Merging person-specific bio-markers for predicting oral cancer recurrence through an ontology

One of the major problems related to cancer treatment is its recurrence. Without knowing in advance how likely the cancer will relapse, clinical practice usually recommends adjuvant treatments that have strong side effects. A way to optimize treatments is to predict the recurrence probability by analyzing a set of bio-markers. The NeoMark European project has identified a set of preliminary bio-markers for the case of oral cancer by collecting a large series of data from genomic, imaging, and clinical evidence. This heterogeneous set of data needs a proper representation in order to be stored, computed, and communicated efficiently. Ontologies are often considered the proper mean to integrate biomedical data, for their high level of formality and for the need of interoperable, universally accepted models. This paper presents the NeoMark system and how an ontology has been designed to integrate all its heterogeneous data. The system has been validated in a pilot in which data will populate the ontology and will be made public for further research.

Estudio molecular de gluteninas de alto y bajo peso molecular en Triticum aestivum ssp. vulgare L. y su relación con la calidad panadera

El trigo blando (Triticum aestivum ssp vulgare L., AABBDD, 2n=6x=42) presenta propiedades viscoélasticas únicas debidas a la presencia en la harina de las prolaminas: gluteninas y gliadinas. Ambos tipos de proteínas forman parte de la red de gluten. Basándose en la movilidad en SDS-PAGE, las gluteninas se clasifican en dos grupos: gluteninas de alto peso molecular (HMW-GS) y gluteninas de bajo peso molecular (LMW-GS). Los genes que codifican para las HMW-GS se encuentran en tres loci del grupo 1 de cromosomas: Glu-A1, Glu-B1 y Glu-D1. Cada locus codifica para uno o dos polipéptidos o subunidades. La variación alélica de las HMW-GS es el principal determinante de de la calidad harino-panadera y ha sido ampliamente estudiado tanto a nivel de proteína como de ADN. El conocimiento de estas proteínas ha contribuido sustancialmente al progreso de los programas de mejora para la calidad del trigo. Comparadas con las HMW-GS, las LMW-GS forman una familia proteica mucho más compleja. La mayoría de los genes LMW se localizan en el grupo 1 de cromosomas en tres loci: Glu-A3, Glu-B3 y Glu-D3 que se encuentran estrechamente ligados a los loci que codifican para gliadinas. El número de copias de estos genes ha sido estimado entre 10-40 en trigo hexaploide, pero el número exacto aún se desconoce debido a la ausencia de un método eficiente para diferenciar los miembros de esta familia multigénica. La nomenclatura de los alelos LMW-GS por electroforesis convencional es complicada, y diferentes autores asignan distintos alelos a la misma variedad lo que dificulta aún más el estudio de esta compleja familia. El uso de marcadores moleculares para la discriminación de genes LMW, aunque es una tarea dificil, puede ser muy útil para los programas de mejora. El objetivo de este trabajo ha sido profundizar en la relación entre las gluteninas y la calidad panadera y desarrollar marcadores moleculares que permitan ayudar en la correcta clasificación de HMW-GS y LMW-GS. Se han obtenido dos poblaciones de líneas avanzadas F4:6 a partir de los cruzamientos entre las variedades ‘Tigre’ x ‘Gazul’ y ‘Fiel’ x ‘Taber’, seleccionándose para los análisis de calidad las líneas homogéneas para HMW-GS, LMW-GS y gliadinas. La determinación alélica de HMW-GS se llevó a cabo por SDS-PAGE, y se complementó con análisis moleculares, desarrollándose un nuevo marcador de PCR para diferenciar entre las subunidades Bx7 y Bx7*del locus Glu-B1. Resumen 2 La determinación alélica para LMW-GS se llevó a cabo mediante SDS-PAGE siguiendo distintas nomenclaturas y utilizando variedades testigo para cada alelo. El resultado no fue concluyente para el locus Glu-B3, así que se recurrió a marcadores moleculares. El ADN de los parentales y de los testigos se amplificó usando cebadores diseñados en regiones conservadas de los genes LMW y fue posteriormente analizado mediante electroforesis capilar. Los patrones de amplificación obtenidos fueron comparados entre las distintas muestras y permitieron establecer una relación con los alelos de LMW-GS. Con este método se pudo aclarar la determinación alélica de este locus para los cuatro parentales La calidad de la harina fue testada mediante porcentaje de contenido en proteína, prueba de sedimentación (SDSS) y alveógrafo de Chopin (parámetros P, L, P/L y W). Los valores fueron analizados en relación a la composición en gluteninas. Las líneas del cruzamiento ‘Fiel’ x ‘Taber’ mostraron una clara influencia del locus Glu-A3 en la variación de los valores de SDSS. Las líneas que llevaban el nuevo alelo Glu-A3b’ presentaron valores significativamente mayores que los de las líneas con el alelo Glu-A3f. En las líneas procedentes del cruzamiento ‘Tigre ’x ‘Gazul’, los loci Glu-B1 y Glu-B3 loci mostraron ambos influencia en los parámetros de calidad. Los resultados indicaron que: para los valores de SDSS y P, las líneas con las HMW-GS Bx7OE+By8 fueron significativamente mejores que las líneas con Bx17+By18; y las líneas que llevaban el alelo Glu-B3ac presentaban valores de P significativamente superiores que las líneas con el alelo Glu-B3ad y significativamente menores para los valores de L . El análisis de los valores de calidad en relación a los fragmentos LMW amplificados, reveló un efecto significativo entre dos fragmentos (2-616 y 2-636) con los valores de P. La presencia del fragmento 2-636 estaba asociada a valores de P mayores. Estos fragmentos fueron clonados y secuenciados, confirmándose que correspondían a genes del locus Glu-B3. El estudio de la secuencia reveló que la diferencia entre ambos se hallaba en algunos SNPs y en una deleción de 21 nucleótidos que en la proteína correspondería a un InDel de un heptapéptido en la región repetida de la proteína. En este trabajo, la utilización de líneas que difieren en el locus Glu-B3 ha permitido el análisis de la influencia de este locus (el peor caracterizado hasta la fecha) en la calidad panadera. Además, se ha validado el uso de marcadores moleculares en la determinación alélica de las LMW-GS y su relación con la calidad panadera. Summary 3 Bread wheat (Triticum aestivum ssp vulgare L., AABBDD, 2n=6x=42) flour has unique dough viscoelastic properties conferred by prolamins: glutenins and gliadins. Both types of proteins are cross-linked to form gluten polymers. On the basis of their mobility in SDS-PAGE, glutenins can be classified in two groups: high molecular weight glutenins (HMW-GS) and low molecular weight glutenins (LMW-GS). Genes encoding HMW-GS are located on group 1 chromosomes in three loci: Glu-A1, Glu-B1 and Glu-D1, each one encoding two polypeptides, named subunits. Allelic variation of HMW-GS is the most important determinant for bread making quality, and has been exhaustively studied at protein and DNA level. The knowledge of these proteins has substantially contributed to genetic improvement of bread quality in breeding programs. Compared to HMW-GS, LMW-GS are a much more complex family. Most genes encoded LMW-GS are located on group 1 chromosomes. Glu-A3, Glu-B3 and Glu-D3 loci are closely linked to the gliadin loci. The total gene copy number has been estimated to vary from 10–40 in hexaploid wheat. However, the exact copy number of LMW-GS genes is still unknown, mostly due to lack of efficient methods to distinguish members of this multigene family. Nomenclature of LMW-GS alleles is also unclear, and different authors can assign different alleles to the same variety increasing confusion in the study of this complex family. The use of molecular markers for the discrimination of LMW-GS genes might be very useful in breeding programs, but their wide application is not easy. The objective of this work is to gain insight into the relationship between glutenins and bread quality, and the developing of molecular markers that help in the allele classification of HMW-GS and LMW-GS. Two populations of advanced lines F4:6 were obtained from the cross ‘Tigre’ x ‘Gazul’ and ‘Fiel’ x ‘Taber’. Lines homogeneous for HMW-GS, LMW-GS and gliadins pattern were selected for quality analysis. The allele classification of HMW-GS was performed by SDS-PAGE, and then complemented by PCR analysis. A new PCR marker was developed to undoubtedly differentiate between two similar subunits from Glu-B1 locus, Bx7 and Bx7*. The allele classification of LMW-GS was initially performed by SDS-PAGE following different established nomenclatures and using standard varieties. The results were not completely concluding for Glu-B3 locus, so a molecular marker system was applied. DNA from parental lines and standard varieties was amplified using primers designed in conserved domains of LMW genes and analyzed by capillary electrophoresis. The pattern of amplification products obtained was compared among samples and related to the protein allele classification. It was possible to establish a correspondence between specific amplification products and almost all LMW alleles analyzed. With this method, the allele classification of the four parental lines was clarified. Flour quality of F4:6 advanced lines were tested by protein content, sedimentation test (SDSS) and alveograph (P, L, P/L and W). The values were analyzed in relation to the lines prolamin composition. In the ‘Fiel’ x ‘Taber’ population, Glu-A3 locus showed an influence in SDSS values. Lines carrying new allele Glu-A3b’, presented a significantly higher SDSS value than lines with Glu-A3f allele. In the ‘Tigre ’x ‘Gazul’ population, the Glu-B1 and Glu-B3 loci also showed an effect in quality parameters, in SDSS, and P and L values. Results indicated that: for SDSS and P, lines with Bx7OE+By8 were significantly better than lines with Bx17+By18; lines carrying Glu-B3ac allele had a significantly higher P values than Glu-B3ad allele values. lines with and lower L The analysis of quality parameters and amplified LMW fragments revealed a significant influence of two peaks (2-616 y 2-636) in P values. The presence of 2-636 peak gave higher P values than 2-616. These fragments had been cloned and sequenced and identified as Glu-B3 genes. The sequence analysis revealed that the molecular difference between them was some SNPs and a small deletion of 21 nucleotides that in the protein would produce an InDel of a heptapeptide in the repetitive region. In this work, the analysis of two crosses with differences in Glu-3 composition has made possible to study the influence of LMG-GS in quality parameters. Specifically, the influence of Glu-B3, the most interesting and less studied loci has been possible. The results have shown that Glu-B3 allele composition influences the alveograph parameter P (tenacity). The existence of different molecular variants of Glu-B3 alleles have been assessed by using a molecular marker method. This work supports the use of molecular approaches in the study of the very complex LMW-GS family, and validates their application in the analysis of advanced recombinant lines for quality studies.

Clones Identification and Genetic Characterization of Garnacha Grapevine by Means of Different PCR-Derived Marker Systems

This study uses PCR-derived marker systems to investigate the extent and distribution of genetic variability of 53 Garnacha accessions coming from Italy, France and Spain. The samples studied include 28 Italian accessions (named Tocai rosso in Vicenza area; Alicante in Sicily and Elba island; Gamay perugino in Perugia province; Cannonau in Sardinia), 19 Spanish accessions of different types (named Garnacha tinta, Garnacha blanca, Garnacha peluda, Garnacha roja, Garnacha erguida, Garnacha roya) and 6 French accessions (named Grenache and Grenache noir). In order to verify the varietal identity of the samples, analyses based on 14 simple sequence repeat (SSR) loci were performed. The presence of an additional allele at ISV3 locus (151 bp) was found in four Tocai rosso accessions and in a Sardinian Cannonau clone, that are, incidentally, chimeras. In addition to microsatellite analysis, intravarietal variability study was performed using AFLP, SAMPL and M-AFLP molecular markers. AFLPs could discriminate among several Garnacha samples; SAMPLs allowed distinguishing few genotypes on the basis of their geographic origin, whereas M-AFLPs revealed plant-specific markers, differentiating all accessions. Italian samples showed the greatest variability among themselves, especially on the basis of their different provenance, while Spanish samples were the most similar, in spite of their morphological diversity.

Molecular characterization of Glu-B3 locus in wheat cultivars and segregating populations

Bread wheat quality constitutes a key trait for the demands of the baking industry as well as the broad consumer preferences. The role of the low molecular weight glutenin subunits (LMW-GS) with regard to bread quality is so far not well understood owing to their genetic complexity and to the use of different nomenclatures and standards for the LMW-GS assignment by different research groups, which has made difficult the undertaking of association studies between genotypes and bread quality. The development of molecular markers to carry out genetic characterization and allele determination is demanding. Nowadays, the most promising LMW gene marker system is based on PCR and high resolution capillary electrophoresis for the simultaneous analysis of the complete multigene family. The molecular analysis of the bread wheat Glu-B3 locus in F2 and F4:6 populations expressed the expected one-locus Mendelian segregation pattern, thus validating the suitability of this marker system for the characterization of LMW-GS genes in segregating populations, allowing for the successful undertaking of studies related to bread-making quality. Moreover, the Glu-B3 allele characterization of standard cultivars with the molecular marker system has revealed its potential as a complementary tool for the allelic determination of this complex multigene family.

Cartografía de QTL asociados a la tolerancia a estrés hídrico en frijol común (Phaseolus vulgaris L.)

Phaseolus vulgaris L. (frijol común o judía) es una leguminosa de gran demanda para la nutrición humana y un producto agrícola muy importante. Sin embargo, la producción de frijol se ve limitada por presiones ambientales como la sequía. En México, el 85% de la cosecha de frijol se produce en la temporada de primavera-verano, principalmente en las regiones del altiplano semiárido con una precipitación anual entre 250 y 400 mm. A pesar del implemento de tecnología en el campo, los factores naturales impiden al agricultor llegar a los rendimientos deseados. El Instituto Nacional de Investigaciones Forestales, Agrícolas y Pecuarias (INIFAP), como instituto de investigación gubernamental en México, tiene como objetivo la mejora de cultivos estratégicos, uno de ellos, P. vulgaris. Los estudios en relación a la sequía se enfocan especialmente en la selección de genotipos tolerantes, los cuales son sometidos en condiciones de estrés y monitoreando parámetros como el rendimiento y peso de semilla, además de algunos indicadores tales como índice de cosecha. El resultado de estos trabajos ha sido la obtención de variedades con mayor tolerancia a la sequía, tales como Pinto Villa y Pinto Saltillo. En los últimos años se ha avanzado notablemente en el conocimiento de las bases moleculares en las respuestas de las plantas al estrés. De acuerdo a diversos estudios se ha demostrado que las plantas bajo estrés por sequía experimentan cambios en la expresión de genes involucrados en la señalización, regulación de la transcripción y la traducción, transporte de agua y la función directa en la protección celular. También se ha observado que el déficit de agua es causado por las temperaturas extremas y la alta concentración de sales, por lo que al nivel molecular, las respuestas al estrés tienen puntos de especificidad y puntos de entrecruzamiento. La sequía puede generar estreses secundarios, tales como el nutricional, oxidativo y osmótico. Sin embargo, es necesario identificar y caracterizar muchos de los componentes involucrados en las respuestas al déficit hídrico, la caracterización de estos genes permitirá tener una mejor comprensión de los mecanismos bioquímicos y fisiológicos involucrados en la tolerancia al estrés. Actualmente, con el apoyo de la biología molecular se han identificado algunos genes que otorgan ventajas para la adaptación a ambientes desfavorables. Por lo que el objetivo del presente trabajo es identificar marcadores genéticos asociados a rasgos fenotípicos con énfasis a la tolerancia a estrés hídrico en P. vulgaris. Una vez establecidos los marcadores asociados al estrés hídrico, es factible considerar su uso para la selección asistida por marcadores en líneas o variedades de frijol de interés para los mejoradores. Se evaluaron 282 familias F3:5 derivadas de la cruza entre los cultivares Pinto Villa y Pinto Saltillo. Las familias se sembraron bajo un diseño simple de látice 17x17, el experimento se llevo acabo en el ciclo primavera-verano del 2010 y 2011, y otoñoinvierno de 2010 en el Campo Experimental Bajío del INIFAP con dos repeticiones para cada tratamiento de humedad (riego completo y sequía terminal). En todos los genotipos se realizó el fenotipado (variables fenotípicas) y el genotipado a través de marcadores moleculares. Los análisis estadísticos se basaron en el análisis de componentes principales (Eigen Analysis Selection Index Method, ESIM), la asociación entre marcadores SNP y el fenotipado (paquete SNPassoc para R) y el análisis de varianza (ANOVA). Los valores ESIM mostraron que las variables de Rendimiento, Días a floración, Días a madurez fisiológica e Índice de cosecha fueron sobresalientes en sequía terminal, por lo que se sugieren tomarse en consideración para los estudios de sequía en P. vulgaris como monitores de evaluación a la resistencia. Se identificaron nueve familias sobresalieron por sus valores ESIM (PV/PS6, 22, 131, 137, 149, 154, 201, 236 y 273), además de presentar valores superiores para el rendimiento en comparación con los parentales. Estos genotipos son candidatos interesantes para realizar estudios de identificación de loci asociados con la respuesta al estrés, y como potenciales parentales en el desarrollo de nuevas variedades de frijol. En los análisis de asociación SNPassoc se identificaron 83 SNPs significativos (p<0,0003) asociados a los rasgos fenotípicos, obteniendo un total de 222 asociaciones, de las cuales predomina el modelo genético de codominancia para las variables Días a floración, Periodo reproductivo y Biomasa total. Treinta y siete SNPs se identificaron a diferentes funciones biológicas a través del análisis de anotación funcional, de los cuales 12 SNPs (9, 18, 28, 39, 61, 69, 80, 106, 115, 128, 136 y 142) sobresalen por su asociación al fenotipado, y cuya anotación funcional indica que se encuentran en genes relacionados a la tolerancia a la sequía, tales como la actividad kinasa, actividad metabólica del almidón, carbohidratos y prolina, respuesta al estrés oxidativo, así como en los genes LEA y posibles factores de transcripción. En el caso de los análisis ANOVA, se identificaron 72 asociaciones entre los SNPs y las variables fenotípicas (F< 3,94E-04). Las 72 asociaciones corresponden a 30 SNPs y 7 variables fenotípicas, de las que predomina Peso de 100 semillas y Periodo reproductivo. Para los rasgos de Rendimiento, Índice de cosecha y Días a madurez fisiológica se presentaron asociaciones con seis SNPs (17, 34, 37, 50, 93 y 107), de los cuales, a los SNP37 y SNP107 fueron identificados a la anotación biológica de protein binding. Por otro lado, los SNP106 y SNP128 asociados al Periodo reproductivo, son genes con actividad kinasa y actividad metabólica del almidón, respectivamente. Para los marcadores tipo AFLP, se identificaron 271 asociaciones (F<2,34E-04). Las asociaciones corresponden a 86 AFLPs con todas las variables fenotípicas evaluadas, de las que predomina peso de 100 semillas, Días a floración y Periodo reproductivo. Debido a que los en los AFLPs no es posible determinar su anotación biológica, se proponen como marcadores potenciales relacionados a la resistencia a la sequía en frijol. Los AFLPs candidatos requieren más estudios tales como la secuenciación de los alelos respectivos, así como la identificación de éstas secuencias en el genoma de referencia y su anotación biológica, entre otros análisis, de esta manera podríamos establecer aquellos marcadores candidatos a la validación para la selección asistida. El presente trabajo propone tanto genotipos como marcadores genéticos, que deben ser validados para ser utilizados en el programa de mejoramiento de P. vulgaris, con el objetivo de desarrollar nuevas líneas o variedades tolerantes a la sequía. ABSTRACT Phaseolus vulgaris L. (common bean or judia) is a legume of great demand for human consumption and an important agricultural product. However, the common bean production is limited by environmental stresses, such as drought. In Mexico, 85% of the common bean crop is produced in the spring-summer season mainly in semiarid highland regions with a rainfall between 250 and 400 mm per year. In spite of the improvement of crop technology, the natural factors hamper getting an optimal yield. The National Institute for Forestry, Agriculture and Livestock (INIFAP) is a government research institute from Mexico, whose main objective is the genetic breeding of strategic crops, like P. vulgaris L. The drought tolerance studies particularly focus on the selection of bean tolerant genotypes, which are subjected to stress conditions, by means of monitoring parameters such as yield and seed weight, plus some agronomic indicators such as harvest index. The results of these works have led to obtain cultivars with higher drought tolerance such as Pinto Villa and Pinto Saltillo. Significant achievements have been recently made in understanding the molecular basis of stress plant responses. Several studies have shown that plants under drought stress present changes in gene expression related to cell signalling, transcriptional and translational regulation, water transport and cell protection. In addition, it has been observed that the extreme temperatures and high salt concentrations can cause a water deficiency so, at the molecular level, stress responses have specific and crossover points. The drought can cause secondary stresses, such as nutritional, oxidative and osmotic stress. It is required the identification of more components involved in the response to water deficit, the characterization of these genes will allow a better understanding of the biochemical and physiological mechanisms involved in stress tolerance. Currently, with the support of molecular biology techniques, some genes that confer an advantage for the crop adaptation to unfavourable environments have been identified. The objective of this study is to identify genetic markers associated with phenotypic traits with emphasis on water stress tolerance in P. vulgaris. The establishment of molecular markers linked to drought tolerance would make possible their use for marker-assisted selection in bean breeding programs. Two hundred and eighty two F3:5 families derived from a cross between the drought resistant cultivars Pinto Villa and Pinto Saltillo were evaluated. The families were sowed under a 17x17 simple lattice design. The experiment was conducted between spring-summer seasons in 2010 and 2011, and autumn-winter seasons in 2010 at the Bajio Experimental Station of INIFAP with two treatments (full irrigation and terminal drought). All families were phenotyped and genotyped using molecular markers. Statistical analysis was based on principal component analysis (Eigen Analysis Selection Index Method, ESIM), association analysis between SNP markers and phenotype (SNPassoc package R) and analysis of variance (ANOVA). The ESIM values showed that seed yield, days to flowering, days to physiological maturity and harvest index were outstanding traits in terminal drought treatment, so they could be considered as suitable parameters for drought-tolerance evaluation in P. vulgaris. Nine outstanding families for the ESIM values were identified (PV/PS6, 22, 131, 137, 149, 154, 201, 236 and 273), in addition, these families showed higher values for seed yield compared to the parental cultivars. These families are promising candidates for studies focused on the identification of loci associated to the stress response, and as potential parental cultivars for the development of new varieties of common bean. In the SNPassoc analysis, 83 SNPs were found significantly associated (p<0.0003) with phenotypic traits, obtaining a total of 222 associations, most of which involved the traits days to flowering, reproductive period and total biomass under a codominant genetic model. The functional annotation analysis showed 37 SNPs with different biological functions, 12 of them (9, 18, 28, 39, 61, 69, 80, 106, 115, 128, 136 and 142) stand out by their association to phenotype. The functional annotation suggested a connection with genes related to drought tolerance, such as kinase activity, starch, carbohydrates and proline metabolic processes, responses to oxidative stress, as well as LEA genes and putative transcription factors. In the ANOVA analysis, 72 associations between SNPs and phenotypic traits (F<3.94E- 04) were identified. All of these associations corresponded to 30 SNPs markers and seven phenotypic traits. Weight of 100 seeds and reproductive period were the traits with more associations. Seed yield, harvest index and days to physiological maturity were associated to six SNPs (17, 34, 37, 50, 93 and 107), the SNP37 and SNP107 were identified as located in protein binding genes. The SNP106 and SNP128 were associated with the reproductive period and belonged to genes with kinase activity and genes related to starch metabolic process, respectively. In the case of AFLP markers, 271 associations (F<2.34E-04) were identified. The associations involved 86 AFLPs and all phenotypic traits, being the most frequently associated weight of 100 seeds, days to flowering and reproductive period. Even though it is not possible to perform a functional annotation for AFLP markers, they are proposed as potential markers related to drought resistance in common bean. AFLPs candidates require additional studies such as the sequencing of the respective alleles, identification of these sequences in the reference genome and gene annotation, before their use in marker assisted selection. This work, although requires further validation, proposes both genotypes and genetic markers that could be used in breeding programs of P. vulgaris in order to develop new lines or cultivars with enhanced drought-tolerance.

Estudio de la estabilidad genética asociada a los procesos de crioconservación de ápices de menta

La crioconservación se ha descrito como una técnica de conservación ex situ a largo plazo que ha sido aplicada con éxito a numerosas especies, y resulta especialmente importante en aquellas con propagación vegetativa, infértiles o amenazadas, en las que sistemas de conservación ex situ más sencillos, como los bancos de semillas, no son posibles. También presenta ventajas frente a la conservación in vitro, ya que logra disminuir o eliminar problemas como la excesiva manipulación del material, evitando los subcultivos periódicos y disminuyendo así el riesgo de contaminaciones y de aparición de variación somaclonal. Sin embargo, someter al material vegetal a los procedimientos que implica la crioconservación provoca distintos estreses. Entre ellos, el estrés oxidativo puede potencialmente producir daños en membranas, proteínas, carbohidratos y en el ADN. En este trabajo se han evaluado diversos sistemas de crioconservación en ápices de Mentha × piperita L., híbrido estéril entre Mentha aquatica L. y Mentha spicata L. Se han utilizado ápices de dos genotipos (‘MEN 186’y ‘MEN 198’) en los cuales se compararon dos técnicas de crioconservación, encapsulación-deshidratación y vitrificación-droplet. El análisis de la supervivencia y capacidad de regeneración del material sometido a los tratamientos de crioconservación, junto con el análisis de la estabilidad genética de dicho material mediante marcadores moleculares (RAPD y AFLP) han permitido comparar los distintos protocolos y tratamientos establecidos. El estudio sobre el tipo de protocolo empleado reveló una mayor variabilidad genética en la técnica de encapsulación-deshidratación, especialmente en el genotipo ‘MEN 186’, ya que ‘MEN 198’ resultó ser más estable en todos los análisis. La inestabilidad encontrada en esta técnica no fue exclusiva de aquellos explantos crioconservados, sino que los pasos previos a la inmersión en nitrógeno líquido (NL) también provocaron variaciones en el ADN. Según el tipo de muestra analizada se encontraron diferencias en la estabilidad: muestras provenientes de callos presentaron una mayor inestabilidad que aquellas de hojas (brotes). Se utilizaron tres medios para la recuperación de los ápices tras la crioconservación con el uso de diferentes combinaciones de reguladores de crecimiento: “Reed” (0,5 mgL-1 6-bencilaminopurina, BAP), “Senula” (0,5 mgL-1 6-dimetilalilamino-purina, 2-iP + 0,1 mgL-1 ácido α-naftalen-acético, ANA) y “Nudos” (0,5 mgL-1 BAP + 0,1 mgL-1ANA). El medio “Reed” produjo un aumento en la supervivencia y recuperación de los ápices en ambos genotipos y técnicas, y disminuyó la formación de callo. Sin embargo, no tuvo un efecto significativo en la estabilidad genética. El medio “Senula” provocó una mayor estabilidad genética en el genotipo más inestable, ‘MEN 186’. Para reducir el daño oxidativo producido durante la encapsulación-deshidratación, e incrementar la recuperación de los ápices manteniendo su estabilidad genética, se comparó el efecto de añadir sustancias antioxidantes en el precultivo de los ápices (ácido ascórbico, vitamina E y glutatión). No se obtuvo la respuesta esperada y estos tratamientos no presentaron efectos significativos tanto en la estabilidad como en la recuperación. Para entender mejor qué sucede durante todo el proceso de encapsulación-deshidratación, se evaluó cada paso del protocolo por separado y su efecto en la estabilidad y la recuperación. Además, se determinó el estado de oxidación en cada etapa mediante la cuantificación de malondialdehído y la detección de la formación de radicales libres (mediante el ensayo del ácido tiobarbitúrico, y sondas fluorescentes específicas, respectivamente). Se determinó que a partir de los primeros pasos se genera estrés oxidativo, el cual aumenta a medida que se avanza por el protocolo hasta la inmersión en nitrógeno líquido. Esto se ve reflejado en la disminución progresiva tanto de la recuperación como de la estabilidad genética. Con el uso de antioxidantes en el precultivo (ácido ascórbico y vitamina E) no se obtuvo un efecto positivo en el mantenimiento de la estabilidad genética, y tan sólo con el uso de vitamina E se observó una recuperación mayor en uno de los pasos estudiados (después de la desecación). Sin embargo, cuando se utilizó ácido ascórbico durante el precultivo o la deshidratación osmótica se consiguió disminuir de forma significativa la formación de MDA y la acumulación del radical superóxido (O2•-) en la mayoría los pasos analizados, aunque esta reducción no parece tener un efecto directo en la estabilidad genética del material recuperado. ABSTRACT Cryopreservation has been described as an effective technique for the long term of ex situ conservation that has been successfully applied to numerous species, and is of especial relevance for those with vegetative propagation, infertile or endangered, in which simpler systems of ex situ conservation, such as seed banking, are not feasible. It also has advantages over in vitro conservation, as it reduces or eliminates excessive material handling, avoids periodic subcultures and thus limits the risk of contamination and the appearance of somaclonal variation. However, plant material is subjected to different treatments involved in the cryopreservation procedures, which impose several stresses. Among them, oxidative stress can potentially cause damage to membranes, proteins, carbohydrates and DNA. In this work, two cryopreservation techniques have been evaluated in Mentha × piperita L. shoot tips, sterile hybrid between Mentha aquatica L. and Mentha spicata L. Two genotypes ('MEN 186' and 'MEN 198') were used to compare two techniques: encapsulation-dehydration and droplet-vitrification. The analysis of survival and recovery capacity of the material after the cryopreservation treatments, and the analysis of the genetic stability by molecular markers (RAPD and AFLP) have enabled the comparison between protocols and treatments. The study of the two cryopreservation procedures revealed a higher genetic variability in the encapsulation-dehydration technique, especially in genotype 'MEN 186', as 'MEN 198' was more stable in all analyses. The instability generated in this technique was not exclusive of cryopreserved explants, pretreatments prior to immersion in NL also caused DNA variations. The type of sampled plant material revealed also differences in the stability: callus samples showed greater instability than shoots. Three different culture media were used for the recovery of shoot tips after cryopreservation, using different combinations of growth regulators: "Reed" (0.5 mgL-1 6-benzylaminopurine, BAP), "Senula" (0.5 mgL-1 6-dimetilalilamino-purine, 2-iP + 0.1 mgL-1 α-naphthalene acetic acid, ANA) and "Nodes" (0.5 mgL-1 BAP + 0.1 mgL-1 ANA). "Reed" medium increased survival and recovery of shoot tips in both genotypes and techniques and decreased callus formation. However, it didn`t have a significant effect on genetic stability. "Senula" medium caused a higher genetic stability in the most unstable genotype, 'MEN 186'. To reduce oxidative damage during encapsulation-dehydration, and increase shoot tip recovery and maintain genetic stability, the effect of added antioxidants (ascorbic acid, vitamin E and glutathione) in the shoot tip preculture medium was studied. These treatments had no significant effect on both stability and recovery. To better understand the events during the encapsulation-dehydration process, the effect of each step of the protocol on stability and recovery was evaluated separately. Moreover, the oxidation level was determined by quantifying malondialdehyde (MDA) formation and detecting free radical accumulation (using the thiobarbituric acid assay, and specific fluorescent probes, respectively). The oxidative stress was detected from the first steps and increased throughout the protocol until the immersion in liquid nitrogen. This was also reflected in the gradual decline of recovery and genetic stability. The use of antioxidants (ascorbic acid and vitamin E) in the shoot tip preculture medium had no effect in maintaining genetic stability; only vitamin E increased recovery in one of the steps studied (after desiccation). However, when ascorbic acid was used during the preculture or during the osmotic dehydration, a significantly decrease was observed in MDA formation and superoxide radical accumulation in most of the steps analyzed, although this reduction did not seem to have a direct effect on the genetic stability of recovered material.

Identification of novel drought-tolerant-associated SNPs in common bean (Phaseolus vulgaris)

Common bean (Phaseolus vulgaris L.) is a leguminous in high demand for human nutrition and a very important agricultural product. Production of common bean is constrained by environmental stresses such as drought. Although conventional plant selection has been used to increase production yield and stress tolerance, drought tolerance selection based on phenotype is complicated by associated physiological, anatomical, cellular, biochemical, and molecular changes. These changes are modulated by differential gene expression. A common method to identify genes associated with phenotypes of interest is the characterization of Single Nucleotide Polymorphims (SNPs) to link them to specific functions. In this work, we selected two drought-tolerant parental lines from Mesoamerica, Pinto Villa, and Pinto Saltillo. The parental lines were used to generate a population of 282 families (F3:5) and characterized by 169 SNPs. We associated the segregation of the molecular markers in our population with phenotypes including flowering time, physiological maturity, reproductive period, plant, seed and total biomass, reuse index, seed yield, weight of 100 seeds, and harvest index in three cultivation cycles. We observed 83 SNPs with significant association (p < 0.0003 after Bonferroni correction) with our quantified phenotypes. Phenotypes most associated were days to flowering and seed biomass with 58 and 44 associated SNPs, respectively. Thirty-seven out of the 83 SNPs were annotated to a gene with a potential function related to drought tolerance or relevant molecular/biochemical functions. Some SNPs such as SNP28 and SNP128 are related to starch biosynthesis, a common osmotic protector; and SNP18 is related to proline biosynthesis, another well-known osmotic protector.

Uso de marcadores moleculares para la racionalización y evaluación de la calidad en las colecciones de Recursos Fitogenéticos de trigo

Esta tesis tiene dos objetivos generales: el primero, analizar el uso de proteínas del endospermo y SSRs para la racionalización de las colecciones de trigo, y el segundo, estudiar la influencia de las proteínas del endospermo, del año de cultivo y del abonado nitrogenado en la calidad en un grupo de variedades locales españolas. Dentro del primer objetivo, se estudió la diversidad genética de la colección de Triticum monococcum L. (escaña menor), y de una muestra de la colección de Triticum turgidum L. (trigo duro) del CRF-INIA, con 2 y 6 loci de gliadinas, y 6 y 24 SSRs, para la escaña menor y el trigo duro, respectivamente. Ambas colecciones presentaron una gran diversidad genética, con una gran diferenciación entre las variedades y pequeña dentro de ellas. Los loci de gliadinas mostraron una gran variabilidad, siendo los loci Gli-2 los más útiles para distinguir variedades. En la escaña menor, las gliadinas presentaron mayor poder de discriminación que los SSRs; aunque en trigo duro los SSRs identificaron más genotipos. El número de alelos encontrado fue alto; 24 y 38 en gliadinas, y 29 y 203 en SSRs, en escaña menor y trigo duro, respectivamente. En trigo duro, se identificaron 17 alelos nuevos de gliadinas lo que demuestra que el germoplasma español es muy singular. En ambas especies, se detectaron asociaciones entre la variación alélica en prolaminas y el origen geográfico y filogenético de las variedades. La utilidad de las proteínas (6 loci de gliadinas, 2 loci de gluteninas y proteína total) y de los SSRs (24 loci) para verificar duplicados, y analizar la variabilidad intraaccesión, se estudió en 23 casos de duplicados potenciales de trigo duro. Los resultados indicaron que tanto los biotipos como las accesiones duplicadas mostraban el mismo genotipo en gliadinas, pocas diferencias o ninguna en las subunidades de gluteninas HMW y proteína total, y diferencias en menos de tres loci de SSRs. El mismo resultado se obtuvo para los biotipos de la colección de T. monococcum. Sin embargo, las discrepancias observadas en algunos casos entre proteínas y SSRs demostraron la utilidad del uso conjunto de ambos tipos de marcadores. Tanto las proteínas como los SSRs mostraron gran concordancia con los caracteres agro-morfológicos, especialmente cuando las diferencias entre los genotipos eran grandes. Sin embargo, los caracteres agro-morfológicos fueron menos discriminantes que los marcadores moleculares. Para el segundo objetivo de la tesis, se analizó la variación alélica en siete loci de prolaminas relacionados con la calidad en trigo duro: Glu-A1 y Glu-B1 de gluteninas HMW, Glu-A3, Glu-B3 y Glu-B2 de gluteninas B-LMW, y Gli-A1 y Gli-B1 de gliadinas. La submuestra analizada incluía variedades locales de todas las provincias españolas donde se ha cultivado tradicionalmente el trigo duro. Todos los loci, excepto el Glu-B2, mostraron gran variabilidad genética, siendo los Glu-3 los más polimórficos. En total, se identificaron 65 alelos, de los que 29 eran nuevos, que representan una fuente importante de variabilidad genética para la mejora de la calidad. Se detectaron diferencias en la composición en prolaminas entre la convar. turgidum y la zona norte, y la convar. durum y la zona sur; el genotipo Glu-B3new-1 - Gli-B1new-1 fue muy común en la convar. turgidum, mientras que el Glu-B3a - Gli-B1c, asociado con mejor calidad, fue más frecuente en la convar. durum. En la convar. turgidum, se observó mayor variabilidad que en la convar. durum, principalmente en los loci Glu-B1 y Glu-B3, lo que indica que esta convariedad puede ser una fuente valiosa de nuevos alelos de gluteninas. Esta submuestra fue evaluada para calidad (contenido en proteína, P, y test de sedimentación, SDSS) con dos dosis de abonado nitrogenado (N), y en dos años diferentes. No se detectaron interacciones Variedad × Año, ni Variedad × N en la calidad. Para la P, los efectos ambientales (año y N) fueron mayores que el efecto de la variedad, siendo, en general, mayor la P con dosis altas de N. La variedad influyó más en el test SDSS, que no se vio afectado por el año ni el N. El aumento del contenido en proteína no influyó significativamente sobre la fuerza del gluten estimada con el SDSS. Respecto a la influencia de las prolaminas en la fuerza del gluten, se confirmó la superioridad del Glu-B3a; aunque también se detectó una influencia alta y positiva de los alelos nuevos Glu-A3new-1, y Glu-B3new-6 y new-9. La no correlación entre el rendimiento (evaluado en un trabajo anterior) y la P, en las variedades adaptadas a bajo N, permitió seleccionar cuatro variedades locales con alto rendimiento y buena fuerza del gluten para producción con bajo N. SUMMARY There are two main objectives in this thesis: The first, to analyse the use of endosperm proteins and SSRs to rationalize the wheat collections, and the second, to study the influence on quality of endosperm proteins, year and nitrogen fertilization in a group of Spanish landraces. For the first objective, we studied the genetic diversity of the collection of Triticum monococcum L. (cultivated einkorn), and of a sample of the collection of Triticum turgidum L. (durum wheat) maintained at the CRF-INIA. Two and 6 gliadin loci, and 6 and 24 SSRs, were used for einkorn and durum wheat, respectively. Both collections possessed a high genetic diversity, being the differentiation large between varieties and small within them. Gliadin loci showed great variability, being the loci Gli-2 the most useful for distinguish among varieties. In einkorn, the gliadins showed higher discrimination power than SSRs; although SSRs identified more genotypes in durum wheat. Large number of alleles were found; 24 and 38 in gliadins, and 29 and 203 in SSRs, for einkorn and durum wheat, respectively. In durum wheat, 17 new alleles of gliadins were identified, which indicate that Spanish durum wheat germplasm is rather unique. Some associations between prolamin alleles and geographical and phylogenetic origin of varieties were found in both species. The value of endosperm proteins (6 gliadin loci, 2 glutenin loci and total protein) and SSRs (24 loci) for validation of duplicates, and monitoring the intra-accession variability, was studied in 23 potential duplicates of durum wheat. The results indicated that biotypes and duplicated accessions showed identical gliadin genotype, few or none differences in HMW glutenin subunits and total protein, and less than three different SSR loci. A similar result was obtained for biotypes of T. monococcum. However, the discrepancies in some cases support the convenience to use together both marker systems. A good concordance among endosperm proteins, agro-morphological traits and SSRs were also found, mainly when differences between genotypes were high. However, agro-morphological traits discriminated less between accessions than molecular markers. For the second objective of the thesis, we analysed the allelic variation at seven prolamin loci, involved in durum wheat quality: Glu-A1 and Glu-B1 of HMW glutenin, Glu-A3, Glu-B3 and Glu-B2 of B-LMW glutenin, and Gli-A1 and Gli-B1 of gliadin. The subsample analysed included landraces from all the Spanish provinces where the crop was traditionally cultivated. All the loci, except for Glu-B2, showed high genetic variability, being Glu-3 the most polymorphic. A total of 65 alleles were studied, 29 of them being new, which represent an important source of variability for quality improvement. Differences in prolamin composition were detected between convar. turgidum and the North zone, and the convar. durum and the South zone; the genotype Glu-B3new-1 - Gli-B1new-1 was very common in the convar. turgidum, while the Glu- B3a - Gli-B1c, associated with better quality, was more frequent in the convar. durum. Higher variability was detected in the convar. turgidum than in the convar. durum, mainly at the Glu-B1 and Glu-B3, showing that this convariety could be a valuable source of new glutenin alleles. The subsample was evaluated for quality (protein content, P, and sedimentation test, SDSS) with two doses of nitrogen fertiliser (N), and in two different years. No significant Variety x Year or Variety x Nitrogen interactions were detected. For P, environmental (year and N) effects were higher than variety effect, being P values , in general, larger with high dose of N. The variety exhibited a strong influence on SDSS test, which was not affected by year and N. Increasing values of P did not significantly influence on gluten strength, estimated with the SDSS. Respect to the prolamin effects on gluten strength, the superiority of Glu-B3a was confirmed; although a high positive effect of the new alleles Glu-A3new-1, and Glu-B3new-6 and new-9 was also detected. The no correlation between yield (evaluated in a previous research) and P, in the landraces adapted to low N, allowed to select four landraces with high yield and high gluten strength for low N production.

Seed image analysis and taxonomy of Diplotaxis DC. (Brassicaceae, Brassiceae)

The genus Diplotaxis, comprising 32 or 34 species, plus several additional infraspecific taxa, displays a considerable degree of heterogeneity in the morphology, molecular markers, chromosome numbers and geographical amplitude of the species. The taxonomic relationships within the genus Diplotaxis were investigated by phenetic characterisation of germplasm belonging to 27 taxa of the genus, because there is an increasing interest in Diplotaxis, since some of its species (D. tenuifolia, D. muralis) are gathered or cultivated for human consumption, whereas others are frequent arable weeds (D. erucoides) in many European vineyards. Using a computer-aided vision system, 33 morpho-colorimetric features of seeds were electronically measured. The data were used to implement a statistical classifier, which is able to discriminate the taxa within the genus Diplotaxis, in order to compare the resulting species grouping with the current infrageneric systematics of this genus. Despite the high heterogeneity of the samples, due to the great intra-population variability, the stepwise Linear Discriminant Analysis method, applied to distinguish the groups, was able to reach over 80% correct identification. The results obtained allowed us to confirm the current taxonomic position of most taxa and suggested the taxonomic position of others for reconsideration.

Probabilistic reasoning with a bayesian DNA device based on strand displacement

We present a computing model based on the DNA strand displacement technique which performs Bayesian inference. The model will take single stranded DNA as input data, representing the presence or absence of a specific molecular signal (evidence). The program logic encodes the prior probability of a disease and the conditional probability of a signal given the disease playing with a set of different DNA complexes and their ratios. When the input and program molecules interact, they release a different pair of single stranded DNA species whose relative proportion represents the application of Bayes? Law: the conditional probability of the disease given the signal. The models presented in this paper can empower the application of probabilistic reasoning in genetic diagnosis in vitro.

Probabilistic reasoning with an enzyme-driven DNA device

We present a biomolecular probabilistic model driven by the action of a DNA toolbox made of a set of DNA templates and enzymes that is able to perform Bayesian inference. The model will take single-stranded DNA as input data, representing the presence or absence of a specific molecular signal (the evidence). The program logic uses different DNA templates and their relative concentration ratios to encode the prior probability of a disease and the conditional probability of a signal given the disease. When the input and program molecules interact, an enzyme-driven cascade of reactions (DNA polymerase extension, nicking and degradation) is triggered, producing a different pair of single-stranded DNA species. Once the system reaches equilibrium, the ratio between the output species will represent the application of Bayes? law: the conditional probability of the disease given the signal. In other words, a qualitative diagnosis plus a quantitative degree of belief in that diagno- sis. Thanks to the inherent amplification capability of this DNA toolbox, the resulting system will be able to to scale up (with longer cascades and thus more input signals) a Bayesian biosensor that we designed previously.

Caracterización de nanopartículas magnéticas y de oro para posibles aplicaciones biomédicas en diagnóstico y terapia

Los nanomateriales han adquirido recientemente un gran interés debido a la gran variedad de aplicaciones que pueden llegar a tener en el ámbito de la biomedicina. Este trabajo recoge las posibilidades tanto diagnósticas como terapéuticas que presentan dos modalidades de nanomateriales: nanopartículas de óxido de hierro y nanopartículas de oro. Para ello, en una primera aproximación se ha llevado a cabo la caracterización de las nanopartículas desde el punto de vista de la biocompatibilidad asociada a su tamaño y al tiempo de contacto o circulación en células y tejidos, ensayada tanto in vitro como in vivo así como la cinética de acumulación de dichas nanopartículas en el organismo vivo. Posteriormente se ha realizado la biofuncionalización de los dos tipos de nanopartículas para reconocer dianas moleculares específicas y poder ser utilizadas en el futuro en dos aplicaciones biomédicas diferentes: diagnóstico de enfermedad de Alzheimer mediante imagen de resonancia magnética y destrucción selectiva de células tumorales mediante hipertermia óptica. ABSTRACT Nanomaterials have recently gained a great interest due to the variety of applications that can have in the field of biomedicine. This work covers both diagnostic and therapeutic possibilities that present two types of nanomaterials: iron oxide nanoparticles and gold nanoparticles. Therefore, in a first approximation it has performed the characterizing of nanoparticles from the standpoint of biocompatibility associated with their size and time of contact or movement in cells and tissues, tested both in vitro and in vivo as well as the kinetics of accumulation of the nanoparticles into the living organism. Subsequently the biofunctionalization of two types of nanoparticles was made to recognize specific molecular targets and can be used in the future in two different biomedical applications: diagnosis of Alzheimer's disease by magnetic resonance imaging and selective destruction of tumor cells by optical hyperthermia.

Mapping Genetic Diversity of Cherimoya (Annona cherimola Mill.): Application of Spatial Analysis for Conservation and Use of Plant Genetic Resources.

There is a growing call for inventories that evaluate geographic patterns in diversity of plant genetic resources maintained on farm and in species' natural populations in order to enhance their use and conservation. Such evaluations are relevant for useful tropical and subtropical tree species, as many of these species are still undomesticated, or in incipient stages of domestication and local populations can offer yet-unknown traits of high value to further domestication. For many outcrossing species, such as most trees, inbreeding depression can be an issue, and genetic diversity is important to sustain local production. Diversity is also crucial for species to adapt to environmental changes. This paper explores the possibilities of incorporating molecular marker data into Geographic Information Systems (GIS) to allow visualization and better understanding of spatial patterns of genetic diversity as a key input to optimize conservation and use of plant genetic resources, based on a case study of cherimoya (Annona cherimola Mill.), a Neotropical fruit tree species. We present spatial analyses to (1) improve the understanding of spatial distribution of genetic diversity of cherimoya natural stands and cultivated trees in Ecuador, Bolivia and Peru based on microsatellite molecular markers (SSRs); and (2) formulate optimal conservation strategies by revealing priority areas for in situ conservation, and identifying existing diversity gaps in ex situ collections. We found high levels of allelic richness, locally common alleles and expected heterozygosity in cherimoya's putative centre of origin, southern Ecuador and northern Peru, whereas levels of diversity in southern Peru and especially in Bolivia were significantly lower. The application of GIS on a large microsatellite dataset allows a more detailed prioritization of areas for in situ conservation and targeted collection across the Andean distribution range of cherimoya than previous studies could do, i.e. at province and department level in Ecuador and Peru, respectively.

Populations genomics analysis of legume host preference for specific rhizobial genotypes in the Rhizobium leguminosarum bv. viciae symbioses

Rhizobium leguminosarum bv. viciae establishes root nodule symbioses with several legume genera. Although most isolates are equally effective in establishing symbioses with all host genera, previous evidence suggests that hosts select specific rhizobial genotypes among those present in the soil. We have used population genomics to further investigate this observation. P. sativum, L. culinaris, V. sativa, and V. faba plants were used to trap rhizobia from a well-characterized soil, and pooled genomic DNAs from one-hundred isolates from each plant were sequenced. Sequence reads were aligned to the R. leguminosarum bv. viciae 3841 reference genome. High overall conservation of sequences was observed in all subpopulations, although several multigenic regions were absent from the soil population. A large fraction (16-22%) of sequence reads could not be recruited to the reference genome, suggesting that they represent sequences specific to that particular soil population. Although highly conserved, the 16S-23S rRNA gene region presented single nucleotide polymorphisms (SNPs) regarding the reference genome, but no striking differences could be found among plant-selected subpopulations. Plant-specific SNP patterns were, however, clearly observed within the nod gene cluster, supporting the existence of a plant preference for specific rhizobial genotypes. This was also shown after genome-wide analysis of SNP patterns.