Masayoshi Shigyo

Genetic mapping of a major gene affecting onion bulb fructan content.

The non-structural dry matter content of onion bulbs consists principally of fructose, glucose, sucrose and fructans. The objective of this study was to understand the genetic basis for the wide variation observed in the relative amounts of these carbohydrates. Bulb carbohydrate composition was evaluated in progeny from crosses between high dry matter storage onion varieties and sweet, low dry matter varieties. When samples were analysed on a dry weight basis, reducing sugar and fructan content exhibited high negative correlations and bimodal segregation suggestive of the action of a major gene. A polymorphic SSR marker, ACM235, was identified which exhibited strong disequilibrium with bulb fructan content in F2:3 families from the ‘W202A’ × ‘Texas Grano 438’ mapping population evaluated in two environments. This marker was mapped to chromosome 8 in the interspecific population ‘Allium cepa × A. roylei’. Mapping in the ‘Colossal Grano PVP’ × ‘Early Longkeeper P12’ F2 population showed that a dominant major gene conditioning high-fructan content lay in the same genomic region. QTL analysis of total bulb fructan content in the intraspecific mapping population ‘BYG15-23’ × ‘AC43’ using a complete molecular marker map revealed only one significant QTL in the same chromosomal region. This locus, provisionally named Frc, may account for the major phenotypic differences in bulb carbohydrate content between storage and sweet onion varieties.

Peroxidase-mediated chlorophyll degradation in horticultural crops

One of the symptoms of senescence in harvested horticultural crops is the loss of greenness that comes with the degradation of chlorophyll (Chl). With senescence, peroxidase, which is involved in Chl degradation, increased greatly in stored horticultural crops. C132-hydroxychlorophyll a, an oxidized form of Chl a, is formed in vitro through Chl oxidation by peroxidase. Peroxidase mediates Chl degradation in the presence of phenolic compounds such as p-coumaric acid and apigenin, which have a hydroxyl group at the p-position. Apparently, not all phenolic compounds are able to degrade Chl in this system, and their effectiveness appears to depend on their molecular configuration. In peroxidase-mediated Chl degradation, peroxidase oxidizes the phenolic compounds with hydrogen peroxide and forms phenoxy radical; then, the phenoxy radical oxidizes Chl and its derivatives to colorless low molecular weight compounds through the formation of C132-hydroxychlorophyll a,a fluorescent Chl catabolite and a bilirubin-like compound as an intermediate. In addition to the phenoxy radical, superoxide anion, which is formed in the peroxidase-catalyzed reaction, might be involved in Chl oxidation. Moreover, Chl degradation by peroxidase seems to occur in the chloroplast and/or the vacuole. The involvement of peroxidase in Chl degradation in senescing horticultural crops is also discussed.

Effects of sugars and abscisic acid on somatic embryogenesis from melon (Cucumis melo L.) expanded cotyledon

Effects of sugars and abscisic acid (ABA) on somatic embryogenesis from melon (Cucumis melo L.) expanded cotyledon and changes in ABA content on cultures on the various sucrose treatment media were investigated. Sucrose induced somatic embryogenesis, but mannitol did not. Two hundred millimolar sucrose treatments were optimal for somatic embryogenesis; the formation of somatic embryos was suppressed at lower or higher sucrose concentrations. The frequency of somatic embryogenesis, however, was increased by the addition of mannitol to the initial medium, even if the sucrose concentration was less than 200 mM. Somatic embryogenesis was enhanced by the addition of 0.5 μM ABA to 200 mM sucrose treatment. In the 200 mM sucrose treatment, in which somatic embryos were easily formed, endogenous levels of ABA in callus increased greatly after 2 weeks of culture. ABA content of embryogenic callus was markedly higher than that of non-embryonic callus at any given period. These results indicate that ABA formation, which was controlled by osmotic conditions induced by the sugars, could be involved in somatic embryogenesis.

Involvement of peroxidase in chlorophyll degradation in stored broccoli (Brassica oleracea L.) and inhibition of the activity by heat treatment

Cell localization of chlorophyll (Chl)-degrading peroxidase and the effects of heat treatment on the activity were determined to elucidate the involvement of the enzyme with yellowing in stored broccoli. Subcellular fractions and intact chloroplasts were prepared by differential and Percoll gradient centrifugation, respectively. In fresh broccoli, only low levels of Chl-degrading peroxidase activity were located in the intact chloroplast, and the highest activity was present in the cytosolic fraction. The Chl content of broccoli without heat treatment decreased greatly after 4 days storage at 15 °C, whereas the content in broccoli treated at 50 °C for 2 h showed almost no change during storage. In microsomal and cytosolic fractions, the activity of C2 (Rf=0.47) isoperoxidase, which is involved in Chl degradation, increased greatly with floret yellowing. The Chl-degrading peroxidase activities in microsomal and cytosolic fractions also increased strongly after 4 days of storage. In addition, the Chl-degrading peroxidase activity as well as C2 isoperoxidase activity was suppressed by heat treatment. These results indicate that heat treatments may be effective in inhibiting senescence in part by suppressing the enhancement of Chl-degrading peroxidase activity in the microsomes and the cytosol.

AlliumMap-A comparative genomics resource for cultivated Allium vegetables

BackgroundVegetables of the genus Allium are widely consumed but remain poorly understood genetically. Genetic mapping has been conducted in intraspecific crosses of onion (Allium cepa L.), A. fistulosum and interspecific crosses between A. roylei and these two species, but it has not been possible to access genetic maps and underlying data from these studies easily.DescriptionAn online comparative genomics database, AlliumMap, has been developed based on the GMOD CMap tool at http://alliumgenetics.org. It has been populated with curated data linking genetic maps with underlying markers and sequence data from multiple studies. It includes data from multiple onion mapping populations as well as the most closely related species A. roylei and A. fistulosum. Further onion EST-derived markers were evaluated in the A. cepa x A. roylei interspecific population, enabling merging of the AFLP-based maps. In addition, data concerning markers assigned in multiple studies to the Allium physical map using A. cepa-A. fistulosum alien monosomic addition lines have been compiled. The compiled data reveal extensive synteny between onion and A. fistulosum.ConclusionsThe database provides the first online resource providing genetic map and marker data from multiple Allium species and populations. The additional markers placed on the interspecific Allium map confirm the value of A. roylei as a valuable bridge between the genetics of onion and A. fistulosum and as a means to conduct efficient mapping of expressed sequence markers in Allium. The data presented suggest that comparative approaches will be valuable for genetic and genomic studies of onion and A. fistulosum. This online resource will provide a valuable means to integrate genetic and sequence-based explorations of Allium genomes.

Direct comparison between genomic constitution and flavonoid contents in Allium multiple alien addition lines reveals chromosomal locations of genes related to biosynthesis from dihydrokaempferol to quercetin glucosides in scaly leaf of shallot (Allium cepa L.)

The extrachromosome 5A of shallot (Allium cepa L., genomes AA) has an important role in flavonoid biosynthesis in the scaly leaf of Allium fistulosum–shallot monosomic addition lines (FF+nA). This study deals with the production and biochemical characterisation of A. fistulosum–shallot multiple alien addition lines carrying at least 5A to determine the chromosomal locations of genes for quercetin formation. The multiple alien additions were selected from the crossing between allotriploid FFA (♀) and A. fistulosum (♂). The 113 plants obtained from this cross were analysed by a chromosome 5A-specific PGI isozyme marker of shallot. Thirty plants were preliminarily selected for an alien addition carrying 5A. The chromosome numbers of the 30 plants varied from 18 to 23. The other extrachromosomes in 19 plants were completely identified by using seven other chromosome markers of shallot. High-performance liquid chromatography analyses of the 19 multiple additions were conducted to identify the flavonoid compounds produced in the scaly leaves. Direct comparisons between the chromosomal constitution and the flavonoid contents of the multiple alien additions revealed that a flavonoid 3′-hydroxylase (F3′H) gene for the synthesis of quercetin from kaempferol was located on 7A and that an anonymous gene involved in the glucosidation of quercetin was on 3A or 4A. As a result of supplemental SCAR analyses by using genomic DNAs from two complete sets of A. fistulosum–shallot monosomic additions, we have assigned F3′H to 7A and flavonol synthase to 4A.

Development of microsatellite markers in cultivated and wild species of sections Cepa and Phyllodolon in Allium

The potential of microsatellite markers for use in genetic studies has been evaluated in Allium cultivated species (Allium cepa, A. fistulosum) and its allied species (A. altaicum, A. galanthum, A. roylei, A. vavilovii). A total of 77 polymerase chain reaction (PCR) primer pairs were employed, 76 of which amplified a single product or several products in either of the species. The 29 AMS primer pairs derived from A. cepa and 46 microsatellites primer pairs from A. fistulosum revealed a lot of polymorphic amplicons between seven Allium species. Some of the microsatellite markers were effective not only for identifying an intraspecific F1 hybrid between shallot and bulb onion but also for applying to segregation analyses in its F2 population. All of the microsatellite markers can be used for interspecific taxonomic analyses among two cultivated and four wild species of sections Cepa and Phyllodolon in Allium. Generally, our data support the results obtained from recently performed analyses using molecular and morphological markers. However, the phylogeny of A. roylei, a threatened species with several favorable genes, was still ambiguous due to its different positions in each dendrogram generated from the two primer sets originated from A. cepa and A. fistulosum.

Integrating transcriptome and target metabolome variability in doubled haploids of Allium cepa for abiotic stress protection

Environmental stress conditions such as drought, heat, salinity, or pathogen infection can have a devastating impact on plant growth and yield, resulting in a need for stress-tolerant crop varieties. Crossbreeding tropical and cultivated onion species produced a hybrid F1 generation possessing genetic and metabolic parental properties that aided abiotic stress tolerance. Targeted metabolite profiling using liquid chromatography–tandem mass spectrometry integrated with transcriptional analysis of their relevant genes provided insights into the metabolic and genomic architecture of the onion doubled haploid (Allium cepa L., DHC), shallot doubled haploid (A. cepa L. Aggregatum group, DHA), and F1 hybrid. Out of a complete set of 113 targeted metabolites, 49 metabolites were found to be statistically significantly different between genotypes: 11 metabolites were characteristic for DHC, 10 for DHA, 14 for F1, and 14 metabolites were shared among the three genotypes. Several key genes and metabolites introgressed in abiotic stress response were up-regulated in DHA and F1 genotypes as compared to DHC. Principal component analysis and Volcano plot analysis revealed that metabolic traits and their relevant genes (namely, amino acid, carbohydrate, flavonoid, and phospholipid biosynthesis) were strongly linked with DHA and F1, reflecting the adaptability of DHA and F1 toward abiotic stress as compared to DHC.

Interspecific hybrids between Allium fistulosum and Allium schoenoprasum reveal carotene-rich phenotype

Interspecific hybridization is an effective method to generate a new crop that gains available functions in a short time. Interspecific hybrids (2n= 16) betweenAllium fistulosumL. (2n= 16) andAllium schoenoprasumL. (2n= 16) were produced by reciprocal crossings through ovary culture, but the hybrids were much fewer in the combination usingA. schoenoprasumas a seed plant. All the hybrids have eight long chromosomes originated fromA.fistulosumand eight short chromosomes originated fromA. schoenoprasum. In addition, the hybridity was confirmed by randomly amplified polymorphic DNA (RAPD) analysis and cleaved amplified polymorphic sequence (CAPS) analysis of the rDNA internal transcribed spacer (ITS) region. The interspecific hybrids showed a vigorous growth habit; their foliage was slightly bloomy and deep green. The hybrids did not form bulbs, but rather propagated vegetatively by tillering. Carotene contents of the hybrids and both parents were quantified using high-performance liquid chromatography (HPLC). The contents of all edible parts of the hybrids were approximately seven times higher than those of either parent. These results indicate that the hybrid is a new and carotene-rich vegetable ofAlliumspecies.

Identification and Biological Activity of Antifungal Saponins from Shallot (Allium cepa L. Aggregatum Group)

The n-butanol extract of shallot basal plates and roots showed antifungal activity against plant pathogenic fungi. The purified compounds from the extract were examined for antifungal activity to determine the predominant antifungal compounds in the extract. Two major antifungal compounds purified were determined to be alliospiroside A (ALA) and alliospiroside B. ALA had prominent antifungal activity against a wide range of fungi. The products of acid hydrolysis of ALA showed a reduced antifungal activity, suggesting that the compounds sugar chain is essential for its antifungal activity. Fungal cells treated with ALA showed rapid production of reactive oxygen species. The fungicidal action of ALA was partially inhibited by a superoxide scavenger, Tiron, suggesting that superoxide anion generation in the fungal cells may be related to the compounds action. Inoculation experiments showed that ALA protected strawberry plants against Colletotrichum gloeosporioides , indicating that ALA has the potential to control anthracnose of the plant.