Green fluorescent protein gene as a tool to examine the efficacy of Agrobacterium-delivered CRISPR/Cas9 reagents to generate targeted mutations in the potato genome

CRISPR/Cas9 has emerged as a simple, yet efficient gene editing tool to generate targeted mutations in desired genes in crops plants. Agrobacterium tumefaciens, a reliable and inexpensive DNA-delivery mechanism into plant cells, has been used for the generation of CRISPR/Cas9-mediated mutations in crop plants, including potato. However, little information is available as to the progression of gene knockout during various stages of culture following the introduction of CRISPR components in this species. In the current study, the green fluorescent protein (gfp) transgene was first introduced in the genome of a potato variety, Yukon Gold. Two GFP-expressing lines, one with a single gfp copy integrated and another with four gfp copies integrated, were subjected to CRISPR/Cas9-mediated mutations in the transgene(s) using three different gRNAs. Disappearance of GFP fluorescence was monitored during the entire culture/regeneration process. Although all three gRNAs successfully knocked out the transgene(s), their efficiencies differed greatly and did not completely match the predicted scores by some guide RNA prediction tools. The nature of mutations in various knockout events was analyzed. Several lines containing four gfp-copies showed four different types of mutations. These findings suggest that it is possible to target all four alleles of a desired native gene in the tetraploid potato.

     

Interactions of 17β-Hydroxysteroid Dehydrogenase Type 10 and Cyclophilin D in Alzheimer's Disease

The nucleus-encoded 17β-hydroxysteroid dehydrogenase type 10 (17β-HSD10) regulates cyclophilin D (cypD) in the mitochondrial matrix. CypD regulates opening of mitochondrial permeability transition pores. Both mechanisms may be affected by amyloid β peptides accumulated in mitochondria in Alzheimer's disease (AD). In order to clarify changes occurring in brain mitochondria, we evaluated interactions of both mitochondrial proteins in vitro (by surface plasmon resonance biosensor) and detected levels of various complexes of 17β-HSD10 formed in vivo (by sandwich ELISA) in brain mitochondria isolated from the transgenic animal model of AD (homozygous McGill-R-Thy1-APP rats) and in cerebrospinal fluid samples of AD patients. By surface plasmon resonance biosensor, we observed the interaction of 17β-HSD10 and cypD in a direct real-time manner and determined, for the first time, the kinetic parameters of the interaction (k_a 2.0?×?10⁵ M¹s^?1, k_d 5.8?×?10⁴ s^?1, and K_D 3.5?×?10^–10 M). In McGill-R-Thy1-APP rats compared to controls, levels of 17β-HSD10–cypD complexes were decreased and those of total amyloid β increased. Moreover, the levels of 17β-HSD10–cypD complexes were decreased in cerebrospinal fluid of individuals with AD (in mild cognitive impairment as well as dementia stages) or with Frontotemporal lobar degeneration (FTLD) compared to cognitively normal controls (the sensitivity of the complexes to AD dementia was 92.9%, that to FTLD 73.8%, the specificity to AD dementia equaled 91.7% in a comparison with the controls but only 26.2% with FTLD). Our results demonstrate the weakened ability of 17β-HSD10 to regulate cypD in the mitochondrial matrix probably via direct effects of amyloid β. Levels of 17β-HSD10–cypD complexes in cerebrospinal fluid seem to be the very sensitive indicator of mitochondrial dysfunction observed in neurodegeneration but unfortunately not specific to AD pathology. We do not recommend it as the new biomarker of AD.

     

Variation in trophic resources in female South American sea lions at a small geographic scale

Difference among colonies in the population structure of otariids can be driven by philopatry and/or by specializations in the foraging ecology of females. In northern Patagonia, the South American sea lion (SASL) shows some degree of spatial genetic structure among colonies from north and south zones. This study aims to explore the isotopic niche of SASL females in the last period of the pregnancy from different colonies of northern Patagonia and to consider whether the fine scale genetic spatial structuring is potentially related to variation in trophic resources. Stable isotope analysis was performed on 101 skin samples of newborn pups in 10 colonies, as a proxy for the feeding ecology of their mothers. Differences among colonies in the metrics studied revealed the plasticity of the species and support individual trophic specialization of SASL females at a small geographic scale. Also, significant differences were found in all isotopic metrics between the north and south zones. Several hypotheses were proposed to explain the differences in SASL females' isotope values (e.g., use of different foraging areas or prey, isotopic baseline variation). Nonetheless, further research is needed to better understand the relation between fine scale genetic structuring and the foraging ecology of SASL females.     

Changing Val-76 towards Kir channels drastically influences the folding and gating properties of the bacterial potassium channel KcsA

All K⁺-channels are stabilized by K⁺-ions in the selectivity filter. However, they differ from each other with regard to their selectivity filter. In this study, we changed specific residue Val-76 in the selectivity filter of KcsA to its counterpart Ile in inwardly rectifying K⁺-channels (Kir). The tetramer was exclusively converted into monomers as determined by conventional gel electrophoresis. However, by perfluoro-octanoic acid (PFO) gel electrophoresis mutant channel was mostly detected as tetramer. Tryptophan fluorescence and acrylamide quenching experiments demonstrated significant alteration in channel folding properties via increase in hydrophilicity of local environment. Furthermore, in planar lipid bilayer experiments V76I exhibited drastically lower conductance and decreased channel open time as compared to the unmodified KcsA. These studies suggest that V76I might contribute to determine the stabilizing, folding and channel gating properties in a selective K⁺-channel.     

Mapping multiple disease resistance genes using a barley mapping population evaluated in Peru, Mexico, and the USA

We used a well-characterized barley mapping population (BCD 47 × Baronesse) to determine if barley stripe rust (BSR) resistance quantitative trait loci (QTL) mapped in Mexico and the USA were effective against a reported new race in Peru. Essentially the same resistance QTL were detected using data from each of the three environments, indicating that these resistance alleles are effective against the spectrum of naturally occurring races at these sites. In addition to the mapping population, we evaluated a germplasm array consisting of lines with different numbers of mapped BSR resistance alleles. A higher BSR disease severity on CI10587, which has a single qualitative resistance gene, in Peru versus Mexico suggests there are differences in pathogen virulence between the two locations. Confirmation of a new race in Peru will require characterization using a standard set of differentials, an experiment that is underway. The highest levels of resistance in Peru were observed when the qualitative resistance gene was pyramided with quantitative resistance alleles. We also used the mapping population to locate QTL conferring resistance to barley leaf rust and barley powdery mildew. For mildew, we identified resistance QTL under field conditions in Peru that are distinct from the Mla resistance that we mapped using specific isolates under controlled conditions. These results demonstrate the long-term utility of a reference mapping population and a well-characterized germplasm array for locating and validating genes conferring quantitative and qualitative resistance to multiple pathogens.     

Longer and Less Overlapping Food Webs in Anthropogenically Disturbed Marine Ecosystems: Confirmations from the Past

The human exploitation of marine resources is characterised by the preferential removal of the largest species. Although this is expected to modify the structure of food webs, we have a relatively poor understanding of the potential consequences of such alteration. Here, we take advantage of a collection of ancient consumer tissues, using stable isotope analysis and SIBER to assess changes in the structure of coastal marine food webs in the South-western Atlantic through the second half of the Holocene as a result of the sequential exploitation of marine resources by hunter-gatherers, western sealers and modern fishermen. Samples were collected from shell middens and museums. Shells of both modern and archaeological intertidal herbivorous molluscs were used to reconstruct changes in the stable isotopic baseline, while modern and archaeological bones of the South American sea lion Otaria flavescens, South American fur seal Arctocephalus australis and Magellanic penguin Spheniscus magellanicus were used to analyse changes in the structure of the community of top predators. We found that ancient food webs were shorter, more redundant and more overlapping than current ones, both in northern-central Patagonia and southern Patagonia. These surprising results may be best explained by the huge impact of western sealing on pinnipeds during the fur trade period, rather than the impact of fishing on fish populations. As a consequence, the populations of pinnipeds at the end of the sealing period were likely well below the ecosystem''s carrying capacity, which resulted in a release of intraspecific competition and a shift towards larger and higher trophic level prey. This in turn led to longer and less overlapping food webs.     

Improved technique for reconstituting incredibly high and soluble amounts of tetrameric K⁺ channel in natural membranes

The reconstitution of large amounts of integral proteins into lipid vesicles is largely prompted by the complexity of most biological membranes and protein stability. We optimized a particular system which maximized the incorporation efficiency of large soluble amounts of KcsA potassium channel in Escherichia coli membranes. The effects of two detergents, octylglucoside and 3-[(cholamidopropyl)-dimethyl-ammonio]-1-propanesulfonate (CHAPS), on KcsA reconstitution were compared. Reconstitution efficiency was found to be incredibly high for CHAPS-treated proteoliposomes followed by dialysis at room temperature. This approach may allow more accurate investigation of integral membrane proteins in their natural membrane environment via biophysical or biochemical techniques.     

Chloroplast and nuclear microsatellite analysis of Aegilops cylindrica

Aegilops cylindrica Host (2n=4x=28, genome CCDD) is an allotetraploid formed by hybridization between the diploid species Ae. tauschii Coss. (2n=2x=14, genome DD) and Ae. markgrafii (Greuter) Hammer (2n=2x=14, genome CC). Previous research has shown that Ae. tauschii contributed its cytoplasm to Ae. cylindrica. However, our analysis with chloroplast microsatellite markers showed that 1 of the 36 Ae. cylindrica accessions studied, TK 116 (PI 486249), had a plastome derived from Ae. markgrafii rather than Ae. tauschii. Thus, Ae. markgrafii has also contributed its cytoplasm to Ae. cylindrica. Our analysis of chloroplast and nuclear microsatellite markers also suggests that D-type plastome and the D genome in Ae. cylindrica were closely related to, and were probably derived from, the tauschii gene pool of Ae. tauschii. A determination of the likely source of the C genome and the C-type plastome in Ae. cylindrica was not possible.     

Effect of population size on the estimation of QTL: a test using resistance to barley stripe rust

The limited population sizes used in many quantitative trait locus (QTL) detection experiments can lead to underestimation of QTL number, overestimation of QTL effects, and failure to quantify QTL interactions. We used the barley/barley stripe rust pathosystem to evaluate the effect of population size on the estimation of QTL parameters. We generated a large (n=409) population of doubled haploid lines derived from the cross of two inbred lines, BCD47 and Baronesse. This population was evaluated for barley stripe rust severity in the Toluca Valley, Mexico, and in Washington State, USA, under field conditions. BCD47 was the principal donor of resistance QTL alleles, but the susceptible parent also contributed some resistance alleles. The major QTL, located on the long arm of chromosome 4H, close to the Mlo gene, accounted for up to 34% of the phenotypic variance. Subpopulations of different sizes were generated using three methods—resampling, selective genotyping, and selective phenotyping—to evaluate the effect of population size on the estimation of QTL parameters. In all cases, the number of QTL detected increased with population size. QTL with large effects were detected even in small populations, but QTL with small effects were detected only by increasing population size. Selective genotyping and/or selective phenotyping approaches could be effective strategies for reducing the costs associated with conducting QTL analysis in large populations. The method of choice will depend on the relative costs of genotyping versus phenotyping. Electronic Supplementary Material Supplementary material is available for this article at     

Map-based analysis of genes affecting the brittle rachis character in tetraploid wheat (Triticum turgidum L.)

The mature spike rachis of wild emmer [Triticum turgidum L. ssp. dicoccoides (Körn. ex Asch. and Graebner) Thell.] disarticulates spontaneously between each spikelet leading to the dispersion of wedge-type diaspores. By contrast, the spike rachis of domesticated emmer (Triticum turgidum L. ssp. turgidum) fails to disarticulate and remains intact until it is harvested. This major distinguishing feature between wild and domesticated emmer is controlled by two major genes, brittle rachis 2 (Br-A2) and brittle rachis 3 (Br-A3) on the short arms of chromosomes 3A and 3B, respectively. Because of their biological and agricultural importance, a map-based analysis of these genes was undertaken. Using two recombinant inbred chromosome line (RICL) populations, Br-A2, on chromosome 3A, was localized to a ~11-cM region between Xgwm2 and a cluster of linked loci (Xgwm666.1, Xbarc19, Xcfa2164, Xbarc356, and Xgwm674), whereas Br-A3, on chromosome 3B, was localized to a ~24-cM interval between Xbarc218 and Xwmc777. Comparative mapping analyses suggested that both Br-A2 and Br-A3 were present in homoeologous regions on chromosomes 3A and 3B, respectively. Furthermore, Br-A2 and Br-A3 from wheat and Btr1/Btr2 on chromosome 3H of barley (Hordeum vulgare L.) also were homoeologous suggesting that the location of major determinants of the brittle rachis trait in these species has been conserved. On the other hand, brittle rachis loci of wheat and barley, and a shattering locus on rice chromosome 1 did not appear to be orthologous. Linkage and deletion-based bin mapping comparisons suggested that Br-A2 and Br-A3 may reside in chromosomal areas where the estimated frequency of recombination was ~ 4.3 Mb/cM. These estimates indicated that the cloning of Br-A2 and Br-A3 using map-based methods would be extremely challenging.