Single-Molecule Real-Time Sequencing of Full-Length Transcriptome and Identification of Genes Related to Male Development in Cannabis sativa

Abstract

Female Cannabis sativa plants have important therapeutic properties. The sex ratio of the dioecious cannabis is approximately 1:1. Cultivating homozygous female plants by inducing female plants to produce male flowers is of great practical significance. However, the mechanism underlying cannabis male development remains unclear. In this study, single-molecule real-time (SMRT) sequencing was performed using a mixed sample of female and induced male flowers from the ZYZM1 cannabis variety. A total of 15,241 consensus reads were identified, and 13,657 transcripts were annotated across seven public databases. A total of 48 lncRNAs with an average length of 986.54 bp were identified. In total, 8202 transcripts were annotated as transcription factors, the most common of which were bHLH transcription factors. Moreover, tissue-specific expression pattern analysis showed that 13 MADS transcription factors were highly expressed in male flowers. Furthermore, 232 reads of novel genes were predicted and enriched in lipid metabolism, and qRT-PCR results showed that CER1 may be involved in the development of cannabis male flowers. In addition, 1170 AS events were detected, and two AS events were further validated. Taken together, these results may improve our understanding of the complexity of full-length cannabis transcripts and provide a basis for understanding the molecular mechanism of cannabis male development.

Keywords: Cannabis sativa; SMRT sequencing; male development.

Figure 1

Sequence length distribution of CDS.

Figure 2

Statistics of lncRNA annotation in CPC, PLEK, CNCI and Pfam databases.

Figure 3

The classification statistics of non-redundant transcripts in NR database.

Figure 4

KOG (a) and GO (b) database function annotation classification statistics.

Figure 5

Heatmap of gene expression of genes associated with starch and sucrose metabolism in different tissues of cannabis (R: root; S: stem; L: leaves; F: female; M: male).

Figure 6

The distribution of TFs family.

Figure 7

Heatmap of gene expression of MADS transcription factors in different tissues of cannabis (R: root; S: stem; L: leaves; F: female; M: male).

Figure 8

The unique pathway number of novel genes in KEGG database.

Figure 9

The gene expression level of three genes (PLD1_2, CER1, KCR1) of lipid metabolism pathway by qRT-PCR. Different letters indicate significant differences among three tissues according to one-way ANOVA test (p < 0.05). FF: female flower; MF: male flower; IMF: induced-male flower.

Figure 10

The distribution and validation of AS events. (a) The distribution of AS events number; and (b) gel electrophoresis validation of AS events PB.870 and PB.4392.