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

Yingjuan Su,Nan Deng,Chen Hou

doi:10.3390/f10111043

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.

Highlights

The order Gnetales comprises three extant genera: Ephedra L., Welwitschia Hook.f. and Gnetum L.The gross morphology of the Gnetales, especially their reproductive organs, dramatically differs from that of non-Gnetalean gymnosperms [1,2,3]
After the deletion of 30,101 short reads (4.2%), the remaining Reads of inserts (ROIs) were classified into 483,475 full-length reads (FLs, 68.1%), comprising 3551 full-length chimeric reads (0.5%) and 480,225 full-length nonchimeric reads (67.6%, FLNC), and 196,816 non-full-length reads (Figure 2A)
Our results show that a total of 1196 long noncoding RNAs (lncRNAs) with the mean length 1258 bp were identified for the major developmental stages of G. luofuense female strobili

Summary

Introduction

The order Gnetales comprises three extant genera: Ephedra L., Welwitschia Hook.f. and Gnetum L.The gross morphology of the Gnetales, especially their reproductive organs, dramatically differs from that of non-Gnetalean gymnosperms [1,2,3]. The order Gnetales comprises three extant genera: Ephedra L., Welwitschia Hook.f. and Gnetum L. The systematic position of the Gnetales remain unresolved among extant seed plant groups, and hypotheses proposed based on morphological and molecular data are constantly contradictory [1,4,5,6,7,8]. A female strobilus of Gnetum is featured by several rolls of involucres—for each roll, multiple reproductive units closely connect to a Forests 2019, 10, 1043; doi:10.3390/f10111043 www.mdpi.com/journal/forests. Forests 2019, 10, 1043 main axis—whereas a male strobilus usually bears one layer of sterile reproductive units and several layers of staminate structures attached by 20–80 microsporangia [1,3,11,12]. Lan et al [18]

Results

Discussion

Conclusion