Comparative Transcriptome Analysis Combining SMRT- and Illumina-Based RNA-Seq Identifies Potential Candidate Genes Involved in Betalain Biosynthesis in Pitaya Fruit

Abstract

To gain more valuable genomic information about betalain biosynthesis, the full-length transcriptome of pitaya pulp from ‘Zihonglong’ (red pulp) and ‘Jinghonglong’ (white pulp) in four fruit developmental stages was analyzed using Single-Molecule Real-Time (SMRT) sequencing corrected by Illumina RNA-sequence (Illumina RNA-Seq). A total of 65,317 and 91,638 genes were identified in ‘Zihonglong’ and ‘Jinghonglong’, respectively. A total of 11,377 and 15,551 genes with more than two isoforms were investigated from ‘Zihonglong’ and ‘Jinghonglong’, respectively. In total, 156,955 genes were acquired after elimination of redundancy, of which, 120,604 genes (79.63%) were annotated, and 30,875 (20.37%) sequences without hits to reference database were probably novel genes in pitaya. A total of 31,169 and 53,024 simple sequence repeats (SSRs) were uncovered from the genes of ‘Zihonglong’ and ‘Jinghonglong’, and 11,650 long non-coding RNAs (lncRNAs) in ‘Zihonglong’ and 11,113 lncRNAs in ‘Jinghonglong’ were obtained herein. qRT-PCR was conducted on ten candidate genes, the expression level of six novel genes were consistent with the Fragments Per Kilobase of transcript per Million mapped reads (FPKM) values. In conclusion, we firstly undertook SMRT sequencing of the full-length transcriptome of pitaya, and the valuable resource that was acquired through this sequencing facilitated the identification of additional betalain-related genes. Notably, a list of novel putative genes related to the synthesis of betalain in pitaya fruits was assembled. This may provide new insights into betalain synthesis in pitaya.

Elucidation of the core betalain biosynthesis pathway in Amaranthus tricolor

“…In recent years, with the elucidation of the central committed steps of the betalain biosynthesis pathway, comparative transcriptome analyses have been intensively applied to identify genes involved in regulating betalain biosynthesis in Caryophyllales 16 , 20 , 30 , 36 , 37 . However, numerous duplication events have led to difficulty in elucidating the functional activities of key enzymes in betalain-pigmented species through annotation 10 .…”

“…Recently, betalain biosynthesis in different pitaya species, such as Hylocereus polyrhizus , Hylocereus costaricensis , Hylocereus undatus , and Hylocereus megalanthus , has been intensively studied through comparative transcriptome analysis 36 , 37 , 41 , 42 . However, further studies remain to be conducted to provide experimental evidence and strengthen the understanding of the roles of candidate genes in betalain biosynthesis.…”

Full-Length Transcriptome Analyses of Genes Involved in Triterpenoid Saponin Biosynthesis of Psammosilene tunicoides Hairy Root Cultures With Exogenous Salicylic Acid

“…RNA-seq has been performed to identify the putative genes involved in terpenoid biosynthesis in multiple plants ( Luo et al, 2011 ; Ma et al, 2016 ; Wang et al, 2017 ; Zhou et al, 2017 ; Choudhri et al, 2018 ; Shan et al, 2020 ). Nevertheless, the short read lengths from RNA-seq restrict the construction of completely assembled transcripts, making it difficult to obtain full-length sequences ( Wu et al, 2019 , 2020 ). As a third-generation sequencing technique, single-molecule real-time (SMRT) sequencing can generate full-length cDNA sequences without post-sequencing assembly, which basically overcomes the limitation of RNA-seq ( Wu et al, 2019 , 2020 ).…”

Elucidation of the Core Betalain Biosynthesis Pathway in Amaranthus tricolor

“…In recent years, with the elucidation of the central committed steps of the betalain biosynthesis pathway, comparative transcriptome analyses have been intensively applied to identify genes involved in regulating betalain biosynthesis in Caryophyllales 16,20,30,36,37 . However, numerous duplication events have led to di culty in elucidating the functional activities of key enzymes in betalain-pigmented species through annotation 10 .…”