Identification of Genomic Regions for Traits Associated with Flowering in Cassava (Manihot esculenta Crantz).

Flowering in cassava (Manihot esculenta Crantz) is crucial for the generation of botanical seed for breeding. However, genotypes preferred by most farmers are erect and poor at flowering or never flower. To elucidate the genetic basis of flowering, 293 diverse cassava accessions were evaluated for flowering-associated traits at two locations and seasons in Uganda. Genotyping using the Diversity Array Technology Pty Ltd. (DArTseq) platform identified 24,040 single-nucleotide polymorphisms (SNPs) distributed on the 18 cassava chromosomes. Population structure analysis using principal components (PCs) and kinships showed three clusters; the first five PCs accounted for 49.2% of the observed genetic variation. Linkage disequilibrium (LD) estimation averaged 0.32 at a distance of ~2850 kb (kilo base pairs). Polymorphism information content (PIC) and minor allele frequency (MAF) were 0.25 and 0.23, respectively. A genome-wide association study (GWAS) analysis uncovered 53 significant marker-trait associations (MTAs) with flowering-associated traits involving 27 loci. Two loci, SNPs S5_29309724 and S15_11747301, were associated with all the traits. Using five of the 27 SNPs with a Phenotype_Variance_Explained (PVE) ≥ 5%, 44 candidate genes were identified in the peak SNP sites located within 50 kb upstream or downstream, with most associated with branching traits. Eight of the genes, orthologous to Arabidopsis and other plant species, had known functional annotations related to flowering, e.g., eukaryotic translation initiation factor and myb family transcription factor. This study identified genomic regions associated with flowering-associated traits in cassava, and the identified SNPs can be useful in marker-assisted selection to overcome hybridization challenges, like unsynchronized flowering, and candidate gene validation.

Flowering and fruit-set in cassava under extended red-light photoperiod supplemented with plant-growth regulators and pruning.

BACKGROUND: Cassava (Manihot esculenta Crantz) is staple food and major source of calories for over 500 million people in sub-Saharan Africa. The crop is also a source of income for smallholder farmers, and has increasing potential for industrial utilization. However, breeding efforts to match the increasing demand of cassava are impeded by its inability to flower, delayed or unsynchronized flowering, low proportion of female flowers and high fruit abortions. To overcome these sexual reproductive bottlenecks, this study investigated the effectiveness of using red lights to extend the photoperiod (RLE), as a gateway to enhancing flowering and fruit set under field conditions. MATERIALS AND METHODS: Panels of cassava genotypes, with non- or late and early flowering response, 10 in each case, were subjected to RLE from dusk to dawn. RLE was further evaluated at low (LL), medium (ML) and high (HL) red light intensities, at ~ ≤ 0.5; 1.0 and 1.5PFD (Photon Flux Density) in µmol m-2 s-1 respectively. Additionally, the effect of a cytokinin and anti-ethylene as plant growth regulators (PGR) and pruning under RLE treatment were examined. RESULTS: RLE stimulated earlier flower initiation in all genotypes, by up to 2 months in the late-flowering genotypes. Height and number of nodes at first branching, particularly in the late-flowering genotypes were also reduced, by over 50%. Number and proportion of pistillate flowers more than doubled, while number of fruits and seeds also increased. Number of branching levels during the crop season also increased by about three. Earlier flowering in many genotypes was most elicited at LL to ML intensities. Additive effects on flower numbers were detected between RLE, PGR and pruning applications. PGR and pruning treatments further increased number and proportion of pistillate flowers and fruits. Plants subjected to PGR and pruning, developed bisexual flowers and exhibited feminization of staminate flowers. Pruning at first branching resulted in higher pistillate flower induction than at second branching. CONCLUSIONS: These results indicate that RLE improves flowering in cassava, and its effectiveness is enhanced when PGR and pruning are applied. Thus, deployment of these technologies in breeding programs could significantly enhance cassava hybridizations and thus cassava breeding efficiency and impact.

Molecular Diversity of Rice Yellow Mottle Virus in Uganda and Relationships with Other Strains from Africa.

Rice yellow mottle virus disease, caused by Rice yellow mottle virus (RYMV), is the most important disease of lowland rice in Uganda. However, little is known about its genetic diversity in Uganda and relationships with other strains elsewhere across Africa. A new degenerate primer pair that targets amplification of the entire RYMV coat protein gene (circa 738 bp) was designed to aid virus variability analysis using RT-PCR and Sanger sequencing. A total of 112 rice leaf samples from plants with RYMV mottling symptoms were collected during the year 2022 in 35 lowland rice fields within Uganda. The RYMV RT-PCR results were 100% positive, and all 112 PCR products were sequenced. BLASTn analysis revealed that all isolates were closely related (93 to 98%) to those previously studied originating from Kenya, Tanzania, and Madagascar. Despite high purifying selection pressure, diversity analysis on 81 out of 112 RYMV CP sequences revealed a very low diversity index of 3 and 1.0% at the nucleotide and amino acid levels, respectively. Except for glutamine, amino acid profile analysis revealed that all 81 Ugandan isolates shared the primary 19 amino acids based on the RYMV coat protein region examined. Except for one isolate (UG68) from eastern Uganda that clustered alone, phylogeny analysis revealed two major clades. The Ugandan RYMV isolates were phylogenetically related to those from the Democratic Republic of Congo, Madagascar, and Malawi but not to RYMV isolates in West Africa. Thus, the RYMV isolates in this study are related to serotype 4, a strain common in eastern and southern Africa. RYMV serotype 4 originated in Tanzania, where evolutionary forces of mutation have resulted in the emergence and spread of new variants. Furthermore, mutations are evident within the coat protein gene of the Ugandan isolates, which may be attributed to changing RYMV pathosystems as a result of rice production intensification in Uganda. Overall, the diversity of RYMV was limited and most noticeably in eastern Uganda.

Use of timelapse photography to determine flower opening time and pattern in banana (Musa spp.) for efficient hand pollination.

Sterility and low seed set in bananas is the main challenge to their conventional genetic improvement. The first step to seed set in a banana breeding program depends on pollination at the right time to ensure effective fertilization. This study aimed at determining bract opening time (BOT) to enhance efficient pollination and seed set in bananas. A Nikon D810 digital camera was set-up to take pictures of growing banana inflorescences at five-minute intervals and time-lapse movies were developed at a speed of 30 frames per second to allow real-time monitoring of BOT. Genotypes studied included wild banana (1), Mchare (2), Matooke (4), Matooke hybrid (1), and plantain (1). Events of bract opening initiated by bract lift for female flowers (P < 0.01) started at 16:32 h and at 18:54 h for male flowers. Start of bract rolling was at 18:51 h among female flowers (P < 0.001) and 20:48 h for male flowers. Bracts ended rolling at 02:33 h and 01:16 h for female and flowers respectively (P < 0.05). Total time of bract opening (from lift to end of rolling) for female flowers was significantly longer than that of male flowers (P < 0.001). On average, the number of bracts subtending female flowers opening increased from one on the first day, to between one and four on the fourth day. The number regressed to one bract on day eight before start of opening of bracts subtending male flowers. There was a longer opening interval between bracts subtending female and male flowers constituting spatial and temporal separation. Bract rolling increased from partial to complete rolling from proximal to the distal end of the inflorescence among female flower. On the other hand, bracts subtending male flowers completely rolled. Differences in BOT of genotypes with the same reference time of assessment may be partly responsible for variable fertility. Hand pollination time between 07:00 and 10:00 h is slightly late thus an early feasible time should be tried.

In silico prediction and segregation analysis of putative virus defense genes based on SSR markers in sweet potato F1 progenies of cultivars 'New Kawogo' and 'Resisto'.

In sweet potato, an anti-virus defense mechanism termed reversion has been postulated to lead to virus freedom from once infected plants. The objectives of this study were to identify anti-virus defense genes and evaluate their segregation in progenies. Reference genes from different plant species were used to assemble transcript sequences of each sweet potato defense gene in silico. Sequences were used for evaluate phylogenetic relationships with similar genes from different plant species, mining respective defense genes and thereafter developing simple sequence repeats (SSRs) for segregation analysis. Eight potential defense genes were identified: RNA dependent RNA polymerases 1, 2, 5, and 6; Argonaute 1, and Dicer-like 1, 2, and 4. Identified genes were differentially related to those of other plants and were observed on different chromosomes. The defense genes contained mono-, di-, tri-, tetra, penta-, and hexa-nucleotide repeat motifs. The SSR markers within progenies were segregated in disomic, co-segregation, nullisomic, monosomic, and trisomic modes. These findings indicate the possibility of deriving and utilizing SSRs using published genomic information. Furthermore, and given that the SSR markers were derived from known genes on defined chromosomes, this work will contribute to future molecular breeding and development of resistance gene analogs in this economically important crop.

Factors influencing reversion from virus infection in sweetpotato.

Viruses limit sweetpotato (Ipomoea batatas) production worldwide. Many sweetpotato landraces in East Africa are, however, largely virus-free. Moreover, some plants infected by the prevalent Sweet potato feathery mottle virus (SPFMV) may be able to revert to virus-free status. In this study, we analysed reversion from SPFMV, Sweet potato virus C, Sweet potato mild mottle virus, Sweet potato chlorotic stunt virus (SPCSV) and Sweet potato leaf curl Uganda virus using the indicator plant I. setosa and PCR/reverse-transcriptase PCR. We also investigated environmental factors (temperature and soil nutrients) that may influence reversion from virus infection. We tested reversion in the East African cultivars New Kawogo, NASPOT 1 and NASPOT 11, and the United States cultivars Resisto and Beauregard. Reverted plants were asymptomatic and virus was undetectable in assayed parts of the plant. After graft inoculation, only the East African cultivars mostly reverted at a high rate and from most viruses though cultivar Beauregard fully reverted following sap inoculation with Sweet potato virus C. None of the tested cultivars fully reverted from single or double infections involving SPCSV, and reversion was only observed in co-infections involving potyviruses. Root sprouts derived from SPFMV-reverted plants were also virus free. Reversion generally increased with increasing temperature and by improved soil nutrition. Overall, these results indicate variation in reversion by cultivar and that the natural ability of sweetpotato plants to revert from viruses is malleable, which has implications for both breeding and virus control.

Mixed Infections of Four Viruses, the Incidence and Phylogenetic Relationships of Sweet Potato Chlorotic Fleck Virus (Betaflexiviridae) Isolates in Wild Species and Sweetpotatoes in Uganda and Evidence of Distinct Isolates in East Africa.

Viruses infecting wild flora may have a significant negative impact on nearby crops, and vice-versa. Only limited information is available on wild species able to host economically important viruses that infect sweetpotatoes (Ipomoea batatas). In this study, Sweet potato chlorotic fleck virus (SPCFV; Carlavirus, Betaflexiviridae) and Sweet potato chlorotic stunt virus (SPCSV; Crinivirus, Closteroviridae) were surveyed in wild plants of family Convolvulaceae (genera Astripomoea, Ipomoea, Hewittia and Lepistemon) in Uganda. Plants belonging to 26 wild species, including annuals, biannuals and perennials from four agro-ecological zones, were observed for virus-like symptoms in 2004 and 2007 and sampled for virus testing. SPCFV was detected in 84 (2.9%) of 2864 plants tested from 17 species. SPCSV was detected in 66 (5.4%) of the 1224 plants from 12 species sampled in 2007. Some SPCSV-infected plants were also infected with Sweet potato feathery mottle virus (SPFMV; Potyvirus, Potyviridae; 1.3%), Sweet potato mild mottle virus (SPMMV; Ipomovirus, Potyviridae; 0.5%) or both (0.4%), but none of these three viruses were detected in SPCFV-infected plants. Co-infection of SPFMV with SPMMV was detected in 1.2% of plants sampled. Virus-like symptoms were observed in 367 wild plants (12.8%), of which 42 plants (11.4%) were negative for the viruses tested. Almost all (92.4%) the 419 sweetpotato plants sampled from fields close to the tested wild plants displayed virus-like symptoms, and 87.1% were infected with one or more of the four viruses. Phylogenetic and evolutionary analyses of the 3'-proximal genomic region of SPCFV, including the silencing suppressor (NaBP)- and coat protein (CP)-coding regions implicated strong purifying selection on the CP and NaBP, and that the SPCFV strains from East Africa are distinguishable from those from other continents. However, the strains from wild species and sweetpotato were indistinguishable, suggesting reciprocal movement of SPCFV between wild and cultivated Convolvulaceae plants in the field.

Genetic variability and evolutionary implications of RNA silencing suppressor genes in RNA1 of sweet potato chlorotic stunt virus isolates infecting sweetpotato and related wild species.

BACKGROUND: The bipartite single-stranded RNA genome of Sweet potato chlorotic stunt virus (SPCSV, genus Crinivirus; Closteroviridae) encodes a Class 1 RNase III (RNase3), a putative hydrophobic protein (p7) and a 22-kDa protein (p22) from genes located in RNA1. RNase3 and p22 suppress RNA silencing, the basal antiviral defence mechanism in plants. RNase3 is sufficient to render sweetpotato (Ipomoea batatas) virus-susceptible and predisposes it to development of severe diseases following infection with unrelated virus. The incidence, strains and gene content of SPCSV infecting wild plant species have not been studied. METHODOLOGY/PRINCIPAL FINDINGS: Thirty SPCSV isolates were characterized from 10 wild Ipomoea species, Hewittia sublobata or Lepistemon owariensis (family Convolvulaceae) in Uganda and compared with 34 local SPCSV isolates infecting sweetpotatoes. All isolates belonged to the East African (EA) strain of SPCSV and contained RNase3 and p7, but p22 was not detected in six isolates. The three genes showed only limited genetic variability and the proteins were under purifying selection. SPCSV isolates lacking p22 synergized with Sweet potato feathery mottle virus (SPFMV, genus potyvirus; Potyviridae) and caused severe symptoms in co-infected sweetpotato plants. One SPCSV isolate enhanced accumulation of SPFMV, but no severe symptoms developed. A new whitefly-transmitted virus (KML33b) encoding an RNase3 homolog (<56% identity to SPCSV RNase3) able to suppresses sense-mediated RNA silencing was detected in I. sinensis. CONCLUSIONS/SIGNIFICANCE: SPCSV isolates infecting wild species and sweetpotato in Uganda were genetically undifferentiated, suggesting inter-species transmission of SPCSV. Most isolates in Uganda contained p22, unlike SPCSV isolates characterized from other countries and continents. Enhanced accumulation of SPFMV and increased disease severity were found to be uncoupled phenotypic outcomes of RNase3-mediated viral synergism in sweetpotato. A second virus encoding an RNase3-like RNA silencing suppressor was detected. Overall, results provided many novel and important insights into evolutionary biology of SPCSV.

Molecular genetic analysis of virus isolates from wild and cultivated plants demonstrates that East Africa is a hotspot for the evolution and diversification of sweet potato feathery mottle virus.

Sweet potato feathery mottle virus (SPFMV, genus Potyvirus) is globally the most common pathogen of cultivated sweet potatoes (Ipomoea batatas; Convolvulaceae). Although more than 150 SPFMV isolates have been sequence-characterized from cultivated sweet potatos across the world, little is known about SPFMV isolates from wild hosts and the evolutionary forces shaping SPFMV population structures. In this study, 46 SPFMV isolates from 14 wild species of genera Ipomoea, Hewittia and Lepistemon (barcoded for the matK gene in this study) and 13 isolates from cultivated sweet potatoes were partially sequenced. Wild plants were infected with the EA, C or O strain, or co-infected with the EA and C strains of SPFMV. In East Africa, SPFMV populations in wild species and sweet potato were genetically undifferentiated, suggesting inter-host transmission of SPFMV. Globally, spatial diversification of the 178 isolates analysed was observed, strain EA being largely geographically restricted to East Africa. Recombination was frequently detected in the 6K2-VPg-NIaPro region of the EA strain, demonstrating a recombination 'hotspot'. Recombination between strains EA and C was rare, despite their frequent co-infections in wild plants, suggesting purifying selection against strain EA/C recombinants. Positive selection was predicted on 17 amino acids distributed over the entire coat protein in the globally distributed strain C, as compared to only four amino acids in the coat protein N-terminus of the EA strain. This selection implies a more recent introduction of the C strain and a higher adaptation of the EA strain to the local ecosystem. Thus, East Africa appears as a hotspot for evolution and diversification of SPFMV.

Recombination and selection pressure in the ipomovirus sweet potato mild mottle virus (Potyviridae) in wild species and cultivated sweetpotato in the centre of evolution in East Africa.

Sweet potato mild mottle virus (SPMMV) is the type member of the genus Ipomovirus (family Potyviridae). SPMMV occurs in cultivated sweetpotatoes (Ipomoea batatas Lam.; Convolvulaceae) in East Africa, but its natural wild hosts are unknown. In this study, SPMMV was detected in 283 (9.8 %) of the 2864 wild plants (family Convolvulaceae) sampled from different agro-ecological zones of Uganda. The infected plants belonged to 21 species that were previously not known to be natural hosts of SPMMV. The size of the SPMMV coat protein (CP) was determined by Western blot analysis, N-terminal protein sequencing and peptide mass fingerprinting. Data implicated a proteolytic cleavage site, VYVEPH/A, at the NIb/CP junction, resulting in a CP of approximately 35 kDa. Nearly complete sequences of 13 SPMMV isolates were characterized. Phylogenetic analysis of non-recombinant CP-encoding sequences placed five isolates from wild species sampled in the central zone of Uganda into a separate cluster. Recombination events were detected in the 5'- and 3'-proximal parts of the genome, providing novel evidence of recombination in the genus Ipomovirus. Thirteen amino acids in the N terminus of the P1 protein were under positive selection, whereas purifying selection was implicated for the HC-Pro-, P3-, 6K1- and CP-encoding regions. These data, supported by previous studies on ipomoviruses, provide indications of an evolutionary process in which the P1 proteinase responds to the needs of adaptation.

Cassava brown streak virus (Potyviridae) encodes a putative Maf/HAM1 pyrophosphatase implicated in reduction of mutations and a P1 proteinase that suppresses RNA silencing but contains no HC-Pro.

The complete positive-sense single-stranded RNA genome of Cassava brown streak virus (CBSV; genus Ipomovirus; Potyviridae) was found to consist of 9,069 nucleotides and predicted to produce a polyprotein of 2,902 amino acids. It was lacking helper-component proteinase but contained a single P1 serine proteinase that strongly suppressed RNA silencing. Besides the exceptional structure of the 5'-proximal part of the genome, CBSV also contained a Maf/HAM1-like sequence (678 nucleotides, 226 amino acids) recombined between the replicase and coat protein domains in the 3'-proximal part of the genome, which is highly conserved in Potyviridae. HAM1 was flanked by consensus proteolytic cleavage sites for ipomovirus NIaPro cysteine proteinase. Homology of CBSV HAM1 with cellular Maf/HAM1 pyrophosphatases suggests that it may intercept noncanonical nucleoside triphosphates to reduce mutagenesis of viral RNA.

Unravelling the genetic diversity of the three main viruses involved in Sweet Potato Virus Disease (SPVD), and its practical implications.

SUMMARY Sweetpotato (Ipomoea batatas) is a widely grown food crop, in which the most important diseases are caused by viruses. Genetic variability of three widely distributed sweetpotato viruses was analysed using data from 46 isolates of Sweet potato feathery mottle virus (SPFMV), 16 isolates of Sweet potato mild mottle virus (SPMMV) and 25 isolates of Sweet potato chlorotic stunt virus (SPCSV), of which 19, seven and six isolates, respectively, are newly characterized. Division of SPFMV into four genetic groups (strains) according to phylogenetic analysis of coat protein (CP) encoding sequences revealed that strain EA contained the East African isolates of SPFMV but none from elsewhere. In contrast, strain RC contained ten isolates from Australia, Africa, Asia and North America. Strain O contained six heterogeneous isolates from Africa, Asia and South America. The seven strain C isolates from Australia, Africa, Asia, and North and South America formed a group that was genetically distant from the other SPFMV strains. SPMMV isolates showed a high level of variability with no discrete strain groupings. SPCSV isolates from East Africa were phylogenetically distant to SPCSV isolates from elsewhere. Only from East Africa were adequate data available for different isolates of the three viruses to estimate the genetic variability of their local populations. The implications of the current sequence information and the need for more such information from most sweetpotato-growing regions of the world are discussed in relation to virus diagnostics and breeding for virus resistance.

Incidence of Viruses and Virus like Diseases of Sweetpotato in Uganda.

Sweetpotato plants were surveyed for viruslike diseases and viruses in the four major agroecological zones of Uganda. Testing of 1,260 sweetpotato plants, of which 634 had virus-like symptoms, showed that virus disease incidence ranged from 2.7% (Soroti district, short grassland-savannah zone) to 20% (Mukono district, tall grass-forest mosaic zone). Sweet potato chlorotic stunt virus (SPCSV), Sweet potato feathery mottle virus (SPFMV), Sweet potato mild mottle virus (SPMMV), and sweet potato chlorotic fleck virus (SPCFV) were serologically detected and positive results confirmed by immunocapture reverse transcriptase polymerase chain reaction (IC-RT-PCR) and subsequent sequence analyses of the amplified fragments, except SPCFV, which lacked sequence information. SPCSV and SPFMV were detected in all the 14 districts surveyed, whereas SPMMV and SPCFV were detected in 13 and 8 districts, respectively. Logistic regression analysis revealed that SPCSV and SPFMV, SPFMV and SPMMV, and SPFMV and SPCFV more frequently occurred together than any other virus combinations or as single virus infections. Co-infections of SPCSV with SPFMV and/or SPMMV were associated with more severe and persistent symptoms than infections with each of the viruses alone. Several plants (11%) displaying viruslike symptoms did not react with the virus antisera used, suggesting that more viruses or viruslike agents are infecting sweetpotatoes in Uganda.