Abstract
Rafflesia cantleyi, known as one of the world's largest flowers, is a specialised holoparasite due to dramatic morphological modifications. It possesses highly reduced vegetative structure and only appears as a flower for sexual reproduction. Moreover, it has an unusual life cycle in that its floral bud development takes up to nine months. In order to fully understand the highly modified floral organ structure and long life cycle of R. cantleyi, we used Illumina sequencing technology (HiSeq) for sequence generation followed by de novo assembly of sequence reads. We obtained the RNA-seq data from three different stages of floral bud, representing the early, mid and advanced developmental stages. These data are available via BioProject accession number PRJNA378435. More than 10.3 Gb raw sequence data were generated, corresponding to 102,203,042 raw reads. Following removal of low-quality reads and trimming of adapter sequences, a total of 91,638,836 reads were obtained. De novo assembly of these sequences using Trinity resulted in 89,690 unique transcripts with an N50 of 1653 bp. The obtained transcriptomic data will be useful for further study to understand the molecular interactions that result in R. cantleyi floral development.
Keywords: Floral bud, Rafflesia, RNA-seq, Transcriptome
| Specifications | |
|---|---|
| Organism | Rafflesia cantleyi |
| Sequencer | Illumina HiSeq™ 2000 |
| Data format | Raw |
| Experimental factors | Floral buds from early, mid and advanced developmental stages |
| Experimental features | Inner tissue of individual floral buds were sampled for RNA extraction, sequencing and de novo transcriptome assembly |
| Consent | Not applicable |
| Sample source location | Raub, Pahang, Malaysia (3° 47′ 24″ N, 101° 51′ 25″E) |
1. Direct link to deposited data
2. Introduction
Rafflesia cantleyi is an endophytic holoparasite plant of the tropical rainforests of South-east Asia belonging to the family Rafflesiaceae, subfamily Rafflesia. It depends entirely on its specific host, Tetrastigma (Vitaceae) [1]. It has no apparent leaves, stems or roots, and only appear as a flower of up to 0.6 m in diameter for sexual reproduction [2]. This large flower includes five highly modified perianth and a petal-derived diaphragm, which together formed a large, bowl-shaped structure called floral chamber [3]. Previous studies have provided insights on its morphological evolution [3], [4]; yet, the floral organs development in this giant flower remains poorly understood.
Rafflesia is also known for its long life cycle, which takes an average of 9 months for the flower to be fully opened [1]. The buds of R. cantleyi first emerge through the bark of Tetrastigma with the bracts still covering them. They then grow progressively and upon maturation, the buds open gradually over a 24 to 48-hour period. Previous work has shed light on the genes potentially involved in the growth and development of the R. cantleyi flower [5]. However, knowledge on the biological processes that occur during R. cantleyi flower transition is still scarce. In this study, we carried out sequencing and de novo assembly of floral bud transcriptome from three developmental stages to provide essential data for further studies on these aspects of R. cantleyi.
3. Experimental design, materials and methods
3.1. Plant materials
Rafflesia cantleyi floral bud samples from different developmental stages were collected from Raub, Pahang, Malaysia. Cross section of the floral buds showed that they represent the early (floral bud stage 1), mid (floral bud stage 2) and advanced (floral bud stage 3) developmental stages. Floral bud stage 1 is made up of undifferentiated masses of cells, floral bud stage 2 contains moderately differentiated and visible internal organs, while floral bud stage 3 consists of more developed internal organs. These R. cantleyi floral buds, attached to their host, Tetrastigma, were carefully dissected from the host plant. The buds were surface sterilised using 10% (v/v) Clorox® solution (1% sodium hypochlorite), followed by three rinses with sterile water.
3.2. RNA isolation and cDNA library construction
Inner tissues of the floral buds were cut and flash-frozen in liquid nitrogen in the field. All samples were then stored at -80 °C until further use. Total RNA was extracted using a modified CTAB extraction protocol based on previously described methods [6], [7], [8]. RNA purity and integrity was measured using the ND-1000 Nanodrop spectrophotometer (Thermo Fisher Scientific Inc., USA) and Agilent 2100 bioanalyzer (Agilent Technologies, USA), respectively. cDNA library preparation was performed using the TruSeq® Stranded Total RNA Library Prep Kit (Illumina, USA) according to the manufacturer's protocol. The libraries were sequenced using the Illumina HiSeq™ 2000 platform.
3.3. Transcriptome de novo assembly
Approximately 102.2 million bp paired-end reads were generated using the Illumina HiSeq™ 2000 platform. Raw reads from all the three data sets were filtered using Trimmomatic [9] (default setting SLIDINGWINDOW:4:5, LEADING:5, TRAILING:5, MINLEN:25) to remove adaptor sequence or low quality reads. De novo assembly of clean reads was performed using Trinity (v2.0.6) [10] with the default k-mer size of 25. Table 1 shows the transcriptome assembly statistics that were generated utilising the TrinityStats.pl script.
Table 1.
Statistics of Rafflesia cantleyi floral bud transcriptome assembly.
| Attributes | Floral bud stage 1 | Floral bud stage 2 | Floral bud stage 3 | Merged assembly |
|---|---|---|---|---|
| Pre-assembly | ||||
| Total raw reads | 36,776,468 | 29,428,456 | 35,998,118 | 102,203,042 |
| Total processed reads | 32,543,575 | 26,453,373 | 32,641,888 | 91,638,836 |
| Post-assembly | ||||
| Number of unigenes | 48,825 | 28,950 | 31,053 | 70,467 |
| Number of unique transcripts | 67,055 | 44,300 | 44,827 | 89,690 |
| N50 (bp) | 1765 | 1972 | 1943 | 1653 |
| Size range (bp) | 200–15,281 | 200–15,218 | 200–16,493 | 200–16,553 |
In conclusion, we have generated and de novo assembled transcriptome sequences from the floral buds of R. cantleyi. These sequences can be used for gene discovery, further studies on the regulation of Rafflesia growth and development, and comparative transcriptome analysis with other Rafflesia species.
Conflicts of interest
The authors declare no conflicts of interest in this study.
Acknowledgements
This work was supported financially by Research University grants from Universiti Kebangsaan Malaysia [grant numbers LAUREATE-2013-001 and GUP-2016-008].
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