Genetic relatedness of Plasmodium falciparum isolates and the origin of allelic diversity at the merozoite surface protein-1 (MSP-1) locus in Brazil and Vietnam

Abstract Background Despite the extensive polymorphism at the merozoite surface protein-1 (MSP-1) locus of Plasmodium falciparum, that encodes a major repetitive malaria vaccine candidate antigen, identical and nearly identical alleles frequently occur in sympatric parasites. Here we used microsatellite haplotyping to estimate the genetic distance between isolates carrying identical and nearly identical MSP-1 alleles. Methods We analyzed 28 isolates from hypoendemic areas in north-western Brazil, collected between 1985 and 1998, and 23 isolates obtained in mesoendemic southern Vietnam in 1996. MSP-1 alleles were characterized by combining PCR typing with allele-specific primers and partial DNA sequencing. The following single-copy microsatellite markers were typed : Polyα, TA42 (only for Brazilian samples), TA81, TA1, TA87, TA109 (only for Brazilian samples), 2490, ARAII, PfG377, PfPK2, and TA60. Results The low pair-wise average genetic distance between microsatellite haplotypes of isolates sharing identical MSP-1 alleles indicates that epidemic propagation of discrete parasite clones originated most identical MSP-1 alleles in parasite populations from Brazil and Vietnam. At least one epidemic clone propagating in Brazil remained relatively unchanged over more than one decade. Moreover, we found no evidence that rearrangements of MSP-1 repeats, putatively created by mitotic recombination events, generated new alleles within clonal lineages of parasites in either country. Conclusion Identical MSP-1 alleles originated from co-ancestry in both populations, whereas nearly identical MSP-1 alleles have probably appeared independently in unrelated parasite lineages.

Low genetic diversity in Wolbachia-Infected Culex quinquefasciatus (Diptera: Culicidae) from Brazil and Argentina

Culex quinquefasciatus is a vector of human pathogens, including filarial nematodes and several viruses. Although its epidemiological relevance is known to vary across geographical regions, an understanding of its population genetic structure is still incipient. In light of this, we evaluated the genetic diversity of Cx. quinquefasciatus and Cx. pipiens x Cx. quinquefasciatus hybrids collected from nine localities in Brazil and one site in Argentina. We used mitochondrial genes cox1 and nd4, along with the coxA and wsp genes of the maternally-inherited Wolbachia endosymbiont. The nd4 fragment was invariant between samples, whilst cox1 exhibited four haplotypes that separated two types of Cx. quinquefasciatus, one clustered in southern Brazil. Low sequence diversity was generally observed, being discussed. Both Brazilian and Argentinian mosquitoes were infected with a single Wolbachia strain. As reported in previous studies with these populations, cox1 and nd4 diversity is not congruent with the population structure revealed by nuclear markers or alar morphology. Future Cx. quinquefasciatus research should, if possible, evaluate mtDNA diversity in light of other markers.

Genetic diversity of the Brazilian Creole cattle Pé-duro assessed by microsatellites and mitochondrial DNA

The objective of this study was to describe the genetic diversity and structure of the largest Pé-duro population by assessing variation at ten autosomal microsatellite (STR) loci and mitochondrial DNA (mtDNA) sequences. The mean expected heterozygosity was 0.755, the mean observed heterozygosity was 0.600 and significant inbreeding coefficient (Fis) and deviations from the Hardy-Weinberg equilibrium in most of analyzed loci demonstrate the impact of inbreeding and homozygosis on this population. A more in-depth genetic analysis could be achieved by expanding the STR list. The analysis of mtDNA provided evidence of ancestral African taurine haplotypes in Pé-duro and excluded maternal Zebuine introgression. In this report, the main Pé-duro population is genetically portrayed by sampling approximately 40% of it. As this herd represents the core of the Pé-duro conservation program, these findings are of outstanding value for the management and preservation of this Brazilian 'native' cattle breed.

RET haplotype, not linked to the C620R activating mutation, associated with Hirschsprung disease in a novel MEN2 family

Hirschsprung disease is a congenital form of aganglionic megacolon that results from cristopathy. Hirschsprung disease usually occurs as a sporadic disease, although it may be associated with several inherited conditions, such as multiple endocrine neoplasia type 2. The rearranged during transfection (RET) proto-oncogene is the major susceptibility gene for Hirschsprung disease, and germline mutations in RET have been reported in up to 50% of the inherited forms of Hirschsprung disease and in 15-20% of sporadic cases of Hirschsprung disease. The prevalence of Hirschsprung disease in multiple endocrine neoplasia type 2 cases was recently determined to be 7.5% and the cooccurrence of Hirschsprung disease and multiple endocrine neoplasia type 2 has been reported in at least 22 families so far. It was initially thought that Hirschsprung disease could be due to disturbances in apoptosis or due to a tendency of the mutated RET receptor to be retained in the Golgi apparatus. Presently, there is strong evidence favoring the hypothesis that specific inactivating haplotypes play a key role in the fetal development of congenital megacolon/Hirschsprung disease. In the present study, we report the genetic findings in a novel family with multiple endocrine neoplasia type 2: a specific RET haplotype was documented in patients with Hirschsprung disease associated with medullary thyroid carcinoma, but it was absent in patients with only medullary thyroid carcinoma. Despite the limited number of cases, the present data favor the hypothesis that specific haplotypes not linked to RET germline mutations are the genetic causes of Hirschsprung disease.

Cryptic species in the cosmopolitan Bugulan eritina complex (Bryozoa,Cheilostomata)

Previous analyses of the mitochondrial gene cytochrome c oxidase subunit 1 (COI) and γ-proteobacterial endosymbiont diversity have suggested that the marine bryozoan Bugula neritina is a complex of three cryptic species, namely Types S, D and N. Types D and N were previously reported to have restricted distributions along California (western USA) and Delaware and Connecticut (eastern USA), respectively, whereas Type S is considered widespread in tropical, subtropical and temperate regions due to anthropogenic transport. Here, Bayesian species delimitation analysis of a data set composed of two mitochondrial (COI and large ribosomal RNA subunit [16S]) and two nuclear genes (dynein light chain roadblock type-2 protein [DYN] and voltage-dependent anion-selective channel protein [VDAC]) demonstrated that Types S, D and N correspond to three biological species. This finding was significantly supported, in spite of the combinations of priors applied for ancestral population size and root age. Furthermore, COI sequences were used to assess the introduction patterns of the cosmopolitan Type S species. Two COI haplotypes of Type S (S1a and S1d) were found occurring at a global scale. Mantel tests showed correlation between these haplotypes and local sea surface temperature tolerance. Accordingly, the distributions of Type S haplotypes may reflect intraspecific temperature tolerance variation, in addition to the role of introduction vectors. Finally, we show that the Type N may also have been introduced widely, as this species was found for the first time in Central California and north-eastern Australia.

Periodontopathogens levels and clinical response to periodontal therapy in individuals with the interleukin-4 haplotype associated with susceptibility to chronic periodontitis

Periodontitis is an inflammatory disease that results from an interaction between dental biofilm agents and the host immune-inflammatory response. Periodontopathogenic organisms, such as Porphyromonas gingivalis, Tannerella forsythia, and Treponema denticola, as well as the host's susceptibility, represented by the host's genetic makeup, are the key factors that influence this complex disease. Recently, we identified haplotypes in the IL4 gene that were associated with chronic periodontitis (CP). This study aimed to evaluate whether subjects with different IL4 haplotypes (TCI/CCI and TTD/CTI) would be differentially colonized by periodontopathogens and whether they would respond differently to non-surgical periodontal therapy. Thirty-nine patients carrying the IL4 haplotype of genetic susceptibility to CP (IL4+) or protection against CP (IL4-) were evaluated. Those groups were further subdivided into individuals with CP (CP IL4+ or CP IL4-) and those that were periodontally healthy (H) (H IL4+ or H IL4-). CP patients were submitted to non-surgical periodontal therapy. Clinical and microbiological analyses were performed considering the data at baseline and 45 and 90 days after periodontal therapy. Periodontopathogens levels were evaluated by absolute quantitative polymerase chain reaction (qPCR). The baseline data revealed that the total levels of periodontopathogens were higher in the CP IL4+ than in the CP IL4- groups. Clinical analyses revealed that the periodontal therapy was equally effective, independent of the subject's IL4 genetic load. The TCI/CCI IL4 haplotype, previously associated with genetic susceptibility to CP, was also associated with increased levels of periodontopathogenic bacteria, but this genetic background did not influence the response to non-surgical periodontal treatment.

Bone mass and the CAG and GGN androgen receptor polymorphisms in young men

[EN] BACKGROUND: To determine whether androgen receptor (AR) CAG (polyglutamine) and GGN (polyglycine) polymorphisms influence bone mineral density (BMD), osteocalcin and free serum testosterone concentration in young men. METHODOLOGY/PRINCIPAL FINDINGS: Whole body, lumbar spine and femoral bone mineral content (BMC) and BMD, Dual X-ray Absorptiometry (DXA), AR repeat polymorphisms (PCR), osteocalcin and free testosterone (ELISA) were determined in 282 healthy men (28.6+/-7.6 years). Individuals were grouped as CAG short (CAG(S)) if harboring repeat lengths of < or = 21 or CAG long (CAG(L)) if CAG > 21, and GGN was considered short (GGN(S)) or long (GGN(L)) if GGN < or = 23 or > 23. There was an inverse association between logarithm of CAG and GGN length and Ward's Triangle BMC (r = -0.15 and -0.15, P<0.05, age and height adjusted). No associations between CAG or GGN repeat length and regional BMC or BMD were observed after adjusting for age. Whole body and regional BMC and BMD values were similar in men harboring CAG(S), CAG(L), GGN(S) or GGN(L) AR repeat polymorphisms. Men harboring the combination CAG(L)+GGN(L) had 6.3 and 4.4% higher lumbar spine BMC and BMD than men with the haplotype CAG(S)+GGN(S) (both P<0.05). Femoral neck BMD was 4.8% higher in the CAG(S)+GGN(S) compared with the CAG(L)+GGN(S) men (P<0.05). CAG(S), CAG(L), GGN(S), GGN(L) men had similar osteocalcin concentration as well as the four CAG-GGN haplotypes studied. CONCLUSION: AR polymorphisms have an influence on BMC and BMD in healthy adult humans, which cannot be explained through effects in osteoblastic activity.

Genetic characterization and molecular identification of the bloodmeal sources of the potential bluetongue vector Culicoides obsoletus in the Canary Islands, Spain

[EN] Background: Culicoides (Diptera: Ceratopogonidae) biting midges are vectors for a diversity of pathogens including bluetongue virus (BTV) that generate important economic losses. BTV has expanded its range in recent decades, probably due to the expansion of its main vector and the presence of other autochthonous competent vectors. Although the Canary Islands are still free of bluetongue disease (BTD), Spain and Europe have had to face up to a spread of bluetongue with disastrous consequences. Therefore, it is essential to identify the distribution of biting midges and understand their feeding patterns in areas susceptible to BTD. To that end, we captured biting midges on two farms in the Canary Islands (i) to identify the midge species in question and characterize their COI barcoding region and (ii) to ascertain the source of their bloodmeals using molecular tools.Methods: Biting midges were captured using CDC traps baited with a 4-W blacklight (UV) bulb on Gran Canaria and on Tenerife. Biting midges were quantified and identified according to their wing patterns. A 688 bp segment of the mitochondrial COI gene of 20 biting midges (11 from Gran Canaria and 9 from Tenerife) were PCR amplified using the primers LCO1490 and HCO2198. Moreover, after selected all available females showing any rest of blood in their abdomen, a nested-PCR approach was used to amplify a fragment of the COI gene from vertebrate DNA contained in bloodmeals. The origin of bloodmeals was identified by comparison with the nucleotide-nucleotide basic alignment search tool (BLAST). Results: The morphological identification of 491 female biting midges revealed the presence of a single morphospecies belonging to the Obsoletus group. When sequencing the barcoding region of the 20 females used to check genetic variability, we identified two haplotypes differing in a single base. Comparison analysis using the nucleotide-nucleotide basic alignment search tool (BLAST) showed that both haplotypes belong to Culicoides obsoletus, a potential BTV vector. As well, using molecular tools we identified the feeding sources of 136 biting midges and were able to confirm that C. obsoletus females feed on goats and sheep on both islands.Conclusions: These results confirm that the feeding pattern of C. obsoletus is a potentially important factor in BTV transmission to susceptible hosts in case of introduction into the archipelago. Consequently, in the Canary Islands it is essential to maintain vigilance of Culicoides-transmitted viruses such as BTV and the novel Schmallenberg virus.

El uso de la información genética en la gestión de la biodiversidad marina de islas oceánicas : el cangrejo moro "Grapsus adscensionis" en la Macaronesia

Máster Oficial en Gestión Costera

Metodi molecolari nello studio della variabilità genetica di Tuber borchii

Tuber borchii (Ascomycota, order Pezizales) is highly valued truffle sold in local markets in Italy. Despite its economic importance, knowledge on its distribution and population variation is scarce. The objective of this work was to investigate the evolutionary forces shaping the genetic structure of this fungus using coalescent and phylogenetic methods to reconstruct the evolutionary history of populations in Italy. To assess population structure, 61 specimens were collected from 11 different Provinces of Italy. Sampling was stratified across hosts and habitats to maximize coverage in native oak and pine stands and both mychorrizae and fruiting bodies were collected. Samples were identified considering anatomo-morphological characters. DNA was extracted and both multilocus (AFLP) and single-locus (18 loci from rDNA, nDNA, and mtDNA) approaches were used to look for polymorphisms. Screening AFLP profiles, both Jaccard and Dice coefficients of similarity were utilized to transform binary matrix into a distance matrix and then to desume Neighbour-Joining trees. Though these are only preliminary examinations, phylogenetic trees were totally concordant with those deriving from single locus analyses. Phylogenetic analyses of the nuclear loci were performed using maximum likelihood with PAUP and a combined phylogenetic inference, using Bayesian estimation with all nuclear gene regions, was carried out. To reconstruct the evolutionary history, we estimated recurrent migration, migration across the history of the sample, and estimated the mutation and approximate age of mutations in each tree using SNAP Workbench. The combined phylogenetic tree using Bayesian estimation suggests that there are two main haplotypes that are difficult to be differentiated on the basis of morphology, of ecological parameters and symbiontic tree. Between these two lineages, that occur in sympatry within T. borchii populations, there is no evidence of recurrent migration. However, migration over the history of the sample was asymmetrical suggesting that isolation was a result of interrupted gene flow followed by range expansion. Low levels of divergence between the haplotypes indicate that there are likely to be two cryptic species within the T. borchii population sampled. Our results suggest that isolation between populations of T. borchii could have led to reproductive isolation between two lineages. This isolation is likely due to sympatric speciation caused by a multiple colonization from different refugia or a recent isolation. In attempting to determinate whether these haplotypes represent separate species or a partition of the same species we applied Biological and Mechanistic species Concepts. Notwithstanding, further analyses are necessary to evaluate if selection favoured premating or post-mating isolation.

Studio molecolare dei meccanismi dell'autoincompatibilità gametofitica in pero europeo (Pyrus communis)

Self-incompatibility (SI) systems have evolved in many flowering plants to prevent self-fertilization and thus promote outbreeding. Pear and apple, as many of the species belonging to the Rosaceae, exhibit RNase-mediated gametophytic self-incompatibility, a widespread system carried also by the Solanaceae and Plantaginaceae. Pear orchards must for this reason contain at least two different cultivars that pollenize each other; to guarantee an efficient cross-pollination, they should have overlapping flowering periods and must be genetically compatible. This compatibility is determined by the S-locus, containing at least two genes encoding for a female (pistil) and a male (pollen) determinant. The female determinant in the Rosaceae, Solanaceae and Plantaginaceae system is a stylar glycoprotein with ribonuclease activity (S-RNase), that acts as a specific cytotoxin in incompatible pollen tubes degrading cellular RNAs. Since its identification, the S-RNase gene has been intensively studied and the sequences of a large number of alleles are available in online databases. On the contrary, the male determinant has been only recently identified as a pollen-expressed protein containing a F-box motif, called S-Locus F-box (abbreviated SLF or SFB). Since F-box proteins are best known for their participation to the SCF (Skp1 - Cullin - F-box) E3 ubiquitine ligase enzymatic complex, that is involved in protein degradation through the 26S proteasome pathway, the male determinant is supposed to act mediating the ubiquitination of the S-RNases, targeting them for the degradation in compatible pollen tubes. Attempts to clone SLF/SFB genes in the Pyrinae produced no results until very recently; in apple, the use of genomic libraries allowed the detection of two F-box genes linked to each S haplotype, called SFBB (S-locus F-Box Brothers). In Japanese pear, three SFBB genes linked to each haplotype were cloned from pollen cDNA. The SFBB genes exhibit S haplotype-specific sequence divergence and pollen-specific expression; their multiplicity is a feature whose interpretation is unclear: it has been hypothesized that all of them participate in the S-specific interaction with the RNase, but it is also possible that only one of them is involved in this function. Moreover, even if the S locus male and female determinants are the only responsible for the specificity of the pollen-pistil recognition, many other factors are supposed to play a role in GSI; these are not linked to the S locus and act in a S-haplotype independent manner. They can have a function in regulating the expression of S determinants (group 1 factors), modulating their activity (group 2) or acting downstream, in the accomplishment of the reaction of acceptance or rejection of the pollen tube (group 3). This study was aimed to the elucidation of the molecular mechanism of GSI in European pear (Pyrus communis) as well as in the other Pyrinae; it was divided in two parts, the first focusing on the characterization of male determinants, and the second on factors external to the S locus. The research of S locus F-box genes was primarily aimed to the identification of such genes in European pear, for which sequence data are still not available; moreover, it allowed also to investigate about the S locus structure in the Pyrinae. The analysis was carried out on a pool of varieties of the three species Pyrus communis (European pear), Pyrus pyrifolia (Japanese pear), and Malus × domestica (apple); varieties carrying S haplotypes whose RNases are highly similar were chosen, in order to check whether or not the same level of similarity is maintained also between the male determinants. A total of 82 sequences was obtained, 47 of which represent the first S-locus F-box genes sequenced from European pear. The sequence data strongly support the hypothesis that the S locus structure is conserved among the three species, and presumably among all the Pyrinae; at least five genes have homologs in the analysed S haplotypes, but the number of F-box genes surrounding the S-RNase could be even greater. The high level of sequence divergence and the similarity between alleles linked to highly conserved RNases, suggest a shared ancestral polymorphism also for the F-box genes. The F-box genes identified in European pear were mapped on a segregating population of 91 individuals from the cross 'Abbé Fétel' × 'Max Red Bartlett'. All the genes were placed on the linkage group 17, where the S locus has been placed both in pear and apple maps, and resulted strongly associated to the S-RNase gene. The linkage with the RNase was perfect for some of the F-box genes, while for others very rare single recombination events were identified. The second part of this study was focused on the research of other genes involved in the SI response in pear; it was aimed on one side to the identification of genes differentially expressed in compatible and incompatible crosses, and on the other to the cloning and characterization of the transglutaminase (TGase) gene, whose role may be crucial in pollen rejection. For the identification of differentially expressed genes, controlled pollinations were carried out in four combinations (self pollination, incompatible, half-compatible and fully compatible cross-pollination); expression profiles were compared through cDNA-AFLP. 28 fragments displaying an expression pattern related to compatibility or incompatibility were identified, cloned and sequenced; the sequence analysis allowed to assign a putative annotation to a part of them. The identified genes are involved in very different cellular processes or in defense mechanisms, suggesting a very complex change in gene expression following the pollen/pistil recognition. The pool of genes identified with this technique offers a good basis for further study toward a better understanding of how the SI response is carried out. Among the factors involved in SI response, moreover, an important role may be played by transglutaminase (TGase), an enzyme involved both in post-translational protein modification and in protein cross-linking. The TGase activity detected in pear styles was significantly higher when pollinated in incompatible combinations than in compatible ones, suggesting a role of this enzyme in the abnormal cytoskeletal reorganization observed during pollen rejection reaction. The aim of this part of the work was thus to identify and clone the pear TGase gene; the PCR amplification of fragments of this gene was achieved using primers realized on the alignment between the Arabidopsis TGase gene sequence and several apple EST fragments; the full-length coding sequence of the pear TGase gene was then cloned from cDNA, and provided a precious tool for further study of the in vitro and in vivo action of this enzyme.

Molecular phylogeography of the Woodland Ringlet (Erebia medusa [Denis and Schiffermüller] 1775) in Europe

Phylogeography is a recent field of biological research that links phylogenetics to biogeography through deciphering the imprint that evolutionary history has left on the genetic structure of extant populations. During the cold phases of the successive ice ages, which drastically shaped species’ distributions since the Pliocene, populations of numerous species were isolated in refugia where many of them evolved into different genetic lineages. My dissertation deals with the phylogeography of the Woodland Ringlet (Erebia medusa [Denis and Schiffermüller] 1775) in Central and Eastern Europe. This Palaearctic butterfly species is currently distributed from central France and south eastern Belgium over large parts of Central Europe and southern Siberia to the Pacific. It is absent from those parts of Europe with mediterranean, oceanic and boreal climates. It was supposed to be a Siberian faunal element with a rather homogeneous population structure in Central Europe due to its postglacial expansion out of a single eastern refugium. An already existing evolutionary scenario for the Woodland Ringlet in Central and Eastern Europe is based on nuclear data (allozymes). To know if this is corroborated by organelle evolutionary history, I sequenced two mitochondrial markers (part of the cytochrome oxydase subunit one and the control region) for populations sampled over the same area. Phylogeography largely relies on the construction of networks of uniparentally inherited haplotypes that are compared to geographic haplotype distribution thanks to recent developed methods such as nested clade phylogeographic analysis (NCPA). Several ring-shaped ambiguities (loops) emerged from both haplotype networks in E. medusa. They can be attributed to recombination and homoplasy. Such loops usually avert the straightforward extraction of the phylogeographic signal contained in a gene tree. I developed several new approaches to extract phylogeographic information in the presence of loops, considering either homoplasy or recombination. This allowed me to deduce a consistent evolutionary history for the species from the mitochondrial data and also adds plausibility for the occurrence of recombination in E. medusa mitochondria. Despite the fact that the control region is assumed to have a lack of resolving power in other species, I found a considerable genetic variation of this marker in E. medusa which makes it a useful tool for phylogeographic studies. In combination with the allozyme data, the mitochondrial genome supports the following phylogeographic scenario for E. medusa in Europe: (i) a first vicariance, due to the onset of the Würm glaciation, led to the formation of several major lineages, and is mirrored in the NCPA by restricted gene flow, (ii) later on further vicariances led to the formation of two sub-lineages in the Western lineage and two sub-lineages in the Eastern lineage during the Last Glacial Maximum or Older Dryas; additionally the NCPA supports a restriction of gene flow with isolation by distance, (iii) finally, vicariance resulted in two secondary sub-lineages in the area of Germany and, maybe, to two other secondary sub-lineages in the Czech Republic. The last postglacial warming was accompanied by strong range expansions in most of the genetic lineages. The scenario expected for a presumably Siberian faunal element such as E. medusa is a continuous loss of genetic diversity during postglacial westward expansion. Hence, the pattern found in this thesis contradicts a typical Siberian origin of E. medusa. In contrast, it corroboratess the importance of multiple extra-Mediterranean refugia for European fauna as it was recently assumed for other continental species.

Genetic structure and connectivity between populations of two common Mediterranean sessile invertebrates

Population genetic and phylogeography of two common mediterranean species were studied in 10 localities located on the coasts of Toscana, Puglia and Calabria. The aim of the study was to verify the extent of genetic breaks, in areas recognized as boundaries between Mediterranean biogeographic sectors. From about 100 sequences obtained from the mitochondrial Cytochrome Oxidase subunit I (COI) gene of Halocynthia papillosa and Hexaplex trunculus genetic diversity, genetic structure at small and large distances and demographic history of both specieswere analyzed. No evidences of genetic breaks were found for the two species in Toscana and Puglia. The genetic structure of H. trunculus evidences the extent of a barrier to gene flow localized in Calabria, which could be represented by the Siculo-Tunisian Strait and the Strait of Messina. The observed patterns showed similar level of gene flow at small distances in both species, although the two species have different larval ecology. These results suggest that other factors, such as currents, local dynamics and seasonal temperatures, influence the connectivity along the Italian peninsula. The geographic distribution of the haplotypes shows that H. papillosacould represent a single genetic pool in expansion, whereas H. trunculus has two distinct genetic pools in expansion. The demographic pattern of the two species suggests that Pleistocene sea level oscillations, in particular of the LGM, may have played a key role in shaping genetic structure of the two species. This knowledge provides basic information, useful for the definition of management plans, or for the design of a network of marine protected areas along the Italian peninsula.

Mappaggio fine di un QTL principale per la resa in granella sul cromosoma 3B di frumento duro

In durum wheat, two major QTL for grain yield (Qyld.idw-2B and Qyld.idw-3B) and related traits were identified in a recombinant population derived from Kofa and Svevo (Maccaferri et al. 2008). To further investigate the genetic and physiological basis of allelic variation for this important trait, the fine mapping of Qyld.idw-2B e Qyld.idw-3B was done during the PhD. In this regard, new molecular markers were added to increase the map resolution in the target interval. For Qyld.idw-2B region COS markers derived from the synteny between wheat and rice/ sorghum /brachypodiu genomes were screened. While for Qyld.idw-3B region SSR, ISBP and COS markers obtained from BAC end-sequences and BAC sequences generated during the construction of the 3B physical map (Paux et al., 2008) were screened. In the RIL population a final map resolution of 2,8 markers/cM for Qyld.idw-2B and 0,6 markers/cM for Qyld.idw-3B were obtained. Eighteen pairs of near-isogenic lines (NILs) for Qyld.idw-3B were obtained from F4:5 heterogeneous inbred families. In order to confirm the phenotypic effect of the QTL all pairs were evaluated in field trials (2010 and 2011) for all traits. Three pairs of NILs, with contrasted haplotypes at the target region, were crossed to produce a large F2 population (ca. 7,500 plants in total) that was screened for the identification of recombinants. A total of 233 homozygous F4:5 segmental isolines were obtained and the phenotypic and genotypic characterization of these materials were done. A fine mapping for Qyld.idw-3B was obtained and the QTL peak was identified in a interval of 0,4 cM. All markers were anchored to the Chinese Spring physical map of chr. 3B, which allowed us to identify the BAC Contigs spanning the QTL region and to assign the QTL peak to Contig 954. Sequencing of this contig has revealed the presence of 42 genes.

Study of variability and genetic structure of European populations of Myotis emarginatus and Myotis capaccinii (Chiroptera, Vespertilionidae)

The Geoffroy’s bat Myotis emarginatus is mainly present in southern, south-eastern and central Europe (Červerný, 1999) and is often recorded from northern Spain (Quetglas, 2002; Flaquer et al., 2004). It has demonstrated the species’ preference for forest. Myotis capaccinii, confined to the Mediterranean (Guille´n, 1999), is classified as ‘vulnerable’ on a global scale (Hutson, Mickleburgh & Racey, 2001). In general, the species preferred calm waters bordered by well-developed riparian vegetation and large (> 5 m) inter-bank distances (Biscardi et al. 2007). In this study we present the first results about population genetic structure of these two species of genus Myotis. We used two methods of sampling: invasive and non-invasive techniques. A total of 323 invasive samples and a total of 107 non-invasive samples were collected and analyzed. For Myotis emarginatus we have individuated for the first time a set of 7 microsatellites, which can work on this species, started from a set developed on Myotis myotis (Castella et al. 2000). We developed also a method for analysis of non-invasive samples, that given a good percentage of positive analyzed samples. The results have highlighted for the species Myotis emarginatus the presence on the European territory of two big groups, discovered by using the microsatellites tracers. On this species, 33 haplotypes of Dloop have been identified, some of them are presented only in some colonies. We identified respectively 33 haplotypes of Dloop and 10 of cytB for Myotis emarginatus and 25 of dloop and 15 of cytB for Myotis capaccinii. Myotis emarginatus’ results, both microsatellites and mtDNA, show that there is a strong genetic flow between different colonies across Europe. The results achieved on Myotis capaccinii are very interesting, in this case either for the microsatellites or the mitochondrial DNA sequences, and it has been highlighted a big difference between different colonies.