Provitamin A biofortification of cassava enhances shelf life but reduces dry matter content of storage roots due to altered carbon partitioning into starch.

Storage roots of cassava (Manihot esculenta Crantz), a major subsistence crop of sub-Saharan Africa, are calorie rich but deficient in essential micronutrients, including provitamin A ß-carotene. In this study, ß-carotene concentrations in cassava storage roots were enhanced by co-expression of transgenes for deoxy-d-xylulose-5-phosphate synthase (DXS) and bacterial phytoene synthase (crtB), mediated by the patatin-type 1 promoter. Storage roots harvested from field-grown plants accumulated carotenoids to ≤50 µg/g DW, 15- to 20-fold increases relative to roots from nontransgenic plants. Approximately 85%-90% of these carotenoids accumulated as all-trans-ß-carotene, the most nutritionally efficacious carotenoid. ß-Carotene-accumulating storage roots displayed delayed onset of postharvest physiological deterioration, a major constraint limiting utilization of cassava products. Large metabolite changes were detected in ß-carotene-enhanced storage roots. Most significantly, an inverse correlation was observed between ß-carotene and dry matter content, with reductions of 50%-60% of dry matter content in the highest carotenoid-accumulating storage roots of different cultivars. Further analysis confirmed a concomitant reduction in starch content and increased levels of total fatty acids, triacylglycerols, soluble sugars and abscisic acid. Potato engineered to co-express DXS and crtB displayed a similar correlation between ß-carotene accumulation, reduced dry matter and starch content and elevated oil and soluble sugars in tubers. Transcriptome analyses revealed a reduced expression of genes involved in starch biosynthesis including ADP-glucose pyrophosphorylase genes in transgenic, carotene-accumulating cassava roots relative to nontransgenic roots. These findings highlight unintended metabolic consequences of provitamin A biofortification of starch-rich organs and point to strategies for redirecting metabolic flux to restore starch production.

Overexpression of Arabidopsis FLOWERING LOCUS T (FT) gene improves floral development in cassava (Manihot esculenta, Crantz).

Cassava is a tropical storage-root crop that serves as a worldwide source of staple food for over 800 million people. Flowering is one of the most important breeding challenges in cassava because in most lines flowering is late and non-synchronized, and flower production is sparse. The FLOWERING LOCUS T (FT) gene is pivotal for floral induction in all examined angiosperms. The objective of the current work was to determine the potential roles of the FT signaling system in cassava. The Arabidopsis thaliana FT gene (atFT) was transformed into the cassava cultivar 60444 through Agrobacterium-mediated transformation and was found to be overexpressed constitutively. FT overexpression hastened flower initiation and associated fork-type branching, indicating that cassava has the necessary signaling factors to interact with and respond to the atFT gene product. In addition, overexpression stimulated lateral branching, increased the prolificacy of flower production and extended the longevity of flower development. While FT homologs in some plant species stimulate development of vegetative storage organs, atFT inhibited storage-root development and decreased root harvest index in cassava. These findings collectively contribute to our understanding of flower development in cassava and have the potential for applications in breeding.

Phenotypic approaches to drought in cassava: review.

Cassava is an important crop in Africa, Asia, Latin America, and the Caribbean. Cassava can be produced adequately in drought conditions making it the ideal food security crop in marginal environments. Although cassava can tolerate drought stress, it can be genetically improved to enhance productivity in such environments. Drought adaptation studies in over three decades in cassava have identified relevant mechanisms which have been explored in conventional breeding. Drought is a quantitative trait and its multigenic nature makes it very challenging to effectively manipulate and combine genes in breeding for rapid genetic gain and selection process. Cassava has a long growth cycle of 12-18 months which invariably contributes to a long breeding scheme for the crop. Modern breeding using advances in genomics and improved genotyping, is facilitating the dissection and genetic analysis of complex traits including drought tolerance, thus helping to better elucidate and understand the genetic basis of such traits. A beneficial goal of new innovative breeding strategies is to shorten the breeding cycle using minimized, efficient or fast phenotyping protocols. While high throughput genotyping have been achieved, this is rarely the case for phenotyping for drought adaptation. Some of the storage root phenotyping in cassava are often done very late in the evaluation cycle making selection process very slow. This paper highlights some modified traits suitable for early-growth phase phenotyping that may be used to reduce drought phenotyping cycle in cassava. Such modified traits can significantly complement the high throughput genotyping procedures to fast track breeding of improved drought tolerant varieties. The need for metabolite profiling, improved phenomics to take advantage of next generation sequencing technologies and high throughput phenotyping are basic steps for future direction to improve genetic gain and maximize speed for drought tolerance breeding.

Retention during processing and bioaccessibility of ß-carotene in high ß-carotene transgenic cassava root.

Cassava is a root crop that serves as a primary caloric source for many African communities despite its low content of ß-carotene (ßC). Carotenoid content of roots from wild type (WT) and three transgenic lines with high ßC were compared after cooking and preparation of nonfermented and fermented flours according to traditional African methods. The various methods of processing all decreased ßC content per gram dry weight regardless of genotype. The greatest loss of ßC occurred during preparation of gari (dry fermentation followed by roasting) from WT and transgenic lines. The quantities of ßC in cooked transgenic cassava root that partitioned into mixed micelles during in vitro digestion and transported into Caco-2 cells were significantly greater than those for identically processed WT root. These results suggest that transgenic high ßC cassava will provide individuals with greater quantities of bioaccessible ßC.

The BioCassava plus program: biofortification of cassava for sub-Saharan Africa.

More than 250 million Africans rely on the starchy root crop cassava (Manihot esculenta) as their staple source of calories. A typical cassava-based diet, however, provides less than 30% of the minimum daily requirement for protein and only 10%-20% of that for iron, zinc, and vitamin A. The BioCassava Plus (BC+) program has employed modern biotechnologies intended to improve the health of Africans through the development and delivery of genetically engineered cassava with increased nutrient (zinc, iron, protein, and vitamin A) levels. Additional traits addressed by BioCassava Plus include increased shelf life, reductions in toxic cyanogenic glycosides to safe levels, and resistance to viral disease. The program also provides incentives for the adoption of biofortified cassava. Proof of concept was achieved for each of the target traits. Results from field trials in Puerto Rico, the first confined field trials in Nigeria to use genetically engineered organisms, and ex ante impact analyses support the efficacy of using transgenic strategies for the biofortification of cassava.

Resistance to the whitefly, Aleurotrachelus socialis, in wild populations of cassava, Manihot tristis.

The levels of resistance in the wild species of cassava, Manihot tristis Muell-Arg. (Malpighiales: Euphorbiaceae), to the whitefly, Aleurotrachelus socialis Bondar (Hemiptera: Alelyrodidae), the most important economic pest in cassava, Manihot esculenta Crantz (Malpighiales: Euphorbiaceae) crops in South America, were estimated under glasshouse conditions. The parameters of the life history of A. socialis were studied on TST-26 and TST-18 accessions of the wild parent and compared with the susceptible (CMC-40) and resistant (MEcu-72) cultivars. The average longevity on the wild accessions (TST-26, 4.1; TST-18, 4.6 days) and oviposition rates (TST-26, 2.0; TST-18, 1.6 eggs/female/2 days) of the A. socialis females were not significantly different from those of MEcu-72 (5.1 days and 3.4 eggs/female/2 days). The longevity and oviposition rates on CMC-40 were highest (11 days and 8.6 eggs/female/2 days). Analyses of the demographic parameters (Ro, r(m); DT) showed a significant impact of the M. tristis accessions on the potential growth of A. socialis. The average survival time of adults that fed on TST-26, TST-18, and MEcu-72 were significantly different from those recorded on the susceptible genotype. Results from this study revealed important levels of resistance to the whitefly A. socialis on the TST-26 and TST-18 accessions due to the marked differences found for longevity and reproduction, which influenced and were consistent with the differences found in the net reproduction rate (Ro), intrinsic growth rate (r(m)) and population doubling time (DT). The combined effect of these parameters indicated that M. tristis accessions were inappropriate hosts for A. socialis.

Quantitative trait loci controlling cyanogenic glucoside and dry matter content in cassava (Manihot esculenta Crantz) roots.

Cassava (Manihot esculenta Crantz) is a starchy root crop grown in the tropics mainly by small-scale farmers even though agro-industrial processing is rapidly increasing. For this processing market improved varieties with high dry matter root content (DMC) is required. Potentially toxic cyanogenic glucosides are synthesized in the leaves and translocated to the roots. Selection for varieties with low cyanogenic glucoside potential (CNP) and high DMC is among the principal objectives in cassava breeding programs. However, these traits are highly influenced by the environmental conditions and the genetic control of these traits is not well understood. An S(1) population derived from a cross between two bred cassava varieties (MCOL 1684 and Rayong 1) that differ in CNP and DMC was used to study the heritability and genetic basis of these traits. A broad-sense heritability of 0.43 and 0.42 was found for CNP and DMC, respectively. The moderate heritabilities for DMC and CNP indicate that the phenotypic variation of these traits is explained by a genetic component. We found two quantitative trait loci (QTL) on two different linkage groups controlling CNP and six QTL on four different linkage groups controlling DMC. One QTL for CNP and one QTL for DMC mapped near each other, suggesting pleiotrophy and/or linkage of QTL. The two QTL for CNP showed additive effects while the six QTL for DMC showed additive effect, dominance or overdominance. This study is a first step towards developing molecular marker tools for efficient breeding of CNP and DMC in cassava.

Discovery of an amylose-free starch mutant in cassava (Manihot esculenta Crantz).

One of the objectives of the cassava-breeding project at CIAT is the identification of clones with special root quality characteristics. A large number of self-pollinations have been made in search of useful recessive traits. During 2006 harvests an S1 plant produced roots that stained brownish-red when treated with an iodine solution, suggesting that it had lower-than-normal levels of amylose in its starch. Colorimetric and DSC measurements indicated low levels (3.4%) and an absence of amylose in the starch, respectively. SDS-PAGE demonstrated the absence of GBSS enzyme in the starch from these roots. Pasting behavior was analyzed with a rapid visco-analyzer and resulted in larger values for peak viscosity, gel breakdown, and setback in the mutant compared with normal cassava starch. Solubility was considerably reduced, while the swelling index and volume fraction of the dispersed phase were higher in the mutant. No change in starch granule size or shape was observed. This is the first report of a natural mutation in cassava that drastically reduces amylose content in root starch.

Genetic diversity and variety composition of cassava on small-scale farms in Uganda: an interdisciplinary study using genetic markers and farmer interviews.

Cassava is a tropical crop and grown for its tuberous starchy roots. In Africa it is mainly cultivated by small-scale farmers who observe, select and name their cassava varieties based on morphology, food, social and economic interest. Here we have used an interdisciplinary approach involving farmer interviews, genetic markers and morphological descriptors to study the composition of cassava varieties on small-scale farms in 11 villages located in three districts in Uganda, the genetic structure within and between these varieties and their morphology. The composition of local, newly introduced and improved varieties differed widely between villages and districts. The Ugandan farmers in our study seemed to adopt improved varieties to a greater extent when there was a nearby market, prevalence of disease epidemics and good extension service. We found considerable genetic variation both within and between cassava varieties though the variation was larger between varieties. However, most local and improved varieties showed predominating genotypes at many loci. Accessions of commonly grown varieties meeting farmers' preferences could therefore be selected and implemented in future breeding programmes involving development, dissemination and adoption. The like-named varieties in different villages were genetically similar, demonstrating farmers' ability to differentiate and maintain the same variety over large areas. However, some varieties with different names in different villages showed both genetic and morphological similarity, suggesting that farmers may rename plants when they are introduced into their fields. The large differences found in variety and genetic composition between villages and districts in Uganda may be a result of the diverse needs and growing conditions characteristic for traditional farming system. This suggests that efforts to conserve and increase the genetic diversity in farmers' fields will require policies tailored to each area.

DArT for high-throughput genotyping of Cassava (Manihot esculenta) and its wild relatives.

Understanding the distribution of genetic diversity within and among individuals, populations, species and gene pools is crucial for the efficient management of germplasm collections. Molecular markers are playing an increasing role in germplasm characterization, yet their broad application is limited by the availability of markers, the costs and the low throughput of existing technologies. This is particularly true for crops of resource-poor farmers such as cassava, Manihot esculenta. Here we report on the development of Diversity Arrays Technology (DArT) for cassava. DArT uses microarrays to detect DNA polymorphism at several hundred genomic loci in a single assay without relying on DNA sequence information. We tested three complexity reduction methods and selected the two that generated genomic representations with the largest frequency of polymorphic clones (PstI/TaqI: 14.6%, PstI/BstNI: 17.2%) to produce large genotyping arrays. Nearly 1,000 candidate polymorphic clones were detected on the two arrays. The performance of the PstI/TaqI array was validated by typing a group of 38 accessions, 24 of them in duplicate. The average call rate was 98.1%, and the scoring reproducibility was 99.8%. DArT markers displayed fairly high polymorphism information content (PIC) values and revealed genetic relationships among the samples consistent with the information available on these samples. Our study suggests that DArT offers advantages over current technologies in terms of cost and speed of marker discovery and analysis. It can therefore be used to genotype large germplasm collections.

An EST resource for cassava and other species of Euphorbiaceae.

Cassava (Manihot esculenta) is a major food staple for nearly 600 million people in Africa, Asia, and Latin America. Major losses in yield result from biotic and abiotic stresses that include diseases such as Cassava Mosaic Disease (CMD) and Cassava Bacterial Blight (CBB), drought, and acid soils. Additional losses also occur from deterioration during the post-harvest storage of roots. To help cassava breeders overcome these obstacles, the scientific community has turned to modern genomics approaches to identify key genetic characteristics associated with resistance to these yield-limiting factors. One approach for developing a genomics program requires the development of ESTs (expressed sequence tags). To date, nearly 23,000 ESTs have been developed from various cassava tissues, and genotypes. Preliminary analysis indicates existing EST resources contain at least 6000-7000 unigenes. Data presented in this report indicate that the cassava ESTs will be a valuable resource for the study of genetic diversity, stress resistance, and growth and development, not only in cassava, but also other members of the Euphorbiaceae family.