Hachimantai City Floricultural Research and Development Center

Hachimantai, Japan

Hachimantai City Floricultural Research and Development Center

Hachimantai, Japan
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Tasaki K.,Iwate Biotechnology Research Center | Higuchi A.,Iwate Biotechnology Research Center | Fujita K.,Iwate Biotechnology Research Center | Watanabe A.,Iwate Biotechnology Research Center | And 7 more authors.
Molecular Breeding | Year: 2017

Double flowers are valuable floral traits in most floricultural plants. We recently revealed that a double-flowered mutant of Gentiana scabra was caused by an insertion of a retrotransposable element (Tgs1) into GsAG1, one of the C-class MADS-box genes in gentian. In this study, we developed a PCR-based molecular DNA marker to distinguish double- and single-flower phenotypes at the young seedling stage in Japanese gentian plants. To test the validity of the markers, 17 F2 populations were produced by selfing F1 plants crossed between the double-flower mutant and seven breeding lines. Multiplex PCR demonstrated that the Tgs1 insertion in GsAG1 cosegregated with the double-flower phenotype in two F2 populations, indicating that the PCR-based DNA marker was useful to discriminate between double- and single-flower phenotypes in advance of flowering in Japanese gentian. Given that Japanese gentians lack variation in flower shape and require a long breeding period, the DNA marker developed here will be helpful for efficient breeding of double-flowered cultivars in the future. © 2017, Springer Science+Business Media Dordrecht.

Nakatsuka T.,Iwate Biotechnology Research Center | Yamada E.,Iwate Biotechnology Research Center | Saito M.,Iwate Biotechnology Research Center | Hikage T.,Hachimantai City Floricultural Research and Development Center | And 2 more authors.
BMC Genomics | Year: 2012

Background: Japanese gentians (Gentiana triflora and Gentiana scabra) are amongst the most popular floricultural plants in Japan. However, genomic resources for Japanese gentians have not yet been developed, mainly because of the heterozygous genome structure conserved by outcrossing, the long juvenile period, and limited knowledge about the inheritance of important traits. In this study, we developed a genetic linkage map to improve breeding programs of Japanese gentians.Results: Enriched simple sequence repeat (SSR) libraries from a G. triflora double haploid line yielded almost 20,000 clones using 454 pyrosequencing technology, 6.7% of which could be used to design SSR markers. To increase the number of molecular markers, we identified three putative long terminal repeat (LTR) sequences using the recently developed inter-primer binding site (iPBS) method. We also developed retrotransposon microsatellite amplified polymorphism (REMAP) markers combining retrotransposon and inter-simple sequence repeat (ISSR) markers. In addition to SSR and REMAP markers, modified amplified fragment length polymorphism (AFLP) and random amplification polymorphic DNA (RAPD) markers were developed. Using 93 BC1 progeny from G. scabra backcrossed with a G. triflora double haploid line, 19 linkage groups were constructed with a total of 263 markers (97 SSR, 97 AFLP, 39 RAPD, and 30 REMAP markers). One phenotypic trait (stem color) and 10 functional markers related to genes controlling flower color, flowering time and cold tolerance were assigned to the linkage map, confirming its utility.Conclusions: This is the first reported genetic linkage map for Japanese gentians and for any species belonging to the family Gentianaceae. As demonstrated by mapping of functional markers and the stem color trait, our results will help to explain the genetic basis of agronomic important traits, and will be useful for marker-assisted selection in gentian breeding programs. Our map will also be an important resource for further genetic analyses such as mapping of quantitative trait loci and map-based cloning of genes in this species. © 2012 Nakatsuka et al.; licensee BioMed Central Ltd.

Nishihara M.,Iwate Biotechnology Research Center | Hikage T.,Hachimantai City Floricultural Research and Development Center | Yamada E.,Iwate Biotechnology Research Center | Nakatsuka T.,Iwate Biotechnology Research Center
Molecular Genetics and Genomics | Year: 2011

We investigated the genetic basis for the derivation of pink coloration in petals from blue flowers in cultivated gentians. Using a revertant blue-flower phenotype that arose spontaneously from a pink-flowered cultivar, we sought to elucidate the molecular mechanism of flower color restoration caused by a suppressor mutation. Detailed sequencing analysis identified three novel deficient flavonoid 3',5'-hydroxylase (F3'5'H) alleles in pinkflowered gentians in addition to two mutations identified previously (Nakatsuka et al. in Mol Genet Genomics 275:231-241, 2006). Among the deficient alleles, one allele that contained a novel miniature inverted-repeat transposable element (GtMITE1) insertion in an intron of F3'5'H was shown to cause missplicing, resulting in abnormal F3'5'H transcripts and the pink-flower phenotype. The other two mutations were identified as a singlenucleotide insertion and gypsy-Ty3 retrotransposon (Tgt1) insertion within exon 1 and exon 2 of the F3'5'H gene, respectively. The blue-flowered revertant mutant contained a single-nucleotide spontaneous mutation immediately 3' of the TAA target site duplication and the GtMITE1 insertion, which caused restoration of normal splicing of F3'5'H and the normal blue-flower phenotype. Transient expression assays in gentian flowers in vivo demonstrated that normal F3'5'H splicing pattern was recovered from missplicing induced by the GtMITE1 insertion by the single- nucleotide substitution. These findings extend our knowledge of genomic evolution by transposable elements and spontaneous mutations in Gentiana species of economic and medical importance. © Springer-Verlag 2011.

Doi H.,Iwate University | Hoshi N.,Iwate Agricultural Research Center | Yamada E.,Iwate Biotechnology Research Center | Yokoi S.,Iwate University | And 3 more authors.
Breeding Science | Year: 2013

Factors affecting reliable plant regeneration from unfertilized ovule culture of gentians (Gentiana spp.) were examined. Cold pretreatment (4°C) of flower buds enhanced or maintained production of embryo-like structure (ELS). When 43 genotypes were surveyed in two different labs, 40 of them produced ELSs ranging from 0.01 to 26.5 ELSs per flower bud. No ELSs could be obtained in three genotypes. A significant correlation (r = 0.64) was observed between the number of ELS per flower and the frequency of responding flower buds. Eight genotypes of G. triflora, which were used as common materials in two different labs, produced ELSs in both labs. The ploidy levels of a total of 1,515 regenerated plantlets were determined, revealing that the majority of these plants consisted of haploids (57.9%) and diploids (34.3%). However, the frequency of haploids and diploids was different between G. triflora and G. scabra, and G. triflora showed higher frequencies of haploids than G. scabra. When haploids were treated with oryzalin for chromosome doubling, diploids and tetraploids were obtained. These results demonstrate that the unfertilized ovule culture technique of gentians is a powerful tool for obtaining haploids and DHs because of its reproducible and reliable nature and application to a wide range of genotypes. ©2013 by JAPANESE SOCIETY OF BREEDING.

Doi H.,Iwate University | Yokoi S.,Iwate University | Hikage T.,Hachimantai City Floricultural Research and Development Center | Nishihara M.,Iwate Biotechnology Center | And 2 more authors.
Plant Cell Reports | Year: 2011

Gynogenesis was investigated on gentian (Gentiana triflora, G. scabra and their hybrids), which is an important ornamental flower. When unfertilized ovules were cultured in 1/2 NLN medium containing a high concentration of sucrose (100 g/l), embryo-like structures (ELS) were induced. Although genotypic variation was observed in ELS induction, all four genotypes produced ELSs ranging from 0. 93 to 0.04 ELSs per flower bud. The ovules collected from flower buds of later stages (just before anthesis or flower anthesis) tended to exhibit higher response. The dark culture condition produced more than four times as many ELSs than in 16-h light condition. A significant number of plantlets were directly regenerated from ELSs on MS regeneration medium. The ploidy levels of 179 regenerated plants were determined by flow cytometry, revealing that the majority of them were diploid (55.9%) and haploid (31.3%). When a total of 54 diploid plants were examined by molecular genetic markers, 52 (96.3%) were considered as doubled haploids (DHs). This is the first report showing successful gynogenesis in gentian. The production of haploids and DHs by unfertilized ovule culture opens a novel prospect in gentian F1 hybrid breeding. © 2011 Springer-Verlag.

Doi H.,Iwate University | Takahashi R.,Hachimantai City Floricultural Research and Development Center | Hikage T.,Hachimantai City Floricultural Research and Development Center | Takahata Y.,Iwate University
Plant Cell, Tissue and Organ Culture | Year: 2010

The overall goal of this study is to develop an anther culture system to produce doubled haploid (DH) lines of gentian (Gentiana triflora), an ornamental flowering plant, for use in an F1 hybrid breeding program. Embryogenesis was induced from anther cultures incubated on half-strength modified Lichter (NLN) medium containing a high concentration of sucrose (130 g/l) and subjected to heat shock treatment. Among the various parameters investigated, anthers collected from buds 9-12 mm in length induced the highest frequency of androgenesis. Moreover, among three genotypes tested, cvs. Ashiro-no-Aki and Ashiro-no-Natsu produced 21.3 and 3.7 embryos per 100 anthers, respectively, whereas, cv. Lovely-Ashiro failed to produce embryos. Among a total of 427 embryos transferred to a regeneration medium consisting of Murashige and Skoog (MS) medium, 138 plants were regenerated. The ploidy levels of regenerants were determined by flow cytometry and chromosome counts, revealing the presence of 5% haploids, 25% diploids, and 70% triploids. Inter simple sequence repeat (ISSR) analysis using the 6PS line obtained following self-pollination of the diploid plant obtained from anther culture confirmed that the diploid plant was indeed a DH. © 2010 Springer Science+Business Media B.V.

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