Statistical method for testing the neutral mutation hypothesis by DNA polymorphism.

Advances in next generation technologies have driven the costs of DNA sequencing down to the point that genotyping-by-sequencing (GBS) is now feasible for high diversity, large genome species. Here, we report a procedure for constructing GBS libraries based on reducing genome complexity with restriction enzymes (REs). This approach is simple, quick, extremely specific, highly reproducible, and may reach important regions of the genome that are inaccessible to sequence capture approaches. By using methylation-sensitive REs, repetitive regions of genomes can be avoided and lower copy regions targeted with two to three fold higher efficiency. This tremendously simplifies computationally challenging alignment problems in species with high levels of genetic diversity. The GBS procedure is demonstrated with maize (IBM) and barley (Oregon Wolfe Barley) recombinant inbred populations where roughly 200,000 and 25,000 sequence tags were mapped, respectively. An advantage in species like barley that lack a complete genome sequence is that a reference map need only be developed around the restriction sites, and this can be done in the process of sample genotyping. In such cases, the consensus of the read clusters across the sequence tagged sites becomes the reference. Alternatively, for kinship analyses in the absence of a reference genome, the sequence tags can simply be treated as dominant markers. Future application of GBS to breeding, conservation, and global species and population surveys may allow plant breeders to conduct genomic selection on a novel germplasm or species without first having to develop any prior molecular tools, or conservation biologists to determine population structure without prior knowledge of the genome or diversity in the species.

/pdf/a-robust-simple-genotyping-by-sequencing-gbs-approach-for-2orahkct9n.pdf

A Robust, Simple Genotyping-by-Sequencing (GBS) Approach for High Diversity Species

BIOE 402. Medical Technology Assessment. 2 or 3 hours. Bioentrepreneur course. Assessment of medical technology in the context of commercialization. Objectives, competition, market share, funding, pricing, manufacturing, growth, and intellectual property; many issues unique to biomedical products. Course Information: 2 undergraduate hours. 3 graduate hours. Prerequisite(s): Junior standing or above and consent of the instructor.

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where A represents the magnetic vector potential, is an integral of the hydromagnetic equations. This -integral made it possible to formulate a variational principle for the force-free magnetic fields. The integral expresses the fact that motions cannot transform a given field in an entirely arbitrary different field, if the conductivity of the medium isconsidered infinite. In this paper we shall show that the full set of hydromagnetic equations admit five more integrals, besides the energy integral, if dissipative processes are absent. These integrals, as we shall presently verify, are I2 =fbHvdV, (2)

Proceedings of the National Academy of Sciences

Although the chloroplast genome contains many noncoding regions, relatively few have been exploited for interspecific phylogenetic and intraspecific phylogeographic studies. In our recent evaluation of the phylogenetic utility of 21 noncoding chloroplast regions, we found the most widely used noncoding regions are among the least variable, but the more variable regions have rarely been employed. That study led us to conclude that there may be unexplored regions of the chloroplast genome that have even higher relative levels of variability. To explore the potential variability of previously unexplored regions, we compared three pairs of single-copy chloroplast genome sequences in three disparate angiosperm lineages: Atropa vs. Nicotiana (asterids); Lotus vs. Medicago (rosids); and Saccharum vs. Oryza (monocots). These three separate sequence alignments highlighted 13 mutational hotspots that may be more variable than the best regions of our former study. These 13 regions were then selected for a more detailed analysis. Here we show that nine of these newly explored regions (rpl32-trnL((UAG)), trnQ((UUG))-5'rps16, 3'trnV((UAC))-ndhC, ndhF-rpl32, psbD-trnT((GGU)), psbJ-petA, 3'rps16-5'trnK((UUU)), atpI-atpH, and petL-psbE) offer levels of variation better than the best regions identified in our earlier study and are therefore likely to be the best choices for molecular studies at low taxonomic levels.

/pdf/comparison-of-whole-chloroplast-genome-sequences-to-choose-2ezw8t9zef.pdf

Comparison of whole chloroplast genome sequences to choose noncoding regions for phylogenetic studies in angiosperms: the tortoise and the hare III

Most eukaryotes have genomes that exhibit high levels of gene redundancy, much of which seems to have arisen from one or more cycles of genome doubling. Polyploidy has been particularly prominent during flowering plant evolution, yielding duplicated genes (homoeologs) whose expression may be retained or lost either as an immediate consequence of polyploidization or on an evolutionary timescale. Expression of 40 homoeologous gene pairs was assayed by cDNA-single-stranded conformation polymorphism in natural (1- to 2-million-yr-old) and synthetic tetraploid cotton (Gossypium) to determine whether homoeologous gene pairs are expressed at equal levels after polyploid formation. Silencing or unequal expression of one homoeolog was documented for 10 of 40 genes examined in ovules of Gossypium hirsutum. Assays of homoeolog expression in 10 organs revealed variable expression levels and silencing, depending on the gene and organ examined. Remarkably, silencing and biased expression of some gene pairs are reciprocal and developmentally regulated, with one homoeolog showing silencing in some organs and the other being silenced in other organs, suggesting rapid subfunctionalization. Duplicate gene expression was examined in additional natural polyploids to characterize the pace at which expression alteration evolves. Analysis of a synthetic tetraploid revealed homoeolog expression and silencing patterns that sometimes mirrored those of the natural tetraploid. Both long-term and immediate responses to polyploidization were implicated. Data suggest that some silencing events are epigenetically induced during the allopolyploidization process.

https://www.pnas.org/content/pnas/100/8/4649.full.pdf

Genes duplicated by polyploidy show unequal contributions to the transcriptome and organ-specific reciprocal silencing

The cotton genus ( Gossypium ) includes approximately 50 species distributed in arid to semi-arid regions of the tropic and subtropics. Included are four species that have independently been domesticated for their fiber, two each in Africa–Asia and the Americas. Gossypium species exhibit extraordinary morphological variation, ranging from herbaceous perennials to small trees with a diverse array of reproductive and vegetative characteristics. A parallel level of cytogenetic and genomic diversity has arisen during the global radiation of the genus, leading to the evolution of eight groups of diploid ( n =13) species (genome groups A–G, and K). The evolutionary history of the genus included multiple episodes of trans-oceanic dispersal, invasion of new ecological niches, and a surprisingly high frequency of natural interspecific hybridization among lineages that are presently both geographically isolated and intersterile. Recent investigations have clarified many aspects of this history, including relationships within and among the eight genome groups, the domestication history of each of the four cultivated species, and the origin of the allopolyploid cottons. Data implicate an origin for Gossypium 5–15 million years ago (mya) and a rapid early diversification of the major genome groups. Allopolyploid cottons appear to have arisen within the last million years, as a consequence of trans-oceanic dispersal of an A-genome taxon to the New World followed by hybridization with an indigenous D-genome diploid. Subsequent to formation, allopolyploids radiated into three modern lineages, including those containing the commercially important species G. hirsutum and G. barbadense . Genome doubling has led to an array of molecular genetic interactions, including inter-locus concerted evolution, differential rates of genomic evolution, inter-genomic genetic transfer, and probable alterations in gene expression. The myriad underlying mechanisms are also suggested to have contributed to both ecological success and agronomic potential.

https://www.fs.fed.us/pnw/pubs/journals/pnw_2002_wendel001.pdf

Polyploidy and the Evolutionary History of Cotton

Organellar DNA sequences are widely used in evolutionary and population genetic studies, however, the conservative nature of chloroplast gene and genome evolution often limits phylogenetic resolution and statistical power. To gain maximal access to the historical record contained within chloroplast genomes, we have adapted multiplex sequencing-by-synthesis (MSBS) to simultaneously sequence multiple genomes using the Illumina Genome Analyzer. We PCR-amplified ∼120 kb plastomes from eight species (seven Pinus, one Picea) in 35 reactions. Pooled products were ligated to modified adapters that included 3 bp indexing tags and samples were multiplexed at four genomes per lane. Tagged microreads were assembled by de novo and reference-guided assembly methods, using previously published Pinus plastomes as surrogate references. Assemblies for these eight genomes are estimated at 88–94% complete, with an average sequence depth of 55× to 186×. Mononucleotide repeats interrupt contig assembly with increasing repeat length, and we estimate that the limit for their assembly is 16 bp. Comparisons to 37 kb of Sanger sequence show a validated error rate of 0.056%, and conspicuous errors are evident from the assembly process. This efficient sequencing approach yields high-quality draft genomes and should have immediate applicability to genomes with comparable complexity.

https://www.fs.fed.us/pnw/olympia/silv/publications/opt/700_CronnEtal2008.pdf

Multiplex sequencing of plant chloroplast genomes using Solexa sequencing-by-synthesis technology

 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 signifi cantly 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 160 000 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 effi ciently obtained for molecular phylogenetics studies

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

Phylogenetic resolution is often low within groups of recently diverged taxa due to a paucity of phylogenetically informative characters. We tested the relative utility of seven noncoding cpDNA regions and a pair of homoeologous nuclear genes for resolving recent divergences, using tetraploid cottons ( Gossypium) as a model system. The five tetraploid species of Gossypium are a monophyletic assemblage derived from an allopolyploidization event that probably occurred within the last 0.5‐2 million years. Previous analysis of cpDNA restriction site data provided only partial resolution within this clade despite a large number of enzymes employed. We sequenced three cpDNA introns (rpl16, rpoC1, ndhA) and four cpDNA spacers (accD-psaI, trnL-trnF, trnT-trnL, atpB-rbcL) for a total of over 7 kb of sequence per taxon, yet obtained only four informative nucleotide substitutions (0.05%) resulting in incomplete phylogenetic resolution. In addition, we sequenced a 1.65-kb region of a homoeologous pair of nuclear-encoded alcohol dehydrogenase (Adh) genes. In contrast with the cpDNA sequence data, the Adh homoeologues yielded 25 informative characters (0.76%) and provided a robust and completely resolved topology that is concordant with previous cladistic and phenetic analyses. The enhanced resolution obtained using the nuclear genes reflects an approximately three- to sixfold increase in nucleotide substitution rate relative to the plastome spacers and introns.

The tortoise and the hare: choosing between noncoding plastome and nuclear Adh sequences for phylogeny reconstruction in a recently diverged plant group

Richard Cronn

Papers

Genes duplicated by polyploidy show unequal contributions to the transcriptome and organ-specific reciprocal silencing

Polyploidy and the Evolutionary History of Cotton

Multiplex sequencing of plant chloroplast genomes using Solexa sequencing-by-synthesis technology

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

The tortoise and the hare: choosing between noncoding plastome and nuclear Adh sequences for phylogeny reconstruction in a recently diverged plant group