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Fil: Velez, Maria Laura. Centro de Investigacion y Extension Forestal Andino Patagonico; Argentina. Consejo Nacional de Investigaciones Cientificas y Tecnicas; Argentina

The aberrant phenotype is characterized by the progressive expression of abnormal traits during vegetative growth affecting leaf thickness, shape and/or plant vigour. These striking morphological abnormalities do not appear to be caused by agronomical practices or pathogen infections. Furthermore, the aberrant phenotype, which is observed in 3-20% of cultivated cauliflowers, is not linked to DNA sequence or structural polymorphisms. To detect candidate genes related to the aberrant phenotype, we used amplified fragment length polymorphism on cDNA approach, sampling normal and aberrant F(1) hybrid plants several times before and after the expression of the aberrant phenotype. This screen led to the detection of 51 differentially expressed transcripts. Twenty-nine of these were homologous to annotated genes in genomic databases. We identified transcripts, which were differentially expressed before the expression of the aberrant trait with homology to genes involved in various abiotic stress responses. A non-specific lipid transfer protein homologue was also identified and given the role that these proteins play in epicuticular wax formation and leaf morphology, it may be implicated in the abnormal leaf shape phenotypes.

Two species, Trifolium glomeratum and T. nigrescens, from Sardinia, Italy, were analysed for genetic diversity using amplified fragment length polymorphism (AFLP). Variation between and within populations was compared between the inbreeder, T. glomeratum, and the outbreeder, T. nigrescens. Four AFLP primer combinations resulted in a total of 292 loci, of which 75% were polymorphic in T. glomeratum and 85% in Trifolium nigrescens. Variation was highest between populations in both species, but the difference between populations was greater in T. glomeratum (Fst = 0.17), compared with T. nigrescens (Fst = 0.02). Cluster analysis and principal coordinates analysis were used to verify the relationships found. The high level of genetic variation within populations in both species is attributed to the movement of sheep between paddocks, the existence of both species in Sardinia for thousands of years and the persistence of a long-lived seedbank due to the production of large numbers of small seeds with high levels of hard seededness.

Mangifera odorata Griff. (Anacardiaceae), was suggested to be a hybrid between M. indica L. and M. foetida Lour. due to morphological intermediacy. Results from this study show that M. indica and M. foetida produced unique amplified fragment length polymorphism (AFLP) profiles. Mangifera odorata did not produce any unique bands. All the M. odorata samples additively inherit bands specific to M. indica and M. foetida, which strongly suggested the hybrid origin. Three major clusters were produced in the phenogram. All samples of M. indica, M. foetida and M. odorata segregated distinctly. Mangifera odorata was closer to M. foetida than to M. indica, indicating that backcrossing with M. foetida might have taken place. AFLP analysis therefore verified the hybrid status of M. odorata.

With 2 figures Abstract In most economically important dioecious plants such as Simmondsia chinensis (Jojoba), sex identification based on flowering can only be made after 1–5 years of plantation. Identification of sex-associated molecular markers for ascertaining sex at seedling stage is a desirable prerequisite for optimal plantation and economical benefits. Jojoba is being developed as a plantation crop in the arid regions of Western India due to the commercial benefits of the unique waxy content of its seeds. In the present study, sex-linked DNA markers have been identified in S. chinensis using the AFLP technique. From sixteen primers combinations used for AFLP analysis, two combinations EcoRI-TAC/MseI-GCG and EcoRI-GC/MseI-GCG resulted in amplification of sex-linked markers. Two male-specific markers of ∼525 bp and ∼325 bp were identified using the primer combinations EcoRI-GC/MseI-GCG and EcoRI-TAC/MseI-GCG, respectively. A female-specific marker of ∼270 bp was also identified with the primer combination EcoRI-TAC/MseI-GCG. In future, SCAR markers developed from these sex-specific markers will provide an effective and reliable method for sex identification.

Phytophthora austrocedrae is a recently discovered pathogen that causes severe mortality of Austrocedrus chilensis in Patagonia. The high level of susceptibility of the host tree, together with the distribution pattern of the pathogen, have led to the hypothesis that P. austrocedrae was introduced into Argentina. The aim of this study was to assess the population structure of P. austrocedrae isolates from Argentina in order to gain an understanding of the origin and spread of the pathogen. Genetic diversity was determined based on amplified fragment length polymorphisms (AFLPs). In total, 48 isolates of P. austrocedrae were obtained from infected A. chilensis trees, representing the geographical range of the host. Four primer combinations were used for the AFLP analysis. Of the 332 scored bands, 12% were polymorphic. Gene diversity (h) ranged from 0·01 to 0·03; the Shannon index (I) ranged from 0·01 to 0·04. A high degree of genetic similarity was observed among the isolates (pairwise S values = 0·958–1; 0·993 ± 0·009, mean ± SD). A frequency histogram showed that most of the isolate pairs were identical. Principal coordinate analysis using three-dimensional plots did not group any of the isolates based on their geographical origin. The low genetic diversity (within and between sites) and absence of population structure linked to geographic origin, together with the aggressiveness of the pathogen and the disease progression pattern, suggest that P. austrocedrae might have been introduced into Argentina. Fil: Velez, Maria Laura. Centro de Investigación y Extensión Forestal Andino Patagónico; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Coetzee, M. P. A.. University of Pretoria; Sudáfrica Fil: Wingfield, M. J.. University of Pretoria; Sudáfrica Fil: Rajchenberg, Mario. Centro de Investigación y Extensión Forestal Andino Patagónico; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Greslebin, Alina Gabriela. Centro de Investigación y Extensión Forestal Andino Patagónico; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

Zusammenfassung. 27 Trichophyton verrucosum-Stamme aus 14 Rinderbestanden in Thuringen und Mecklenburg-Vorpommern wurden unter Einbeziehung von sechs T. verrucosum-Referenzstammen, unter anderem auch die sogenannten album-, discoides- und ochraceum-Varietaten, kulturmorphologisch und molekularbiologisch (PCR-Fingerprinting, AFLP-Analyse sowie Sequenzieung der ITS-Region der rDNA) gepruft. Trotz groser kulturmorphologischer Variabilitat der lsolate und ihrer moglichen Zuordnung zu vier verschiedenen Kolonietypen waren alle gepruften T. verrucosum-Stamme genotypisch identisch. Auch die zwei mit Gelbpigment wachsenden Feldisolate, die moghcherweise als ochraceum-Varietat angesehen werden konnten, sowie der ochrceum-Referenzstamm konnten mittels der drei genannten molekularbiologischen Methoden nicht differenziert werden. Somit ergeben sich weder Hinweise fur die eigenstandige taxonomische Stellung der drei T. verrucosum-Varietaten noch konnte die vermutete Infektion mit ochraceum-Stammen in Rinderbestanden als Ursache fur das Versagen der Impfprophylaxe mit verschiedenen T. verrucosum-Impfstoffen und fur das gehaufte Auftreten sogenannter Impfversager bestatigt werden. Es wird vielmehr vermutet, das neben der unzureichenden Impfstammpflege vor allem Anwendungsfehler, insbesondere in der luckenlosen, systematischen Immunprophylaxe in der Rinderherde, ursachlich fur das gehaufte Auftreten erkrankter Rinder in gegen Trichophytie geimpften Bestanden verantwortlich sind. Summary. Twenty-seven strains of Trichophyton verrucosum from 14 cattle herds in the Federal States of Thuringia and Mecklenburg-Vorpommern were examined by culture morphological and molecular biological (PCR fingerprinting, AFLP analysis sequencing of ITS region) methods. Six reference strains of the same species, among them the so-called album andochraceum varieties, were also included. Despite great variability in terms of culture morphology, which suggested their possible classification into 4 different colony types, all T. verrucosum isolates were genotypically almost identical. Even the 2 field isolates growing with yellow pigment, which could possibly be regarded as belonging to the ochraceum variety, could not be differentiated using molecular biological methods. The results do not provide indications of a separate taxonomic position of the 3 T. verrucosum varieties. Furthermore, there is no evidence confirming the suspected infection of cattle herds with ochraceum strains as the cause of the failure of immune prophylaxis using various T. verrucosum vaccines. The frequent occurrence of animals not responding to vaccination could not be explained either. It should be assumed that the main factors responsible for this situation include poor handling of the vaccine strains and errors in application, especially the absence of continuous and systematic immune prophylaxis in the herds.

Analysis of amplified fragment length polymorphism (AFLP) has the potential to become a powerful new DNA fingerprinting technique for studying genetic relationships and genetic diversity in arthropods. Since DNA of high quality is a crucial prerequisite for AFLP analysis we evaluated the applicability of six protocols (one fast and four complex methods with phenol-chloroform treatments as well as one CTAB-based method) for extracting DNA from insect material and three additional DNA purification steps. The most rapid DNA isolation method did not produce DNA suitable for AFLP analysis. Among four complex methods tested, two protocols resulted in comparatively low yields of DNA that was therefore not used as template for AFLP analysis. The other two complex methods with phenol treatments and a CTAB-based DNA extraction protocol provided DNA suitable for AFLP assay. An additional purification of the DNA using spermine precipitation revealed a few extra bands in an AFLP gel that were masked in unpurified DNA. Therefore spermine precipitation is recommended for AFLP templates.