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- Main results from the EU-funded project "evaluation and utilization of pineapple genetic resources from the amazon to breed resistant varieties"Publication . D'Eeckenbrugge, G. Coppens; Cabral, J. R. S.; Matos, A. P. de; Carlier, Jorge; Leitão, José; Duval, M. F.; Noyer, J. L.; Ferreira, F. R.; Leal, F.; Maggioni, L.; Suarez, Z.This project was aimed at obtaining knowledge and tools for the development of cultivars resistant to the main diseases in South America, by (i) characterizing and evaluating available genetic resources, (ii) studying the genetic structure of the genera Ananas and Pseudanas (iii) developing and applying resistance screening techniques for important pests and disease in the region, (iv) studying the heredity of agronomic traits, and (v) testing the potential of partial inbreeds in breeding. In addition, a standard database is being established in collaboration with IPGRI, to promote information on germplasm exchange. The project, which started in 1997 and ended in 2001, has produced an inventory of the germplasm held in its collections, allowing exchange and repatriation of lost germplasm. The list of descriptors has been revised. Multivariate analyses of the morphological data have produced consistent results. Molecular characterization, based on RFLP of genomic DNA and PCR-RFLP of cpDNA has confirmed the proximity of Ananas and Pseudananas, the absence of interspecific barriers in Ananas, the importance of a geographic component of variation, and the existence of a higher genetic diversity in the North of South America. Pineapple taxonomy has been revised. Genetic mapping was based on isozyme, RAPD, AFLP and ISSR markers and was carried out on a hybrid progeny between A. bracteatus and A. comosus, following the pseudotestcross approach. It resulted in a first map of 390 and 174 DNA markers distributed in 46 and 31 linkage groups for these two species. New sources of resistance to fusariosis have been identified, some of which show other interesting traits for the Brazilian producers. According to the first inheritance studies, this resistance seems monogenic and dominant, a result still to be confirmed. Selfprogenies obtained from the cultivars Perolera, Primavera, and Roxo de Tefé, showed inbreeding depression, but normal segregation for major leaf traits (spininess, colour). Subsequent hybridization of the inbreeds restored vigour.
- Molecular characterization of Phaseolus vulgaris L. genotypes included in Bulgarian collection by ISSR and AFLP (TM) analysesPublication . Svetleva, D.; Pereira, G.; Carlier, Jorge; Cabrita, L.; Leitão, J. M.; Genchev, D.Seventy-eight (33 Bulgarian and 45 foreign) common bean (Phaseolus vulgaris L.) genotypes included in Bulgarian collection were screened for ISSR (Inter Simple Sequence Repeats) and AFLP (TM) (Amplified Fragment Length Polymorphism) markers. ISSR analysis was performed with 13 primers, and 55 (36.7%) out of the 150 bands observed were polymorphic. One hundred and sixty-four AFLP (TM) fragments were obtained with three primer combinations, of which 54 (32.9%) were polymorphic. UPGMA (Unweighted Pair Group Method Arithmetic Averages) analysis was performed using DICE coefficient and dendrograms were constructed using either the data from each method (ISSR and AFLP (TM)) separately or combined in a single joint matrix. Despite some genetic heterogeneity observed in both pools (Middle American origin: M and Andean origin: A) the genotypes were separated in to main groups: one gathering genotypes mainly from pool M and the other more genotypes from pool A. Moreover, Bulgarian genotypes were spread over the two groups suggesting that they are not subject to genetic erosion and that the Phaseolus genetic diversity is conserved. (c) 2006 Elsevier B.V. All rights reserved.
- Population genetic structure of Cistus ladanifer L. (Cistaceae) and genetic differentiation from co-occurring Cistus speciesPublication . Carlier, Jorge; Leitão, J. M.; Fonseca, FilomenaAmplified fragment length polymorphism and inter simple sequence repeat markers were used to assess the genetic structure of Cistus ladanifer and its genetic differentiation from co-occurring Cistus species. Clear genetic differentiation was found between two populations corresponding to the morphological description of Cistus palhinhae and the 23 Cistus ladanifer populations analyzed. However, the magnitude of differentiation (DICE coefficients > 0.98) appears to be more in agreement with the taxonomic designation of Cistus ladanifer ssp. sulcatus, which has been proposed by some authors, and not C. palhinhae. Dendrogram analysis of the genetic relationships among Cistus ladanifer, Cistus populifolius, Cistus monspeliensis, Cistus crispus, Cistus libanotis, Cistus salvifolius and Cistus albidus, with Halimium halimifolium as an outgroup, showed two main clusters. Taxa composition of these clusters concurs with the taxonomic classification proposed for Iberian species on the basis of flower color. One cluster comprised species with pink flowers (C. crispus and C. albidus, P = 81.3%) classified into the subgenus Cistus, and the other cluster contained the remaining five species (P = 77.9%) that have white flowers and are classified into two subgenera, Leucocistus (C. ladanifer, C. populifolius, C. salvifolius and C. monspeliensis) and Halimioides (C. libanotis). Within the subgenera Leucocistus, C. ladanifer was found to be distinct and can be separated into the section Ladanium, while the remaining species that are grouped together in a branch (P = 74.8%) can be separated into the section Ledonia.
- Molecular markers in genetic variability assessment, cultivar fingerprinting and hybrid identification in fruit and vegetablesPublication . Leitão, José; Elisiario, P. J.; Cabrita, L. F.; Justo, E. M.; Pereira, G. M.; Carlier, Jorge; Farinho, M. J.Isozyme, RADP and AFLP markers were used in the UCTA, University of Algarve, for several studies in fruit tree and vegetable species. In Citrus, isozyme analysis was used for assessing genetic variability of traditional cultivars, for distinguishing nucellars from zigotics scion and rootstock cultivars, for parentage determination of hybrid plants and to identify mistakes occurring in commercial nurseries. In figs (Ficus carica L.), isozyme and RAPD analyses allowed the detection of errors in a field collection, and jointly with AFLP analysis were used to discriminate and fingerprint germplasm accessions. In almond (Prunus dulcis Webb) isozyme, RAPD and AFLP markers were utilised to discriminate among cultivars. Recently, several programs aiming to identify molecular markers linked to resistance genes and to construct genomic maps in pineapple, Brassica sp., peas and chickpeas, were initiated in our laboratory.
- Mapping of a locus for adult plant resistance to downy mildew in broccoli (Brassica oleracea convar. italica)Publication . Farinho, M.; Coelho, P.; Carlier, Jorge; Svetleva, D.; Monteiro, A.; Leitão, J. M.The identification of the gene Pp523, conferring downy mildew resistance to adult plants of broccoli (Brassica oleracea convar. italica), led to the construction of a genetic map that included this resistance locus, 301 amplified fragment length polymorphisms, 55 random amplified polymorphic DNAs, 46 inter-simple sequence repeats, three simple sequence repeats, four other PCR markers and a flower colour locus, all gathered into nine major linkage groups. Nineteen additional molecular markers were clustered into one group of four markers, one group of three markers and six pairs of markers. The map spans over 731.9 cM, corresponding to 89.5% of the 818 cM estimated to be the total genome length. A significant number of the mapped markers, 19.3%, showed distorted segregation. The average distance between mapped adjacent markers is 1.64 cM, which places this map among the densest published to date for this species. Using bulked segregant analysis, we identified a group of molecular markers flanking and closely linked in coupling to the resistance gene and included these in the map. Two markers linked in coupling, OPK17_980 and AT.CTA_133/134, are located at 3.1 cM and 3.6 cM, respectively, at each side from the resistance gene. These markers can be used for marker-assisted selection in breeding programs aiming at the introgression of this gene in susceptible B. oleracea genotypes. The fine mapping of the genomic region surrounding the Pp523 resistance gene is currently being carried out, a basic condition for its isolation via positional cloning.
- Genetic mapping of DNA markers in pineapplePublication . Carlier, Jorge; Nancheva, D.; Leitão, José; d'Eeckenbrugge, G. C.Two genetic maps of DNA-markers (RAPD, AFLP and ISSR) of Ananas comosus var. bracteatus and A. comosus var. comosus have been previously published by our team. These maps were constructed at the Laboratory of Genetics and Plant Breeding of FERN, University of Algarve, using an F1 mapping population derived from a cross performed in Martinique between the two botanical varieties. However, the use of a F1 population, the small size of this population and the use of molecular markers with dominant inheritance allowed only markers that were heterozygous in the parents to be included in those maps and prevented the construction of an integrated high-density map. A new genetic map, intended to cover all the pineapple genome, is currently under construction using a mapping population of 142 F2 plants. This new map will integrate the already published maps and will include markers that had remained unlinked, as they were polymorphic but homozygous in parental genotypes, as well as newly identified markers. At the present time, the new map consists of seven linkage groups (> 40 cM) that integrate markers from both parents, 21 groups (> 40 cM) with markers mostly from one parent, six groups (< 40 cM and > 25 cm) with markers from one parent, and 12 smaller (< 25 cM) linkage groups, covering approximately 62% of the pineapple genome.
- Genetic maps of RAPD, AFLP and ISSR markers in Ananas bracteatus and A-comosus using the pseudo-testcross strategyPublication . Carlier, Jorge; Reis, A.; Duval, M. F.; d'Eeckenbrugge, G. C.; Leitão, J. M.Genetic maps of random amplified polymorphic DNA (RAPD), amplified fragment length polymorphisms (AFLP) and inter simple sequence repeats (ISSR) markers in pineapple (2n = 2x = 50) are reported for the first time. On the basis of a segregating population of 46 F1 individuals from a cross Ananas comosus x A. bracteatus, genetic maps of these two species were constructed using the two-way pseudo-testcross approach. The A. bracteatus map consists of 335 markers (60 RAPDs, 264 AFLPs and 11 ISSRs) assembled into 50 linkage groups, 26 of them with at least four markers. The A. comosus map consists of 157 markers (33 RAPDs, 115 AFLPs, eight ISSRs and the 'piping' trait locus) organized into 30 linkage groups, 18 of them with at least four markers. These maps cover, respectively, 57.2% of the A. bracteatus genome estimated as 3693 cM long, and 31.6% of the A. comosus genome calculated as 4146 cM. A rough estimate of 120 and 127 kbp/cM on average was found for the relationship between physical and genetic distance for A. bracteatus and A. comosus, respectively.
- Mapeamento genético do ananaseiro (Ananas comosus (L.) Merrilll)Publication . Carlier, Jorge Daniel Dias; Leitão, José Manuel Peixoto TeixeiraThe main aim of this thesis was the construction of a Pineapple (Ananas comosus (L.) Merrill) genetic reference map. Based on an (A. comosus var. comosus X Ananas comosus var. bracteatus) F1 population, two genetic maps, one for each progenitor, were assembled. Subsequently, a new map integrating markers from both progenitors was constructed on the basis of an F2 population. Using markers segregating simultaneously among the F1 and F2 populations, an integrated map was constructed gathering 783 molecular markers (588 AFLP, 137 RAPD, 57 ISSR, and the PGM isozyme locus) and the morphological trait locus “piping” into 77 linkage groups. In view of the facts that (1) in the integrated map, 31 linkage groups (a number close to the 25 pineapple chromosomes) are longer than 50cM; (2) that the different maps span over 60.7% to 91.4% of the genome, and; (3) that marker density is moderately high, with 4.16 cM average distance between markers, it is legitimate to suppose that these first genetic maps of pineapple will be very useful for construction of physical maps, for genome sequencing and for gene isolation via map-based cloning in this crop species.
- Identification of RAPD markers linked to the Fin and P loci in Phaseolus vulgaris L.Publication . Apostolova, E.; Svetleva, D.; Carlier, Jorge; Leitão, JoséBulked segregant analysis was used to identify random amplified polymorphic DNA RAPD markers linked to the genes for indeterminate growth habit (Fin) and seed color (P) in common bean (Phaseolus vulgaris L.) using an F2 population derived from the cross A 195 × Kristal 137. The variety A 195 is characterized by determinate growth habit and brown colored seeds, while Kristal 137 exhibits indeterminate growth habit and white colored seeds. Among the 140 RAPD primers used to discriminate between two parental genotypes and between two bulks of DNA, respectively, from determinate and indeterminate plants, only 64 (45.71%) amplified polymorphic markers. Nineteen primers amplified clear polymorphic bands and were further tested among 100 F 2 plants. The analysis of the genetic linkage between analyzed molecular markers and the loci for indeterminate growth habit and seed color was performed using the program Join Map 3.0. The logarithm of odds (LOD) of 8.0 and 10 were used as thresholds for linkage grouping, respectively of locus Fin and locus P. One RAPD marker, AA18-600-P1, was found linked to the Fin locus that determines plant habit type and mapped at 17.6 cM apart from it (rec. freq. = 0.162). Five RAPD markers were found linked to the P locus, with distances to this locus varying from 4.1 cM (marker AB18 1500-P2, rec. freq.= 0.034) to 42.6 cM (marker Q03700-P1).