CLAW: An automated Snakemake workflow for the assembly of chloroplast genomes from long-read data.

Chloroplasts are photosynthetic organelles in algal and plant cells that contain their own genome. Chloroplast genomes are commonly used in evolutionary studies and taxonomic identification and are increasingly becoming a target for crop improvement studies. As DNA sequencing becomes more affordable, researchers are collecting vast swathes of high-quality whole-genome sequence data from laboratory and field settings alike. Whole tissue read libraries sequenced with the primary goal of understanding the nuclear genome will inadvertently contain many reads derived from the chloroplast genome. These whole-genome, whole-tissue read libraries can additionally be used to assemble chloroplast genomes with little to no extra cost. While several tools exist that make use of short-read second generation and third-generation long-read sequencing data for chloroplast genome assembly, these tools may have complex installation steps, inadequate error reporting, poor expandability, and/or lack scalability. Here, we present CLAW (Chloroplast Long-read Assembly Workflow), an easy to install, customise, and use Snakemake tool to assemble chloroplast genomes from chloroplast long-reads found in whole-genome read libraries (https://github.com/aaronphillips7493/CLAW). Using 19 publicly available reference chloroplast genome assemblies and long-read libraries from algal, monocot and eudicot species, we show that CLAW can rapidly produce chloroplast genome assemblies with high similarity to the reference assemblies. CLAW was designed such that users have complete control over parameterisation, allowing individuals to optimise CLAW to their specific use cases. We expect that CLAW will provide researchers (with varying levels of bioinformatics expertise) with an additional resource useful for contributing to the growing number of publicly available chloroplast genome assemblies.

Physiological and structural traits contribute to thermotolerance in wild Australian cotton species.

BACKGROUND AND AIMS: Five species of cotton (Gossypium) were exposed to 38°C days during early vegetative development. Commercial cotton (Gossypium hirsutum) was contrasted with four wild cotton species (G. australe, G. bickii, G. robinsonii and G. sturtianum) that are endemic to central and northern Australia. METHODS: Plants were grown at daytime maxima of 30°C or 38°C for 25 d, commencing at the four-leaf stage. Leaf areas and shoot biomass were used to calculate relative rates of growth and specific leaf areas. Leaf gas exchange measurements revealed assimilation and transpiration rates, as well as electron transport rates (ETR) and carboxylation efficiency (CE) in steady-state conditions. Finally, leaf morphological traits (mean leaf area and leaf shape were quantified), along with leaf surface decorations, imaged using scanning electron microscopy. KEY RESULTS: Shoot morphology was differentially affected by heat, with three of the four wild species growing faster at 38°C than at 30°C, whereas early growth in G. hirsutum was severely inhibited by heat. Areas of individual leaves and leaf numbers both contributed to these contrasting growth responses, with fewer, smaller leaves at 38°C in G. hirsutum. CO2 assimilation and transpiration rates of G. hirsutum were also dramatically reduced by heat. Cultivated cotton failed to achieve evaporative cooling, contrasting with the transpiration-driven cooling in the wild species. Heat substantially reduced ETR and CE in G. hirsutum, with much smaller effects in the wild species. We speculate that leaf shape, as assessed by invaginations of leaf margins, and leaf size contributed to heat dispersal differentially among the five species. Similarly, reflectance of light radiation was also highly distinctive for each species. CONCLUSIONS: These four wild Australian relatives of cotton have adapted to hot days that are inhibitory to commercial cotton, deploying a range of physiological and structural adaptations to achieve accelerated growth at 38°C.

Imprinted gene alterations in the kidneys of growth restricted offspring may be mediated by a long non-coding RNA.

Altered epigenetic mechanisms have been previously reported in growth restricted offspring whose mothers experienced environmental insults during pregnancy in both human and rodent studies. We previously reported changes in the expression of the DNA methyltransferase Dnmt3a and the imprinted genes Cdkn1c (Cyclin-dependent kinase inhibitor 1C) and Kcnq1 (Potassium voltage-gated channel subfamily Q member 1) in the kidney tissue of growth restricted rats whose mothers had uteroplacental insufficiency induced on day 18 of gestation, at both embryonic day 20 (E20) and postnatal day 1 (PN1). To determine the mechanisms responsible for changes in the expression of these imprinted genes, we investigated DNA methylation of KvDMR1, an imprinting control region (ICR) that includes the promoter of the antisense long non-coding RNA Kcnq1ot1 (Kcnq1 opposite strand/antisense transcript 1). Kcnq1ot1 expression decreased by 51% in growth restricted offspring compared to sham at PN1. Interestingly, there was a negative correlation between Kcnq1ot1 and Kcnq1 in the E20 growth restricted group (Spearman's ρ = 0.014). No correlation was observed between Kcnq1ot1 and Cdkn1c expression in either group at any time point. Additionally, there was a 11.25% decrease in the methylation level at one CpG site within KvDMR1 ICR. This study, together with others in the literature, supports that long non-coding RNAs may mediate changes seen in tissues of growth restricted offspring.