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.     

SNPs,SSRs and inferences on cassava’s origin

Despite its importance as a staple food throughout the tropics, the root crop cassava (it Manihot esculenta ssp. esculenta) has traditionally not been a major focus of research. One basic question about cassava that remained unresolved until recently concerns the crop’s origin. This paper describes analyses of SNPs (single nucleotide polymorphisms) and SSR (simple sequence repeat) variation as a means of tracing cassava’s evolutionary and geographical origins. Genetic diversity was examined in a sample of 20 cassava varieties that are representative of germplasm diversity within the crop, and in 212 individuals collected from wild populations of two closely related Manihot species. SNP and indel variation was examined in portions of two low copy nuclear genes, BglA and Hnl. Inferences from these genes were compared to those from previously examined loci, including the low copy nuclear gene G3pdh and 5 SSR loci. For all genes examined, SNPs and SSR alleles are shared between domesticated cassava and a specific geographical subset of wild Manihot populations, which suggests the following: (1) Cassava was likely domesticated from a single wild Manihot species, M. esculenta ssp. flabellifolia, rather than from multiple hybridizing species, as traditionally believed; and (2) the crop most likely originated in the southern Amazon basin.     

Evolution under domestication: contrasting functional morphology of seedlings in domesticated cassava and its closest wild relatives

Although cassava (Manihot esculenta ssp. esculenta) is asexually propagated, farmers incorporate plants from seedlings into planting stocks. These products of sex are exposed to selection, which in agricultural environments should favour rapid growth. To examine whether seedling morphology has evolved under domestication, we compared domesticated cassava, its wild progenitor (M. esculenta ssp. flabellifolia) and their sister species (M. pruinosa) under controlled conditions. Field observations complemented laboratory study. In both wild taxa, the hypocotyl did not elongate (hypogeal germination) and cotyledons remained enclosed in the testa. In domesticated cassava, the hypocotyl elongated (epigeal germination), and cotyledons emerged and became foliaceous. The difference in hypocotyl elongation was fixed, whereas cotyledon morphology varied with environmental conditions in M. pruinosa. Comparative analysis suggests that epigeal germination is primitive in Manihot, that the lineage including wild ancestors of cassava evolved hypogeal germination--which confers greater tolerance to risks in their savanna environment--and that with domestication, there was a reversion to epigeal germination and photosynthetic cotyledons, traits conferring high initial growth rates in agricultural habitats.     

Breeding cassava for apomixis

Apomixis genes have been successfully transferred to cassava (Manihot esculenta) by hybridizing it with the wild species, M. glaziovii. The interspecific hybrid of cassava and M. glaziovii was exposed to open pollination during three subsequent generations. Seven sibs and the maternal progenitor of the fourth generation were genotyped using five microsatellite loci previously developed for cassava. All sibs were identical with each other and with their maternal progenitor. Sibs from M. glaziovii proved to be identical when examined by the same microsatellite loci. This evidence leads to the conclusion that apomixis does occur in wild-cassava relatives and apparently has played an important role in Manihot speciation. This is the first report of nearly 100% apomixis.     

Population history of Manihot esculenta (Euphorbiaceae) inferred from nuclear DNA sequences

The nature of gene flow in plants -- including the propensity for interspecific introgression -- makes them interesting candidates for phylogeographical analysis. Plant phylogeography studies have been limited, however, by the availability of suitable intraspecific variation. In this study, DNA sequence variation from a nuclear gene [Glyceraldehyde 3-phosphate dehydrogenase; (G3pdh)] was used to examine the population history of Manihot esculenta ssp. flabellifolia and a potentially hybridizing species, M. pruinosa. These species occur in the rainforest-savanna ecotone adjoining the Amazon basin, a region believed to have undergone major habitat shifts since the Pleistocene. Geographical distributions of the G3pdh haplotypes indicate genetic isolation-by-distance across the range of M. esculenta ssp. flabellifolia. However, there is greater genetic similarity between northeastern and western populations than would be expected given the present species distribution. A nested clade analysis suggests that northeastern and western populations were connected by gene flow until relatively recently, when they became fragmented. This inferred fragmentation event is consistent with post-Pleistocene habitat shifts proposed for the Amazon basin. At the interspecific level, haplotype sharing with M. pruinosa may reflect either recent interspecific introgression or incomplete lineage sorting between these closely related species.     

Genetic diversity of traditional South American Landraces of Cassava (Manihot esculenta Crantz): an analysis using microsatellites

The extent and structure of the genetic variability of traditional varieties of cassava (Manihot esculenta Crantz) have been little documented, despite considerable evidence for this crop? great varietal diversity in traditional agroecosystems. We used microsatellite markers to assess the genetic structure of traditional landraces of sweet and bitter cassava collected from five South American sites. As reference, we used a sample of 38 accessions from a world collection of cultivated cassava. For a total of 10 loci examined, we found 15 alleles that were not represented in this sample. Ten of these had been previously detected in wild Manihot species. The geographical structure of genetic variability was weak, but the genetic differentiation between bitter and sweet landraces was significant, suggesting that each form had evolved separately after domestication. Our results showed that traditional landraces form an important source of genetic diversity and merit more attention from managers of crop genetic resources.     

Insights on the evolution of a vegetatively propagated crop species

The opportunity for gene flow between a vegetatively propagated crop and its wild relatives is expected to be much lower than for seed-propagated crops, since sexual reproduction in the crop occurs only infrequently. A study by Duputié and colleagues now demonstrates evidence of sexual reproduction between a vegetatively propagated crop and a closely related wild congener. Working in French Guiana, these workers have documented a hybrid zone arising from introgression between cassava (Manihot esculenta ssp. esculenta, Euphorbiaceae), which is propagated by stem cuttings, and wild Manihot populations growing in close proximity. Patterns of heterozygosity suggest that there are little-to-no barriers to reproduction between the crop and these wild populations. Previous work by these researchers has documented the importance of occasional sexual reproduction for the development of cassava varieties in traditional Amerindian farming systems. Taken together with their previous work, these new findings suggest that gene flow between wild Manihot populations and cassava plants could potentially play a much greater role in the crop's evolution than previously thought.     

Characterization of microsatellite markers in cassava based on microsatellite-AFLP technique

We developed molecular markers for cassava based on the microsatellite-amplified fragment length polymorphism (M-AFLP) technique. Twenty primer pairs were developed and used for the analysis of 48 samples of Manihot species, consisting of M. esculenta (33), M. esculenta ssp flabellifolia (3), M. chlorosticta (3), M. carthaginensis (3), M. filamentosa (3), and M. tristis (3). Nine microsatellite loci that were polymorphic among these Manihot species were identified, giving 32 polymorphic alleles and from two to seven alleles per locus. Unbiased and direct count heterozygosity varied from 0.0233 to 0.7924 and 0.0000 to 0.7083, respectively. There was significant deviation (P < 0.05) from Hardy-Weinberg equilibrium at five loci. Genotypic data from the Manihot species were subjected to genetic diversity analysis. We found that M. chlorosticta and M. esculenta ssp flabellifolia were the closest populations, while M. filamentosa and M. esculenta ssp flabellifolia were the most divergent. Considering within M. esculenta, the samples from Nigeria and Fiji were the most closely related, while those from Venezuela and of unknown origin were the most divergent. We conclude that the M-AFLP technique is an effective method for generating microsatellite markers that are useful for genetic diversity analysis in Manihot species.     

Informal "seed" systems and the management of gene flow in traditional agroecosystems: the case of cassava in Cauca, Colombia

Our ability to manage gene flow within traditional agroecosystems and their repercussions requires understanding the biology of crops, including farming practices' role in crop ecology. That these practices' effects on crop population genetics have not been quantified bespeaks lack of an appropriate analytical framework. We use a model that construes seed-management practices as part of a crop's demography to describe the dynamics of cassava (Manihot esculenta Crantz) in Cauca, Colombia. We quantify several management practices for cassava--the first estimates of their kind for a vegetatively-propagated crop--describe their demographic repercussions, and compare them to those of maize, a sexually-reproduced grain crop. We discuss the implications for gene flow, the conservation of cassava diversity, and the biosafety of vegetatively-propagated crops in centers of diversity. Cassava populations are surprisingly open and dynamic: farmers exchange germplasm across localities, particularly improved varieties, and distribute it among neighbors at extremely high rates vis-à-vis maize. This implies that a large portion of cassava populations consists of non-local germplasm, often grown in mixed stands with local varieties. Gene flow from this germplasm into local seed banks and gene pools via pollen has been documented, but its extent remains uncertain. In sum, cassava's biology and vegetative propagation might facilitate pre-release confinement of genetically-modified varieties, as expected, but simultaneously contribute to their diffusion across traditional agroecosystems if released. Genetically-modified cassava is unlikely to displace landraces or compromise their diversity; but rapid diffusion of improved germplasm and subsequent incorporation into cassava landraces, seed banks or wild populations could obstruct the tracking and eradication of deleterious transgenes. Attempts to regulate traditional farming practices to reduce the risks could compromise cassava populations' adaptive potential and ultimately prove ineffectual.     

Biological parameters of Bemisia tabaci (Gennadius) biotype B (Hemiptera: Aleyrodidae) on Jatropha gossypiifolia, commercial (Manihot esculenta) and wild cassava (Manihot flabellifolia and M. carthaginensis) (Euphorbiaceae)

Bemisia tabaci (Gennadius) is one of the most important pests of cassava in Africa and several countries of Asia due to the damage caused by direct feeding, the excretion of honeydew, and its capacity as a vector of cassava mosaic geminivirus. There is a general consensus that B. tabaci is a complex of morphologically indistinguishable populations with different biotypes. In the Americas, the polyphagous biotype B does not appear to feed on cassava. Recent studies indicate that it is possible, however, for biotype B to gradually adapt to cassava using phylogenetically related hosts. Therefore, the possibility that some wild species of cassava constitute intermediate hosts in the adaptation process may lead to the establishment of biotype B on commercial varieties of Manihot esculenta. In here, we evaluated Jatropha gossypiifolia, two wild species of cassava (Manihot flabellifolia and M. carthaginensis) and a commercial cassava variety (MCol 2063) as hosts of biotype B. The highest oviposition rate (2.7 eggs /two days) occurred on M. esculenta, although the development time (44 d) was the longest when compared to M. carthaginensis and J. gossypiifolia. About 60% of the population could reproduce on the wild cassava species vs. 55% on J. gossypiifolia and 27.5% on the commercial variety. Our data suggest that J. gossypiifolia is a suitable host and the wild species M. carthaginensis can constitute a potential intermediate host in the adaptation of biotype B to commercial varieties of cassava.     

Microsatellite genetic variation in small and isolated populations of Magnolia sieboldii ssp. japonica

Magnolia sieboldii ssp. japonica, distributed mainly in western Japan, is restricted to high elevation areas (1000-2000 m above sea level) and usually forms small isolated populations. Four microsatellite loci were assayed for 19 populations from six regions spanning the range of distribution, and the levels and distribution of genetic variation were estimated. All four loci were variable, with a total of 39 alleles, but the overall level of microsatellite genetic variation was low, especially compared with a related species, M. obovata. Genetic structure in M. sieboldii was characterised by low intrapopulational genetic variation (A = 3.74 and H(o) = 0.366 on average) and high genetic differentiation even among regional populations. Highly significant isolation-by-distance (IBD) models at the short distance were detected. Genetic drift and limited gene flow was considered to be important in determining the genetic structure within regions. Total genetic differentiation was remarkably high (F(ST) = 0.488 and R(ST) = 0.538), suggesting genetic barriers among regions. Neighbour-joining dendrograms relating the 19 populations, and further analysis on the IBD models, revealed that a stepwise mutation model was more suited than an infinite allele model to explain the genetic differentiation among regions. It is suggested that mutation at microsatellite loci might be influential in generating the genetic differentiation among regions. These results showed the potential of hypervariable microsatellite loci to evaluate the effects of genetic drift and population isolation within regions, and to detect genetic distinctiveness, in spite of the loss of overall genetic variation in M. sieboldii.     

Population genetic structure of wild and hatchery black rockfish Sebastes inermis in Korea, assessed using cross-species microsatellite markers

The population structure of the black rockfish, Sebastes inermis (Sebastidae), was estimated using 10 microsatellite loci developed for S. schlegeli on samples of 174 individuals collected from three wild and three hatchery populations in Korea. Reduced genetic variation was detected in hatchery strains [overall number of alleles (N(A)) = 8.07; allelic richness (A(R)) = 7.37; observed heterozygosity (H(O)) = 0.641] compared with the wild samples (overall N(A) = 8.43; A(R) = 7.83; H(O) = 0.670), but the difference was not significant. Genetic differentiation among the populations was significant (overall F(ST) = 0.0237, P < 0.05). Pairwise F(ST) tests, neighbor-joining tree, and principal component analyses showed significant genetic heterogeneity among the hatchery strains and between wild and hatchery strains, but not among the wild populations, indicating high levels of gene flow along the southern coast of Korea, even though the black rockfish is a benthic, non-migratory marine species. Genetic differentiation among the hatchery strains could reflect genetic drift due to intensive breeding practices. Thus, in the interests of optimal resource management, genetic variation should be monitored and inbreeding controlled within stocks in commercial breeding programs. Information on genetic population structure based on cross-species microsatellite markers can aid in the proper management of S. inermis populations.     

Cassava: an appraisal of its phytochemistry and its biotechnological prospects

The present state of knowledge of the phytochemistry of small molecules isolated from the roots and leaves of cassava, Manihot esculenta Crantz (Euphorbiaceae), is reviewed. Cassava roots are an important source of dietary and industrial carbohydrates, mainly eaten as a source of starch, forming the staple food to over 500 million; additionally, the roots have value as a raw material for industrial starch production and for animal feed giving the crop high economic value, but it suffers markedly from post-harvest physiological deterioration (PPD). The hydroxycoumarins scopoletin and its glucoside scopolin as well as trace quantities of esculetin and its glucoside esculin are identified from cassava roots during PPD. The biotechnological prospects for cassava are also reviewed including a critical appraisal of transgenic approaches for crop improvement, together with its use for bioethanol production, due to cassava's efficient ability to fix carbon dioxide into carbohydrate.     

Unmanaged sexual reproduction and the dynamics of genetic diversity of a vegetatively propagated crop plant, cassava (Manihot esculenta Crantz), in a traditional farming system

Occurrence of intervarietal or interspecific natural crosses has been reported for many crop plants in traditional farming systems, underlining the potential importance of this source of genetic exchange for the dynamics of genetic diversity of crop plants. In this study, we use microsatellite loci to investigate the role of volunteer seedlings (plants originating from unmanaged sexual reproduction) in the dynamics of genetic diversity of cassava (Manihot esculenta Crantz), a vegetatively propagated crop, in a traditional farming system in Guyana. A previous field study showed that farmers incorporate such plants into the germplasm for vegetative propagation, and that many of them are likely to be assigned by farmers to recognized varieties. Under strict vegetative propagation clonality of varieties is expected. The high proportion of polyclonal varieties observed suggests that incorporation of seedlings into the germplasm for propagation is a frequent event. The molecular variability assessed with microsatellite markers shows that there is high differentiation among heterozygous varieties, whereas populations of seedlings do not depart from the proportions expected under Hardy-Weinberg assumptions. Assignment of seedlings to a recognized variety on the basis of morphological similarity greatly increases genetic diversity within the variety. We argue that recombination and gene flow play a major role in the dynamics of genetic diversity of cassava in traditional farming systems. Documenting unmanaged sexual reproduction and its genetic consequences is a prerequisite for defining strategies of in situ conservation of crop plant genetic resources.     

Genetic variation of wild and hatchery populations of the catla Indian major carp (Catla catla Hamilton 1822: Cypriniformes, Cyprinidae) revealed by RAPD markers

Genetic variation is a key component for improving a stock through selective breeding programs. Randomly amplified polymorphic DNA (RAPD) markers were used to assess genetic variation in three wild population of the catla carp (Catla catla Hamilton 1822) in the Halda, Jamuna and Padma rivers and one hatchery population in Bangladesh. Five decamer random primers were used to amplify RAPD markers from 30 fish from each population. Thirty of the 55 scorable bands were polymorphic, indicating some degree of genetic variation in all the populations. The proportion of polymorphic loci and gene diversity values reflected a relatively higher level of genetic variation in the Halda population. Sixteen of the 30 polymorphic loci showed a significant (p < 0.05, p < 0.01, p < 0.001) departure from homogeneity and the F(ST) values in the different populations indicated some degree of genetic differentiation in the population pairs. Estimated genetic distances between populations were directly correlated with geographical distances. The unweighted pair group method with averages (UPGMA) dendrogram showed two clusters, the Halda population forming one cluster and the other populations the second cluster. Genetic variation of C. catla is a useful trait for developing a good management strategy for maintaining genetic quality of the species.     

AFLP analysis of relationships among cassava and other Manihot species

 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. Received: 4 November 1996 / Accepted: 20 December 1996     

Genetic diversity of root anatomy in wild and cultivated Manihot species

An anatomical study of roots was conducted on two wild Manihot species, namely M. glaziovii and M. fortalezensis, and two cassava varieties, M. esculenta Crantz variety UnB 201 and M. esculenta variety UnB 122, to identify taxonomic differences in primary growth. Anatomical characters of cassava roots have been rarely investigated. Their study may help cassava breeders to identify varieties with economically important characters, such as tolerance to drought. We investigated tap and lateral adventitious roots of two specimens of each clone or species. Free-hand cross-sections of roots were drawn; these had been clarified with 20% sodium hypochlorite solution, stained with 1% safranin-alcian blue ethanolic solution, dehydrated in ethanol series and butyl acetate and mounted in synthetic resin. Anatomical differences among Manihot species and varieties were found in the epidermal and exodermal cell shape and wall thickness, content of cortical parenchyma, and number of xylem poles. Wall thickness of the epidermis and exodermis of tap root were similar in all species, while in the lateral root there were differences in cell shape and wall thickness. Epidermal cells with thick walls were found in the tap root of all species and in lateral roots of cassava varieties. This character is apparently associated with tolerance to drought and disease. The variation in the number of xylem poles of cassava varieties was larger (4-8) than in wild species (4-6), and appears to support the hybrid origin of cassava.     

AFLP assessment of genetic variability in cassava accessions (Manihot esculenta) resistant and susceptible to the cassava bacterial blight (CBB).

Cassava bacterial blight (CBB) is caused by Xanthomonas axonopodis pv. manihotis (Xam). Resistance is found in Manihot esculenta and, in addition, has been introgressed from a wild relative, M. glaziovii. The resistance is thought to be polygenic and additively inherited. Ninety-three varieties of M. esculenta (Crantz) were assessed by AFLPs for genetic diversity and for resistance to CBB. AFLP analysis was performed using two primer combinations and a 79.2% level of polymorphism was found. The phenogram obtained showed between 74% and 96% genetic similarity among all cassava accessions analysed. The analysis permitted the unique identification of each individual. Two Xam strains were used for resistance screening. Variation in the reaction of cassava varieties to Xam strains was observed for all plant accessions. The correlation of resistance to both strains, had a coefficient of 0.53, suggesting the independence of resistance to each strain. Multiple correspondence analysis showed a random distribution of the resistance/susceptibility response with respect to overall genetic diversity as measured by AFLP analysis. A total heterozygosity index was calculated to determine the diversity within clusters as well as among them. Our results demonstrate that resistance to CBB is broadly distributed in cassava germplasm and that AFLP analysis is an effective and efficient means of providing quantitative estimates of genetic similarities among cassava accessions.     

Population genetics of geographically restricted and widespread species of Myrica (Myricaceae)

Allozyme variation of 11 putative loci in five populations of the rare Myrica adenophora Hance, and four populations of its widespread congeneric species, M. rubra (Lour.) Sieb. & Zucc. was studied. Among the 21 alleles studied, no unique allele was detected for M. adenophora, whereas M. rubra had 3 alleles not found in the former species. In terms of genetic diversity, populations of the rare species contained fewer alleles per locus (1.5 versus 1.7), fewer effective number of alleles per locus (1.12 versus 1.20), fewer number of alleles per polymorphic locus (2.14 versus 2.46), lower percentage of polymorphic loci (30.9 versus 40.9), and lower expected heterozygosity (0.106 versus 0.163) than populations of the widespread species. Genetic distances within species average 0.043 for M. adenophora and 0.045 for M. rubra, and between species ranged from 0.052 to 0.177, with a mean of 0.103, which agrees with the very similar gross morphologies of these two species. Intrapopulation differentiation was similar in both species: G(ST) = 0.152 for M. adenophora, and 0.146 for M. rubra, whereas estimated gene flow based on G(ST) values were moderate in these two species (Nm = 1.39 versus 1.46). We inferred that M. rubra and M. adenophora are a progenitor-derivative species pair that emerged before migrating into Taiwan during the last glacial period. We consider the Hengchun population (Chiupeng, Hsuhai, and Chufengpi) and Taitung population (Tienkuan and Lanshan) of M. adenophora which probably arose from two subsets of the genome of M. rubra. Genetic drift was inferred to be one of the forces shaping the observed genetic structure in M. adenophora and M. rubra.     

Wild manihot species do not possess C4 photosynthesis

Cultivated cassava (Manihot esculenta) has a higher rate of photosynthesis than is usual for C3 plants and photosynthesis is not light saturated. For these reasons it has been suggested that cultivated cassava could be derived from wild species possessing C4 photosynthesis. The natural abundance of 13C and activities of phosphoenolpyruvate carboxylase and phosphoglycolate phosphatase were measured in leaves of 20 wild cassava species to test this hypothesis. All the species studied, including M. flabellifolia the potential wild progenitor of cultivated cassava, clearly exhibited C3 not C4 characteristics.