Development of molecular markers from the GDPS promoter sequence for floral terpenoid scent in Phalaenopsis orchids

Abstract

Phalaenopsis orchids are important crops with high economic value worldwide However breeding of scented Phalaenopsis orchids are with difficulties and thus the breeders usually have little successful targets on breeding orchids with floral scent For time saving molecular markers detecting the scent production is needed Phalaenopsis bellina has sweet floral fragrance consisted of monoterpenes and it usually is a seed or pod parent for breeding scent Phalaenopsis orchids Previously P bellina geranyl diphosphate synthase (PbGDPS) is identified as the key enzyme for monoterpenes biosynthesis In addition the GDPS promoter from 10 native Phalaenopsis species were isolated and analyzed They were very similar except the presence or absence of a tandem repeat sequences In previous study in scented Phalaenopsis species the tandem repeat sequences were complete and the GDPS was expressed However in scentless species the incomplete tandem repeat sequences were detected and without GDPS expression In this study for developing the molecular marker from the tandem repeat sequences of GDPS promoter to detect the scent of monoterpene production GDPS promoters of 14 native species Phalaenopsis spp were amplified and analyzed The PCR-amplified tandem repeat fragments in the promoter region showed 2 fragments of 400 bp and 300 bp Sequence analysis showed that they were very similar except the presence or absence of two complete sets of tandem repeats The 400-bp fragment contains the complete tandem repeats of repeat 1 and repeat 2 and each repeat can be subdivided as ABCD and A’B’C’D’ regions The 300-bp fragment has a deletion of the repeat 1 and contains only the A’B’C’D’ regions However in some native species the deletion of tandem repeat sequences did not affect the expression of GDPS Furthermore a population with 25 F1 progenies and their putative parents were analyzed for the development of molecular markers for assessing floral scent Intriguingly the single nucleotide polymorphisms (SNPs) occurred in the D region of tandem repeat in GDPS promoter might cause low production of monoterpene However no mutations were detected in the A’ region of the 400-bp fragment and the D’ region of the 300-bp fragment for all 14 native species regardless with or without scent Most SNPs and large deletion existed in the repeat 1 rather than repeat 2 These results suggest that the repeat 2 is more conserved than the repeat 1 and may play an important role in orchid evolution for scent traits In addition the tandem repeat sequences of GDPS promoter may have potential to develop as molecular markers and apply for the early detection of the floral scent and facilitates scent orchid breeding in the future However the commercial cultivars which contain the parenthood of P schilleriana P aphrodite subsp formosana and P amboinensis var common were not suitable because their results were not so consistent with those in this study

Date of Award	2016 Jan 22
Original language	English
Supervisor	Hong-Hwa Chen (Supervisor)