The complete chloroplast genome of greater duckweed (Spirodela polyrhiza 7498) using PacBio long reads: Insights into the chloroplast evolution and transcription regulation

- The complete chloroplast genome of greater duckweed (Spirodela polyrhiza.
- 7498) using PacBio long reads: insights into the chloroplast evolution and transcription regulation.
- The chloroplast genome, as an efficient solar-powered reactor, is an invaluable resource to study biodiversity and to carry foreign genes.
- The chloroplast genome sequencing has become routine and less expensive with the delivery of high-throughput sequencing technologies, allowing us to deeply investigate genomics and transcriptomics of duckweed organelles..
- Results: Here, the complete chloroplast genome of Spirodela polyrhiza 7498 (SpV2) is assembled by PacBio sequencing.
- A number of 37 RNA editing sites are recognized to have cytosine (C) to uracil (U) substitutions, eight of which are newly defined including six from the intergenic regions and two from the coding sequences of rpoC2 and ndhA genes.
- Conclusions: The understanding of the chloroplast genomics and the transcriptomics of S.polyrhiza would greatly facilitate the study of phylogenetic evolution and the application of genetically engineering duckweeds..
- 2020 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0.
- Full list of author information is available at the end of the article.
- They are phylogenetically located at the early-diverging monocots of the Alismatale order.
- The chloroplast genome has dual characteristics of sequence variation and conservation, which are widely applied in the studies of population genetics and phylogenetic relationships.
- The chloroplast genome is one of the three genetic systems in- cluding nuclei, mitochondria, and plastids in plants that possesses both eukaryote-like introns and prokaryote-like operons [6].
- One broad hypothesis is that the chloroplast is derived from an initial engulfment and integration of a free-living cyanobacterium into a host cell around 1.5 bil- lion years ago [7].
- The chloroplast is also a vital organelle for plants, playing a crucial role by converting solar energy to carbohydrates through photosynthesis, and promoting their growth and starch accumulation..
- In 2008, the first duckweed chloroplast genome (L.minor) was sequenced by Sanger sequencing [11].
- In the meanwhile, the duckweed nuclear genomes have become more complete with the expan- sion of sequencing technology.
- Long-read sequencing, such as SMRT (Single Molecule Real-Time) technology emerged in 2009 [17] has been widely applied in sequen- cing the chloroplast genomes with the improved con- tiguity and accuracy.
- The studies of annotating chloroplast genome and gene structure at the transcriptomic and post- transcriptomic levels were limited, which were involved in a series of RNA regulation and process, such as RNA splicing, 5′- and 3′-end modification, and RNA editing and turnover [18].
- Most previous studies relied on the sequence alignment and computer prediction to deter- mine the intron boundary and the possible RNA editing sites, which need to be confirmed by PCR and se- quenced one by one [19, 20].
- With the high-throughput RNA-seq data with a read length of 75 bp, 66 RNA edit- ing in Spirodela chloroplast genome were defined at the genome-wide level [21].
- In this study, we improved and validated the chloroplast genome of S.polyrhiza assembled by PacBio sequencing reads with retrieval of two repeated fragments compared with the last version.
- The integration of full- length cDNAs from isoform sequencing allowed us to dis- cover new RNA editing sites, to detect introns, and to define poly-cistrons similar to prokaryotic transcripts in Spirodela chloroplast.
- The understanding of the chloro- plast genomics and the transcriptomics of S.polyrhiza would facilitate the study of phylogenetic evolution and the application of genetically engineering the solar reactor of chloroplasts..
- Chloroplast genome assembly, validation and annotation The last version of the complete chloroplast genome of S.polyrhiza 7498 (SpV1) was sequenced on a SOLiD platform and published in 2011 (GenBank accession number: JN .
- Because of the limitations of the second-generation sequencing technology with short reads (50 bp), the assembly of SpV1 was tedious and challenging to resolve boundaries of IR regions, resulting in 3 genomic breakage and 52 small gaps (Table 1)..
- generating long reads with the mean length of 10,789 bp..
- After bioinformatic filtering, a total of 239,086 high- quality long reads were selected to be chloroplast related sequences, which were used to run the chloroplast gen- ome de novo assembly.
- The chloroplast genome with long-read assembly exhibited the typical quadripartite structure, a pair of inverted repeat regions (IRs) of 31, 844 bp separated by a large single copy (LSC) of 91,210 Table 1 The comparative statistics of the chloroplast genome.
- 1 Gene map of the chloroplast genome of S.polyrhiza 7498.
- The darker area in the inner circle indicates the GC content.
- 2), indicating high accuracy of the assembled gen- ome.
- The chloroplast genome was annotated as 107 unique genes, including 78 protein-coding genes, 25 tRNAs and 4 rRNAs.
- A coverage plot was demonstrated by re-mapping the Pac- Bio reads to the chloroplast genome, showing an even distribution across the genome with a mean coverage of 7837 times (Fig.
- Surprisingly, the recovered se- quences were the copies of the downstream nucleotides, which could be a failure of genome assembly in SpV1 due to short reads of second-generation sequencing..
- Such limitation could be easily conquered by the nature of PacBio long reads with the spanning of the ambiguous repeats..
- The full-length cDNAs generated by PacBio isoform se- quencing allowed us to define the chloroplast transcript structures.
- We found that the early- diverging monocot of Amborella had the longest atpF introns (1825 bp), whereas the dicot of tobacco had the shortest one (1250 bp), indicating that introns might play roles in genomic diversity during the chloroplast evolu- tion (Fig.
- RNA editing analysis.
- Here, with isoform sequences, we defined 37 RNA editing sites, including 30 sites that occurred in protein-coding sequences, one in intron and six in non-coding regions (Additional file 1: Table S3)..
- The RNA editing efficiency had a range of 21 to 100%.
- Combined with known and newly discovered RNA editing sites, there were 74 in total, 62 of which occurred in gene regions, whereas the Ndh gene showed the most heavily edited sites (33 sites) (Additional file 1: Figure S2).
- a The x-axis shows the chloroplast genome of S.polyrhiza .
- b The sequence alignment of two versions of S.polyrhiza 7498 chloroplast genomes.
- The event of RNA editing in Spirodela rpoC2 was consist- ent with rice and tobacco, whereas the C-to-U conversion in ndhA made Spirodela keep the conserved amino acid of L as other plants (Additional file 1: Figure S3)..
- As we knew, the size of the chloroplast genome was compact, but it played a critical role in photosynthesis in the survival of plants.
- The pattern of co-transcription in the chloroplast of duckweed may enhance the work efficiency of transcription-translation factors like RNA polymerase..
- With the announce- ment of the launch of PacBio Sequel II system, it gener- ates 8-times more data and makes sequencing more affordable.
- No matter how hard scientists try to remove organellar DNA from the total DNA (including nuclear, mitochondria and chloroplast DNA), chloroplast gen- ome still can be assembled from the left “purified” DNA as a side project of the whole genome sequencing study due to its high copy number [33].
- Our trial confirmed that two pairs of repeats in the coding sequence of ycf2 gene were filled in the assembly of the chloroplast gen- ome of S.polyrhiza.
- The phylogenetic analysis suggested that ycf2 gene was evolved from the membrane-bound AAA-protease FtsH of the ancestral endosymbiont [34]..
- The gene structures of introns and operons remained unknown, although some RNA editing sites were detected by using high-throughput RNA-seq [21].
- transcripts without assembly from PacBio isoform se- quencing (Iso-Seq), it is advantageous for gene annota- tion, identification of introns, RNA editing and operons in chloroplasts.
- represent the early-diverging monocot of the phylogen- etic tree with their small and simple plant bodies, which is challenging to identify species by merely counting on morphology for non-experts.
- Still, it is necessary to verify the potential of the utilization of ndhA intron itself or with other markers to distinguish intra- and inter-species in duckweeds..
- RNA editing and its evolution.
- RNA editing is a post-transcriptional modification that broadly exists in land plants from hornworts and ferns to seed plants.
- We could not detect RNA editing sites in the Spirodela chloroplast genome all at once only using one technique.
- Aligned length are longer than the original sequence length because of the addition of the aligned gaps.
- The controls of the intergenic region of atpF- atpH a and the coding sequence of rbcL b are also included.
- The gene order in the operon is based on the full-length transcript.
- Genome Position means the location of operon in the new version of S.polyrhiza 7498 chloroplast genome.
- Here, long reads using PacBio isoform sequencing identified 37 RNA editing sites.
- Excluding overlapped sites from two platforms, there are 74 RNA editing in Spirodela, more than twice of those in rice (35 sites) and maize (26 sites) [40].
- The early-branching flowering plant of Amborella trichopoda was found to have 138 sites of RNA editing.
- It was proposed that early-branching flowering plants carried more abundant chloroplast RNA editing, whereas there was a tremen- dous decrease in RNA editing frequencies during flower- ing plant evolution [41].
- Such knowledge about operon structures would enable engineering new pathways in a simulated operon via a single transformation event into the chloroplast genome..
- Large amounts of foreign pro- tein accumulation were observed in these transgenic lines, showing that the chloroplast posttranscriptional.
- With the evidence of full-length cDNA generated from PacBio isoform sequencing, we accur- ately detect nine introns, 37 RNA editing sites and nine operons.
- The complete chloroplast genome of Arabidopsis thaliana (NC_000932.1) was downloaded from NCBI as a reference genome.
- The chloroplast genome of S.polyrhiza was assembled using a Perl-based software named “Organelle_PBA”.
- The chloroplast genome was annotated with the tool GeSeq (https://chlorobox.mpimp-golm.mpg.de/.
- A circular map of the annotated genome was illustrated by using Organellar Genome DRAW (OGDRAW) (https://.
- The chloroplast genomes of Amborella trichopoda (NC Nicotiana tabacum (NC Arabidopsis thaliana (NC Oryza sativa Japonica Group (NC Zea mays (NC Lemna minor (DQ400350), Wolffiella lin- gulata 7289 (JN160604) and Wolffia australiana 7733.
- Intron, RNA editing and operon analysis.
- The full-length transcript generated by PacBio isoform sequencing were mapped to the previous and new chloroplast genome of S.polyrhiza by Gmap (version .
- The RNA editing sites, introns and op- erons were identified through the mapped data and visu- alized under IGV (Integrative Genomics Viewer) (version .
- Annotated gene list in the chloroplast of SpV2.
- The list of RNA editing sites in SpV2.
- Bioinformatic pipeline of chloroplast genome assembly and annotation.
- The distribution of RNA editing events in the chloroplast genes of S.polyrhiza .
- Graph shows the number of currently detected RNA editing sites in protein coding genes.
- The sequences included RNA editing sites are shown before RNA editing.
- The start, RNA editing and end locations are listed above the alignment.
- The chloroplast genome assembly and annotation (SpV2) were deposited in GenBank under the accession number of MN419335..
- Species identification of Conyza bonariensis assisted by chloroplast genome sequencing.
- Complete sequence of the duckweed (Lemna minor) chloroplast genome: structural organization and phylogenetic relationships to other angiosperms.
- Generating a high-confidence reference genome map of the greater duckweed by integration of cytogenomic, optical mapping, and Oxford Nanopore technologies.
- RNA editing in plants: a comprehensive survey of bioinformatics tools and databases.
- RNA editing in chloroplasts of Spirodela polyrhiza, an aquatic Monocotelydonous species.
- Implications of the plastid genome sequence of typha (typhaceae, poales) for.
- DNA barcoding of the Lemnaceae, a family of aquatic monocots.
- Complex processing patterns of mRNAs of the large ATP synthase operon in Arabidopsis chloroplasts.
- Organization and post-transcriptional processing of the psb B operon from chloroplasts of Populus deltoides.
- Characterization of the psbH precursor RNAs reveals a precise endoribonuclease cleavage site in the psbT/psbH intergenic region that is dependent on psbN gene expression.
- Cotranscription of the S10- and spc-like operons in spinach chloroplasts and identification of three of their gene products.
- The two largest chloroplast genome-encoded open reading frames of higher plants are essential genes..
- The complete chloroplast genome sequence of watercress (Nasturtium officinale R.
- RNA editing in plants and its evolution.
- Frequent chloroplast RNA editing in early- branching flowering plants: pilot studies on angiosperm-wide coexistence of editing sites and their nuclear specificity factors.
- Overexpression of the Bt cry2Aa2 operon in chloroplasts leads to formation of insecticidal crystals.

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