Tadayuki Wako

Genetic variation and phylogenetic relationships in East and South Asian melons, Cucumis melo L., based on the analysis of five isozymes

Genetic structure and phylogenetic relationships in East and South Asian melons were analyzed, based on the geographical variation of five isozymes. The analysis of Indian melon accessions showed a continuous variation in seed length, ranging from 4 to 13 mm. Most of the East Asian melons, vars. makuwa and conomon, were classified as the small seed type with seed length shorter than 9 mm. The frequency of the small seed type increased from the west to the east in India. Allelic variation was detected at a total of nine loci of five isozymes among 114 melon accessions. Gene diversity calculated for the nine loci indicated that Indian melon was rich in genetic variation, which decreased from India towards the east. Clear geographical variation was detected in two enzymes, APS and6-PGDH. Pgd-11 and Ap-31 were frequent in India and Myanmar, while most of the melons in Laos, China, Korea and Japan carried Pgd-13 and Ap-33, except var. inodorusin China. Among the latter two alleles, the frequency of Ap-33 was more than 50% in the small seed type in north and east India, indicating that vars. makuwa and conomon were related to the small seed type in these areas. It was also suggested that the small seed type with wet tolerance originated in central India and was selected under wet condition in the east.

Genetic mapping of AFLP markers in Japanese bunching onion (Allium fistulosum)

SummaryThe first genetic linkage map of Japanese bunching onion (Allium fistulosum) based primarily on AFLP markers was constructed using reciprocally backcrossed progenies. They were 120 plants each of (P1)BC1 and (P2)BC1 populations derived from a cross between single plants of two inbred lines: D1s-15s-22 (P1) and J1s-14s-20 (P2). Based on the (P2)BC1 population, a linkage map of P1 was constructed. It comprises 164 markers – 149 amplified fragment length polymorphisms (AFLPs), 2 cleaved amplified polymorphic sequences (CAPSs), and 12 simple sequence repeats (SSRs) from Japanese bunching onion, and 1 SSR from bulb onion (A. cepa) – on 15 linkage groups covering 947 centiMorgans (cM). The linkage map of P2 was constructed with the (P1)BC1 population and composed of 120 loci – 105 AFLPs, 1 CAPS, and 13 SSRs developed from Japanese bunching onion and 1 SSR from bulb onion – on 14 linkage groups covering 775 cM. Both maps were not saturated but were considered to cover the majority of the genome. Nine linkage groups in P2 map were connected with their counterparts in P1 map using co-dominant anchor markers, 13 SSRs and 1 CAPS.

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.

Inheritance mode of male sterility in bunching onion (Allium fistulosum L.) accessions

Cytoplasmic male sterility (CMS) is an indispensable trait for F1 hybrid seed production in bunching onion (Allium fistulosum L.). Expansion of the cytoplasmic diversity of F1 hybrid cultivars by introduction of various CMS resources has great potential to eliminate vulnerability to cytoplasm type-specific diseases. This study aimed to evaluate appearance frequency of male sterile plants in several bunching onion accessions and to identify CMS resources. In eight (‘Nogiwa Aigara’, ‘Bansei Hanegi’, ‘Amarume’, ‘Kimnung’, ‘Zhangqiu’, ‘INT/CHN/1990/GOTOU’, ‘Natsunegi’ and ‘Guangzhou’) of 135 accessions collected from Japan, China, Mongolia, Korea and Taiwan, male sterile plants appeared with varied frequencies from 1.7% (‘Nogiwa Aigara’ and ‘Bansei Hanegi’) to 24.5% (‘Zhangqiu’). The inheritance mode of Zhangqiu- and Guangzhou-derived male sterility was confirmed to be CMS by sib-crossings and interbreed crossings. Microscopic examination of microsporogenesis in the CMS plants revealed that microspore protoplasm rapidly degenerated without mitotic division after the release of microspores from tetrads. The CMS germplasm described here would be useful for the development of “A” lines to be used in F1 hybrid seed production of bunching onion. Male fertility in ‘Nogiwa Aigara’, ‘Bansei Hanegi’, ‘Kimnung’, ‘INT/CHN/1990/GOTOU’ and ‘Natsunegi’ was verified to be controlled by a single fertility restoration locus.

SSR-tagged breeding scheme for allogamous crops: a trial in bunching onion ( Allium fistulosum )

In several autogamous and vegetatively propagated crops, DNA markers have been used for cultivar identification. However, allogamous crops such as bunching onion (Allium fistulosum L.) are recalcitrant to marker-aided cultivar identification, as well as hybrid seed purity tests, due to the high degree of genetic heterogeneity within each cultivar. To aid cultivar identification and ensure its accuracy in bunching onion, we proposed the “SSR-tagged breeding” scheme in our previous study. The feasibility of this scheme was investigated here using a landrace of bunching onion with two populations tagged with two or four selected SSR markers. Compared with a control population, no significant differences were detected in the agronomic traits of the SSR-tagged populations. The targeted SSR loci were genetically uniform within each population whereas other loci maintained high heterogeneity. These results demonstrate that the SSR-tagged breeding scheme, even with a very small number of markers, is efficient for the identification of newly bred cultivars, and consequently for F1 purity tests, in allogamous crops in which inbreeding depression is as severe as in bunching onion.

Activation of Immune Responses in Mice by an Oral Administration of Bunching Onion (Allium fistulosum) Mucus

Bunching onion [Allium fistulosum L. (Liliaceae)] secretes mucus in the cavities of its green leaves. The effects of the mucus, which is consumed as food, were examined. The mucus augmented the production of tumor necrosis factor (TNF)-α and monocyte chemotactic protein (MCP)-1 from RAW 264 cells and of interleukin (IL)-12 from J774.1 cells; however, extracts from green leaves and white sheaths did not. An oral administration of this mucus to mice augmented the immune functions of peritoneal cells by increasing TNF-α and IL-12 production and phagocytosis. It also augmented interferon (IFN)-γ production from spleen cells and natural killer (NK) activity. These results suggest that an oral administration of the A. fistulosum mucus can enhance natural immunity.

Modes of inheritance of two apomixis components, diplospory and parthenogenesis, in Chinese chive (Allium ramosum) revealed by analysis of the segregating population generated by back-crossing between amphimictic and apomictic diploids

To investigate the mode of inheritance of apomixis in Chinese chive, the degrees of diplospory and parthenogenesis were evaluated in F1 and BC1 progenies derived from crosses between amphimictic and apomictic diploids (2n = 16, 2x). The F1 population was generated by crossing three amphimictic diploids 94Mo13, 94Mo49 and 94Mo50 with an apomictic diploid KaD2 and comprised 110 diploids and 773 triploids. All the diploid F1 plants examined were completely or highly eusporous and completely syngamic. All the triploid F1 plants examined were highly diplosporous and highly parthenogenetic. KaD2 could not transmit its high level of apomixis via monoploid pollen grains. The BC1 population, generated by crossing 94Mo49 with apomictic triploids found in the F1 offspring, exhibited heteroploidy; it comprised haploid, diploid, triploid, tetraploid and various aneuploid individuals. In this generation, clear segregation was observed between diplospory and parthenogenesis. Analysis of the BC1 population suggests that diplospory and parthenogenesis are each controlled by single dominant genes, D and P, respectively. However, all the BC1 plants characterized as parthenogenetic were diplosporous. The absence of phenotypically eusporous parthenogenetic plants can be explained by assuming that the presence of diplospory gene is a prerequisite for the parthenogenesis gene expression in Chinese chive.

Mapping of quantitative trait loci for bolting time in bunching onion (Allium fistulosum L.)

Late bolting is one of the most important agronomic traits in bunching onion (Allium fistulosum L.) because it affects the yield and quality of the harvested products during the spring and early summer. However, genetic and molecular studies of bolting time in bunching onion have not been reported thus far. To understand the genetic basis of bolting time in bunching onion plants, we evaluated the bolting time of two F2:3 populations derived from crosses between cultivars differing in bolting time and conducted quantitative trait loci (QTL) analysis based on the bunching onion genetic linkage map. When the KiC population derived from a cross between the ever-flowering line Ki and the late-bolting line C was grown under field conditions, two QTLs associated with bolting time were repeatedly detected on the linkage groups 1a on chromosome 1 (Chr. 1a) and 2a on chromosome 2 (Chr. 2a). However, the QTL on Chr. 1a was not detected when the KiC population was grown in a heated greenhouse under unvernalized conditions. A single QTL with major effect was identified exclusively on the linkage group Chr. 2a in the SaT03 population derived from a cross between early-bolting line Sa03 and late-bolting T03 evaluated under field conditions. QTL located on Chr. 2a in both populations were linked to the same marker loci, suggesting that these regions were strongly related. Simple sequence repeat loci linked to these QTLs had significant effects on bolting time in both populations.

Screening and incorporation of rust resistance from Allium cepa into bunching onion (Allium fistulosum) via alien chromosome addition

Bunching onion (Allium fistulosum L.; 2n = 16), bulb onion (Allium cepa L. Common onion group), and shallot (Allium cepa L. Aggregatum group) cultivars were inoculated with rust fungus, Puccinia allii, isolated from bunching onion. Bulb onions and shallots are highly resistant to rust, suggesting they would serve as useful resources for breeding rust resistant bunching onions. To identify the A. cepa chromosome(s) related to rust resistance, a complete set of eight A. fistulosum - shallot monosomic alien addition lines (MAALs) were inoculated with P. allii. At the seedling stage, FF+1A showed a high level of resistance in controlled-environment experiments, suggesting that the genes related to rust resistance could be located on shallot chromosome 1A. While MAAL, multi-chromosome addition line, and hypoallotriploid adult plants did not exhibit strong resistance to rust. In contrast to the high resistance of shallot, the addition line FF+1A+5A showed reproducibly high levels of rust resistance.