Are GES carbapenemases underdiagnosed? An allelic discrimination assay for their accurate detection and differentiation.

GES (Guiana Extended Spectrum) carbapenemases belong to "minor class A carbapenemases" and its prevalence could be underestimated due to the lack of specific tests. The aim of this study was to develop an easy PCR method to differentiate between GES ß-lactamases with or without carbapenemase activity, based on an allelic discrimination system of SNPs that encode E104K and G170S mutations, without need of sequencing. Two pair of primers and Affinity Plus probes, labeled with different fluorophores; FAM/IBFQ and YAK/IBFQ, were designed for each one of the SNPs. This allelic discrimination assay allows to detect in real time the presence of all type of GES- ß-lactamases, being able to differentiate between carbapenemases and extended-spectrum ß-lactamase (ESBL), through a quick PCR test that avoid costly sequencing approaches and could help to decrease the current underdiagnosis of minor carbapenemases that scape of phenotypic screenings.

A linkage disequilibrium perspective on the genetic mosaic of speciation in two hybridizing Mediterranean white oaks.

We analyzed the genetic mosaic of speciation in two hybridizing Mediterranean white oaks from the Iberian Peninsula (Quercus faginea Lamb. and Quercus pyrenaica Willd.). The two species show ecological divergence in flowering phenology, leaf morphology and composition, and in their basic or acidic soil preferences. Ninety expressed sequence tag-simple sequence repeats (EST-SSRs) and eight nuclear SSRs were genotyped in 96 trees from each species. Genotyping was designed in two steps. First, we used 69 markers evenly distributed over the 12 linkage groups (LGs) of the oak linkage map to confirm the species genetic identity of the sampled genotypes, and searched for differentiation outliers. Then, we genotyped 29 additional markers from the chromosome bins containing the outliers and repeated the multilocus scans. We found one or two additional outliers within four saturated bins, thus confirming that outliers are organized into clusters. Linkage disequilibrium (LD) was extensive; even for loosely linked and for independent markers. Consequently, score tests for association between two-marker haplotypes and the 'species trait' showed a broad genomic divergence, although substantial variation across the genome and within LGs was also observed. We discuss the influence of several confounding effects on neutrality tests and review the evolutionary processes leading to extensive LD. Finally, we examine how LD analyses within regions that contain outlier clusters and quantitative trait loci can help to identify regions of divergence and/or genomic hitchhiking in the light of predictions from ecological speciation theory.

Detecting the footprints of divergent selection in oaks with linked markers.

Genome scans are increasingly used to study ecological speciation, providing a useful genome-wide perspective on divergent selection in the presence of gene flow. Here, we compare current approaches to detect footprints of divergent selection in closely related species. We analyzed 192 individuals from two interfertile European temperate oak species using 30 nuclear microsatellites from eight linkage groups. These markers present little intraspecific differentiation and can be used in combination to assign individual genotypes to species. We first show that different outlier detection tests give somewhat different results, possibly due to model constraints. Second, using linkage information for these markers, we further characterize the signature of divergent selection in the presence of gene flow. In particular, we show that recombination estimates for regions with outlier markers are lower than those for a control region, in line with a prediction from ecological speciation theory. Most importantly, we show that analyses at the haplotype level can distinguish between truly divergent (bi-directional) selection and positive selection in one of the two species, offering a new and improved method for characterizing the speciation process.

Gene flow and hybridisation in a mixed oak forest (Quercus pyrenaica Willd. and Quercus petraea (Matts.) Liebl.) in central Spain.

Oaks are long-standing models for the study of gene flow and hybridisation. Temperate (Quercus petraea) and sub-Mediterranean (Quercus pyrenaica) oaks coexist in central Spain, showing remarkable differences in population size and structure. Q. petraea has a scattered distribution in central Spain, where it is at one of the southernmost limits of its range, and forms low-density stands; in contrast, Q. pyrenaica is widespread in the region. We selected a mixed population of the two species ( approximately 13 ha, 176 adults and 96 saplings) to compare the patterns of gene flow within each species and the extent of introgression between them. Using five nuclear microsatellite markers, we performed a parentage analysis and found considerable immigration from outside the stand ( approximately 38% for Q. petraea and approximately 34% for Q. pyrenaica), and estimated average seed-dispersal distances of 42 and 14 m for Q. petraea and Q. pyrenaica, respectively. Introgression between species was also estimated using our microsatellite battery. First, we developed a multivariate discriminant approach and, second, we compared our results with a widely used clustering method (STRUCTURE). Both analyses were consistent with a low level of introgression between Q. petraea and Q. pyrenaica. Indeed, only 15 adult trees, approximately 8.5%, were identified as putative hybrids when both methods of analysis were combined. Hybrids may be most common in contact zones due merely to physical proximity.

RFLP maps of rye chromosomes 6R and 7R including terminal C-bands.

A F2 mapping population was created from a cross between 'UC-90' and E-line ryes (Secale cereale L.), two lines that showed polymorphism for eight C-band loci. Clones from rye, as well as other grasses, were used as probes. RFLP maps of rye chromosomes 6R and 7R were generated that include the 6RS and 6RL terminal C-bands and the 7RS terminal C-band. The 6R map spans 230 cM and includes 9 loci. The 7R map covers 225 cM and includes 21 loci. Segregation distortion was detected for several chromosomal regions. Heterochromatic C-bands did not appear to be responsible for the distortion.

Physical mapping of 5S rDNA reveals a new locus on 3R and unexpected complexity in a rye translocation used in chromosome mapping.

Using fluorescence in situ hybridization (FISH) with probe pScT7, three different 5S rDNA loci were detected in the satellite of rye chromosome 1R (5SDna-R1) and in the short arms of chromosomes 3R (5SDna-R3) and 5R (5SDna-R2) respectively. All three loci showed polymorphism for the hybridization signal intensity. In order to determine the localization of these rye 5S rDNA multigene loci with higher precision within the corresponding chromosome arms, the probe pScT7 was physically mapped by FISH in relation to the following five translocations (Wageningen Tester Set): T850W (1RS/4RL), T248W (1RS/6RS), T273W (1RS/5RL), T305W (2RS/5RS) and T240W (3RS/5RL). Accurate physical maps of the translocation breakpoints had previously been made using electron microscope analysis of spread pachytene synaptonemal complexes of heterozygotes for the different translocations. The results indicate that locus 5SDna-R3 is located between the breakpoint of translocation T240W and the telomere, whereas locus 5SDna-R2 is located between the breakpoint of translocation T305W and the centromere, the hybridization of probe pScT7 on T305W translocated chromosomes demonstrating the complex nature of this translocation. On the other hand, the simultaneous detection of probes pScT7 and pTA71 (18S-5.8S-26S rDNA) with two different fluorochromes, indicated that the breakpoints of translocations T850W and T248W are located between loci Nor-R1 and 5SDna-R1.

Physical mapping of translocation breakpoints in rye by means of synaptonemal complex analysis.

A physical map including 40 translocation breakpoints has been constructed in rye by means of synaptonemal complex (SC) analysis of well-paired pachytene quadrivalents. The chromosome arms involved in such translocations were previously identified either from mitotic C-banding analysis or from the meiotic configurations observed in the progenies of crosses with a rye line having multiple chromosome rearrangements. The synaptonemal complexes formed by some translocation homozygotes were also analyzed, the relative pachytene SC length of their translocated chromosomes being compared to that observed in the corresponding translocation heterozygotes. In the translocations in which the position of the breakpoint could be well defined from mitotic C-banding analysis, a good correspondence between the relative position of the point showing partner exchange in the pachytene quadrivalents and the actual location of the breakpoint was established. It is concluded that the mapping of translocation breakpoints by SC analysis of pachytene quadrivalents provides a more accurate estimate of the position of the breakpoints than that obtained from mitotic C-banding analysis, due to the lack of evenly-distributed interstitial C-bands in most rye chromosomes. The distribution of the breakpoints along the chromosomes in relation to their spontaneous or induced origin is also discussed.

Genetic mapping of cytological and isozyme markers on chromosomes 1R, 3R, 4R and 6R of rye.

Cytogenetic maps involving chromosomes 1R, 3R, 4R and 6R have been developed from the analysis of offspring of crosses between multiple heterozygous rye plants. The maps include isozyme loci GpiR1, Mdh-R1 and Pgd2 (located in chromosome 1R), Mdh-R2 (located in chromosome 3R), Pgm-R1 (located in chromosome 4R) and Aco-R1 (located in chromosome 6R). Various telomeric and interstitial C-bands of these four chromosomes, the centromere split of chromosome 3R, and translocation TR01 were used as cytological markers. By means of electron microscope analysis of spread pachytene synaptonemal complexes, the breakpoint of TR01 was physically mapped in chromosome arms 4RS and 6RL. From the linkage data, conclusions were derived concerning the cytological locations of the isozyme loci and the physical extent of the evolutive translocations involving chromosome arm 6RL.

Genetic linkage between cytological markers and the seed storage protein lociSec2[Gli-R2] andSec3[Glu-R1] in rye.

In order to reach a higher accuracy concerning the cytological locations of the rye seed storage protein lociSec2[Gli-R2] andSec3[Glu-R1] located within chromosome arms 2RS and 1RL, respectively, the linkage relationships between the following loci were analyzed: isozyme lociGpi-R1,Mdh-R1, andPgd2, translocationT273W (Wageningen tester set, involving chromosome arms 1RS and 5RL), the telomere C-bands of chromosome arms 1RL (tL1), 2RS (tS2), and 5RS (tS5), and three interstitial C-bands in chromosome arm 1RS (iS1), in the middle of chromosome arm 1RL (iL1), and in the middle of chromosome arm 2RL (iL2), respectively. The data indicated that locusSec3 is located in the distal half of chromosome arm 1RL (between C-bandiL1 and locusPgd2), while locusSec2 is located a short distance (2.9 ± 1.4%) from the telomere C-band of chromosome arm 2RS.

A cytogenetic map on the entire length of rye chromosome 1R, including one translocation breakpoint, three isozyme loci and four C-bands.

A cytogenetic map of the whole 1 R chromosome of rye has been made, with distances between adjacent markers shorter than 50% recombination. Included in the map are isozyme loci Gpi-R1, Mdh-R1 and Pgd2, the telomere C-bands of the short arm (ts1) and the long arm (tl1), two interstitial C-bands in the short arm proximal to the nuclear organizing region (NOR) (is1) and in the middle of the long arm (il1), respectively, and translocation T273W (Wageningen tester set). By means of electron microscope analysis of spread pachytene synaptonemal complexes, the breakpoint of this translocation was physically mapped in the short arm of 1R, proximal to NOR, and in the long arm of 5R (contrary to previous assumptions). The data indicated the marker order: ts1 - Gpi-R1 - is1 - T273W/Mdh-R1 - il1 - Pgd2 - tl1. A comparison between genetic and physical maps revealed that recombination is mainly restricted to the distal regions of both arms. For the translocation T273W, in heterozygotes no recombinants were observed between the translocation breakpoint and its two adjacently located markers (is1 and Mdh-R1), but recombination was not reduced in the distal regions of the chromosome. The segregations of several other isozyme and C-band markers also analyzed in the investigation presented here were consistent with observations of earlier authors concerning chromosome asignment and linkage relationships.