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.

Effects of forage type on diversity in bacterial pellets isolated from liquid and solid phases of the rumen content in sheep and goats.

Two sheep and two goats, fitted with a ruminal cannula, received two diets composed of 30% concentrate and 70% of either alfalfa hay (AL) or grass hay (GR) as forage in a two-period crossover design. Solid and liquid phases of the rumen were sampled from each animal immediately before feeding and 4 h post-feeding. Pellets containing solid associated bacteria (SAB) and liquid associated bacteria (LAB) were isolated from the corresponding ruminal phase and composited by time to obtain 2 pellets per animal (one SAB and one LAB) before DNA extraction. Denaturing gradient gel electrophoresis (DGGE) analysis of 16S ribosomal DNA was used to analyze bacterial diversity. A total of 78 and 77 bands were detected in the DGGE gel from sheep and goats samples, respectively. There were 18 bands only found in the pellets from sheep fed AL-fed sheep and 7 found exclusively in samples from sheep fed the GR diet. In goats, 21 bands were found only in animals fed the AL diet and 17 were found exclusively in GR-fed ones. In all animals, feeding AL diet tended (P < 0.10) to promote greater NB and SI in LAB and SAB pellets compared with the GR diet. The dendrogram generated by the cluster analysis showed that in both animal species all samples can be included in two major clusters. The four SAB pellets within each animal species clustered together and the four LAB pellets grouped in a different cluster. Moreover, SAB and LAB clusters contained two clear subclusters according to forage type. Results show that in all animals bacterial diversity was more markedly affected by the ruminal phase (solid vs. liquid) than by the type of forage in the diet.

Biodiversidad de frijol (Phaseolus vulgaris L.) en Honduras. Caracterización agromorfológica, molecular y ecogeográfica

En el presente trabajo se ha llevado a cabo un estudio de la biodiversidad del frijol común (Phaseolus vulgaris L.) en Honduras, que es el segundo de los cultivos de granos básicos en importancia. Dicho estudio se ha realizado mediante una caracterización agromorfológica, molecular y ecogeográfica en una selección de 300 accesiones conservadas en el banco de germoplasma ubicado en la Escuela Agrícola Panamericana (EAP) El Zamorano, y que se colectaron en 13 departamentos del país durante el periodo de 1990 a 1994. Estas accesiones fueron colectadas cuatro años antes del acontecimiento del huracán Mitch, el cual a su paso afectó al 96% del área total cultivable en su momento, lo cual nos hace considerar que la biodiversidad de razas locales (landraces) de frijol común existentes in situ fueron severamente afectadas. Los trabajos dirigidos a analizar la biodiversidad de razas locales de frijol común en Honduras son escasos, y este trabajo se constituye como el primero que incluye una amplia muestra a ser estudiada a través de una caracterización en tres aspectos complementarios (agromorfológico, molecular y ecogeográfico). Se evaluaron 32 caracteres agromorfológicos, 12 cuantitativos y 20 cualitativos, en distintas partes de la planta. Se establecieron las correlaciones entre los caracteres agromorfológicos y se elaboró un dendrograma con los mismos, en el que se formaron ocho grupos, en parte relacionados principalmente con los colores y tamaños de la semilla. Mediante el análisis de componentes principales se estudiaron los caracteres de más peso en cada uno de los tres primeros componentes. Asimismo, se estudiaron las correlaciones entre caracteres, siendo las más altas la longitud y anchura de la hoja, días a madurez y a cosecha y longitud y peso de semilla. Por otra parte, el mapa de diversidad agromorfológica mostró la existencia de tres zonas con mayor diversidad: en el oeste (en los departamentos de Santa Bárbara, Lempira y Copán), en el centro-norte (en los departamentos de Francisco Morazán, Yoro y Atlántida) y en el sur (en el departamento de El Paraíso y al sur de Francisco Morazán). Para la caracterización molecular partimos de 12 marcadores de tipo microsatélite, evaluados en 54 accesiones, que fueron elegidas por constituir grupos que compartían un mismo nombre local. Finalmente, se seleccionaron los cuatro microsatélites (BM53, GATS91, BM211 y PV-AT007) que resultaron ser más polimórficos e informativos para el análisis de las 300 accesiones, con los que se detectaron un total de 119 alelos (21 de ellos únicos o privados de accesión) y 256 patrones alélicos diferentes. Para estudiar la estructura y relaciones genéticas en las 300 accesiones se incluyeron en el análisis tres controles o accesiones de referencia, pertenecientes dos de ellas al acervo genético Andino y una al Mesoamericano. En el dendrograma se obtuvieron 25 grupos de accesiones con idénticas combinaciones de alelos. Al comparar este dendrograma con el de caracteres agromorfológicos se observaron diversos grupos con marcada similitud en ambos. Un total de 118 accesiones resultaron ser homogéneas y homocigóticas, a la vez que representativas del grupo de 300 accesiones, por lo que se analizaron con más detalle. El análisis de la estructura genética definió la formación de dos grupos, supuestamente relacionados con los acervos genéticos Andino (48) y Mesoamericano (61), y un reducido número de accesiones (9) que podrían tener un origen híbrido, debido a la existencia de un cierto grado de introgresión entre ambos acervos. La diferenciación genética entre ambos grupos fue del 13,3%. Asimismo, 66 de los 82 alelos detectados fueron privados de grupo, 30 del supuesto grupo Andino y 36 del Mesoamericano. Con relación al mapa de diversidad molecular, presentó una distribución bastante similar al de la diversidad agromorfológica, detectándose también las zonas de mayor diversidad genética en el oeste (en los departamentos de Lempira y Santa Bárbara), en el centro-norte (en los departamentos de Yoro y Atlántida) y en el sur (en el departamento de El Paraíso y al sur de Francisco Morazán). Para la caracterización ecogeográfica se seleccionaron variables de tipo bioclimático (2), geofísico (2) y edáfico (8), y mediante el método de agrupamiento de partición alrededor de los medoides, la combinación de los grupos con cada uno de los tres tipos de variables definió un total de 32 categorías ecogeográficas en el país, detectándose accesiones en 16 de ellas. La distribución de las accesiones previsiblemente esté relacionada con la existencia de condiciones más favorables al cultivo de frijol. En el mapa de diversidad ecogeográfica, nuevamente, se observaron varias zonas con alta diversidad tanto en el oeste, como en el centro-norte y en el sur del país. Como consecuencia del estudio realizado, se concluyó la existencia de una marcada biodiversidad en el material analizado, desde el punto de vista tanto agromorfológico como molecular. Por lo que resulta de gran importancia plantear la conservación de este patrimonio genético tanto ex situ, en bancos de germoplasma, como on farm, en las propias explotaciones de los agricultores del país, siempre que sea posible. ABSTRACT In the present work we have carried out a study of the biodiversity of the common bean (Phaseolus vulgaris L) in Honduras, which is the second of the basic grain crops in importance. This study was conducted through agro-morphological, molecular and ecogeographical characterization of a selection of 300 accessions conserved in the genebank located in the ‘Escuela Agrícola Panamericana (EAP) El Zamorano’ that were collected in 13 departments of the country during the 1990 to 1994 period. These accessions were collected four years before the occurrence of Mitch hurricane, which affected 96% of the total cultivable area at the time, which makes us to consider that the biodiversity of local landraces of common bean existing in situ were severely affected. The work aimed to analyze the biodiversity of local races of common bean in Honduras are scarce, and this work constitutes the first to include a large sample to be studied through a characterization on three complementary aspects (agromorphological, molecular and ecogeographical). Thirty two agromorphological characters, 12 quantitative and 20 qualitative, in various parts of the plant were evaluated. Correlations between agromorphological characters were established and a dendrogram with them was constructed, in which eight groups were formed, in part mainly related to the colors and sizes of the seeds. By principal component analysis the characters with more weight in each of the first three components were studied. Also, correlations between characters were studied, the highest of them being length and leaf width, days to maturity and harvest, and seed length and weight. Moreover, the map of agromorphological diversity showed the existence of three areas with more diversity: the west (departments of Santa Barbara, Copan and Lempira), the center-north (departments of Francisco Morazán, Yoro and Atlántida) and the south (department of El Paraiso and south of Francisco Morazán). For molecular characterization we started with 12 microsatellite markers, evaluated in 54 accessions, which were chosen because they formed groups that shared the same local name. Finally, four microsatellites (BM53, GATS91, BM211 and PV-AT007) were selected for the analysis of 300 accessions, since they were the most polymorphic and informative. They gave a total of 119 alleles (21 of them unique or private for the accession) and 256 different allelic patterns. To study the structure and genetic relationships in the 300 accessions, three controls or accessions of reference were included in the analysis: two of them belonging to the Andean gene pool and one to the Mesoamerican. In the dendrogram, 25 accession groups with identical allele combinations were obtained. Comparing this dendrogram to the obtained with agromorphological characters, several groups with marked similarity in both were observed. A total of 118 accessions were homozygous and homogeneous, while representing the group of 300 accessions, therefore they were analyzed in more detail. The analysis of the genetic structure defined the formation of two groups, supposedly related to the Andean (48) and the Mesoamerican (61) gene pools, and a small number of accessions (9) which may have a hybrid origin, due to the existence of some degree of introgression between both gene pools. Genetic differentiation between both groups was 13.3%. Also, 66 of the 82 detected alleles were private or unique for the group, 30 of the supposed Andean group and 36 of the Mesoamerican. With relation to the map of molecular diversity, it showed a quite similar distribution to the agromorphological, also detecting the areas of greatest genetic diversity in the west (departments of Lempira and Santa Bárbara), in the center-north (departments Atlántida and Yoro) and in the south (departments of El Paraíso and south of Francisco Morazán). For the ecogeographical characterization, bioclimatic (2), geophysical (2) and edaphic (8) variables were selected, and by the method of clustering partition around the medoids, the combination of the groups to each of the three types of variables defined a total of 32 ecogeographical categories in the country, having accessions in 16 of them. The distribution of accessions is likely related to the existence of more favorable conditions for the cultivation of beans. The map of ecogeographical diversity, again, several areas with high diversity both in the west and in the center-north and in the south of the country were observed. As a result of study, the existence of marked biodiversity in the analyzed material was concluded, both from the agromorphological and from the molecular point of view. Consequently it is very important to propose the conservation of this genetic heritage both ex situ, in genebanks, as on farm, in the holdings of the farmers of the country, whenever possible.