Toxigenic profile and AFLP variability of Alternaria alternata and Alternaria infectoria occurring on wheat

The objectives of this study were to evaluate the ability to produce alternariol (AOH), alternariol monomethyl ether (AME) and tenuazonic acid (TA) by A. alternata and A. infectoria strains recovered from wheat kernels obtained from one of the main production area in Argentina; to confirm using AFLPs molecular markers the identify of the isolates up to species level, and to evaluate the intra and inter-specific genetic diversity of these two Alternaria species. Among all the Alternaria strains tested (254), 84% of them were able to produce mycotoxins. The most frequent profile of toxin production found was the co-production of AOH and AME in both species tested. TA was only produced by strains of A. alternata. Amplified fragment polymorphism (AFLPs) analysis was applied to a set of 89 isolates of Alternaria spp (40 were A. infectoria and 49 were A. alternata) in order to confirm the morphological identification. The results showed that AFLPs are powerful diagnostic tool for differentiating between A. alternata and A. infectoria. Indeed, in the current study the outgroup strains, A. tenuissima was consistently classified. Characteristic polymorphic bands separated these two species regardless of the primer combination used. Related to intraspecific variability, A. alternata and A. infectoria isolates evaluated seemed to form and homogeneous group with a high degree of similarity among the isolates within each species. However, there was more scoreable polymorphism within A. alternata than within A. infectoria isolates. There was a concordance between morphological identification and separation up to species level using molecular markers. Clear polymorphism both within and between species showed that AFLP can be used to asses genetic variation in A. alternata and A. infectoria. The most important finding of the present study was the report on AOH and AME production by A. infectoria strains isolated from wheat kernels in Argentina on a semisynthetic media for the first time. Also, specific bands for A. alternata and A. infectoria have been identified; these may be useful for the design of specific PCR primers in order to differentiate these species and to detect them in cereals.

Genetic diversity in cultivated carioca common beans based on molecular marker analysis

A wide array of molecular markers has been used to investigate the genetic diversity among common bean species. However, the best combination of markers for studying such diversity among common bean cultivars has yet to be determined. Few reports have examined the genetic diversity of the carioca bean, commercially one of the most important common beans in Brazil. In this study, we examined the usefulness of two molecular marker systems (simple sequence repeats - SSRs and amplified fragment length polymorphisms - AFLPs) for assessing the genetic diversity of carioca beans. The amount of information provided by Roger's modified genetic distance was used to analyze SSR data and Jaccards similarity coefficient was used for AFLP data. Seventy SSRs were polymorphic and 20 AFLP primer combinations produced 635 polymorphic bands. Molecular analysis showed that carioca genotypes were quite diverse. AFLPs revealed greater genetic differentiation and variation within the carioca genotypes (Gst = 98 percent and Fst = 0.83, respectively) than SSRs and provided better resolution for clustering the carioca genotypes. SSRs and AFLPs were both suitable for assessing the genetic diversity of Brazilian carioca genotypes since the number of markers used in each system provided a low coefficient of variation. However, fingerprint profiles were generated faster with AFLPs, making them a better choice for assessing genetic diversity in the carioca germplasm.

Caracterización molecular de tres líneas de Cavia porcellus mediante la aplicación de AFLP / Molecular characterization of three populations of Cavia porcellus with AFLP markers

Los marcadores moleculares son una herramienta eficaz para determinar variabilidad genética entre y dentro de poblaciones, pero en el caso de Cavia porcellus, no existen reportes referentes al uso de estas técnicas. Con los marcadores moleculares AFLP´s (Amplified Fragment Length Polimorphism), se analizaron tres poblaciones, dos criollas y una mejorada genéticamente, sometida a selección durante varias generaciones y obtenida a partir del cruzamiento entre animales peruanos y nativos de Nariño. Para obtener los marcadores moleculares AFLP´s (Amplified Fragment Lenght Polimorphism), se utilizaron en total cinco combinaciones de cebadores, tres combinaciones recomendadas para el orden Rodenthia y dos por la casa fabricante del Kit, de las cuales sólo una de ellas, con 116 loci, permitió establecer diferencias entre las poblaciones estudiadas, de acuerdo con el valor de distancia genética insesgada de Nei (p<0.01). Las dos poblaciones criollas constituyeron un grupo estrechamente relacionado y distante de la población mejorada genéticamente, lo que indica que los animales importados absorbieron al criollo. De acuerdo con los valores de heterocigosidad promedio, que variaron entre 0.48% y 14.48%, y el porcentaje de polimorfismo que osciló entre 0.00% y 39.65%, se deduce una baja variabilidad intrapoblacional, siendo la población mejorada genéticamente la más polimórfica. La baja variabilidad entre los animales mejorados, se explica por la intensa selección a la que han sido sometidos, mientras que en los núcleos criollos este fenómeno puede atribuirse al bajo tamaño efectivo en las dos poblaciones. Los resultados de esta investigación sugieren un replanteamiento de los programas de mejoramiento genético y conservación de los recursos genéticos autóctonos en la región.

Molecular markers are a powerful tool to determine genetic variability within and among populations, but for the Cavia porcellus there are no reports on the use of these techniques. Three populations, two native and another one, genetically improved which was obtained by crossing native and Peruvian animals and submitted to genetic selection through several generations were analyzed by means of AFLP markers. Five primer’s combinations recommended for Rodenthia were used, but only one allowed to establish significant differences (p<0.01) according to unbiased Nei´s distance Value. Both native populations were grouped in a cluster genetically distant from the genetically improved animals. This showed that foreign animals absorbed the native populations. The average heterosigosity between 0.48% and 14.48% and the percentage of polymorphisms between 0.00% and 39.65% allow to conclude that there was a low variability between the populations, but the population genetically improved was the most polymorphic. The low variability within the improved animals it can be explained because of the intensive selection procedures use with them, whereas within the native populations can be explained because of their very low populations effective size. These results suggest that there is a need to restate the genetic improvement and preservation programs of the native Cavia porcellus in the southwest region of Colombia.

Polimorfismos en la longitud de fragmentos amplificados (AFLP´s) a partir de muestras de sangre almacenadas en tarjetas FTA®para la especie Cavia porcellus Lin. (Rodentia: Caviidae). / Amplified Fragments Length Polymorphisms (AFLP´s) from blood samples stored in FTA® cards for Cavia porcellus

Una manera eficaz de establecer el grado de variabilidad entre y dentro de poblaciones, es a través del análisis de polimorfismos de ADN con marcadores moleculares como los AFLP`s. En este artículo se presenta una metodología que combina la utilización de tarjetas de FTA® (Whatman Bioscience, Cambridge) para colección y conservación de muestras de sangre, con los procedimientos de extracción de ADN y obtención de marcadores AFLP´s, aspectos sobre los cuales no existen antecedentes para la especie Cavia porcellus. Se utilizaron muestras de ADN procedentes de tres poblaciones, dos criollas y una mejorada genéticamente obtenida a partir de un pie de cría procedente del Perú y sometida a selección en Colombia durante varias generaciones. Todos los animales procedieron de la Granja “Botana”, propiedad de la Universidad de Nariño, Pasto-Colombia. Para la detección de polimorfismos en la longitud de los fragmentos (AFLP`s) se utilizaron uno, tres y cinco discos FTA® de 1.2 mm, cada disco con aproximadamente 25 ng de ADN. Los ensayos indicaron que los mejores productos de amplificación, para la visualización de AFLP´s, se obtuvieron de muestras con tres discos de FTA por individuo, lo que sugiere que con esta metodología,75 ng de ADN por animal son suficientes para detectar polimorfismos de alta calidad en el genoma de Cavia porcellus. Se recomienda el uso de las tarjetas de FTA para el estudio genético de poblaciones de Cavia porcellus, con las modificaciones metodológicas descritas en este artículo para marcadores AFLP´s.

A methodology that includes the use of FTA® (Whatman Bioscience, Cambridge) to collect and store animals` blood samples and the procedures to extract and to get AFLP markers is presented in this paper. A review of the literature indicates that there are no reports concerning both aspects for the Cavia porcellus case. To reach our goal blood samples of three populations – Two native ones and other genetically improved- were obtained through heart puncture. This blood was stored in the FTA cards in order to extract, purify, amplify and analyze their DNA forms. All of the animals came from “Botana” farm of the Universidad de Nariño, located in Pasto, Colombia. For amplifying the AFLP one, three and five 1.2 mm FTA disks of approximately 75 ng of DNA per disk where used. The tests indicated that the best products to amplify and to visualize the AFLP where those ones obtained from samples of three FTA disks per animal. This suggests that 75 ng of DNA per animal is enough to generate AFLP of high quality in the Cavia porcellus` genome. We recommend the use of FTA cards to carry out genetic analyses in the Cavia porcellus, including the methodology modifications presented in this paper.