-PCR verification. The specific primers for 11 genes are shown in Table S3. Expression level is relative to the housekeeping gene Actin of highbush blueberry[32]. qPCR was conducted using the TB Green® Fast qPCR Mix(Takara, Japan) according to manufacturer's requirements.The 2−ΔΔCt method was used to calculate the relative gene expression[33]. The experiment carried out three biological repetitions.