Scientists at Huazhong Agricultural University in China and collaborating institutions recently published results of an Iso-Seq analysis of allotetraploid cotton. The team’s findings are expected to be particularly useful for functional genomics, driving advances for cotton breeders as well as research biologists.
“A global survey of alternative splicing in allopolyploid cotton: landscape, complexity and regulation” was published in New Phytologist by lead authors Maojun Wang and Pengcheng Wang, senior author Xianlong Zhang, and collaborators. Existing genome assemblies for polyploidy cotton were not “released with a well-annotated transcript isoform set,” the scientists write, “and so the extent and differences in [alternative splicing (AS)] of homoeologous gene transcripts remain poorly understood.”
To remedy the situation, the team used SMRT Sequencing to analyze RNA in 12 plant samples across six tissues, including root, leaf, and petal, among others. They focused on the allotetraploid Gossypium barbadense because “allotetraploid cottons contribute to the vast majority of fibre yield every year, and their recently published genome sequences of allotetraploid cottons are of interest to both breeders and genome biologists,” they explain. As part of this project, the scientists developed a new Iso-Seq data analysis pipeline geared toward complex genomes. “This includes methods for quality control of raw data, classification of transcripts, clustering and transcriptome analysis,” they note. The tool is available for public use with step-by-step instructions.
Data analysis revealed nearly 177,000 unique, full-length transcripts of almost 45,000 gene models. Aligning to the reference genome and comparing with its annotation allowed the team to extend genes at both 5’ and 3’ ends and expand the number of transcripts linked to each gene. The previous annotation had just 20% of genes associated with more than one transcript, while the SMRT Sequencing annotation boosted that to 57%.
“These data led us to identify 15,102 fibre-specific AS events and estimate that c. 51.4% of homoeologous genes produce divergent isoforms in each subgenome,” the scientists report. Among other key findings: thanks to alternative splicing, the same gene can be regulated differently by various microRNAs.
“This study provides a rich resource of transcript isoforms for the cotton community, evolutionary biologists and provides a useful reference for other species,” the scientists conclude. “These results will facilitate future functional genomics studies and enhance our understanding of AS in polyploid species.”