Myriam C. Duque

Cross-species amplification of cassava (Manihot esculenta) (Euphorbiaceae) microsatellites: allelic polymorphism and degree of relationship

Microsatellite amplification was performed on cassava (Manihot esculenta) and six other different species (all wild) of the Manihot genus. We used ten pairs of microsatellite primers previously developed from cassava, detecting 124 alleles in a sample of 121 accessions of the seven species. The number of alleles per locus ranged from four to 21 alleles, and allelic diversity was greater in the wild species than in cassava. Seventy-nine alleles, including unique ones, were detected in the wild species but were not found in the crop. The lower level of heterozygosity in some wild species probably resulted from a combination of fine-scale differentiation within the species and the presence of null alleles. Overall, microsatellite primers worked across the genus, but, with increasing genetic distance, success in amplifying loci tended to decrease. No accession of M. aesculifolia, M. carthaginensis, and M. brachyloba presented a banding pattern at locus Ga-140; neither did one appear for M. aesculifolia at locus Ga-13. Previous work with amplified fragment length polymorphism (AFLP) markers and this microsatellite analysis show that these three wild taxa are the most distant relatives of the crop, whereas the wild forms M. esculenta subsp. flabellifolia and M. esculenta subsp. peruviana appear to be the closest.

AFLP analysis of relationships among cassava and other Manihot species

Abstract Despite the worldwide importance of cultivated cassava (M. esculenta Crantz) its origin and taxonomic relationships with other species in the genus have not been clearly established. We evaluated a representative sample of the crop’s diversity and six wild taxa with AFLPs to estimate genetic relationships within the genus. Groupings of accessions of each species by data analysis corresponded largely with their previous taxonomic classifications. A mixed group, consisting of Manihot esculenta subsp. flabellifolia and M. esculenta subsp. peruviana, was most similar to cassava, while M. aesculifolia, M. brachyloba, and M. carthaginensis were more distant. Species-specific markers, which may be useful in germ-plasm classification or introgression studies, were suggested by the unique presence of AFLP products in samples of each of the three wild species. Heterogeneity of similarities among individuals of certain species suggested the existence of intraspecific gene pools, a hypothesis that was supported by morphological or ecogeographic evidence with varying degrees of success. Quantitative assessment of genetic diversity revealed greater homogeneity among cassava accessions than among itsclosest wild relatives. The demonstration of unique genetic diversity in the two M. esculenta subspecies and their genetic similarity to the crop supports the hypothesis that these materials may be the ancestors of cassava.

Genetic diversity, seed size associations and population structure of a core collection of common beans (Phaseolus vulgaris L.)

Cultivated common bean germplasm is especially diverse due to the parallel domestication of two genepools in the Mesoamerican and Andean centers of diversity and introgression between these gene pools. Classification into morphological races has helped to provide a framework for utilization of this cultivated germplasm. Meanwhile, core collections along with molecular markers are useful tools for organizing and analyzing representative sets of these genotypes. In this study, we evaluated 604 accessions from the CIAT core germplasm collection representing wide genetic variability from both primary and secondary centers of diversity with a newly developed, fluorescent microsatellite marker set of 36 genomic and gene-based SSRs to determine molecular diversity and with seed protein analysis to determine phaseolin alleles. The entire collection could be divided into two genepools and five predominant races with the division between the Mesoamerica race and the Durango–Jalisco group showing strong support within the Mesoamerican genepool and the Nueva Granada and Peru races showing less diversity overall and some between-group admixture within the Andean genepool. The Chile race could not be distinguished within the Andean genepool but there was support for the Guatemala race within the Mesoamerican genepool and this race was unique in its high level of diversity and distance from other Mesoamerican races. Based on this population structure, significant associations were found between SSR loci and seed size characteristics, some on the same linkage group as the phaseolin locus, which previously had been associated with seed size, or in other regions of the genome. In conclusion, this study has shown that common bean has very significant population structure that can help guide the construction of genetic crosses that maximize diversity as well as serving as a basis for additional association studies.

Microsatellite characterization of Andean races of common bean (Phaseolus vulgaris L.)

The Andean gene pool of common bean (Phaseolus vulgaris L.) has high levels of morphological diversity in terms of seed color and size, growth habit and agro-ecological adaptation, but previously was characterized by low levels of molecular marker diversity. Three races have been described within the Andean gene pool: Chile, Nueva Granada and Peru. The objective of this study was to characterize a collection of 123 genotypes representing Andean bean diversity with 33 microsatellite markers that have been useful for characterizing race structure in common beans. The genotypes were from both the primary center of origin as well as secondary centers of diversity to which Andean beans spread and represented all three races of the gene pool. In addition we evaluated a collection of landraces from Colombia to determine if the Nueva Granada and Peru races could be distinguished in genotypes from the northern range of the primary center. Multiple correspondence analyses of the Andean race representatives identified two predominant groups corresponding to the Nueva Granada and Peru races. Some of the Chile race representatives formed a separate group but several that had been defined previously as from this race grouped with the other races. Gene flow was more notable between Nueva Granada and Peru races than between these races and the Chile race. Among the Colombian genotypes, the Nueva Granada and Peru races were identified and introgression between these two races was especially notable. The genetic diversity within the Colombian genotypes was high, reaffirming the importance of this region as an important source of germplasm. Results of this study suggest that the morphological classification of all climbing beans as Peru race genotypes and all bush beans as Nueva Granada race genotypes is erroneous and that growth habit traits have been mixed in both races, requiring a re-adjustment in the concept of morphological races in Andean beans.

AFLP fingerprinting : An efficient technique for detecting genetic variation of Xanthomonas axonopodis pv. manihotis

Xanthomonas axonopodis pv. manihotis (Xam) is the causative agent of cassava bacterial blight (CBB), a worldwide disease that is particularly destructive in South America and Africa. CBB is controlled essentially through the use of resistant varieties. To develop an appropriate disease management strategy, the genetic diversity of the pathogens populations must be assessed. Until now, the genetic diversity of Xam was characterized by RFLP analyses using ribotyping, and plasmid and genomic Xam probes. We used AFLP (amplified fragment length polymorphism), a novel PCR-based technique, to characterize the genetic diversity of Colombian Xam isolates. Six Xam strains were tested with 65 AFLP primer combinations to identify the best selective primers. Eight primer combinations were selected according to their reproducibility, number of polymorphic bands and polymorphism detected between Xam strains. Forty-seven Xam strains, originating from different Colombian ecozones, were analysed with the selected combinations. Results obtained with AFLP are consistent with those obtained with RFLP, using plasmid DNA as a probe. Some primer combinations differentiated Xam strains that were not distinguished by RFLP analyses, thus AFLP fingerprinting allowed a better definition of the genetic relationships between Xam strains.

Molecular characterization by AFLPs of Capsicum germplasm from the Amazon department in Colombia, characterization by AFLPs of Capsicum

Using AFLPs, 71 peppers (Capsicum) accessions from the species C. chinense Jacq., C. baccatum L., C. annuum L. and C. frutescens L. from indigenous communities of the Amazon Department (Colombia) were studied to assess the genetic diversity of the collections, and delineate species gene groups. Ten additional accessions were included as a reference species group. Three clusters were identified in the Amazonian accessions by Multiple Correspondence Analyses (MCA) and a dendrogram from the UPGMA analyses of Nei – Li genetic similarity. The clusters correspond to gene groups of the species Capsicum chinense (the majority of the accessions), C. baccatum and the complex annuum - frutescens. A fourth cluster corresponds to the reference accession C. pubescens. The MCA analyses accounted for 95% of the total variation. The total genetic variation was low (Ht 0.119) and a genetic diversity index (Gst) of 0.331 was obtained. This suggests a limited genetic diversity of the accessions and a close relatedness of the species. This study is the first molecular marker assessment of genetic diversity for peppers from the Colombian Amazon, and provides information of biodiversity that can be employed in the preservation and use of Capsicum germplasm.

Mapping EST-derived SSRs and ESTs involved in resistance to bacterial blight in Manihot esculenta

Cassava (Manihot esculenta Crantz) is a major root crop widely grown in the tropics. Cassava bacterial blight, caused by Xanthomonas axonopodis pv. manihotis (Xam), is an important disease in Latin America and Africa resulting in significant losses. The preferred control method is the use of resistant genotypes. Mapping expressed sequence tags (ESTs) and determining their co-localization with quantitative trait loci (QTLs) may give additional evidence of the role of the corresponding genes in resistance or defense. Twenty-one EST-derived simple sequence repeats (SSRs) were mapped in 16 linkage groups. ESTs showing similarities with candidate resistance genes or defense genes were also mapped using strategies such as restriction fragment length polymorphisms, cleaved amplified polymorphic sequences, and allele-specific primers. In total, 10 defense-related genes and 2 bacterial artificial chromosomes (BACs) containing resistance gene candidates (RGCs) were mapped in 11 linkage groups. Two new QTLs associated with resistance to Xam strains CIO121 and CIO151 were detected in linkage groups A and U, respectively. The QTL in linkage group U explained 61.6% of the phenotypic variance and was associated with an RGC-containing BAC. No correlation was found between the new EST-derived SSRs or other mapped ESTs and the new or previously reported QTLs.

Genetic Structure and Population Dynamics of Xanthomonas axonopodis pv. manihotis in Colombia from 1995 to 1999

ABSTRACT Restriction fragment length polymorphisms (RFLPs) were used to study the population genetics and temporal dynamics of the cassava bacterial pathogen Xanthomonas axonopodis pv. manihotis. The population dynamics were addressed by comparing samples collected from 1995 to 1999 from six locations, spanning four different edaphoclimatic zones (ECZs). Forty-five different X. axonopodis pv. manihotis RFLP types or haplotypes were identified between 1995 and 1999. High genetic diversity of the X. axonopodis pv. manihotis strains was evident within most of the fields sampled. In all but one site, diversity decreased over time within fields. Haplotype frequencies significantly differed over the years in all but one location. Studies of the rate of change of X. axonopodis pv. manihotis populations during the cropping cycle in two sites showed significant changes in the haplotype frequencies but not composition. However, variations in pathotype composition were observed from one year to the next at a single site in ECZs 1 and 2 and new pathotypes were described after 1997 in these ECZs, thus revealing the dramatic change in the pathogen population structure of X. axonopodis pv. manihotis. Disease incidence was used to show the progress of cassava bacterial blight in Colombia during the 5-year period in different ecosystems. Low disease incidence values were correlated with low rainfall in 1997 in ECZ 1.

Random amplified polymorphic DNA analysis of Anopheles nuneztovari (Diptera: Culicidae) from Western and northeastern Colombia.

Random amplified polymorphic DNA (RAPD) markers were used to analyze 119 DNA samples of three Colombian Anopheles nuneztovari populations to study genetic variation and structure. Genetic diversity, estimated from heterozygosity, averaged 0.34. Genetic flow was greater between the two populations located in Western Colombia (F ST: 0.035; Nm: 6.8) but lower between these two and the northeastern population (F ST: 0.08; Nm: 2.8). According to molecular variance analysis, the genetic distance between populations was significant (phi ST 0.1131, P < 0.001). The variation among individuals within populations (phi ST 0.8869, P < 0.001)was also significant, suggesting a greater degree of population subdivision, not considered in this study. Both the parameters evaluated and the genetic flow suggest that Colombian An. nuneztovari populations are co-specific.

Evaluating purple passion fruit (Passiflora edulis Sims f. edulis) genetic variability in individuals from commercial plantations in Colombia

This study evaluated P. edulis f. edulis intraspecific variation at DNA level using AFLPs and SSRs. About 60 specimens were collected from Colombian commercial plantations in the departments of Antioquia, Boyacá, Cundinamarca, Huila, Quindío and Tolima. Ten specimens were also included from Corpoica’s La Selva research center Passiflora germplasm bank. A non-commercial purple passion fruit plant and three species from the Passiflora genus (Passiflora maliformis, Passiflora edulis f. flavicarpa and Passiflora ligularis) were included as controls. The six most informative ones were selected from the initial screening of 49 different AFLP primer combinations; 52–92 DNA fragments for each combination in each genotype (total 419 fragments) were obtained by using these six combinations. 17 SSR primers reported in P. edulis f. flavicarpa and Passiflora alata were tested on purple passion fruit. Eight of them were successfully amplified but none showed polymorphism. Relationships among individuals were assessed on AFLP data using cluster analysis by Dice coefficient, UPGMA algorithm and the multiple correspondence analysis ordination method based on Greenacre measurement. The similarity coefficients ranged from 0.75 to 1.00 giving an average of 0.96 within the different purple passion fruit materials, showing low genetic variability for the purple passion fruit material used in this study.