Full-Length Transcriptome Assembly of Italian Ryegrass Root Integrated with RNA-Seq to Identify Genes in Response to Plant Cadmium Stress

Abstract

Cadmium (Cd) is a toxic heavy metal element. It is relatively easily absorbed by plants and enters the food chain, resulting in human exposure to Cd. Italian ryegrass (Lolium multiflorum Lam.), an important forage cultivated widely in temperate regions worldwide, has the potential to be used in phytoremediation. However, genes regulating Cd translocation and accumulation in this species are not fully understood. Here, we optimized PacBio ISO-seq and integrated it with RNA-seq to construct a de novo full-length transcriptomic database for an un-sequenced autotetraploid species. With the database, we identified 2367 differentially expressed genes (DEGs) and profiled the molecular regulatory pathways of Italian ryegrass with Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis in response to Cd stress. Overexpression of a DEG LmAUX1 in Arabidopsis thaliana significantly enhanced plant Cd concentration. We also unveiled the complexity of alternative splicing (AS) with a genome-free strategy. We reconstructed full-length UniTransModels using the reference transcriptome, and 29.76% of full-length models had more than one isoform. Taken together, the results enhanced our understanding of the genetic diversity and complexity of Italian ryegrass under Cd stress and provided valuable genetic resources for its gene identification and molecular breeding.

Integration of early disease-resistance phenotyping, histological characterization, and transcriptome sequencing reveals insights into downy mildew resistance in impatiens

“…The full-length transcriptome not only provided full-length transcript sequences for genes but also provided insights into the AS events in Impatiens spp. In consistent with other crops such as cotton 61 , rice 62 , and Italian ryegrass 63 , the retained intron type contributed the majority of AS events in Impatiens . On the basis of Iso-seq isoforms and in combination with RNA-Seq assemblies, we constructed a reference transcriptome for Impatiens spp.…”

Phytoremediation of Cadmium: Physiological, Biochemical, and Molecular Mechanisms

“…Out of 2367 DEGs, the overexpression of the LmAUX1 gene significantly enhanced the Cd concentration in A. thaliana. The transcriptome analysis helped in the construction of full-length UniTransModels; out of this, 26.76% had isoforms [262]. RNA-seq analysis of Pokeweed revealed 10.63 Gb transcriptomic data consist of up to 97,000 unigenes covering 72 metabolic pathways.…”

Transcriptome sequencing of cochleae from constant-frequency and frequency-modulated echolocating bats

“…In these earlier studies, the reference used for quantification of gene expression was from a de novo assembly based on the short RNA-seq reads which may contain many artificial transcripts 11 . The PacBio single-molecule real-time isoform sequencing (Iso-seq) can generate full-length (FL) sequences of all transcripts without the need for assembly 12 , which has been integrated with RNA-seq for transcriptome quantification in multiple studies 12,13 . PacBio Iso-seq is also used to detect alternative splicing events without the help of a reference genome sequence 14 and to identify previously unannotated transcripts 15 .…”