Characterization of simple sequence repeats (SSRs) from Phlebotomus papatasi (Diptera: Psychodidae) expressed sequence tags (ESTs)

<p>Abstract</p> <p>Background</p> <p><it>Phlebotomus papatasi </it>is a natural vector of <it>Leishmania major</it>, which causes cutaneous leishmaniasis in many countries. Simple sequence repeats (SSRs), or microsatellites, are common in eukaryotic genomes and are short, repeated nucleotide sequence elements arrayed in tandem and flanked by non-repetitive regions. The enrichment methods used previously for finding new microsatellite loci in sand flies remain laborious and time consuming; <it>in silico </it>mining, which includes retrieval and screening of microsatellites from large amounts of sequence data from sequence data bases using microsatellite search tools can yield many new candidate markers.</p> <p>Results</p> <p>Simple sequence repeats (SSRs) were characterized in <it>P. papatasi </it>expressed sequence tags (ESTs) derived from a public database, National Center for Biotechnology Information (NCBI). A total of 42,784 sequences were mined, and 1,499 SSRs were identified with a frequency of 3.5% and an average density of 15.55 kb per SSR. Dinucleotide motifs were the most common SSRs, accounting for 67% followed by tri-, tetra-, and penta-nucleotide repeats, accounting for 31.1%, 1.5%, and 0.1%, respectively. The length of microsatellites varied from 5 to 16 repeats. Dinucleotide types; AG and CT have the highest frequency. Dinucleotide SSR-ESTs are relatively biased toward an excess of (AX)n repeats and a low GC base content. Forty primer pairs were designed based on motif lengths for further experimental validation.</p> <p>Conclusion</p> <p>The first large-scale survey of SSRs derived from <it>P. papatasi </it>is presented; dinucleotide SSRs identified are more frequent than other types. EST data mining is an effective strategy to identify functional microsatellites in <it>P. papatasi</it>.</p

Identification, characterization and utilization of EST-derived genic microsatellite markers for genome analyses of coffee and related species

Genic microsatellites or EST–SSRs derived from expressed sequence tags (ESTs) are desired because these are inexpensive to develop, represent transcribed genes, and often a putative function can be assigned to them. In this study we investigated 2,553 coffee ESTs (461 from the public domain and 2,092 in-house generated ESTs) for identification and development of genic microsatellite markers. Of these, 2,458 ESTs (all >100 bp in size) were searched for SSRs using MISA—search module followed by stackPACK clustering that revealed a total of 425 microsatellites in 331 (13.5%) non-redundant ESTs/consensus sequences suggesting an approximate frequency of 1 SSR/2.16 kb of the analysed coffee transcriptome. Identified microsatellites mainly comprised of di-/tri-nucleotide repeats, of which repeat motifs AG and AAG were the most abundant. A total of 224 primer pairs could be designed from the non-redundant SSR-positive ESTs (excluding those with only mononucleotide repeats) for possible use as potential genic markers. Of this set, a total of 24 (10%) primer pairs were tested and 18 could be validated as usable markers. Sixteen of these markers revealed moderate to high polymorphism information content (PIC) across 23 genotypes of C. arabica and C. canephora, while 2 markers were found to be monomorphic. All the markers also showed robust cross-species amplifications across 14 Coffea and 4 Psilanthus species. The apparent broad cross-species/ genera transferability was further confirmed by cloning and sequencing of the amplified alleles. Thus, the study provides an insight about the frequency and distribution of SSRs in coffee transcriptome, and also demonstrates the successful development of genic-SSRs. It is expected that the potential markers described here would add to the repertoire of DNA markers needed for genetic studies in cultivated coffee and also related taxa that constitute the important secondary genepool for coffee improvement

A White Campion (Silene latifolia) floral expressed sequence tag (EST) library: annotation, EST-SSR characterization, transferability, and utility for comparative mapping

<p>Abstract</p> <p>Background</p> <p>Expressed sequence tag (EST) databases represent a valuable resource for the identification of genes in organisms with uncharacterized genomes and for development of molecular markers. One class of markers derived from EST sequences are simple sequence repeat (SSR) markers, also known as EST-SSRs. These are useful in plant genetic and evolutionary studies because they are located in transcribed genes and a putative function can often be inferred from homology searches. Another important feature of EST-SSR markers is their expected high level of transferability to related species that makes them very promising for comparative mapping. In the present study we constructed a normalized EST library from floral tissue of <it>Silene latifolia </it>with the aim to identify expressed genes and to develop polymorphic molecular markers.</p> <p>Results</p> <p>We obtained a total of 3662 high quality sequences from a normalized <it>Silene </it>cDNA library. These represent 3105 unigenes, with 73% of unigenes matching genes in other species. We found 255 sequences containing one or more SSR motifs. More than 60% of these SSRs were trinucleotides. A total of 30 microsatellite loci were identified from 106 ESTs having sufficient flanking sequences for primer design. The inheritance of these loci was tested via segregation analyses and their usefulness for linkage mapping was assessed in an interspecific cross. Tests for crossamplification of the EST-SSR loci in other <it>Silene </it>species established their applicability to related species.</p> <p>Conclusion</p> <p>The newly characterized genes and gene-derived markers from our <it>Silene </it>EST library represent a valuable genetic resource for future studies on <it>Silene latifolia </it>and related species. The polymorphism and transferability of EST-SSR markers facilitate comparative linkage mapping and analyses of genetic diversity in the genus <it>Silene</it>.</p

Microsatellite characterization and marker development from massive sequencing data of the blenny Salaria pavo

Tese de mestrado. Biologia (Bioinformática e Biologia Computacional). Universidade de Lisboa, Faculdade de Ciências, 2011No blenídeo Salaria pavo o comportamento reprodutor de fêmeas e machos encontra-se modulado de acordo com a disponibilidade de ninhos no seu habitat. O sistema de acasalamento é promíscuo e os cuidados parentais às posturas são prestados exclusivamente por parte do macho. Nas populações de substrato rochoso onde existe uma grande disponibilidade de ninhos, os machos nidificam em cavidades na rocha e cortejam activamente as fêmeas, enquanto as fêmeas assumem um papel mais passivo. Por outro lado, a população da Ria Formosa apresenta consideráveis modificações ao padrão observado em costas rochosas. Nesta lagoa costeira, os substratos de nidificação são escassos e os únicos ninhos existentes são encontrados em tijolos utilizados na delimitação de áreas de cultivo de bivalves. A escassez de ninhos leva a que nesta população haja uma reversão dos papéis sexuais, com as fêmeas a cortejarem intensamente os machos e a competirem entre si pelo acesso a ninhos, e ao surgimento de tácticas alternativas de reprodução, com o aparecimento de machos parasitas. Estes machos apresentam um tamanho menor daqueles que nidificam e imitam tanto a morfologia como o comportamento de corte das fêmeas de modo a conseguirem aproximar-se do ninho e fertilizar as posturas durante episódios de desova. Estas tácticas alternativas de reprodução são sequenciais e os machos que apresentam uma táctica parasita numa época de reprodução normalmente adquirem um ninho na época seguinte. De forma a perceber a evolução e manutenção deste tipo de sistemas é necessário estimar o número de ovos que são fertilizados por machos parasitas. Actualmente a forma mais eficaz de fazer testes de paternidades é usando marcadores genéticos, que podem ou não ser complementados com observações de campo. De entre os vários marcadores existentes, o mais utilizado para este tipo de estudos é o microssatélite devido à elevada reproductibilidade dos resultados obtidos. Os microssatélites são motivos de 1 a 6 nucleótidos repetidos em tandem, altamente polimórficos e facilmente reproduzidos por PCR (Polymerase Chain Reation). Até recentemente, o isolamento de novos microssatélites para uma espécie dependia essencialmente da construção de bibliotecas genómicas enriquecidas para determinados microssatélites ou da reutilização de microssatélites de espécies próximas, tendo ambos os processos taxas de insucesso elevadas. Com o rápido desenvolvimento e disponibilização das tecnologias de sequenciação massiva à comunidade científica, uma nova forma de detectar e isolar microssatélites surgiu, a pesquisa in silico. De entre as várias plataformas de sequenciação massiva, a mais indicada para espécies que ainda não tenham sequências referência (genoma), é a pirosequenciação, por sequenciar fragmentos suficientemente longos (≈400 pb) que permitem a assemblagem de novo. Neste trabalho, o objectivo principal foi o de isolar e determinar o polimorfismo dos primeiros marcadores genéticos desenvolvidos para esta espécie. Para tal o transcriptoma deste blenídeo, que já se encontra sequenciado por pirosequenciação mas ainda não disponível nas bases de dados públicas, foi utilizado em conjunto com uma ferramenta bioinformática de pesquisa de microssatélites. Cerca de 640,000 sequências foram alinhadas de forma a se obter as 62,038 sequências consensus (unigenes com um tamanho médio de 452 pb) que foram utilizadas para fazer uma anotação funcional do transcriptoma e para a pesquisa de microssatélites. A anotação funcional foi realizada recorrendo a um algoritmo de alinhamento de sequências e procura de similaridades, BLAST (Basic Local Alignment Search Tool), mais concretamente o BLASTX, uma vez que se utilizou sequências nucleótidicas para pesquisas de semelhança na base de dados não redundante de sequências proteicas do NCBI. Utilizando um e-value<10-5, apenas 31% dos unigenes ficaram anotados funcionalmente e 21% tiveram termos do Gene Ontology atribuídos. Apesar de a percentagem de unigenes anotados ter sido baixa, a anotação realizada tem significado biológico uma vez que 80% das anotações obtidas foram provenientes de dezoito espécies de peixes. A pesquisa por microssatélites in silico resultou em 4,190 microssatélites identificados em 3,670 unigenes, usando como parâmetros de pesquisa microssatélites perfeitos com um mínimo de 6 repetições para todos os tipos de microssatélites (di-, tri-, tetra-, penta- e hexanucleótidos). Como acontece noutras espécies de peixes, os dinucleótidos são o tipo de microssatélites que se encontram em maior frequência (79%), seguidos dos trinucleótidos (19%). Os restantes tipos de microssatélites encontram-se em menor frequência correspondendo a apenas 6.5% do total de microssatélites. Qualquer que seja o processo utilizado para a obtenção de microssatélites, será sempre necessário testá-los e aplicá-los num conjunto de amostras de ADN de forma a poder avaliar o seu polimorfismo. Uma vez que neste trabalho interessava isolar microssatélites polimórficos, foram aplicadas duas estratégias de selecção dos microssatélites. A primeira estratégia utilizada baseou-se no conhecimento a priori do grau de polimorfismo dos microssatélites na população. Para tal, as sequências individuais que compõem o unigene na região do microssatélite foram manualmente curadas in silico de forma a avaliar o seu grau de polimorfismo. No total, 737 microssatélites revelaram ser polimórficos, dos quais 727 eram dinucleótidos e 6 trinucleótidos, com um número máximo de alelos observado in silico de 4 alelos. Para além do grau de polimorfismo também se teve em consideração nesta estratégia se o microssatélite estava num unigene anotado e com termos GO atribuídos e se era possível desenvolver um par de primers que o amplificassem. Depois da filtragem dos microssatélites pelos parâmetros mencionados anteriormente obteve-se uma lista de 97 dinucleótidos dos quais 33 foram seleccionados para aplicação (média de 8.5 repetições). A segunda estratégia para a selecção de microssatélites teve exclusivamente em conta o tamanho da repetição do microssatélite. Estudos anteriores apontam que microssatélites com mais repetições tendem a ser mais polimórficos e, desta forma, foram seleccionados 29 microssatélites para aplicação que continham em média 12 repetições. Para além dos 63 microssatélites seleccionados, outros 3 microssatélites isolados em Lipophrys pholis, pertencente à mesma subfamília do blenídeo Salaria pavo, foram também testados numa amostra de ADN de Salaria pavo para testar a sua amplificação heteróloga. De modo a testar a viabilidade de aplicação destes microssatélites para estudos de genética de populações, quarenta e um microssatélites, cujos primers amplificaram um só fragmento com o tamanho esperado, tiveram o seu primer forward marcado com fluorescência para a genotipagem de 20 indivíduos provenientes da população da ilha da Culatra (Portugal) e 6 indivíduos provenientes das ilhas de Formentera (Espanha) e Borovac (Croácia). Depois de analisados os resultados obtidos, 28 microssatélites isolados em Salaria pavo e 1 microssatélite isolado em Lipophrys pholis ficaram validados para futuros estudos. Na população da Culatra todos os microssatélites, à excepção de 5, microssatélites revelaram ser polimórficos. O número de alelos variou entre 2 e 12 alelos e a heterozigosidade observada e esperada variou entre 0.05 a 0.85 e 0.05 a 0.79 respectivamente. O número médio de alelos e a heterozigosidade esperada foi superior nos microssatélites seleccionados usando a segunda estratégia (6.5 e 0.62) comparativamente à primeira estratégia (3.54 e 0.40). Dois microssatélites revelaram estar em desequilíbrio de Hardy-Weinberg e 2 pares de microssatélites em desequilíbrio de linkage. Todos os microssatélites amplificaram nas amostras de ADN das populações de Formentera e Borovac. Tendo em conta os resultados obtidos, a segunda estratégia revelou ser mais eficiente para a selecção de microssatélites com maior taxa de polimorfismo. Apesar de os microssatélites monomórficos encontrados na população da Culatra terem sido isolados com base na primeira estratégia será necessário aumentar o número de indivíduos genotipados de forma a confirmar-se o grau de polimorfismo observado in silico.Next-generation sequencing is providing researchers with a relatively fast and affordable option for developing microsatellite loci for non-model organisms. The number of studies using this approach is fast-growing and a new focus has been given to the development of microsatellites from cDNA due to their potential in targeting candidate genes (type I markers). When the microsatellite polymorphism is of interest, developing microsatellites can become time-consuming due to the numerous primer pairs to be tested for polymorphism by polymerase chain reaction (PCR) in the focal species. Assemblies have a new potential not yet fully explored for microsatellite mining and evaluation, which can help improve the polymorphism rates obtained. Their high sequence coverage enables to access the microsatellite polymorphism in silico, if the DNA library sequenced was obtained from a pool of DNA from various individuals of the focal species. Therefore, in this study the transcriptome assembly obtained with pyrosequencing for the blenny Salaria pavo, was mined for microsatellites and their polymorphism manually evaluated in silico. Two strategies emerged for microsatellite selection and application in a sample of 26 individuals from the islands of Culatra, Formentera and Borovac. Microsatellites were selected based on their in silico polymorphism and annotation results (first strategy) or based only on their repetition length (second strategy). From a set of 63 microsatellite loci isolated in Salaria pavo sequences, 28 were validated plus one microsatellite from Lipophrys pholis. All microsatellites, except 5, revealed to be polymorphic on the 20 individuals genotyped from Culatra Island, the focal population of study. With the results obtained in this work, the second strategy revealed to be more efficient in yielding polymorphic microsatellites than the first strategy (average number of alleles was 6.5 and 3.54 respectively). Nevertheless, merging these two strategies in future studies may help improving the polymorphism results and at the same time develop type I markers

Maternal effects on oocyte quality in farmed Atlantic halibut (Hippoglossus hippoglossus L.)

Atlantic halibut (Hippoglossus hippoglossus) oocyte quality is highly variable and one of the major bottlenecks during fry-production for on-growth in commercial Atlantic halibut farming. In this study, the effect of maternally derived oocyte constituents (i.e. yolk components and mRNAs) on oocyte quality (i.e fertilisation, embryonic hatching and normal blastomere symmetry) in farmed Atlantic halibut has been investigated. Atlantic halibut embryos and larvae depend on nutritional yolk components until larval first feeding. The importance of yolk n-3 fatty acids for oocyte quality was confirmed. However, highest positive correlations with oocyte quality were found for the less studied fatty acids dihomo-γ-linolenic acid (DGLA, 20:3n6) and docosapentaenoic acid (DHA, 20:5n3) that are known to compete with two of the most abundant fatty acids, arachidonic acid (ARA, 20:4n6)and docosahexaenoic acid (DPA, 22:5n3), respectively during fatty acid metabolism. High methionine and aspartic concentrations, amino acids essential to eukaryotic protein synthesis, were found to influence oocyte quality positively while no significant correlations were found between oocyte folate concentrations and oocyte quality. Before activation of zygotic transcription, maternal mRNAs control cell divisions and embryonic patterning. Due to the limited available genomic information on Atlantic halibut maternal transcripts, an expressed sequence tag (EST) maternal library containing 2,341high quality ESTs was created by suppressive subtractive hybridization (SSH). The maternal library constitutes an EST pool to identify suitable Atlantic halibut reference genes and identify differentially expressed maternal genes in high and low quality Atlantic halibut oocytes. To perform reliable quantification of gene expression by qPCR, stable reference genes have to be used to normalize target gene expression. Tubb2/Actb and Tbb2/Fau were identified as the best two-gene normalization factors during Atlantic halibut embryonic and larval development. Either of these normalization factors can be used for future developmental gene expression studies in Atlantic halibut. Tubb2/Actb was further used as reference gene during this study. Poor embryonic hatching success was found to not be correlated with a general decrease in oocyte maternal transcript abundance but with low transcript levels of specific maternal transcripts by qPCR. The majority of genes showed either no or very minor correlations between their transcript levels and oocyte quality parameters (Fertilisation: 13-93 %, embryonic hatching: 1-94 %). However, maternal transcript levels of three genes, most likely involved in nuclear protein and mRNA transport, growth factor regulation, and embryonic patterning, correlated with oocyte quality. Further, a new Atlantic halibut 4x44k oligonucleotide microarray was constructed and used to identify 192 strictly maternal genes during Atlantic halibut embryonic development and 20 differentially expressed genes between high and low quality oocytes, involved in immune response, metabolism, RNA transcription, protein degradation, cell signalling and the cytoskeleton. Microarray validation confirmed its suitability for future gene expression studies during Atlantic halibut embryonic development. The identified maternal genes in this study can serve as a pool for future in-depth studies of embryonic gene expression to advance the knowledge of important developmental processes such as germ cell development, growth and immune response in Atlantic halibut. Some of these may serve as possible markers for Atlantic halibut oocyte quality due to their high expression differences between high and low quality oocytes. Future nutritional studies on Atlantic halibut broodstock should focus on the identified yolk constituents acting positively on oocyte quality

Image-based tissue informatics - TAA induced hepatotoxicity model

Master'sMASTER OF SCIENC

Long-term effects of oestrogenic effluent exposure on wild fish populations

This thesis was submitted for the award of Doctor of Philosophy and was awarded by Brunel University London.Freshwater streams in the developed world are becoming increasingly dominated by treated wastewater. Continually discharged into most surface waters, these effluents contain a suite of bioactive man-made chemicals, including steroid and non-steroid oestrogens, which have been found to feminise male fish, skew sex ratios, and cause reproductive failure. However, the consequences of reproductive disruption remain poorly explored at the population level. This thesis was initiated to evaluate how oestrogenic contaminants might influence the population ecology of a common cyprinid, the roach (Rutilus rutilus). An investigation encompassing population structure, multigenerational exposure and the role of additional drivers of fish population dynamics was undertaken to contextualise the effects of oestrogenic effluents on wild fish populations. Population genetic analysis of UK roach found they exhibit moderately high levels of genetic diversity and significant intra-river genetic structure. Genetically differentiated local subpopulations indicate little interbreeding and limited gene flow, consistent with a typical metapopulation that has not been homogenised by restocking. Similarly, my thesis demonstrates no significant relationship between effluent exposure and Ne (effective population size) or genetic diversity of roach populations, albeit a 65% reduction in Ne is possible at highly polluted sites. River stretches contaminated with high levels of effluent can support breeding populations, which recruit successfully with minimal immigration from less contaminated sites. Multigenerational effects of effluent exposure on roach were also evaluated experimentally using reproductive success from breeding adults over three generations. Lifelong exposure to 100% treated effluent resulted in feminised phenotypes (ovarian cavities and intersex condition) in males but no observable effect on females. Additionally, despite gonadal disruption in males and effluent exposure of their mothers, I found no detrimental effect on their ability to compete with control fish. Instead, reproductive success was primarily determined by body size. A novel approach considering additional fish population drivers suggests that genetic diversity and species diversity decline in parallel with an increasing presence of disturbed land, when combined with geographical isolation. In conclusion, group assemblage and genetic structure of fish populations appears multi-causal and cannot be disaggregated, such that a single environmental characteristic can be shown to drive patterns of population success.NERC (NE/G019355/1) and DEFRA