Assessing the effect of sample storage time on viral detection using a rapid and cost-effective CTAB-based extraction method.

BACKGROUND: Cassava leaf samples degrade quickly during storage and transportation from distant areas. Proper sampling and efficient, low-cost storage methods are critical for obtaining sufficient quality DNA and RNA for plant virus epidemiology and improving disease control understanding. This is useful when samples are collected from remote areas far from a laboratory or in developing countries where money and materials for virus diagnostics are scarce. RESULTS: The effect of sample storage duration on nucleic acid (N.A.) quality on virus detection was investigated in this study. A simple, rapid, and cost-effective CTAB-based approach (M3) for single N.A. extraction was optimized and tested alongside two existing CTAB-based methods (M1 and M2) for N.A. extraction from fresh and herbarium cassava leaves stored for; 1, 8, 26, and 56 months. The amount and quality of DNA and RNA were determined using Nanodrop 2000 c U.V.-vis Spectrophotometer and agarose gel electrophoreses. The sample degradation rate was estimated using a simple mathematical model in Matlab computational software. The results show no significant difference in mean DNA concentration between M1 and M2 but a significant difference between M3 and the other two methods at p < 0.005. The mean DNA concentration extracted using M3 was higher for 1 and 8 months of leave storage. M3 and M2 produced high concentrations at 26 and 56 months of leave storage. Using a developed scale for quality score, M3 and M2 produced high-quality DNA from fresh samples. All methods produced poor-quality DNA and RNA at 8 and 26 months of leave storage and no visual bands at the age of 56 months. Statistically, there was a significant difference in the mean DNA quality between M1 and M2, but there was no significant difference between M3 and the other two methods at p < 0.005. However, Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV) were readily detected by RT-PCR from RNA isolated using M3. The quality of DNA declined per storage time at 0.0493 and 0.0521/month, while RNA was 0.0678 and 0.0744/month. Compared to the existing two methods, modified CTAB extracted enough high-quality N.A. in one-third the time of the existing two methods. CONCLUSION: Our method provides cost-effective, quick, and simple processing of fresh and dry samples, which will quicken and guide the decision on when and what type of sample to process for plant disease management and surveillance actions.

Cassava brown streak virus evolves with a nucleotide-substitution rate that is typical for the family Potyviridae.

The ipomoviruses (family Potyviridae) that cause cassava brown streak disease (cassava brown streak virus [CBSV] and Uganda cassava brown streak virus [UCBSV]) are damaging plant pathogens that affect the sustainability of cassava production in East and Central Africa. However, little is known about the rate at which the viruses evolve and when they emerged in Africa - which inform how easily these viruses can host shift and resist RNAi approaches for control. We present here the rates of evolution determined from the coat protein gene (CP) of CBSV (Temporal signal in a UCBSV dataset was not sufficient for comparable analysis). Our BEAST analysis estimated the CBSV CP evolves at a mean rate of 1.43 × 10-3 nucleotide substitutions per site per year, with the most recent common ancestor of sampled CBSV isolates existing in 1944 (95% HPD, between years 1922 - 1963). We compared the published measured and estimated rates of evolution of CPs from ten families of plant viruses and showed that CBSV is an average-evolving potyvirid, but that members of Potyviridae evolve more quickly than members of Virgaviridae and the single representatives of Betaflexiviridae, Bunyaviridae, Caulimoviridae and Closteroviridae.

SEGS-1 a cassava genomic sequence increases the severity of African cassava mosaic virus infection in Arabidopsis thaliana.

Cassava is a major crop in Sub-Saharan Africa, where it is grown primarily by smallholder farmers. Cassava production is constrained by Cassava mosaic disease (CMD), which is caused by a complex of cassava mosaic begomoviruses (CMBs). A previous study showed that SEGS-1 (sequences enhancing geminivirus symptoms), which occurs in the cassava genome and as episomes during viral infection, enhances CMD symptoms and breaks resistance in cassava. We report here that SEGS-1 also increases viral disease severity in Arabidopsis thaliana plants that are co-inoculated with African cassava mosaic virus (ACMV) and SEGS-1 sequences. Viral disease was also enhanced in Arabidopsis plants carrying a SEGS-1 transgene when inoculated with ACMV alone. Unlike cassava, no SEGS-1 episomal DNA was detected in the transgenic Arabidopsis plants during ACMV infection. Studies using Nicotiana tabacum suspension cells showed that co-transfection of SEGS-1 sequences with an ACMV replicon increases viral DNA accumulation in the absence of viral movement. Together, these results demonstrated that SEGS-1 can function in a heterologous host to increase disease severity. Moreover, SEGS-1 is active in a host genomic context, indicating that SEGS-1 episomes are not required for disease enhancement.

Genetic diversity and SNP's from the chloroplast coding regions of virus-infected cassava.

Cassava is a staple food crop in sub-Saharan Africa; it is a rich source of carbohydrates and proteins which currently supports livelihoods of more than 800 million people worldwide. However, its continued production is at stake due to vector-transmitted diseases such as Cassava mosaic disease and Cassava brown streak disease. Currently, the management and control of viral diseases in cassava relies mainly on virus-resistant cultivars of cassava. Thus, the discovery of new target genes for plant virus resistance is essential for the development of more cassava varieties by conventional breeding or genetic engineering. The chloroplast is a common target for plant viruses propagation and is also a potential source for discovering new resistant genes for plant breeding. Non-infected and infected cassava leaf samples were obtained from different locations of East Africa in Tanzania, Kenya and Mozambique. RNA extraction followed by cDNA library preparation and Illumina sequencing was performed. Assembling and mapping of the reads were carried out and 33 partial chloroplast genomes were obtained. Bayesian phylogenetic analysis from 55 chloroplast protein-coding genes of a dataset with 39 taxa was performed and the single nucleotide polymorphisms for the chloroplast dataset were identified. Phylogenetic analysis revealed considerable genetic diversity present in chloroplast partial genome among cultivated cassava of East Africa. The results obtained may supplement data of previously selected resistant materials and aid breeding programs to find diversity and achieve resistance for new cassava varieties.

Expansion of the cassava brown streak pandemic in Uganda revealed by annual field survey data for 2004 to 2017.

Cassava brown streak disease (CBSD) is currently the most devastating cassava disease in eastern, central and southern Africa affecting a staple crop for over 700 million people on the continent. A major outbreak of CBSD in 2004 near Kampala rapidly spread across Uganda. In the following years, similar CBSD outbreaks were noted in countries across eastern and central Africa, and now the disease poses a threat to West Africa including Nigeria - the biggest cassava producer in the world. A comprehensive dataset with 7,627 locations, annually and consistently sampled between 2004 and 2017 was collated from historic paper and electronic records stored in Uganda. The survey comprises multiple variables including data for incidence and symptom severity of CBSD and abundance of the whitefly vector (Bemisia tabaci). This dataset provides a unique basis to characterize the epidemiology and dynamics of CBSD spread in order to inform disease surveillance and management. We also describe methods used to integrate and verify extensive field records for surveys typical of emerging epidemics in subsistence crops.

Tree Lab: Portable genomics for Early Detection of Plant Viruses and Pests in Sub-Saharan Africa.

In this case study we successfully teamed the PDQeX DNA purification technology developed by MicroGEM, New Zealand, with the MinION and MinIT mobile sequencing devices developed by Oxford Nanopore Technologies to produce an effective point-of-need field diagnostic system. The PDQeX extracts DNA using a cocktail of thermophilic proteinases and cell wall-degrading enzymes, thermo-responsive extractor cartridges and a temperature control unit. This closed system delivers purified DNA with no cross-contamination. The MinIT is a newly released data processing unit that converts MinION raw signal output into nucleotide base called data locally in real-time, removing the need for high-specification computers and large file transfers from the field. All three devices are battery powered with an exceptionally small footprint that facilitates transport and setup. To evaluate and validate capability of the system for unbiased pathogen identification by real-time sequencing in a farmer's field setting, we analysed samples collected from cassava plants grown by subsistence farmers in three sub-Sahara African countries (Tanzania, Uganda and Kenya). A range of viral pathogens, all with similar symptoms, greatly reduce yield or destroy cassava crops. Eight hundred (800) million people worldwide depend on cassava for food and yearly income, and viral diseases are a significant constraint to its production. Early pathogen detection at a molecular level has great potential to rescue crops within a single growing season by providing results that inform decisions on disease management, use of appropriate virus-resistant or replacement planting. This case study presented conditions of working in-field with limited or no access to mains power, laboratory infrastructure, Internet connectivity and highly variable ambient temperature. An additional challenge is that, generally, plant material contains inhibitors of downstream molecular processes making effective DNA purification critical. We successfully undertook real-time on-farm genome sequencing of samples collected from cassava plants on three farms, one in each country. Cassava mosaic begomoviruses were detected by sequencing leaf, stem, tuber and insect samples. The entire process, from arrival on farm to diagnosis, including sample collection, processing and provisional sequencing results was complete in under 3 h. The need for accurate, rapid and on-site diagnosis grows as globalized human activity accelerates. This technical breakthrough has applications that are relevant to human and animal health, environmental management and conservation.

Immunohistochemical localization of Cassava brown streak virus and its morphological effect on cassava leaves.

The localization of Cassava brown streak virus (CBSV) in cassava (Manihot esculenta) leaf tissues was determined and cellular morphological changes in CBSV-infected tissues were evaluated. CBSV-symptomatic leaves were screened with CBSV-specific primers using reverse-transcriptase polymerase chain reaction. Immunohistochemical reactions showed precipitation in CBSV-infected but not CBSV-free tissues, demonstrating successful localization of CBSV. Microscopic inspection showed significantly larger (P < 0.001) midribs in CBSV-infected compared with control (uninfected) leaves. Viral accumulation occurred in middle and lower but rarely in young upper leaves. This immunohistochemical method for virus localization will be invaluable for efficient screening of CBSV and for breeding resistant cassava.

First report of Cassava brown streak viruses on wild plant species in Mozambique.

Cassava brown streak disease (CBSD) caused by Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV) is the main constraint to cassava (Manihot esculenta Crantz) production in Mozambique. Using RT-PCR to amplify partial coat protein nucleotide sequences, we detected for the first time the occurrence of CBSV in two non-cassava perennial wild plant species: Zanha africana (Radlk.) Exell. and Trichodesma zeylanicum (Burm.f.) R.Br., that occur widely within and near cassava fields in Nampula, Zambezia, Niassa and Cabo Delgado provinces. In addition, we also detected CBSV and UCBSV in Manihot carthaginensis subsp. glaziovii (Müell-Arg.) Allem., a wild cassava relative. These findings were verified in biological assays through mechanical inoculation of CBSV to T. zeylanicum, albeit at low rates of infection. Phylogenetic analysis clustered the CBSV isolates from the non-cassava plant species with those from cultivated cassava, with high sequence homology among CBSV (91.0-99.6%) and with UCBSV (84-92%) isolates. These results provide definitive evidence of a wider host range for CBSV and UCBSV in Mozambique, indicating that these viruses are not restricted to cultivated cassava. Our findings are key to understanding the epidemiology of CBSD and will aid in the development of sustainable management strategies for the disease.

Resistance of advanced cassava breeding clones to infection by major viruses in Uganda.

Cassava brown streak disease (CBSD) and cassava mosaic disease (CMD) are two viral diseases that cause severe yield losses in cassava of up to 100%, thereby persistently threatening food and income security in sub-Saharan Africa. For effective management of these diseases, there is a critical need to develop and deploy varieties with dual resistance to CBSD and CMD. In this study, we determined the response of advanced breeding lines to field infection by cassava brown streak viruses (CBSVs) and cassava mosaic begomoviruses (CMBs). This aim helped in identifying superior clones for downstream breeding. In total, 220 cassava clones, three in uniform yield trials (UYTs) and 217 in a crossing block trial (CBT), were evaluated for virus and disease resistance. Field data were collected on disease incidence and severity. To detect and quantify CBSVs, 448 and 128 leaf samples from CBSD symptomatic and symptomless plants were analyzed by reverse transcription PCR and real-time quantitative PCR, respectively. In addition, 93 leaf samples from CMD symptomatic plants in the CBT were analyzed by conventional PCR using CMB species-specific primers. In the CBT, 124 (57%) cassava clones did not express CMD symptoms. Of the affected plants, 44 (55%) had single African cassava mosaic virus infection. Single Cassava brown streak virus (CBSV) infections were more prevalent (81.6%) in CBT clones than single Ugandan cassava brown streak virus (UCBSV) infection (3.2%). Of the three advanced clones in the UYT, NAROCASS 1 and NAROCASS 2 had significantly lower (P < 0.05) CBSD severity, incidence, and CBSV load than MH04/0300. In the UYT, only 22% of samples tested had CBSVs, and all showed a negative result for CMBs. The low disease incidence, severity, and viral load associated with NAROCASS 1 and NAROCASS 2 is evidence of their tolerance to both CBSD and CMD. Therefore, these two cassava clones should be utilized in CBSD and CMD management in Uganda, including their utilization as progenitors in further virus resistance breeding.

Localization of cassava brown streak virus in Nicotiana rustica and cassava Manihot esculenta (Crantz) using RNAscope® in situ hybridization.

BACKGROUND: Cassava brown streak disease (CBSD) has a viral aetiology and is caused by viruses belonging to the genus Ipomovirus (family Potyviridae), Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV). Molecular and serological methods are available for detection, discrimination and quantification of cassava brown streak viruses (CBSVs) in infected plants. However, precise determination of the viral RNA localization in infected host tissues is still not possible pending appropriate methods. RESULTS: We have developed an in situ hybridization (ISH) assay based on RNAscope® technology that allows the sensitive detection and localization of CBSV RNA in plant tissues. The method was initially developed in the experimental host Nicotiana rustica and was then further adapted to cassava. Highly sensitive and specific detection of CBSV RNA was achieved without background and hybridization signals in sections prepared from non-infected tissues. The tissue tropism of CBSV RNAs appeared different between N. rustica and cassava. CONCLUSIONS: This study provides a robust method for CBSV detection in the experimental host and in cassava. The protocol will be used to study CBSV tropism in various cassava genotypes, as well as CBSVs/cassava interactions in single and mixed infections.

The first transcriptomes from field-collected individual whiteflies ( Bemisia tabaci, Hemiptera: Aleyrodidae): a case study of the endosymbiont composition.

Background:Bemisia tabaci species ( B. tabaci), or whiteflies, are the world's most devastating insect pests. They cause billions of dollars (US) of damage each year, and are leaving farmers in the developing world food insecure. Currently, all publically available transcriptome data for B. tabaci are generated from pooled samples, which can lead to high heterozygosity and skewed representation of the genetic diversity. The ability to extract enough RNA from a single whitefly has remained elusive due to their small size and technological limitations. Methods: In this study, we optimised a single whitefly RNA extraction procedure, and sequenced the transcriptome of four individual adult Sub-Saharan Africa 1 (SSA1) B. tabaci. Transcriptome sequencing resulted in 39-42 million raw reads. De novo assembly of trimmed reads yielded between 65,000-162,000 Contigs across B. tabaci transcriptomes. Results: Bayesian phylogenetic analysis of mitochondrion cytochrome I oxidase (mtCOI) grouped the four whiteflies within the SSA1 clade. BLASTn searches on the four transcriptomes identified five endosymbionts; the primary endosymbiont Portieraaleyrodidarum and four secondary endosymbionts: Arsenophonus, Wolbachia, Rickettsia, and Cardinium spp. that were predominant across all four SSA1 B. tabaci samples with prevalence levels of between 54.1 to 75%. Amino acid alignments of the NusG gene of P. aleyrodidarum for the SSA1 B. tabaci transcriptomes of samples WF2 and WF2b revealed an eleven amino acid residue deletion that was absent in samples WF1 and WF2a. Comparison of the protein structure of the NusG protein from P. aleyrodidarum in SSA1 with known NusG structures showed the deletion resulted in a shorter D loop. Conclusions: The use of field-collected specimens means time and money will be saved in future studies using single whitefly transcriptomes in monitoring vector and viral interactions. Our method is applicable to any small organism where RNA quantity has limited transcriptome studies.

African ancestry of New World, Bemisia tabaci-whitefly species.

Bemisia tabaci whitefly species are some of the world's most devastating agricultural pests and plant-virus disease vectors. Elucidation of the phylogenetic relationships in the group is the basis for understanding their evolution, biogeography, gene-functions and development of novel control technologies. We report here the discovery of five new Sub-Saharan Africa (SSA) B. tabaci putative species, using the partial mitochondrial cytochrome oxidase 1 gene: SSA9, SSA10, SSA11, SSA12 and SSA13. Two of them, SSA10 and SSA11 clustered with the New World species and shared 84.8‒86.5% sequence identities. SSA10 and SSA11 provide new evidence for a close evolutionary link between the Old and New World species. Re-analysis of the evolutionary history of B. tabaci species group indicates that the new African species (SSA10 and SSA11) diverged from the New World clade c. 25 million years ago. The new putative species enable us to: (i) re-evaluate current models of B. tabaci evolution, (ii) recognise increased diversity within this cryptic species group and (iii) re-estimate divergence dates in evolutionary time.

Unusual occurrence of a DAG motif in the Ipomovirus Cassava brown streak virus and implications for its vector transmission.

Cassava is the main staple food for over 800 million people globally. Its production in eastern Africa is being constrained by two devastating Ipomoviruses that cause cassava brown streak disease (CBSD); Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV), with up to 100% yield loss for smallholder farmers in the region. To date, vector studies have not resulted in reproducible and highly efficient transmission of CBSV and UCBSV. Most virus transmission studies have used Bemisia tabaci (whitefly), but a maximum of 41% U/CBSV transmission efficiency has been documented for this vector. With the advent of next generation sequencing, researchers are generating whole genome sequences for both CBSV and UCBSV from throughout eastern Africa. Our initial goal for this study was to characterize U/CBSV whole genomes from CBSD symptomatic cassava plants sampled in Kenya. We have generated 8 new whole genomes (3 CBSV and 5 UCBSV) from Kenya, and in the process of analyzing these genomes together with 26 previously published sequences, we uncovered the aphid transmission associated DAG motif within coat protein genes of all CBSV whole genomes at amino acid positions 52-54, but not in UCBSV. Upon further investigation, the DAG motif was also found at the same positions in two other Ipomoviruses: Squash vein yellowing virus (SqVYV), Coccinia mottle virus (CocMoV). Until this study, the highly-conserved DAG motif, which is associated with aphid transmission was only noticed once, in SqVYV but discounted as being of minimal importance. This study represents the first comprehensive look at Ipomovirus genomes to determine the extent of DAG motif presence and significance for vector relations. The presence of this motif suggests that aphids could potentially be a vector of CBSV, SqVYV and CocMov. Further transmission and ipomoviral protein evolutionary studies are needed to confirm this hypothesis.

Deep Sequencing Reveals a Divergent Ugandan cassava brown streak virus Isolate from Malawi.

Illumina sequencing of RNA from a cassava cutting from northern Malawi produced a genome of Ugandan cassava brown streak virus (UCBSV-MW-NB7_2013). Sequence comparisons revealed stronger similarity to an isolate from nearby Tanzania (93.4% pairwise nucleotide identity) than to those previously reported from Malawi (86.9 to 87.0%).

Variability in P1 gene redefines phylogenetic relationships among cassava brown streak viruses.

BACKGROUND: Cassava brown streak disease is emerging as the most important viral disease of cassava in Africa, and is consequently a threat to food security. Two distinct species of the genus Ipomovirus (family Potyviridae) cause the disease: Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV). To understand the evolutionary relationships among the viruses, 64 nucleotide sequences from the variable P1 gene from major cassava producing areas of east and central-southern Africa were determined. METHODS: We sequenced an amplicon of the P1 region of 31 isolates from Malawi and Tanzania. In addition to these, 33 previously reported sequences of virus isolates from Uganda, Kenya, Tanzania, Malawi and Mozambique were added to the analysis. RESULTS: Phylogenetic analyses revealed three major P1 clades of Cassava brown streak viruses (CBSVs): in addition to a clade of most CBSV and a clade containing all UCBSV, a novel, intermediate clade of CBSV isolates which has been tentatively called CBSV-Tanzania (CBSV-TZ). Virus isolates of the distinctive CBSV-TZ had nucleotide identities as low as 63.2 and 63.7% with other members of CBSV and UCBSV respectively. CONCLUSIONS: Grouping of P1 gene sequences indicated for distinct sub-populations of CBSV, but not UCBSV. Representatives of all three clades were found in both Tanzania and Malawi.

Viruses infecting common bean (Phaseolus vulgaris L.) in Tanzania: A review on molecular characterization, detection and disease management options.

Common bean (Phaseolus vulgaris L.) is a major legume crop, serving as a main source of dietary protein and calories and generating income for many Tanzanians. It is produced in nearly all agro-ecological zones of Tanzania. However, the average yields are low (<1000 kg/ha), which is attributed to many factors including virus diseases. The most important viruses of common bean in Tanzania are Bean common mosaic virus (BCMV) and Bean common mosaic necrosis virus (BCMNV) but other viruses have also been reported. There has never been a review of common bean virus diseases in the country, and the lack of collated information makes their management difficult. Therefore, this review focuses on (1) occurrence of different viruses of common bean in Tanzania, (2) molecular characterization of these viruses, (3) detection tools for common bean viruses in Tanzania and (4) available options for managing virus diseases in the country. Literature and nucleotide sequence database searches revealed that common bean diseases are inadequately studied and that their causal viruses have not been adequately characterized at the molecular level in Tanzania. Increased awareness on common bean virus diseases in Tanzania is expected to result into informed development of strategies for management of the same and thus increased production, which in turn has implication on nutrition and income.

Cassava brown streak virus has a rapidly evolving genome: implications for virus speciation, variability, diagnosis and host resistance.

Cassava is a major staple food for about 800 million people in the tropics and sub-tropical regions of the world. Production of cassava is significantly hampered by cassava brown streak disease (CBSD), caused by Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV). The disease is suppressing cassava yields in eastern Africa at an alarming rate. Previous studies have documented that CBSV is more devastating than UCBSV because it more readily infects both susceptible and tolerant cassava cultivars, resulting in greater yield losses. Using whole genome sequences from NGS data, we produced the first coalescent-based species tree estimate for CBSV and UCBSV. This species framework led to the finding that CBSV has a faster rate of evolution when compared with UCBSV. Furthermore, we have discovered that in CBSV, nonsynonymous substitutions are more predominant than synonymous substitution and occur across the entire genome. All comparative analyses between CBSV and UCBSV presented here suggest that CBSV may be outsmarting the cassava immune system, thus making it more devastating and harder to control.

Analyses of Twelve New Whole Genome Sequences of Cassava Brown Streak Viruses and Ugandan Cassava Brown Streak Viruses from East Africa: Diversity, Supercomputing and Evidence for Further Speciation.

Cassava brown streak disease is caused by two devastating viruses, Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV) which are frequently found infecting cassava, one of sub-Saharan Africa's most important staple food crops. Each year these viruses cause losses of up to $100 million USD and can leave entire families without their primary food source, for an entire year. Twelve new whole genomes, including seven of CBSV and five of UCBSV were uncovered in this research, doubling the genomic sequences available in the public domain for these viruses. These new sequences disprove the assumption that the viruses are limited by agro-ecological zones, show that current diagnostic primers are insufficient to provide confident diagnosis of these viruses and give rise to the possibility that there may be as many as four distinct species of virus. Utilizing NGS sequencing technologies and proper phylogenetic practices will rapidly increase the solution to sustainable cassava production.

Meristem micropropagation of cassava (Manihot esculenta) evokes genome-wide changes in DNA methylation.

There is great interest in the phenotypic, genetic and epigenetic changes associated with plant in vitro culture known as somaclonal variation. In vitro propagation systems that are based on the use of microcuttings or meristem cultures are considered analogous to clonal cuttings and so widely viewed to be largely free from such somaclonal effects. In this study, we surveyed for epigenetic changes during propagation by meristem culture and by field cuttings in five cassava (Manihot esculenta) cultivars. Principal Co-ordinate Analysis of profiles generated by methylation-sensitive amplified polymorphism revealed clear divergence between samples taken from field-grown cuttings and those recovered from meristem culture. There was also good separation between the tissues of field samples but this effect was less distinct among the meristem culture materials. Application of methylation-sensitive Genotype by sequencing identified 105 candidate epimarks that distinguish between field cutting and meristem culture samples. Cross referencing the sequences of these epimarks to the draft cassava genome revealed 102 sites associated with genes whose homologs have been implicated in a range of fundamental biological processes including cell differentiation, development, sugar metabolism, DNA methylation, stress response, photosynthesis, and transposon activation. We explore the relevance of these findings for the selection of micropropagation systems for use on this and other crops.