Cloning of the Papaya Chromoplast-Specific Lycopene β-Cyclase, CpCYC-b, Controlling Fruit Flesh Color Reveals Conserved Microsynteny and a Recombination Hot Spot

Abstract

Carotenoid pigments in fruits are indicative of the ripening process and potential nutritional value. Papaya (Carica papaya) fruit flesh color is caused by the accumulation of lycopene or b-carotenoids in chromoplasts. It is a distinct feature affecting nutritional composition, fruit quality, shelf life, and consumer preference. To uncover the molecular basis of papaya flesh color, we took map-based cloning and candidate gene approaches using integrated genetic and physical maps. A DNA marker tightly linked to flesh color colocalized on a contig of the physical map with a cDNA probe of the tomato (Solanum lycopersicum) chromoplast-specific lycopene b-cyclase, CYC-b. Candidate gene sequences were obtained from amplified fragments and verified by sequencing two bacterial artificial chromosomes containing the two alleles. Sequence comparison revealed a 2-bp insertion in the coding region of the recessive red flesh allele resulting in a frame-shift mutation and a premature stop codon. A color complementation test in bacteria confirmed that the papaya CpCYC-b is the gene controlling fruit flesh color. Sequence analysis of wild and cultivated papaya accessions showed the presence of this frame-shift mutation in all red flesh accessions examined. Evaluation of DNA markers near CpCYC-b revealed a recombination hot spot, showing that CpCYC-b is located in a gene-rich region with a recombination rate at 3.7 kb per centimorgan, more than 100-fold higher than the genome average at 400 kb per centimorgan. Conserved microsynteny of the CpCYC-b region is indicated by colinearity of two to four genes between papaya, Arabidopsis (Arabidopsis thaliana), grape (Vitis vinifera), and tomato. Our results enhanced our understanding of papaya flesh color inheritance and generated new tools for papaya improvement.

Chromoplast Differentiation: Current Status and Perspectives

“…As expected, genes involved in carotenoid biosynthesis, such as lycopene β -cyclase ( CYCB ), are up-regulated during chromoplast formation in many plants, including citrus fruit (Alquezar et al 2009); the wild species of tomato, Solanum habrochaites ( Dalal et al 2010 ); safron ( Ahrazem et al 2010 ); papaya fruit ( Blas et al 2010 ); and carrot ( Chen et al 2001 ). On the contrary, genes involved in photosynthetic activity are generally down-regulated during chromoplast formation ( Cheung et al 1993 ).…”

Papaya Nutritional Analysis

“…A backcross program linked with microsatellite marker selection was demonstrated for introgression of desirable genes into an elite recurrent parent (Ramos et al 2011 ). A comprehensive high-density genetic linkage map for papaya is available and useful for mapping quantitative trait loci and for marker-assisted selection (Blas et al 2010 ). In addition, transgenic techniques have been successful, as proven with the "Rainbow" papaya, genetically modifi ed for resistance to papaya ringspot virus (PRSV).…”

“…Differentially expressed gene transcripts associated with fruit ripening have been described, including several related to vitamin C, biotin and folate biosynthesis, and precursors for carotenoids (Fabi et al 2010 ). Genes involved in papaya carotenoid biosynthesis have been identifi ed and characterized (Skelton et al 2006 ;Blas et al 2010 ;Devitt et al 2010 ;Yan et al 2011 ). Papaya fl esh color is controlled by a single genetic locus, with yellow dominant over red (Devitt et al 2010 ).…”

A wild origin of the loss‐of‐function lycopene beta cyclase (CYC‐b) allele in cultivated, red‐fleshed papaya (Carica papaya)

“…Th e causative mutation for red fruit color is a recessive, loss-of-function mutation caused by a "TT" frameshift insertion in the coding region of the CYC-b gene for chromoplast-specifi c lycopene beta cyclase, that catalyzes the conversion of lycopene to beta-carotene ( Blas et al, 2010 ;Devitt et al, 2010 ). It is the accumulation of lycopene that gives red-fl eshed papaya, as well as other red-fl eshed fruit, such as tomato, their color ( Giuliano, 2014 ).…”

“…It is the accumulation of lycopene that gives red-fl eshed papaya, as well as other red-fl eshed fruit, such as tomato, their color ( Giuliano, 2014 ). Red fruit color is a likely target for cultivar improvement since some consumers prefer red-fl eshed papaya and call them "strawberry papaya" ( Blas et al, 2010 ).…”