Chloroplast Genomics And Transcriptomics In Duckweeds By PacBio Long-reads

Duckweeds(Lemnaceae)are higher aquatic flowering plants distributed all over the world.The chloroplast genome,as an efficient solar powered reactor,is an invaluable resource to study biodiversity and is a potential carrier to engineer foreign genes.The chloroplast genome sequencing has become routine using long-read sequencing technology,allowing us to deeply investigate the genomics and transcriptomics of duckweed organelles feasible.Here,the complete chloroplast genome of Spirodela polyrhiza 7498 was assembled by Pac Bio.The length of 168,956 bp circular genome was composed of a pair of inverted repeats(IRs)of 31,844 bp separated by a large single copy(LSC)of 91,210 bp and a small single copy(SSC)of14,058 bp.We annotated a total of 107 unique genes,including 78 proteincoding genes,25 t RNA genes and 4 r RNA genes.Compared to the previous chloroplast genome of S.polyrhiza assembled from short reads,two repeated fragments in ycf2 gene of 45 bp and 48 bp respectively were retrieved demonstrating Pac Bio long reads could resolve the difficulty of assembling of repeats and the integrity were greatly improved.Furthermore,with the full-length c DNA generated from Pac Bio isoform,seven genes were detected to have type-II introns(ycf3,clp P,atp F,rpo C1,rpl2,rps12 and ndh A).The ndh A intron had 50% more polymorphism than the species-barcoding marker of atp F-atp H,giving sufficient genetic distance to discriminate close species.RNA editing is the process of changing the genetic information C to U at the m RNA level,which is also an important post-transcriptional regulation phenomenon in the chloroplast.A number of 37 RNA editing sites were recognized to have C to U substitutions,eight of which were newly defined.Most of the new RNA editing sites were silent due to the location of the inter-genetic region,except the sites in rpo C2 and ndh A genes could change amino acids.In addition,chloroplasts also have prokaryotic transcription characteristics,like operons,which means a single m RNA could encode a number of proteins.We identified nine operon classes strongly supported by transcriptomic data.It was found that the operons contained multiple subunit genes encoding the same functional complex including ATP synthase,photosynthesis system,ribosomal proteins,et.al.In short,we successfully obtained and improved the chloroplast genome of duckweed using long reads and deeply understood the intron,RNA editing and operon by full-length transcript data would greatly facilitate the study of phylogenetic evolution and chloroplast genetic engineering.