Full-length transcriptome analysis of Phytolacca americana and its congener P. icosandra and gene expression normalization in three Phytolaccaceae species

Abstract

Background: Phytolaccaceae species in China are not only ornamental plants but also perennial herbs that are closely related to human health. However, both large-scale full-length cDNA sequencing and reference gene validation of Phytolaccaceae members are still lacking. Therefore, single-molecule real-time sequencing technology was employed to generate full-length transcriptome in invasive Phytolacca americana and non-invasive exotic P. icosandra. Based on the transcriptome data, RT-qPCR was employed to evaluate the gene expression stability in the two plant species and another indigenous congener P. acinosa.

Results: Total of 19.96 Gb and 19.75 Gb clean reads of P. americana and P. icosandra were generated, including 200,857 and 208,865 full length non-chimeric (FLNC) reads, respectively. Transcript clustering analysis of FLNC reads identified 89,082 and 98,448 consensus isoforms, including 86,989 and 96,764 high-quality ones. After removing redundant reads, 46,369 and 50,220 transcripts were obtained. Based on structure analysis, total 1675 and 1908 alternative splicing variants, 25,641 and 31,800 simple sequence repeats (SSR) as well as 34,971 and 36,841 complete coding sequences were detected separately. Furthermore, 3574 and 3833 lncRNA were predicted and 41,676 and 45,050 transcripts were annotated respectively. Subsequently, seven reference genes in the two plant species and a native species P. acinosa were selected and evaluated by RT-qPCR for gene expression analysis. When tested in different tissues (leaves, stems, roots and flowers), 18S rRNA showed the highest stability in P. americana, whether infested by Spodoptera litura or not. EF2 had the most stable expression in P. icosandra, while EF1-α was the most appropriate one when attacked by S. litura. EF1-α showed the highest stability in P.acinosa, whereas GAPDH was recommended when infested by S. litura. Moreover, EF1-α was the most stable one among the three plant species whenever germinating seeds or flowers only were considered.

Conclusion: Full-length transcriptome of P. americana and P. icosandra were produced individually. Based on the transcriptome data, the expression stability of seven candidate reference genes under different experimental conditions was evaluated. These results would facilitate further exploration of functional and comparative genomic studies in Phytolaccaceae and provide insights into invasion success of P. americana.

Keywords: Full-length transcriptome analysis; Phytolaccaceae; RT-qPCR; Reference gene evaluation; SMRT sequencing.

Fig. 1

Homologous species distribution of P. americana and P. icosandra annotated based on the Nr database. a, P. americana; b, P. icosandra

Fig. 2

Classification of the transcripts annotated by the Gene Ontology (GO)

Fig. 3

Venn diagram of the number of lncRNAs predicted by CPC, CNCI, CPAT and Pfam. a, P. americana; b, P. icosandra

Fig. 4

Classification of predicted transcription factors

Fig. 5

RNA transcription levels of seven candidate reference genes in P. americana, P.icosandra and P. acinosa. The expression level of candidate reference genes in total samples (n = 99) was presented as cycle threshold number (Ct-value) and explained by box and whisker plots. The asterisks represented the minimum and maximum Ct value. The squares indicated the 25th and 75th percentiles, and the median was represented by a bar across the square

Fig. 6

Pairwise variation analyzed by geNorm to determine the optimal number of reference genes for accurate normalization. A threshold value of 0.15 was suggested for valid normalization. If the value of V_n/n + 1 (pairwise variation) is less than 0.15, then n reference genes in combination are recommended for gene normalization. If the value of V_n/n + 1 is more than 0.15, then V_{n + 1/n + 2} should be taken into account. Pam: P. americana; Pic: P. icosandra; Pac: P. acinosa; LSRF: different tissues of leaves, stems, roots and flowers; GS: germinating seeds of these three plant species; F: flowers of these three plant species; LSR: different tissues of leaves, stems and roots; I: infested by S. litura of third instar