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- Phytogeographical origin of Madeiran common beans based on phaseolin patternsPublication . Silva, Emanuel Marques da; Correia, Anísia Soraia Abreu; Lopes, Nuno Alexandre Amaral; Nóbrega, Humberto Gil Moreira; Ganança, José Filipe Teixeira; Domingues, Ana Maria; Khadem, Manhaz; Slaski, Jan Jacek; Carvalho, Miguel Ângelo Almeida Pinheiro deThe objective of this work was to determine the geographic origin of the Madeiran common bean (Phaseolus vulgaris) gene pool. Phaseolin patterns of 50 accessions representing the diversity of common bean collected in Madeira, Portugal, and conserved in the ISOPlexis Germplasm Bank, were analysed using the Experion automated electrophoresis system, based on lab-on-a-chip technology. Five common bean standard varieties with typical phaseolin patterns were used to determine the phytogeographical origin of the Madeiran common bean accessions. Ninety two percent of the accessions exhibited a phaseolin pattern consistent with the one of common bean types belonging to the Andean gene pool, while the origin of the remaining 8% of the accessions was indistinguishable. The application of a similarity coefficient of 85%, based on Pearson correlations, increases the number of accessions with uncertain pattern. The analytical approach used permitted the determination of the origin of the common bean gene pool, which is Andean in 98% of the cases, and clustering of the observed variability among the Madeiran common beans.
- Viral diagnosis in cultivars of Ipomoea batatas (L.) LamPublication . Andrade, Carlos G.; Silva, Emanuel M. da; Ragonezi, Carla; Carvalho, Miguel Â. A. Pinheiro deIpomoea batatas (L.) Lam. commonly known as sweet potato, is an important staple food worldwide, mainly due to its high nutritional value and yield. However, vegetative reproduction of sweet potato makes it more susceptible to viral infections, which threatens its productivity, quality, and difficult long-term preservation in germplasm banks. Also, it can act as a virus reservoir infecting the rest of the plant accessions in the bank collections. Hence, this work aimed to screen Begomovirus, Potyvirus, and Carlavirus infections in 16 traditional sweet potato cultivars from the germplasm collection of the ISOPlexis Germplasm Bank, Madeira, Portugal. The infection prevalence by these viruses among cultivars was 81.25%, 25.00%, and 6.25%, respectively; being ISOP1011 the only accession coinfected by Potyvirus and Carlavirus. The accessions ISOP1006, ISOP1010, and ISOP1047 were also coinfected by Begomovirus and Potyvirus, highlighting their vulnerability to viral infections. The ISOP1005 and ISOP1027 accessions were the only ones not infected by any of these viruses. The analysis of the partial sequence obtained from the Carlavirus detected in the accession ISOP1011, revealed the existence of an ORF that encodes for 93 amino acids of the catalytic domain of an RNA-directed RNA polymerase related to the Tymovirus protein family, as could be confirmed by comparison with proteins stored in UniProtKB. Multiple sequence alignment with these proteins showed that Motifs A and B of the catalytic domain were conserved. The search for sequence similarity with sequences deposited in GenBank reported a high sequence identity with Sweet potato yellow mottle virus (SPYMV) and Sweet potato chlorotic fleck virus (SPCFV). However, the 9-11% discrepancy in nucleotide sequence identity and a phylogenetic analysis carried out using the maximum probability method suggests the virus isolated from ISOP1011 is a new divergent strain of the SPCFV species.
- Phenotyping the ideotypes of two maize landraces from Madeira archipelago using morpho-agronomic traits and zein patternPublication . Carvalho, Miguel Â. A. Pinheiro de; Sardinha, Carmen M. S.; Gouveia, Carla S.S.; Silva, Emanuel M. da; Nóbrega, Humberto G. M.; Ganança, José F. T.; Nóbrega, Sónia R.; Santos, Teresa M. M. dosMadeira Archipelago holds specific maize genetic resources whose populations, representing field diversity, were previously classified into four main landrace groups. The ISOPs 0061 and 0070, with common names of “Milho Sequeiro” and “Milho Branco” yellow and white maize, respectively, represent the ideotypes of two of these landraces. These ideotypes have now been analyzed using morpho-agronomic and biochemical traits, to phenotype these landraces. The variation of nine of the ten morpho-agronomic traits was used to separate the landraces ideotypes. However, the seven traits of seed biochemical composition did not allow to segregate the landraces ideotypes. This is not the case of zein pattern, which polymorphism shows to be specific for each ideotype. The original ideotypes populations were regenerated twice under low input conditions. Zein patterns of the original and regenerated accessions were compared, using two electrophoresis techniques. The lab-on-a-chip electrophoresis showed that the standard SDS-PAGE zein pattern with six fractions could be separated into eighteen (ISOP 0061) and twenty (ISOP 0070) protein bands. In contrast, no significant changes were detected in the zein pattern structure of the initial and regenerated accessions of both landraces ideotypes. The chip electrophoresis showed to be a suitable technique to screen and characterize a large number of individuals and accessions of the germplasm collections, due to its reproducibility. In particular, the zein patterns can be used to phenotype ideotypes and establish a formula representing their structure, allowing to detect changes in landrace structure, occurring as a result of gene bank management actions.
- Molecular identification and VOMs characterization of Saccharomyces cerevisiae strains isolated from Madeira region winery environmentsPublication . Castillo, Mariangie; Silva, Emanuel da; Câmara, José S.; Khadem, MahnazThe quality and typical characteristic of wines depends, among other factors, on the volatile organic metabolites (VOMs) that are biosynthesized by yeasts, mainly Saccharomyces cerevisiae species. The yeast strain influences the diversity and proportions of the VOMs produced during the fermentation process, as the genetic predisposition of the strains is a by-product of selective adaptation to the ecosystem. The present work reports the characterization of S. cerevisiae strains isolated from grape must, used in the Demarcated Region of Madeira (DRM) for winemaking. Yeast species were identified by amplification and by restriction fragment length polymorphism (RFLP) analysis of the region 5.8S-internal transcribed spacers (PCR-RFLP of 5.8S-ITS) of ribosomal DNA (rDNA). The strains identification was performed by analyzing the RFLP pattern of mitochondrial DNA (RFLP-mtDNA). The representative strains were selected for the characterization of the volatile profile through headspace solid-phase microextraction (HS-SPME) followed by gas chromatography-mass spectrometry (GC-MS) analysis. A total of 77 VOMs were identified. Higher alcohols, esters, and fatty acids were the major chemical families representing 63%, 16%, and 9%, respectively, in strain A and 54%, 23%, and 15% in strain B. The results indicate the influence of the strain metabolism in the production of VOMs, many of which probably participate in the aroma of the corresponding wines.
- Genetic variability of high molecular weight glutenin subunits in bread wheat from continental Portugal, Madeira and Canary IslandsPublication . Santos, Teresa M. M. dos; Nóbrega, Humberto; Ganança, José Filipe Teixeira; Silva, Emanuel; Afonso, Desire; Gutiérres, Abílio F. M.; Slaski, Jan J.; Khadem, Manhaz; Carvalho, Miguel Â. A. Pinheiro deThe genetic variability of high molecular weight glutenin subunits (HMWGS) composition at the Glu-1 loci in bread wheat (Triticum aestivum L.) was studied electrophoretically using the SDS–PAGE in 3,470 individuals representing 159 populations originated from the Canary Islands (Spain), the Archipelago of Madeira (Portugal) and the continental Portugal. A total of 25 alleles were detected, resulting in 69 different allele combinations. The geographical distribution of the high molecular weight glutenin alleles confirms historical data regarding circulation of wheat germplasm between the Iberian Peninsula and Madeira and between Madeira and the Canary Islands and vice versa.
- Aluminium tolerance in bean traditional cultivars from MadeiraPublication . Domingues, Ana Maria; Silva, Emanuel da; Freitas, Gregório; Ganança, José Filipe; Nóbrega, Humberto; Slaski, Jan J.; Carvalho, Miguel Ângelo Pinheiro deCommon bean (Phaseolus vulgaris L.) is the most important legume crop in the world, providing low-cost, high quality protein, minerals and dietary fiber for human nutrition. The crop was originated from diversity centers in America and exhibits adaptation abilities to different environmental conditions, including soil with low pH. Acid soils occupy 30% of the agro ecosystem areas in the world. In Madeira, acid Andosols and unsatured Cambisols are the dominant groups of soils. Generally, under acidic and infertile conditions, besides of H+ toxicity, soluble aluminium (Al) is the most important abiotic factor limiting plant development and crop productivity. In the field, the hidden roots are also affected and the reduction of root growth under Al stress can be clearly observed in early stages. Seedlings of fifty bean accessions from the Archipelago of Madeira were tested under controlled conditions in the presence of 50 mM Al at pH 4.4. In general, the tested germplasm appeared to be sensitive or very sensitive to Al toxicity. However, fifteen traditional cultivars clearly exhibited elevated Al-tolerance, with an average root relative elongation (RRE) exceeding 50%, while top six accessions surpassed the 60% RRE mark. The Madeira bean germplasm is a valuable resource for sustainable crop production in acid soils and it could be used as parental lines in breeding programs aimed for Al tolerance in common beans.
- Microchip electrophoretic analysis of Phaseolin patterns and Its comparison with currently used SDS-PAGE techniquesPublication . Silva, Emanuel Marques da; Santos, Teresa Maria Marques dos; Ganança, José Filipe Teixeira; Slaski, Jan Jacek; Carvalho, Miguel Â. A. Pinheiro deThe goal of this work was to compare reproducibility of phaseolin patterns of common bean obtained by two electrophoretic protein separation techniques including the conventional SDS-PAGE and an automated chip electrophoresis system. Five standard cultivars of common bean provided by the United States Department of Agriculture (Beltsville, Maryland) that represented five phaseolin types, T (Tendergreen), C (Contender) and S (Sanilac), B (Boyaca) and P (Pampa), were used in this study. Comparison of the phaseolin patterns revealed that the chip-on-a-lab electrophoresis provided a good reproducibility. The phaseolin polymorphism included four to seven polypeptides typical for the pattern composition of the T, C and S types. The polymorphism of the B and P patterns was also established. Phaseolin polypeptides separated by the microchip electrophoresis exhibited differences with respect to the molecular weights and electrophoretic mobility as compared to the SDS-PAGE technique. This phenomenon could be attributed to the absence of a solid separation phase in the microchip electrophoresis. Moreover, this technique has potential to substantially accelerate screening of large bean germplasm collections since it allows for the accurate analysis of the higher number of individual plants within accessions than the conventional, tedious and time consuming SDS-PAGE method.
- Evidences of organic acids exudation in aluminium stress responses of two Madeiran wheat (Triticum aestivum L.) landracesPublication . Rodrigues, Marta; Ganança, José F.T.; Silva, Emanuel M. da; Santos, Teresa M. M. dos; Slaski, Jan J.; Zimny, Janusz; Carvalho, Miguel Â. A. Pinheiro deTwo wheat (Triticum aestivum L.) Madeiran landraces were subjected to 100 μM and 200 μM of aluminium (Al) in hydroponic culture, assessing the organic acid exudation role in plant’s responses to this metal. Samples of initial landrace populations (F0), F3 and haplodiploid lines (DH) were evaluated using standard tests: eriochrome cyanine R staining, root elongation and callose accumulation in roots. Root exudates were obtained to determine if the accumulation of malic and citric acids in hydroponic medium was a response to Al exposure. Additionally, the presence of ALMT1 gene was determined using five microsatellite markers. Standard tests confirmed that ISOP 76 was Al tolerant and ISOP 239, Al susceptible. ISOP 76, in the presence of 100 μM Al, exuded substantially more malic acid (12.87 to 43.33 mg/L), than ISOP 239 (3.65 to 7.72 mg/L). The levels of both organic acid exudation were substantially lower in ISOP 239 than in the ISOP 76. In the presence of 200 μM Al, ISOP 76 F0 shows a higher root elongation ratio (better tolerates Al), but the DH line was the one that exuded higher content of malic acid. Different gene alleles and promoters were detected in both landraces. Molecular differences could explain the observed dissimilarity in organic acid exudation response to Al stress.