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, 18 (1), 806

Transcriptome Assembly in Suaeda Aralocaspica to Reveal the Distinct Temporal gene/miRNA Alterations Between the Dimorphic Seeds During Germination

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Transcriptome Assembly in Suaeda Aralocaspica to Reveal the Distinct Temporal gene/miRNA Alterations Between the Dimorphic Seeds During Germination

Lei Wang et al. BMC Genomics.

Abstract

Background: Dimorphic seeds from Suaeda aralocaspica exhibit different germination behaviors that are thought to be a bet-hedging strategy advantageous in harsh and unpredictable environments. To understand the molecular mechanisms of Suaeda aralocaspica dimorphic seed germination, we applied RNA sequencing and small RNA sequencing for samples collected at three germination stages.

Results: A total of 79,414 transcripts were assembled using Trinity, of which 57.67% were functionally annotated. KEGG enrichment unveiled that photosynthesis and flavonol biosynthesis pathways were activated earlier in brown seed compared with black seed. Gene expression analysis revealed that nine candidate unigenes in gibberellic acid and abscisic acid signal transduction and 23 unigenes in circadian rhythm-plant pathway showed distinct expression profiles to promote dimorphic seed germination. 194 conserved miRNAs comprising 40 families and 21 novel miRNAs belonging to 20 families in Suaeda aralocaspica were identified using miRDeep-P and Mfold. The expression of miRNAs in black seed was suppressed at imbibition stage. Among the identified miRNAs, 59 conserved and 13 novel miRNAs differentially expressed during seed germination. Of which, 43 conserved and nine novel miRNAs showed distinct expression patterns between black and brown seed. Using TAPIR, 208 unigenes were predicted as putative targets of 35 conserved miRNA families and 17 novel miRNA families. Among functionally annotated targets, genes participated in transcription regulation constituted the dominant category, followed by genes involved in signaling and stress response. Seven of the predicted targets were validated using 5' rapid amplification of cDNA ends or real-time quantitative reverse transcription-PCR.

Conclusions: Our results indicate that specific genes and miRNAs are regulated differently between black and brown seed during germination, which may contribute to the different germination behaviors of Suaeda aralocaspica dimorphic seeds in unpredictable variable environments. Our results lay a solid foundation for further studying the roles of candidate genes and miRNAs in Suaeda aralocaspica dimorphic seed germination.

Keywords: De novo assembly; Dimorphic seed; Euhalophyte; Germination; Non-deep dormant; Non-dormant; Suaeda aralocaspica; Transcriptome.

Conflict of interest statement

Ethics approval and consent to participate

Freshly matured fruits of Suaeda aralocaspica were collected from plants in a natural population (44o14’ N; 87o44’ E; 445 m a.s.l) growing at the Fukang Desert Ecosystem Observation and Experimental Station in Xinjiang Province, China in early October 2013. Fruits were dried naturally for ten days under ambient room conditions. After that, seeds were separated from the dried plant material and sorted into black and brown seeds. S. aralocaspica is a plant species that is not listed under Convention on International Trade in Endangered Species of Wild Fauna and Flora (https://cites.org/eng/app/appendices.php). No ethics approval is required for collection of S. aralocaspica samples or performing experiments on S. aralocaspica samples.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Figures

Fig. 1
Fig. 1
Comparative mRNA and miRNA transcriptome of Suaeda aralocaspica between black and brown seed during germination. a Venn diagram of the number of differentially expressed unigenes (left) and miRNAs (right) in black (green) and brown (yellow) seed germination. b The number of differentially expressed unigenes (upper) and miRNAs (lower) in black (left) and brown (right) seeds. Aquamarine color bars refer to up-regulated unigenes/miRNAs, salmon color bars refer to down-regulated unigenes/miRNAs. c Expression of the differentially expressed unigenes (left) and miRNAs (right) identified in Suaeda aralocaspica seed germination. BlDS represents black dry seed, BlIS represents black imbibed seed, BlS represents seedlings germinated from black seed, BrDS represents brown dry seed, BrIS represents brown imbibed seed, BrS represents seedlings germinated from brown seed
Fig. 2
Fig. 2
The significantly overrepresented KEGG pathways identified by enrichment analysis for differentially expressed unigenes. a Differentially expressed unigenes during Suaeda aralocaspica dimorphic seed germination. b Differentially expressed unigenes in the comparison between black and brown dry seed. X-axis represents the base 10 logarithm of the enrichment p-value, y-axis represents the term of enriched KEGG pathways. The number of differentially expressed unigenes in each pathway is indicated at the end of each bar. Up means up-regulated unigenes in black dry seed, Down means down-regulated unigenes in black dry seed
Fig. 3
Fig. 3
Distinct expression patterns of abscisic acid and gibberellic acid-related genes between black and brown seed. Red lines represent the transcript levels in black seed, green lines represent the transcript levels in brown seed. The transcript levels were determined by reads per kilobase of transcript per million mapped reads (RPKM). DS represents dry seed, IS represents imbibed seed, S represents seedlings of Suaeda aralocaspica. * means significant differences (p < 0.05) in IS vs. DS or S vs. IS, and # means significant differences (p < 0.05) in S vs. DS. The abbreviations of abscisic acid/gibberellic acid gene names were listed with parentheses
Fig. 4
Fig. 4
Heat maps of 23 clock genes in black (a) and brown (b) seed during germination. In the heatmaps, RPKM (reads per kilobase of transcript per million mapped reads) value of each gene was replaced by log2RPKM. Colors ranged from blue to red, corresponding to low to high expressions. BlDS represents black dry seed, BlIS represents black imbibed seed, BlS represents seedlings germinated from black seed, BrDS represents brown dry seed, BrIS represents brown imbibed seed, BrS represents seedlings germinated from brown seed. * means significant differences (p < 0.05) in IS vs. DS or S vs. IS and # means significant differences (p < 0.05) in S vs. DS. The abbreviations of clock gene names were listed with parentheses
Fig. 5
Fig. 5
Length distribution of small RNA sequences in dry seed, imbibed seed, and seedling three libraries. BlDS represents black dry seed, BlIS represents black imbibed seed, BlS represents seedlings germinated from black seed, BrDS represents brown dry seed, BrIS represents brown imbibed seed, BrS represents seedlings germinated from brown seed. nt means nucleotides
Fig. 6
Fig. 6
The number of members in each conserved miRNA family in Suaeda aralocaspica
Fig. 7
Fig. 7
Functional classifications of predicted targets of conserved (a) and novel (b) miRNAs in Suaeda aralocaspica. Only the functionally annotated target genes are shown. The number of targets in each category is shown under the iterm
Fig. 8
Fig. 8
Validation of miRNA-guided target unigene cleavage. Partial sequences from target genes were aligned with the corresponding miRNAs. Each top strand (blue) represents a miRNA-homologous site in the target gene and each bottom strand (red) represents the aligned sequence of miRNA. Red arrows indicate the observed miRNA cleavage sites following 5′ RACE analysis, with the frequency of clones shown

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