Resistance of Pinus Sibirica clones to insect conobionts in the Yelbashinsky nursery of JSC «Berdsky forestry» in Novosibirsk region

The inter-clonal variability of Siberian cedar in resistance to insect conobionts was studied. The authors revealed that the main pests of cones and seeds are cone moth Dioryctria abietella Schiff and giant cone moth Eupithecia abietaria Goeze in the archives of plus-tree clones of Novosibirsk region (Yelbashinsky Nursery of JSC «Berdsky forestry», Iskitimsky district). The moth’s infestation of buds was 23.4%, and the infestation of buds by the moth was 2.0%. Selection of cedar for resistance to cone moths can be an effective way to increase yield at breeding and seed production facilities of this breed based on the study of inter-clonal variability in the degree of cone moth damage and other traits. Selection for resistance to cone moth disease will not significantly change stem wood produc- tivity due to the absence of a reliable interclonal correlation between the degree of cone damage and tree size. The authors described the result of the findings of the inter-clone correlation, consisting of a significant positive correlation between cone size and «granularity» and stem height and diameter. This result was against the background of the complete absence of any correlation between cone damage by conobionts and tree and cone size. The authors also concluded that selection for stem wood productivity in cedar would be accompanied by an increase in cone size and «granularity» (and vice versa), while selection for resistance to conobionts will not lead to significant changes in other vegetative and generative traits.

Dissemination of blaNDM–1 Gene Among Several Klebsiella pneumoniae Sequence Types in Mexico Associated With Horizontal Transfer Mediated by IncF-Like Plasmids

Nosocomial infections caused by multidrug-resistant (MDR) Klebsiella pneumoniae are a major health problem worldwide. The aim of this study was to describe NDM-1-producing K. pneumoniae strains causing bacteremia in a tertiary referral hospital in Mexico. MDR K. pneumoniae isolates were screened by polymerase chain reaction for the presence of resistance genes. In resistant isolates, plasmids were identified and conjugation assays were performed. Clonal diversity and the sequence types were determined by pulsed-field gel electrophoresis and multilocus sequence typing. A total of 80 K. pneumoniae isolates were collected from patients with bacteremia over a 1-year period. These isolates showed a level of resistance of 59% (47/80) to aztreonam, 56–60% (45–48/80) to cephalosporins, 54% (43/80) to colistin and 12.5% (10/80) to carbapenems. The carbapenem resistant isolates were blaNDM–1 carriers and negative for blaKPC, blaNDM, blaIMP, blaVIM and blaOXA–48–like carbapenemases genes. Conjugative plasmids IncFIIA and IncF group with sizes of 82–195 kbp were carriers of blaNDM–1, blaCTX–M–15, blaTEM–1, aac(6′)-Ib and/or aac(3′)-IIa. Clonal variability and nine different multilocus sequence types were detected (ST661, ST683, ST1395, ST2706, ST252, ST1198, ST690, ST1535, and ST3368) for the first time in the isolates carrying blaNDM–1 in Mexico. This study demonstrates that blaNDM–1 has remained within this hospital in recent years and suggests that it is currently the most prevalent carbapenemase among K. pneumoniae MDR strains causing bacteremia in Mexico. The horizontal transfer of blaNDM–1 gene through IncF-like plasmids among different clones demonstrates the dissemination pathway of antimicrobial resistance and underscore the need for strong and urgent joint measures to control the spread of NDM-1 carbapenemase in the hospital.

Establishment of a highly efficient gene disruption strategy to analyze and manipulate lipid co-regulatory networks

SUMMARYGene disruption has been drastically facilitated by recent genome editing tools. While the efficiency of gene disruption in cell culture has improved, clone isolation remains routinely performed to obtain fully mutated cells, potentially leading to artifacts due to clonal variances in cellular phenotypes. Here we report GENF, a highly efficient strategy to disrupt genes without isolating clones, which can be multiplexed. We obtained reliable lipidomics datasets from mutant cells generated with GENF, which was impossible when using clones. Through this, we found that an enzyme involved in congenital generalized lipodystrophy regulates glycerophospholipids with specific acyl-chains. We also demonstrate the possibility to dissect complex lipid co-regulatory networks and provide some common mechanisms explaining the adaptations to altered lipid metabolism. GENF is likely to contribute to all experimental setups affected by clonal variability and should be especially useful for -omics approaches.

Mitotic interhomolog recombination drives genomic diversity in diatoms

Diatoms, an evolutionarily successful group of microalgae, display high levels of intraspecific variability in natural populations. However, the process generating such diversity is unknown. Here we estimated the variability within a natural diatom population and subsequently mapped the genomic changes arising within cultures clonally propagated from single diatom cells. We demonstrate that genome rearrangements and mitotic recombination between homologous chromosomes underlie clonal variability, resulting in haplotype diversity accompanied by the appearance of novel protein variants and loss of heterozygosity resulting in the fixation of alleles. The frequency of interhomolog mitotic recombination exceeds 4 out of 100 cell divisions and increases under environmental stress. We propose that this plastic response in the interhomolog mitotic recombination rate increases the evolutionary potential of diatoms, contributing to their ecological success.One Sentence SummaryRecombination between homologous chromosomes in diatom vegetative cells leads to extensive genomic diversity in clonal populations.

An approach for accelerated isolation of genetically manipulated cell clones with reduced clonal variability

Genomic editing methods, such as the CRISPR/Cas9 system, are routinely used to study gene function in somatic cells. Due to the heterogeneity of mutations, it is necessary to purify cell clones grown in high dilution until colony formation, which can be a time consuming process. Here we tested a modified approach in which we seeded cells in high dilution, together with non-edited carrier cells. As a comparison, cells were also grown in high dilution with conditioned medium from a high density culture. When using carrier cells or conditioned medium, the formation of cell colonies is accelerated. Additionally, clones grown with carrier cells are more similar to the parental lines in terms of their tumorigenic properties. Surprisingly, key signaling cascades are very divergent between clones isolated from low density cultures, even with conditioned medium, in contrast to clones isolated with carrier cells. Thus, our study uncovers a significant limitation using the common approach of isolating cell clones following genetic modifications and suggests an alternative method that mitigates the problem of heterogeneity of gene expression between clones.

Clonal variability for vulnerability to cavitation and other drought-related traits in Hevea brasiliensis Müll. Arg.

Selection for drought-tolerant clones has become a major challenge in rubber breeding programs undertaken to ensure the sustainability of natural rubber production, as rubber plantations are expanding in drought-prone areas. Xylem vulnerability to cavitation is a trait related to drought-induced mortality. It can be rapidly evaluated without subjecting plant materials to drought stress, making it useful in large-scale screening for drought tolerance in the near future. We first compared the most widely used techniques for measuring vulnerability to cavitation (air pressurization and Cavitron) on this species, and the effect of sample conditions (size, age and sunlight exposure), in order to ensure reliable analysis. Secondly, ten rubber clones were compared for their xylem vulnerability to cavitation in branches and petioles, and for other traits related to drought response, including stomatal response and leaf shedding occurring during a simulated drought. We also tested the plasticity of vulnerability to cavitation on two clones grown in three locations with contrasting precipitation regimes. We found no clonal variability and a small phenotypic plasticity for xylem vulnerability to cavitation in branches. However, clonal differences in xylem vulnerability to cavitation were found in petioles, and clones also showed differences in stomatal response and in leaf shedding behavior in response to a simulated drought. Our study suggests a genetic canalization for vulnerability to cavitation in organs critical for survival, such as branches, whereas there are clonal differences for traits related to drought avoidance: vulnerability to cavitation of petioles, leaf shedding behavior and stomatal response. The insights gained in this study for screening rubber tree clones for drought tolerance is also discussed.