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Annonaceae represent the largest extant family among the early divergent angiosperms. Despite the long-standing interest in its evolutionary and taxonomic aspects, cytogenetic studies on this family remain extremely few even on economically important species. With this study, we realized a detailed characterization of the chromosomes of Annona cherimola (2n = 14) by a combination of in situ hybridization techniques, fluorochrome banding, and karyomorphological analysis. FISH revealed that 45S and 5S rDNA sites are co-localized in correspondence to the secondary constrictions of the SAT-chromosome pair. Some hypotheses on the organization of the linked 45S and 5S rDNA repeats have been made. FISH with Arabidopsis-type telomeric arrays demonstrated that the A. cherimola telomeres are constituted by TTTAGGG sequences and that they are exclusively localized at the extremities of chromosomes. An insight into the chromosome structure of A. cherimola was obtained by the self-GISH procedure which revealed highly repeated DNA sequences localized in the centromeric regions of all chromosomes. The correspondence of s-GISH signals with DAPI banding suggests that these sequences are the principal component of the centromeric heterochromatin of this species. The karyotype of A. cherimola here described is proposed as the basic reference karyotype for successive investigations in Annonaceae. Key words: Annona cherimola, karyotyping, FISH, GISH, telomeres, ribosomal genes, repetitive DNA. Résumé: Les Annonacées constituent la plus grande famille existante parmi les angiospermes primitives. En dépit d'un intérêt de longue date portant sur les aspects évolutifs et taxonomiques, les études cytogénétiques au sein de cette famille sont très peu nombreuses, même chez les espèces d'importance économique. Dans ce travail, les auteurs ont réalisé une caractérisation détaillée des chromosomes chez l'Annona cherimola (2n = 14) en combinant diverses approches d'hybridation in situ, de coloration des bandes à l'aide de fluorochromes et une analyse caryo-morphologique. Les analyses FISH ont révélé que les sites d'ADNr 45S et 5S co-localisent avec les sites de constriction secondaire chez la paire de chromosomes SAT. Certaines hypothèses sur l'organisation des répétitions liées d'ADNr 45S et 5S ont été formulées. Les analyses FISH au moyen des répétitions télomériques du type retrouvé chez Arabidopsis ont permis de démontrer que les télomères de l'A. cherimola sont constitués de séquences TTTAGGG et que ces séquences sont uniquement présentes aux extrémités des chromosomes. Un aperçu de la structure des chromosomes chez l'A. cherimola a été obtenu au moyen d'une approche auto-GISH, laquelle a révélé que des séquences très fortement répétées sont localisées au sein des régions centromériques de tous les chromosomes. La proche correspondance entre les signaux s-GISH et les bandes colorées au DAPI suggère que ces séquences constituent la principale composante de l'hétérochromatine chez cette espèce. Les auteurs proposent le caryotype de l'A. cherimola décrit dans ce travail comme caryotype de référence de base pour les futures investigations chez les Annonacées. [Traduit par la Rédaction] Mots-clés : Annona cherimola, caryotypage, FISH, GISH, télomères, gènes ribosomiques, ADN répété., Introduction The Annonaceae are a pantropical family of flowering trees, shrubs, and lianas, predominantly growing in tropical and subtropical ecosystems where they play an important ecological role in terms of [...]

Plantago is a large genus with a worldwide distribution characterized by three basic sets, x = 6 is the most common and is considered the ancestral set from which x = 5 and x = 4 derived following mechanisms of descending dysploidy. Advanced molecular cytogenetic methods are important for understanding evolutionary relationships but in Plantago their use has been rather limited. In this study the karyotypes of Plantago lanceolata L. (2n = 12), Plantago major L. (2n = 12) and Plantago coronopus L. (2n = 10) were characterized by using rDNA FISH, GISH, fluorochrome banding and chromosome morphology to obtain markers useful for evaluating their genomic affinity. The rDNA FISH mapping produced three distinct patterns but with evident similarities such as the chromosome position of 45S rDNA sites and the colocalization of 45S and 5S rDNA. Reciprocal GISH showed a low level of genome affinity between P. lanceolata and P. major and a total absence of homology between them and P. coronopus. This result supports the exclusion of the 2n = 12 species as possible ancestors of P. coronopus. This study confirms that a systematic use of in situ hybridization techniques, and in particular GISH, provide valid information for understanding the origin of species and can resolve many taxonomic and phylogenetic problematics. [ABSTRACT FROM AUTHOR]

Changes in chromosome number have played an important role in the evolution of the genus Trifolium. Along with a few species of polyploid origin there are several cases of dysploidy as evidenced by the presence of four basic chromosome numbers (x = 8,7,6,5). Trifolium subterraneum and Trifolium israeliticum are related species with chromosome complements 2n = 16 and 2n = 12, respectively. Although they represent an interesting case of speciation based on chromosome number reduction, no attempts to demonstrate their cytogenetic affinity have been carried out to date. With this study we performed a comparative cytogenetic study with the purpose of clarifying the evolutionary relationship between these species and to verify whether genomic rearrangements, other than modification of the chromosome number, are associated with the speciation process. Although karyomorphological analysis supports the hypothesis that chromosome rearrangements had a role in the reduction of the chromosome number, the physical mapping of the rDNA sequences revealed a significant remodelling of the 45S and 5S rDNA sites that greatly contributed to the differentiation of the 2n = 16 and 2n = 12 karyotypes. The nucleotide analysis of 5S rDNA repeats confirmed that the two species are related but constitute distinct entities. The observed genomic changes lead to the hypothesis that the 2n = 12 species is the result of an evolutionary pathway that passed through intermediate forms. It cannot be excluded that the most direct ancestor of T. israeliticum is a species with 2n = 14. Key words: Trifolium subterraneum, Trifolium israeliticum, karyotype, dysploidy, rDNA, FISH. Les changements survenus dans le nombre de chromosomes ont joue un grand role dans revolution au sein du genre Trifolium. En plus de quelques especes d'origine polyploide, il y a plusieurs cas de dysploidie puisque quatre nombres chromosomiques de base y sont rapportes (x = 8,7,6,5). Trifolium subterraneum et Trifolium israeliticum sont des especes apparentees ayant 2n = 16 et 2n = 12 chromosomes respectivement. Bien qu'ils constituent un cas interessant de speciation fondee sur une reduction du nombre de chromosomes, aucune etude visant a demontrer leur affinite cytogenetique n'a ete realisee a ce jour. Dans ce travail, les auteurs ont mene une etude cytogenetique comparee dans le but de clarifier les relations evolutionnaires entre ces especes et de verifier si des rearrangements genomiques, autres que la modification du nombre de chromosomes, seraient associes au processus de speciation. Bien que l'analyse caryomorphologique supporte l'hypothese que les rearrangements chromosomiques ont joue un role dans la reduction du nombre de chromosomes, la cartographie physique des ADNr a revele une modification importante des sites d'ADNr 45S et 5S, laquelle aurait contribue a la differenciation des caryotypes 2n = 16et 2n = 12. L'analyse des sequences repetees d'ADNr 5S a confirme que les deux especes sont apparentees mais constituent des entites distinctes. Les changements genomiques observes ont mene a l'hypothese que l'espece a 2n = 12 est le produit d'un parcours evolutionnaire ayant connu des formes intermediaires. Il n'est pas exclu que l'ancetre le plus immediat du T. israeliticum ait ete une espece a 2n = 14. [Traduit par la Redaction] Mots-cles: Trifolium subterraneum, Trifolium israeliticum, caryotype, dysploidie, ADNr, FISH., Introduction Trifolium L. is one of the most important genera of the family Fabaceae. It includes about 250 species some of which are of high agricultural value through being cultivated [...]

The rising interest in medicinal plants has brought several species of the genus Echinacea to the attention of many scientists. Echinacea angustifolia, E. pallida, and E. purpurea are the most important for their immunological properties, well known and widely used by the native Americans. The three species are easily distinguishable on the basis of their morphological characteristics, but it would be difficult, if not impossible, to distinguish them in commercial preparations of ground, dry plant parts of E. purpurea (the most valuable species for chemotherapeutic properties) mixed with the other two species. Species-specific molecular markers could be useful to address this issue. In the present work, using fresh material collected from cultivated Echinacea spp., AFLP analysis was used to discriminate the three species and to detect species-specific DNA fragments. By using 14 primer combinations it was possible to detect a total of 994 fragments, of which 565 were polymorphic. Overall, 89 fragments were unique to E. purpurea, 32 to E. angustifolia, and 26 to E. pallida. E+CAC/M+AAT or E+CAC/M+AGC alone provided 13, 9, and 4 or 7, 5, and 5 specific fragments for E. purpurea, E. angustifolia, and E. pallida, respectively. A validation trial to confirm the results was carried out on bulked samples of 23 accessions covering most of the genetic diversity of the three species. The results are discussed in terms of practical applications in the field of popular medicine, detecting frauds, and implications for the genus Echinacea. Key words: Echinacea pallida, E. purpurea, E. angustifolia, AFLP, species identification, commercial frauds. L'interet croissant pour les plantes medicinales a attire l'attention de plusieurs chercheurs sur le genre Echinacea. Les especes E. angustifolia, E. pallida et E. purpurea sont les plus importantes pour leurs proprietes immunologiques, bien connues et largement utilisees par les amerindiens. Les trois especes sont faciles a distinguer sur la base de leurs caracteristiques morphologiques, mais il serait difficile, voire impossible, de les distinguer au sein de preparations commerciales composees de parties seches broyees de FE. purpurea (la plus prisee des especes pour ses proprietes chimiotherapeutiques), melees avec celles provenant des deux autres especes. Des marqueurs moleculaires permettant de distinguer les especes seraient utiles dans ce cas. Dans le present travail, a partir de materiel frais d'echinacees cultivees, des marqueurs AFLP ont ete employes pour differencier les trois especes et pour detecter des amplicons specifiques des chacune. A l'aide de 14 combinaisons d'amorces, 994 amplicons ont ete detectes au total, dont 565 etaient polymorphes. Globalement, 89 amplicons etaient uniques a FE. purpurea, 32 a PE. angustifolia et 26 a FE. pallida. Les combinaisons E+CAC/M+AAT et E+CAC/M+AGC ont permis, a elles seules, de fournir respectivement 13, 9 et 4 ou 7, 5 et 5 marqueurs specifiques des especes E. purpurea, E. angustifolia et E. pallida. Un travail de validation pour confirmer ces resultats a ete mene sur des echantillons melanges de 23 accessions couvrant l'essentiel de la diversite genetique au sein de ces trois especes. Les resultats sont discutes en fonction des applications pratiques dans le domaine des medecines douces, de la detection des fraudes et des implications pour le genre Echinacea. Mots-cles : Echinacea pallida, E. purpurea, E. angustifolia, AFLP, identification des especes, fraudes commerciales., [Traduit par la Redaction] Introduction The genus Echinacea Moench (Compositae) is native to the prairies of North America, from which it spreads from southern Canada to Texas and Georgia, but [...]

Chromosome number variations play an important role in the genus Medicago. In addition to polyploidy there are cases of dysploidy as evidenced by two basic numbers, x = 8 and x = 7, the latter limited to five annual species having 2n = 14. Annuals are diploid with the exception of Medicago scutellata and Medicago rugosa which have 2n = 30 and are considered the result of crosses between the 2n = 16 and 2n = 14 species. However, this hypothesis has never been tested. This study was carried out to investigate the 2n = 14 and 2n = 30 karyotypes and verify the allopolyploid origin of M. scutellata and M. rugosa. Fluorescence in situ hybridization (FISH) of rDNA probes and genomic in situ hybridization (GISH) were performed. FISH showed that all five diploids with 2n = 14 have one pair of 45S and one pair of 5S rDNA sites. M. scutellata displayed four sites of 45S and four sites of 5S rDNA, while in M. rugosa only one pair of each of these sites was found. GISH did not produce signals useful to identify the presumed progenitors with 14 chromosomes. This result suggests alternative evolutionary pathways, such as the formation of tetraploids (2n = 32) and subsequent dysploidy events leading to the chromosome number reduction. [ABSTRACT FROM AUTHOR]

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To analyse cellular response to O3, the tolerant Arabidopsis thaliana genotype Col-0 was exposed to O3 fumigation (300 ppb) for 6 h and the modulation of gene expression during the treatment (3 h after the beginning of the treatment, T3 h) and the recovery phase (6 h from the end of the treatment, T12 h) assessed by gene chip microarray and real-time reverse transcriptase (RT)-PCR analyses. The Arabidopsis transcriptional profile is complex, as new genes (i.e. reticuline oxidase) and pathways, other than those already reported as O3-responsive, appear to be involved in the O3 response. The steady-state transcript levels of several WRKY genes were increased in O3-treated plants and the W-box was the cis-element over-represented in the promoter region of T3 h up-regulated genes. The fact that the W-box element was also over-represented in almost all T3 h-induced receptor-like kinases (RLKs) suggests a WRKY-mediated control of RLKs under O3 stress and a mechanicistic similarity with the pathogen-induced transcriptional responses. We investigated the molecular and physiological implications of our findings in relation to O3-induced plant stress response. [ABSTRACT FROM AUTHOR]

Cytogenetic research in Medicago sativa subsp. sativa L., the cultivated tetraploid alfalfa (2n = 4x = 32), has lagged behind other crops mostly due to the small size and the uniform morphology of its chromosomes. However, in the last decades, the development of molecular cytogenetic techniques based on in situ hybridization has largely contributed to overcoming these limitations. The purpose of this study was to extend our knowledge about the chromosome structure of alfalfa by using a combination of genomic in situ hybridization (GISH) and fluorescence in situ hybridization (FISH) techniques. The results of self-GISH (sGISH) suggested that a substantial part of the repetitive fraction of the genome of subsp. sativa is constituted by tandem repeats typical of satellite DNA. The coincidence of sGISH and C-banding patterns supported this assumption. The FISH mapping of the Arabidopsis-type TTTAGGG telomeric repeats demonstrated, for the first time, that the alfalfa telomeres consist of this type of sequence and revealed a massive presence of interstitial telomeric repeats (ITRs). In the light of this finding M. sativa appears to be a suitable material for studying the origin and function of such extra telomeric repeats. To further exploit this result, investigation will be extended to the diploid subspp. coerulea and falcata in order to explore possible connections between the distribution of ITRs, the ploidy level, and the evolutionary pathway of the taxa. [ABSTRACT FROM AUTHOR]