Utilizing PacBio Iso-Seq for Novel Transcript and Gene Discovery of Abiotic Stress Responses in Oryza sativa L.

Abstract

The wide natural variation present in rice is an important source of genes to facilitate stress tolerance breeding. However, identification of candidate genes from RNA-Seq studies is hampered by the lack of high-quality genome assemblies for the most stress tolerant cultivars. A more targeted solution is the reconstruction of transcriptomes to provide templates to map RNA-seq reads. Here, we sequenced transcriptomes of ten rice cultivars of three subspecies on the PacBio Sequel platform. RNA was isolated from different organs of plants grown under control and abiotic stress conditions in different environments. Reconstructed de novo reference transcriptomes resulted in 37,500 to 54,600 plant-specific high-quality isoforms per cultivar. Isoforms were collapsed to reduce sequence redundancy and evaluated, e.g., for protein completeness (BUSCO). About 40% of all identified transcripts were novel isoforms compared to the Nipponbare reference transcriptome. For the drought/heat tolerant aus cultivar N22, 56 differentially expressed genes in developing seeds were identified at combined heat and drought in the field. The newly generated rice transcriptomes are useful to identify candidate genes for stress tolerance breeding not present in the reference transcriptomes/genomes. In addition, our approach provides a cost-effective alternative to genome sequencing for identification of candidate genes in highly stress tolerant genotypes.

Physiological and molecular attributes contribute to high night temperature tolerance in cereals

“…Given the importance of N22 as a HNT‐tolerant model cultivar, the genome (Stein et al, 2018) and the transcriptome of N22 from control and abiotic stress conditions were sequenced on the PacBio Sequel platform and reconstructed as template for mapping RNA‐seq reads (Schaarschmidt, Fischer, et al, 2020). This provides tools for the identification of new candidate genes that are important for HNT tolerance and are currently overlooked in molecular studies because a larger number of N22 specific genes are absent from common reference genomes and transcriptomes.…”

“…These genes could then be integrated into high yielding varieties using, for example, the CRISPR/CAS technology to improve HNT tolerance. As an example, the newly identified gene B12288 was up‐regulated in N22 during combined heat and drought stress (Schaarschmidt, Fischer, et al, 2020). This gene is a homolog of RAB21, a member of the dehydrin family of Late Embryogenesis Abundant (LEA) proteins controlling cellular membrane traffic, and was in N22 more similar to genes from wild rice species than from O. sativa ssp.…”

“…This gene is a homolog of RAB21, a member of the dehydrin family of Late Embryogenesis Abundant (LEA) proteins controlling cellular membrane traffic, and was in N22 more similar to genes from wild rice species than from O. sativa ssp. japonica (Schaarschmidt, Fischer, et al, 2020).…”

A high resolution single molecule sequencing-based Arabidopsis transcriptome using novel methods of Iso-seq analysis

“…For example, a recent study of Iso-seq of nine tissues in rice covered only ca. one-third of rice genes [36]. Coverage of the other genes and transcripts in AtRTD3 came from Araport11 and, primarily, from AtRTD2 due to its far greater transcript diversity [39].…”

“…), and medicinal plants (e.g. Zanthoxylum, safflower, Salvia) [15,16,[25][26][27][28][29][30][31][32][33][34]17,[35][36][37][38][18][19][20][21][22][23][24]. The majority of these applications of PacBio sequencing investigated transcriptome diversity and complexity and determined transcription start sites, AS events and APA sites.…”