The rolling circle amplification and next generation sequencing approaches reveal genome wide diversity of Kenyan cassava mosaic geminivirus

Rolling circle amplification is a simple approach of enriching populations of single-stranded DNA plant begomovirus genomes (genus, Begomovirus; family,  Geminiviridae). This is an innovative approach that utilizes the robustness of the bacteriophage phi29 DNA polymerase used in circle amplification, together with deep sequencing using Illumina Miseq and bioinformatics to assess population  diversity of begomoviruses in naturally infected cassava. The approach is suitable for detecting rare members in a population in begomoviral populations in situation where mixtures of isolates, strains, and multiple species occur. The main objectives were to increase the sensitivity of detection of next generation sequencing by enriching it using rolling circle amplification then determination of the diversity of  the cassava mosaic geminivirus. This was done by total nucleic acids isolated from symptomatic, field cassava infected plants, then using rolling circle amplification to multiply the less abundant viral  sequences. Enriched and non-enriched virus-libraries were subjected to deep sequencing using Illumina Miseq. Using  bioinformatic CLC Genomics 5.5.1 software programs the quality assessment of reads and contig assembly of viral sequences. This was done through de novo and reference-guided assembly. The identity and diversities of the begomoviral sequences were compared with sequences in Sanger sequencing of viral components deposited in the NCBI Gene Bank. In this study we have demonstrated that RCA increases the chances of detecting the virus by approximately 10 to 1000 fold and wide genome diversity of cassava mosaic geminivirus in various cassava growing zones in Kenya were detected. In conclusion, this approach described herein is simple and will enhance the exploration of begomovirus diversities from cassava infected plants, irrespective of their viral abundance. This will make it possible for routine screening of field samples as the cost of deep sequencing NGS is decreasing and the advances of bioinformatic software development become enhanced. This is the first report of the RCA-Illumina-NGS approach to explore cassava infected with begomoviruses under field conditions and their diversities. Key words: Illumina Miseq sequencing, geminivirus, ssDNA viruses, viral sequence enrichment, de novo genome assembly, rolling cycle amplification (RCA)

Detection of latent infection by Ralstonia solanacearum in potato (Solanum tuberosum) using stems instead of tubers

The potential of using stems for the detection of latent infection caused by Ralstonia solanacearum (Rs) was studied. Forty plants each were collected from four farms with bacterial wilt incidence below 4% intwo growing seasons (season A and season B of 2005). The tubers of all the selected plants including 10 cm of the all lower stems were collected. Samples were taken to the laboratory for indexing againstR. solanacearu (Rs) using ELISA techniques. The Rs status of each of the composite samples of all the tubers and of stems was determined and then correlation coefficients computed. There was a notabledifference in the percentage number of samples per farm with particular categories of R. solanacearum status. When stems were compared to tubers for detection of Rs, an average r – value of 0.4 wasobtained when r-values for the four different farms were averaged. The lowest r-value recorded was 0.2 while the highest was 0.5. When individual farms were considered it was only in one farm out of the fourthat r was not significant (p = 0.2). Overall the r-value was significant (p < 0.05). These results indicate that there is scope for adoption of stems as an alternative sample to tubers for indexing against R.solanacearum in potato tuber seed certification schemes more so in screening for presence of R. solanacearum in seed potato fields. However, although significant, the low r-value calls for moreinvestigations to be done prior to final recommendation on use of stems from potato fields

Estimation of genetic diversity of the Kenyan yam (Dioscorea spp.) using microsatellite markers

Yam landraces in Kenya have not been fully characterized both at morphological and molecular level. Application of molecular markers can overcome this bottleneck. 187 accessions comprising of 166 yam landraces and 21 Yam DNA samples from IITA, Nigeria were extracted from leaf samples grown at Muguga and genotyped at BeCA. DNA was extracted using CTAB method. Twelve (12) primer pairs were used for genotyping and PCR products detected on ABI-3730 capillary system. Data was analyzed for genetic diversity, ordination and analysis of molecular variance with GenAIEx software. A total of 131 alleles were amplified with a minimum of two alleles and a maximum of 13 alleles per primer with a minimum allele size of 64 bp and a maximum of 368 bp. Accessions from Eastern province had the highest number of unique alleles. Shannon’s information index (I) was 0.1444 for West African samples and 0.2366 for Central province. Accession dispersion revealed four clusters with no distinct geographical pattern. Dense clustering of accessions was an indication of genetic relatedness. Analysis of molecular variance revealed that most variation of 88% (P&lt;0.010) was found within populations or provinces. The simple sequence repeats (SSR) markers were polymorphic and were able to discriminate local yam landraces.Keywords: Genetic diversity, microsatellite, yam, KenyaAfrican Journal of Biotechnology Vol. 12(40), pp. 5845-585

Morphological diversity of mango germplasm from the upper Athi River region of Eastern Kenya: An analysis based on non- fruit descriptors

Phenotypic variation in plants can be evaluated by morphological characterization using visual attributes. Fruits have been the major descriptors for identification of different varieties of fruit crops. However, even in their absence, farmers, breeders and interested stakeholders require to distinguish between different mango varieties. This study aimed at determining diversity in mango germplasm from the Upper Athi River (UAR) and providing useful alternative descriptors for the identification of different mango varieties in the absence of fruits. A total of 20 International Plant Genetic Resources Institute (IPGRI) descriptors for mango were selected for use in the visual assessment of 98 mango accessions from 15 sites of the UAR region of eastern Kenya. Purposive sampling was used to identify farmers growing diverse varieties of mangoes. Evaluation of the descriptors was performed on-site and the data collected were then subjected to multivariate analysis including Principal Component Analysis (PCA) and Cluster analysis, one- way analysis of variance (ANOVA) and Chi square tests. Results classified the accessions into two major groups corresponding to indigenous and exotic varieties. The PCA showed the first six principal components accounting for 75.12% of the total variance. A strong and highly significant correlation was observed between the color of fully grown leaves, leaf blade width, leaf blade length and petiole length and also between the leaf attitude, color of young leaf, stem circumference, tree height, leaf margin, growth habit and fragrance. Useful descriptors for morphological evaluation were 14 out of the selected 20; however, ANOVA and Chi square test revealed that diversity in the accessions was majorly as a result of variations in color of young leaves, leaf attitude, leaf texture, growth habit, leaf blade length, leaf blade width and petiole length traits. These results reveal that mango germplasm in the UAR has significant diversity and that other morphological traits apart from fruits can be useful in morphological characterization of mango.Keywords: Mango, morphological characterization, Principal Component Analysis, IPGRI, eastern Keny

Evaluating diversity among Kenyan papaya germplasm using simple sequence repeat markers

Papaya is an important fruit crop, produced in Kenya for local consumption and export. Despite a history of varietal introductions, no attempts concerned on developing varieties suited to Kenyan conditions have been documented. The objective of this study was to provide information on the diversity of germplasm available in Kenya, as a precursor to systematic plant breeding program. Forty two papaya accessions were collected from farmers’ fields located in Coast, Rift Valley, Western, Nyanza, Central and Eastern provinces. Genetic diversity was determined using seven simple sequence repeat (SSR) markers, computing allelic richness and frequency, expected heterozygosity and cluster analysis. Results indicated that themarkers were highly polymorphic among the accessions, with polymorphicinformation content (PIC) varying from 0.75 to 0.852 with an average of 0.81. The genetic similarity among the 42 papaya accessions ranged from 0.764 to 0.932 with an average of 0.844 showing that most papaya accessions used in this study were closely related. About 96.9% of the pair-wise comparisons among papaya accessions exhibited genetic similarity greater than 0.802, while less than 4% (3.1%) showed genetic similarity lower than 0.802. The phylogenetic analysis grouped the 42accessions into two main clusters A and B. Cluster A had four sub-clusters while cluster B had one cluster. Accessions from Coast, and some from Rift Valley Provinces, presented the highest variation, being scattered throughout the tree, with little or no differentiation from most accessions, whereas some accessions from Coast regrouped in clusters A (iv) and B. The genetic differences among the accessions revealed by the formation of distinct clusters suggest significant genetic variability emanation from varying sources of the papaya germplasm in Kenya. Although the level of genetic diversity revealed by SSR markers in this study is sufficient todistinguish between breeding lines for varietal protection, the rather narrow genetic diversity demonstrated indicates the need to introduce new germplasm or use other techniques such as mutation and genetic engineering to provide breeding materials for the future improvement of papaya in Kenya.Key words: Kenya, papaya, genetic diversity

Morphological diversity of Kenyan papaya germplasm

Papaya is one of the major fruit crops of the tropical regions of the world. It shows considerable phenotypic variation in morphological and horticultural traits that can be utilized in its genetic improvement. In Kenya, wide range of papaya germplasm exists and has not been characterized. Therefore, there is difficulty in differentiating the papaya accessions in the different regions of Kenya. Characterization of papaya germplasm is normally accomplished by use of morphological descriptors, hence as a first step, a germplasm collection from within Kenya was gathered and its morphological diversity was assessed. The papaya germplasm was collected from Coast, Nyanza, Western, Rift Valley, Eastern and Central provinces of Kenya and characterized in the field using morphological descriptors based on fruit, flower, stem and leaf characteristics. The morphological characters were recorded andmorphological data from sixty accessions were submitted to principal component and Neighbor- Joining cluster analysis. Accessions from Coastal, Western, Rift Valley and Nyanza provinces showed the widest morphological diversity with those from Eastern and Central provinces showing the least diversity. Fruit shape, fruit diameter, tree habit, leaf size and flower color showed the greatest variation in principal component analysis. The high diversity observed within the accessions points to ample possibilities of obtaining desirable trait combinations in specific cultivars.Keywords: Kenya, papaya, germplasm, morphological characterizatio

Biotechnology in plant nutrient management for agricultural production in the tropics: The research link

The potential benefits of biotechnology are extraordinary and traverse sectors like agriculture, environment, health, industry, bio-informatics, and human resource development. In agriculture, biotechnology research has helped to improve the understanding of diseases, to improve the diagnosis and treatment of diseases, to improve resistance to herbicides/insects/pests/diseases/drought, to improve crop varieties and yields, marker assisted selection breeding, to develop new uses foragricultural products, to facilitate early maturation and to improve food and feed nutritional value. The uncertainties and risks of biotechnology are yet to be fully understood but its possibilities are yet alsoto be fully exploited for agricultural production. Research has currently linked plant nutrition to biotechnology through plants modifications to obtain improved photosynthetic system and enhanced nutrient uptake. Due to the corresponding higher physiological efficiency of the improved crops via the biotechnological modifications, plant nutrient management can be adjusted appropriately. These adjustments ultimately lead to other potential benefits in agriculture that include reduced labour andcapital inputs, improved environmental protection and strengthened rural economies, which can translate into sustained agricultural production

Cassava starch as an alternative cheap gelling agent for the in vitro micro-propagation of potato (Solanum tuberosum L.)

The potential of cassava starch as an alternative and cheap gelling agent for potato in vitro culture micro-propagation media was investigated. A two-factor experiment in randomized complete blockdesign was conducted. Four levels of gelling agents; 10% (w/v) cassava starch, 8% cassava starch mixed with 0.25% agar, 0.8% agar and a liquid medium, were evaluated using three selected Kenyanpotato cultivars (Tigoni, Asante and Kenya Sifa). Cassava starch at 10% gave adequate support of explants, though it had low viscosity and softened at 42 days after explant inoculation. Cassava starchmixed with 0.25% agar provided the same firmness as 0.8% agar and maintained gel integrity throughout the culturing period of 84 days. Survival in- and ex vitro was lowest in liquid medium culture. Potato transplants from the liquid medium and cassava starch gelled medium had similar (p > 0.05) mean number of nodes and biomass. These mean values were significantly higher compared to the transplants from the agar gelled medium. The use of 10% cassava starch reduced cost by 42.5% in comparison with use of agar

Table sugar as an alternative low cost medium component for in vitro micro-propagation of potato (Solanum tuberosum L.)

Most developing countries are limited in maximizing tissue culture technology due to the overhead costs involved. In view of this, the aim of this research was to evaluate alternative cheap sources ofcarbon and energy in potato culture media in order to reduce the overall cost of micro-propagation. A randomized complete block design was used to compare laboratory grade sucrose with two types oflocal commercial table sugar, specifically white and brown sugar. Three selected Kenyan potato cultivars, Tigoni, Asante and Kenya Sifa were cultured on full strength Murashige and Skoog (MS) medium at 3% (w/v) in combination with the 3 different sugars. The variation in growth performance of the cultivars was then observed. Plantlet survival of 100% was recorded after four subculture generations on all sugars for all the cultivars. The mean number of nodes per plantlet was significantly higher in brown sugar for cultivars Kenya Sifa and Asante. Brown sugar enhanced significantly higher mean number of roots per plantlet after four subculture generations for all cultivars. There was nosignificant difference in percentage of plantlet survival after transplanting for cultivars Asante and Kenya Sifa but significantly lower for cultivar Tigoni on grade sucrose medium. Results also showedthat table sugar not only enhanced micro-propagation but also significantly lowered the production input costs by 34 to 51% when compared with the analytical grade sucrose

Characterization of Kenyan sweet potato genotypes for SPVD resistance and high dry matter content using simple sequence repeat markers

Simple sequence repeat (SSR) markers were used to characterize Kenyan sweet potato genotypes for resistance to the sweet potato virus disease (SPVD) and high dry matter content. Eighty nine (89)genotypes with a mean symptom severity score of between 1 and 1.5 were selected following graft inoculation with SPVD-infected scions and characterized using 6 SSR primers. The 6 SSR primer pairshad an average polymorphic information content (PIC) of 0.47. The average number of alleles within the 89 genotypes across the 6 loci was 13.52. Cluster analyses revealed a 50% variation among the 89genotypes. The dendrogram did not reveal any unique clustering of the genotypes according to dry matter content and reaction to SPVD. The genetic differences among the SPVD resistant genotypes andthose with high dry matter revealed by the distinct groups suggest a significant genetic variability and the presence of different sources of resistance to SPVD and high dry matter. This characterization willgive valuable information for breeders and serve as a baseline for efficient development of new cultivars resistant to SPVD and containing high dry matter