Developing the Protocol Infrastructure for DNA Sequencing Natural History Collections

Intentionally preserved biological material in natural history collections represents a vast repository of biodiversity. Advances in laboratory and sequencing technologies have made these specimens increasingly accessible for genomic analyses, offering a window into the genetic past of species and often permitting access to information that can no longer be sampled in the wild. Due to their age, preparation and storage conditions, DNA retrieved from museum and herbarium specimens is often poor in yield, heavily fragmented and biochemically modified. This not only poses methodological challenges in recovering nucleotide sequences, but also makes such investigations susceptible to environmental and laboratory contamination. In this paper, we review the practical challenges associated with making the recovery of DNA sequence data from museum collections more routine. We first review key operational principles and issues to address, to guide the decision-making process and dialogue between researchers and curators about when and how to sample museum specimens for genomic analyses. We then outline the range of steps that can be taken to reduce the likelihood of contamination including laboratory set-ups, workflows and working practices. We finish by presenting a series of case studies, each focusing on protocol practicalities for the application of different mainstream methodologies to museum specimens including: (i) shotgun sequencing of insect mitogenomes, (ii) whole genome sequencing of insects, (iii) genome skimming to recover plant plastid genomes from herbarium specimens, (iv) target capture of multi-locus nuclear sequences from herbarium specimens, (v) RAD-sequencing of bird specimens and (vi) shotgun sequencing of ancient bovid bone samples

A large-scale species level dated angiosperm phylogeny for evolutionary and ecological analyses.

Phylogenies are a central and indispensable tool for evolutionary and ecological research. Even though most angiosperm families are well investigated from a phylogenetic point of view, there are far less possibilities to carry out large-scale meta-analyses at order level or higher. Here, we reconstructed a large-scale dated phylogeny including nearly 1/8th of all angiosperm species, based on two plastid barcoding genes, matK (incl. trnK) and rbcL. Novel sequences were generated for several species, while the rest of the data were mined from GenBank. The resulting tree was dated using 56 angiosperm fossils as calibration points. The resulting megaphylogeny is one of the largest dated phylogenetic tree of angiosperms yet, consisting of 36,101 sampled species, representing 8,399 genera, 426 families and all orders. This novel framework will be useful for investigating different broad scale research questions in ecological and evolutionary biology

Variation in Zymoseptoria tritici virulence genes in field isolates

The fungus Zymoseptoria tritici is the causal agent of Septoria tritici blotch (STB), oneof the most economically devastating diseases of wheat (Triticum aestivum)particularly in the humid countries of Northern-Western Europe, including Ireland. Themanagement of STB is a problem due to the ability of the pathogen to adapt toenvironmental changes, to overcome resistant varieties and fungicide insensitivity .Little is known about the molecular and cellular strategies used by the pathogen tocause disease. This study aimed to identify candidate virulence genes and search fordifferences in gene expression and polymorphic variants between two Irish isolates(IPO553 and IPO560), and the Dutch isolate IPO323, that may impact on theprogression of STB. We sequenced the transcriptome of the Z. tritici isolates IPO323,IPO553 and IPO560 obtained from infected wheat seedlings at 7 dpi. The RNAsequencing allowed the identification of 9556 genes expressed in all the fungal isolates.This includes 820 genes encode proteins predicted to be secreted during wheatpathogenesis, while 363 of them are Small Secreted Peptides (SSPs), some of whichare highly expressed at 7 days post infection (dpi), suggesting that these genes mayplay a role in wheat pathogenesis. Twelve genes encoding SSPs were significantlydifferentially expressed between Z. tritici isolates, which may be involved inintraspecific variation in STB. This includes several genes encoding SSPs, interestingly,with no functional annotation. We also performed a genomic approach to search forSingle Nucleotide Polymorphisms (SNPs) in the Irish isolates IPO553 and IPO560,compared to the reference genome isolate IPO323. Focusing on SSPs exhibitingsignificant differences in gene expression, eleven of these exhibit Single NucleotidePolymorphisms (SNPs) in the Irish isolates IPO553 and IPO560, in comparison to thereference genome isolate IPO323. We also evaluated variations in disease severity andprogression in the Z. tritici isolates IPO323, IPO553, IPO560. The Irish isolate IPO553was found to be the most virulent, exhibiting a higher percentage of pycnidia coverageat 21 dpi and causing early disease symptoms compared to the isolates IPO323 andIPO560. Six candidate virulence genes were cloned and their expression profile wasanalysed during the progression of disease. All six genes were expressed in all Z. triticiisolates at 7 dpi, but most of them were highly expressed in the Irish isolate IPO553compared to the other isolates.Check date.issued and date.embargo: Flexible delayed release embargo added by autho

<i>In Vitro</i> Morphogenesis of <i>Arabidopsis</i> to Search for Novel Endophytic Fungi Modulating Plant Growth

<div><p>Fungal endophytes have shown to affect plant growth and to confer stress tolerance to the host; however, effects of endophytes isolated from water plants have been poorly investigated. In this study, fungi isolated from stems (stem-E) and roots (root-E) of <i>Mentha aquatica</i> L. (water mint) were identified, and their morphogenetic properties analysed on <i>in vitro</i> cultured <i>Arabidopsis</i> (L.) Heynh., 14 and 21 days after inoculation (DAI). Nineteen fungi were analysed and, based on ITS analysis, 17 isolates showed to be genetically distinct. The overall effect of water mint endophytes on <i>Arabidopsis</i> fresh (FW) and dry weight (DW) was neutral and positive, respectively, and the increased DW, mainly occurring 14 DAI, was possibly related to plant defence mechanism. Only three fungi increased both FW and DW of <i>Arabidopsis</i> at 14 and 21 DAI, thus behaving as plant growth promoting (PGP) fungi. E-treatment caused a reduction of root depth and primary root length in most cases and inhibition-to-promotion of root area and lateral root length, from 14 DAI. Only <i>Phoma macrostoma</i>, among the water mint PGP fungi, increased both root area and depth, 21 DAI. Root depth and area 14 DAI were shown to influence DWs, indicating that the extension of the root system, and thus nutrient uptake, was an important determinant of plant dry biomass. Reduction of <i>Arabidopsis</i> root depth occurred to a great extent when plants where treated with stem-E while root area decreased or increased under the effects of stem-E and root-E, respectively, pointing to an influence of the endophyte origin on root extension. <i>M</i>. <i>aquatica</i> and many other perennial hydrophytes have growing worldwide application in water pollution remediation. The present study provided a model for directed screening of endophytes able to modulate plant growth in the perspective of future field applications of these fungi.</p></div

Closest match of fungal isolate ITS sequence inferred from Blastn search in GenBank.

<p>Closest match of fungal isolate ITS sequence inferred from Blastn search in GenBank.</p

Endophyte effects on fresh weights.

<p>(a, b) Boxplots illustrating variability of fresh weight in E-treated and control (C) <i>Arabidopsis</i> plants 14 (a) and 21 (b) DAI. The reference hatched line represents the median of controls. Differences were considered significant at a probability level of *: p<0.05; **: <0.01; and ***: <0.001. (c, d) Pooled data for controls (C) and plants treated with all (E), stem (stem-E) and root (root-E) endophytes and 14 (a) and 21 (b) DAI.</p

Endophyte effects on root areas.

<p>(a, b) Boxplots illustrating root area variability in E-treated and control (C) <i>Arabidopsis</i> plants 14 (a) and 21 (b) DAI. The reference hatched line represents the median of controls. Differences were considered significant at a probability level of *: p<0.05; **: <0.01; and ***: <0.001. (c, d) Pooled data for controls (C) and plants treated with all (E), stem (stem-E) and root (root-E) endophytes and 14 (a) and 21 (b) DAI.</p

Endophyte effects on dry weights.

<p>(a, b) Boxplots illustrating variability of dry weight in E-treated and control (C) <i>Arabidopsis</i> plants 14 (a) and 21 (b) DAI. The reference hatched line represents the median of controls. Differences were considered significant at a probability level of *: p<0.05; **: <0.01; and ***: <0.001. (c, d) Pooled data for controls (C) and plants treated with all (E), stem (stem-E) and root (root-E) endophytes and 14 (a) and 21 (b) DAI.</p

Endophyte effects on percentage dry weights.

<p>(a, b) Boxplots illustrating variability of percentage dry weight in E-treated and control (C) <i>Arabidopsis</i> plants 14 (a) and 21 (b) DAI. The reference hatched line represents the median of controls. Differences were considered significant at a probability level of *: P<0.05; **: <0.01; and ***: <0.001. (c, d) Pooled data for controls (C) and plants treated with all (E), stem (stem-E) and root (root-E) endophytes and 14 (a) and 21 (b) DAI.</p