Masahiro Fujimori

Identification of QTL controlling adventitious root formation during flooding conditions in teosinte (Zea mays ssp. huehuetenangensis) seedlings

Adventitious root formation (ARF) at the soil surface is one of the most important adaptations to soil flooding or waterlogging. Quantitative trait loci (QTL) controlling ARF under flooding condition were identified in a 94 F2 individual population by crossing maize (Zea mays L., B64) × teosinte (Z. mays ssp. huehuetenangensis). A base-map was constructed using 66 SSR and 42 AFLP markers, covering 1,378 cM throughout all ten maize chromosomes. The ARF capacity for seedlings was determined by evaluating the degree of root formation at the soil surface following flooding for 2 weeks. ARF showed continuous variation in the F2 population. Interval mapping and composite interval mapping analyses revealed that the QTL for ARF was located on chromosome 8 (bin 8.05). Utilising a selective genotyping strategy with an additional 186 F2 population derived from the same cross combination and 32 AFLP primer combinations, regions on chromosomes 4 (bin 4.07) and 8 (bin 8.03) were found to be associated with ARF. Z. mays ssp. huehuetenangensis contributed all of the QTL detected in this study. Results of the study suggest a potential for transferring waterlogging tolerance to maize from Z. mays ssp. huehuetenangensis.

Increased resistance to crown rust disease in transgenic Italian ryegrass (Lolium multiflorum Lam.) expressing the rice chitinase gene

We introduced the rice chitinase (Cht-2; RCC2) gene into calli of Italian ryegrass (Lolium multiflorum Lam.), with a hygromycin phosphotransferase (HPT) gene as a selectable marker, by particle bombardment. Hygromycin-resistant calli were selected and transferred to regeneration medium for shoot formation. Polymerase chain reaction (PCR) analysis revealed regenerants containing the HPT gene. The RCC2 gene was detected in 65.5% of those regenerants. Southern hybridization detected both HPT and RCC2 genes and indicated that the transgenic plants were independently transformed. Expression of the RCC2 gene in the transgenic plants was confirmed by Northern hybridization, reverse transcription–PCR and Western blotting. Bioassay of detached leaves indicated increased resistance to crown rust (Puccinia coronata) in transgenic plants, which exhibited higher chitinase activity than a nontransgenic plant.

Development of EST-derived CAPS and AFLP markers linked to a gene for resistance to ryegrass blast (Pyricularia sp.) in Italian ryegrass (Lolium multiflorum Lam.)

Ryegrass blast, also called gray leaf spot, is caused by the fungus Pyricularia sp. It is one of the most serious diseases of Italian ryegrass (Lolium multiflorum Lam.) in Japan. We analyzed segregation of resistance in an F1 population from a cross between a resistant and a susceptible cultivar. The disease severity distribution in the F1 population suggested that resistance was controlled by a major gene (LmPi1). Analysis of amplified fragment length polymorphisms with bulked segregant analysis identified several markers tightly linked to LmPi1. To identify other markers linked to LmPi1, we used expressed sequence tag-cleaved amplified polymorphic sequence (EST-CAPS) markers mapped in a reference population of Italian ryegrass. Of the 30 EST-CAPS markers screened, one marker, p56, flanking the LmPi1 locus was found. The restriction pattern of p56 amplification showed a unique fragment corresponding to the resistant allele at the LmPi1 locus. A linkage map constructed from the reference population showed that the LmPi1 locus was located in linkage group 5 of Italian ryegrass. Genotype results obtained from resistant and susceptible cultivars indicate that the p56 marker is useful for introduction of the LmPi1 gene into susceptible germplasm in order to develop ryegrass cultivars with enhanced resistance to ryegrass blast.

Screening of Regenerable Genotypes of Italian Ryegrass (Lolium multiflorum Lam.)

Abstract We screened regenerable genotypes from 12 cultivars of Italian ryegrass (Lolium multiflorum Lam.) through tissue culture of mature seeds, shoot tips and root tips. Although three morphological types of calli, friable, watery, and compact with fine root hair were observed, green shoots regenerated only from the friable calli. The highest frequency of the formation of regenerable callus from mature seeds was 1.4% in the diploid cultivar Waseaoba and 1.1% in the tetraploid cultivar Meritra. The plants regenerated from mature seed-derived calli were maintained and propagated aseptically in vitro, and the method of regenerating plantlets via callus culture was established by using their shoot tips. Sixty-three seeds of Waseaoba and 44 seeds of Meritra were aseptically sown, each seedling (genotype) was propagated in vitro, and the shoot tip from each genotype was subjected to tissue culture. More than 22% of these genotypes formed regenerable calli. From these results, we conclude that the shoot tip of the in vitro-preserved plantlet is useful for producing the regenerable calli routinely. The information obtained in the present study is useful for establishing a reliable transformation system for Italian ryegrass.