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Multi-lineages of Shiikuwasha (Citrus depressa Hayata) evaluated by using whole chloroplast genome sequences and its bio-diversity in Okinawa, Japan.

ABSTRACT: Shiikuwasha (Citrus depressa Hayata) is distributed from the South-west of the Japanese archipelago to Taiwan. In this study, re-sequencing against the orange (C. sinensis (L.) Osbeck) chloroplast genome was applied to one superior landrace of Shiikuwasha cultivated in Oku ward, Okinawa, Japan. The chloroplast genome of the landrace was estimated to comprise 160,118 bp, including 48 indels and 71 nucleotide substitutions against the reference genome. The presumptive chloroplast indels were confirmed by subsequent experiments, and these identified multiple maternal lineages among other landraces. Some of the orange SSR markers were available for genotyping of other superior landraces and were able to distinguish among them. These molecular markers were then applied for evaluation of genetic diversity among wild and cultivated Shiikuwasha accessions. Except for Oku ward, the cultivated populations were found to have lost their genetic diversity in comparison with wild populations. Groves in Oku ward maintained, or showed even higher genetic diversity than wild accessions in the surrounding areas by the force of villagers.

SUBMITTER: Ishikawa R

PROVIDER: S-EPMC5010300 | biostudies-literature | 2016 Sep

REPOSITORIES: biostudies-literature

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Multi-lineages of Shiikuwasha (Citrus depressa Hayata) evaluated by using whole chloroplast genome sequences and its bio-diversity in Okinawa, Japan.

Ishikawa Ryuji R Badenoch Nathan N Miyagi Kunimasa K Medoruma Kaname K Osada Toshiki T Onishi Masayuki M

Breeding science 20160709 4

Shiikuwasha (Citrus depressa Hayata) is distributed from the South-west of the Japanese archipelago to Taiwan. In this study, re-sequencing against the orange (C. sinensis (L.) Osbeck) chloroplast genome was applied to one superior landrace of Shiikuwasha cultivated in Oku ward, Okinawa, Japan. The chloroplast genome of the landrace was estimated to comprise 160,118 bp, including 48 indels and 71 nucleotide substitutions against the reference genome. The presumptive chloroplast ind ...[more]

PMID: 27795674

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Project description:BackgroundFlavonoids are secondary metabolites that play significant roles in plant cells. In particular, polymethoxy flavonoids (PMFs), including nobiletin, have been reported to exhibit various health-supporting properties such as anticancer, anti-inflammatory, and anti-pathogenic properties. However, it is difficult to utilize PMFs for medicinal and dietary use because plant cells contain small amounts of these compounds. Biosynthesis of PMFs in plant cells is carried out by the methylation of hydroxyl groups of flavonoids by O-methyltransferases (FOMT), and many kinds of FOMTs with different levels of substrate specificity and regioselectivity are cooperatively involved in this biosynthesis.ResultsIn this study, we isolated five genes encoding FOMT (CdFOMT1, 3, 4, 5, and 6) from Citrus depressa, which is known to accumulate nobiletin in the peels of its fruits. The genes encoded Mg(2+)-independent O-methyltransferases and showed high amino acid sequence similarity (60-95 %) with higher plant flavonoid O-methyltransferases. One of these genes is CdFOMT5, which was successfully expressed as a soluble homodimer enzyme in Escherichia coli. The molecular mass of the recombinant CdFOMT5 subunit was 42.0 kDa including a 6× histidine tag. The enzyme exhibited O-methyltransferase activity for quercetin, naringenin, (-)-epicatechin, and equol using S-adenosyl-L-methionine (SAM) as a methyl donor, and its optimal pH and temperature were pH 7.0 and 45 °C, respectively. The recombinant CdFOMT5 demonstrated methylation activity for the 3-, 5-, 6-, and 7-hydroxyl groups of flavones, and 3,3',5,7-tetra-O-methylated quercetin was synthesized from quercetin as a final product of the whole cell reaction system. Thus, CdFOMT5 is a O-methyltransferase possessing a broad range of substrate specificity and regioselectivity for flavonoids.ConclusionsFive FOMT genes were isolated from C. depressa, and their nucleotide sequences were determined. CdFOMT5 was successfully expressed in E. coli cells, and the enzymatic properties of the recombinant protein were characterized. Recombinant CdFOMT5 indicated O-methyltransferase activity for many flavonoids and a broad regioselectivity for quercetin as a substrate. Whole-cell biocatalysis using CdFOMT5 expressed in E. coli cells was performed using quercetin as a substrate, and 3,3',5,7-tetramethylated quercetin was obtained as the final product.

Dataset Information

Multi-lineages of Shiikuwasha (Citrus depressa Hayata) evaluated by using whole chloroplast genome sequences and its bio-diversity in Okinawa, Japan.

Publications

Multi-lineages of Shiikuwasha (<i>Citrus depressa</i> Hayata) evaluated by using whole chloroplast genome sequences and its bio-diversity in Okinawa, Japan.

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