The genetic structure of Australasian green turtles (Chelonia mydas): exploring the geographical scale of genetic exchange

Ecological and genetic studies of marine turtles generally support the hypothesis of natal homing, but leave open the question of the geographical scale of genetic exchange and the capacity of turtles to shift breeding sites. Here we combine analyses of mitochondrial DNA (mtDNA) variation and recapture data to assess the geographical scale of individual breeding populations and the distribution of such populations through Australasia. We conducted multiscale assessments of mtDNA variation among 714 samples from 27 green turtle rookeries and of adult female dispersal among nesting sites in eastern Australia. Many of these rookeries are on shelves that were flooded by rising sea levels less than 10 000 years (c. 450 generations) ago. Analyses of sequence variation among the mtDNA control region revealed 25 haplotypes, and their frequency distributions indicated 17 genetically distinct breeding stocks (Management Units) consisting either of individual rookeries or groups of rookeries in general that are separated by more than 500 km. The population structure inferred from mtDNA was consistent with the scale of movements observed in long-term mark-recapture studies of east Australian rookeries. Phylogenetic analysis of the haplotypes revealed five clades with significant partitioning of sequence diversity (Phi = 68.4) between Pacific Ocean and Southeast Asian/Indian Ocean rookeries. Isolation by distance was indicated for rookeries separated by up to 2000 km but explained only 12% of the genetic structure. The emerging general picture is one of dynamic population structure influenced by the capacity of females to relocate among proximal breeding sites, although this may be conditional on large population sizes as existed historically across this region.

Isolation and characterization of microsatellite loci from Macadamia

Thirty-three microsatellite loci were isolated for the Australian rainforest tree Macadamia integrifolia. Genotyping across a test panel of 43 commercial cultivars generated an average polymorphic information content of 0.480. Five loci showed no polymorphism across cultivars. Significant linkage disequilibrium was detected in 10 pairwise comparisons, including two pairs of loci identified from the same clone sequence. The 33 microsatellite loci represent a significant tool for genome mapping and population genetic studies.

Understanding Isolation and Change in Island Human Populations through a study of Indigenous Cultural Patterns in the Gulf of Carpentaria

This paper presents a set of hypotheses to explain the cultural differences between Aboriginal people of the North and South Wellesley Islands, Gulf of Carpentaria and to characterise the relative degree and nature of their isolation and cultural change over a 10,000-year time-scale. This opportunity to study parallelisms and divergences in the cultural and demographic histories of fisher-hunter-gatherers arises from the comparison of three distinct cultural groupings: (a) the Ganggalida of the mainland, (b) the Lardil and Yangkaal of the North Wellesley Islands, and (c) the Kaiadilt of the South Wellesley Islands. Despite occupying similar island environments and despite their languages being as closely related as for example, the West Germanic languages, there are some major differences in cultural, economic and social organization as well as striking genetic differences between the North and South Wellesley populations. This paper synthesizes data from linguistics, anthropology, archaeology, genetics and environmental science to present hypotheses of how these intriguing differences were generated, and what we might learn about early processes of marine colonization and cultural change from the Wellesley situation.

Genetic structure and male-mediated gene flow in the ghost bat (Macroderma gigas)

The Australian ghost bat is a large, opportunistic carnivorous species that has undergone a marked range contraction toward more mesic, tropical sites over the past century. Comparison of mitochondrial DNA (mtDNA) control region sequences and six nuclear microsatellite loci in 217 ghost bats from nine populations across subtropical and tropical Australia revealed strong population subdivision (mtDNA phi(ST) = 0.80; microsatellites URST = 0.337). Low-latitude (tropical) populations had higher heterozygosity and less marked phylogeographic structure and lower subdivision among sites within regions (within Northern Territory [NT] and within North Queensland [NQ]) than did populations at higher latitudes (subtropical sites; central Queensland [CQ]), although sampling of geographically proximal breeding sites is unavoidably restricted for the latter. Gene flow among populations within each of the northern regions appears to be male biased in that the difference in population subdivision for mtDNA and microsatellites (NT phi(ST) = 0.39, URST = 0.02; NQ phi(ST) = 0.60, URST = -0.03) is greater than expected from differences in the effective population size of haploid versus diploid loci. The high level of population subdivision across the range of the ghost bat contrasts with evidence for high gene flow in other chiropteran species and may be due to narrow physiological tolerances and consequent limited availability of roosts for ghost bats, particularly across the subtropical and relatively arid regions. This observation is consistent with the hypothesis that the contraction of the species' range is associated with late Holocene climate change. The extreme isolation among higher-latitude populations may predispose them to additional local extinctions if the processes responsible for the range contraction continue to operate.

Isolation, characterization and discovery of new alleles of sHsp genes in durum wheat (Triticum durum Desf.)

Gli organismi vegetali mostrano una notevole capacità di adattamento alle condizioni di stress e lo studio delle componenti molecolari alla base dell'adattamento in colture cerealicole di interesse alimentare, come il frumento, è di particolare interesse per lo studio di varietà che consentano una buona produzione con basso input anche in condizioni ambientali non ottimali. L'esposizione delle colture cerealicole a stress termico durante determinate fasi del ciclo vitale influisce negativamente sulla resa e sulla qualità, a questo fine è necessario chiarire le basi genetiche e molecolari della termotolleranza per identificare geni e alleli vantaggiosi da impiegare in programmi di incrocio volti al miglioramento genetico. Numerosi studi dimostrano il coinvolgimento delle sHSP a localizzazione cloroplastica (in frumento sHSP26) nel meccanismo di acquisizione della termotolleranza e la loro interazione con diverse componenti del fotosistema II (PSII) che determinerebbe un’azione protettiva in condizioni di stress termico e altri tipi di stress. Lo scopo del progetto è quello di caratterizzare in frumento duro nuove varianti alleliche correlate alla tolleranza a stress termico mediate l'utilizzo del TILLING (Target Induced Local Lesion In Genome), un approccio di genetica inversa che prevede la mutagenesi e l'identificazione delle mutazioni indotte in siti di interesse. Durante la tesi sono state isolate e caratterizzate 3 sequenze geniche complete per smallHsp26 denominate TdHsp26-A1; TdHsp26-A2; TdHsp26-B1 e un putativo pseudogene denominato TdHsp26-A3. I geni isolati sono stati usati come target in analisi di TILLING in due popolazioni di frumento duro mutagenizzate con EMS (EtilMetanoSulfonato). Nel nostro studio sono stati impiegati due differenti approcci di TILLING: un approccio di TILLING classico mediante screening con High Resolution Melting (HRM) e un approccio innovativo che sfrutta un database di TILLING recentemente sviluppato. La popolazione di mutanti cv. Kronos è stata analizzata per la presenza di mutazioni in tutti e tre i geni individuati mediante ricerca online nel database di TILLING, il quale sfrutta la tecnica dell’exome capture sulla popolazione di TILLING seguito da sequenziamento ad alta processività. Attraverso questa tecnica sono state individuate, nella popolazione mutagenizzata di frumento duro cv. Kronos, 36 linee recanti mutazioni missenso. Contemporaneamente lo screening con HRM, effettuato su 960 genotipi della libreria di TILLING di frumento duro cv. Cham1 ha consentito di individuare mutazioni in una regione di 211bp di interesse funzionale del gene TdHsp26-B1, tra le quali 3 linee mutanti recanti mutazioni missenso in omozigosi. Alcune mutazioni missenso individuate sui due geni TdHsp26-A1 e TdHsp26-B1 sono state confermate in vivo nelle piante delle rispettive linee mutanti generando marcatori codominanti KASP (Kompetitive Allele Specific PCR) con cui è stato possibile verificare anche il grado di zigosità di tali mutazioni. Al fine di ridurre il numero di mutazioni non desiderate nelle linee risultate più interessanti, è stato eseguito il re-incrocio dei mutanti con i relativi parentali wild type ed inoltre sono stati generati alcuni doppi mutanti che consentiranno di comprendere meglio i meccanismi molecolari presieduti da questa classe genica. Gli individui F1 degli incroci sono stati poi genotipizzati con i medesimi marcatori KASP specifici per la mutazione di interesse per verificare la buona riuscita dell’incrocio. Questo approccio ha permesso di individuare ed implementare risorse genetiche utili ad intraprendere studi funzionali relativi al ruolo di smallHSP plastidiche implicate nella acquisizione di termotolleranza in frumento duro e di generare marcatori potenzialmente utili in futuri programmi di breeding.

The isolation, characterization, and application of WRKY genes as useful molecular markers in tropical trees

The improvement of tropical tree crops using conventional breeding methods faces challenges due to the length of time involved. Thus, like most crops, there is an effort to utilize molecular genetic markers in breeding programs to select for desirable agronomic traits. Known as marker assisted breeding or marker assisted selection, genetic markers associated with a phenotype of interest are used to screen and select material reducing the time necessary to evaluate candidates. As the focus of this research was improving disease resistance in tropical trees, the usefulness of the WRKY gene superfamily was investigated as candidates for generating useful molecular genetic markers. WRKY genes encode plant-specific transcriptional factors associated with regulating plants' responses to both biotic and abiotic stress. ^ One pair of degenerate primers amplified 48 WRKY gene fragments from three taxonomically distinct, economically important, tropical tree crop species: 18 from Theobroma cacao L., 21 from Cocos nucifera L. and 9 from Persea americana Mill. Several loci from each species were polymorphic because of single nucleotide substitutions present within a putative non-coding region of the loci. Capillary array electrophoresis-single strand conformational polymorphism (CAE-SSCP) mapped four WRKY loci onto a genetic linkage map of a T. cacao F2 population segregating for resistance to witches' broom disease. Additionally, PCR primers specific for four T. cacao loci successfully amplified WRKY loci from 15 members of the Byttneriae tribe. A method was devised to allow the reliable discrimination of alleles by CAE-SSCP using only the mobility assigned to the sample peaks. Once this method was validated, the diversity of three WRKY loci was evaluated in a germplasm collection of T. cacao . One locus displayed high diversity in the collection, with at least 18 alleles detected from mobility differences of the product peaks. The number of WRKY loci available within the genome, ease of isolation by degenerate PCR, codominant segregation demonstrated in the F2 population, and usefulness for screening germplasm collections and closely related wild species demonstrates that the WRKY superfamily of genes are excellent candidates for developing a number of genetic molecular markers for breeding purposes in tropical trees. ^

The isolation and characterization of tescalcin, a novel gene differentially expressed in the mouse testis during the early stages of gonadal differentiation

Gonadal development is an ideal model to study organogenesis because a variety of developmental processes can be studied during the differentiation of the bipotential primordium into testis or ovary. To better understand this process, Representational Difference Analysis of cDNA was used to identify genes that are differentially expressed in mouse gonads at 13.5 days post-coitus. The analysis led to the identification of three testis specific genes and a sequence that was only expressed in the ovary. The male genes identified: renin, Col9a3, and a novel gene termed tescalcin had patterns of expression that suggested a role in testis determination. ^ Studies of the tescalcin gene revealed that it is organized into eight exons and seven introns. The gene was located at 64 cM in mouse chromosome 5, where it spans approximately 35 Kb. Three mRNA variants resulting from alternative splicing of intron 5 were identified in mouse tissues. Gel mobility shift assays demonstrated that Sp1 and Sp3 from Y-1, msc-1, and MIN-6 cells nuclear extracts bind the GC-boxes within the tescalcin proximal promoter. Bisulfite sequencing analysis of tescalcin CpG island revealed that it is differentially methylated in male and female mouse embryonic gonads, and that hypermethylation of this region represses expression of tescalcin in the β-TC3 cell line. ^ The major tescalcin mRNA encodes a protein with 214 amino acids that contains a consensus EF-hand Ca2+-binding domain and an N-myristoylation motif. The amino acid sequence of tescalcin is highly conserved among various species, and it showed the highest homology with calcineurin B homologous proteins 1 and 2, and calcineurin B. Western blot analysis using antibodies generated against the tescalcin protein confirmed its presence in specific mouse tissues and cell lines. Immunohistochemical analysis of mouse embryos confirmed the pattern of expression of tescalcin mRNA in fetal testis. Using pull-down assays, glyceraidehydes-3-phosphate dehydrogenase was identified as an interacting and potential functional partner of tescalcin. ^ The identification and characterization of tescalcin as a novel embryonic testicular marker will contribute to the elucidation of the genetic pathways involved in testis development and likely to the understanding of pathological conditions such as sex reversal and infertility. ^

Genetic and Molecular Basis of Encapsulation and Capsule Diversity in Kingella kingae

Kingella kingae is a bacterial pathogen that is increasingly recognized as an etiology of septic arthritis, osteomyelitis, bacteremia, and endocarditis in young children. The pathogenesis of K. kingae disease starts with bacterial adherence to the respiratory epithelium of the posterior pharynx. Previous work has identified type IV pili and a trimeric autotransporter protein called Knh (Kingella NhhA homolog) as critical factors for adherence to human epithelial cells. Additional studies established that the presence of a polysaccharide capsule interferes with Knh-mediated adherence. Given the inhibitory role of capsule during adherence we sought to uncover the genes involved in capsule expression to understand how capsule is elaborated on the cell surface. Additionally, this work aimed to further characterize capsule diversity among K. kingae clinical isolates and to investigate the relationship between capsule type and site of isolation.

We first set out to identify the carbohydrates present in the K. kingae capsule present in the prototype strain 269-492. Glycosyl composition and NMR analysis of surface extractable polysaccharides demonstrated two distinct polysaccharides, one consisting of GalNAc and Kdo with the structure →3)-β-GalpNAc-(1→5)-β-Kdop-(2→ and the other containing galactose alone with the structure →5)-β-Galf-(1→.

To discern the two polysaccharides we disrupted the ctrA gene required for surface localization of the K. kingae polysaccharide capsule and observed a loss of GalNAc and Kdo but no effect on the presence of Gal in bacterial surface extracts. In contrast, deletion of the pamABCDE locus involved in production of a reported galactan exopolysaccharide eliminated Gal but had no effect on the presence of GalNAc and Kdo in surface extracts. These results established that K. kingae strain KK01 produces a polysaccharide capsule with the structure →3)-β-GalpNAc-(1→5)-β-Kdop-(2→ and a separate exopolysaccharide with the structure →5)-β-Galf-(1→.

Having established that K. kingae produces a capsule comprised of GalNAc and Kdo, we next set out to identify the genetic determinants of capsule through a transposon mutagenesis screen. In addition to the previously identified ctrABCD operon, lipA, lipB, and a putative glycosyltransferase termed csaA (capsule synthesis region A gene A) were found to be essential for the production of surface-localized capsule. The ctr operon, lipA, lipB, and csaA were found to be present at unlinked locations throughout the genome, which is atypical for gram-negative organisms that elaborate a capsule dependent on an ABC-type transporter for surface localization. Through examining capsule localization in the ctrA, lipA, lipB, and csaA mutant strains, we determined that the ctrABCD, lipA/lipB, and csaA gene products respectively function in capsule export, assembly, and synthesis, respectively. The GalNAc transferase and Kdo transferase domains found in CsaA further support its role in catalyzing the synthesis of the GalNAc-Kdo capsule in the K. kingae prototype strain.

To investigate the capsule diversity that exists in K. kingae we screened a panel of strains isolated from patients with invasive disease or healthy carriers for the csaA capsule synthesis locus. We discovered that Kingella kingae expresses one of 4 capsule synthesis loci (csa, csb, csc, or csd) associated with a capsule consisting of Kdo and GalNAc (type a), Kdo and GlcNAc (type b), Kdo and ribose (type c), and GlcNAc and galactose (type d), respectively. Cloning of the csa, csb, csc, or csd locus into the empty flanking gene region in a non-encapsulated mutant (creation of an isogenic capsule swap) was sufficient to produce either the type a, type b, or type c capsule, respectively, further supporting the role of these loci in expression of a specific polysaccharide linkage. Capsule type a and capsule type b accounted for 96% of invasive strains. Conversely, capsule type c and capsule type d were found disproportionately among carrier isolates, suggesting that capsule type is important in promoting invasion and dissemination.

In conclusion, we discovered that Kingella kingae expresses a polysaccharide capsule and an exopolysaccharide on its surface that require distinct genetic loci for surface localization. Further investigation into genetic determinants of encapsulation revealed the loci ctrABCD, lipA/lipB, and a putative glycosyltransferase are required for capsule expression, with the gene products having roles in capsule export, assembly, and synthesis, respectively. The putative glycosyltransferase CsaA was determined to be a bifunctional enzyme with both GalNAc-transferase and Kdo-transferase activity. Furthermore, we discovered a total of 4 capsule types expressed in clinical isolates of K. kingae, each with a distinct capsule synthesis locus. The variation in the proportion of capsule types found between invasive strains and carriage strains suggest that capsule type is important in promoting invasion and dissemination. Taken together, this work expands our knowledge of the capsule types expressed among K. kingae carrier and invasive isolates and provides insights into the common genetic determinants of capsule expression. These contributions may lead to selecting clinically relevant capsule types to develop into a capsule based vaccine to prevent K. kingae colonization.

Acoustic and Magnetic Techniques for the Isolation and Analysis of Cells in Microfluidic Platforms

Cancer comprises a collection of diseases, all of which begin with abnormal tissue growth from various stimuli, including (but not limited to): heredity, genetic mutation, exposure to harmful substances, radiation as well as poor dieting and lack of exercise. The early detection of cancer is vital to providing life-saving, therapeutic intervention. However, current methods for detection (e.g., tissue biopsy, endoscopy and medical imaging) often suffer from low patient compliance and an elevated risk of complications in elderly patients. As such, many are looking to “liquid biopsies” for clues into presence and status of cancer due to its minimal invasiveness and ability to provide rich information about the native tumor. In such liquid biopsies, peripheral blood is drawn from patients and is screened for key biomarkers, chiefly circulating tumor cells (CTCs). Capturing, enumerating and analyzing the genetic and metabolomic characteristics of these CTCs may hold the key for guiding doctors to better understand the source of cancer at an earlier stage for more efficacious disease management.

The isolation of CTCs from whole blood, however, remains a significant challenge due to their (i) low abundance, (ii) lack of a universal surface marker and (iii) epithelial-mesenchymal transition that down-regulates common surface markers (e.g., EpCAM), reducing their likelihood of detection via positive selection assays. These factors potentiate the need for an improved cell isolation strategy that can collect CTCs via both positive and negative selection modalities as to avoid the reliance on a single marker, or set of markers, for more accurate enumeration and diagnosis.

The technologies proposed herein offer a unique set of strategies to focus, sort and template cells in three independent microfluidic modules. The first module exploits ultrasonic standing waves and a class of elastomeric particles for the rapid and discriminate sequestration of cells. This type of cell handling holds promise not only in sorting, but also in the isolation of soluble markers from biofluids. The second module contains components to focus (i.e., arrange) cells via forces from acoustic standing waves and separate cells in a high throughput fashion via free-flow magnetophoresis. The third module uses a printed array of micromagnets to capture magnetically labeled cells into well-defined compartments, enabling on-chip staining and single cell analysis. These technologies can operate in standalone formats, or can be adapted to operate with established analytical technologies, such as flow cytometry. A key advantage of these innovations is their ability to process erythrocyte-lysed blood in a rapid (and thus high throughput) fashion. They can process fluids at a variety of concentrations and flow rates, target cells with various immunophenotypes and sort cells via positive (and potentially negative) selection. These technologies are chip-based, fabricated using standard clean room equipment, towards a disposable clinical tool. With further optimization in design and performance, these technologies might aid in the early detection, and potentially treatment, of cancer and various other physical ailments.

Genetic Analysis of Mitotic Recombination in Saccharomyces cerevisiae

Mitotic genome instability can occur during the repair of double-strand breaks (DSBs) in DNA, which arise from endogenous and exogenous sources. Studying the mechanisms of DNA repair in the budding yeast, Saccharomyces cerevisiae has shown that Homologous Recombination (HR) is a vital repair mechanism for DSBs. HR can result in a crossover event, in which the broken molecule reciprocally exchanges information with a homologous repair template. The current model of double-strand break repair (DSBR) also allows for a tract of information to non-reciprocally transfer from the template molecule to the broken molecule. These “gene conversion” events can vary in size and can occur in conjunction with a crossover event or in isolation. The frequency and size of gene conversions in isolation and gene conversions associated with crossing over has been a source of debate due to the variation in systems used to detect gene conversions and the context in which the gene conversions are measured.

In Chapter 2, I use an unbiased system that measures the frequency and size of gene conversion events, as well as the association of gene conversion events with crossing over between homologs in diploid yeast. We show mitotic gene conversions occur at a rate of 1.3x10-6 per cell division, are either large (median 54.0kb) or small (median 6.4kb), and are associated with crossing over 43% of the time.

DSBs can arise from endogenous cellular processes such as replication and transcription. Two important RNA/DNA hybrids are involved in replication and transcription: R-loops, which form when an RNA transcript base pairs with the DNA template and displaces the non-template DNA strand, and ribonucleotides embedded into DNA (rNMPs), which arise when replicative polymerase errors insert ribonucleotide instead of deoxyribonucleotide triphosphates. RNaseH1 (encoded by RNH1) and RNaseH2 (whose catalytic subunit is encoded by RNH201) both recognize and degrade the RNA in within R-loops while RNaseH2 alone recognizes, nicks, and initiates removal of rNMPs embedded into DNA. Due to their redundant abilities to act on RNA:DNA hybrids, aberrant removal of rNMPs from DNA has been thought to lead to genome instability in an rnh201Δ background.

In Chapter 3, I characterize (1) non-selective genome-wide homologous recombination events and (2) crossing over on chromosome IV in mutants defective in RNaseH1, RNaseH2, or RNaseH1 and RNaseH2. Using a mutant DNA polymerase that incorporates 4-fold fewer rNMPs than wild type, I demonstrate that the primary recombinogenic lesion in the RNaseH2-defective genome is not rNMPs, but rather R-loops. This work suggests different in-vivo roles for RNaseH1 and RNaseH2 in resolving R-loops in yeast and is consistent with R-loops, not rNMPs, being the the likely source of pathology in Aicardi-Goutières Syndrome patients defective in RNaseH2.

Contemporary population genetic structure and phylogeography of pink salmon

Thesis (Master's)--University of Washington, 2016-06

DNA extraction and RAPD markers to assess the genetic similarity among Gelidium sesquipedale (Rhodophyta) populations

A simple method developed for genomic DNA isolation from fungus was tested on the red alga, Gelidium sesquipedale (Clem.) Born et Thur., which is commercially exploited for its high sulfated polysaccharide (agar) content. This method is faster, cheaper, and less toxic than conventional phenol/chloroform methods. Random amplified polymorphic DNA (RAPD) amplifications were performed successfully without the necessity of purifying the DNA. RAPD markers were used to investigate the genetic similarity among three natural populations of G. sesquipedale from southern Portugal. Bulked-genomic DNA samples of 15 different individuals were made in each population. These can be conceived of as a sample of the population DNA. Of the 62 primers screened, 41 produced bands and 22 revealed polymorphisms. Genetic similarities among populations were high. Populations that are further away from each other have the lowest similarity coefficients, whereas the intermediate Ingrina population, located on the south coast, showed higher genetic similarity with the Odeceixe population located on the southwest coast, than with the Sao Rafael southern population. This suggests a higher genetic flow between Odeceixe and Ingrina or the result may be a founder effect in the sense that the species has propagated from the east coast to the south coast of Portugal. We conclude that the use of this isolation method with RAPD analysis is appropriate to characterize the genetic variability of this commercial species along its geographical distribution. Large sample sizes can be screened at a relatively low cost. Finding genetic markers for commercial populations of C. sesquipedale may be of industrial interest.

Connectivity patterns inferred from the genetic structure of white seabream (Diplodus sargus L.)

Themarine environment seems, at first sight, to be a homogeneousmediumlacking barriers to species dispersal. Nevertheless, populations of marine species show varying levels of gene flow and population differentiation, so barriers to gene flow can often be detected. Weaimto elucidate the role of oceanographical factors ingenerating connectivity among populations shaping the phylogeographical patterns in the marine realm, which is not only a topic of considerable interest for understanding the evolution ofmarine biodiversity but also formanagement and conservation of marine life. For this proposal,we investigate the genetic structure and connectivity between continental and insular populations ofwhite seabreamin North East Atlantic (NEA) and Mediterranean Sea (MS) aswell as the influence of historical and contemporary factors in this scenario using mitochondrial (cytochrome b) and nuclear (a set of 9 microsatellite) molecular markers. Azores population appeared genetically differentiated in a single cluster using Structure analysis. This result was corroborated by Principal Component Analysis (PCA) and Monmonier algorithm which suggested a boundary to gene flow, isolating this locality. Azorean population also shows the highest significant values of FST and genetic distances for both molecular markers (microsatellites and mtDNA). We suggest that the breakdown of effective genetic exchange between Azores and the others' samples could be explained simultaneously by hydrographic (deep water) and hydrodynamic (isolating current regimes) factors acting as barriers to the free dispersal of white seabream(adults and larvae) and by historical factors which could be favoured for the survival of Azorean white seabream population at the last glaciation. Mediterranean islands show similar genetic diversity to the neighbouring continental samples and nonsignificant genetic differences. Proximity to continental coasts and the current system could promote an optimal larval dispersion among Mediterranean islands (Mallorca and Castellamare) and coasts with high gene flow.

DNA extraction and RAPD markers to assess the genetic similarity among Gelidium sesquipedale (Rhodophyta) populations

A simple method developed for genomic DNA isolation from fungus was tested on the red alga, Gelidium sesquipedale (Clem.) Born et Thur., which is commercially exploited for its high sulfated polysaccharide (agar) content. This method is faster, cheaper, and less toxic than conventional phenol/chloroform methods. Random amplified polymorphic DNA (RAPD) amplifications were performed successfully without the necessity of purifying the DNA. RAPD markers were used to investigate the genetic similarity among three natural populations of G. sesquipedale from southern Portugal. Bulked-genomic DNA samples of 15 different individuals were made in each population. These can be conceived of as a sample of the population DNA. Of the 62 primers screened, 41 produced bands and 22 revealed polymorphisms. Genetic similarities among populations were high. Populations that are further away from each other have the lowest similarity coefficients, whereas the intermediate Ingrina population, located on the south coast, showed higher genetic similarity with the Odeceixe population located on the southwest coast, than with the Sao Rafael southern population. This suggests a higher genetic flow between Odeceixe and Ingrina or the result may be a founder effect in the sense that the species has propagated from the east coast to the south coast of Portugal. We conclude that the use of this isolation method with RAPD analysis is appropriate to characterize the genetic variability of this commercial species along its geographical distribution. Large sample sizes can be screened at a relatively low cost. Finding genetic markers for commercial populations of C. sesquipedale may be of industrial interest.