Revealing the developmental dynamics in male strobilus transcriptome of Gnetum luofuense using nanopore sequencing technology

Abstract

Gnetum is a pantropical distributed gymnosperm genus. As being dioecious, Gnetum species apply female and male strobili to attract and provide nutrition to insect pollinators. Due to its unique gross morphology, a Gnetum male strobilus receives much attention in previous taxonomic and evolutionary studies. However, underlying molecular mechanisms that control male strobilus development and pollination adaptation have not been well studied. In the present study, nine full-length transcriptomes were sequenced from three developmental stages of the G. luofuense male strobili using Oxford Nanopore Technologies. In addition, weighted gene co-expression network analysis (WGCNA), and RT-qPCR analysis were performed. Our results show that a total of 3138 transcription factors and 466 long non-coding RNAs (lncRNAs) were identified, and differentially expressed lncRNAs and TFs reveal a dynamic pattern during the male strobilus development. Our results show that MADS-box and Aux/IAA TFs were differentially expressed at the three developmental stages, suggesting their important roles in the regulation of male strobilus development of G. luofuense. Results of WGCNA analysis and annotation of differentially expressed transcripts corroborate that the male strobilus development of G. luofuense is closely linked to plant hormone changes, photosynthesis, pollination drop secretion and reproductive organ defense. Our results provide a valuable resource for understanding the molecular mechanisms that drive organ evolution and pollination biology in Gnetum.

Figure 1

Different developmental stages of male strobili in Gnetum luofuense C.Y. Cheng. A white bar represents 1 cm. The photograph was made by C.H.

Figure 2

Identification of open reading frames (ORFs) and lncRNAs based on nine full-length transcriptome. (A) Length distribution of ORFs detected in all full-length transcripts. (B) Venn diagram showing the number of lncRNAs identified using four different approaches: CPC (Coding Potential Calculator), CNCI (Coding-Non-Coding Index), CPAT (Coding Potential Assessment Tool), and Pfam (Protein Family). (C) Functional classification and numbers of four lncRNA types. (D) Venn diagram showing the overlap in the differential expressed lncRNAs among the three different developmental stages of G. luofuense male strobili. (E) Representatives of predicted interaction networks among lncRNAs and their target genes. Solid lines and dotted lines represent the expression regulation by the lncRNAs in cis and in trans, respectively. (F) KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway annotations of cis- and trans-regulated genes regulated by the detected lncRNAs.

Figure 3

Weighted correlation network analysis (WGCNA) of co-expressed transcripts. (A) Hierarchical clustering tree showing co-expression modules based on WGCNA analysis. Each branch in the phylogenetic tree corresponds to an individual gene, and highly interconnected genes are grouped into seven modules. The different colored rows below the phylogeny indicate differentially expressed transcripts in G. luofuense. (B) Module–trait relationship with physiological indexes. The number represents the correlation coefficient about modules with physiological indexes. The number in the bracket means p-value (C) KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway annotations of co-expressed genes in the largest five modules.

Figure 4

Detected differentially expressed transcripts (DETs) and their annotation. (A) Numbers of DETs between different developmental stages of G. luofuense male strobili. (B) Venn diagram showing the overlap in DETs between different developmental stages of male strobili. (C) KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway annotations of up-regulated transcripts between FA and FC. (D) KEGG pathway annotation of down-regulated transcripts between FA and FC. (E) Two clusters identified based on expression levels of DETs from FA to FC. (F) GO enrichment amongst the two clusters with orange boxes denoting significant enrichment of GO annotation.

Figure 5

(A) A partial list of transcription factors (top 20 gene families) identified in nine full-length transcriptome of G. luofuense male strobili. (B) Transcription factors highly expressed in FA, FB, and FC with numbers in brackets. (C) RT–qPCR of eight genes of interests that encode differentially expressed transcription. Expression of the ACTIN gene used as an internal control was also shown. Counts per million (CPM) values from Nanopore sequencing are indicated on the left y-axis, and relative RT-qPCR expression levels are indicated on the right y-axis.