A molecular genetic map of cassava (Manihot esculenta Crantz)

 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. Received: 5 July 1996/Accepted: 22 November 1996     

Genetic mapping of resistance to bacterial blight disease in cassava (Manihot esculenta Crantz)

Cassava bacterial blight (CBB), caused by Xanthomonas axonopodis pv. manihotis (Xam), is a major disease of cassava (Manihot esculenta Crantz) in Africa and South America. Planting resistant varieties is the preferred method of disease control. Recent genetic mapping of an F₁ cross (TMS 30572 × CM 2177–2) led to the construction of the first molecular genetic map of cassava. To better understand the genetics of resistance to CBB, we evaluated individuals of the F₁ cross for CBB resistance by controlled greenhouse inoculations and visually assessed symptoms on days 7, 15, and 30 days after inoculation, using a scale where 0 = no disease and 5 = maximum susceptibility. Five Xam strains were used: CIO-84, CIO-1, CIO-136, CIO-295, and ORST X-27. Area under the disease progress curve (AUDPC) was used as a quantitative measure of resistance in QTL analysis by single-marker regression. Based on the AUDPC values, eight QTLs (quantitative trait loci), located on linkage groups B, D, L, N, and X of the female-derived framework map, were found to explain 9–20% of the phenotypic variance of the crop’s response to the five Xam strains. With the male-derived framework map, four QTLs on linkage groups G and C explained 10.7–27.1% of the variance. A scheme to confirm the usefulness of these markers in evaluating segregating populations for resistance to CBB is proposed. Received: 20 September 1999 / Accepted: 30 December 1999     

Genetic characterization of cassava (Manihot esculenta Crantz) genotypes using agro-morphological and single nucleotide polymorphism markers

Physiology and Molecular Biology of Plants - Dearth of information on extent of genetic variability in cassava limits the genetic improvement of cassava genotypes in Sierra Leone. The aim of this...     

AFLP analysis of African cassava (Manihot esculenta Crantz) germplasm resistant to the cassava mosaic disease (CMD)

Amplified fragment length polymophism was assessed in 20 land races and nine elite lines of cassava from Africa, resistant and susceptible to the cassava mosaic disease (CMD). Eleven accessions from a representative core collection from Latin America, previously studied by AFLPs, were included as a reference. AFLP data from all accessions was analyzed by both the unweighted pair group mean average (UPGMA) and multiple cluster analysis (MCA) methods of analysis. Genetic differentiation between clusters and the coefficient of genetic differentiation was also calculated. Results reveal a genetic divergence between African and Latin American accessions, although some overlap was found between them. African land races resistant to CMD, were also found to be genetically differentiated from susceptible land races and from resistant elite lines. AFLP analysis identified a considerable number of duplicates in the African accessions, suggesting a sizeable percentage of redundancy. A unique AFLP fragment, found in a relatively high frequency in African accessions, but absent in the Latin American accessions, was found to be associated with branching pattern by QTL mapping in an F1 progeny derived from African and Latin American parents. The likely source and the utility of the unique AFLP fragment in understanding the processes of genetic divergence in Africa is discussed. Received: 15 May 1999 / Accepted: 28 July 1999     

Gene-based microsatellites for cassava (Manihot esculenta Crantz): prevalence, polymorphisms, and cross-taxa utility

Background  

Cassava (Manihot esculenta Crantz), a starchy root crop grown in tropical and subtropical climates, is the sixth most important crop in the world after wheat, rice, maize, potato and barley. The repertoire of simple sequence repeat (SSR) markers for cassava is limited and warrants a need for a larger number of polymorphic SSRs for germplasm characterization and breeding applications.     

In vitro flowering in cassava (Manihot esculenta Crantz)

Meristem-derived plantlets of cassava (Manihot esculenta Crantz) were induced to flower in vitro. Five genotypes out of 13 consistently responded to our culture conditions giving rise to male or female flowers. Male flowers contained anthers in which meiosis occurred and apparently normal pollen grains were formed.     

Photosynthetic responses of cassava cultivars (Manihot esculenta Crantz) from different habitats to temperature

Maximum photosynthetic CO₂ exchange rates (P_n) of single attached leaves were determined for several cassava cultivars selected from different habitats and grown in pots outdoors at CIAT, Colombia, S.A. P_n rates were in a narrow range of 22 to 26 mol CO₂ m^–2s^–1 for all cultivars tested when measured at high photon flux density, normal air, optimum temperature and with low leaf-air vapor pressure differences. For all tested cultivars (9 cvs.), there was a broad optimum temperature for P_n between 25 to 35°C. At temperatures below and above this range P_n declined in all cultivars with P_n rates reaching 80% of maximum at 20 and 40°C. P_n temperature coefficient (Q₁₀) from 15–25°C was 1.6±0.2 across cultivars. No consistent relation existed between P_n, optimum temperature, and the original habitat.     

In vitro propagation of cassava (Manihot esculenta Crantz)

A method is presented for the rapid in vitro propagation of cassava (Manihot esculenta Crantz). Nodal explants were induced to grow as multiple-shoot cultures on a medium containing 1.0 M 6-benzylamino purine (BAP), supplemented with 0.25 M -naphthaleneacetic acid (NAA). Nodes were removed from the shoots after three weeks of growth and subcultured on fresh culture medium. An average of 7.0 nodes were produced from each explanted node after three weeks in culture. Nodal explants were transferred to a medium containing 2.5 M indole-3-butyric acid (IBA) to improve root initiation on the developing plantlets. Plant establishment was possible upon transfer to soil. In vitro propagation offers enhanced rates of multiplication over more conventional methods of propagation. In addition, in vitro propagation facilitates the storage and international exchange of cassava germplasm.     

REVIEW ARTICLE: Cyanogenesis in cassava (Manihot esculenta Crantz)

Cassava is the most agronomically important of the cyanogeniccrops. Linamarin, the predominant cyanogenic glycoside in cassava,can accumulate to concentrations as high as 500 mg kg^–1fresh weight in roots and to higher levels in leaves. Recently,the pathway of linamarin synthesis and the cellular site oflinamarin storage have been determined. In addition, the cyanogenicenzymes, linamarase and hydroxynitrile lyase, have been characterizedand their genes cloned. These results, as well as studies onthe organ- and tissue-specific localization of linamarase andhydroxy-nitrile lyase, allow us to propose models for the regulationof cyanogenesis in cassava. There remain, however, many unansweredquestions regarding the tissue-specific synthesis, transport,and accumulation of cyanogenic glycosides. The resolution ofthe sequestions will facilitate the development of food processing,biochemical and transgenic plant approaches to reducing thecyanogen content of cassava foods. Key words: Cyanide, cyanogenic glycosides, linamarin, cyanogens     

木薯体细胞胚胎发生及植株再生研究

对木薯体细胞胚胎发生的影响因素进行了优化研究。结果表明,基因型对木薯体细胞胚胎发生影响很大,在供试的六个品种中,“华南 8 号”的体细胞胚胎发生率和产胚量最高,分别为 65% 和19个;侧芽茎尖为最佳外植体,体细胞胚胎发生的最佳培养基为MS +0.5mg/L CuSO4 + 4 mg/L 2,4-D。同时,对木薯体细胞胚再生成完整植株的主要影响因素作了分析,建立了一个高效的植株再生体系。     

Regeneration of transgenic cassava plants (Manihot esculenta Crantz) through Agrobacterium-mediated transformation of embryogenic suspension cultures

A protocol was developed for Agrobacterium-mediated transformation of embryogenic suspension cultures of cassava. The bacterial strain ABI containing the binary vector pMON977 with the nptII gene as selectable marker and an intron-interrupted uidA gene (encoding β-glucuronidase) as visible marker was used for the experiments. Selection of transformed tissue with paromomycin resulted in the establishment of antibiotic-resistant, β-glucuronidase-expressing lines of friable embryogenic callus, from which embryos and subsequently plants were regenerated. Southern blot analysis demonstrated stable integration of the uidA gene into the cassava genome in five lines of transformed embryogenic suspension cultures and in two plant lines.     

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 crop's present day variability.     

Cytogenetics of Manihot esculenta Crantz (cassava) and eight related species

Thirty-nine cultivars of cassava and eight related wild species of Manihot were analyzed in this work for number, morphology and size of chromosomes, prophase condensation pattern and the structure of the interphase nucleus. In four accessions, the chromosome size was measured and in some others, the number of secondary constrictions, meiotic behavior, C-band pattern, CMA/DAPI bands, nucleoli number and the location of 5S and 18S-5.8S-28S rDNA sites were also observed. All investigated accessions showed a similar karyotype with 2n = 36, small metacentric to submetacentric chromosomes. Two pairs of terminal secondary constrictions were observed in the chromosome complement of each accession except Manihot sp. 1, which presented two proximal secondary constrictions. The prophase chromosome condensation pattern was proximal and the interphase nuclei structure was areticulate to semi-reticulate. The meiosis, investigated in seven cultivars and four wild species, was regular, displaying 18 bivalents. C-banding revealed heterochromatin in 9 or 10 chromosomes. The analysis with fluorochromes frequently showed four chromosome pairs with a single CMA+ terminal or subterminal band and a few other chromosomes with DAPI+ unstable bands. Six 45S rDNA sites were revealed by FISH, which seemed to colocalize with six CMA+ bands. Only one chromosome pair presented a 5S rDNA site. The maximum nucleoli number observed per nucleus was also six. These data suggest that all Manihot species present a very similar chromosome complement.     

Microsatellites in Cassava (Manihot esculenta Crantz): discovery, inheritance and variability

 Fourteen microsatellites containing GA-repeats were isolated and characterized in cassava (Manihot esculenta Crantz, Euphorbiaceae). Microsatellite heterozygosity (h) was estimated in 48 accessions using (³²P)-end-labeled primers and in more than 500 accessions using fluorescence-based genotyping. Heterozygosity values ranged from 0.00 to 0.88 and the number of alleles detected varied from 1 to 15. The reproducibility of allele sizing was also assessed using fluorescence-based genotyping. The average inter-gel size difference was 1.03 nucleotides. Chi-square tests (χ²) were performed to analyse segregation distortion and the linkage between alleles segregating from either or both parents in an F₁ mapping population. Most microsatellite loci segregated in the expected 1 : 1, 1 : 2 : 1 or 1 : 1 : 1 : 1 ratio. Linkage was detected between loci segregating from either parent, and segregation distortion from the male parent was detected for locus GA-131. Approximately 80% of the microsatellites detected one or two alleles per accession, suggesting a low degree of microsatellite locus duplication, an unexpected finding for a putative allopolyploid, highly heterozygous species. The high h values of most microsatellites, their amplification in other Manihot taxa and their suitability for high-throughput, fluorescence-based genotyping, make microsatellites the marker of choice for germplasm characterization and saturation of the cassava map. Received: 4 September 1997 / Accepted 16 March 1998     

Effect of medium salt concentration on differentiation and maturation of somatic embryos of cassava (Manihot esculenta Crantz)

Culture of cassava somatic embryos on media with an altered macro- and micro-nutrient salt concentration affected embryo development and germination capability. In the tests, quarter-, half-, full- or double-strength Murashige and Skoog (MS) media were compared. The maximum number of somatic embryos differentiated from a proliferative nodular embryogenic callus (NEC) on either half- or full-strength MS medium, and the greatest numbers of cotyledonary stage embryos were formed on full-strength MS medium. Developed somatic embryos were then desiccated above a saturated K2SO4 solution for 10 d. After transfer to germination medium, embryos that had developed on half- and full-strength MS medium yielded 8.3 and 8.6 germinants g(-1) NEC tissue, respectively. For this important but often disregarded culture factor, either half- or full-strength MS medium is recommended for both the differentiation and development of cassava somatic embryos that are capable of germination.     

Response of cassava (Manihot esculenta Crantz) to water stress and fertilization

Experiments done in Santander de Quilichao (Cauca, Colombia) on two cassava cultivars indicated that cassava had at least three defence mechanisms against water deficit, enabling it to assimilate and store photosynthates in roots, even during prolonged droughts. These mechanisms include partial stomatal closure, ability of leaves to maintain reasonable net photosynthetic rate for long periods of water stress, reduced leaf area, and exploration of water from deep soil layers. While cassava responded positively to fertilization, no significant statistical differences were found between treatments of stress and non-stress, confirming cassava's ability to tolerate soil water deficit. This revised version was published online in June 2006 with corrections to the Cover Date.     

Improvements of cyclic somatic embryogenesis of cassava (Manihot esculenta Crantz)

Summary In cassava a cyclic system of somatic embryogenesis was developed. Primary (torpedo shaped or germinated) embryos, originating from leaf lobes, could only be obtained after culture on solid medium. Cyclic embryos, originating from embryos, could be obtained in both liquid and on solid medium. The production of embryos in liquid medium was distinctly higher, faster and more synchronized than on solid medium. Lower densities and fragmentation of starting embryos improved the production significantly. The highest production found was 32.1 embryos per initial embryo. In all treatments the explants initiated multiple embryos. The production of single embryos was achieved by pressing starting embryos through a fine meshed sieve, indicating that embryos can be produced from a piece of tissue with a restricted number of cells. The shoot conversion rate of embryos from liquid medium was comparable with that of embryos from solid medium.Abbreviations BM Basal Medium - MIE medium volume per initial embryo - E/IE number of Embryos per Initial Embryo     

Minor root rot pathogens of cassava (Manihot esculenta Crantz) in Nigeria

Many different species of fungi are often isolated from rotted cassava root tubers and pathogenicity studies have often implicated Botryodiplodia theobromae and Fusarium solani as the major causal pathogens. Consequently, more attention has often been focused on Botryodiplodia theobromae and Fusarium solani with little or no attention on the other minor pathogens. Considering the increasing importance of cassava to the Nigerian economy and the fact that minor root rot pathogens of cassava today could become major tomorrow, the aim of this research is to determine the incidence, pathogenicity and symptoms of the minor root rot pathogens in cassava from cassava fields within the derived savanna and the humid forest of Nigeria. Isolation of associated fungi was done on rotted root samples and the pathogenicity of these isolates were established by inoculating them into healthy cassava tuberous roots and subsequently reisolating them from resulting rotted tissue. The less frequently isolated fungi where Macrophomina sp., Trichoderma sp., Aspergillus niger, Aspergillus flavus, Sclerotium rolfsii and Fungus ‘A’ (a yet to be identified fungus). Repeated experiments confirmed a constant relationship between inoculated fungus and the resulting rotted tissue colour. The root rot tissue colours associated with inoculated pathogens in the laboratory were identical with the pathogens colony colour on potato dextrose agar.     

Response of cassava (Manihot esculenta Crantz) to water stress and fertilization

Experiments done in Santander de Quilichao (Cauca, Colombia) on two cassava cultivars indicated that cassava had at least three defence mechanisms against water deficit, enabling it to assimilate and store photosynthates in roots, even during prolonged droughts. These mechanisms include partial stomatal closure, ability of leaves to maintain reasonable net photosynthetic rate for long periods of water stress, reduced leaf area, and exploration of water from deep soil layers. While cassava responded positively to fertilization, no significant statistical differences were found between treatments of stress and non-stress, confirming cassava's ability to tolerate soil water deficit.     

Characterization of cyanogenic beta-glucosidase (linamarase) from cassava (Manihot esculenta Crantz)

Linamarase (EC 3.2.1.21) was purified from cassava petiole, stem, and root cortex by ammonium sulfate precipitation, column chromatography on Sepharose 6B, and chromatofocusing. The last step resolved the enzyme from each source into three forms with pI values of 4.3, 3.3, and 2.9. Each form was found to be oligomeric, consisting of one kind of subunit, Mr 63,000. The major isozyme with a pI of 4.3 from petiole showed a Km for linamarin of 0.6 mM and possessed both beta-glucosidase and beta-fucosidase activities. The former was sensitive to inhibition by delta-gluconolactone, isopropyl-beta-D-thioglucoside, and HgCl2, whereas the latter was inhibited by Tris ion.