Molecular analysis of evolutionary relationships of Phaseolus dumosus with the gene pool of common bean

Valuable genetic variation for bean breeding programs is held within the common bean secondary gene pool which consists of Phaseolus albescens, P. coccineus, P. costaricensis, and P. dumosus. However, the use of close relatives for bean improvement is limited due to the lack of knowledge about genetic variation and genetic plasticity of many of these species. Characterisation and analysis of the genetic diversity is necessary among beans' wild relatives; in addition, conflicting phylogenies and relationships need to be understood and a hypothesis of a hybrid origin of P. dumosus needs to be tested. This thesis research was orientated to generate information about the patterns of relationships among the common bean secondary gene pool, with particular focus on the species Phaseolus dumosus. This species displays a set of characteristics of agronomic interest, not only for the direct improvement of common bean but also as a source of valuable genes for adaptation to climate change. Here I undertake the first comprehensive study of the genetic diversity of P. dumosus as ascertained from both nuclear and chloroplast genome markers. A germplasm collection of the ancestral forms of P. dumosus together with wild, landrace and cultivar representatives of all other species of the common bean secondary gene pool, were used to analyse genetic diversity, phylogenetic relationships and structure of P. dumosus. Data on molecular variation was generated from sequences of cpDNA loci accD-psaI spacer, trnT-trnL spacer, trnL intron and rps14-psaB spacer and from the nrDNA the ITS region. A whole genome DArT array was developed and used for the genotyping of P. dumosus and its closes relatives. 4208 polymorphic markers were generated in the DArT array and from those, 742 markers presented a call rate >95% and zero discordance. DArT markers revealed a moderate genetic polymorphism among P. dumosus samples (13% of polymorphic loci), while P. coccineus presented the highest level of polymorphism (88% of polymorphic loci). At the cpDNA one ancestral haplotype was detected among all samples of all species in the secondary genepool. The ITS region of P. dumosus revealed high homogeneity and polymorphism bias to P. coccineus genome. Phylogenetic reconstructions made with Maximum likelihood and Bayesian methods confirmed previously reported discrepancies among the nuclear and chloroplast genomes of P. dumosus. The outline of relationships by hybridization networks displayed a considerable number of interactions within and between species. This research provides compelling evidence that P. dumosus arose from hybridisation between P. vulgaris and P. coccineus and confirms that P. costaricensis has likely been involved in the genesis or backcrossing events (or both) in the history of P. dumosus. The classification of the specie P. persistentus was analysed based on cpDNA and ITS sequences, the results found this species to be highly related to P. vulgaris but not too similar to P. leptostachyus as previously proposed. This research demonstrates that wild types of the secondary genepool carry a significant genetic variation which makes this a valuable genetic resource for common bean improvement. The DArT array generated in this research is a valuable resource for breeding programs since it has the potential to be used in several approaches including genotyping, discovery of novel traits, mapping and marker-trait associations. Efforts should be made to search for potential populations of P. persistentus and to increase the collection of new populations of P. dumosus, P. albescens and P. costaricensis that may provide valuable traits for introgression into common bean and other Phaseolus crops.

Proteomic and phosphoproteomic analysis of polyethylene glycol-induced osmotic stress in root tips of common bean (Phaseolus vulgaris L.)

Previous studies have shown that polyethylene glycol (PEG)-induced osmotic stress (OS) reduces cell-wall (CW) porosity and limits aluminium (Al) uptake by root tips of common bean (Phaseolus vulgaris L.). A subsequent transcriptomic study suggested that genes related to CW processes are involved in adjustment to OS. In this study, a proteomic and phosphoproteomic approach was applied to identify OS-induced protein regulation to further improve our understanding of how OS affects Al accumulation. Analysis of total soluble proteins in root tips indicated that, in total, 22 proteins were differentially regulated by OS; these proteins were functionally categorized. Seventy-seven per- cent of the total expressed proteins were involved in metabolic pathways, particularly of carbohydrate and amino acid metabolism. An analysis of the apoplastic proteome revealed that OS reduced the level of five proteins and increased that of seven proteins. Investigation of the total soluble phosphoproteome suggested that dehydrin responded to OS with an enhanced phosphorylation state without a change in abundance. A cellular immunolocalization analysis indicated that dehydrin was localized mainly in the CW. This suggests that dehydrin may play a major protective role in the OS-induced physical breakdown of the CW structure and thus maintenance of the reversibility of CW extensibility during recovery from OS. The proteomic and phosphoproteomic analyses provided novel insights into the complex mechanisms of OS-induced reduction of Al accumulation in the root tips of common bean and highlight a key role for modification of CW structure.

Physiological and molecular analysis of the interaction between aluminium toxicity and drought stress in common bean (Phaseolus vulgaris)

Aluminium (Al) toxicity and drought are two major factors limiting common bean (Phaseolus vulgaris) production in the tropics. Short-term effects of Al toxicity and drought stress on root growth in acid, Al-toxic soil were studied, with special emphasis on Al-drought interaction in the root apex. Root elongation was inhibited by both Al and drought. Combined stresses resulted in a more severe inhibition of root elongation than either stress alone. This result was different from the alleviation of Al toxicity by osmotic stress (-0.60 MPa polyethylene glycol) in hydroponics. However, drought reduced the impact of Al on the root tip, as indicated by the reduction of Al-induced callose formation and MATE expression. Combined Al and drought stress enhanced up-regulation of ACCO expression and synthesis of zeatin riboside, reduced drought-enhanced abscisic acid (ABA) concentration, and expression of NCED involved in ABA biosynthesis and the transcription factors bZIP and MYB, thus affecting the regulation of ABA-dependent genes (SUS, PvLEA18, KS-DHN, and LTP) in root tips. The results provide circumstantial evidence that in soil, drought alleviates Al injury, but Al renders the root apex more drought-sensitive, particularly by impacting the gene regulatory network involved in ABA signal transduction and cross-talk with other phytohormones necessary for maintaining root growth under drought.

Alteration of cell-wall porosity is involved in osmotic stress-induced enhancement of aluminium resistance in common bean (Phaseolus vulgaris L.)

Aluminium (Al) toxicity and drought are the two major abiotic stress factors limiting common bean production in the tropics. Using hydroponics, the short-term effects of combined Al toxicity and drought stress on root growth and Al uptake into the root apex were investigated. In the presence of Al stress, PEG 6000 (polyethylene glycol)-induced osmotic (drought) stress led to the amelioration of Al-induced inhibition of root elongation in the Al-sensitive genotype VAX 1. PEG 6000 (>> PEG 1000) treatment greatly decreased Al accumulation in the 1 cm root apices even when the roots were physically separated from the PEG solution using dialysis membrane tubes. Upon removal of PEG from the treatment solution, the root tips recovered from osmotic stress and the Al accumulation capacity was quickly restored. The PEG-induced reduction of Al accumulation was not due to a lower phytotoxic Al concentration in the treatment solution, reduced negativity of the root apoplast, or to enhanced citrate exudation. Also cell-wall (CW) material isolated from PEG-treated roots showed a low Al-binding capacity which, however, was restored after destroying the physical structure of the CW. The comparison of the Al(3+), La(3+), Sr(2+), and Rb(+) binding capacity of the intact root tips and the isolated CW revealed the specificity of the PEG 6000 effect for Al. This could be due to the higher hydrated ionic radius of Al(3+) compared with other cations (Al(3+) >> La(3+) > Sr(2+) > Rb(+)). In conclusion, the results provide circumstantial evidence that the osmotic stress-inhibited Al accumulation in root apices and thus reduced Al-induced inhibition of root elongation in the Al-sensitive genotype VAX 1 is related to the alteration of CW porosity resulting from PEG 6000-induced dehydration of the root apoplast.

Physical and chemical properties of starch and flour from different common bean (Phaseolus vulgaris L.) cultivar.

The physical, chemical and pasting properties of the flour and isolated starches from six different bean cultivars (Phaseolus vulgaris L.) were investigated in order to obtain information for application in new products. The protein and total starch contents of the bean flours ranged from 17.72 to 20.27% and from 39.68 to 43.78%, respectively. The bean starches had low amounts of proteins, lipids and ash and showed an amylose content ranging between 45.32 and 51.11% and absolute density values between 1.55 and 1.78 g.cm?3. The bean starch granules were round to oval with a smooth surface. Results viscoamylographic profiles of the starches and flours showed the possibility of selecting cultivars for specific applications according to these characteristics.

Drought impact on rainfed common bean production areas in Brazil.

Common bean production in Goiás, Brazil is concentrated in the same geographic area, but spread acrossthree distinct growing seasons, namely, wet, dry and winter. In the wet and dry seasons, common beansare grown under rainfed conditions, whereas the winter sowing is fully irrigated. The conventional breed-ing program performs all varietal selection stages solely in the winter season, with rainfed environmentsbeing incorporated in the breeding scheme only through the multi environment trials (METs) wherebasically only yield is recorded. As yield is the result of many interacting processes, it is challengingto determine the events (abiotic or biotic) associated with yield reduction in the rainfed environments(wet and dry seasons). To improve our understanding of rainfed dry bean production so as to produceinformation that can assist breeders in their efforts to develop stress-tolerant, high-yielding germplasm,we characterized environments by integrating weather, soil, crop and management factors using cropsimulation models. Crop simulations based on two commonly grown cultivars (Pérola and BRS Radi-ante) and statistical analyses of simulated yield suggest that both rainfed seasons, wet and dry, can bedivided in two groups of environments: highly favorable environment and favorable environment. Forthe wet and dry seasons, the highly favorable environment represents 44% and 58% of production area,respectively. Across all rainfed environment groups, terminal and/or reproductive drought stress occursin roughly one fourth of the seasons (23.9% for Pérola and 24.7% for Radiante), with drought being mostlimiting in the favorable environment group in the dry TPE. Based on our results, we argue that eventhough drought-tailoring might not be warranted, the common bean breeding program should adapttheir selection practices to the range of stresses occurring in the rainfed TPEs to select genotypes moresuitable for these environments.

Major loci controlling resistance to the angular leaf spot of common bean.

Based on the evidences presented in this paper, results from classical genetic studies, fine-mapping information and physical position analysis using the reference genome sequence of P. vulgaris, the BIC Genetic Committee has formally accepted the proposed new gene symbols.

Resistance to Fusarium wilt in common bean cultivars and lines in pre-commercial stage.

The objective of this study was to identify common bean cultivars with resistance to Fusarium wilt.

Phenotypic and molecular characterization of common bean genotypes for resistance to anthracnose.

This study aimed to perform phenotypic and molecular characterization of cultivars and breeding lines of common bean for resistance to anthracnose.

Cooking time of common bean: genetic parameters and selection of elite lines.

The objectives of this study were to investigate the genetic variability and select elite lines for CT, since these lines aggregate essential agronomic traits.

Progeny tests for common bean isolines resistant to diseases with the aid molecular markers.

The aim of this study was progeny tests in segregating populations for resistance genes to these three diseases.

Pyramided lines of "carioca" common bean and their reaction to Pseudocercospora griseola.

The present work aimed to characterize lines produced by the Breeding Program of Common Bean (PMGF) of the Federal University of Viçosa (UFV), called ?Ruda R3? and ?Pérola R1?, in reaction to different races of P. griseola.

Marker-assisted selection integrated to the embrapa common bean breeding program.

The establishment of a specific Marker-Assisted Selection Facility at the Embrapa Rice and Beans Biotechnology Laboratory, in 2014, has better supported the routine analysis with molecular markers demanded by the Embrapa Common Bean Breeding Program. In addition, it has also supported other Embrapa plant breeding programs, such as rice and cotton.

Productivity, adaptability and stability of production of special grain common bean lines in different environments of Minas Gerais, Brazil.

This study aimed to select special grain bean lines with high productivity, adaptability and stability of production, evaluated in different environments of the Minas Gerais State, Brazil.