Heredity of seventeen isozyme loci in cassava (Manihot esculenta Crantz)

Summary Starch gel electrophoresis was used to assess isozyme polymorphism in two Manihot species. Crude extracts were obtained from leaves and pollen. Ten enzymes were examined for their polymorphism in a germplasm collection of 365 cultivated plus 109 wild accessions, mainly from Africa. The inheritance of these enzymes was examined using 13 intra and interspecific progenies. Seventeen polymorphic loci were found for the ten enzyme systems, with 59 alleles. All the markers showed disomic heredity and three linkage groups were identified.     

Somatic embryogenesis from floral tissue of cassava (Manihot esculenta Crantz)

The aim of this study was to examine the embryogenic potential of floral material of the cassava cultivar MCOL 1505. Macerated immature inflorescences were found to be highly embryogenic, with almost 78% of the explants producing somatic embryos. Somatic embryos were also produced from whole male florets and half florets although at much lower rates. No regeneration was obtained from anther, microspore or floret wall tissue. Somatic embryos derived from immature inflorescences were regenerated via organogenesis and the plants derived from this process were assessed in terms of phenotype and ploidy level. If haploid plants could be produced by this method, this would have significant implications in assisting traditional cassava breeding, as this would allow homozygosity to be reached more rapidly. In a crop such as cassava, which is highly heterozygous in nature, the use of haploids in a breeding programme could considerably shorten the time taken to produce new desirable cultivars. This is the first report on plant regeneration through somatic embryogenesis from floral tissue of cassava. This revised version was published online in July 2006 with corrections to the Cover Date.     

RAPD-based genetic diversity study of fifty cassava (Manihot esculenta Crantz) genotypes

Fifty cassava clones were studied using RAPD technique. They included landraces from the Wenchi, Nkoranza, Dormaa Ahenkoro and Asonafo districts of the Brong Ahafo region of Ghana and three improved varieties. Genetic diversity of these genotypes was studied using four primers, OPK-01, OPR-02, OPR-09 and OPJ-14. A total of 41 different bands were detected. Levels of polymorphic fragments detected by the four primers ranged from 90% to 100%. By pooling bands from individual accessions together, mean number of fragments per accession per primer ranged from 5.50±1.04 for the Improved cultivars to 7.00±0.71 for populations of landraces from Dormaa. Mean frequencies of fragments not detected by the primers for the accessions were 0.524±0.12, 0.460±0.12, 0.561±0.12 and 0.523±0.12 for landraces from Wenchi, Nkoranza, Dormaa Ahenkro, Asonafo and the Improved varieties, respectively. The grand mean frequency of individuals showing fragments not present in populations was 0.522±0.10. Genetic diversity estimates ranged from 0.290 to 0.425 (mean 0.352±0.05) for primer OPK-01, 0.001 to 0.381 (mean 0.309±0.06) for primer OPR-02, 0.335 to 0.344 (mean 0.283±0.04) for primer OPR-09 and 0.152 to 0.352 (mean 0.261±0.07) for primer OPJ-14. Within the accessions mean gene diversity estimates were 0.316±0.03, 0.293±0.09, 0.331±0.02, 0.322±0.07 and 0.247±0.03 for accessions from Wenchi, Nkoranza, Dormaa Ahenkro, Asonafo districts and the Improved varieties, respectively. Interpopulational genetic divergence ranged from 0.069 to 0.203 (mean 0.119±0.04). Rate of nucleotide substitution among the landraces was 9.8 per cent per site per year, while that for the Improved varieties was 15 per cent. This revised version was published online in August 2006 with corrections to the Cover Date.     

Characterization and genetic distance analysis of cassava (Manihot esculenta Crantz) germplasm from Mozambique using RAPD fingerprinting

Twenty-eight cassava genotypes from Mozambique, along with seven genotypes from Angola, Madagascar, Nigeria, Togo, Columbia, and Thailand for comparison, were fingerprinted using random amplified polymorphic DNA (RAPD) analysis. The Mozambican material represented a wide range of landraces. A total of 311 scored RAPD loci were used to calculate genetic distances between the genotypes. This revealed an average genetic distance of 3.1% between all the germplasm. The average genetic distance between the Mozambiquen genotypes was 2.7%, whilst the seven accessions from the other countries showed an average distance of 3.4%. Neighbor-joining (NJ) method cluster analysis of the genetic distance yielded a tree that did not indicate a relationship between geographic distribution and genetic diversity. This revised version was published online in August 2006 with corrections to the Cover Date.     

Histological analysis of embryo-sac formation and detection of meiotic diplospory in cassava (Manihot esculenta Crantz)

Summary Embryo-sac formation in six cultivated Asian cassava clones and one unknown wild type, was histologically investigated to determine apomictic potential in the genus Manihot. All the 6 clones were found to possess one single sexual embryo-sac containing 6–8 nuclei at maturity. However, one clone, Rayong 3 possessed two functional embryo-sacs at 12 hours after controlled pollination (HACP). The larger embryo-sac was 6-nucleate (all 3 nuclei of the egg apparatus, 1 polar and 2 antipodal cells) and located towards the false micropylar or the nucellar beak region. The smaller embryo-sac also contained a total of 6 nuclei and approximately 10 m³ or 1/2 the volume of the former and located towards the chalazal pole. Percentage sexuality estimates indicated a 100% sexual reproduction in embryo-sacs of all the clones analysed. However, apomeiotic relationships indicate a low (0.3%) degree of meiotic diplosporous embryo-sac formation, thus suggesting facultative apomixis in cv. Rayong 3. This is the first embryological identification and evidence of apomeiosis and apomictic potential in cassava.Contribution from Plant Breeding Laboratory, Applied Genetics & Biotechnology Division, Faculty of Agriculture, Miyazaki University, No. 91.     

The closest wild relatives of cassava ( Manihot esculenta Crantz)

The origin of cassava has long eluded researchers. The botanical origin, the geographical origin, the area of domestication, all are disputed matters. The argument advanced here is that, once the wild ancestor is identified, speculation about the taxonomy and the geographical cradle of the root crop will narrow considerably, as a number of species and geographical areas will automatically become excluded from consideration in matters of classification, domestication and evolution. Current knowledge about the three subjects shows that discoveries on the botanical origin of manioc have progressed a great deal, discoveries on the geographical origin are in progress, and discoveries on the area where the beginning of cultivation happened are incipient. In this paper, five Brazilian Manihot species are suggested as the closest wild relatives of cassava. One of them (M. esculenta ssp. flabellifolia) is regarded as the wild progenitor of modern cultivars and thus part of the primary genepool of the root crop. Another species (M. pruinosa) is regarded as the nearest species to the GP1 of cassava and can hardly be separated from the wild strain M. esculenta ssp. flabellifolia on morphological grounds. This revised version was published online in July 2006 with corrections to the Cover Date.     

Mating system in an experimental garden composed of cassava (Manihot esculenta Crantz) ethnovarieties

Crossing patterns were investigated in an experimental garden of ethnovarieties ofManihot esculenta (Euphorbiaceae) inPiracicaba, São Paulo, Brazil. A model of evolutionary dynamics for cassava presupposes genetic recombination by means of crossing within cassava gardens as a source that amplifies genetic diversity. Quantitative analysis of mating system parameters was performed using progeny arrays assayed for eight allozyme markers. The multilocus outcrossing rate (t _m)estimate (0.915±0.04)revealed that outcrossing was prevalent, but that a low level of self-pollination also occurred. The multilocus outcrossing rate ranged from 0.69 to 1.00 among eight varieties. The high value found for the outcrossing rate indicates that the ethnovarieties studied are preferentially allogamous. Genetic recombination occurred through crossing within the cassava garden, in agreement with an assumption of the model of evolutionary dynamics for this species.     

Efficient production of transgenic plants by Agrobacterium-mediated transformation of cassava (Manihot esculenta Crantz)

An efficient and reproducible method was developed for Agrobacterium-mediated transformation of embryogenic suspension cultures of cassava. LBA4404(pTOK233), containing the nptII, hph and gus marker genes, was used in the experiments. Chemical selection by means of kanamycin was used to establish 1037antibiotic resistant callus lines, of which 526 showed GUS expression. Of the 241 callus lines that were transferred to maturation medium 219formed somatic embryos. Thirty-seven of the 38 lines that were transferred to germination medium produced plants. GUS-positive plants could be obtained from 31 lines; in 14 of those lines 100% of the produced plants were GUS-positive, the remaining 17 lines yielded GUS-positive plants at an average of 72%. The transgenic nature of these plants was confirmed by Southern blot analysis. This revised version was published online in July 2006 with corrections to the Cover Date.     

Nuclear DNA content and in vitro induced somatic polyploidization cassava (Manihot esculenta Crantz) breeding

Summary The diploid (2C) amount of DNA in cassava (Manihot esculenta Crantz) is 1.67 picograms (pg) per cell nucleus. This value corresponds to 772 mega-base pairs in the haploid genome. The size of the nuclear genome in cassava is very small in comparison with other Angiosperms. Flow cytometry techniques were used to screen ploidy levels in a large population of in vitro plantlets treated with colchicine and oryzalin (3,5-dinitro-N⁴,N-dipropylsulphate). Culture of axillary node cuttings for 48 hours in liquid medium supplemented with 2.5 to 5.0 mM colchicine in combination with 2% dimethyl sulfoxide (DMSO) resulted in a high frequency (23 to 42%) of non-chimeric tetraploids in the V₃ generation. Although mixoploidy may persist in as many as four cycles of vegetative propagation of node cuttings, solid (non-chimeric) tetraploids can be identified by flow cytometry among in vitro plantlets and then rapidly propagated for field testing. A somatic polyploidization system is proposed for implementation in cassava breeding programmes.Dedicated to the memory of the late Dr. Novak. Correspondence to M. van Duren     

Production of embryogenic tissues and regeneration of transgenic plants in cassava (Manihot esculenta Crantz)

Disorganised embryogenic tissues have been utilised as target tissues for transgene insertion and transgenic plant regeneration in cassava (Manihot esculenta). The production of friable embryogenic callus in fourteen geographically diverse cassava cultivars, from which eleven were established as embryogenic suspension cultures, is reported. Embryogenic tissues were similar in nature in all cultivars tested although there was variation in the time required to generate friable callus and the growth rates of suspension cultures. Regeneration of plants has been achieved from eight cultivars but varied significantly in efficiency, with cv. TMS 60444and Line 2 from Zimbabwe being the most responsive. Tissues from the remaining eight cultivars became arrested at globular and torpedo stages of regeneration indicating that they most likely process an inherent ability to produce plants but require further research to allow this to be realised. Significant numbers of transgenic plants containing transgenes for putative resistance to important viral diseases of cassava in addition to visual marker genes have been regenerated. Transgenic plants from three the cultivars TMS 60444, Bonoua Rouge and M.Col 1505 were recovered after particle bombardment of embryogenic suspension cultures. Correlation's have been made between abnormal leaf morphology and plant vigour with the use of embryogenic suspension cultures for transgene insertion. As an result friable embryogenic callus is now being successfully utilsed as the target tissue for genetic transformation and plant regeneration at ILTAB. This revised version was published online in July 2006 with corrections to the Cover Date.     

A cytogenetic study of bilateral sexual polyploidization in cassava (Manihot esculenta Crantz)

Microsporogenesis and megasporogenesis were cytogenetically and histologically analysed in three cassava clones:‘Rayong 1′,‘Rayong 60′,‘M. mga’ and two hybrid lines,‘OMR 3641‐1’ and ‘OMR 3641‐1’ to elucidate the evolution of sexual polyploids in cassava. At telophase II, formation of 17‐21 micronuclei per pollen cell plate was observed in 16 out of 351 cell plates in ‘M. mga’. Micronuclei were observed at low (0.3‐2.3%) frequencies, at the sporad stage in all clones. Monads, dyads, triads and tetrads, which are established sources of high ploidy levels were observed at low (2.6%) and high (22.2%) frequencies. Megasporogenesis in ‘Rayong 1’ and ‘Rayong 60’ showed a lack of second meiotic divisions after a successful first division that resulted in partly unreduced embryo sacs with 2n eggs, suggesting another unrecognized and, as yet, unreported source of sexual polyploid formation in cassava. Meiotic abnormalities during microsporogenesis and megasporogenesis are implicated as being responsible for the formation of mixoploids (triploids and tetraploids) in cassava breeding programmes. A cytogenetic mechanism resulting in bilateral sexual polyploids through different gametic fertilization pathways in cassava is suggested and its role in breeding is briefly discussed.     

Evaluation of cassava (Manihot esculenta Crantz) germplasm collections using RAPD markers

Summary Deterioration of cotton, Gossypium hirsutum L., seed can adversely affect stands, seedling vigor, and subsequent performance of plants, but deterioration can be lessened by improved genetic resistance. This study was conducted to evaluate the relationship of physical and germination characteristics of cotton seed and to determine the inheritance of resistance to seed deterioration. Physical (weight, volume, density, and imbibition rate) and germination (germination of non-deteriorated seed and deteriorated seed) characteristics were determined for eight cotton genotypes. In addition, imbibition rate and the germination characteristics were determined for all possible F₁, including reciprocals, combinations of the eight genotypes. Variation among the parents for germination of deteriorated seed and imbibition rate were significantly, highly correlated (r=–0.97) and intrinsically related. Resistance to seed deterioration tended to increase as seed weight and volume decreased. Significant genetic effects were found for imbibition rate and germination of both non-deteriorated and deteriorated seed. However, general combining ability (GCA) accounted for only 21% of the variation in crosses sums of squares for germination of non-deteriorated seed. In contrast, GCA accounted for 90% of the variation for germination of deteriorated seed and imbibition rate. A breeding approach involving selection of early generation seed for ability to survive hot water treatment, then evaluating later generations for imbibition rate is suggested as a means to improve resistance to seed deterioration in cotton.     

Genetic linkage map of cassava (Manihot esculenta Crantz) based on AFLP and SSR markers

To generate a genetic linkage map of cassava ( Manihot esculenta Crantz), 58 F₁ progenies from a cross between Rayong 90 (female) and Rayong 5 (male) were examined in amplification fragment length polymorphism (AFLP) and simple sequence repeat (SSR) analyses. A total of 469 polymorphic markers consisting of 378 AFLPs generated from 76 primer combinations and 91 SSRs were identified. These markers were analyzed using the joinmap ^® 3.0 package program to construct a genetic linkage map. A total of 33 linkage groups of a common map were constructed from 119 AFLPs and 18 SSRs, spanning 1095 cM with an average of 7.99 cM between markers. The genetic linkage map generated in this study will be useful for genetic studies in cassava particularly for the identification of genetic markers linked to traits of interest, although the complex cassava genome suggests that maybe a long term objective.     

Leaf Waxes of Cassava (Manihot Esculenta Crantz) in Relation to Ecozone and Resistance to Xanthomonas Blight

Summary To elucidate the role of leaf surface structures as first barriers to confer resistance to bacterial blight, leaf stomata and their occlusion with leaf waxes were studied in cassava genotypes. For the first time, cassava leaf waxes were quantitatively and qualitatively analysed. Comparing the susceptible and resistant standard genotypes BEN86052 and TMS30572, respectively, the total quantity of triterpenes was significantly higher in the resistant genotype, grown in three ecozones of Benin. In cuticular leaf waxes of seven cassava genotypes the triterpenes beta amyrins, epi-taraxerol, taraxerone and taraxerol were dominant constituents across genotypes, and alkanes (C₂₅-C₃₃) and acids (C₃₀ and C₃₂) occurred in minor concentrations. Comparing seven genotypes, no clear relation between resistance or ecozones and total quantities of the major wax constituents was observed. Only the highly resistant genotype TMS30572 showed high triterpene levels irrespective of ecozone. Scanning electron-microscopy revealed a regular distribution of waxes at the abaxial leaf surface, covering and occluding stomatal pores of susceptible and resistant genotypes, while on the adaxial leaf surface waxes were in form of crystalloids and did not occlude the stomata. The number of stomata on the abaxial surfaces was about 7–11 fold higher than on the adaxial surfaces, where stomata were located along the midrib and major veins. No significant differences in stomata number were observed between genotypes varying in resistance to bacterial blight. It is suggested, that stomata on the adaxial surface might be portals of entry for the bacteria.     

Isozyme variation in taro (Colocasia esculenta (L.) Schott) from Asia and Oceania

Summary Isozyme variation was studied in 1,417 cultivars and wild forms of taro collected in Asia and Oceania. Seven polymorphic enzyme systems (MDH, IDH, PGI, 6-PGD, ME, SkDH, and ADH) revealed 143 isozyme phenotypes, or zymotypes, each uniquely characterized by the presence or absence of 56 electromorphs. Results showed greater isozyme variation in Asia than in Oceania, with Indonesia being the area of greatest diversity. No correlations were found between zymotypes and morphotypes or ploidy levels (as described by other investigators). Multivariate analyses of the isozyme data indicated that the majority of the Indonesian cultivars were different from the Philippine and Oceanian taro cultivars. Oceanian cultivars constituted a continuum of clusters and are thought to have originated from a narrow genetic base introduced from Indonesia. If taro breeding is to have any future in Oceania, it is important to exchange genotypes to broaden the base of existing breeding programmes.     

The effect of cassava mosaic disease on the genetic diversity of cassava in Uganda

Summary Cassava (Manihot esculenta) is a tropical crop that is grown in Africa, Latin America and Southeast Asia. Cassava was introduced from Latin America into West and East Africa at two independent events. In Uganda a serious threat to cassava's survival is the cassava mosaic disease (CMD). Uganda has had two notable CMD epidemics since the introduction of cassava in the 1850s causing severe losses. SSR markers were used to study the effect of CMD on the genetic diversity in five agroecologies in Uganda with high and low incidence of CMD. Surprisingly, high gene diversity was detected. Most of the diversity was found within populations, while the diversity was very small among agroecological zones and the high and low CMD incidence areas. The high genetic diversity suggests a mechanism by which diversity is maintained by the active involvement of the Ugandan farmer in continuously testing and adopting new genotypes that will serve their diverse needs. However, in spite of the high genetic diversity we found a loss of rare alleles in areas with high CMD incidence. To study the effect of the introgression history on the gene pool the genetic differentiation between East and West Africa was also studied. Genetic similarities were found between the varieties in Uganda and Tanzania in East Africa and Ghana in West Africa. Thus, there is no evidence for a differentiation of the cassava gene pool into a western and an eastern genetic lineage. However, a possible difference in the genetic constitution of the introduced cassava into East and West Africa may have been diminished by germplasm movement.     

Mapping wound-response genes involved in post-harvest physiological deterioration (PPD) of cassava (Manihot esculenta Crantz)

The genome locations of the wound-response genes that were expressedduring the post-harvest physiological deterioration (PPD) of cassava, suchas phenylalanine ammonia lyase, -1.3 glucanase, hydroxyprolinerich glycoprotein, catalase, 1-aminocyclopropane 1-carboxylate, cysteineprotease inhibitor, aspartic protease, a partial cDNA for serine/threonineprotein kinase and peroxidase, have been identified on the frameworkmolecular genetic map of cassava. Also, molecular markers linked toputative quantitative trait loci (QTLs) influencing PPD of cassava weremapped using an F₁mapping population derived from elite parentallines (TMS 30572 × cm 2177-2). A molecular linkage map previouslyconstructed based on the segregation of 240 RFLP, 100 RAPD, 85microsatellite and five isoenzyme markers on 144 F₁ individuals wasused for the QTL mapping.A set of 10 molecular markers with a significant association with putativeQTLs for PPD were identified based on probability values < 0.005in order to minimize the detection of false positives. Based on single-markerregression, eight putative QTLs located on the linkage groups G, P, L, U,and X of the female-derived framework map were found to explain between 5–12% of the phenotypic variance of the PPD. In the male-derived frameworkmap, two putative QTLs on linkage groups C and L explained 13% and11% of this variance, respectively. This study thus identified the majorgenome regions of cassava related to physiological post-harvestdeterioration, thereby providing tools for the identification of gene(s)controlling this trait.     

Assessing genetic diversity in the cassava (Manihot esculenta Crantz) germplasm collection in Brazil using PCR-based markers

Knowledge of the origin, organization and nature of the cassava (Manihot esculenta Crantz) germplasm collection in Brazil is incomplete due to lack of critical information on several aspects of the collection. This study verifies the utility of SSR-primed PCR markers for germplasm assessment and then utilizes these markers as well as RAPD's to characterize the Brazilian collection. We specifically address the following questions: 1)what is the relationship of morphologically closely related species to cultivated cassava? 2) What is the genetic diversity of cultivars within and between different habitats in Brazil? 3) Do agronomic traits and molecular markers reveal the same relationship among cassava accessions? 4) How complete is the Brazilian cassava collection and how well is it represented in the Word Core Collection of cassava, maintained by CIAT? Results of the interspecific studies of cassava and its wild relatives confirms the close relationship of cassava, Manihot esculenta ssp. esculenta to Manihot esculenta ssp. flabellifolia as well identifying several other closely related wild species. Next, PCR-based markers indicate a strong grouping of varieties related to the region of cultivation in Brazil. Moreover, important regions such as Cerrados and Amazon are relatively poorly represented in germplasm collections. Interestingly, the relationships of accessions based on agronomic traits are not fully congruent with relationships revealed with RAPD markers. Finally, the genetic diversity of the Brazilian cassava collection is not fully represented in the Core of the Word Core Collection of CIAT.