Combination of long-read and short-read sequencing provides comprehensive transcriptome and new insight for Chrysanthemum morifolium ray-floret colorization

Abstract

Chrysanthemum morifolium is one of the most popular ornamental plants globally. Owing to its large and complex genome (around 10 Gb, segmental hexaploid), it has been difficult to obtain comprehensive transcriptome, which will promote to perform new breeding technique, such as genome editing, in C. morifolium. In this study, we used single-molecule real-time (SMRT) sequencing and RNA-seq technologies, combined them with an error-correcting process, and obtained high-coverage ray-floret transcriptome. The SMRT-seq data increased the ratio of long mRNAs containing complete open-reading frames, and the combined dataset provided a more complete transcriptomic data than those produced from either SMRT-seq or RNA-seq-derived transcripts. We finally obtained ‘Sei Arabella’ transcripts containing 928,645 non-redundant mRNA, which showed 96.6% Benchmarking Universal Single-Copy Orthologs (BUSCO) score. We also validated the reliability of the dataset by analyzing a mapping rate, annotation and transcript expression. Using the dataset, we searched anthocyanin biosynthesis gene orthologs and performed a qRT-PCR experiment to assess the usability of the dataset. The assessment of the dataset and the following analysis indicated that our dataset is reliable and useful for molecular biology. The combination of sequencing methods provided genetic information and a way to analyze the complicated C. morifolium transcriptome.

Novel transcriptional activator TAC3 regulates age-dependent floral transition in Chinese fir (Cunninghamia lanceolata)

“…For downstream analysis in Chinese fir, after the adapter sequences and low-quality sequences of raw reads were deleted, clean reads were assembled using Trinity (v2.8.5) 57 . CD-hit was used to classify transcripts, corset was used to remove redundancy, and BUSCO (Benchmarking Universal Single Copy Orthologs, V 3.0.2) was used for the evaluation of the quality of the constructed transcriptome sequences 58, 59 . Then, these sequences were annotated by commonly used databases, including NCBI protein non-redundant database (NR), and Gene Ontology (GO) (http://www.geneontology.org/).…”

Overcoming Difficulties in Molecular Biological Analysis through a Combination of Genetic Engineering, Genome Editing, and Genome Analysis in Hexaploid Chrysanthemum morifolium

“…Although we could obtain many isoforms from RNA-Seq by Illumina, we should also recognize many artifacts and chimeric structures in the data. In chrysanthemum, RNA-Seq analysis was performed for both purposes, expression analysis and comprehensive transcript construction [68,69], even if short-and long-read NGS technology could not solve the complexity of transcripts in chrysanthemum because of many heterogenous transcripts. In combination with the accumulating genomic information, transcriptome data will be a powerful tool for molecular breeding in Chrysanthemum.…”