Yoshiko Shimono

Genetic Analysis of Putative Triploid Miscanthus Hybrids and Tetraploid M. sacchariflorus Collected from Sympatric Populations of Kushima, Japan

Miscanthus  ×giganteus, a triploid hybrid between tetraploid M. sacchariflorus and diploid M. sinensis, has considerable potential as a bioenergy crop. Currently only one genotype is widely cultivated, increasing its vulnerability to diseases during production. Finding new hybrids is important to broaden genetic resources of M. ×giganteus. Three putative triploid hybrids were discovered in a sympatric population of tetraploid M. sacchariflorus and diploid M. sinensis in Kushima, Japan. The hybrid nature of the triploids was determined by morphological analysis and sequencing the ribosomal DNA internal transcribed spacer (ITS) region. The triploids had awns on their florets, which is a common characteristic of diploid M. sinensis, and sheath hairs, which is typical of tetraploid M. sacchariflorus. All triploids showed heterozygosity in their ribosomal DNA ITS sequences. Based on these results, it is confirmed that the triploids are hybrids and novel genotypes of M. ×giganteus. Natural crossing between tetraploid M. sacchariflorus × diploid M. sinensis may also lead to the production of tetraploid hybrids. ITS analysis of tetraploid plants showed that one maternal parent of the triploid hybrids, K-Ogi-1, had heterozygous ITS, which was different than the other analyzed tetraploid, M. sacchariflorus. Thus, K-Ogi-1 was likely of hybrid origin. These tetraploid hybrids can also be utilized as parents in M. ×giganteus breeding. Since all hybrids identified in this study had tetraploid M. sacchariflorus as maternal parents, collecting and analyzing seeds from tetraploid M. sacchariflorus in sympatric areas could be an effective strategy to identify natural Miscanthus hybrids that can be used as bioenergy crops.

Phylogeography of Mugwort (Artemisia indica), a Native Pioneer Herb in Japan

Many phylogeographic studies of various tree species have been conducted to elucidate the locations of refugia and the colonization patterns during the Pleistocene. However, only a few large-scale phylogeographic studies have been conducted on herbaceous plants, especially scarce on herbs that are adapted to disturbance. Artemisia indica is a fast-growing perennial herb found in open habitats. To examine the basic information on the genetic structure of this species, we investigated the chloroplast DNA variation within and among populations across Japan. We detected 26 haplotypes in 604 individuals from 28 Japanese populations. The haplotype A1 had wide geographic distribution, and its close relatives were locally present. Some putative ancestral lineages were found mainly in the Kyushu region. This may be because several lineages migrated from the Eurasian continent to the northern coast in Kyushu via the Korean peninsula during the Pleistocene, and the A1 haplotype expanded northward, whereas others remained in southern areas. Phylogenetic distant haplotypes were present mainly in the Kanto region. Because the geographic distribution pattern of these haplotypes in this region is believed to be unnatural, these haplotypes may be derived from commercial sources for re-vegetation during the last few decades.

Copy Number Variation in Acetolactate Synthase Genes of Thifensulfuron-Methyl Resistant Alopecurus aequalis (Shortawn Foxtail) Accessions in Japan

Severe infestations of Alopecurus aequalis (shortawn foxtail), a noxious weed in wheat and barley cropping systems in Japan, can occur even after application of thifensulfuron-methyl, a sulfonylurea (SU) herbicide. In the present study, nine accessions of A. aequalis growing in a single wheat field were tested for sensitivity to thifensulfuron-methyl. Seven of the nine accessions survived application of standard field rates of thifensulfuron-methyl, indicating that severe infestations likely result from herbicide resistance. Acetolactate synthase (ALS) is the target enzyme of SU herbicides. Full-length genes encoding ALS were therefore isolated to determine the mechanism of SU resistance. As a result, differences in ALS gene copy numbers among accessions were revealed. Two copies, ALS1 and ALS2, were conserved in all accessions, while some carried two additional copies, ALS3 and ALS4. A single-base deletion in ALS3 and ALS4 further indicated that they represent pseudogenes. No differences in ploidy level were observed between accessions with two or four copies of the ALS gene, suggesting that copy number varies. Resistant plants were found to carry a mutation in either the ALS1 or ALS2 gene, with all mutations causing an amino acid substitution at the Pro197 residue, which is known to confer SU resistance. Transcription of each ALS gene copy was confirmed by reverse transcription PCR, supporting involvement of these mutations in SU resistance. The information on the copy number and full-length sequences of ALS genes in A. aequalis will aid future analysis of the mechanism of resistance.

Non-target-site mechanism of glyphosate resistance in Italian ryegrass (Lolium multiflorum): NTSR to glyphosate in ryegrass

In Shizuoka Prefecture, Japan, glyphosate-resistant Lolium multiflorum is a serious problem on the levees of rice paddies and in wheat fields. The mechanism of resistance of this biotype was analyzed. Based on LD50, the resistant population was 2.8–5.0 times more resistant to glyphosate than the susceptible population. The 5-enolpyruvyl-shikimate-3-phosphate synthase (EPSPS) gene sequence of the resistant biotype did not show a non-synonymous substitution at Pro106, and amplification of the gene was not observed in the resistant biotype. The metabolism and translocation of glyphosate were examined 4 days after application through the direct detection of glyphosate and its metabolite aminomethylphosphonic acid (AMPA) using liquid chromatograph-tandem mass spectrometer (LC-MS/MS). AMPA was not detected in either biotype in glyphosate-treated leaves or the other plant parts. The respective absorption rates of the susceptible and resistant biotypes were 37.90 3.63% and 41.09 3.36%, respectively, which were not significantly different. The resistant biotype retained more glyphosate in a glyphosate-treated leaf (91.36 1.56% of absorbed glyphosate) and less in the untreated parts of shoots (5.90 1.17%) and roots (2.76 0.44%) compared with the susceptible biotype, 79.58 3.73%, 15.77 3.06% and 4.65 0.89%, respectively. The results indicate that the resistance mechanism is neither the acquisition of a metabolic system nor limiting the absorption of glyphosate but limited translocation of the herbicide in the resistant biotype of L. multiflorum in Shizuoka Prefecture.

The Expansion Route of Ryegrasses (Lolium spp.) into Sandy Coasts in Japan

Although an increasing number of investigations have been made into the evolution of alien species once introduced, few studies have identified the invasion routes of these introduced species. Because multiple introductions are common in invasive species, failing to take into account the introduced lineages can be misleading when studying evolutionary change in alien species after they begin to extend their ranges. In Japan, diverse lineages of ryegrasses (Lolium spp.) were introduced as forage crops and contaminants in trading grain and have expanded to sandy coasts. We studied the expansion route of populations established along the coasts of three geographic regions within Japan by comparing variations in morphology and nuclear microsatellite and chloroplast DNA in the two habitats where ryegrasses were first introduced: croplands and international seaports. Chloroplast DNA haplotypes did not differ significantly among habitats and regions, but the coastal and seaport populations displayed similar microsatellite genetic compositions and morphological characteristics. Our results revealed that coastal populations originated from seaport populations derived from contaminants. Selective forces from the past, including domestication and naturalization, may have assisted the introduced lineages in colonizing new habitats. Nomenclature: Italian ryegrass, Lolium multiflorum L.; rigid ryegrass, Lolium rigidum Gaudin.