Erianthus germplasm collection in Thailand: genetic structure and phylogenetic aspects of tetraploid and hexaploid accessions.

BACKGROUND: The genus Erianthus, which belongs to the "Saccharum complex", includes C4 warm-season grasses. Erianthus species are widely distributed throughout Southeast Asia, East Asia and South Asia. Erianthus arundinaceus (Retz.) Jeswiet is highly adaptable to the environment, has a high percentage of dry matter, and is highly productive. Recently, this species has attracted attention as a novel bioenergy crop and as a breeding material for sugarcane improvement. Such interest in E. arundinaceus has accelerated the collection and conservation of its genetic resources, mainly in Asian countries, and also evaluation of morphological, agricultural, and cytogenetic features in germplasm collections. In Thailand, genetic resources of E. arundinaceus have been collected over the past 20 years and their phenotypic traits have been evaluated. However, the genetic differences and relatedness of the germplasms are not fully understood. RESULTS: A set of 41 primer pairs for nuclear simple sequence repeats (SSRs) developed from E. arundinaceus were used to assess the genetic diversity of 121 Erianthus germplasms collected in Thailand; of these primer pairs, 28 detected a total of 316 alleles. A Bayesian clustering approach with these alleles classified the accessions into four main groups, generally corresponding to the previous classification based on phenotypic analysis. The results of principal coordinate analysis and phylogenetic analysis of the 121 accessions on the basis of the SSR markers showed the same trend as Bayesian clustering, whereas sequence variations of three non-coding regions of chloroplast DNA revealed eight haplotypes among the accessions. The analysis of genetic structure and phylogenetic relationships, however, found some accessions whose classification contradicted the results of previous phenotypic classification. CONCLUSIONS: The molecular approach used in this study characterized the genetic diversity and relatedness of Erianthus germplasms collected across Thailand. This knowledge would allow efficient maintenance and conservation of the genetic resources of this grass and would help to use Erianthus species as breeding materials for development of novel bioenergy crops and sugarcane improvement.

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.

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.

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.

Transgenic superroots of Lotus corniculatus can be regenerated from superroot-derived leaves following Agrobacterium-mediated transformation.

Super-growing roots (superroots; SR), which have been established in the legume species Lotus corniculatus, are a fast-growing root culture that allows continuous root cloning, direct somatic embryogenesis and mass regeneration of plants under entirely growth regulator-free culture conditions. These features are unique for non-hairy root cultures, and they are now stably expressed since the culture was isolated more than 10 years ago (1997). Attempts to achieve direct and stable transformation of SR turned out to be unsuccessful. Making use of the supple regeneration plasticity of SR, we are reporting here an indirect transformation protocol. Leaf explants, derived from plants regenerated from SR, were inoculated with Agrobacterium tumefaciens strain LBA4404 harboring the binary vector pBI121, which contains the neomycin phosphotransferase II (NPTII) and beta-glucuronidase (GUS) genes as selectable and visual markers, respectively. After co-cultivation, the explants were selected on solidified MS medium with 0.5 mg/L benzylamino purine (BAP), 100 mg/L kanamycin and 250 mg/L cefotaxime. Kanamycin-resistant calli were transferred to liquid rooting medium. The newly regenerated, kanamycin-resistant roots were harvested and SR cultures re-established, which exhibited all the characteristics of the original SR. Furthermore, kanamycin-resistant roots cultured onto solidified MS medium supplemented with 0.5 mg/L BAP produced plants at the same rate as control SR. Six months after gene transfer, PCR analysis and histochemical locating indicated that the NPTII gene was integrated into the genome and that the GUS gene was regularly expressed in leaves, roots and nodules, respectively. The protocol makes it now possible to produce transformed SR and nodules as well as transgenic plants from transformed SR.

Green, herbicide-resistant plants by particle inflow gun-mediated gene transfer to diploid bahiagrass (Paspalum notatum).

We have established an efficient particle-bombardment transformation protocol for the diploid non-apomictic genotype of the warm season forage crop Paspalum notatum (bahiagrass). A vector containing a herbicide resistance gene (bar) together with the GUS reporter gene was used in transformation experiments. The bar gene confers resistance to the herbicide bialaphos. An improved culture system, highly regenerative callus, dense in compact polyembryogenic clusters, was produced on medium with a high CuSO4 content at elevated temperature. Target tissue (360 calli) produced under these conditions yielded 52 rooted plants on herbicide-containing medium, and 22 of these plants were PCR-positive. DNA gel blot analysis revealed a copy number of 1-5 for the GUS gene in different independent transformants. There was no correlation between copy number and GUS activity. While conventional cultures yielded exclusively albino plants on herbicide-containing medium, improved culture conditions for the target tissue resulted in the recovery of 100% green transgenic plants. All green herbicide-resistant regenerants were morphological normal and fertile.