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

Stephanie Schaarschmidt,Bruno Huettel,Julia Bailey-Serres,Axel Fischer,Endang M. Septiningsih,S. V. Krishna Jagadish,Rejbana Alam,Lovely Mae F. Lawas,Ellen Zuther,Dirk K. Hincha

doi:10.3390/ijms21218148

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.

Highlights

Global climate change is causing an increase in the severity and frequency of abiotic stress conditions such as heat, drought and high night temperatures that all have a strong negative impact on crop yield [1–5]
Pooled RNA samples were sequenced on the Pacific Bioscience (PacBio) Sequel I platform on two or three Single-Molecule Real-Time (SMRT) cells per cultivar (Table 2)
Sequence raw data was processed with the software IsoSeq3 using the steps ccs and lima, resulting in between 460,340 and 736,747 full-length non-chimeric reads (FLNC, containing 5 primer, 3 primer and poly(A) tail) for the combined SMRT cells per cultivar

Summary

Introduction

Global climate change is causing an increase in the severity and frequency of abiotic stress conditions such as heat, drought and high night temperatures that all have a strong negative impact on crop yield [1–5]. The sequences obtained in the 3000 Rice Genomes Project [8] were mapped against the Nipponbare genome, excluding all sequences that could not be mapped to this reference [11] This may have led to the loss of genetic information that is specific to the non-japonica subspecies. The phosphate-starvation tolerance gene OsPSTOL1, the deepwater escape genes OsSNORKEL1/2 and the submergence tolerance gene OsSUB1A were identified in the genomes of aus cultivars. These genes are absent in the genome sequence of the japonica reference cultivar Nipponbare

Methods

Results

Discussion

Conclusion