Masumi Ebina

Analysis of expressed sequence tags in apomictic guineagrass (Panicum maximum).

Apomixis is an intriguing asexual mode of reproduction, because it produces maternal clones that permit vegetative reproduction through seeds. Guineagrass (Panicum maximum) has both facultative aposporous apomixis and obligate sexual modes of reproduction. Despite the importance of apomixis in guineagrass, expressed sequence tags (ESTs) for this condition have not been studied in this species. We constructed a guineagrass cDNA library from two aposporous strains, Ku5954 and GM64-3A, and utilized them as microarray probes. To find genes uniquely expressed in the immature pistils of apomicts, we performed a microarray analysis using target RNA from another apomict, OKI64. Of the 4608 probes in the microarray, only 394 showed clear gene expression in the immature pistils. Of the 394 expressed probes, 196 were successfully sequenced. Of these, 181 had significant homology with other species, including 10 ESTs with matches in a pistil cDNA library from another aposporous species, Cenchrus ciliaris. Of the remaining ESTs, three showed significant homology only with animal database sequences and the other 12 ESTs showed no homology with any previously registered sequence. In reverse-transcriptase PCR and real-time quantitative PCR, nine ESTs reliably detected ovary-specific gene expression. Of these, three revealed aposporous ovary-specific genes expressed in the early developmental stage, suggesting that these could be apomixis-related genes.

Particle inflow gun-mediated transformation of multiple-shoot clumps in rhodes grass (Chloris gayana).

Rhodes grass (Chloris gayana) is one of the most important warm-season forage grasses. It is cultivated in tropical and subtropical parts of the world and is mostly used for grazing and hay production. We have established a particle-bombardment transformation protocol for rhodes grass using multiple-shoot clumps (MSCs) as the target tissue. A vector pAHC25 containing a herbicide-resistance gene (bar) together with the beta-glucuronidase (GUS) gene was used in transformation experiments. The most efficient recovery of bialaphos-resistant tissue was achieved when the bombarded MSCs were first cultured for 15 d on bialaphos-free medium before being subjected to selection pressure. The resistant tissues regenerated transgenic plants that displayed GUS gene expression. Under optimized conditions, 251 target pieces yielded 46 transgenic plants from 4 independent transgenic lines.

Complete chloroplast genomes of Erianthus arundinaceus and Miscanthus sinensis: comparative genomics and evolution of the Saccharum complex.

The genera Erianthus and Miscanthus, both members of the Saccharum complex, are of interest as potential resources for sugarcane improvement and as bioenergy crops. Recent studies have mainly focused on the conservation and use of wild accessions of these genera as breeding materials. However, the sequence data are limited, which hampers the studies of phylogenetic relationships, population structure, and evolution of these grasses. Here, we determined the complete chloroplast genome sequences of Erianthus arundinaceus and Miscanthus sinensis by using 454 GS FLX pyrosequencing and Sanger sequencing. Alignment of the E. arundinaceus and M. sinensis chloroplast genome sequences with the known sequence of Saccharum officinarum demonstrated a high degree of conservation in gene content and order. Using the data sets of 76 chloroplast protein-coding genes, we performed phylogenetic analysis in 40 taxa including E. arundinaceus and M. sinensis. Our results show that S. officinarum is more closely related to M. sinensis than to E. arundinaceus. We estimated that E. arundinaceus diverged from the subtribe Sorghinae before the divergence of Sorghum bicolor and the common ancestor of S. officinarum and M. sinensis. This is the first report of the phylogenetic and evolutionary relationships inferred from maternally inherited variation in the Saccharum complex. Our study provides an important framework for understanding the phylogenetic relatedness of the economically important genera Erianthus, Miscanthus, and Saccharum.