Sérgio Augusto Morais Carbonell

Development, characterization, and comparative analysis of polymorphism at common bean SSR loci isolated from genic and genomic sources

Microsatellites or SSRs (single sequence repeats) have been used to construct and integrate genetic maps in crop species, including Phaseolus vulgaris. In the present study, 3 cDNA libraries generated by the Bean EST project (http://lgm.esalq.usp.br/BEST/), comprising a unigene collection of 3126 sequences and a genomic microsatellite-enriched library, were analyzed for the presence of SSRs. A total of 219 expressed sequence tags (ESTs) were found to carry 240 SSRs (named EST-SSR), whereas 714 genomic sequences contained 471 SSRs (named genomic-SSR). A subset of 80 SSRs, 40 EST-SSRs, and 40 genomic-SSRs were evaluated for molecular polymorphism in 23 genotypes of cultivated beans from the Mesoamerican and Andean genetic pools, including Brazilian cultivars and 2 related species. Of the common bean genotypes, 31 EST-SSR loci were polymorphic, yielding 2-12 alleles as compared with 26 polymorphic genomic-SSRs, accounting for 2-7 alleles. Cluster analysis from data using both genic and genomic-SSR revealed a clear separation between Andean and Mesoamerican beans. The usefulness of these loci for distinguishing bean genotypes and genetic mapping is discussed.

Structure of genetic diversity among common bean ( Phaseolus vulgaris L.) varieties of Mesoamerican and Andean origins using new developed microsatellite markers

A common bean genomic library was constructed using the ‘IAC-UNA’ variety enriched for (CT) and (GT) for microsatellite motifs. From 1,209 sequenced clones, 714 showed microsatellites distributed over 471 simple and 243 compound motifs. GA/CT and GT/CA were the most frequent motifs found among these sequences. A total of 123 microsatellites has been characterized. Out of these, 87 were polymorphic (73.7%), 33 monomorphic (26.8%), and 3 (2.4%) did not amplify at all. In a sample of 20 common bean materials selected from the Agronomic Institute Germplasm Bank, the number of alleles per locus varied 2–9, with an average of 2.82. The polymorphic information content (PIC) of each marker varied from 0.05 to 0.83, with a 0.45 average value. Cluster and principal coordinate analysis of the microsatellite data were consistent with the original assignment of the germplasm accessions into the Andean and Mesoamerican gene pools of common bean. Low polymorphism levels detected could be associated with the domestication process. These microsatellites could be a valuable resource for the bean community because of their use as new markers for genetic studies.

Estabilidade de cultivares e linhagens de feijoeiro em diferentes ambientes no estado de São Paulo

The present study had the objective of a genotypic evaluation of common bean lines in the state of Sao Paulo. Genotypic values of the lines for each location and for the environment mean of all locations were predicted as well. Studies on the adaptability and stability of the predicted genotypic values were realized by the BLUP procedure. The cultivar Grauna and line GEN 96A31 presented an average superiority of 10% over the general mean of the 15 environments. These values were computed including the penalization of the lines for the instability across locations and simultaneously crediting the response capacity (adaptability) to improved environments, using intrinsic properties of the MHPRVG method (Harmonic mean of the relative performance of the genetic values). The methods MHPRVG, Lin & Binns and Annicchiarico selected the same lines. Moreover, the results provided by the method MHPRVG were in the same measurement scale of the evaluated trait and were directly interpreted as genetic values simultaneously, for yield, stability and adaptability.

Inheritance of growth habit detected by genetic linkage analysis using microsatellites in the common bean (Phaseolus vulgaris L.)

The genetic linkage map for the common bean (Phaseolus vulgaris L.) is a valuable tool for breeding programs. Breeders provide new cultivars that meet the requirements of farmers and consumers, such as seed color, seed size, maturity, and growth habit. A genetic study was conducted to examine the genetics behind certain qualitative traits. Growth habit is usually described as a recessive trait inherited by a single gene, and there is no consensus about the position of the locus. The aim of this study was to develop a new genetic linkage map using genic and genomic microsatellite markers and three morphological traits: growth habit, flower color, and pod tip shape. A mapping population consisting of 380 recombinant F10 lines was generated from IAC-UNAxa0×xa0CAL143. A total of 871 microsatellites were screened for polymorphisms among the parents, and a linkage map was obtained with 198 mapped microsatellites. The total map length was 1865.9xa0cM, and the average distance between markers was 9.4xa0cM. Flower color and pod tip shape were mapped and segregated at Mendelian ratios, as expected. The segregation ratio and linkage data analyses indicated that the determinacy growth habit was inherited as two independent and dominant genes, and a genetic model is proposed for this trait.

Genetic diversity in common bean accessions evaluated by means of morpho-agronomical and RAPD data

Germplasm banks store genotype samples, improved varieties, landraces and wild species, all generically denominated accessions. The importance of characterizing germplasm banks is based on the identification and knowledge of relevant traits for genetic improvement and ex situ germplasm conservation. Thus, the present study had as aim the evaluation of the genetic diversity among 220 accessions of a Brazilian common bean germplasm bank of the Instituto Agronomico de Campinas (IAC) by means of 23 morpho-agronomical descriptors and 19 RAPD loci. These accessions correspond to genotypes from the Andean and Middle American gene pool as well as from cultivars derived from common bean improvement programs. The Middle American accessions and the improved cultivars were clustered into one group, distinct from the one formed by the Andean accessions. In relation to the molecular data, 47% of the genetic similarity was detected among the Middle American accessions, and similar results were observed for the improved cultivars (50%). The Andean accessions revealed 60% of genetic similarity. The cluster constituted by the improved cultivars and the Middle American genotypes differed, basically, in tegument color. Both molecular and morpho-agronomical data sets were equally effective to quantify and organize the genetic diversity of common bean accessions. This information may be useful to direct crosses and for the proper organization of the IAC germplasm bank.

Estabilidade fenotípica de linhagens de feijoeiro em três épocas de plantio no Estado de São Paulo

Aiming at knowing the yield potential behavior of selected common bean lines based on data of phenotypical stability, two experimental sets were carried out in the three traditional growing seasons: rainy (September-October), dry (January-February) and winter (May-June). The first set, with 47 trials, was made up of 16 experimental lines and the check Carioca 80SH cultivar; the second, with 44 trials, was made up of 11 lines and the checks Carioca 80SH and Rosinha G2 cultivars, allocated to the three growing seasons above cited. The experimental design was a randomized complete block with five replications and plots constituted of a line of 5 m of length. Joint (general and seasonal) and single (per season) statistical analyses were performed on the yield data. Phenotypical stability and responsiveness were estimated by Cruz et al. (1989). Several adapted lines in unfavorable environments were observed in at least a growing season. Several genotypes showed high responsiveness or stability. The results indicated the feasibility of recommending cultivars through the selection of the best adapted and responsive experimental lines, specific for each growing season.

A whole genome DArT assay to assess germplasm collection diversity in common beans

A Diversity Arrays Technology (DArT) marker system was applied for the first time to common beans (Phaseolus vulgaris L.) and tested on 89 accessions from the IAC and CIAT common bean breeding programs, as well as on landraces from the FAO collection. Seven frequently used restriction endonucleases were tested in combination with a rare-cutting restriction enzyme, PstI, to evaluate their suitability for DArT technology. Two restriction enzyme combinations (PstI/BstNI and PstI/TaqI) were selected to evaluate polymorphisms and PstI/BstNI, which yielded the most polymorphisms, was used to construct the final array. Genotyping was done by labelling the genomic representations with the fluorescent nucleotides cy3-dUTP and cy5-dUTP. The poly-linker fragment was labelled with 6-FAM and used as a control treatment and standard to determine the amount of DNA spotted on the array for each clone. DArTsoft version 7.3 software was used to analyse, identify and score polymorphic markers. Arrays containing individual fragments from these representations generated DArT fingerprints with a genotype call rate of 97.1% and a scoring reproducibility of at least 99.9%. A total of 2,501 polymorphic markers were found. Neighbour-joining distance matrices were used to create dispersion graphs that distinguished the two major gene pools of common beans and classified the accessions as either Andean or Mesoamerican. A principal coordinate analysis of the DArT marker results explained 82% of the total data variation. These results show that the DArT platform was accurate for studying the genetic diversity of the common bean and efficient for the large-scale detection of polymorphisms. These properties make marker technology a choice for future experiments. This is the first report to describe the use of DArT technology for genotyping the common bean.

Genome-Wide Association Studies of Anthracnose and Angular Leaf Spot Resistance in Common Bean ( Phaseolus vulgaris L.)

The common bean (Phaseolus vulgaris L.) is the world’s most important legume for human consumption. Anthracnose (ANT; Colletotrichum lindemuthianum) and angular leaf spot (ALS; Pseudocercospora griseola) are complex diseases that cause major yield losses in common bean. Depending on the cultivar and environmental conditions, anthracnose and angular leaf spot infections can reduce crop yield drastically. This study aimed to estimate linkage disequilibrium levels and identify quantitative resistance loci (QRL) controlling resistance to both ANT and ALS diseases of 180 accessions of common bean using genome-wide association analysis. A randomized complete block design with four replicates was performed for the ANT and ALS experiments, with four plants per genotype in each replicate. Association mapping analyses were performed for ANT and ALS using a mixed linear model approach implemented in TASSEL. A total of 17 and 11 significant statistically associations involving SSRs were detected for ANT and ALS resistance loci, respectively. Using SNPs, 21 and 17 significant statistically associations were obtained for ANT and angular ALS, respectively, providing more associations with this marker. The SSR-IAC167 and PvM95 markers, both located on chromosome Pv03, and the SNP scaffold00021_89379, were associated with both diseases. The other markers were distributed across the entire common bean genome, with chromosomes Pv03 and Pv08 showing the greatest number of loci associated with ANT resistance. The chromosome Pv04 was the most saturated one, with six markers associated with ALS resistance. The telomeric region of this chromosome showed four markers located between approximately 2.5 Mb and 4.4 Mb. Our results demonstrate the great potential of genome-wide association studies to identify QRLs related to ANT and ALS in common bean. The results indicate a quantitative and complex inheritance pattern for both diseases in common bean. Our findings will contribute to more effective screening of elite germplasm to find resistance alleles for marker-assisted selection in breeding programs.

Mapping of angular leaf spot resistance QTL in common bean (Phaseolus vulgaris L.) under different environments

BackgroundCommon bean (Phaseolus vulgaris L.) is the most important grain legume for human diet worldwide and the angular leaf spot (ALS) is one of the most devastating diseases of this crop, leading to yield losses as high as 80%. In an attempt to breed resistant cultivars, it is important to first understand the inheritance mode of resistance and to develop tools that could be used in assisted breeding. Therefore, the aim of this study was to identify quantitative trait loci (QTL) controlling resistance to ALS under natural infection conditions in the field and under inoculated conditions in the greenhouse.ResultsQTL analyses were made using phenotypic data from 346 recombinant inbreed lines from the IAC-UNA x CAL 143 cross, gathered in three experiments, two of which were conducted in the field in different seasons and one in the greenhouse. Joint composite interval mapping analysis of QTL x environment interaction was performed. In all, seven QTLs were mapped on five linkage groups. Most of them, with the exception of two, were significant in all experiments. Among these, ALS10.1DG,UC presented major effects (R2 between 16% - 22%). This QTL was found linked to the GATS11b marker of linkage group B10, which was consistently amplified across a set of common bean lines and was associated with the resistance. Four new QTLs were identified. Between them the ALS5.2 showed an important effect (9.4%) under inoculated conditions in the greenhouse. ALS4.2 was another major QTL, under natural infection in the field, explaining 10.8% of the variability for resistance reaction. The other QTLs showed minor effects on resistance.ConclusionsThe results indicated a quantitative inheritance pattern of ALS resistance in the common bean line CAL 143. QTL x environment interactions were observed. Moreover, the major QTL identified on linkage group B10 could be important for bean breeding, as it was stable in all the environments. Thereby, the GATS11b marker is a potential tool for marker assisted selection for ALS resistance.

Quantitative analysis of race-specific resistance to Colletotrichum lindemuthianum in common bean

Abstract Molecular genetic maps continue to play a major role in breeding of crop species. The common bean genetic map of the recombinant inbred line population IAC-UNAxa0×xa0CAL 143 (UC) has been used to detect loci controlling important agronomic traits in common bean. In the current study, new microsatellite markers were added to the UC map and the linkage analysis was refined using current genomic resources of common bean, in order to identify quantitative resistance loci (QRL) associated with different races of the anthracnose pathogen. A single race inoculation was conducted in greenhouse using four plants per plot. Both race-specific and joint-adjusted disease severity means, obtained from linear-mixed model, were used to perform multiple interval mapping (MIM) and multi-trait MIM (MTMIM). In total, 13 and 11 QRL were identified by MIM and MTMIM analyses, respectively; with nine being observed in both analyses. ANT02.1UC and ANT07.1UC showed major effects on resistance both for MIM and MTMIM. Common major QRL for resistance to the three anthracnose races were expected, since high genetic pairwise-correlation was observed between the race-specific and joint-adjusted disease severity means. Therewith, both ANT02.1 and ANT07.1 can be regarded as valuable targets for marker-assisted selection; and so, putative genes potentially involved in the resistance response were identified in these QRL regions. Minor effect QRL were also observed, showing differential affects either on race-specific or multi-trait analyses and may play a role on durable horizontal resistance. These results contribute to a better understanding of the host-pathogen interaction and to breeding for enhancing resistance to Colletotrichum lindemuthianum in common bean.