Fernando Angel

A molecular genetic map of cassava (Manihot esculenta Crantz)

Abstract A genetic linkage map of cassava has been constructed with 132 RFLPs, 30 RAPDs, 3 microsatellites, and 3 isoenzyme markers segregating from the heterozygous female parent of an intraspecific cross. The F1 cross was made between ‘TMS 30572’ and ‘CM 2177-2’, elite cassava cultivars from Nigeria and Colombia, respectively. The map consists of 20 linkage groups spanning 931.6 cM or an estimated 60% of the cassava genome. Average marker density is 1 per 7.9 cM. Since the mapping population is an F1 cross between heterozygous parents, with unique alleles segregating from either parent, a second map was constructed from the segregation of 107 RFLPs, 50 RAPDs, 1 microsatellite, and 1 isoenzyme marker from the male parent. Comparison of intervals in the male-and female-derived maps, bounded by markers heterozygous in both parents, revealed significantly less meiotic recombination in the gametes of the female than in the male parent. Six pairs of duplicated loci were detected by low-copy genomic and cDNA sequences used as probes. Efforts are underway to saturate the cassava map with additional markers, to join the male- and female-derived maps, and to elucidate genome organization in cassava.

Genome mapping in cassava improvement: Challenges, achievements and opportunities

Breeding goals of yield increases, root quality improvement, and disease resistance in cassava are considerably slowed down by biological characteristics of the crop, which includes a long growth cycle, a heterozygous genetic background and a poor knowledge of the organization of crop diversity. These factors severely hamper the speed and ease of moving around useful genes in cassava. The consequences are that cassava production fails to keep up with demand, especially in regions where over90% of yield is consumed as food, leading to an increase in acreage of cassava fields mostly into marginal lands. The advent of molecular markers,genome studies and plant genetic transformation holds promise of providing ways around breeding obstacles in long growth cycle and heterozygous crops. A number of these new tools, including a molecular genetic map, markers linked to disease resistance genes, and marker-aided studies of complex traits now exist or are being developed for cassava at CIAT. Large scale sequencing and mapping of expressed sequence tags(ESTs) have been initiated, towards a transcript map of cassava and the implementation of the candidate-gene approach to complex trait mapping. A cassava bacterial artificial chromosome (BAC) library has also been constructed to expedite positional cloning of genes, known only by their phenotypes and their position relative to markers on a molecular genetic map and complementation studies of candidate loci. Studies of genes that control traits of agronomic importance, and their allelic diversity in nature,provides powerful tools for understanding the basis of crop performance and improvement.

Genetic variation analysis of the genus Passiflora L. using RAPD markers

Genetic analysis based on Random Amplified Polymorphic DNA (RAPD) was carried out on 52 accessions representing 14 species of the genus Passiflora L. using 50 random 10-mer primers. A dendrogram constructed using the Dice similarity coefficient and the UPGMA algorithm based on 626 reproducible polymorphic products ranging in size from 2.8 to 0.3 Kb revealed high levels of variation within and among species, and clustering of accessions according to species. Similarity coefficients ranged from 0.929 to 0.075 showing a diverse genepool in the genus. Large intraspecific variation was found in P. ligularis and P. adenopoda while P. edulis and P. maliformis exhibited a low level of intraspecific variation. The clusters based on RAPD markers correlate fairly well to the present classification scheme based on morphological description with two exceptions. The subgenus Passiflora was split into P. edulis and the rest of the members of the subgenus Passiflora separated by the subgenus Tacsonia; secondly the two species of the subgenus Decaloba, P. adenopoda and P. coriacea, were not clustered together on the dendrogram of genetic relationships.

Variability of chloroplast DNA and nuclear ribosomal DNA in cassava (Manihot esculenta Crantz) and its wild relatives

Chloroplast DNA (cp) and nuclear ribosomal DNA (rDNA) variation was investigated in 45 accessions of cultivated and wild Manihot species. Ten independent mutations, 8 point mutations and 2 length mutations were identified, using eight restriction enzymes and 12 heterologous cpDNA probes from mungbean. Restriction fragment length polymorphism analysis defined nine distinct chloroplast types, three of which were found among the cultivated accessions and six among the wild species. Cladistic analysis of the cpDNA data using parsimony yielded a hypothetical phylogeny of lineages among the cpDNAs of cassava and its wild relatives that is congruent with morphological evolutionary differentiation in the genus. The results of our survey of cpDNA, together with rDNA restriction site change at the intergenic spacer region and rDNA repeat unit length variation (using rDNA cloned fragments from taro as probe), suggest that cassava might have arisen from the domestication of wild tuberous accessions of some Manihot species, followed by intensive selection. M. esculenta subspp flabellifolia is probably a wild progenitor. Introgressive hybridization with wild forms and pressures to adapt to the widely varying climates and topography in which cassava is found might have enhanced the crops present day variability.

Stability of cassava plants at the DNA level after retrieval from 10 years of in vitro storage

SummaryCassava (Manihot esculenta Crantz) germplasm collections are conventionally maintained by continuous vegetative propagation in the field. Tissue culture techniques provide a more convenient way to conserve germplasm. The cassava in vitro gene bank held in trust at CIAT comprises nearly 6000 accessions. A study was carried out to determine whether any DNA rearrangements resulting from in vitro storage under slow growth could be detected by molecular analysis in retrieved plants. RFLPs with homologous probes, RAPDs with twenty primers and DNA fingerprinting with M13 probe were tested to detect variation at DNA level in cassava plants after ten-years in vitro storage. The molecular marker data obtained in this study supports the stability of the cassava germplasm under the in vitro storage conditions described in this work.

Variability of chloroplast DNA in the genus Passiflora L.

Restriction fragment analysis was used to determine the variation of chloroplast DNA (cpDNA) among and within different species of Passiflora. Total DNAs of 35 accessions, representing 12 species, were digested with four restriction endonucleases. Southern blots of the restriction digests were then probed with eight different cpDNA fragments from Petunia and mung bean. Two hundred fragments were scored, and used to estimate the genetic distance among the species according to UPGMA (Unweighted Pair Group Method using Aritmetic Averages) analysis. Overall, the analysis successfully separated species according to the present classification scheme based on morphological description demonstrating profound cpDNA diversity among the 12 species evaluated. Intraspecific variation was observed in four of seven species that were represented by more than two accessions.

Toward the construction of a molecular map of cassava (Manihot esculenta Crantz): comparison of restriction enzymes and probe sources in detecting RFLPs

The construction of a detailed genetic map of cassava (Manihot esculenta Crantz), classified as a tetraploid species, depends on the ability of cloned sequences to detect polymorphisms. As a first step in developing this map, 200 cloned nuclear sequences generated with different restriction enzymes were hybridized to total digested DNA from eleven cultivated lines and one wild Manihot species, M. aesculifolia. Polymorphism was detected less frequently with both BamHI and EcoRI genomic probes than with PstI, HindIII and XbaI genomic probes. DNA digested with HpaII, DraI and TaqI displayed less polymorphism, whereas DNA digested with EcoRI and EcoRV displayed more polymorphism like that found in lettuce, rice and tomato (Landry et al., 1987; McCouch et al., 1988; Miller and Tanksley, 1990). Four-cutter restriction enzymes displayed less frequency of polymorphism when compared with six-cutter restriction enzymes. Polymorphism displayed by DraI was extremely low, indicating that regions rich in adenine and thymine may not be hot spots for mutation in cassava. Polymorphism detected between cultivated genotypes and M. aesculifolia was dramatically higher than that found among cultivated genotypes.