Combined use of gliadins and SSRs to analyse the genetic variability of the Spanish collection of cultivated diploid wheat (Triticum monococcum L. ssp. monococcum)

This work studied the combined use of gliadins and SSRs to analyse inter- and intra-accession variability of the Spanish collection of cultivated einkorn (Triticum monococcum L. ssp. monococcum) maintained at the CRF-INIA. In general, gliadin loci presented higher discrimination power than SSRs, reflecting the high variability of the gliadins. The loci on chromosome 6A were the most polymorphic with similar PIC values for both marker systems, showing that these markers are very useful for genetic variability studies in wheat. The gliadin results indicated that the Spanish einkorn collection possessed high genetic diversity, being the differentiation large between varieties and small within them. Some associations between gliadin alleles and geographical and agro-morphological data were found. Agro-morphological relations were also observed in the clusters of the SSRs dendrogram. A high concordance was found between gliadins and SSRs for genotype identification. In addition, both systems provide complementary information to resolve the different cases of intra-accession variability not detected at the agro-morphological level, and to identify separately all the genotypes analysed. The combined use of both genetic markers is an excellent tool for genetic resource evaluation in addition to agro-morphological evaluation.

Validation of microsatellite markers for cytotype discrimination in the model grass Brachypodium distachyon

Brachypodium distachyon (2n = 2x = 10) is a small annual grass species where the existence of three different cytotypes (10, 20 and 30 chromosomes) has long been regarded as a case of autopolyploid series, with x = 5. However, it has been demonstrated that the cytotypes assumed to be polyploids represent two separate Brachypodium species recently named as B. stacei (2n = 2x = 20) and B. hybridum (2n = 4x = 30). The aim of this study was to find a PCR-based alternative approach that could replace standard cytotyping methods (i. e., chromosome counting and flow cytometry) to characterize each of the three Brachypodium species. We have analyzed with four microsatellite (SSR) markers eighty-three Brachypodium distachyon-type lines from varied locations in Spain, including the Balearic and Canary Islands. Within this set of lines, 64, 4 and 15 had 10, 20 and 30 chromosomes, respectively. The surveyed markers produced cytotype-specific SSR profiles. So, a single amplification product was generated in the diploid samples, with non-overlapping allelic ranges between the 2n = 10 and 2n = 20 cytotypes, whereas two bands, one in the size range of each of the diploid cytotypes, were amplified in the 2n = 30 lines. Furthermore, the remarkable size difference obtained with the SSR ALB165 allowed the identification of the Brachypodium species by simple agarose gel electrophoresis.

Taxonomía y caracterización del arbusto forrajero Atriplex halimus L.

El objetivo de esta Tesis Doctoral ha sido la revisión de la taxonomía infraespecífica de la especie Atriplex halimus L., la delimitación del área geográfica que ocupan sus taxones y su caracterización morfológica. En base a las citas bibliográficas y material de herbario consultado se ha trazado un mapa de distribución de la especie, que delimita su distribución como planta espontánea a la Cuenca Mediterránea, desierto de Siria, Macizo de Hoggar y Sahara Occidental. El resto debe considerarse poblaciones naturalizadas o cultivadas. La taxonomía actual, basada en relaciones filogenéticas, nos indica que lo adecuado es separar las poblaciones de A. halimus en dos clados que se caracterizan por tener diferente nivel de ploidía. Para definir el área de distribución de los clados diploide y tetraploide se ha determinado la ploidía de 19 poblaciones en el espacio comprendido entre las dos áreas de distribución conocidas. Y como resultado el área de distribución de ambos clados puede definirse trazando una recta que une el estrecho de Gibraltar con Estambul, las poblaciones al sur de esta línea son tetraploides a las que habría que añadir las poblaciones del este y sur de la isla de Cerdeña que son también tetraploides. Se han determinado cuales son los parámetros morfológicos que permiten caracterizar ambos clados, para lo cual se han estudiado 52 poblaciones que comprenden toda el área de distribución de la especie. Se han evaluado caracteres cualitativos y cuantitativos de valvas fructíferas, plántulas cultivadas en cámara y arbustos de 2 a 4 años situados en la misma parcela. El único elemento fiable para determinar la pertenencia al clado diploide o tetraploide es la medición del nivel de ploidía. Ninguno de los caracteres morfológicos estudiados es plenamente fiable para determinar si los individuos de una población pertenecen al clado diploide o tetraploide a pesar de realizarse las mediciones sobre poblaciones cultivadas en igualdad de condiciones de suelo y clima, y en condiciones muy favorables para la expresión de caracteres cualitativos y cuantitativos. Los caracteres cualitativos observados que guardan una mayor correlación con el nivel de ploidía son: la base de las hojas de las plántulas, el porte del arbusto, la ramificación, el ángulo de inserción de las ramas y la longitud de la inflorescencia. Respecto a los datos cuantitativos los más relevantes han sido, la longitud de los cotiledones y la longitud del limbo de la 1ª, 3ª o 5ª hoja de la plántula o la razón entre este valor y la anchura del limbo. ABSTRACT The aim of this Thesis has been in the revision of the infraspecies taxa of Atriplex halimus L., the delimitation of their natural growing area and their morphological characterization. Using references to relevant literature and consultation of herbariums, it has been possible to map the distribution of the species as spontaneous plant in the Mediterranean Basin, Desert of Syria, Massif of Hoggar and Western Sahara. The remaining populations must be considered as naturalized or cultivated. The current taxonomy, based on phylogenetic relationships, indicates that A.halimus should be divided in two clades. These clades are characterized by their different ploidy level. Samples of nineteen populations collected from localizations between the defined distribution area of both clades have been characterized for their ploidy level. As a result, it can be seen that the line of clade separation can be trazed from Istambul tothe Strait of Gibraltar, where the populations north to this line are classified as diploid and those south of the line are clsassified as tetraploid. The only exception of this rule is in Sardinia, hwre populations to the West and South of the Island are also classified as tetraploid. The morphological parameters, which include the characterization of both clades, have been defined, and the study of fifty-two populations from the entire distribution area according to these parameters have been analysed. The qualitative and quantitative characteristics of the fruit valves have been evaluated, with seedlings grown in a culture chamber, and 2-4 years old shrubs grown in the same orchard. None of the morphological characters under study are fully reliable to determine whether individuals in a population belong to clade diploid or tetraploid. This is despite measurements being made on populations grown under the same conditions of soil and climate, and in very favorable conditions for the expression of qualitative and quantitative traits. The qualitative characteristics that show a higher correlation with the ploidy level are: the base of the leaves of the seedlings, the bearing bush, the branch arrangement, the angle of insertion of the branches and the inflorescence length. With regards to the quantitative data the most relevant were the length of the cotyledons and the length of the blade of the 1st, 3rd or 5th leaves of the seedling, and the ratio of any of this values and the leaf blade width. The only reliable way to determine the diploid or tetraploid clade membership is the measurement of ploidy level.

Environmental niche variation and evolutionary diversification of the Brachypodium distachyon grass complex species in their native circum-Mediterranean range

PREMISE OF THE STUDY: We conducted environmental niche modeling (ENM) of the Brachypodium distachyon s.l. complex, a model group of two diploid annual grasses ( B. distachyon , B. stacei ) and their derived allotetraploid ( B. hybridum) , native to the circum-Mediterranean region. We (1) investigated the ENMs of the three species in their native range based on present and past climate data; (2) identifi ed potential overlapping niches of the diploids and their hybrid across four Quaternary windows; (3) tested whether speciation was associated with niche divergence/conservatism in the complex species; and (4) tested for the potential of the polyploid outperforming the diploids in the native range. M ETHODS: Geo-referenced data, altitude, and 19 climatic variables were used to construct the ENMs. We used paleoclimate niche models to trace the potential existence of ancestral gene fl ow among the hybridizing species of the complex. KEY RESULTS: Brachypodium distachyon grows in higher, cooler, and wetter places, B. stacei in lower, warmer, and drier places, and B. hybridum in places with intermediate climatic features. Brachypodium hybridum had the largest niche overlap with its parent niches, but a similar distribution range and niche breadth. C ONCLUSIONS: Each species had a unique environmental niche though there were multiple niche overlapping areas for the diploids across time, suggesting the potential existence of several hybrid zones during the Pleistocene and the Holocene. No evidence of niche divergence was found, suggesting that species diversifi cation was not driven by ecological speciation but by evolutionary history, though it could be associated to distinct environmental adaptations.