Research Notes : United States : Superoxide dismutase (SOD) in soybean

We are using vertical polyacrylamide gel electrophoresis (Davis, 1964) and a staining system modified after Beauchamp and Fridovich (1971) to study superoxide dismutase polymorphisms in the subgenus soja. This staining system generates superoxide radical; hence, it is specific for SOD activity. We re-solve up to 9 SOD bands in dry or germinating soybean cotyledons, and in leaves

Revisiting Recombination Signal in the Tick-Borne Encephalitis Virus: A Simulation Approach

<div><p>The hypothesis of wide spread reticulate evolution in Tick-Borne Encephalitis virus (TBEV) has recently gained momentum with several publications describing past recombination events involving various TBEV clades. Despite a large body of work, no consensus has yet emerged on TBEV evolutionary dynamics. Understanding the occurrence and frequency of recombination in TBEV bears significant impact on epidemiology, evolution, and vaccination with live vaccines. In this study, we investigated the possibility of detecting recombination events in TBEV by simulating recombinations at several locations on the virus’ phylogenetic tree and for different lengths of recombining fragments. We derived estimations of rates of true and false positive for the detection of past recombination events for seven recombination detection algorithms. Our analytical framework can be applied to any investigation dealing with the difficult task of distinguishing genuine recombination signal from background noise. Our results suggest that the problem of false positives associated with low detection <i>P</i>-values in TBEV, is more insidious than generally acknowledged. We reappraised the recombination signals present in the empirical data, and showed that reliable signals could only be obtained in a few cases when highly genetically divergent strains were involved, whereas false positives were common among genetically similar strains. We thus conclude that recombination among wild-type TBEV strains may occur, which has potential implications for vaccination with live vaccines, but that these events are surprisingly rare.</p></div

Synoptic diagram presenting the methods and analytical framework deployed during in the simulation.

<p>The analytical protocol designed to estimate the rates of true and false positive for the detection of recombination in simulated data consists in several steps: STEPS 1–3 derive the parameters for the simulation from empirical sequence alignments. STEPS 4–8 simulate alignments that are similar to the empirical data in term of tree topology, divergence dates, number of strains, number of sites and rate heterogeneity among sites. Several stochastic processes are added in order to model lineage specific substitution rate variation (STEP 4) and model the effect of purifying selection (STEP 6). The simulated datasets with and without recombinations are finally analyzed with RDP4 (STEP 9).</p

Collection localities for the E-sequences used to build the tree in Fig I in S1 file.

<p>Strains origins for three clades of interest (W1, FE4 and FE5) are indicated with arrows. Because strain origins are reported with various levels of precision (from local to national level), this map should only be used as an indication of the patchy record of TBEV genetic diversity. This map also shows that the territory comprised between the Irkutsk and Zabaikalsky regions represent a hot spot of genetic diversity with the co-circulation of X1-, X2-, FE- and S-strains. Some well known foci such as the presence of the S-TBEV in Finland and the isolation of all three sub-types in Estonia and Latvia are not included on the map as the associated sequences are too short to yield reliable phylogenetic signal. Sampling intensity is given in number of sequences collected in the same locality. The map was generated using an equidistant cylindrical projection with the Basemap toolkit available from the python package Matplotlib (<a href="http://matplotlib.org/basemap/users/cyl.html?highlight=cylindrical" target="_blank">http://matplotlib.org/basemap/users/cyl.html?highlight=cylindrical</a>).</p

Range of durations between the recombination tMRCA of the donor and receiver clades for the simulated events.

<p>Range of durations between the recombination tMRCA of the donor and receiver clades for the simulated events.</p

Reappraisal of the RDP4 identified putative recombination events.

<p>Reappraisal of the RDP4 identified putative recombination events.</p

Supplementary Material

SUPPLEMENTARY 1., 2. Plant material and GenBank accessions for nuclear and chloroplast sequences respectively. SUPPLEMENTARY 3. Material and methods for sequencing the plant material with a description of all primers designed for this study SUPPLEMENTARY 4. Illustration of the clone filtering procedure for identifying putative PCR-recombinant sequences and cross taxon contamination. All clones for the octoploid F. mirabilis (specimen 13673) were used to construct a neighborNet (uncorrected p-distances) in SplitsTree4. From ploidal level, four distinct sequences are expected. Each group highlighted in green was composed of several nearly identical sequences and was retained for phylogenetic analysis. Red sequences, connected to the main frame of the network with zero-length terminal edges, were considered to be putative PCR recombinants and were therefore discarded. A sufficient number of clones was not recovered for the blue group, which was further investigated with clade specific primers. SUPPLEMENTARY 5. Description of the phylogenetic analyses deployed for inferring gene trees. SUPPLEMENTARY 6. Details of the BEAST analysis dating the Dicentra-Corydalis split. SUPPLEMENTARY 7. Description and results of the simulation that assessed the performance of the coalescent stochasticity test. SUPPLEMENTARY 8. Table that reports the result of the coalescent stochasticity test for the sets of sequences derived from the individual small Ne and large Ne analyses of coalescent stochasticity tests. Here, all the sequences in a set are analyzed together and the inference Ne is increased until the full set passes the coalescent stochasticity test. SUPPLEMENTARY 9. Tanglegram inferred with RAxML from sequences of the primary set. Nuclear (a) and chloroplast (b) topologies correspondences were visualized in Dendroscope 3 (Huson and Scornavacca 2012). SUPPLEMENTARY 10. Genome tree inferred with BEAST. The maximum clade credibility chronogram was built from all nuclear homoeologues (except sequences from F. bastardii), together with chloroplast haplotypes from the primary set plus those that passed the substitution model error test. PP ≥ 0.70 are indicated below branches. Icons indicate the chloroplast sequences that were added to the analysis at the different steps of genome tree reconstruction

Chloroplast alignment Supplementary Figure 10.

Chloroplast alignment used for constructing BEAST phylogeny in Supplementary Figure 10

Nuclear alignment for Figures 5, 6 and Supplementary Figure 10

Nuclear alignment used for Figures 5, 6 (STEM analyses )and Supplementary Figure 10 (BEAST analysis

The 50 loci proposed with the respective linkage group, gene reference in Mt3.0 annotation, captured portion of the gene (bp positions in the reference sequence), alignment length, substitution rate ([subst./site/year] E-9),G-C content, number and % of parsimony-informative (PI) sites, % of exon based on the reference sequence, Consistency index (CI) and Retention index (RI).

<p>The 50 loci proposed with the respective linkage group, gene reference in Mt3.0 annotation, captured portion of the gene (bp positions in the reference sequence), alignment length, substitution rate ([subst./site/year] E-9),G-C content, number and % of parsimony-informative (PI) sites, % of exon based on the reference sequence, Consistency index (CI) and Retention index (RI).</p