Going beyond consensus genome sequences: An innovative SNP-based methodology reconstructs different Ugandan cassava brown streak virus haplotypes at a nationwide scale in Rwanda

Cassava is grown by small-scale farmers and consumed by over 200 million people in sub-Saharan Africa mainly as staple food. An emerging viral disease Cassava brown streak disease (CBSD) caused by Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV), has severely affected cassava production and resulted in poor quality tubers. The disease severely affecting root quality both for domestic use and marketing and has become a real threat to the livelihoods of the millions of poor in both eastern and southern African countries. There is also increase in the number of CBSV and UCBSV sequences available now, and a need to have primers that would detect all isolates of CBSV and UCBSV is required, particularly for field surveys. Two sets of virus-specific primers, CBSVF2 & CBSVR7 and CBSVF2 & CBSVR8, were developed to specifically detect CBSV and UCBSV respectively on six glass house maintained isolates and field collected samples from coastal Kenya. Positive samples produced expected PCR products of 345 bp and 441 bp using CBSVF2 & CBSVR7 and CBSVF2 & CBSVR8, respectively. In addition, our study further confirmed the genetic diversity of these viruses by sequencing over 41 new samples from Kenya as well as Tanzania and Zanzibar. The coat protein, partial HAM1 and 3' UTR regions of CBSV and UCBSV were amplified using CBSVF2 & CBSVR1 and CBSVF2 & CBSVR2, respectively. The PCR products of ~1.6 Kb obtained were extracted from gel, cloned and sequenced. The sequences of the UCBSV and CBSV isolates varied in length; 1678 nucleotide for UCBSV isolates and 1615 nucleotide for CBSV isolates. CBSV and UCBSV sequences were used to the construct phylogenetic trees by comparing with other sequences from GenBank. The phylogenetic tree clustered the CBSD isolates in two groups reflecting the two virus species causing CBSD. Based on the available sequences (~1600 bases), the CBSV group shared 93.7% nucleotide identities, UCBSV 93.1%, and there was ~70% identity between the two groups. However, the percentage nucleotide identities for coat protein nucleotide sequences were slightly greater than those observed for the sequences involving partial HAM1 and 3'UTR region. CBSV group shared 94.4% nucleotide identity, UCBSV 93.5% and ~74% identity between the two groups. In conclusion, this study has shown the genetic diversity between CBSV and UCBSV isolates collected from Kenya with those from Tanzania and Zanzibar.Keywords: Brown streak virus, cassava, Kenya

Cassava, a vital global food source, faces a threat from Cassava Brown Streak Disease (CBSD). CBSD results from two viruses: Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV). These viruses frequently pose challenges to the traditional symptom-based 1-5 phenotyping method due to its limitations in terms of accuracy and objectivity. Quantitative polymerase chain reaction (qPCR) offers precise virus quantification, although high costs hinder its widespread adoption. In this research, we utilized qPCR to measure the viral titer/load of CBSV and UCBSV. The objectives were to evaluate titer variability within the Cycle 2 (C2) population in two different environments, establish connections between viral titers and CBSD severity scores from the 1-5 scoring method, perform Genome-Wide Association Studies (GWAS) to identify genomic regions associated with CBSV and UCBSV titers, and investigate the functional annotated genes. The results demonstrated a significantly higher prevalence of CBSV (50.2%) in clones compared to UCBSV (12.9%) with mixed infections in some cases. Genotypic effects, particularly concerning UCBSV, were significant, with genotype-by-environment effects primarily influencing CBSV titer. GWAS Studies identified genomic regions associated with CBSV and UCBSV titers. Twenty-one SNP markers on chromosomes 10, 13, 17, and 18 exhibited significant associations with CBSV titer, collectively explaining 43.14% of the phenotypic variation. Additionally, 25 SNP markers on chromosomes 1, 2, 4, 5, 8, 11, 12, 13, 16, and 18 were associated with UCBSV titer, and explained 70.71% of the phenotypic variation. No shared genomic regions were identified between CBSV and UCBSV viral titers. Gene ontology analysis also revealed diverse gene functions, especially in transport and catalytic activities. These findings enhance our understanding of virus prevalence, genetics, and molecular functions in cassava plants, offering valuable insights for targeted breeding strategies.

Infection of plant cells by viral pathogens triggers RNA silencing, an innate antiviral defense mechanism. In response to infection, small RNAs (sRNAs) are produced that associate with Argonaute (AGO)-containing silencing complexes which act to inactivate viral genomes by posttranscriptional gene silencing (PTGS). Deep sequencing was used to compare virus-derived small RNAs (vsRNAs) in cassava genotypes NASE 3, TME 204 and 60444 infected with the positive sense single-stranded RNA (+ssRNA) viruses cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV), the causal agents of cassava brown streak disease (CBSD). An abundance of 21–24nt vsRNAs was detected and mapped, covering the entire CBSV and UCBSV genomes. The 21nt vsRNAs were most predominant, followed by the 22 nt class with a slight bias toward sense compared to antisense polarity, and a bias for adenine and uracil bases present at the 5′-terminus. Distribution and frequency of vsRNAs differed between cassava genotypes and viral genomes. In susceptible genotypes TME 204 and 60444, CBSV-derived sRNAs were seen in greater abundance than UCBSV-derived sRNAs. NASE 3, known to be resistant to UCBSV, accumulated negligible UCBSV-derived sRNAs but high populations of CBSV-derived sRNAs. Transcript levels of cassava homologues of AGO2, DCL2 and DCL4, which are central to the gene-silencing complex, were found to be differentially regulated in CBSV- and UCBSV-infected plants across genotypes, suggesting these proteins play a role in antiviral defense. Irrespective of genotype or viral pathogen, maximum populations of vsRNAs mapped to the cytoplasmic inclusion, P1 and P3 protein-encoding regions. Our results indicate disparity between CBSV and UCBSV host-virus interaction mechanisms, and provide insight into the role of virus-induced gene silencing as a mechanism of resistance to CBSD.

Cassava brown streak disease (CBSD) has major impacts on yield and quality of the tuberous roots of cassava in Eastern and Central Arica. At least two Potyviridae species cause the disease: Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV). Cloned viral genome sequences known as infectious clones (ICs) have been important in the study of other viruses, both as a means of standardising infectious material and characterising viral gene function. IC construction is often technically challenging for Potyviridae due to sequence instability in E. coli. Here, we evaluate three methods for the construction of infectious clones for CBSD. Whilst a simple IC for in vitro transcription was made for UCBSV isolate ‘Kikombe’, such an approach failed to deliver full-length clones for CBSV isolates ‘Nampula’ or ‘Tanza’, necessitating more complex approaches for their construction. The ICs successfully generated symptomatic infection in the model host N. benthamiana and in the natural host cassava. This shows that whilst generating ICs for CBSV is still a technical challenge, a structured approach, evaluating both in vitro and in planta transcription systems should successfully deliver ICs, allowing further study into the symptomology and virulence factors in this important disease complex.

A confined field trial was established to determine durability of RNAi-mediated resistance to Cassava brown streak disease (CBSD). Stem cuttings were obtained from field-grown cassava plants of cv 60444 transgenic for construct p718, consisting of an 894 bp inverted repeat sequence from the Ugandan Cassava brown streak virus (UCBSV) coat protein. Plants were established from three transgenic lines previously shown to provide complete resistance to UCBSV and differing levels of protection to the non-homologous virus species Cassava brown streak virus (CBSV), and grown for 11 months. CBSD symptoms were observed on shoots and storage roots of all non-transgenic cv 60444 control plants and transgenic lines p718–002 and p718–005, but not on p718–001. RT-PCR diagnostic showed tissues of plant lines p718–002 and p718–005 to be infected with CBSV, but free of UCBSV. All leaves and roots of p718–001 plants were to carry no detectable levels of either pathogen. Plants of cv 60444 in this field trial showed severe cassava mosaic disease symptoms, indicating that presence of replicating geminiviruses did not cause significant suppression of RNAi-mediated resistance to CBSD. Resistance to CBSD across a vegetative cropping cycle confirms earlier field data, and provides an important step in proof of concept for application of RNAi technology to control of CBSD under conditions encountered in farmers’ fields.

Cassava brown streak disease (CBSD), caused by cassava brown streak virus (CBSV; Ipomovirus brunusmanihotis) and Ugandan cassava brown streak virus (UCBSV; Ipomovirus manihotis) (family Potyviridae, genus Ipomovirus), is increasingly becoming a threat to cassava production in several parts of Africa, especially in Eastern, Central and Southern Africa. In Kenya, the disease continues to wreak havoc on cassava production leading to a significant reduction in crop yields and economic losses of up to USD 1 billion. Variation in virus populations make the control of CBSD challenging as virus genomic variation can affect the accuracy of diagnostic tests, lead to resistance breaking isolates and jeopardize strategies of breeding for resistance. CBSV and UCBSV populations obtained from cassava fields in Kenya were characterized. In total, 44 new complete sequences of CBSV and UCBSV were assembled and 40 sequences successfully submitted to GenBank. Single Nucleotide Polymorphism (SNP) analysis revealed that the cylindrical inclusion protein (CI) is the most stable region across the genome of CBSV and UCBSV. In contrast, protein 1 (PI) and the coat protein (CP) were the most hypervariable regions. Phylogenetic analysis showed three major geographical groupings for both UCBSV and CBSV isolates, suggesting a continued spread of the viruses through human-mediated movement of infected planting materials. The data obtained in this study can support the development of disease management strategies through improved molecular diagnostic tests and targets for breeding for resistance against CBSD.

A time-course transcriptome analysis of two cassava varieties that are either resistant or susceptible to cassava brown streak disease (CBSD) was conducted using RNASeq, after graft inoculation with Ugandan cassava brown streak virus (UCBSV). From approximately 1.92 billion short reads, the largest number of differentially expressed genes (DEGs) was obtained in the resistant (Namikonga) variety at 2 days after grafting (dag) (3887 DEGs) and 5 dag (4911 DEGs). At the same time points, several defense response genes (encoding LRR-containing, NBARC-containing, pathogenesis-related, late embryogenesis abundant, selected transcription factors, chaperones, and heat shock proteins) were highly expressed in Namikonga. Also, defense-related GO terms of ‘translational elongation’, ‘translation factor activity’, ‘ribosomal subunit’ and ‘phosphorelay signal transduction’, were overrepresented in Namikonga at these time points. More reads corresponding to UCBSV sequences were recovered from the susceptible variety (Albert) (733 and 1660 read counts per million (cpm)) at 45 dag and 54 dag compared to Namikonga (10 and 117 cpm respectively). These findings suggest that Namikonga’s resistance involves restriction of multiplication of UCBSV within the host. These findings can be used with other sources of evidence to identify candidate genes and biomarkers that would contribute substantially to knowledge-based resistance breeding.

In recent years, numerous single nucleotide polymorphism (SNP) panel methods to genotype non-invasive faecal samples have been developed. However, none of these existing methods fit all of the criteria necessary to make a SNP panel broadly usable for conservation projects in any country-cost effective, streamlined lab protocol and user-friendly open-source bioinformatics protocols for panel design and analysis. Here, we present such a method and display its utility by developing a multiplex PCR SNP panel for conducting individual ID of snow leopards, Panthera uncia, from faecal samples. The SNP panel we present consists of 144 SNPs and utilises next-generation sequencing technology. We validate our SNP panel with paired tissue and faecal samples from zoo individuals, showing a minimum of 96.7% accuracy in allele calls per run. We then generate SNP data from 235 field-collected faecal samples from across Pakistan to show that the panel can reliably identify individuals from low-quality faecal samples of unknown age and is robust to contamination. We also show that our SNP panel has the capability to identify first-order relatives among sampled zoo individuals and provides insights into the geographic origin of samples. This SNP panel will empower the snow leopard research community in their efforts to assess local and global snow leopard population sizes. More broadly, we present a SNP panel development method that can be used for any species of interest for which adequate genomic reference data is available.

BackgroundGenotype imputation is a key element of the implementation of genomic selection within the New Zealand sheep industry, but many factors can influence imputation accuracy. Our objective was to provide practical directions on the implementation of imputation strategies in a multi-breed sheep population genotyped with three single nucleotide polymorphism (SNP) panels: 5K, 50K and HD (600K SNPs).ResultsImputation from 5K to HD was slightly better (0.6 %) than imputation from 5K to 50K. Two-step imputation from 5K to 50K and then from 50K to HD outperformed direct imputation from 5K to HD. A slight loss in imputation accuracy was observed when a large fixed reference population was used compared to a smaller within-breed reference (including all 50K genotypes on animals from different breeds excluding those in the validation set i.e. to be imputed), but only for a few animals across all imputation scenarios from 5K to 50K. However, a major gain in imputation accuracy for a large proportion of animals (purebred and crossbred), justified the use of a fixed and large reference dataset for all situations. This study also investigated the loss in imputation accuracy specifically for SNPs located at the ends of each chromosome, and showed that only chromosome 26 had an overall imputation (5K to 50K) accuracy for 100 SNPs at each end higher than 60 % (r2). Most of the chromosomes displayed reduced imputation accuracy at least at one of their ends. Prediction of imputation accuracy based on the relatedness of low-density genotypes to those of the reference dataset, before imputation (without running an imputation software) was also investigated. FIMPUTE V2.2 outperformed BEAGLE 3.3.2 across all imputation scenarios.ConclusionsImputation accuracy in sheep breeds can be improved by following a set of recommendations on SNP panels, software, strategies of imputation (one- or two-step imputation), and choice of the animals to be genotyped using both high- and low-density SNP panels. We present a method that predicts imputation accuracy for individual animals at the low-density level, before running imputation, which can be used to restrict genomic prediction only to the animals that can be imputed with sufficient accuracy.

Cassava is an important food security and industrial crop. Its production is constrained by viral diseases such as cassava mosaic disease (CMD) and cassava brown streak disease (CBSD), caused by cassava mosaic begomoviruses (CMBs) and ipomoviruses, respectively. In recent years, CMBs have been associated with satellite DNAs. Food security status of cassava coupled with high demand for cassava as feed and industrial uses has been the driving force for scientists and the research community in Africa and beyond. In this review, cassava production, uses of cassava, production constraints, begomoviruses, satellite DNAs, Bemisia tabaci, cassava mosaic disease (CMD), Cassava brown streak virus (CBSV), current and future efforts in cassava production and research are discussed. This was done in an effort to create a knowledge pool that can promote cassava food security status and mitigate disease and yield loss.

Simple SummaryBemisia tabaci is an important vector that transmits cassava brown streak viruses and cassava mosaic begomoviruses that cause cassava brown streak and cassava mosaic diseases, respectively. In 2013 and 2015 we carried out a study to determine the genetic variability within the Bemisia tabaci complex associated with cassava in Zambia. This investigation made use of mitochondrial cytochrome oxidase I gene sequences of samples collected from selected provinces of Zambia. We found three population subgroups (SGs): SSA1-SG1, SSA1-SG2 and SSA1-SG3 within the sub-Saharan Africa 1 (SSA1) genetic group. Whitefly abundance and the incidence of cassava mosaic disease were both greatest in Western Province, in which the SSA1-SG1 subgroup predominated. Establishing which genetic groups and populations of the B. tabaci species complex are associated with cassava mosaic disease and their distribution in the country is key to guiding the strategic deployment of resources to monitor disease spread and ensure food security for millions of cassava-dependent households.Bemisia tabaci is an important vector of cassava brown streak viruses and cassava mosaic begomoviruses, the causal agents of cassava brown streak disease and cassava mosaic disease (CMD), respectively. A study was carried out to determine the genetic variability of B. tabaci associated with cassava and the occurrence of CMD in Zambia in 2013 and 2015. Phylogenetic analysis showed the presence of only the sub-Saharan Africa 1 (SSA1) genetic group in Zambia. The SSA1 population had three population subgroups (SGs): SSA1-SG1, SSA1-SG2 and SSA1-SG3. All three SSA1 population subgroups occurred in Western Province. However, only SSA1-SG3 occurred in Eastern Province, while only SSA1-SG1 occurred in North Western and Luapula Provinces. Adult B. tabaci were most abundant in Western Province in 2013 (11.1/plant) and 2015 (10.8/plant), and least abundant (0.2/plant) in Northern Province in both 2013 and 2015. CMD was prevalent in all seven provinces surveyed, with the highest incidence recorded in Lusaka Province in both 2013 (78%) and 2015 (83.6%), and the lowest in Northern Province in both 2013 (26.6%) and 2015 (29.3%). Although SSA1-SG1 occurred at greater abundances than the other subgroups, there was no direct association demonstrated between whitefly subgroup and incidence of CMD. Establishing which B. tabaci genetic groups and populations are associated with CMD and their distribution in the country is a key factor in guiding the development of CMD control strategies for cassava-dependent households.

This datasheet on Ugandan cassava brown streak virus covers Identity, Hosts/Species Affected, Further Information.

Imputation of genotypes from different single nucleotide polymorphism panels in dairy cattle

Cassava (Manihot esculenta Crantz) production and productivity in Africa is affected by two viral diseases; cassava mosaic disease (CMD) and cassava brown streak disease (CBSD). Induced mutagenesis of totipotent/embryogenic tissues or in vitro plant material can lead to the generation of CMD and/or CBSD tolerant mutants. To massively produce non-chimeric plants timely and with less labor, totipotent cells or tissues are a pre-requisite. This study aimed to determine the effect of gamma radiation on the proliferation and growth of friable embryogenic callus (FEC) and in vitro nodal cuttings respectively. To obtain FEC, 2-6 mm sized leaf lobes of nine cassava genotypes were plated on Murashige and Skoog (MS) basal media supplemented with varying levels (37, 50, 70, 100) μM of picloram for production of organized embryogenic structures (OES). The OES of five cassava genotypes (Alado, CV-60444, NASE 3, NASE 13 and TME 204) were crushed and plated in Gresshoff and Doy (GD) basal media in combination with the amino acid tyrosine in varying concentrations for FEC production. FEC from five cassava genotypes and in vitro nodal cuttings of nine genotypes were irradiated using five different gamma doses (0, 5, 10, 15, 20 and 25 Gy) at a dose rate of 81Gy/hr. The lethal dose (LD)50 was determined using the number of roots produced and flow cytometry was done to determine the ploidy status of plants. The highest production of OES was noted in Alado across varying picloram concentrations, while TME 204 obtained the highest amount of FEC. The irradiated FEC gradually died and by 28 days post irradiation, FEC from all five cassava genotypes were lost. Conversely, the irradiated in vitro nodal cuttings survived and some produced roots, while others produced callus. The LD50 based on number of roots varied from genotype to genotype, but plants remained diploid post-irradiation. Accordingly, the effect of gamma irradiation on Ugandan cassava genotypes (UCGs) was genotype-dependent. This information is foundational for the use of in vitro tissues as target material for cassava mutation breeding.

A novel panel of single nucleotide polymorphisms (SNPs) used in the classification of ancestral origin is proposed. The panel is motivated by recent results in human identification and categorization using soft biometrics. The panel makes use of SNPs related to the height and ear size of an individual. The classifier proposed in this thesis is a standard maximum-likelihood classifier that matches an individual to the ancestral profile closest to the individual. This method produces results with lower error rates than other proposed methods.;This thesis proposes a new distinct panel of 55 single nucleotide polymorphisms for determining ancestral origin. This panel was later reduced to two panels of 18SNPs and 22SNPs respectively. This new approach is producing a high success rate, and has great promise for future progress. This panel is unique in that all prior research has made use of SNPs related to skin pigmentation since this is considered the most readily identifiable difference between different ancestral groups. We chose to use the four common ancestry groups of African American, Asian, European, and Sub-Saharan African.;The proposed panel of 55 SNPs had a successful classification rate of 98% when tested on simulated data, while the 22 SNP panel when tested on real data had a successful classification rate of 86% due to SNP availability issues in the real data. Using a knockout procedure, the 55 SNP panel was reduced to an 18 SNP panel that had a successful classification rate of 99.8% on simulated data.