Premise of the study: Phylogenetic inference is moving to large multilocus data sets, yet there remains uncertainty in the choice of marker and sequencing method at low taxonomic levels. To address this gap, we present a method for enriching long loci spanning intron-exon boundaries in the genus Heuchera. Methods: Two hundred seventy-eight loci were designed using a splice-site prediction method combining transcriptomic and genomic data. Biotinylated probes were designed for enrichment of these loci. Reference-based assembly was performed using genomic references; additionally, chloroplast and mitochondrial genomes were used as references for off-target reads. The data were aligned and subjected to coalescent and concatenated phylogenetic analyses to demonstrate support for major relationships. Results: Complete or nearly complete (>99%) sequences were assembled from essentially all loci from all taxa. Aligned introns showed a fourfold increase in divergence as opposed to exons. Concatenated analysis gave decisive support to all nodes, and support was also high and relationships mostly similar in the coalescent analysis. Organellar phylogenies were also well-supported and conflicted with the nuclear signal. Discussion: Our approach shows promise for resolving a recent radiation. Enrichment for introns is highly successful with little or no sequencing dropout at low taxonomic levels despite higher substitution and indel frequencies, and should be exploited in studies of species complexes.
Phytochromes are red-light photoreceptors in plants that regulate key life cycle processes, yet their evolutionary origins are not well understood. Using transcriptomic and genomic data, Li et al. find that canonical plant phytochromes originated in a common ances…
Chromosome-specific painting is a powerful technique in molecular cytogenetic and genome research. We developed an oligonucleotide (oligo)-based chromosome painting technique in cucumber (Cucumis sativus) that will be applicable in any plant species with a sequenced genome. Oligos specific to a single chromosome of cucumber were identified using a newly developed bioinformatic pipeline and then massively synthesized de novo in parallel. The synthesized oligos were amplified and labeled with biotin or digoxigenin for use in fluorescence in situ hybridization (FISH). We developed three different probes with each containing 23,000–27,000 oligos. These probes spanned 8.3–17 Mb of DNA on targeted cucumber chromosomes and had the densities of 1.5–3.2 oligos per kilobases. These probes produced FISH signals on a single cucumber chromosome and were used to paint homeologous chromosomes in other Cucumis species diverged from cucumber for up to 12 million years. The bulked oligo probes allowed us to track a single chromosome in early stages during meiosis. We were able to precisely map the pairing between cucumber chromosome 7 and chromosome 1 of Cucumis hystrix in a F1 hybrid. These two homeologous chromosomes paired in 71% of prophase I cells but only 25% of metaphase I cells, which may provide an explanation of the higher recombination rates compared to the chiasma frequencies between homeologous chromosomes reported in plant hybrids.
The 4D organization of the interphase nucleus, or the 4D Nucleome (4DN), reflects a dynamical interaction between 3D genome structure and function and its relationship to phenotype. We present initial analyses of the human 4DN, capturing genome-wide structure using chromosome conformation capture and 3D imaging, and function using RNA-sequencing. We introduce a quantitative index that measures underlying topological stability of a genomic region. Our results show that structural features of genomic regions correlate with function with surprising persistence over time. Furthermore, constructing genome-wide gene-level contact maps aided in identifying gene pairs with high potential for coregulation and colocalization in a manner consistent with expression via transcription factories. We additionally use 2D phase planes to visualize patterns in 4DN data. Finally, we evaluated gene pairs within a circadian gene module using 3D imaging, and found periodicity in the movement of clock circadian regulator and period circadian clock 2 relative to each other that followed a circadian rhythm and entrained with their expression.
The Bronze Age of Eurasia (around 3000–1000 BC) was a period of major cultural changes. However, there is debate about whether these changes resulted from the circulation of ideas or from human migrations, potentially also facilitating the spread of languages and certain phenotypic traits. We investigated this by using new, improved methods to sequence low-coverage genomes from 101 ancient humans from across Eurasia. We show that the Bronze Age was a highly dynamic period involving large-scale population migrations and replacements, responsible for shaping major parts of present-day demographic structure in both Europe and Asia. Our findings are consistent with the hypothesized spread of Indo-European languages during the Early Bronze Age. We also demonstrate that light skin pigmentation in Europeans was already present at high frequency in the Bronze Age, but not lactose tolerance, indicating a more recent onset of positive selection on lactose tolerance than previously thought. View full text
* Application of whole-genome capture (WGC) methods to ancient DNA (aDNA) promises to increase efficiency of ancient genome sequencing. * We compared the performance of two recent WGC methods in enriching human aDNA within Illumina libraries built using both double-stranded and single-stranded build protocols. Although both methods effectively enriched aDNA, we observed consistent differences between the methods, providing the opportunity to further explore parameters influencing WGC experiments. * Our results suggest bait length has a potential influence on library enrichment. Moreover, we show WGC biases against shorter molecules that are enriched in single-stranded libraries preparation protocols. Lastly, we document the effect of WGC on features including clonality, GC composition and repetitive DNA content. * Our findings provide insights relevant to those planning WGC on aDNA and suggest future tests and optimization to improve WGC efficiency.
The development of various target enrichment methods in combination with next generation sequencing techniques has greatly facilitated the use of partially degraded DNA samples in genetic studies. We employed the MYbaits target enrichment system in combination with Ion Torrent sequencing on a broad range of DNA quality, extracted from tissues obtained from both natural history archives and through various opportunistic sampling methods, to sequence the mitogenome of 11 mobulid rays and two closely related species. Mobulids are large, elusive pelagic filter feeders, for which conservation concerns have recently be raised in connection to their vulnerable life histories and increasing fishing pressure. We show that the MYbaits target enrichment method can be used to effectively sequence large parts of the mitogenome from heavily degraded DNA samples, and provide a time and cost effective alternative for genetic studies of rare and/or difficult to sample species.
• Premise of the study: The sunflower genus Helianthus has long been recognized as economically significant, containing species of agricultural and horticultural importance. Additionally, this genus displays a large range of phenotypic and genetic variation, making Helianthus a useful system for studying evolutionary and ecological processes. Here we present the most robust Helianthus phylogeny to date, laying the foundation for future studies of this genus. • Methods: We used a target enrichment approach across 37 diploid Helianthus species/subspecies with a total of 103 accessions. This technique garnered 170 genes used for both coalescent and concatenation analyses. The resulting phylogeny was additionally used to examine the evolution of life history and growth form across the genus. • Key results: Coalescent and concatenation approaches were largely congruent, resolving a large annual clade and two large perennial clades. However, several relationships deeper within the phylogeny were more weakly supported and incongruent among analyses including the placement of H. agrestis, H. cusickii, H. gracilentus, H. mollis, and H. occidentalis. • Conclusions: The current phylogeny supports three major clades including a large annual clade, a southeastern perennial clade, and another clade of primarily large-statured perennials. Relationships among taxa are more consistent with early phylogenies of the genus using morphological and crossing data than recent efforts using single genes, which highlight the difficulties of phylogenetic estimation in genera known for reticulate evolution. Additionally, conflict and low support at the base of the perennial clades may suggest a rapid radiation and/or ancient introgression within the genus.
Oligonucleotide microarrays allow the production of complex custom oligonucleotide libraries for nucleic acid detection–based applications such as fluorescence in situ hybridization (FISH). We have developed a PCR-free method to make single-stranded DNA (ssDNA) fluorescent probes through an intermediate RNA library. A double-stranded oligonucleotide library is amplified by transcription to create an RNA library. Next, dye- or hapten-conjugate primers are used to reverse transcribe the RNA to produce a dye-labeled cDNA library. Finally the RNA is hydrolyzed under alkaline conditions to obtain the single-stranded fluorescent probes library. Starting from unique oligonucleotide library constructs, we present two methods to produce single-stranded probe libraries. The two methods differ in the type of reverse transcription (RT) primer, the incorporation of fluorescent dye, and the purification of fluorescent probes. The first method employs dye-labeled reverse transcription primers to produce multiple differentially single-labeled probe subsets from one microarray library. The fluorescent probes are purified from excess primers by oligonucleotide-bead capture. The second method uses an RNA:DNA chimeric primer and amino-modified nucleotides to produce amino-allyl probes. The excess primers and RNA are hydrolyzed under alkaline conditions, followed by probe purification and labeling with amino-reactive dyes. The fluorescent probes created by the combination of transcription and reverse transcription can be used for FISH and to detect any RNA and DNA targets via hybridization.
The main objective of this project was to generate a large single nucleotide polymorphism (SNP) marker resource for later saturation of the genetic linkage map and fine mapping of quantitative trait loci (QTL). Another objective of this project was to learn more about basic crocodile biology, namely immune function and stress, and the underlying genetic function to evaluate their incorporation into CrocPLAN. This report describes the development of new phenotypic trait panels for farmed saltwater crocodiles. Among these is the major crocodilian stress hormone, corticosterone (CORT), which should be useful for the development of animal welfare standards and the eventual selection of individuals in the future. Immune assays, some of which have never been previously used in crocodilians, were employed in this project to assess immune function. These immune assays, which are relatively easy to perform and cheap, could be employed in any farming setting to assess immune function in the future. Levels of estradiol (ESTR) and testosterone (TEST) are also detailed in this report, for the first time in the saltwater crocodile. At the same time as trying to expedite industry adoption of genetic improvement programs, it was necessary to expand on the current selection criteria available to gain a deeper insight into the breeding objectives already defined from RIRDC Project US-109A. The traits added were corticosterone (the main crocodilian stress hormone), two immune parameters, two sex hormones (testosterone and estradiol), two behaviour characters and four skin quality traits. Simultaneously, some of these traits could be used to gauge current industry practices which are set out in the “Code of Practice on the humane treatment of wild and farmed Australian crocodiles”. I am pleased to report that the lowest levels of corticosterone ever reported in saltwater crocodiles were found certifying the recommendations set out in the “code of practice”.
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