Integrated SMRT Technology with UMI RNA-Seq Reveals the Hub Genes in Stamen Petalody in Camellia oleifera

Abstract

Male sterility caused by stamen petalody is a key factor for a low fruit set rate and a low yield of Camellia oleifera but can serve as a useful genetic tool because it eliminates the need for artificial emasculation. However, its molecular regulation mechanism still remains unclear. In this study, transcriptome was sequenced and analyzed on two types of bud materials, stamen petalody mutants and normal materials, at six stages of stamen development based on integrated single-molecule real-time (SMRT) technology with unique molecular identifiers (UMI) and RNA-seq technology to identify the hub genes responsible for stamen petalody in C. oleifera. The results show that a large number of alternative splicing events were identified in the transcriptome. A co-expression network analysis of MADSs and all the differentially expressed genes between the mutant stamens and the normal materials showed that four MADS transcription factor genes, CoSEP3.1, CoAGL6, CoSEP3.2, and CoAP3, were predicted to be the hub genes responsible for stamen petalody. Among these four, the expression patterns of CoAGL6 and CoSEP3.2 were consistently high in the mutant samples, but relatively low in the normal samples at six stages, while the patterns of CoSEP3.1 and CoAP3 were initially low in mutants and then were upregulated during development but remained relatively high in the normal materials. Furthermore, the genes with high connectivity to the hub genes showed significantly different expression patterns between the mutant stamens and the normal materials at different stages. qRT-PCR results showed a similar expression pattern of the hub genes in the RNA-seq. These results lay a solid foundation for the directive breeding of C. oleifera varieties and provide references for the genetic breeding of ornamental Camellia varieties.