Molecular Basis of the Waxy Endosperm Starch Phenotype in Broomcorn Millet (Panicum miliaceum L.)

Waxy varieties of the tetraploid cereal broomcorn millet (Panicum miliaceum L.) have endosperm starch granules lacking detectable amylose. This study investigated the basis of this phenotype using molecular and biochemical methods. Iodine staining of starch granules in 72 plants from 38 landrace accessions found 58 nonwaxy and 14 waxy phenotype plants. All waxy types were in plants from Chinese and Korean accessions, a distribution similar to that of the waxy phenotype in other cereals. Granule-bound starch synthase I (GBSSI) protein was present in the endosperm of both nonwaxy and waxy individuals, but waxy types had little or no granule-bound starch synthase activity compared with the wild types. Sequencing of the GBSSI (Waxy) gene showed that this gene is present in two different forms (L and S) in P. miliaceum, which probably represent homeologues derived from two distinct diploid ancestors. Protein products of both these forms are present in starch granules. We identified three polymorphisms in the exon sequence coding for mature GBSSI peptides. A 15-bp deletion has occurred in the S type GBSSI, resulting in the loss of five amino acids from glucosyl transferase domain 1 (GTD1). The second GBSSI type (L) shows two sequence polymorphisms. One is the insertion of an adenine residue that causes a reading frameshift, and the second causes a cysteine–tyrosine amino acid polymorphism. These mutations appear to have occurred in parallel from the ancestral allele, resulting in three GBSSI-L alleles in total. Five of the six possible genotype combinations of the S and L alleles were observed. The deletion in the GBSSI-S gene causes loss of protein activity, and there was 100% correspondence between this deletion and the waxy phenotype. The frameshift mutation in the L gene results in the loss of L-type protein from starch granules. The L isoform with the tyrosine residue is present in starch granules but is nonfunctional. This loss of function may result from the substitution of tyrosine for cysteine, although it could not be determined whether the cysteine isoform of L represents the functional type. This is the first characterization of mutations that occur in combination in a functionally polyploid species to give a fully waxy phenotype

Rapid Sequencing of the Bamboo Mitochondrial Genome Using Illumina Technology and Parallel Episodic Evolution of Organelle Genomes in Grasses

Background: Compared to their counterparts in animals, the mitochondrial (mt) genomes of angiosperms exhibit a number of unique features. However, unravelling their evolution is hindered by the few completed genomes, of which are essentially Sanger sequenced. While next-generation sequencing technologies have revolutionized chloroplast genome sequencing, they are just beginning to be applied to angiosperm mt genomes. Chloroplast genomes of grasses (Poaceae) have undergone episodic evolution and the evolutionary rate was suggested to be correlated between chloroplast and mt genomes in Poaceae. It is interesting to investigate whether correlated rate change also occurred in grass mt genomes as expected under lineage effects. A time-calibrated phylogenetic tree is needed to examine rate change. Methodology/Principal Findings: We determined a largely completed mt genome from a bamboo, Ferrocalamus rimosivaginus (Poaceae), through Illumina sequencing of total DNA. With combination of de novo and reference-guided assembly, 39.5-fold coverage Illumina reads were finally assembled into scaffolds totalling 432,839 bp. The assembled genome contains nearly the same genes as the completed mt genomes in Poaceae. For examining evolutionary rate in grass mt genomes, we reconstructed a phylogenetic tree including 22 taxa based on 31 mt genes. The topology of the wellresolved tree was almost identical to that inferred from chloroplast genome with only minor difference. The inconsistency possibly derived from long branch attraction in mtDNA tree. By calculating absolute substitution rates, we found significan

High-Throughput Sequencing of Six Bamboo Chloroplast Genomes: Phylogenetic Implications for Temperate Woody Bamboos (Poaceae: Bambusoideae)

BACKGROUND: Bambusoideae is the only subfamily that contains woody members in the grass family, Poaceae. In phylogenetic analyses, Bambusoideae, Pooideae and Ehrhartoideae formed the BEP clade, yet the internal relationships of this clade are controversial. The distinctive life history (infrequent flowering and predominance of asexual reproduction) of woody bamboos makes them an interesting but taxonomically difficult group. Phylogenetic analyses based on large DNA fragments could only provide a moderate resolution of woody bamboo relationships, although a robust phylogenetic tree is needed to elucidate their evolutionary history. Phylogenomics is an alternative choice for resolving difficult phylogenies. METHODOLOGY/PRINCIPAL FINDINGS: Here we present the complete nucleotide sequences of six woody bamboo chloroplast (cp) genomes using Illumina sequencing. These genomes are similar to those of other grasses and rather conservative in evolution. We constructed a phylogeny of Poaceae from 24 complete cp genomes including 21 grass species. Within the BEP clade, we found strong support for a sister relationship between Bambusoideae and Pooideae. In a substantial improvement over prior studies, all six nodes within Bambusoideae were supported with ≥0.95 posterior probability from Bayesian inference and 5/6 nodes resolved with 100% bootstrap support in maximum parsimony and maximum likelihood analyses. We found that repeats in the cp genome could provide phylogenetic information, while caution is needed when using indels in phylogenetic analyses based on few selected genes. We also identified relatively rapidly evolving cp genome regions that have the potential to be used for further phylogenetic study in Bambusoideae. CONCLUSIONS/SIGNIFICANCE: The cp genome of Bambusoideae evolved slowly, and phylogenomics based on whole cp genome could be used to resolve major relationships within the subfamily. The difficulty in resolving the diversification among three clades of temperate woody bamboos, even with complete cp genome sequences, suggests that these lineages may have diverged very rapidly

DNA Barcode Sequence Identification Incorporating Taxonomic Hierarchy and within Taxon Variability

For DNA barcoding to succeed as a scientific endeavor an accurate and expeditious query sequence identification method is needed. Although a global multiple–sequence alignment can be generated for some barcoding markers (e.g. COI, rbcL), not all barcoding markers are as structurally conserved (e.g. matK). Thus, algorithms that depend on global multiple–sequence alignments are not universally applicable. Some sequence identification methods that use local pairwise alignments (e.g. BLAST) are unable to accurately differentiate between highly similar sequences and are not designed to cope with hierarchic phylogenetic relationships or within taxon variability. Here, I present a novel alignment–free sequence identification algorithm–BRONX–that accounts for observed within taxon variability and hierarchic relationships among taxa. BRONX identifies short variable segments and corresponding invariant flanking regions in reference sequences. These flanking regions are used to score variable regions in the query sequence without the production of a global multiple–sequence alignment. By incorporating observed within taxon variability into the scoring procedure, misidentifications arising from shared alleles/haplotypes are minimized. An explicit treatment of more inclusive terminals allows for separate identifications to be made for each taxonomic level and/or for user–defined terminals. BRONX performs better than all other methods when there is imperfect overlap between query and reference sequences (e.g. mini–barcode queries against a full–length barcode database). BRONX consistently produced better identifications at the genus–level for all query types

Multigenic phylogeny and analysis of tree incongruences in Triticeae (Poaceae)

Background: Introgressive events (e.g., hybridization, gene flow, horizontal gene transfer) and incomplete lineage sorting of ancestral polymorphisms are a challenge for phylogenetic analyses since different genes may exhibit conflicting genealogical histories. Grasses of the Triticeae tribe provide a particularly striking example of incongruence among gene trees. Previous phylogenies, mostly inferred with one gene, are in conflict for several taxon positions. Therefore, obtaining a resolved picture of relationships among genera and species of this tribe has been a challenging task. Here, we obtain the most comprehensive molecular dataset to date in Triticeae, including one chloroplastic and 26 nuclear genes. We aim to test whether it is possible to infer phylogenetic relationships in the face of (potentially) large-scale introgressive events and/or incomplete lineage sorting; to identify parts of the evolutionary history that have not evolved in a tree-like manner; and to decipher the biological causes of genetree conflicts in this tribe. Results: We obtain resolved phylogenetic hypotheses using the supermatrix and Bayesian Concordance Factors (BCF) approaches despite numerous incongruences among gene trees. These phylogenies suggest the existence of 4-5 major clades within Triticeae, with Psathyrostachys and Hordeum being the deepest genera. In addition, we construct a multigenic network that highlights parts of the Triticeae history that have not evolved in a tree-lik

Macroevolutionary Patterns in the Aphidini Aphids (Hemiptera: Aphididae): Diversification, Host Association, and Biogeographic Origins

, the most diverse genus in the family. We used a combined dataset of one nuclear and four mitochondrial DNA regions. A molecular dating approach, calibrated with fossil records, was used to estimate divergence times of these taxa.Most generic divergences in Aphidini occurred in the Middle Tertiary, and species-level divergences occurred between the Middle and Late Tertiary. The ancestral state of host use for Aphidini was equivocal with respect to three states: monoecy on trees, heteroecy, and monoecy on grasses. The ancestral state of Rhopalosiphina likely included both heteroecy and monoecy, whereas that of Aphidina was most likely monoecy. The divergence times of aphid lineages at the generic or subgeneric levels are close to those of their primary hosts. The species-level divergences in aphids are consistent with the diversification of the secondary hosts, as a few examples suggest. The biogeographic origin of Aphidini as a whole was equivocal, but the major lineages within Aphidina likely separated into Nearctic, Western Palearctic, and Eastern Palearctic regions.Most generic divergences in Aphidini occurred in the Middle Tertiary when primary hosts, mainly in the Rosaceae, were diverging, whereas species-level divergences were contemporaneous with diversification of the secondary hosts such as Poaceae in the Middle to Late Tertiary. Our results suggest that evolution of host alternation within Aphidini may have occurred during the Middle Tertiary (Oligocene) when the secondary hosts emerged

A Functional Phylogenomic View of the Seed Plants

A novel result of the current research is the development and implementation of a unique functional phylogenomic approach that explores the genomic origins of seed plant diversification. We first use 22,833 sets of orthologs from the nuclear genomes of 101 genera across land plants to reconstruct their phylogenetic relationships. One of the more salient results is the resolution of some enigmatic relationships in seed plant phylogeny, such as the placement of Gnetales as sister to the rest of the gymnosperms. In using this novel phylogenomic approach, we were also able to identify overrepresented functional gene ontology categories in genes that provide positive branch support for major nodes prompting new hypotheses for genes associated with the diversification of angiosperms. For example, RNA interference (RNAi) has played a significant role in the divergence of monocots from other angiosperms, which has experimental support in Arabidopsis and rice. This analysis also implied that the second largest subunit of RNA polymerase IV and V (NRPD2) played a prominent role in the divergence of gymnosperms. This hypothesis is supported by the lack of 24nt siRNA in conifers, the maternal control of small RNA in the seeds of flowering plants, and the emergence of double fertilization in angiosperms. Our approach takes advantage of genomic data to define orthologs, reconstruct relationships, and narrow down candidate genes involved in plant evolution within a phylogenomic view of species' diversification