Dormancy-associated microRNAs in sweet cherry (Prunus avium L.): a first draft. Evelyn Sánchez1, Karin Rothkegel2, Christian Montes1, Paola Andrade1, Pablo Cid1, Andrea M. Almeida3 and Humberto Prieto1* 1Instituto
de Investigaciones Agropecuarias, La Platina Station, Santiago, Chile. 2Centro de Biotecnología Vegetal, Universidad Andrés Bello, Santiago, Chile. 3FONDAP Center for Genome Regulation, Santiago, Chile. *Corresponding author:
[email protected].
Abstract During the winter, sweet cherry (Prunus avium L.) has a restricted growth period of both floral and vegetative buds in which they enter a dormant state. Dormancy is defined as the inability to resume meristem growth under favorable conditions. To be able to exit from this state, an extended cold period, termed the chilling requirement, is needed. Recent information about this species allows for the molecular understanding of dormancy and involves several pathways, including epigenetic modification and gene expression. In this latter group of events, microRNAs (miRNAs) play relevant roles. During dormancy, flowering transition is controlled by two main miRNA families (miR156 and miR172), which have been identified in the juvenile-to-adult and vegetative-to-reproductive transitions, respectively. The miR159, miR319, miR390, and miR399 families have also been described as playing key roles in flowering. In the present study, which is understood as part of a general dormancy study of this species, we present preliminary results about candidate miRNA species during sweet cherry dormancy. Massive small RNA sequencing experiments were conducted with small RNA samples from field trees. Previously identified 21- and 22-nt small RNAs were filtered. The candidate molecules were then aligned to microRNA and reference Prunus spp. genomic databases in order to generate a primary set of candidate miRNAs involved in the chilling and dormancy processes; these are currently undergoing experimental verification.
Materials and Methods Floral Cherry buds
RNA integrity Assessment and quantification
Plant Material
RNA isolation
• Para-Dormancy (0 C.H.) • Endo-Dormancy (853 C.H.) • Eco-Dormancy (909 C.H.)
Three replicates per condition
Library Construction and Sequencing
RNA Quality Number (RQN) >7.5
5’ adapter
3’ adapter
3’ ligation Total RNA or Purified small RNA
3’ adapter
5’ ligation 5’ adapter
small RNA Fragment
3’ adapter
RT-PCR 1ST Strand Synthesis
PCR Amplification
Gel Purification
• Library validation by capillary electrophoresis. • Library quantification by qPCR
Results
Hierarchical cluster analysis of smallRNAs accumulation, in Prunus avium buds with different chilling hours. This analysis was made with nine sequencing libraries, in three different stages of cold accumulation. The short sequences obtained were treated in accordance with the bioinformatic pipeline described above, identifying new miRNA precursors.
• Sequencing on MiSeq. • Raw Data extraction.
BioInformatic analysis
909 C.H.
Raw Data Extraction
miRNA
miRNA Discovery miRPREfeR Pipeline
Bowtie REAPER • •
Short sequences alignment versus reference genome
Trimming Filtering (quality and lenght)
miRBase
Winter
Autumn
Differential expression analysis (annotated) Bioconductor and R.
853 C.H.
Differential expression analysis (non annotated) Bioconductor and R.
SAMTools Identifying candidate regions
RNALfold
Bowtie
Hairpin and Folding detection
Repeated sequences removal and ncRNA (Rfam, RepBase, TIGR).
miRNA loci Prediction
Sample quality control DESeq2 Normalization
0 C.H.
Hierarchical cluster
Unmapped reads siRNA
Bowtie2 mapping
March Bowtie2 Target gene search (P. persica and P. mume)
April
May
June
July
August
RNA-precursor-862 (Pav-miR156-like) accumulation Scheme. This diagram shown a representation of temporal accumulation of Pav-miR156-like between autumn and winter (South Hemisphere).
Work funded by Grant(s): Development and application of genetic engineering tools to enhance cherry (Prunus avium) breeding FONDEF G09I1008 and Biofrutales S.A.
