| Miscanthus (subtribe Saccharinae, tribe Andropogoneae, family Poaceae) is a genus of temperate perennial C4 grasses. This study (chaper 2) combines information on genome size and ploidy levels and DNA sequence variation to classify Miscanthus accessions to aid potential biomass crop improvement programs and to investigate the evolution of the genus. We observed that Miscanthus accessions fell into 4 groups, including section Miscanthus, section Triarrhena and two groups intermediate between two sections, based on morphology and genome size. Sixteen simple sequence repeat (SSR) primer pairs were selected based on amplification and polymorphism across three genera, Miscanthus, Saccharum and Erianthus. Morphology, genome size and SSR genotyping of 42 accessions including diploid and triploid interspecific hybrid progeny suggested that there are three Miscanthus species (M. sinensis, M. sacchariflorus and M. x giganteus), one M. sacchariflorus variety and one putative hybrid among Miscanthus accessions, which were clearly separated from the other two genera. The species status of M. floridulus remains in question. The evolution of Miscanthus and related genera is discussed based on genome size, ploidy level, cluster analysis and geographical distribution.;In chapter 3, a cross between two M. sinensis accessions, 'Grosse fontaine' and 'Undine' was made to produce 221 segrating progeny as a mapping population. Simple sequence repeat (SSR) markers from sugarcane expressed sequence tags (EST) and genomic sequences were screened in the two parental M. sinensis accessions. Single nucleotide polymorphism (SNP) markers from deep transcriptome sequencing (RNAseq) were also used for map construction. A total of 210 SSR markers and 658 single nucleotide polymorphism (SNP) markers were validated via segregation in the full sib F1 mapping population. A genetic map for M. sinensis was constructed that was resolved into 19 linkage groups, the haploid chromosome number expected from cytological evidence. Comparative genomic analysis revealed genome-wide duplication in Miscanthus relative to S. bicolor, with subsequent insertional fusion of a pair of chromosomes. The utility of the map is confirmed by the identification of two paralogous C 4-pyruvate, phosphate dikinase (C4-PPDK) loci in Miscanthus, at positions syntenic to the single orthologous gene in sorghum. The M. sinensis map and comparative mapping with sorghum suggests that the genus Miscanthus experienced an ancestral tetraploidy and chromosome fusion prior to its diversification, but after its divergence from the closely related sugarcane clade. The genetic map for Miscanthus is useful in biological discovery and breeding efforts to improve this emerging biofuel crop, and also provide a valuable resource for understanding genomic responses to tetraploidy and chromosome fusion.;In chapter 4, artificial genome doubling of various Miscanthus accessions with antimototic agents was used to understand the phenotypic responses to whole genome duplication in Miscanthus. Interspecific manipulation of ploidy levels is also a potential strategy for Miscanthus crop improvement to generate superior germplasm and to circumvent reproductive barriers for the introduction of new genetic variation into core germplasm. Therefore, synthetic autotetraploid lines of M. sacchariflorus and M. sinensis, and autoallohexaploid M. x giganteus were produced in tissue culture from oryzalin treatments to seed- and immature inflorescence-derived callus lines. Genome doubling of diploid M. sinensis, M. sacchariflorus, and triploid M. x giganteus to generate tetraploid and hexaploid lines was confirmed by stomata size, nuclear DNA content, and chromosome counts. A putative pentaploid line was also identified among the M. x giganteus synthetic polyploid lines by nuclear DNA content and chromosome counts. Comparisons of phenotypic performance of synthetic polyploid lines with their diploid and triploid progenitors in the greenhouse found species-specific differences in plant tiller number, height, and flowering time among the doubled lines. Stem diameter tended to increase after polyploidization but there were no significant improvement in biomass traits. (Abstract shortened by UMI.). |
