Betalains not only play important roles in ranging in color from red to violet to yellow in plants,but also have high nutritional value and positive effects in health and disease by high antioxidant and anti-inflammatory capabilities.However,except for the cloning of single gene in the betalain pathway in model plant,the biosynthetic pathway of betalains remains to be fully clarified.Red pulp pitaya(H.polyrhizus)is the only fruit containing abundant betalains for consumer.Currently,no information is available about genes involved in betalain biosynthesis in H.polyrhizus.In the present research,two cDNA libraries of pitaya pulps with two different coloration stages(white and red pulp stages)of Guanhuahong(H.polyrhizus)was constructed.A total of about 12 Gb raw RNA-Seq data was generated and was de novo assembled into 122,677 transcripts with an average length of 1,183bp and an N50 value of 2,008.Approximately 99.99%of all transcripts were annotated based on seven public databases.A total of 8,871 transcripts were significantly regulated.Thirty-three candidate transcripts related to betalain biosynthesis were obtained from the transcriptome data.Transcripts encoding enzymes involved in betalain biosynthesis were analyzed using RT-qPCR at the whole pulp coloration stages of H.polyrhizus.Nine key transcripts of betalain biosynthesis were identified.They were assigned to four kinds of genes in betalain biosynthetic pathway,including tyrosinase,4,5-DOPA dioxygenase extradiol,cytochrome P450 and glucosyltransferase.Based on the expression analyses and published documents,we proposed a preliminary pathway of betalain biosynthesis in pitaya.Further work such as enzyme activity analyses and genetic transformation are being carried out to elucidate their roles in betalain biosynthesis of pitaya.