Comparative transcriptomics analysis reveals defense mechanisms of Manihot esculenta Crantz against Sri Lanka Cassava MosaicVirus.

BACKGROUND: Cassava mosaic disease (CMD), caused by Sri Lankan cassava mosaic virus (SLCMV) infection, has been identified as a major pernicious disease in Manihot esculenta Crantz (cassava) plantations. It is widespread in Southeast Asia, especially in Thailand, which is one of the main cassava supplier countries. With the aim of restricting the spread of SLCMV, we explored the gene expression of a tolerant cassava cultivar vs. a susceptible cassava cultivar from the perspective of transcriptional regulation and the mechanisms underlying plant immunity and adaptation. RESULTS: Transcriptomic analysis of SLCMV-infected tolerant (Kasetsart 50 [KU 50]) and susceptible (Rayong 11 [R 11]) cultivars at three infection stages-that is, at 21 days post-inoculation (dpi) (early/asymptomatic), 32 dpi (middle/recovery), and 67 dpi (late infection/late recovery)-identified 55,699 expressed genes. Differentially expressed genes (DEGs) between SLCMV-infected KU 50 and R 11 cultivars at (i) 21 dpi to 32 dpi (the early to middle stage), and (ii) 32 dpi to 67 dpi (the middle stage to late stage) were then identified and validated by real-time quantitative PCR (RT-qPCR). DEGs among different infection stages represent genes that respond to and regulate the viral infection during specific stages. The transcriptomic comparison between the tolerant and susceptible cultivars highlighted the role of gene expression regulation in tolerant and susceptible phenotypes. CONCLUSIONS: This study identified genes involved in epigenetic modification, transcription and transcription factor activities, plant defense and oxidative stress response, gene expression, hormone- and metabolite-related pathways, and translation and translational initiation activities, particularly in KU 50 which represented the tolerant cultivar in this study.

Collection, genotyping and virus elimination of cassava landraces from Tanzania and documentation of farmer knowledge.

Cassava (Manihot esculenta Crantz.) has been a vital staple and food security crop in Tanzania for several centuries, and it is likely that its resilience will play a key role in mitigating livelihood insecurities arising from climate change. The sector is dominated by smallholder farmers growing traditional landrace varieties. A recent surge in virus diseases and awareness in the commercial potential of cassava has prompted a drive to disseminate improved varieties in the country. These factors however also threaten the existence of landraces and associated farmer knowledge. It is important that the landraces are conserved and utilized as the adaptive gene complexes they harbor can drive breeding for improved varieties that meet agro-ecological adaptation as well as farmer and consumer needs, thereby improving adoption rates. Here we report on cassava germplasm collection missions and documentation of farmer knowledge in seven zones of Tanzania. A total of 277 unique landraces are identified through high-density genotyping. The large number of landraces is attributable to a mixed clonal/sexual reproductive system in which the soil seed bank and incorporation of seedlings plays an important role. A striking divergence in genetic relationships between the coastal regions and western regions is evident and explained by (i) independent introductions of cassava into the country, (ii) adaptation to prevailing agro-ecological conditions and (iii) farmer selections according to the intended use or market demands. The main uses of cassava with different product profiles are evident, including fresh consumption, flour production, dual purpose incorporating both these uses and longer-term food security. Each of these products have different trait requirements. Individual landraces were not widely distributed across the country with limited farmer-to-farmer diffusion with implications for seed systems.

Kinship networks of seed exchange shape spatial patterns of plant virus diversity.

By structuring farmers' informal networks of seed exchange, kinship systems play a key role in the dynamics of crop genetic diversity in smallholder farming systems. However, because many crop diseases are propagated through infected germplasm, local seed systems can also facilitate the dissemination of seedborne pathogens. Here, we investigate how the interplay of kinship systems and local networks of germplasm exchange influences the metapopulation dynamics of viruses responsible for the cassava mosaic disease (CMD), a major threat to food security in Africa. Combining anthropological, genetic and plant epidemiological data, we analyzed the genetic structure of local populations of the African cassava mosaic virus (ACMV), one of the main causal agents of CMD. Results reveal contrasted patterns of viral diversity in patrilineal and matrilineal communities, consistent with local modes of seed exchange. Our results demonstrate that plant virus ecosystems have also a cultural component and that social factors that shape regional seed exchange networks influence the genetic structure of plant virus populations.

Protein Cross-Interactions for Efficient Photosynthesis in the Cassava Cultivar SC205 Relative to Its Wild Species.

The underlying mechanisms of the higher photosynthetic efficiency of cultivated cassava relative to its wild species are poorly understood. In the present study, proteins in leaves and chloroplasts were analyzed to compare the differences among the cultivar SC205, its wild ancestor W14, and the related species Glaziovii. The functions of differential proteins are associated with 10 ontology groups including photosynthesis, carbohydrate and energy metabolism, as well as potential signal pathway. The protein-protein networks among 41 differential proteins showed that PGK1 is a hub protein and protein cross-interactions affected the differentiation of photosynthetic rate. Anatomy patterns and PEPC detection suggested that SC205 has more C4 photosynthesis characteristics than Glaziovii and W14. Finally, a mechanism model of the efficient photosynthesis was proposed based on the remarkable variations in photosynthetic parameters and protein functions in the domestic cultivars.

The Class III Peroxidase (POD) Gene Family in Cassava: Identification, Phylogeny, Duplication, and Expression.

The class III peroxidase (POD) enzymes participate in plant development, hormone signaling, and stress responses. However, little is known about the POD family in cassava. Here, we identified 91 cassava POD genes (MePODs) and classified them into six subgroups using phylogenetic analysis. Conserved motif analysis demonstrated that all MePOD proteins have typical peroxidase domains, and gene structure analysis showed that MePOD genes have between one and nine exons. Duplication pattern analysis suggests that tandem duplication has played a role in MePOD gene expansion. Comprehensive transcriptomic analysis revealed that MePOD genes in cassava are involved in the drought response and postharvest physiological deterioration. Several MePODs underwent transcriptional changes after various stresses and related signaling treatments were applied. In sum, we characterized the POD family in cassava and uncovered the transcriptional control of POD genes in response to various stresses and postharvest physiological deterioration conditions. These results can be used to identify potential target genes for improving the stress tolerance of cassava crops.

Identification and expression analysis of MinD gene involved in plastid division in cassava.

Cassava is a tropical crop known for its starchy root and excellent properties. Considering that starch biosynthesis in the amyloplast is affected by its division, it appears conceivable that the regulation of plastid division plays an important role in starch accumulation. As a member of the Min system genes, MinD participated in the spatial regulation of the position of the plastid division site.In our studies, sequence analysis and phylogenetic analysis showed that MeMinD has been highly conserved during the evolutionary process. Subcellular localisation indicated that MeMinD carries a chloroplast transit peptide and was localised in the chloroplast. Overexpression of MeMinD resulted in division site misplacement and filamentous formation in E. coli, indicating that MeMinD protein was functional across species. MeMinD exhibited different spatial and temporal expression patterns which was highly expressed in the source compared to that in the sink organ.

Capturing Biochemical Diversity in Cassava ( Manihot esculenta Crantz) through the Application of Metabolite Profiling.

Cassava ( Manihot esculenta Crantz) is the predominant staple food in Sub-Saharan Africa (SSA) and an industrial crop in South East Asia. Despite focused breeding efforts for increased yield, resistance, and nutritional value, cassava breeding has not advanced at the same rapidity as other staple crops. In the present study, metabolomic techniques were implemented to characterize the chemotypes of selected cassava accessions and assess potential resources for the breeding program. The metabolite data analyzed was applied to describe the biochemical diversity available in the panel, identifying South American accessions as the most diverse. Genotypes with distinct phenotypic traits showed a representative metabolite profile and could be clearly identified, even if the phenotypic trait was a root characteristic, e.g., high amylose content.

A comparison study of five different methods to measure carotenoids in biofortified yellow cassava (Manihot esculenta).

The most commonly used method for measuring carotenoid concentration is high-performance liquid chromatography (HPLC). Nevertheless, easier, quicker, and less costly proxy methods exist. We aimed to determine the diagnostic performance of several proxy methods: the spectrophotometer, iCheck Carotene, and near-infrared spectroscopy using both a desktop (dNIRS) and a portable (pNIRS) device for the measurement of total carotenoid concentration (TCC) and all-trans-ß-carotene concentration (trans-BC) in 30 fresh cassava (Manihot esculenta Crantz) storage roots in comparison with HPLC. The spectrophotometer presented the highest predictability for TCC, followed by iCheck, dNIRS, and pNIRS. The dNIRS showed the highest predictability and agreement for trans-BC. The pNIRS showed the poorest repeatability and greatest underestimations compared with HPLC. The agreement between all methods was lower for higher carotenoid concentration, with the exception of the spectrophotometer. According to our results, and for screening purposes, the measurement of carotenoids in fresh cassava roots can be carried out by spectrophotometer, iCheck Carotene and NIRS methods depending on the availability of equipment.

The Late Embryogenesis Abundant Protein Family in Cassava (Manihot esculenta Crantz): Genome-Wide Characterization and Expression during Abiotic Stress.

Late embryogenesis abundant (LEA) proteins, as a highly diverse group of polypeptides, play an important role in plant adaptation to abiotic stress; however, LEAs from cassava have not been studied in cassava. In this study, 26 LEA members were genome-wide identified from cassava, which were clustered into seven subfamily according to evolutionary relationship, protein motif, and gene structure analyses. Chromosomal location and duplication event analyses suggested that 26 MeLEAs distributed in 10 chromosomes and 11 MeLEA paralogues were subjected to purifying selection. Transcriptomic analysis showed the expression profiles of MeLEAs in different tissues of stem, leaves, and storage roots of three accessions. Comparative transcriptomic analysis revealed that the function of MeLEAs in response to drought may be differentiated in different accessions. Compared with the wild subspecies W14, more MeLEA genes were activated in cultivated varieties Arg7 and SC124 after drought treatment. Several MeLEA genes showed induction under various stresses and related signaling treatments. Taken together, this study demonstrates the transcriptional control of MeLEAs in tissue development and the responses to abiotic stress in cassava and identifies candidate genes for improving crop resistance to abiotic stress.

Community phytosanitation to manage cassava brown streak disease.

Cassava viruses are the major biotic constraint to cassava production in Africa. Community-wide action to manage them has not been attempted since a successful cassava mosaic disease control programme in the 1930s/40s in Uganda. A pilot initiative to investigate the effectiveness of community phytosanitation for managing cassava brown streak disease (CBSD) was implemented from 2013 to 2016 in two communities in coastal (Mkuranga) and north-western (Chato) Tanzania. CBSD incidence in local varieties at the outset was >90%, which was typical of severely affected regions of Tanzania. Following sensitization and monitoring by locally-recruited taskforces, there was effective community-wide compliance with the initial requirement to replace local CBSD-infected material with newly-introduced disease-free planting material of improved varieties. The transition was also supported by the free provision of additional seed sources, including maize, sweet potato, beans and cowpeas. Progress of the initiative was followed in randomly-selected monitoring fields in each of the two locations. Community phytosanitation in both target areas produced an area-wide reduction in CBSD incidence, which was sustained over the duration of the programme. In Chato, maximum CBSD incidence was 39.1% in the third season, in comparison with an incidence of >60% after a single season in a control community where disease-free planting material was introduced in the absence of community phytosanitation. Kriging and geospatial analysis demonstrated that inoculum pressure, which was a function of vector abundance and the number of CBSD-infected plants surrounding monitored fields, was a strong determinant of the pattern of CBSD development in monitored fields. In the first year, farmers achieved yield increases with the new varieties relative to the local variety baseline of 94% in Chato (north-west) and 124% in Mkuranga (coast). Yield benefits of the new material were retained up to the final season in each location. The new variety (Mkombozi) introduced under community phytosanitation conditions in Chato yielded 86% more than the same variety from the same source planted in the no-phytosanitation control location. Although there was an 81% reduction in CBSD incidence in the new variety Kiroba introduced under community phytosanitation compared to control conditions in Mkuranga, there was no concomitant yield increase. Variety Kiroba is known to be tolerant to the effects of CBSD, and tuberous roots of infected plants are frequently asymptomatic. Community phytosanitation has the potential to deliver area-wide and sustained reductions in the incidence of CBSD, which also provide significant productivity gains for growers, particularly where introduced varieties do not have high levels of resistant/tolerance to CBSD. The approach should therefore be considered as a potential component for integrated cassava virus management programmes, particularly where new cassava plantations are being established in areas severely affected by CBSD.

Case of escape in cassava, Manihot esculenta Crantz.

Two cassava escapes where collected from cultivated fields near natural habitat in Bolivia. They are described morphologically and analyzed cytogenetically in this study. It is suggested that they are the product of backcrosses of cassava interspecific hybrids with the cultigen itself, and that selective conditions have developed in which certain forms of cassava segregates have adapted to grow wildly in natural habitats near cassava fields. These segregates may hybridize with cultivated cassava upon coming in contact with such varieties. Because these escapes have incorporated useful genes from the wild into their genetic structure, they could be used for cassava improvement since their genetic barriers with other forms of cassava are very weak.

Genetic diversity in cassava landraces grown on farms in Alta Floresta-MT, Brazil.

Brazil is considered one of the domestication centers of cassava (Manihot esculenta), containing a large part of the biological diversity and traditional knowledge of the species. The aim of the present study was to evaluate the genetic diversity of cassava landraces grown by farmers in the north of Mato Grosso State, Brazil, using inter simple sequence repeat (ISSR) molecular markers. The study was carried out in the municipality of Alta Floresta, MT, on farms located in two rural areas. Seventeen cassava landraces were selected. The DNA was extracted and polymerase chain reaction amplifications were performed using 15 ISSR primers. Genetic similarity estimates were calculated using Jaccard's index and the generated matrix was used for clustering the genotypes by using UPGMA and Tocher's methods. The 15 ISSR primers amplified 120 fragments, revealing 61.67% polymorphism. The polymorphism information content ranged from 0.04 to 0.61, averaging 0.39. The most similar genotypes were AF5 and AF8, whereas the least similar were AF1 and AF16. The UPGMA clustering method formed five groups. Group I included twelve landraces, Group II contained two, and the other groups contained one landrace each. Tocher's method resulted in six groups: 12 landraces clustered in one group, and the other groups each contained one landrace. The ISSR markers proved efficient in revealing genetic diversity among the cassava landraces. The landraces grown by farmers in the two rural areas of Alta Floresta have a great variability and, thus, can be exploited in programs for breeding and preservation of the species.

Sequencing wild and cultivated cassava and related species reveals extensive interspecific hybridization and genetic diversity.

Cassava (Manihot esculenta) provides calories and nutrition for more than half a billion people. It was domesticated by native Amazonian peoples through cultivation of the wild progenitor M. esculenta ssp. flabellifolia and is now grown in tropical regions worldwide. Here we provide a high-quality genome assembly for cassava with improved contiguity, linkage, and completeness; almost 97% of genes are anchored to chromosomes. We find that paleotetraploidy in cassava is shared with the related rubber tree Hevea, providing a resource for comparative studies. We also sequence a global collection of 58 Manihot accessions, including cultivated and wild cassava accessions and related species such as Ceará or India rubber (M. glaziovii), and genotype 268 African cassava varieties. We find widespread interspecific admixture, and detect the genetic signature of past cassava breeding programs. As a clonally propagated crop, cassava is especially vulnerable to pathogens and abiotic stresses. This genomic resource will inform future genome-enabled breeding efforts to improve this staple crop.

Comparative Proteome Analysis of the Tuberous Roots of Six Cassava (Manihot esculenta) Varieties Reveals Proteins Related to Phenotypic Traits.

Cassava (Manihot esculenta Crantz) is a staple food and an important source of starch, and the attributes of its tuberous root largely depend on the variety. The proteome of cassava has been investigated; however, to date, no study has focused on varieties that reveal the molecular basis of phenotypical characteristics. Therefore, we aimed to compare the proteome of the tuberous roots of six cassava varieties that differed in carbohydrates, carotenoids, and resistance to diseases, among other attributes. Two-dimensional gels showed 146 differential spots between the varieties, and the functional roles of some differential proteins were correlated to phenotypic characteristics of the varieties, such as the amount of carbohydrates or carotenoids and the resistance to biotic or abiotic stresses. The results obtained here highlight elements that might help to direct the improvement of new cultivars of cassava, which is an economically and socially relevant crop worldwide.

Comparative petiole anatomy of cassava (Manihot) species.

In this study, we describe the petiole anatomy of six wild cassava (Manihot) species, one hybrid, and two cultivars of Manihot esculenta, in order to identify their dominant anatomical patterns and relate them to possible adaptations to abiotic factors in the Cerrado biome. The median parts of several petiole samples were transversally and longitudinally sectioned and stained. The results include data for the taxonomic classification of the genus, including distinctive anatomical characteristics of hybrid varieties of cassava and wild species, such as the presence/absence of trichomes and a hypodermis, layer type and number in the cortex, number of vascular bundles, cell types in the pith, and type of organization. Morphological analysis revealed differences in length and shape of the petiole insertion. The presence of trichomes, a hypodermis, the amount and type of supporting tissue in the cortex, as well as gelatinous fibers, may be related to drought tolerance.

Metabolomic, enzymatic, and histochemical analyzes of cassava roots during postharvest physiological deterioration.

BACKGROUND: Under postharvest physiological deterioration cassava root tubers alter the expression of biosynthetic pathways of certain primary and secondary metabolites, as well as the activity of some scavenging enzymes. Therefore, in this study we hypothesized that cassava cultivars differ as to their physiological responses to deterioration and their biochemical profiles can be an indicative of the tolerance or susceptibility to deterioration. RESULTS: The results corroborate the working hypothesis, revealing that high Levels of phenolic acids, scopoletin, carotenoids, proteins, and augmented activities of guaiacol peroxidase and hydrogen peroxide in non-stored cassava roots can be used as potential biomarkers of cassava deterioration. CONCLUSIONS: Cassava physiological deterioration depends on cultivar and many compounds are up and downregulated during storage time. Secondary metabolites, enzymes, scopoletin, scavenging reactive oxygen species, and acidic polysaccharides are activated as responses to the physiological stress induced in root tubers.

Tracking crop varieties using genotyping-by-sequencing markers: a case study using cassava (Manihot esculenta Crantz).

BACKGROUND: Accurate identification of crop cultivars is crucial in assessing the impact of crop improvement research outputs. Two commonly used identification approaches, elicitation of variety names from farmer interviews and morphological plant descriptors, have inherent uncertainty levels. Genotyping-by-sequencing (GBS) was used in a case study as an alternative method to track released varieties in farmers' fields, using cassava, a clonally propagated root crop widely grown in the tropics, and often disseminated through extension services and informal seed systems. A total of 917 accessions collected from 495 farming households across Ghana were genotyped at 56,489 SNP loci along with a "reference library" of 64 accessions of released varieties and popular landraces. RESULTS: Accurate cultivar identification and ancestry estimation was accomplished through two complementary clustering methods: (i) distance-based hierarchical clustering; and (ii) model-based maximum likelihood admixture analysis. Subsequently, 30% of the identified accessions from farmers' fields were matched to specific released varieties represented in the reference library. ADMIXTURE analysis revealed that the optimum number of major varieties was 11 and matched the hierarchical clustering results. The majority of the accessions (69%) belonged purely to one of the 11 groups, while the remaining accessions showed two or more ancestries. Further analysis using subsets of SNP markers reproduced results obtained from the full-set of markers, suggesting that GBS can be done at higher DNA multiplexing, thereby reducing the costs of variety fingerprinting. A large proportion of discrepancy between genetically unique cultivars as identified by markers and variety names as elicited from farmers were observed. Clustering results from ADMIXTURE analysis was validated using the assumption-free Discriminant Analysis of Principal Components (DAPC) method. CONCLUSION: We show that genome-wide SNP markers from increasingly affordable GBS methods coupled with complementary cluster analysis is a powerful tool for fine-scale population structure analysis and variety identification. Moreover, the ancestry estimation provides a framework for quantifying the contribution of exotic germplasm or older improved varieties to the genetic background of contemporary improved cultivars.

Apparent similarity, underlying homoplasy: Morphology and molecular phylogeny of the North American clade of Manihot.

PREMISE OF THE STUDY: Morphologically diverse clades are useful for detecting adaptive morphological evolution. Each of their variants may have evolved once or several times, suggesting that their repeated appearance may be due to environmental pressures. The North American Manihot species are an excellent system to detect possible adaptations and to assess the effect of mono- or polyphyly on classification. With 20 species, this group includes growth forms from tuberous herbs to trees. The monophyly of this group and its relationship with the economically important M. esculenta were tested for the first time with complete sampling of North American species. METHODS: We carried out maximum likelihood and Bayesian phylogenetic analyses on a matrix of 3662 bp from chloroplast (psbA-trnH, trnL-trnF) and nuclear loci (PEPC and two paralogous copies of G3pdh). We included all North American Manihot species, Manihotoides pauciflora, and published sequences from 34 South American species. KEY RESULTS: Our results support monophyly of the North American Manihot group. Its taxonomic sections are paraphyletic, and three to four growth forms evolved repeatedly. Manihotoides pauciflora is nested within North American Manihot species. Some PEPC and G3pdh clones grouped with clones of other species and not with clones from their own species. CONCLUSIONS: North and South American Manihot species are sister clades. Paraphyly of North American sections suggests that taxonomic revision is warranted. The position of Manihotoides pauciflora confirms that Manihotoides should remain subsumed within Manihot. Most growth forms likely evolved repeatedly in this group. The behavior of PEPC and G3pdhNA clones is probably due to incomplete lineage sorting.

Classification of cassava genotypes based on qualitative and quantitative data.

We evaluated the genetic variation of cassava accessions based on qualitative (binomial and multicategorical) and quantitative traits (continuous). We characterized 95 accessions obtained from the Cassava Germplasm Bank of Embrapa Mandioca e Fruticultura; we evaluated these accessions for 13 continuous, 10 binary, and 25 multicategorical traits. First, we analyzed the accessions based only on quantitative traits; next, we conducted joint analysis (qualitative and quantitative traits) based on the Ward-MLM method, which performs clustering in two stages. According to the pseudo-F, pseudo-t2, and maximum likelihood criteria, we identified five and four groups based on quantitative trait and joint analysis, respectively. The smaller number of groups identified based on joint analysis may be related to the nature of the data. On the other hand, quantitative data are more subject to environmental effects in the phenotype expression; this results in the absence of genetic differences, thereby contributing to greater differentiation among accessions. For most of the accessions, the maximum probability of classification was >0.90, independent of the trait analyzed, indicating a good fit of the clustering method. Differences in clustering according to the type of data implied that analysis of quantitative and qualitative traits in cassava germplasm might explore different genomic regions. On the other hand, when joint analysis was used, the means and ranges of genetic distances were high, indicating that the Ward-MLM method is very useful for clustering genotypes when there are several phenotypic traits, such as in the case of genetic resources and breeding programs.

Analysis of different strategies adapted by two cassava cultivars in response to drought stress: ensuring survival or continuing growth.

Cassava is one of the most drought-tolerant crops, however, the underlying mechanism for its ability to survive and produce under drought remains obscure. In this study, two cassava cultivars, SC124 and Arg7, were treated by gradually reducing the soil water content. Their responses to the drought stress were examined through their morphological and physiological traits and isobaric tags for relative and absolute quantitation (iTRAQ)-based proteomic analysis. SC124 plants adapted a 'survival' mode under mild drought stress as evidenced by early stomatal closure and a reduction in the levels of various photosynthetic proteins and photosynthetic capacity, resulting in early growth quiescence. In contrast, Arg7 plants underwent senescence of older leaves but continued to grow, although at a reduced rate, under mild drought. SC124 plants were more capable of surviving prolonged severe drought than Arg7. The iTRAQ analysis identified over 5000 cassava proteins. Among the drought-responsive proteins identified in the study were an aquaporin, myo-inositol 1-phosphate synthases, and a number of proteins involved in the antioxidant systems and secondary metabolism. Many proteins that might play a role in signalling or gene regulation were also identified as drought-responsive proteins, which included several protein kinases, two 14-3-3 proteins, several RNA-binding proteins and transcription factors, and two histone deacetylases. Our study also supports the notion that linamarin might play a role in nitrogen reallocation in cassava under drought.