NGS-Indel Coder v2.0: A Streamlined Pipeline to Code Indel Characters in Phylogenomic Data.

Hypothesized evolutionary insertions and deletions in nucleic acid sequences (indels) contain significant phylogenetic information and can be integrated in phylogenomic analyses. However, assemblies of short reads obtained from next-generation sequencing (NGS) technologies can contain errors that result in falsely inferred indels that need to be detected and omitted to avoid inclusion in phylogenetic analysis. Here, we detail the commands that comprise a new version of the NGS-Indel Coder pipeline, which was developed to validate indels using assembly read depth.

Settling a family feud: a high-level phylogenomic framework for the Gentianales based on 353 nuclear genes and partial plastomes.

PREMISE: Comprising five families that vastly differ in species richness-ranging from Gelsemiaceae with 13 species to the Rubiaceae with 13,775 species-members of the Gentianales are often among the most species-rich and abundant plants in tropical forests. Despite considerable phylogenetic work within particular families and genera, several alternative topologies for family-level relationships within Gentianales have been presented in previous studies. METHODS: Here we present a phylogenomic analysis based on nuclear genes targeted by the Angiosperms353 probe set for approximately 150 species, representing all families and approximately 85% of the formally recognized tribes. We were able to retrieve partial plastomes from off-target reads for most taxa and infer phylogenetic trees for comparison with the nuclear-derived trees. RESULTS: We recovered high support for over 80% of all nodes. The plastid and nuclear data are largely in agreement, except for some weakly to moderately supported relationships. We discuss the implications of our results for the order's classification, highlighting points of increased support for previously uncertain relationships. Rubiaceae is sister to a clade comprising (Gentianaceae + Gelsemiaceae) + (Apocynaceae + Loganiaceae). CONCLUSIONS: The higher-level phylogenetic relationships within Gentianales are confidently resolved. In contrast to recent studies, our results support the division of Rubiaceae into two subfamilies: Cinchonoideae and Rubioideae. We do not formally recognize Coptosapelteae and Luculieae within any particular subfamily but treat them as incertae sedis. Our framework paves the way for further work on the phylogenetics, biogeography, morphological evolution, and macroecology of this important group of flowering plants.

Enabling evolutionary studies at multiple scales in Apocynaceae through Hyb-Seq.

PREMISE: Apocynaceae is the 10th largest flowering plant family and a focus for study of plant-insect interactions, especially as mediated by secondary metabolites. However, it has few genomic resources relative to its size. Target capture sequencing is a powerful approach for genome reduction that facilitates studies requiring data from the nuclear genome in non-model taxa, such as Apocynaceae. METHODS: Transcriptomes were used to design probes for targeted sequencing of putatively single-copy nuclear genes across Apocynaceae. The sequences obtained were used to assess the success of the probe design, the intrageneric and intraspecific variation in the targeted genes, and the utility of the genes for inferring phylogeny. RESULTS: From 853 candidate nuclear genes, 835 were consistently recovered in single copy and were variable enough for phylogenomics. The inferred gene trees were useful for coalescent-based species tree analysis, which showed all subfamilies of Apocynaceae as monophyletic, while also resolving relationships among species within the genus Apocynum. Intraspecific comparison of Elytropus chilensis individuals revealed numerous single-nucleotide polymorphisms with potential for use in population-level studies. DISCUSSION: Community use of this Hyb-Seq probe set will facilitate and promote progress in the study of Apocynaceae across scales from population genomics to phylogenomics.

A draft genome and transcriptome of common milkweed (Asclepias syriaca) as resources for evolutionary, ecological, and molecular studies in milkweeds and Apocynaceae.

Milkweeds (Asclepias) are used in wide-ranging studies including floral development, pollination biology, plant-insect interactions and co-evolution, secondary metabolite chemistry, and rapid diversification. We present a transcriptome and draft nuclear genome assembly of the common milkweed, Asclepias syriaca. This reconstruction of the nuclear genome is augmented by linkage group information, adding to existing chloroplast and mitochondrial genomic resources for this member of the Apocynaceae subfamily Asclepiadoideae. The genome was sequenced to 80.4× depth and the draft assembly contains 54,266 scaffolds ≥1 kbp, with N50 = 3,415 bp, representing 37% (156.6 Mbp) of the estimated 420 Mbp genome. A total of 14,474 protein-coding genes were identified based on transcript evidence, closely related proteins, and ab initio models, and 95% of genes were annotated. A large proportion of gene space is represented in the assembly, with 96.7% of Asclepias transcripts, 88.4% of transcripts from the related genus Calotropis, and 90.6% of proteins from Coffea mapping to the assembly. Scaffolds covering 75 Mbp of the Asclepias assembly formed 11 linkage groups. Comparisons of these groups with pseudochromosomes in Coffea found that six chromosomes show consistent stability in gene content, while one may have a long history of fragmentation and rearrangement. The progesterone 5ß-reductase gene family, a key component of cardenolide production, is likely reduced in Asclepias relative to other Apocynaceae. The genome and transcriptome of common milkweed provide a rich resource for future studies of the ecology and evolution of a charismatic plant family.

NGS-Indel Coder: A pipeline to code indel characters in phylogenomic data with an example of its application in milkweeds (Asclepias).

Targeted genome sequencing approaches allow characterization of evolutionary relationships using a considerable number of nuclear genes and informative characters. However, most phylogenomic analyses only utilize single nucleotide polymorphisms (SNPs). Studies at the species level, especially in groups that have recently radiated, often recover low amounts of phylogenetically informative variation in coding regions, and require non-coding sequences, which are richer in indels, to resolve gene trees. Here, NGS-Indel Coder, a pipeline to detect and omit false positive indels inferred from assemblies of short read sequence data, was developed to resolve the relationships among and within major clades of the American milkweeds (Asclepias), which are the result of a rapid and recent evolutionary radiation, and whose phylogeny has been difficult to resolve. This pipeline was applied to a Hyb-Seq data set of 768 loci including targeted exons and flanking intron regions from 33 milkweed species. Robust species tree inference was improved by excluding small alignment partitions (<100 bp) that increased gene tree ambiguity and incongruence. To further investigate the robustness of indel coding, data sets that included small and large indels were explored, and species trees derived from concatenated loci versus coalescent methods based on gene trees were compared. The phylogeny of Asclepias obtained using nuclear data was well resolved, and phylogenetic information from indels improved resolution of specific nodes. The Temperate North American, Mexican Highland, and Incarnatae clades were well supported as monophyletic. Asclepias coulteri, which has been considered part of the Sonoran Desert clade based on plastome analyses, was placed as sister to all the other milkweed species studied here, rather than as a member of that clade. Two groups within the Temperate North American and Mexican clades were not resolved, and the inferred relationships strongly conflicted when comparing results based on data sets that did or did not include indel characters. This new pipeline represents a step forward in making maximal use of the information content in phylogenomic data sets.

Repeated translocation of a gene cassette drives sex-chromosome turnover in strawberries.

Turnovers of sex-determining systems represent important diversifying forces across eukaryotes. Shifts in sex chromosomes-but conservation of the master sex-determining genes-characterize distantly related animal lineages. Yet in plants, in which separate sexes have evolved repeatedly and sex chromosomes are typically homomorphic, we do not know whether such translocations drive sex-chromosome turnovers within closely related taxonomic groups. This phenomenon can only be demonstrated by identifying sex-associated nucleotide sequences, still largely unknown in plants. The wild North American octoploid strawberries (Fragaria) exhibit separate sexes (dioecy) with homomorphic, female heterogametic (ZW) inheritance, yet sex maps to three different chromosomes in different taxa. To characterize these turnovers, we identified sequences unique to females and assembled their reads into contigs. For most octoploid Fragaria taxa, a short (13 kb) sequence was observed in all females and never in males, implicating it as the sex-determining region (SDR). This female-specific "SDR cassette" contains both a gene with a known role in fruit and pollen production and a novel retrogene absent on Z and autosomal chromosomes. Phylogenetic comparison of SDR cassettes revealed three clades and a history of repeated translocation. Remarkably, the translocations can be ordered temporally due to the capture of adjacent sequence with each successive move. The accumulation of the "souvenir" sequence-and the resultant expansion of the hemizygous SDR over time-could have been adaptive by locking genes into linkage with sex. Terminal inverted repeats at the insertion borders suggest a means of movement. To our knowledge, this is the first plant SDR shown to be translocated, and it suggests a new mechanism ("move-lock-grow") for expansion and diversification of incipient sex chromosomes.

Evolution on the backbone: Apocynaceae phylogenomics and new perspectives on growth forms, flowers, and fruits.

PREMISE OF THE STUDY: We provide the largest phylogenetic analyses to date of Apocynaceae in terms of taxa and molecular data as a framework for analyzing the evolution of vegetative and reproductive traits. METHODS: We produced maximum-likelihood phylogenies of Apocynaceae using 21 plastid loci sampled from 1045 species (nearly 25% of the family) and complete plastomes from 73 species. We reconstructed ancestral states and used model comparisons in a likelihood framework to analyze character evolution across Apocynaceae. KEY RESULTS: We obtained a well-supported phylogeny of Apocynaceae, resolving poorly understood tribal and subtribal relationships (e.g., among Amsonieae and Hunterieae, within Asclepiadeae), rejecting monophyly of Melodineae and Odontadenieae, and placing previously unsampled and enigmatic taxa (e.g., Pycnobotrya). We provide new insights into the evolution of Apocynaceae, including frequent shifts between herbaceousness and woodiness, reversibility of twining, integrated evolution of the corolla and gynostegium, and ancestral baccate fruits. CONCLUSIONS: Increased sampling and selection of best-fitting models of evolution provide more resolved and robust estimates of phylogeny and character evolution than obtained in previous studies. Evolutionary inferences are sensitive to choice of phylogenetic frameworks and models.

Evolution at the tips: Asclepias phylogenomics and new perspectives on leaf surfaces.

PREMISE OF THE STUDY: Leaf surface traits, such as trichome density and wax production, mediate important ecological processes such as anti-herbivory defense and water-use efficiency. We present a phylogenetic analysis of Asclepias plastomes as a framework for analyzing the evolution of trichome density and presence of epicuticular waxes. METHODS: We produced a maximum-likelihood phylogeny using plastomes of 103 species of Asclepias. We reconstructed ancestral states and used model comparisons in a likelihood framework to analyze character evolution across Asclepias. KEY RESULTS: We resolved the backbone of Asclepias, placing the Sonoran Desert clade and Incarnatae clade as successive sisters to the remaining species. We present novel findings about leaf surface evolution of Asclepias-the ancestor is reconstructed as waxless and sparsely hairy, a macroevolutionary optimal trichome density is supported, and the rate of evolution of trichome density has accelerated. CONCLUSIONS: Increased sampling and selection of best-fitting models of evolution provide more resolved and robust estimates of phylogeny and character evolution than obtained in previous studies. Evolutionary inferences are more sensitive to character coding than model selection.

Evolution of pyrrolizidine alkaloid biosynthesis in Apocynaceae: revisiting the defence de-escalation hypothesis.

Plants produce specialized metabolites for their defence. However, specialist herbivores adapt to these compounds and use them for their own benefit. Plants attacked predominantly by specialists may be under selection to reduce or eliminate production of co-opted chemicals: the defence de-escalation hypothesis. We studied the evolution of pyrrolizidine alkaloids (PAs) in Apocynaceae, larval host plants for PA-adapted butterflies (Danainae, milkweed and clearwing butterflies), to test if the evolutionary pattern is consistent with de-escalation. We used the first PA biosynthesis specific enzyme (homospermidine synthase, HSS) as tool for reconstructing PA evolution. We found hss orthologues in diverse Apocynaceae species, not all of them known to produce PAs. The phylogenetic analysis showed a monophyletic origin of the putative hss sequences early in the evolution of one Apocynaceae lineage (the APSA clade). We found an hss pseudogene in Asclepias syriaca, a species known to produce cardiac glycosides but no PAs, and four losses of an HSS amino acid motif. APSA clade species are significantly more likely to be Danainae larval host plants than expected if all Apocynaceae species were equally likely to be exploited. Our findings are consistent with PA de-escalation as an adaptive response to specialist attack.

Draft Genome Sequence of Mentha longifolia and Development of Resources for Mint Cultivar Improvement.

The genus Mentha encompasses mint species cultivated for their essential oils, which are formulated into a vast array of consumer products. Desirable oil characteristics and resistance to the fungal disease Verticillium wilt are top priorities for the mint industry. However, cultivated mints have complex polyploid genomes and are sterile. Breeding efforts, therefore, require the development of genomic resources for fertile mint species. Here, we present draft de novo genome and plastome assemblies for a wilt-resistant South African accession of Mentha longifolia (L.) Huds., a diploid species ancestral to cultivated peppermint and spearmint. The 353 Mb genome contains 35 597 predicted protein-coding genes, including 292 disease resistance gene homologs, and nine genes determining essential oil characteristics. A genetic linkage map ordered 1397 genome scaffolds on 12 pseudochromosomes. More than two million simple sequence repeats were identified, which will facilitate molecular marker development. The M. longifolia genome is a valuable resource for both metabolic engineering and molecular breeding. This is exemplified by employing the genome sequence to clone and functionally characterize the promoters in a peppermint cultivar, and demonstrating the utility of a glandular trichome-specific promoter to increase expression of a biosynthetic gene, thereby modulating essential oil composition.

Phylogenetic marker development for target enrichment from transcriptome and genome skim data: the pipeline and its application in southern African Oxalis (Oxalidaceae).

Phylogenetics benefits from using a large number of putatively independent nuclear loci and their combination with other sources of information, such as the plastid and mitochondrial genomes. To facilitate the selection of orthologous low-copy nuclear (LCN) loci for phylogenetics in nonmodel organisms, we created an automated and interactive script to select hundreds of LCN loci by a comparison between transcriptome and genome skim data. We used our script to obtain LCN genes for southern African Oxalis (Oxalidaceae), a speciose plant lineage in the Greater Cape Floristic Region. This resulted in 1164 LCN genes greater than 600 bp. Using target enrichment combined with genome skimming (Hyb-Seq), we obtained on average 1141 LCN loci, nearly the whole plastid genome and the nrDNA cistron from 23 southern African Oxalis species. Despite a wide range of gene trees, the phylogeny based on the LCN genes was very robust, as retrieved through various gene and species tree reconstruction methods as well as concatenation. Cytonuclear discordance was strong. This indicates that organellar phylogenies alone are unlikely to represent the species tree and stresses the utility of Hyb-Seq in phylogenetics.

Intragenomic polymorphisms among high-copy loci: a genus-wide study of nuclear ribosomal DNA in Asclepias (Apocynaceae).

Despite knowledge that concerted evolution of high-copy loci is often imperfect, studies that investigate the extent of intragenomic polymorphisms and comparisons across a large number of species are rarely made. We present a bioinformatic pipeline for characterizing polymorphisms within an individual among copies of a high-copy locus. Results are presented for nuclear ribosomal DNA (nrDNA) across the milkweed genus, Asclepias. The 18S-26S portion of the nrDNA cistron of Asclepias syriaca served as a reference for assembly of the region from 124 samples representing 90 species of Asclepias. Reads were mapped back to each individual's consensus and at each position reads differing from the consensus were tallied using a custom perl script. Low frequency polymorphisms existed in all individuals (mean = 5.8%). Most nrDNA positions (91%) were polymorphic in at least one individual, with polymorphic sites being less frequent in subunit regions and loops. Highly polymorphic sites existed in each individual, with highest abundance in the "noncoding" ITS regions. Phylogenetic signal was present in the distribution of intragenomic polymorphisms across the genus. Intragenomic polymorphisms in nrDNA are common in Asclepias, being found at higher frequency than any other study to date. The high and variable frequency of polymorphisms across species highlights concerns that phylogenetic applications of nrDNA may be error-prone. The new analytical approach provided here is applicable to other taxa and other high-copy regions characterized by low coverage genome sequencing (genome skimming).

Hyb-Seq: Combining target enrichment and genome skimming for plant phylogenomics.

PREMISE OF THE STUDY: Hyb-Seq, the combination of target enrichment and genome skimming, allows simultaneous data collection for low-copy nuclear genes and high-copy genomic targets for plant systematics and evolution studies. • METHODS AND RESULTS: Genome and transcriptome assemblies for milkweed (Asclepias syriaca) were used to design enrichment probes for 3385 exons from 768 genes (>1.6 Mbp) followed by Illumina sequencing of enriched libraries. Hyb-Seq of 12 individuals (10 Asclepias species and two related genera) resulted in at least partial assembly of 92.6% of exons and 99.7% of genes and an average assembly length >2 Mbp. Importantly, complete plastomes and nuclear ribosomal DNA cistrons were assembled using off-target reads. Phylogenomic analyses demonstrated signal conflict between genomes. • CONCLUSIONS: The Hyb-Seq approach enables targeted sequencing of thousands of low-copy nuclear exons and flanking regions, as well as genome skimming of high-copy repeats and organellar genomes, to efficiently produce genome-scale data sets for phylogenomics.

Phylogenetic signal detection from an ancient rapid radiation: Effects of noise reduction, long-branch attraction, and model selection in crown clade Apocynaceae.

Crown clade Apocynaceae comprise seven primary lineages of lianas, shrubs, and herbs with a diversity of pollen aggregation morphologies including monads, tetrads, and pollinia, making them an ideal group for investigating the evolution and function of pollen packaging. Traditional molecular systematic approaches utilizing small amounts of sequence data have failed to resolve relationships along the spine of the crown clade, a likely ancient rapid radiation. The previous best estimate of the phylogeny was a five-way polytomy, leaving ambiguous the homology of aggregated pollen in two major lineages, the Periplocoideae, which possess pollen tetrads, and the milkweeds (Secamonoideae plus Asclepiadoideae), which possess pollinia. To assess whether greatly increased character sampling would resolve these relationships, a plastome sequence data matrix was assembled for 13 taxa of Apocynaceae, including nine newly generated complete plastomes, one partial new plastome, and three previously reported plastomes, collectively representing all primary crown clade lineages and outgroups. The effects of phylogenetic noise, long-branch attraction, and model selection (linked versus unlinked branch lengths among data partitions) were evaluated in a hypothesis-testing framework based on Shimodaira-Hasegawa tests. Discrimination among alternative crown clade resolutions was affected by all three factors. Exclusion of the noisiest alignment positions and topologies influenced by long-branch attraction resulted in a trichotomy along the spine of the crown clade consisting of Rhabdadenia+the Asian clade, Baisseeae+milkweeds, and Periplocoideae+the New World clade. Parsimony reconstruction on all optimal topologies after noise exclusion unambiguously supports parallel evolution of aggregated pollen in Periplocoideae (tetrads) and milkweeds (pollinia). Our phylogenomic approach has greatly advanced the resolution of one of the most perplexing radiations in Apocynaceae, providing the basis for study of convergent floral morphologies and their adaptive value.

Molecular phylogenetics of Amorpha (Fabaceae): an evaluation of monophyly, species relationships, and polyploid origins.

Amorpha L. (false indigos and lead plants) is a North American legume genus of 16 species of shrubs, which is most diverse in the southeastern United States and distinctive due to the reduction of the corolla to a single petal. Most species have limited distributions, but the tetraploid A. fruticosa species complex is widely distributed and its range overlaps those of all of the other species. Morphological variation in the genus is characterized by gradation of characters among species and it has been the subject of repeated taxonomic study due to the difficulty in delimiting species, especially among A. fruticosa and allies. This study presents the first phylogenetic and network analyses for evaluation of relationships amongst Amorpha species based on three non-coding plastome regions (trnD-trnT, trnH-psbA, petN-psbM) and two low-copy nuclear genes (CNGC5, minD). Plastid DNA analyses supported a monophyletic Amorpha with Parryella filifolia and Errazurizia rotundata as successive sister lineages; however, nuclear gene analyses supported the nesting of these two species and thus a paraphyletic Amorpha. Relationships among species of Amorpha were best resolved in the plastid DNA phylogeny and in most cases were concordant with expectations based on morphology. Relationships based on the nuclear gene phylogenies were less clear due to lack of informative variation (CNGC5) or conflict in the data set (minD). The origins of A. fruticosa were unclear, but the plastid phylogeny revealed that this species shares the same or similar plastid haplotype as other species in a geographic region. Putative recombination of diploid species' alleles was evident in the minD-like network. Phenotypic plasticity in combination with gene flow into this species from different diploids, or even tetraploids, across its range may account for the incredible morphological diversity of the A. fruticosa species complex. Putative progenitors for two other suspected allotetraploid species, A. confusa and A. crenulata, were identified as A. fruticosa and A. herbacea.

Horizontal transfer of DNA from the mitochondrial to the plastid genome and its subsequent evolution in milkweeds (apocynaceae).

Horizontal gene transfer (HGT) of DNA from the plastid to the nuclear and mitochondrial genomes of higher plants is a common phenomenon; however, plastid genomes (plastomes) are highly conserved and have generally been regarded as impervious to HGT. We sequenced the 158 kb plastome and the 690 kb mitochondrial genome of common milkweed (Asclepias syriaca [Apocynaceae]) and found evidence of intracellular HGT for a 2.4-kb segment of mitochondrial DNA to the rps2-rpoC2 intergenic spacer of the plastome. The transferred region contains an rpl2 pseudogene and is flanked by plastid sequence in the mitochondrial genome, including an rpoC2 pseudogene, which likely provided the mechanism for HGT back to the plastome through double-strand break repair involving homologous recombination. The plastome insertion is restricted to tribe Asclepiadeae of subfamily Asclepiadoideae, whereas the mitochondrial rpoC2 pseudogene is present throughout the subfamily, which confirms that the plastid to mitochondrial HGT event preceded the HGT to the plastome. Although the plastome insertion has been maintained in all lineages of Asclepiadoideae, it shows minimal evidence of transcription in A. syriaca and is likely nonfunctional. Furthermore, we found recent gene conversion of the mitochondrial rpoC2 pseudogene in Asclepias by the plastid gene, which reflects continued interaction of these genomes.

Navigating the tip of the genomic iceberg: Next-generation sequencing for plant systematics.

PREMISE OF THE STUDY: Just as Sanger sequencing did more than 20 years ago, next-generation sequencing (NGS) is poised to revolutionize plant systematics. By combining multiplexing approaches with NGS throughput, systematists may no longer need to choose between more taxa or more characters. Here we describe a genome skimming (shallow sequencing) approach for plant systematics. METHODS: Through simulations, we evaluated optimal sequencing depth and performance of single-end and paired-end short read sequences for assembly of nuclear ribosomal DNA (rDNA) and plastomes and addressed the effect of divergence on reference-guided plastome assembly. We also used simulations to identify potential phylogenetic markers from low-copy nuclear loci at different sequencing depths. We demonstrated the utility of genome skimming through phylogenetic analysis of the Sonoran Desert clade (SDC) of Asclepias (Apocynaceae). KEY RESULTS: Paired-end reads performed better than single-end reads. Minimum sequencing depths for high quality rDNA and plastome assemblies were 40× and 30×, respectively. Divergence from the reference significantly affected plastome assembly, but relatively similar references are available for most seed plants. Deeper rDNA sequencing is necessary to characterize intragenomic polymorphism. The low-copy fraction of the nuclear genome was readily surveyed, even at low sequencing depths. Nearly 160000 bp of sequence from three organelles provided evidence of phylogenetic incongruence in the SDC. CONCLUSIONS: Adoption of NGS will facilitate progress in plant systematics, as whole plastome and rDNA cistrons, partial mitochondrial genomes, and low-copy nuclear markers can now be efficiently obtained for molecular phylogenetics studies.

Building a model: developing genomic resources for common milkweed (Asclepias syriaca) with low coverage genome sequencing.

BACKGROUND: Milkweeds (Asclepias L.) have been extensively investigated in diverse areas of evolutionary biology and ecology; however, there are few genetic resources available to facilitate and compliment these studies. This study explored how low coverage genome sequencing of the common milkweed (Asclepias syriaca L.) could be useful in characterizing the genome of a plant without prior genomic information and for development of genomic resources as a step toward further developing A. syriaca as a model in ecology and evolution. RESULTS: A 0.5× genome of A. syriaca was produced using Illumina sequencing. A virtually complete chloroplast genome of 158,598 bp was assembled, revealing few repeats and loss of three genes: accD, clpP, and ycf1. A nearly complete rDNA cistron (18S-5.8S-26S; 7,541 bp) and 5S rDNA (120 bp) sequence were obtained. Assessment of polymorphism revealed that the rDNA cistron and 5S rDNA had 0.3% and 26.7% polymorphic sites, respectively. A partial mitochondrial genome sequence (130,764 bp), with identical gene content to tobacco, was also assembled. An initial characterization of repeat content indicated that Ty1/copia-like retroelements are the most common repeat type in the milkweed genome. At least one A. syriaca microread hit 88% of Catharanthus roseus (Apocynaceae) unigenes (median coverage of 0.29×) and 66% of single copy orthologs (COSII) in asterids (median coverage of 0.14×). From this partial characterization of the A. syriaca genome, markers for population genetics (microsatellites) and phylogenetics (low-copy nuclear genes) studies were developed. CONCLUSIONS: The results highlight the promise of next generation sequencing for development of genomic resources for any organism. Low coverage genome sequencing allows characterization of the high copy fraction of the genome and exploration of the low copy fraction of the genome, which facilitate the development of molecular tools for further study of a target species and its relatives. This study represents a first step in the development of a community resource for further study of plant-insect co-evolution, anti-herbivore defense, floral developmental genetics, reproductive biology, chemical evolution, population genetics, and comparative genomics using milkweeds, and A. syriaca in particular, as ecological and evolutionary models.

Development of microsatellite markers in Lupinus luteus (Fabaceae) and cross-species amplification in other lupine species.

PREMISE OF THE STUDY: Microsatellite primers were developed in Lupinus luteus L., an emerging temperate protein crop, to investigate genetic diversity, population structure, and to facilitate the generation of better yellow lupine varieties. • METHODS AND RESULTS: Thirteen polymorphic primer sets were evaluated in a European and Eastern European accession collection of L. luteus. The primers amplified di-, tri-, and tetranucleotide repeats with 2-4 alleles per locus. These revealed a moderate to low level of genetic variation, as indicated by an average observed heterozygosity of 0.0126. Select loci also amplified successfully in the closely related species L. hispanicus Boiss. & Reut. and in the New World species L. mutabilis Sweet. • CONCLUSIONS: These results indicate the utility of primers for the study of genetic diversity across L. luteus populations and related lupine species. The use of these microsatellite markers will facilitate the implementation of several molecular breeding strategies in yellow lupine.

Conservation genetics of Amorpha georgiana (Fabaceae), an endangered legume of the Southeastern United States.

Amorpha georgiana (Fabaceae) is an endangered legume species found in longleaf pine savannas in the Southeastern United States. Approximately 900 individuals and 14 populations remain, most of which are concentrated in North Carolina. Eleven microsatellite loci were used to explore genetic diversity, population structure and recent population bottlenecks using genotypic data from 132 individuals collected at ten different localities. Although A. georgiana is quite rare, it exhibited high levels of genetic diversity (17.7 alleles/locus; H(o) = 0.65, H(E) = 0.75). Most of the genetic variation was found within rather than between populations of this species. The single remaining Georgia population was well differentiated from populations of the Carolinas (F(ST) > 0.1), which had weaker structure among them (F(ST) < 0.1). Only a geographically disjunct population showed strong evidence of a recent population bottleneck, perhaps due to a recent founder event. Hybridization with A. herbacea was also detected. For conservation management plans, A. georgiana populations in each geographic region (North Carolina, South Carolina and Georgia) plus a disjunct population in North Carolina (Holly Shelter) should be treated as separate management units for which in situ conservation, including habitat restoration and use of prescribed burns, should ensure persistence of this species and preservation of its evolutionary potential.