Akira Nakatsuka

Spatial and temporal expression of chalcone synthase and dihydroflavonol 4-reductase genes in the Asiatic hybrid lily

Abstract cDNA clones of three chalcone synthase (CHS) genes (LhCHSA, LhCHSB and LhCHSC) and one dihydroflavonol 4-reductase (DFR) gene (LhDFR) were isolated, and spatial and temporal expression of these genes was analyzed for two Asiatic hybrid lily cultivars, ‘Montreux’ (pink tepals with spots) and ‘Connecticut King’ (yellow tepals without spots). Pink tepals and tepal spots contain the same kind of anthocyanin, and yellow tepals have no anthocyanin. In both cultivars, the expression of all four genes was not detected in unpigmented leaves, stems and white bulbscales, but was detected in anthocyanin pigmentation-induced bulbscales, indicating that these genes are transcriptionally active in both cultivars. In flower organs, three CHS genes were expressed in anthocyanin-pigmented tepals, filaments and pistils of ‘Montreux’, and in unpigmented filaments and pistils of ‘Connecticut King’. Pigmented anthers accumulated only LhCHSC mRNA in both cultivars. LhDFR was abundantly expressed in pigmented tepals, anthers, filaments and pistils of ‘Montreux’, and in pigmented anthers of ‘Connecticut King’. These results indicate that different expression of these genes, especially of the LhDFR gene, controls the distinct pigmentation pattern in flower organs in both cultivars. In three F1 plants with many spots on yellow or white tepals, derived from a cross between ‘Montreux’ and ‘Connecticut King’, anthocyanin accumulation in tepal spots was accompanied by the LhCHSA and LhDFR expression, indicating that the expression of CHS and DFR genes is independently controlled between tepals and tepal spots.

PCR-based molecular markers in Asiatic hybrid lily

PCR-based DNA markers in Asiatic hybrid lily were examined. Random primers with various lengths (10-, 12-, 15- and 20-base) were tested in randomly amplified polymorphic DNA (RAPD) reactions with total DNA from two cultivars, ‘Montreux’ and ‘Connecticut King’. Thirty-three out of 61 (54%) 15-base primers and 14 out of 21 (67%) 20-base primers generated polymorphic fragments whereas 17 out of 145 (12%) and 4 out of 24 (17%) of the 10- and 12-base primers, respectively, amplified them. The efficiency of the RAPD reaction increased with increasing primer length. We examined inter-simple sequence repeat (ISSR) reactions using 3 0 -anchored simple sequence repeat (ASSR) primers, and found 33 out of 63 (52%) primers that amplified polymorphic bands between the two cultivars. We repeated all RAPD and ISSR reactions at least two times and obtained the same DNA fingerprints, indicating high reproducibility obtained with these reactions. All polymorphic fragments were inherited into their F1 hybrids, indicating that these DNA markers are genetically stable. # 2002 Elsevier Science B.V. All rights reserved.

Expressed sequence tags from persimmon at different developmental stages

Persimmon (Diospyros kaki Thunb.) is an important fruit in Asian countries, where it is eaten as a fresh fruit and is also used for many other purposes. To understand the molecular mechanism of fruit development and ripening in persimmon, we generated a total of 9,952 expressed sequence tags (ESTs) from randomly selected clones of two different cDNA libraries. One cDNA library was derived from fruit of “Saijo” persimmon at an early stage of development, and the other from ripening fruit. These ESTs were clustered into 6,700 non-redundant sequences. Of the 6,700 non-redundant sequences evaluated, the deduced amino acid sequences of 4,356 (65%) showed significant homology to known proteins, and 2,344 (35%) showed no significant similarity to any known proteins in Arabidopsis databases. We report comparison of genes identified in the two cDNA libraries and describe some putative genes involved in proanthocyanidin and carotenoid synthesis. This study provides the first global overview of a set of genes that are expressed during fruit development and ripening in persimmon.

Inheritance of the narrow leaf mutation in traditional Japanese evergreen azaleas

Morphological mutations have been admired and preserved in the classical cultivars of the Japanese evergreen azalea since the Edo era (1603–1867). One such mutation is the presence of narrow leaves and petals. In this study, we investigated the characteristics and inheritance patterns of the narrow leaf-type mutational form of Rhododendron macrosepalum ‘Seigaiha’, R. kaempferi ‘Kin-kujyaku’, and R. kiusianum Yanagi-ba mutant. The leaves of all cultivars showed weak development in the transverse plane and thickened margins. Subsequently, based on this qualitative character, the inheritance of the narrow leaf mutation was investigated. F1 seedlings of cross combinations between normal and narrow leaf-type plants had normal leaf-type. All F1 progenies of cross combinations between narrow leaf-type plants had narrow leaves like their parents. In addition, seedlings obtained from backcross and sib-cross tests using narrow leaf mutants segregated into normal and narrow leaf-types at a 1:1 and 3:1 ratio, respectively. These results indicate that the narrow leaf trait of three different azalea cultivars, ‘Seigaiha’, ‘Kin-kujyaku’, and R. kiusianum Yanagi-ba mutant, is probably a recessive mutation caused by a single, common gene.

Chimeric inheritance of organelle DNA in variegated leaf seedlings from inter-subgeneric crossing of azalea

The inheritance of organelle DNA was investigated using PCR–RFLP markers in reciprocal cross combinations of inter-subgeneric azalea hybrids between evergreen azaleas (Rhododendron nakaharai and its hybrids) and fragrant deciduous azaleas (R. arborescens and R. viscosum) for the purpose of breeding fragrant evergreen azaleas. The hybrid progenies included green leaf, pale green leaf, variegated leaf and albino seedlings. Most viable green leaf seedlings had inherited ptDNA from the deciduous parent and non-viable albino and pale green leaf seedlings had inherited ptDNA from the evergreen parent. On the other hand, variegated leaf seedlings had chimerically inherited ptDNA from both parents. Their green leaf segments had ptDNA from the deciduous parent, and the pale green and white segments had biparental or maternal ptDNA depending on the progeny. In this study, we obtained interesting inter-subgeneric azalea hybrid progenies that had chimerically inherited organelle DNA and had different colored leaf segments corresponding to the composition of ptDNA from each parent. These results suggest that variegated leaf progenies with chimeric ptDNA from both parents can be subsistent, whereas albino seedlings resulting from plastome–genome incompatibility between the plastid genome from evergreen azalea and the nuclear genome from deciduous azalea are non-viable.

Long-lasting Corolla Cultivars in Japanese Azaleas: A Mutant AP3/DEF Homolog Identified in Traditional Azalea Cultivars from More Than 300 Years Ago

Floral shape in higher plants typically requires genetic regulation through MADS transcription factors. In Japan, hundreds of azalea cultivars including flower shape mutations have been selected from the diversity of endogenous species and natural hybrids since the early 17th century, the Edo era (1603–1867). The long-lasting trait, known as “Misome-shō” in Japanese, has been identified in several species and cultivar groups of evergreen azaleas (Rhododendron L.) from three hundred years ago in Japan. However, the natural mutation conferring the long-lasting trait in azalea remains unknown. Here, we showed MADS-box gene mutations in long-lasting flowers, R. kaempferi ‘Nikkō-misome,’ R. macrosepalum ‘Kochō-zoroi,’ R. indicum ‘Chōjyu-hō,’ and R. × hannoense ‘Amagi-beni-chōjyu.’ All of the long-lasting flowers exhibited small-sized corollas with stomata during long blooming. In the long-lasting flowers, transcript of the APETALA3 (AP3)/DEFICIENS (DEF) homolog was reduced, and an LTR-retrotransposon was independently inserted into exons 1, 2, and 7 or an unknown sequence in exon 1 in gDNA of each cultivar. This insertion apparently abolished the normal mRNA sequence of the AP3/DEF homolog in long-lasting flowers. Also, long-lasting flowers were shown from F2 hybrids that had homozygous ap3/def alleles. Therefore, we concluded that the loss of function of the AP3/DEF homolog through a transposable element insertion may confer a stable long-lasting mutation in evergreen azaleas.