Explore the vast range of titles available.

The last several years have seen revolutionary advances in DNA sequencing technologies with the advent of next-generation sequencing (NGS) techniques. NGS methods now allow millions of bases to be sequenced in one round, at a fraction of the cost relative to traditional Sanger sequencing. As costs and capabilities of these technologies continue to improve, we are only beginning to see the possibilities of NGS platforms, which are developing in parallel with online availability of a wide range of biological data sets and scientific publications and allowing us to address a variety of questions not possible before. As techniques and data sets continue to improve and grow, we are rapidly moving to the point where every organism, not just select “model organisms”, is open to the power of NGS. This volume presents a brief synopsis of NGS technologies and the development of exemplary applications of such methods in the fields of molecular marker development, hybridization and introgression, transcriptome investigations, phylogenetic and ecological studies, polyploid genetics, and applications for large genebank collections.

Capsaicinoids are compounds synthesized exclusively in the genus Capsicum and are responsible for the burning sensation experienced when consuming hot pepper fruits. To date, only one gene, AT3, a member of the BAHD family of acyltransferases, is currently known to have a measurable quantitative effect on capsaicinoid biosynthesis. Multiple AT3 paralogs exist in the Capsicum genome, but their evolutionary relationships have not been characterized well. Recessive alleles at this locus result in absence of capsaicinoids in pepper fruit. To explore the evolution of AT3 in Capsicum and the Solanaceae, we sequenced this gene from diverse Capsicum genotypes and species, along with a number of representative solanaceous taxa. Our results revealed that the coding region of AT3 is highly conserved throughout the family. Further, we uncovered a tandem duplication that predates the diversification of the Solanaceae taxa sampled in this study. This pair of tandem duplications were designated AT3-1 and AT3-2. Sequence alignments showed that the AT3-2 locus, a pseudogene, retains regions of amino acid conservation relative to AT3-1. Gene tree estimation demonstrated that AT3-1 and AT3-2 form well supported, distinct clades. In C. rhomboideum, a non-pungent basal Capsicum species, we describe a recombination event between AT3-1 and AT3-2 that modified the putative active site of AT3-1, also resulting in a frame-shift mutation in the second exon. Our data suggest that duplication of the original AT3 representative, in combination with divergence and pseudogene degeneration, may account for the patterns of sequence divergence and punctuated amino acid conservation observed in this study. Further, an early rearrangement in C. rhomboidium could account for the absence of pungency in this Capsicum species.

As a first step in innate immunity, pattern recognition receptors (PRRs) recognize the distinct pathogen and herbivore-associated molecular patterns and mediate activation of immune responses, but specific steps in the evolution of new PRR sensing functions are not well understood. We employed comparative genomic and functional analyses to define evolutionary events leading to the sensing of the herbivore-associated peptide inceptin (In11) by the PRR inceptin receptor (INR) in legume plant species. Existing and de novo genome assemblies revealed that the presence of a functional INR gene corresponded with ability to respond to In11 across ~53 million years (my) of evolution. In11 recognition is unique to the clade of Phaseoloid legumes, and only a single clade of INR homologs from Phaseoloids was functional in a heterologous model. The syntenic loci of several non-Phaseoloid outgroup species nonetheless contain non-functional INR-like homologs, suggesting that an ancestral gene insertion event and diversification preceded the evolution of a specific INR receptor function ~28 my ago. Chimeric and ancestrally reconstructed receptors indicated that 16 amino acid differences in the C1 leucine-rich repeat domain and C2 intervening motif mediate gain of In11 recognition. Thus, high PRR diversity was likely followed by a small number of mutations to expand innate immune recognition to a novel peptide elicitor. Analysis of INR evolution provides a model for functional diversification of other germline-encoded PRRs. The health status of a plant depends on the immune system it inherits from its parents. Plants have many receptor proteins that can recognize distinct molecules from insects and microbes, and trigger an immune response. Inheriting the right set of receptors allows plants to detect certain threats and to cope with diseases and pests. Soybeans, chickpeas and other closely-related crop plants belong to a family of plants known as the legumes. Previous studies have found that, unlike other plants, some legumes are able to respond to oral secretions from caterpillars. These plants have a receptor known as INR that binds to a molecule called inceptin in the secretions. However, it remained unclear how or when INR evolved. To address this gap, Snoeck et al. tested immune responses to inceptin in the leaves of 22 species of legume. The experiments revealed that only members of a subgroup of legumes called the Phaseoloids were able to recognize the molecule. Analyzing the genomes of several legume species revealed that the gene encoding INR first emerged around 28 million years ago. Among the descendants of the legumes that first evolved this receptor, only the crop plant soybean and a few other species were unable to respond to inceptin. The genomic data indicated that these species had in fact lost the gene encoding INR over evolutionary time. Snoeck et al. then combined data from genes encoding modern-day receptors to reconstruct the sequence of building blocks that make up the 28-million-year-old version of INR. This ancestral receptor was able to respond to inceptin in the caterpillar secretion, whereas an older version of the protein, which had a slightly different set of building blocks, could not. This suggests that INR evolved the ability to respond to inceptin as a result of small mutations in the gene encoding a more ancient receptor. The work of Snoeck et al. reveals how the Phaseoloids evolved to respond to caterpillars, and how this ability has been lost in soybeans and other members of the subgroup. In the future, these findings may aid plant breeding or genetic engineering approaches for enhancing soybeans and other crops resistance to caterpillar pests.

Horizontal gene transfer (HGT) involves the nonsexual transmission of genetic material across species boundaries. Although often detected in prokaryotes, examples of HGT involving animals are relatively rare, and any evolutionary advantage conferred to the recipient is typically obscure. We identified a gene (HhMAN1) from the coffee berry borer beetle, Hypothenemus hampei, a devastating pest of coffee, which shows clear evidence of HGT from bacteria. HhMAN1 encodes a mannanase, representing a class of glycosyl hydrolases that has not previously been reported in insects. Recombinant HhMAN1 protein hydrolyzes coffee berry galactomannan, the major storage polysaccharide in this species and the presumed food of H. hampei. HhMAN1 was found to be widespread in a broad biogeographic survey of H. hampei accessions, indicating that the HGT event occurred before radiation of the insect from West Africa to Asia and South America. However, the gene was not detected in the closely related species H. obscurus (the tropical nut borer or \"false berry borer\"), which does not colonize coffee beans. Thus, HGT of HhMAN1 from bacteria represents a likely adaptation to a specific ecological niche and may have been promoted by intensive agricultural practices.

Background The medicinal legume Mucuna pruriens (L.) DC. has attracted attention worldwide as a source of the anti-Parkinson’s drug L-Dopa. It is also a popular green manure cover crop that offers many agronomic benefits including high protein content, nitrogen fixation and soil nutrients. The plant currently lacks genomic resources and there is limited knowledge on gene expression, metabolic pathways, and genetics of secondary metabolite production. Here, we present transcriptomic resources for M. pruriens , including a de novo transcriptome assembly and annotation, as well as differential transcript expression analyses between root, leaf, and pod tissues. We also develop microsatellite markers and analyze genetic diversity and population structure within a set of Indian germplasm accessions. Results One-hundred ninety-one million two hundred thirty-three thousand two hundred forty-two bp cleaned reads were assembled into 67,561 transcripts with mean length of 626 bp and N50 of 987 bp. Assembled sequences were annotated using BLASTX against public databases with over 80% of transcripts annotated. We identified 7,493 simple sequence repeat (SSR) motifs, including 787 polymorphic repeats between the parents of a mapping population. 134 SSRs from expressed sequenced tags (ESTs) were screened against 23  M. pruriens accessions from India, with 52 EST-SSRs retained after quality control. Population structure analysis using a Bayesian framework implemented in fastSTRUCTURE showed nearly similar groupings as with distance-based (neighbor-joining) and principal component analyses, with most of the accessions clustering per geographical origins. Pair-wise comparison of transcript expression in leaves, roots and pods identified 4,387 differentially expressed transcripts with the highest number occurring between roots and leaves. Differentially expressed transcripts were enriched with transcription factors and transcripts annotated as belonging to secondary metabolite pathways. Conclusions The M. pruriens transcriptomic resources generated in this study provide foundational resources for gene discovery and development of molecular markers. Polymorphic SSRs identified can be used for genetic diversity, marker-trait analyses, and development of functional markers for crop improvement. The results of differential expression studies can be used to investigate genes involved in L-Dopa synthesis and other key metabolic pathways in M. pruriens .

It is widely recognized that many genes and lineages do not adhere to a molecular clock, yet molecular clocks are commonly used to date divergences in comparative genomic studies. We test the application of a molecular clock across genes and lineages in a phylogenetic framework utilizing 12 genes linked in a 1-Mb region on chromosome 13 of soybean (Glycine max); homoeologous copies of these genes formed by polyploidy in Glycine; and orthologous copies in G. tomentella, Phaseolus vulgaris, and Medicago truncatula. We compare divergence dates estimated by two methods each in three frameworks: a global molecular clock with a single rate across genes and lineages using full and approximate likelihood methods based on synonymous substitutions, a gene-specific clock assuming rate constancy over lineages but allowing a different rate for each gene, and a relaxed molecular clock where rates may vary across genes and lineages estimated under penalized likelihood and Bayesian inference. We use the cumulative variance across genes as a means of quantifying precision. Our results suggest that divergence dating methods produce results that are correlated, but that older nodes are more variable and more difficult to estimate with precision and accuracy. We also find that models incorporating less rate heterogeneity estimate older dates of divergence than more complex models, as node age increases. A mixed model nested analysis of variance testing the effects of framework, method, and gene found that framework had a significant effect on the divergence date estimates but that most variation among dates is due to variation among genes, suggesting a need to further characterize and understand the evolutionary phenomena underlying rate variation within genomes, among genes, and across lineages.

Background Kudzu, Pueraria montana var. lobata , is a woody vine native to Southeast Asia that has been introduced globally for cattle forage and erosion control. The vine is highly invasive in its introduced areas, including the southeastern US. Modern molecular marker resources are limited for the species, despite its importance. Transcriptomes for P. montana var. lobata and a second phaseoloid legume taxon previously ascribed to genus Pueraria , Neustanthus phaseoloides, were generated and mined for microsatellites and single nucleotide polymorphisms. Results Roche 454 sequencing of P. montana var. lobata and N. phaseoloides transcriptomes produced read numbers ranging from ~ 280,000 to ~ 420,000. Trinity assemblies produced an average of 17,491 contigs with mean lengths ranging from 639 bp to 994 bp. Transcriptome completeness, according to BUSCO, ranged between 64 and 77%. After vetting for primer design, there were 1646 expressed simple sequence repeats (eSSRs) identified in P. montana var. lobata and 1459 in N. phaseoloides. From these eSSRs, 17 identical primer pairs, representing inter-generic phaseoloid eSSRs, were created. Additionally, 13 primer pairs specific to P. montana var. lobata were also created. From these 30 primer pairs, a final set of seven primer pairs were used on 68 individuals of P. montana var. lobata for characterization across the US, China, and Japan. The populations exhibited from 20 to 43 alleles across the seven loci. We also conducted pairwise tests for high-confidence SNP discovery from the kudzu transcriptomes we sequenced and two previously sequenced P. montana var. lobata transcriptomes. Pairwise comparisons between P. montana var. lobata ranged from 358 to 24,475 SNPs, while comparisons between P. montana var. lobata and N. phaseoloides ranged from 5185 to 30,143 SNPs. Conclusions The discovered molecular markers for kudzu provide a starting point for comparative genetic studies within phaseoloid legumes. This study both adds to the current genetic resources and presents the first available genomic resources for the invasive kudzu vine. Additionally, this study is the first to provide molecular evidence to support the hypothesis of Japan as a source of US kudzu and begins to narrow the origin of US kudzu to the central Japanese island of Honshu.

Pediomelum is a genus endemic to North America comprising about 26 species, including the megalanthum complex, which consists of Pediomelum megalanthum and its varieties retrorsum and megalanthum , Pediomelum mephiticum , and the recently described Pediomelum verdiense and Pediomelum pauperitense . Historically, species of the megalanthum complex have been variably recognized at the species or variety levels, dependent upon the relative importance of morphological characters as diagnostic of species. Ten quantitative morphological characters regarded as diagnostic at the species level were analyzed using multivariate morphometrics across these taxa in order to examine the discriminatory power of these characters to delineate species and to aid in species delimitation. The analyses support the recognition of Pediomelum megalanthum , Pediomelum mephiticum , and Pediomelum verdiense at the species level, Pediomelum retrorsum as a variety under Pediomelum megalanthum , and suggest the sinking of Pediomelum pauperitense into Pediomelum verdiense . The findings of the present study help quantify the power of certain characters at delimiting taxa and provide a basis for taxonomic revision of the Pediomelum megalanthum complex.

Polyploidy is a widespread speciation mechanism, particularly in plants. Estimating the time of origin of polyploid species is important for understanding issues such as gene loss and changes in regulation and expression among homoeologous copies that coexist in a single genome owing to polyploidy. Polyploid species can originate in various ways; the effects of mode of origin, genetic system, and sampling on estimates of the age of polyploid origin using distances between alleles of polyploids and their diploid progenitors, or between homoeologous loci in a polyploid genome, are explored. Even in the simplest cases, simulations confirm that different loci are expected to give very different estimates of the date of origin. The time of polyploid origin is at least as old as the time estimated from comparison of an allele sampled from the polyploid with the most closely related allele in the diploid progenitor. The polyploidy literature often does not make clear the longstanding observation that the divergence of homoeologous copies in an allopolyploid tracks the divergence of diploid species, not the origin of the polyploid. Estimating the date of origin of a polyploid is difficult, and in some circumstances impossible. Skepticism about dates of polyploid origins is clearly warranted.

Two groups independently and seemingly simultaneously published the new combination of Pediomelum pariense, the Paria River breadroot. Both names were published in the second-quarter issues of separate journals in 1986. Same issue dates have led to confusion over which name takes precedence and is thus accepted. However, one issue was delayed in printing. Evidence is provided herein for acceptance of Pediomelum pariense (S.L. Welsh & N.D. Atwood) J.W. Grimes over Pediomelum pariense (S.L. Welsh & N.D. Atwood) S.L. Welsh & N.D. Atwood.