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Geum geniculatum (Rosaceae), bent avens, is a perennial herb restricted to the high elevations of three mountaintops near the North Carolina/Tennessee border (USA). Although geographically restricted, occurrences on these mountaintops can have up to hundreds and occasionally thousands of individuals. While Geum geniculatum has been censused thoroughly, some sites have not been visited in over a decade, and formal biological studies are lacking. To understand genetic variation within the species and connectivity among populations, individuals were sampled from each of three extant mountain locations and were genotyped using 14 microsatellite markers previously developed for other congeners. Results show that G. geniculatum displays high genetic diversity, and that each mountain acts as a highly structured metapopulation with moderate interpopulation differentiation. Based on the high numbers of private alleles and results of an M-ratio test, genetic drift is likely driving structure and differentiation among metapopulations. Results will inform management of the species, and suggest that transfer of seeds or genetic material among sites should be minimized.

Premise Microsatellite markers were developed in the federally endangered species Liatris helleri (Asteraceae) to evaluate species boundaries with closely related congeners within the genus. Methods and results Using Illumina data, 17 primer pairs were developed in populations of L. helleri. The primers amplified motifs from tri- to hexanucleotide repeats with one to 17 alleles per locus. Primers were also tested for cross-amplification in L. aspera, L. microcephala, and L. pycnostachya. Conclusions The developed primers for L. helleri serve as a novel genetic tool for future investigations in this genus, allowing for more explicit species delineation as well as population genetic analyses.

Maize (Zea maysssp.mays) is not only one of the world’s most important crops, but it also is a powerful tool for studies of genetics, genomics, and cytology. The genome of maize shows the unmistakable signature of an ancient hybridization event followed by whole genome duplication (allopolyploidy), but the parents of this event have been a mystery for over a century, since studies of maize cytogenetics began. Here we show that the whole genome duplication event preceded the divergence of the entire genusZeaand its sister genusTripsacum. One genome was donated, in whole or in part, by a plant related to the modern African generaUrelytrumandVossia, although genomic rearrangement has been extensive. The other genome donor is less well-supported, but may have been related to the modernRottboellia-Hemarthriaclade, which is also African. ThusZeaandTripsacumtogether represent a New World radiation derived from African ancestors.

Highly repetitive regions have historically posed a challenge when investigating sequence variation and content. High-throughput sequencing has enabled researchers to use whole-genome shotgun sequencing to estimate the abundance of repetitive sequence, and these methodologies have been recently applied to centromeres. Previous research has investigated variation in centromere repeats across eukaryotes, positing that the highest abundance tandem repeat in a genome is often the centromeric repeat. To test this assumption, we used shotgun sequencing and a bioinformatic pipeline to identify common tandem repeats across a number of grass species. We find that de novo assembly and subsequent abundance ranking of repeats can successfully identify tandem repeats with homology to known tandem repeats. Fluorescent in-situ hybridization shows that de novo assembly and ranking of repeats from non-model taxa identifies chromosome domains rich in tandem repeats both near pericentromeres and elsewhere in the genome.

The role of polyploidy, particularly allopolyploidy, in plant diversification is a subject of debate. Whole-genome duplications precede the origins of many major clades (e.g., angiosperms, Brassicaceae, Poaceae), suggesting that polyploidy drives diversification. However, theoretical arguments and empirical studies suggest that polyploid lineages may actually have lower speciation rates and higher extinction rates than diploid lineages. We focus here on the grass tribe Andropogoneae, an economically and ecologically important group of C4 species with a high frequency of polyploids. A phylogeny was constructed for ca. 10% of the species of the clade, based on sequences of four concatenated low-copy nuclear loci. Genetic allopolyploidy was documented using the characteristic pattern of double-labeled gene trees. At least 32% of the species sampled are the result of genetic allopolyploidy and result from 28 distinct tetraploidy events plus an additional six hexaploidy events. This number is a minimum, and the actual frequency could be considerably higher. The parental genomes of most Andropogoneae polyploids diverged in the Late Miocene coincident with the expansion of the major C4 grasslands that dominate the earth today. The well-documented whole-genome duplication in Zea mays ssp. mays occurred after the divergence of Zea and Sorghum. We find no evidence that polyploidization is followed by an increase in net diversification rate; nonetheless, allopolyploidy itself is a major mode of speciation.

An extensive survey of the standing genetic variation in natural populations is among the priority steps in developing a species into a model system. In recent years, green foxtail (Setaria viridis), along with its domesticated form foxtail millet (S. italica), has rapidly become a promising new model system for C4 grasses and bioenergy crops, due to its rapid life cycle, large amount of seed production and small diploid genome, among other characters. However, remarkably little is known about the genetic diversity in natural populations of this species. In this study, we survey the genetic diversity of a worldwide sample of more than 200 S. viridis accessions, using the genotyping-by-sequencing technique. Two distinct genetic groups in S. viridis and a third group resembling S. italica were identified, with considerable admixture among the three groups. We find the genetic variation of North American S. viridis correlates with both geography and climate and is representative of the total genetic diversity in this species. This pattern may reflect several introduction/dispersal events of S. viridis into North America. We also modelled demographic history and show signal of recent population decline in one subgroup. Finally, we show linkage disequilibrium decay is rapid (

Geum radiatum is a federally endangered high-elevation rock-outcrop endemic herb that is widely recognized as a hexaploid and a relic species. Little is known about G. radiatum genetic diversity, population interactions, or the effect of past augmentations of populations. This study sampled every known population of G. radiatum and used microsatellite markers to measure genetic diversity and population structure. The analysis demonstrates that there is interconnectedness and structure among populations. In addition, the analysis was able to differentiate transplanted individuals and identify putative anthropogenically admixed individuals within augmented populations. Geum radiatum exhibits diversity within and among populations and current gene flow connects the northern populations. This information provides a greater understanding of the genetic sustainability of G. radiatum and what conservation efforts will most help this imperiled species to survive.

Sample sequence analysis was employed to investigate the repetitive DNAs that were most responsible for the evolved variation in genome content across seven panicoid grasses with >5-fold variation in genome size and different histories of polyploidy. In all cases, the most abundant repeats were LTR retrotransposons, but the particular families that had become dominant were found to be different in the Pennisetum, Saccharum, Sorghum and Zea lineages. One element family, Huck, has been very active in all of the studied species over the last few million years. This suggests the transmittal of an active or quiescent autonomous set of Huck elements to this lineage at the founding of the panicoids. Similarly, independent recent activity of Ji and Opie elements in Zea and of Leviathan elements in Sorghum and Saccharum species suggests that members of these families with exceptional activation potential were present in the genome(s) of the founders of these lineages. In a detailed analysis of the Zea lineage, the combined action of several families of LTR retrotransposons were observed to have approximately doubled the genome size of Zea luxurians relative to Zea mays and Zea diploperennis in just the last few million years. One of the LTR retrotransposon amplification bursts in Zea may have been initiated by polyploidy, but the great majority of transposable element activations are not. Instead, the results suggest random activation of a few or many LTR retrotransposons families in particular lineages over evolutionary time, with some families especially prone to future activation and hyper-amplification.

Eleven populations of witchweed, Striga hermonthica, were collected in four regions of Mali and investigated with 12 microsatellite markers. Extensive genetic diversity was observed, with most plants heterozygous for most markers. Allelic diversity was broadly distributed across populations with little genetic differentiation and large amounts of gene flow. Nearby fields of pearl millet and sorghum were found to have indistinguishable witchweed populations. Some population structure was apparent, but did not correlate with the local environment or host genotype, suggesting that seed transportation or other human-driven variables act to differentiate central Malian S. hermonthica populations from southern Malian populations.

A model of vicariance speciation has been proposed to explain the proliferation of psammophilic (sand loving) species in several endemic genera of scorpions. The current distribution of Paruroctonus utahensis (Williams, 1968) populations on isolated sand dunes seems to fit this model; therefore, this species was examined to test the model for psammophilic population distribution and haplotype variation. A portion of the mitochondrial rDNA gene was sequenced from 44 individuals representing 7 populations of P. utahensis. Three individuals of Paruroctonus gracilior (Hoffmann, 1931) and 1 individual of Paruroctonus boquillas Sissom and Henson, 1998, were used for outgroup comparison. A maximum parsimony heuristic search indicated that the basal populations were in the south and derived populations occurred in the northeastern portion of the range. A nested clade analysis found a statistically significant correlation between haplotypes and their geographical distribution at several clade levels. The biological cause of this association was best explained by allopatric fragmentation. Our data supported the vicariance model of isolation by fragmentation of a larger habitat to explain the variation seen among populations of P. utahensis.

Premise of the study: Microsatellite primers were developed for a widespread limestone endemic sedge, Carex eburnea, to facilitate investigation of the genetic diversity and phylogeography of this taxon and its closest relative, C. mckittrickensis. Methods and Results: Forty-eight primer pairs were designed from Illumina sequence data and screened for suitability. Fourteen of these primer pairs were polymorphic and generated one to seven alleles per locus. Cross-species amplifications were conducted for all four members of Carex sect. Albae. Conclusions: These primer pairs can be used to assess the genetic diversity and population structure in future studies of C. eburnea and C. mckittrickensis, and likely in other members of Carex sect. Albae.

Background and Aims Although monocotyledonous plants comprise one of the two major groups of angiosperms and include .65 000 species, comprehensive genome analysis has been focused mainly on the Poaceae (grass) family. Due to this bias, most of the conclusions that have been drawn for monocot genome evolution are based on grasses. It is not known whether these conclusions apply to many other monocots.† Methods To extend our understanding of genome evolution in the monocots, Asparagales genomic sequence data were acquired and the structural properties of asparagus and onion genomes were analysed. Speci?cally, several available onion and asparagus bacterial arti?cial chromosomes (BACs) with contig sizes .35 kb were annotated and analysed, with a particular focus on the characterization of long terminal repeat (LTR) retrotransposons.† Key Results The results reveal that LTR retrotransposons are the major components of the onion and garden aspara-gus genomes. These elements are mostly intact (i.e. with two LTRs), have mainly inserted within the past 6 million years and are piled up into nested structures. Analysis of shotgun genomic sequence data and the observation of two copies for some transposable elements (TEs) in annotated BACs indicates that some families have become particu-larly abundant, as high as 4–5 % (asparagus) or 3–4 % (onion) of the genome for the most abundant families, as also seen in large grass genomes such as wheat and maize.† Conclusions Although previous annotations of contiguous genomic sequences have suggested that LTR retrotran-sposons were highly fragmented in these two Asparagales genomes, the results presented here show that this was largely due to the methodology used. In contrast, this current work indicates an ensemble of genomic features similar to those observed in the Poaceae.

We generated a high-quality reference genome sequence for foxtail millet (Setaria italica). The ~400-Mb assembly covers ~80% of the genome and >95% of the gene space. The assembly was anchored to a 992-locus genetic map and was annotated by comparison with >1.3 million expressed sequence tag reads. We produced more than 580 million RNA-Seq reads to facilitate expression analyses. We also sequenced Setaria viridis, the ancestral wild relative of S. italica, and identified regions of differential single-nucleotide polymorphism density, distribution of transposable elements, small RNA content, chromosomal rearrangement and segregation distortion. The genus Setaria includes natural and cultivated species that demonstrate a wide capacity for adaptation. The genetic basis of this adaptation was investigated by comparing five sequenced grass genomes. We also used the diploid Setaria genome to evaluate the ongoing genome assembly of a related polyploid, switchgrass (Panicum virgatum).

Premise of the study: Twenty microsatellite loci were developed for the federally threatened species Hexastylis naniflora (Aristolochiaceae) to examine genetic diversity and to distinguish this species from co-occurring congeners, H. heterophylla and H. minor. Methods and Results: Next-generation sequencing approaches were used to identify microsatellite loci and design primers. One hundred fifty-two primer pairs were screened for repeatability, and 20 of these were further characterized for polymorphism. In H. naniflora, the number of alleles identified for polymorphic loci ranged from two to 23 (mean ∼8.8), with a mean heterozygosity of 0.39. Conclusions: These 16 polymorphic primers for H. naniflora will be useful tools in species identification and quantifying genetic diversity within the genus.

Maize (Zea mays or corn), both a major food source and an important cytogenetic model, evolved from a tetraploid that arose about 4.8 million years ago (Mya). As a result, maize has extensive duplicated regions within its genome. We have sequenced the two copies of one such region, generating 7.8 Mb of sequence spanning 17.4 cM of the short arm of chromosome 1 and 6.6 Mb (25.6 cM) from the long arm of chromosome 9. Rice, which did not undergo a similar whole genome duplication event, has only one orthologous region (4.9 Mb) on the short arm of chromosome 3, and can be used as reference for the maize homoeologous regions. Alignment of the three regions allowed identification of syntenic blocks, and indicated that the maize regions have undergone differential contraction in genic and intergenic regions and expansion by the insertion of retrotransposable elements. Approximately 9% of the predicted genes in each duplicated region are completely missing in the rice genome, and almost 20% have moved to other genomic locations. Predicted genes within these regions tend to be larger in maize than in rice, primarily because of the presence of predicted genes in maize with larger introns. Interestingly, the general gene methylation patterns in the maize homoeologous regions do not appear to have changed with contraction or expansion of their chromosomes. In addition, no differences in methylation of single genes and tandemly repeated gene copies have been detected. These results, therefore, provide new insights into the diploidization of polyploid species. ©2006 by Cold Spring Harbor Laboratory Press.

Background Many plant genomes are resistant to whole-genome assembly due to an abundance of repetitive sequence, leading to the development of gene-rich sequencing techniques. Two such techniques are hypomethylated partial restriction (HMPR) and methylation spanning linker libraries (MSLL). These libraries differ from other gene-rich datasets in having larger insert sizes, and the MSLL clones are designed to provide reads localized to "epigenetic boundaries" where methylation begins or ends. Results A large-scale study in maize generated 40,299 HMPR sequences and 80,723 MSLL sequences, including MSLL clones exceeding 100 kb. The paired end reads of MSLL and HMPR clones were shown to be effective in linking existing gene-rich sequences into scaffolds. In addition, it was shown that the MSLL clones can be used for anchoring these scaffolds to a BAC-based physical map. The MSLL end reads effectively identified epigenetic boundaries, as indicated by their preferential alignment to regions upstream and downstream from annotated genes. The ability to precisely map long stretches of fully methylated DNA sequence is a unique outcome of MSLL analysis, and was also shown to provide evidence for errors in gene identification. MSLL clones were observed to be significantly more repeat-rich in their interiors than in their end reads, confirming the correlation between methylation and retroelement content. Both MSLL and HMPR reads were found to be substantially gene-enriched, with the Sal I MSLL libraries being the most highly enriched (31% align to an EST contig), while the HMPR clones exhibited exceptional depletion of repetitive DNA (to ~11%). These two techniques were compared with other gene-enrichment methods, and shown to be complementary. Conclusion MSLL technology provides an unparalleled approach for mapping the epigenetic status of repetitive blocks and for identifying sequences mis-identified as genes. Although the types and natures of epigenetic boundaries are barely understood at this time, MSLL technology flags both approximate boundaries and methylated genes that deserve additional investigation. MSLL and HMPR sequences provide a valuable resource for maize genome annotation, and are a uniquely valuable complement to any plant genome sequencing project. In order to make these results fully accessible to the community, a web display was developed that shows the alignment of MSLL, HMPR, and other gene-rich sequences to the BACs; this display is continually updated with the latest ESTs and BAC sequences.