RT-PCR analysis of differentially expressed transcripts in leaf tissues of non-transgenic ‘Aurora’ (WT) and transgenic ‘VcFT-Aurora’ (Tr).

<p><i>VcACTIN</i> is the internal control. M: 1 Kb ladder.</p

Differential expression of blueberry flowering pathway genes in leaf, bud, and flower tissues of ‘Legacy’.

<p>Differential expression of blueberry flowering pathway genes in leaf, bud, and flower tissues of ‘Legacy’.</p

Transcript Profile of Flowering Regulatory Genes in <i>VcFT</i>-Overexpressing Blueberry Plants - Fig 2

<p><b>Comparison of transcript expression profiles between ‘VcFT-Aurora’ and ‘Aurora’ to identify differentially expressed genes (a, b) and isoforms (c, d) in leaf tissues. a, c,</b><i>MA</i> plot for differential expression analysis generated by EdgeR: for each gene or isoform, the log₂(fold change) (log₂(Aurora/VcFT-Aurora)) between the two samples is plotted (<i>A</i>, <i>y</i> axis) against the gene’s log₂(average expression) in the two samples (<i>M</i>, <i>x</i> axis). <b>b, d,</b> Volcano plot reporting false discovery rate (−logFDR, <i>y</i> axis) as a function of log₂(fold change) between the samples (logFC, <i>x</i> axis). Genes or isoforms that are identified as significantly differentially expressed at most 0.1% FDR are colored in red.</p

Comparison of flowering between ‘VcFT-Aurora’ and ‘Aurora’ plants.

<p><b>a,</b> Continuous flowering of non-vernalized one-year old ‘VcFT-Aurora’ (left) alongside ‘Aurora’ plants with no flowers (right). Arrows show flowers. <b>b,</b> Floral bud formation on the new shoots of ‘VcFT-Aurora’. <b>c,</b> Vegetative buds, but not floral buds, appeared on new shoots of ‘Aurora’. <b>d,</b> Fully vernalized ‘VcFT-Aurora’ flowered normally.</p

Transcript Profile of Flowering Regulatory Genes in <i>VcFT</i>-Overexpressing Blueberry Plants

<div><p>In order to identify genetic components in flowering pathways of highbush blueberry (<i>Vaccinium corymbosum</i> L.), a transcriptome reference composed of 254,396 transcripts and 179,853 gene contigs was developed by assembly of 72.7 million reads using Trinity. Using this transcriptome reference and a query of flowering pathway genes of herbaceous plants, we identified potential flowering pathway genes/transcripts of blueberry. Transcriptome analysis of flowering pathway genes was then conducted on leaf tissue samples of transgenic blueberry cv. Aurora (‘VcFT-Aurora’), which overexpresses a blueberry <i>FLOWERING LOCUS T</i>-like gene (<i>VcFT</i>). Sixty-one blueberry transcripts of 40 genes showed high similarities to 33 known flowering-related genes of herbaceous plants, of which 17 down-regulated and 16 up-regulated genes were identified in ‘VcFT-Aurora’. All down-regulated genes encoded transcription factors/enzymes upstream in the signaling pathway containing <i>VcFT</i>. A blueberry <i>CONSTANS</i>-<i>LIKE 5</i>-like (<i>VcCOL5</i>) gene was down-regulated and associated with five other differentially expressed (DE) genes in the photoperiod-mediated flowering pathway. Three down-regulated genes, <i>i</i>.<i>e</i>., a <i>MADS-AFFECTING FLOWERING 2</i>-like gene (<i>VcMAF2</i>), a <i>MADS-AFFECTING FLOWERING 5</i>-like gene (<i>VcMAF5</i>), and a <i>VERNALIZATION1</i>-like gene (<i>VcVRN1</i>), may function as integrators in place of <i>FLOWERING LOCUS C</i> (<i>FLC</i>) in the vernalization pathway. Because no <i>CONSTAN1</i>-like or <i>FLOWERING LOCUS C</i>-like genes were found in blueberry, <i>VcCOL5</i> and <i>VcMAF2</i>/<i>VcMAF5</i> or <i>VRN1</i> might be the major integrator(s) in the photoperiod- and vernalization-mediated flowering pathway, respectively. The major down-stream genes of <i>VcFT</i>, <i>i</i>.<i>e</i>., <i>SUPPRESSOR of Overexpression of Constans 1</i>-like (<i>VcSOC1</i>), <i>LEAFY</i>-like (<i>VcLFY</i>), <i>APETALA1</i>-like (<i>VcAP1</i>), <i>CAULIFLOWER 1</i>-like (<i>VcCAL1</i>), and <i>FRUITFULL</i>-like (<i>VcFUL</i>) genes were present and showed high similarity to their orthologues in herbaceous plants. Moreover, overexpression of <i>VcFT</i> promoted expression of all of these <i>VcFT</i> downstream genes. These results suggest that <i>VcFT’s</i> down-stream genes appear conserved in blueberry.</p></div

Flowering pathways in blueberry.

<p>Arrows: promotion. T-signs: inhibition. Solid lines: results of this study. Dashed lines: proposed correlation.</p

Statistic analysis of blueberry transcriptome reference developed using Trinity.

<p>Nt: nucleotide.</p

Differential expression of blueberry flowering pathway genes in ‘VcFT-Aurora’ leaves and their putative pathways.

<p>Differential expression of blueberry flowering pathway genes in ‘VcFT-Aurora’ leaves and their putative pathways.</p

Numbers of up- and down-regulated genes, relative to SUC, in DD and DF grown cells, and relative to DF, in DD grown cells, by COG category.

<p>The numbers in red are total genes in each COG. Some genes are assigned to multiple COG categories and counted in each.</p

Flagellar Assembly: down-regulated genes compared to the SUC transcriptome.

<p>Flagellar Assembly: down-regulated genes compared to the SUC transcriptome.</p