Highly efficient multiplex PCR of noninvasive DNA does not require pre-amplification

Among the key issues determining success of a study employing molecular genetics tools in wildlife monitoring or research is a large enough set of highly informative genetic markers and a reliable, cost effective method for their analysis. While optimized commercial genotyping kits have been developed for humans and domestic animals, such protocols are rare in wildlife research. We developed a highly optimized multiplex PCR that genotypes 12 microsatellite loci and a sex determination locus in brown bear (Ursus arctos) faecal samples in a single multiplex PCR and a single sequencer run. We used this protocol to genotype 1053 faecal samples of bears from the Dinaric population, and obtained useful genotypes for 88% of the samples, a very high success rate. The new protocol outperformed the multiplex pre-amplification strategy used in a previous study of 473 faecal samples with a 78.4% success rate. On a subset of 182 samples we directly compared the performance of both approaches, and found no advantage of the multiplex pre-amplification. While pre-amplification protocols might still improve PCR success and reliability on a small fraction of low-quality samples, the higher costs and workload do not justify their use when analysing reasonably fresh non-invasive material. Moreover, the high number of multiplexed loci in the new protocol makes it comparable to commercially developed genotyping kits developed for domestic animals and humans.     

The use of Tn5 transposable elements in a gene trapping strategy for the protozoan Leishmania

The use of transposable elements as a gene-trapping strategy is a powerful tool for gene discovery. Herein we describe the development of a transposable system, based on the bacterial Tn5 transposon, which has been used successfully in Leishmania braziliensis. The transposon carries the neomycin phosphotransferase gene, which is expressed only when inserted in-frame with a Leishmania gene present in the target DNA. Four cosmid clones from a L. braziliensis genomic library were used as targets in transposition reactions and four insertional libraries were constructed and transfected in L. braziliensis. Clones resistant to G418 were selected and analysed by immunofluorescence in order to identify the subcellular localisation of the protein coded by the trapped gene. A definitive subcellular localisation for neomycin phosphotransferase/targeted protein fusion was not obtained in any of the four Leishmania clones investigated. However, the constructed transposable element is highly efficient considering the frequency of insertion in large targets and is therefore a useful tool for functional genetic studies in Leishmania. Our data confirm the utility of the Tn5 transposon system for insertion of sequencing priming sites into target DNA. Furthermore, the high frequency of insertion and even distribution are important in studying genomic regions bearing long and polymorphic repetitive sequences.     

Family of Tc1-like elements from fish genomes and horizontal transfer

The involvement of horizontal transfer (HT) in the evolution of vertebrate transposable elements (TEs) is a matter of an ongoing debate. The phylogenetic relationships between Tc1 TEs, based on limited dataset have been previously used to infer a case of Tc1 HT between the genomes of fish and frogs. Here this hypothesis has been critically evaluated by the experimental approach including comparative data on the range of fish species available today. The distribution of a Tc1 subfamily of TE in selected fish species was investigated by PCR with a single primer complementary to ITRs and showed that they are widespread in the studied 17 fish species. They belong to five different subfamilies of Tc1 TEs, as revealed by the comparison with current genomic data for fish and amphibians. The original hypothesis would get much weaker support from the current data, although at least one novel potential and more convincing case of HT was identified between genomes of Perciformes fish. An interesting case of recombination-driven mobilisation of a degenerated TE by distantly related TE from different subfamily was discovered in the genome of pike. The occurrence of such cases widens the range of TE elements identifiable with the employed experimental approach. Further similar studies would help to explain the evolution of the multiple Tc1 lineages including species for which full genome sequences will not be available soon.     

Spatial and temporal patterns of movement and migration at spawning aggregations of red hind, <Emphasis Type="Italic">Epinephelus guttatus</Emphasis>, in the U.S. Virgin Islands

This study compared the spatial and temporal patterns of red hind, Epinephelus guttatus, movement and migration from annual spawning aggregations on St Thomas (STT) and St Croix (STX), United States Virgin Islands. Around STT E. guttatus migrated 6–33 km from a functional spawning migration area of 500 km² and around STX E. guttatus migrated 5–18 km from an area of 90 km². Similarities between sites were found in regards to timing of movement, temporal and spatial changes in sex ratios, annual and lunar predictability and were synchronized with environmental cues. E. guttatus spawning aggregations in the Virgin Islands occur between the winter solstice (i.e., after December 20) and about February 20 of any year and show a distinctive peak 20–40 days after the winter solstice. Spawning typically occurred during periods of declining seawater temperature and slacking currents within a temperature range of 26–27.5°C and current speed of 2.5–3.5 cm s⁻¹. Males arrived early to spawning sites and stayed longer than females. These gender-based behavioral patterns are important to E. guttatus reproductive dynamics and must be factored into future studies and the design of fisheries regulations to ensure sustainability of spawning aggregation sites. The predictability of E. guttatus spawning aggregations relative to the winter solstice will be extremely beneficial for defining the temporal and spatial aspects of area closures. The consistency and synchrony of movement and migration will improve both the efficiency of planning research and monitoring programs and directing enforcement activities during critical time periods. Applying this knowledge strategically will maximize the limited resources available for research and enforcement and lead to greater protection of spawning aggregations.     

A set of epitope-tagging integration vectors for functional analysis in Saccharomyces cerevisiae

Functional analysis of genes from Saccharomyces cerevisiae has been the major goal after determination of genome sequences. Even though several tools for molecular-genetic analyses have been developed, only a limited number of reliable genetic tools are available to support functional assay at protein level. Epitope tagging is a powerful tool for detecting, purifying, and functional studying of proteins. But systematic tagging systems developed with integration vectors are not available. Here, we have constructed a set of integration vectors allowing a translational fusion of interested proteins to the four different epitope tags (HA, Myc, Flag, and GFP). To confirm function and expression of C-terminal-tagged proteins, we used Cdc11, a component of the septin filament that encircles the mother bud neck and consists of five major proteins: Cdc3, Cdc10, Cdc11, Cdc12, and Sep7. The tagged version of Cdc11 expressed under its endogenous promoter was found to be physiologically functional, as evidenced by localization at the neck and suppression of the growth defect associated with the temperature-sensitive mutation of cdc11-6. The expressed proteins were efficiently detected with antibodies against Cdc11 or the epitopes. When immunoprecipitated with anti-Myc antibody, each septin protein tagged with Myc was effectively copurified with other septin components, indicating formation of a stable septin complex. Because the modules of the tags were located under the same array of eighteen restriction sites on integration vectors containing four different markers (HIS3, TRP1, LEU2, or URA3), this tagging system provides efficient multiple tagging and stable expression of a gene of interest.     

Cotton genome mapping with new microsatellites from Acala ‘Maxxa’ BAC-ends

Fine mapping and positional cloning will eventually improve with the anchoring of additional markers derived from genomic clones such as BACs. From 2,603 new BAC-end genomic sequences from Gossypium hirsutum Acala ‘Maxxa’, 1,316 PCR primer pairs (designated as MUSB) were designed to flank microsatellite or simple sequence repeat motif sequences. Most (1164 or 88%) MUSB primer pairs successfully amplified DNA from three species of cotton with an average of three amplicons per marker and 365 markers (21%) were polymorphic between G. hirsutum and G. barbadense. An interspecific RIL population developed from the above two entries was used to map 433 marker loci and 46 linkage groups with a genetic distance of 2,126.3 cM covering approximately 45% of the cotton genome and an average distance between two loci of 4.9 cM. Based on genome-specific chromosomes identified in G. hirsutum tetraploid (A and D), 56.9% of the coverage was located on the A subgenome while 39.7% was assigned to the D subgenome in the genetic map, suggesting that the A subgenome may be more polymorphic and recombinationally active than originally thought. The linkage groups were assigned to 23 of the 26 chromosomes. This is the first genetic map in which the linkage groups A01 and A02/D03 have been assigned to specific chromosomes. In addition the MUSB-derived markers from BAC-end sequences markers allows fine genetic and QTL mapping of important traits and for the first time provides reconciliation of the genetic and physical maps. Limited QTL analyses suggested that loci on chromosomes 2, 3, 12, 15 and 18 may affect variation in fiber quality traits. The original BAC clones containing the newly mapped MUSB that tag the QTLs provide critical DNA regions for the discovery of gene sequences involved in biological processes such as fiber development and pest resistance in cotton. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Molecular Tagging and Mapping of Quantitative Trait Loci for Lint Percentage and Morphological Marker Genes in Upland Cotton

Using 219 F2 Individuals developed by crossing the genetic standard line TM-1 and the multiple dominant marker line T586 In Gossyplum hirsutum L., a genetic linkage map with 19 linkage groups was constructed based on simple sequence repeat （SSR） markers. Compared with our tetraploid backboned molecular genetic map from a （TM-1xHal 7124）xTM-1 BC1 population, 17 of the 19 I｜nkage groups were combined and anchored to 12 chromosomes （sub-genomes）. Of these groups, four morphological marker genes In T586 had been mapped Into the molecular linkage map. Meanwhile, three quantitative trait loci for lint percentage were tagged and mapped separately on the A03 linkage group and chromosome 6.