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Pitaya (Hylocereus) is the most economically important fleshy-fruited tree of the Cactaceae family that is grown worldwide, and it has attracted significant attention because of its betalain-abundant fruits. Nonetheless, the lack of a pitaya reference genome significantly hinders studies focused on its evolution, as well as the potential for genetic improvement of this crop. Herein, we employed various sequencing approaches, namely, PacBio-SMRT, Illumina HiSeq paired-end, 10× Genomics, and Hi-C (high-throughput chromosome conformation capture) to provide a chromosome-level genomic assembly of ‘GHB’ pitaya (H. undatus, 2n = 2x = 22 chromosomes). The size of the assembled pitaya genome was 1.41 Gb, with a scaffold N50 of ~127.15 Mb. In total, 27,753 protein-coding genes and 896.31 Mb of repetitive sequences in the H. undatus genome were annotated. Pitaya has undergone a WGT (whole-genome triplication), and a recent WGD (whole-genome duplication) occurred after the gamma event, which is common to the other species in Cactaceae. A total of 29,328 intact LTR-RTs (~696.45 Mb) were obtained in H. undatus, of which two significantly expanded lineages, Ty1/copia and Ty3/gypsy, were the main drivers of the expanded genome. A high-density genetic map of F1 hybrid populations of ‘GHB’ × ‘Dahong’ pitayas (H. monacanthus) and their parents were constructed, and a total of 20,872 bin markers were identified (56,380 SNPs) for 11 linkage groups. More importantly, through transcriptomic and WGCNA (weighted gene coexpression network analysis), a global view of the gene regulatory network, including structural genes and the transcription factors involved in pitaya fruit betalain biosynthesis, was presented. Our data present a valuable resource for facilitating molecular breeding programs of pitaya and shed novel light on its genomic evolution, as well as the modulation of betalain biosynthesis in edible fruits.

Background A suitable reference gene is an important prerequisite for guarantying accurate and reliable results in quantitative real-time PCR (qRT-PCR) analyses. However, there is no absolute universality in reference genes among different species. It’s hard to find an ideal reference gene to fit for different tissues and growth periods. Pitaya ( Hylocereus ) is commercially produced as a new fruit crop at a large scale in tropical and subtropical regions. To date, there is no report on the identification of the most reliable reference genes for qRT-PCR normalization in pitaya. Results In this study, six candidate reference genes i.e. Actin(1) , GAPDH , UBC(1) , UBC(2) EF1 - α(1) and histone(1) were selected from thirty-nine typical candidate reference genes to determine the most stable reference genes for qRT-PCR normalization in different tissues, temperature stresses and fruit developmental stages of pitaya. Among the six candidate reference genes, Actin(1) and EF1 - α(1) were the most stable gene according to calculations of three statistical methods (GeNorm, NormFinder and BestKeeper) while UBC(1) and UBC(2) showed the lowest expression stability. The six candidate reference genes were further validated by comparing expression profiles of key genes related to betalain biosynthesis at flesh coloration stages of Guanhuahong ( Hylocereus monacanthus ) and Guanhuabai ( H. undatus ) pitayas. Actin(1) was recommended the best reference gene for accurate normalization of qRT-PCR data. Conclusions In this study, the stability of the selected reference genes for normalizing the qRT-PCR data were identified from pitaya. Actin(1) was the most stably expressed genes in different tissues and fruit developmental stages in pitaya. The present work provides the first data of reference gene identification for pitaya and will facilitate further studies in molecular biology and gene function on Hylocereus and other closely related species. Electronic supplementary material The online version of this article (10.1186/s13007-019-0455-3) contains supplementary material, which is available to authorized users.

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Red dragon fruit or red pitaya (Hylocereus polyrhizus) is the only edible fruit that contains betalains. The color of betalains ranges from red and violet to yellow in plants. Betalains may also serve as an important component of health-promoting and disease-preventing functional food. Currently, the biosynthetic and regulatory pathways for betalain production remain to be fully deciphered. In this study, isobaric tags for relative and absolute quantitation (iTRAQ)-based proteomic analyses were used to reveal the molecular mechanism of betalain biosynthesis in H. polyrhizus fruits at white and red pulp stages, respectively. A total of 1946 proteins were identified as the differentially expressed between the two samples, and 936 of them were significantly highly expressed at the red pulp stage of H. polyrhizus. RNA-seq and iTRAQ analyses showed that some transcripts and proteins were positively correlated; they belonged to “phenylpropanoid biosynthesis”, “tyrosine metabolism”, “flavonoid biosynthesis”, “ascorbate and aldarate metabolism”, “betalains biosynthesis” and “anthocyanin biosynthesis”. In betalains biosynthesis pathway, several proteins/enzymes such as polyphenol oxidase, CYP76AD3 and 4,5-dihydroxy-phenylalanine (DOPA) dioxygenase extradiol-like protein were identified. The present study provides a new insight into the molecular mechanism of the betalain biosynthesis at the posttranscriptional level.

Background MicroRNAs (miRNAs) and their regulatory functions in anthocyanin, carotenoid, and chlorophyll accumulation have been extensively characterized in many plant species. However, the miRNA regulatory mechanism in betalain biosynthesis remains mostly unknown. Results In this study, 126 conserved miRNAs and 41 novel miRNAs were first isolated from Hylocereus monacanthus, among which 95 conserved miRNAs belonged to 53 miRNA families. Thirty-four candidate miRNAs related to betalain biosynthesis were differentially expressed. The expression patterns of those differential expressed miRNAs were analyzed in various pitaya tissues by RT-qPCR. A significantly negative correlation was detected between the expression levels of half those miRNAs and corresponding target genes. Target genes of miRNAs i.e. Hmo-miR157b-HmSPL6-like, Hmo-miR160a-Hpcyt P450-like3, Hmo-miR6020-HmCYP71A8-like, Hmo-novel-2-HmCYP83B1-like, Hmo-novel-15-HmTPST-like, Hmo-miR828a-HmTT2-like, Hmo-miR858-HmMYB12-like, Hmo-miR858-HmMYBC1-like and Hmo-miR858-HmMYB2-like were verified by 5′RACE and transient expression system in tobacco. Conclusions Hmo-miR157b, Hmo-miR160a, Hmo-miR6020 Hmo-novel-2, Hmo-novel-15, Hmo-miR828a and Hmo-miR858 play important roles in pitaya fruit coloration and betalain accumulation. Our findings provide new insights into the roles of miRNAs and their target genes of regulatory functions involved in betalain biosynthesis of pitaya.

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