Whole-genome sequencing and analysis of two azaleas, Rhododendron ripense and Rhododendron kiyosumense.

To enhance the genomics and genetics of azalea, the whole-genome sequences of two species of Rhododendron were determined and analysed in this study: Rhododendron ripense, the cytoplasmic donor and ancestral species of large-flowered and evergreen azalea cultivars; and Rhododendron kiyosumense, a native of Chiba prefecture (Japan) seldomly bred and cultivated. A chromosome-level genome sequence assembly of R. ripense was constructed by single-molecule real-time sequencing and genetic mapping, while the genome sequence of R. kiyosumense was assembled using the single-tube long fragment read sequencing technology. The R. ripense genome assembly contained 319 contigs (506.7 Mb; N50 length: 2.5 Mb) and was assigned to the genetic map to establish 13 pseudomolecule sequences. On the other hand, the genome of R. kiyosumense was assembled into 32,308 contigs (601.9 Mb; N50 length: 245.7 kb). A total of 34,606 genes were predicted in the R. ripense genome, while 35,785 flower and 48,041 leaf transcript isoforms were identified in R. kiyosumense through Iso-Seq analysis. Overall, the genome sequence information generated in this study enhances our understanding of genome evolution in the Ericales and reveals the phylogenetic relationship of closely related species. This information will also facilitate the development of phenotypically attractive azalea cultivars.

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-sho" 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 'Nikko-misome,' R. macrosepalum 'Kocho-zoroi,' R. indicum 'Chojyu-ho,' and R. × hannoense 'Amagi-beni-chojyu.' 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.

Involvement of negative feedback regulation in wound-induced ethylene synthesis in 'Saijo' persimmon.

Wounding is one of the most effective stress signals to induce ethylene synthesis in persimmon (Diospyros kaki Thunb.). We found that wound-induced ethylene biosynthesis is subjected to negative feedback regulation in mature 'Saijo' persimmon fruit since ethylene production was enhanced by 1-methylcyclopropene (1-MCP) (an inhibitor of ethylene perception) pretreatment, which was approximately 1.8 fold of that in control tissues (without 1-MCP pretreatment). Wound-induced 1-aminocyclopropane-1-carboxylate (ACC) synthase activity and DK-ACS2 gene expression were substantially increased by 1-MCP pretreatment after 12 h, which resulted in much higher ACC content in 1-MCP pretreated tissues than that in a control after 24 h. These results indicated that wound-induced DK-ACS2 gene expression was negatively regulated by ethylene in mature persimmon fruit. However, 1-MCP pretreatment had no effect on DK-ACO1 gene expression, suggesting the independence of wound-induced DK-ACO1 on ethylene. Out of accord with DK-ACO1 gene expression, ACC oxidase activity was enhanced 48 h after wounding in 1-MCP pretreated tissues, reaching a peak 1.5-fold higher than that in control tissues at 60 h.

Ty1-copia group retrotransposons in persimmon (Diospyros kaki Thunb.).

We cloned and characterized Ty1-copia group retrotransposons in persimmon (Diospyros kaki Thunb.). Genomic DNA or methyl jasmonate (MJA)-treated cDNA were used as templates to amplify the reverse transcriptase region of Ty1-copia group retrotransposons. About 280 bp fragments were amplified and cloned, and 97 clones were sequenced. Forty-nine clones included frameshift or the stop codon, or both. Among 48 clones containing complete reading frames, 42 clones had unique nucleotide sequences. Alignment and phylogenetic analysis of putative amino acid sequences in the 42 clones indicated that these clones (named Tdk; retroTransposon in Diospyros kaki) fell into seven subgroups and six ungrouped sequences, indicating high sequence heterogeneity in Tdk clones. Phylogenetic analysis comparing unrelated plant species shows that some Tdk clones are more closely related to Ty1-copia group retrotransposons in the orders Solanales and Sapindales rather than to other Tdk clones. Southern blot analysis using Tdk2B, Tdk4c, Tdk6Ac, Tdk12K and Tdk13G clones as probes showed that persimmon and its related species, D. lotus, D. lotus var. glabba, D. oleifera, D. rhombifolia and D. virginiana, contained multiple Tdk-like sequences, indicating that homologous elements exist in other Diospyros species.