PacBio Long-Read Sequencing Reveals the Transcriptomic Complexity and Aux/IAA Gene Evolution in Gnetum (Gnetales)

Abstract

The genus Gnetum includes pantropical trees, shrubs and lianas, with unresolved phylogenetic relationships with other seed plant groups. Despite the reference genome for this genus being recently published, the molecular mechanisms that regulate the reproductive organ development of Gnetum remain unclear. A previous study showed that indole-3-acetic acid is involved in the regulation of female strobili of Gnetum, while the diversity and evolution of indole-3-acetic acid-related genes—the Aux/IAA genes—have never been investigated in Gnetales. Thus, a pooled sample from different developmental stages of female strobili in Gnetum luofuense C.Y. Cheng was sequenced using PacBio single-molecular long-read technology (SMRT) sequencing. PacBio SMRT sequencing generated a total of 53,057 full-length transcripts, including 2043 novel genes. Besides this, 10,454 alternative splicing (AS) events were detected with intron retention constituting the largest proportion (46%). Moreover, 1196 lncRNAs were identified, and 8128 genes were found to possess at least one poly (A) site. A total of 3179 regulatory proteins, including 1413 transcription factors (e.g., MADS-box and bHLHs), 477 transcription regulators (e.g., SNF2), and 1289 protein kinases (e.g., RLK/Pelles) were detected, and these protein regulators probably participated in the female strobili development of G. luofuense. In addition, this is the first study of the Aux/IAA genes of the Gnetales, and we identified 6, 7 and 12 Aux/IAA genes from Gnetum luofuense, Welwitschia mirabilis, and Ephedra equistina, respectively. Our phylogenetic analysis reveals that Aux/IAA genes from the gymnosperms tended to cluster and possessed gene structures as diverse as those in angiosperms. Moreover, the Aux/IAA genes of the Gnetales might possess higher molecular evolutionary rates than those in other gymnosperms. The sequencing of the full-length transcriptome paves the way to uncovering molecular mechanisms that regulate reproductive organ development in gymnosperms.