La especie albaricoquero (P. armeniaca L.), originaria de China y Asia Central, se cultiva en España principalmente en Murcia y Valencia. La aparición y extensión del virus de la Sharka ha puesto de manifiesto la necesidad de obtener nuevas variedades resmoleculares que se han definido útiles en los procesos de selección en programas de mejora: RFLPs (Restriction Fragment Lenght Polimorphism), RAPDs (Random Amplified Polimorphic DNA) y AFLPs (Amplified Fragment Length Polimorphism).Con ellos se ha realizado un estudio de diversidad genética, utilizando 16 variedades de albaricoquero procedentes de Francia, España y Norteamérica, que incluyen variedades resistentes y susceptibles a Sharka. Algunas de ellas se están utilizando como genitores en el programa de mejora del IVIA. Se ha elaborado el primer mapa genómico de la especie albaricoquero, basado en los marcadores moleculares RAPDs y AFLPs. Se ha estudiado la herencia del carácter resistencia a Sharka, y se ha establecido la hipótesis de herencia basada en 2 genes independientes.Con el fin de obtener marcadores moleculares ligados a los caracteres autoincompatibilidad y androesterilidad en albaricoquero, se ha utilizado la técnica del Bulk Segregation Analysis (BSA) combinada con marcadores tipo RAPD. / Hurtado Ruiz, MA. (1999). Obtención de marcadores moleculares para su aplicación a la mejora genética del albaricoquero [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/5426 / Palancia

Patterns of sperm usage in multiply-mated females have profound fitness consequences for males, and create strong selective pressure on male behavior. In the cooperative theridiid spider Anelosimus studiosus Hentz 1850 adult males are tolerated in females' webs, and females have been observed to mate multiply with different males. In this experiment, virgin females were mated with two different males on consecutive days under controlled conditions to determine paternity patterns and behavioral responses of males to non-virgin females. The paternity of broods was analyzed using randomly amplified polymorphic DNA (RAPDs). Fifteen broods were analyzed and complete first male sperm precedence was found. Mating behavior differed between first and second males with the first males attempting fewer intromissions, but having a longer total time of intromission. This suggests that the second males are either prevented from normal copulation, or are reacting to the different condition of the females. The sperm precedence pattern is discussed with respect to its ramifications for male behavior, juvenile inclusive fitness, and the evolution of cooperative behavior.

ABSTRACT GENETIC VARIABILITY IN HYDRASTIS CANADENSIS L. USING RAPD ANALYSIS FEBRUARY 2009 KERRY J. KELLEY, B.A. MOUNT HOLYOKE COLLEGE M.A. UNIVERSITY OF MASSACHUSETTS AMHERST Directed by: Professor Lyle Craker Hydrastis canadensis L. (goldenseal) is an endangered perennial wildflower species native to eastern North America. In this study, several populations of goldenseal, (both cultivated and wild type) were analyzed for genetic variability. The samples were collected from plant populations in North Carolina, Ohio, Pennsylvania and West Virginia and preserved using silica gel during collection. Random amplified polymorphic DNA (RAPD) analysis technique was used to generate DNA profiles from individual plants and to estimate genetic variability between groups (cultivated and wild type), among populations within groups and within populations using analysis of molecular variance (AMOVA) and a UPGMA clustering phenogram. Our results demonstrate that the bulk of genetic diversity may be within and among populations, but not between groups. This indicates the need for preservation and conservation efforts at the population level. The next step would be to study goldenseal populations more in depth for underlying causes of the genetic variability observed in this study. Further study of genetic variability with different molecular markers may be needed to clarify the level of diversity for the species at the group level. Increased knowledge of genetic variability and the identification of accessions of goldenseal would prove useful for reintroduction and cultivation strategies.

Anther-derived monoploids of Solanum phureja were utilized to investigate factors essential to an efficient method of regenerating doubled monoploids (DMs). The presence of silver thiosulfate (STS) in the MS basal medium did not affect the frequency of cells with 2x nuclei but increased the proportion of cells with 1x nuclei and decreased the proportion with 4x nuclei. Results indicated that STS lower the occurrence of endopolyploid nuclei. The production of DMs was not affected by the presence of STS in MS basal medium on which the source of leaf explants was maintained. The incubation of leaf cultures in the dark or light during the callus induction phase did not influence the subsequent regenerative ability of the monoploids. However, there was a significant genotype by incubation condition interaction. Overnight incubation on MS medium with benzyladenine (BA) pulse prior to transfer to regeneration medium did not affect regeneration. Field evaluation showed various responses of DMs in terms of growth and yield compared to their anther donors or corresponding F₁ progeny. Female fertility was observed in a majority of the DMs verifying their applicability as parental genotypes in practical breeding. Efforts to generate potato hybrids based on selection of genetically diverse parents using RAPD markers and to develop high yielding diploid potato germplasm that may be instrumental in new cultivar development were addressed. Genetic diversity among in vitro plantlets of S. phureja monoploids (which represent DM maternal genotypes) and diploid heterozygous pollinators (ID lines) was estimated using RAPD markers. Field evaluation revealed significant differences among fourteen F₁ hybrids of S. phureja DM x ID with respect to total tuber number, total tuber yield, average tuber weight and vigor. Using simple matching or Jaccard coefficients, the largest parental genetic distance was associated with the highest total tuber yield among the progenies of DM parents. Based on our results, RAPDs have the potential to facilitate the identification of diverse parents to maximize the expression of heterosis in S. phureja hybrids. SSR markers were used to analyze the genetic composition of anther-derived potato plants. Anther-derived monoploids exhibited a single allele as expected. Both homozygous and heterozygous diploids were identified. SSRs were also utilized to study allelic segregation in an F₁ population. Results of this experiment revealed Mendelian inheritance of SSR alleles. / Ph. D.

Fusarium oxysporum complex causes wilt disease in a wide variety of plants and are grouped into formae speciales based on their host range. Twenty-one isolates of the complex which represented F. oxysporum, f. sp. cubense, f. sp. lycopersici, f. sp. passiflorae, and f. sp. capsici were assessed for genetic diversity using RFLPs of the IGS region, RAPD-PCR, and DNA sequencing of ITS1- ITS2 and 5.8S rRNA gene. RAPD amplification with primer OPR5 generated 42 polymorphic bands and cluster analysis showed that the population is genetically heterogeneous. Comparison of the banding patterns both visually and by phenetic analysis suggests high level of genetic variation among the isolates and sub-divided them into six major groups. However, there was no correlation between RAPD-PCR banding pattern and f. spp. RFLPs produced by digestion with restriction endonucleases, BglI, SmaI, and SalI were used to further analyse the IGS region and identified several IGS haplotypes which did not differentiate among f. spp. Banding patterns and phenetic analysis generated do not showed clear separation among f. spp. and do not support separation based on host. DNA sequences of 5.8S rRNA gene and flanking intergenic transcribed spacers of several f. spp. from F. oxysporum were analyzed in order to detect molecular marker intraspecific for the f. spp. Primers ITS4/ITS5 showed good specificity for the species and yielded a unique fragment of approximately 550-570 bp. DNA bases determined in a Megabace1000 sequencer were further aligned and cladograms reconstructed with ClustalX (1.83) and Mega2 (2.1), respectively. ITS analysis grouped strains into several clusters based on NJ and UPGMA. The results suggested that the region could be used as a genetic marker to resolve relationships among f. spp. of F.oxysporum, however, it was too conserved for comparisons within a population of Foc. Overall, the ITS 2 was more variable than the ITS1 region and 5.8S rRNA gene was not parsimonicaly informative. Sequences of 18 isolates representing Fusarium oxysporum, including a human pathogenic one and another associated to trees, was chosen from GenBank and combined with our sequences. Phylogenetic reconstruction was not compatible with the separation of the species into f. spp. and agreed with previous reports of independent evolutionary origins within f. spp. Electronic diagnostic using Blastn could be used as a Bioinformatic tool identify Fusarium at genus level, only. Our results questioned the predicte value of the forma specialis naming system in the separation of different f. spp. in F.oxysporum complex and suggest the investigation of more reliable systems to identify the pathogen population. / Fundação de Amparo a Pesquisa do Estado de Alagoas / O complexo Fusarium oxysporum é responsável por murchas em uma variedade de plantas e seus representantes são agrupados em formae speciales, de acordo com a sua patogenicidade a hospedeiros específicos. A diversidade genética de 21 isolados do complexo, representados por F. oxysporum, f. sp. cubense, f. sp. lycopersici, f. sp. passiflorae e f. sp. capsici, foi avaliada utilizando RFLPs da região IGS, RAPD-PCR e sequenciamento de DNA da região ITS1-ITS2 e do gene 5,8S rRNA. Amplificação RAPD com o iniciador OPR5 gerou 42 bandas polimórficas e a análise de agrupamento demonstrou que a população é geneticamente heterogênea. Comparações dos perfis genéticos gerados pelas análises visual e fenética sugerem um alto nível de variação genética entre os isolados, que foram sub-divididos em seis grupos principais. Entretanto, não houve correlação entre os perfis de banda RAPD-PCR e f. spp. Análise dos RFLPs, produzidos pela digestão com enzimas de restrição, BglI, SmaI e SalI da região IGS, identificou vários haplotipos. Perfis de bandas e análise fenética geradas não mostraram separação clara entre as f. spp. e não dá suporte à separação baseada em hospedeiros. Seqüências de DNA do gene 5,8S rRNA e da região espaçadora ITS de diversas f. spp. de F. oxysporum foram analisadas visando a detecção de um marcador molecular intraespecífico para as f. spp. Os iniciadores ITS4/ITS5 mostraram alta especificidade para a espécie e geraram uma banda única de aproximadamente 550-570 pb. Bases de DNA foram determinadas em um seqüenciador MegaBace1000, alinhadas com o programa ClustalX (1.83) e geraram cladogramas a partir do programa Mega2 (2.1), utilizando os métodos NJ e UPGMA, que agrupou os isolados em vários grupos. Os resultados sugerem que a região, apesar de pouco variável, poderia ser utilizada como um marcador molecular para resolver relações entre f. spp. de F. oxysporum. Entretanto, ela foi altamente conservada para comparações dentro da população de Foc estudada. Em geral, a região ITS2 foi mais variável que a ITS1 e o gene 5,8S rRNA não foi parsimonicamente informativo. Seqüências de 18 isolados representando F.oxysporum, inclusive de um isolado patogênico a humanos e de outro associado a árvores, foram selecionadas do GenBank e combinadas com nossas seqüências. Reconstrução filogenética também não foi compatível com a separação de espécies em f. spp. e concorda com relatos anteriores de origens evolucionárias independentes dentro das f. spp. O diagnóstico eletrônico através da ferramenta de Bioinformática blastn identificou Fusarium em nível de gênero. Os resultados questionam o valor preditivo do sistema denominado forma specialis dentro do complexo F. oxysporum e sugere a investigação de sistemas mais confiáveis para identificação de populações do patógeno.

<p>The phylogeography of a wide ranging temperate species, the adder, <i>Vipera berus</i>, was investigated using several genetic tools, with special emphasis on the post-glacial colonisation pattern of Fennoscandia. The area was colonised from two directions by adder populations representing different glacial refugia. The two populations meet in three places and the main contact zone is situated in Northern Finland. The two other contact zones are the result of dispersal across the Baltic Sea to the Umeå archepelago and South-Western Finland. Asymmetrically distributed nuclear genetic variation compared to mitochondrial DNA in the northern contact zone suggests a skewed gene flow from the east to the west across the zone. This pattern might reflect differences in dispersal among sexes and lineages, or may be accounted for by a selective advantage for nuclear variation of eastern origin among Fennoscandian adders.</p><p>The phylogeographic pattern for adders across the entire species range was addressed by sequencing part of the mitochondrial genome and scoring microsatellite markers. The adder can be divided into three major genetic groups. One group is confined to the Balkan peninsula harbouring the distribution range of <i>V. b. bosniensis</i>. A second, well differentiated group is restricted to the Southern Alps. These two areas have probably served as refugia for adders during a number of ice ages for the adders. The third group is distributed across the remainder of the species’ range, from extreme Western Europe to Pacific Russia and can be further divided into one ancestral group inhabiting the Carpathians refugial area, and three more recent groups inhabiting areas west, north and east of the Alps. The adder provides an example of a species where the Mediterranean areas are housing endemic populations, rather than the sources for post-glacial continental colonisation. Continent-wide colonisation has instead occurred from up to three cryptic northern refugia. </p>

The phylogeography of a wide ranging temperate species, the adder, Vipera berus, was investigated using several genetic tools, with special emphasis on the post-glacial colonisation pattern of Fennoscandia. The area was colonised from two directions by adder populations representing different glacial refugia. The two populations meet in three places and the main contact zone is situated in Northern Finland. The two other contact zones are the result of dispersal across the Baltic Sea to the Umeå archepelago and South-Western Finland. Asymmetrically distributed nuclear genetic variation compared to mitochondrial DNA in the northern contact zone suggests a skewed gene flow from the east to the west across the zone. This pattern might reflect differences in dispersal among sexes and lineages, or may be accounted for by a selective advantage for nuclear variation of eastern origin among Fennoscandian adders. The phylogeographic pattern for adders across the entire species range was addressed by sequencing part of the mitochondrial genome and scoring microsatellite markers. The adder can be divided into three major genetic groups. One group is confined to the Balkan peninsula harbouring the distribution range of V. b. bosniensis. A second, well differentiated group is restricted to the Southern Alps. These two areas have probably served as refugia for adders during a number of ice ages for the adders. The third group is distributed across the remainder of the species’ range, from extreme Western Europe to Pacific Russia and can be further divided into one ancestral group inhabiting the Carpathians refugial area, and three more recent groups inhabiting areas west, north and east of the Alps. The adder provides an example of a species where the Mediterranean areas are housing endemic populations, rather than the sources for post-glacial continental colonisation. Continent-wide colonisation has instead occurred from up to three cryptic northern refugia.

Diploid potato populations between a primitive cultivated species, <I>Solanum phureja</I>, and a weedy species, <I>S. chacoense</I>, were used to examine the segregation of microsatellite markers and three traits in backcrosses. Two of the traits, anther culture competence and 2<I>n</I> pollen production, originated from <I>S. phureja</I> whereas the third, leptine production (a specific glycoalkaloid known to convey resistance to the Colorado potato beetle) originated from <I>S. chacoense</I>. Using CP2, a self-incompatible F₁ hybrid originating from a cross between <I>S. chacoense</I> clone 80-1 and <I>S. phureja</I> clone 1-3, three populations were developed: 1-3 x CP2 (PBCp), CP2 x 1-3 (PBCc), and CP2 x 80-1 (CBC). For the microsatellite study, four simple sequence repeat (SSR) primer pairs that amplified fragments within potato sequences found in the GenBank were used to look at segregation ratios in our backcross populations and to eliminate possible spurious genotypes bearing non-parental alleles in these populations. Seventeen spurious genotypes were discarded from PBCp; none was found in PBCc or CBC. Two SSR loci showed skewed segregation in PBCp (favoring transmissnion of the allele originally found in 80-1), PBCc showed normal segregation at all loci, and CBC showed distorted segregation at one locus (revealing a deficiency of homozygotes). In the study of anther culture, three components of ACR were investigated in a preliminary study: 1) embryos produced per anther (EPA), 2) embryo regeneration rate and 3) percentage of monoploids (2<I>n</I>=1<I>x</I>=12) among regenerants. CP2 was intermediate, 80-1 was low, and 1-3 was high for ACR. Only EPA was selected for further characterization in our populations. PBCp (78 genotypes) and CBC (57 genotypes), were characterized for anther culture response ACR/EPA in a series of studies. Nine high and ten low selections were identified in CBC, and ten high and ten low selections were identified in PBCp. EPA selections were used for bulk segregant analysis (BSA) using 214 RAPD primers. Two bands, one amplified by OPQ-10 and another by OPZ-4 were linked in coupling and in repulsion, respectively, to ACR in PBCp. One band amplified by OPW-14 primer was linked in coupling to ACR in CBC. One-way ANOVAs for data from remaining genotypes of the populations verified linkage of the markers to ACR/EPA. For 2n pollen production, a total of 77 PBCp genotypes was characterized; 80-1 produces low % 2<I>n</I> pollen, and 1-3 produces high % 2<I>n</I> pollen. Pollen samples were stained with propidium iodide and examined by flow cytometry. The frequency of 2n pollen varied continuously from 1.7 % to 40.6 % among the 41 genotypes that flowered sufficiently to allow three separate pollen collections. Variation due to the environment was observed where the frequency of 2n pollen appeared greater over a range of genotypes on single collection days. BSA could not be used due to limited population size and a low number of selections at the extremes of the distribution of phenotypes. The continuous variation for 2<I>n</I> pollen production suggests multigenic control of the trait. In the study of leptine content in reciprocal backcross populations, 87 genotypes within PBCp, and 42 genotypes within PBCc were characterized using gas chromatography of leaf samples. CP2 was intermediate, 1-3 had zero, and 80-1 was high for leptine content in the foliage. Leptines were present in low levels in 43 of 87 genotypes in PBCp, indicating simple genetic control. In PBCc, only 7 of 42 genotypes expressed leptines, generally at a higher level than in PBCp, indicating cytoplasmic inheritance. Ten high and ten nil selections within PBCp, and seven high and eight nil selections within PBCc were used for BSA using 214 RAPD primers. Three primers OPQ-2, OPT-16 and OPT-20 amplified bands segregating with high bulks in both populations. These markers were linked in coupling to leptine content in PBCp. Linkage was verified by ANOVAs for leptine content in the entire population. / Ph. D.

El concepto de alimento funcional se emplea para describir a aquellos alimentos adicionados con ingredientes de diversas clases y orígenes que pueden ejercer efectos beneficiosos sobre quien los ingiere. Este concepto surgido en Japón ha ido popularizándose y expandiéndose hacia otros continentes como Europa fundamentalmente debido a la preocupación sobre la nutrición, la dieta y la salud de la sociedad actual. Los probióticos representan una gran área dentro de los alimentos funcionales y se ha intensificado la investigación para desarrollar productos probióticos y profundizar en el conocimiento de los efectos sobre la salud humana. Los probióticos se definen como "suplementos alimentarios microbianos vivos que afectan de forma ventajosa al animal huésped mejorando su equilibrio intestinal microbiano". Son microorganismos que estimulan las funciones protectoras del tracto digestivo, y también son conocidos como bioterapéuticos, bioprotectores o bioprofilácticos, ya que se utilizan para prevenir las infecciones entéricas y gastrointestinales. Un microorganismo probiótico efectivo debe poseer una serie de características: no ha de ser no patógeno ni tóxico, debe ejercer efectos beneficiosos sobre la salud de quien lo ingiere, tener origen humano, ha de ser tecnológicamente utilizable, ha de presentar un elevado porcentaje de células viables, debe ser capaz de sobrevivir a la flora intestinal, ha de permanecer viable durante su almacenamiento en refrigeración, y tener capacidad de adherirse a la superficie mucosa, etc. El establecimiento de criterios de selección y controles de calidad para productos probióticos se considera una prioridad debido a la rápida incorporación de estos productos en el mercado y su distribución en el ámbito internacional sin la existencia previa de una normativa comúnmente aceptada. En los últimos años ha aumentado el interés por los productos elaborados con microorganismos probióticos, pertenecientes a los géneros Lactobacillus y Bifidobac / Collado Amores, MC. (2005). Caracterización de cepas del género Bifidobacterium con carácter probiótico [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/1907 / Palancia