Mitsuru Gau

First RFLP linkage map of red clover (Trifolium pratense L.) based on cDNA probes and its transferability to other red clover germplasm

We constructed a genetic linkage map of red clover (Trifolium pratense L., 2n=2x=14) using RFLP markers from cDNA probes of a backcrossed mapping population, and investigated the transferability of the markers to other red clover germplasm. The map contains 157 RFLP markers and one morphological marker on seven linkage groups. The total map distance was 535.7xa0cM and the average distance between two markers was 3.4xa0cM. All of the cDNA probes of the map were hybridized to the fragments of genomic DNA from 12 plants derived from three varieties, and 87% of the cDNA probes detected polymorphic bands that corresponded to those of mapping parents. This result indicated that RFLP markers on the present map were transferable to the genome analysis of other red clover germplasm. This is the first report to construct a linkage map of Trifolium species; it should provide fundamental and useful genetic information relevant to the breeding of red clover and genus Trifolium.

Mapping candidate QTLs related to plant persistency in red clover.

Red clover (Trifolium pratense L.) is a diploid (2nxa0=xa014), self-incompatible legume that is widely cultivated as a forage legume in cold geographical regions. Because it is a short-lived perennial species, improvement of plant persistency is the most important objective for red clover breeding. To develop a marker-assisted selection (MAS) approach for red clover, we identified candidate QTLs related to plant persistency. Two full-sib mapping populations, 272xa0×xa0WF1680 and HRxa0×xa0R130, were used for QTL identification. Resistance to Sclerotinia trifoliorum and Fusarium species, as well as to winter hardiness, was investigated in the laboratory and in field experiments in Moscow region (Russia), and Sapporo (Japan). With the genotype data derived from microsatellite and other DNA markers, candidate QTLs were identified by simple interval mapping (SIM), Kruskal–Wallis analysis (KW analysis) and genotype matrix mapping (GMM). A total of 10 and 23 candidate QTL regions for plant persistency were identified in the 272xa0×xa0WF1680 and the HRxa0×xa0R130 mapping populations, respectively. The QTLs identified by multiple mapping approaches were mapped on linkage group (LG) 3 and LG6. The significant QTL interactions identified by GMM explained the higher phenotypic variation than single effect QTLs. Identification of haplotypes having positive effect QTLs in each parent were first demonstrated in this study for pseudo-testcross mapping populations in plant species using experimental data.

Breeding potential of the backcross progenies of a hybrid between Trifolium medium × T. pratense to T. pratense

In this study, T. medium (zigzag clover, 2n = 80 and T. pratense (red clover, 2n = 28) were hybridized and backcrossed four times to T. pratense (BC1, BC2, BC3and BC4), and the seed production and vigor of the backcross progenies were assessed under field conditions. Both female and pollen fertility of the backcross progenies increased as the backcross generations advanced. Female and pollen fertility of BC4 were 21.3 and 65.3%, respectively. When honeybees were used as pollinators, almost all BC4 plants produced mature seeds in the field. Most of the BC1, BC2, and BC3 plants died by the end of the fourth year in the field, whereas 61% of the BC4 plants survived for 4 yr. The number of chromosomes in 79% of the BC4 plants was more than 28, which is greater than that of T. pratense. Differences in leaflet size and shape between BC4 plants and T. pratense suggest that genes from T. medium were expressed in the BC4 plants. These results indicate that BC4 plants may be useful for the improvement of the persis...

The Coupling of Differential Display and AFLP Approaches for Nonradioactive mRNA Fingerprinting

We have modified the differential display of 3′-end restriction fragments of cDNAs technique by combining it with the amplified fragment length polymorphism (AFLP) approach and silver staining. Modified oligo-dT primers were used for a reverse transcription step. ds cDNA was digested with the Mse I restriction enzyme and then ligated with an AFLP adapter. The modified template was amplified with oligo-dT primers in a preamplification step (asymmetric PCR) that enriched the template for 3′-end sequences; subsequently, the enriched template was amplified with an AFLP primer having a selective extension and an anchored oligo-dT primer (conventional PCR step). We demonstrated that the asymmetric preamplification step facilitates the preferential amplification of 3′-end fragments and the resulting PCR products can be clear resolved on silver-stained gel. The presented procedure takes advantages of silver-stained gels, generates reproducible display patterns, and allows reliable reamplification of isolated fragments which contain both upstream and downstream primer sequences.

Changes in chloroplast FtsH-like gene during cold acclimation in alfalfa (Medicago sativa)

Summary A cold-induced mRNA for chloroplast FtsH-like protease was identified from cDNA array analysis of alfalfa mRNA (Medicago sativa L., cv. Rambler). Expression of this gene was analyzed under different light and temperature regimes. Exposing plants to low temperatures or to high light conditions increased the FtsH levels in both mRNA and protein in alfalfa leaves. The cold-induced accumulation of FtsH is not part of a general response to stressful conditions, since no increased accumulation was detected in response to treatment with ABA, NaCl or desiccation, while exposure to high temperature resulted in a reduction in transcript level. The low-temperature induction of FtsH transcript in dark-grown seedlings suggests that low temperature and light independently regulate expression of the gene.

Discovery of Natural Interspecific Hybrids Between Miscanthus Sacchariflorus and Miscanthus Sinensis in Southern Japan: Morphological Characterization, Genetic Structure, and Origin

Natural Miscanthus grasses are useful for improving biomass production. We found a population of putative triploid interspecific hybrids between Miscanthus sacchariflorus and Miscanthus sinensis in southern Kyushu, Japan. This study aims to investigate its morphological variation, genetic structure, and origin. Miscanthus plants were collected from 114 points, mainly beside a river along a distance of 2.8xa0km in the Tashiro–Fumoto area. They resembled M. sacchariflorus but showed morphology intermediate between the two species. They had a nuclear DNA content corresponding to that of a hybrid between tetraploid M. sacchariflorus and diploid M. sinensis, and had species-specific alleles from both species revealed by DNA marker analysis. This indicates that the plants are triploid hybrids between M. sacchariflorus and M. sinensis. Genotyping using simple sequence repeat markers revealed only four genotypes among the hybrid population, of which two accounted for most plants. The genotypes showed mostly discrete geographical distributions. The two major genotypes showed contrasting phenotypes in pollen viability and in frequency of awns in florets. Some seeds collected from the population germinated and the seedlings showed a wide range of nuclear DNA content from diploid to tetraploid. In this area, many M. sinensis plants also grew, but we could not find M. sacchariflorus. The hybrid Miscanthus might be selected due to its improved adaptability introduced from M. sinensis. Furthermore, genetic and phenotypic characterization suggests the polyphyletic origin and clonal propagation of this population. Such partially fertile hybrids could be interesting for the improvement of Miscanthus as a biomass crop.

Root-shoot relationships in four strains of field-grown Erianthus arundinaceus at seedling stage

Abstract The production of cellulosic bioethanol from non-edible plants is a potential countermeasure against global warming. Erianthus species provide cellulosic raw material for bioethanol because they have high biomass productivity and high tolerance to environmental stress, associated with their large, deep root systems. However, it is difficult to select Erianthus species for breeding by direct observation of their root systems because the roots are widely dispersed in the soil. Instead, we examined shoot morphological traits that could be closely related to root morphology to find effective reference indices for selection. The potential to evaluate root structure and function in Erianthus according to bleeding rate was also examined. An analysis of root–shoot relationships in seedlings indicated that root number and mean length were closely related to stem number and diameter, respectively. These results suggest that root–shoot relationships may provide useful criteria for selective breeding of root systems in Erianthus.