The population structure of wild sorghum species in agro-ecological zones of Western Kenya

There is need to understand the genetic structure of wild sorghums that grow alongside cultivated traditional sorghum varieties in order to assess the potential effect of crop genes in wild populations. In this study, 175 wild sorghum samples were collected from 13 agroecological zones (AEZs) from three counties in Western Kenya and genotyped using microsattelite markers. Crop alleles were observed in wild sorghum populations. The range of allelic frequencies varied from low (˂0.4), to moderate (0.4-0.7) and to high (0.7) in the AEZs. Wild sorghum populations had moderate to high expected heterozygosity (HE) values of between 0.453 in LM1 to 0.715 in LM2. Differences in the magnitude of diversity was significant in the counties (Busia HE = 0.59 – 0.71; Homabay HE = 0.58-0.68 and Siaya HE = 0.45-0.59) but not distinct among the AEZs. Whole population FIS, FST and FIT values were low at 0.15, 0.16 and 0.29, respectively indicating low level of inbreeding, low genetic differentiation of the population and low to moderate deviation from Hardy–Weinberg (HW) equilibrium respectively. The deviation from HW equilibrium was significant in some wild populations from Siaya and Busia. Intra-population diversity (HS) was larger than inter-population diversity (DST) in 13 populations from the sampled AEZs, indicating the importance of gene flow between populations of wild sorghums. Heterozygosity values under mutation drift equilibrium (HEQ) varied under infinite allele model (IAM), two–phase model (TPM) and the step wise mutation model (SMM). However, significant population bottlenecks were absent in the wild sorghums. Presence of significant geographic county clusters and lack of significance on AEZ clusters indicate that human activities have had more influence on the distribution and diversity of wild sorghums than the prevailing climatic conditions. Efforts towards physical and genetic containment of crops genes need to be enhanced for successful ecologically sensitive confined field trials and future adoption of transgenics in cropping systems.Keywords: Diversity, Sorghum bicolor, Sorghum halepense, Sorghum sudanense, microsatellite loci.Abbreviation: AEZ, Agro-ecological zone; DST, inter-population gene diversity; FIS, fixation index; FIT, index of deviation from HW equilibrium; FST, degree of population differentiation; GST, proportion of inter-population gene diversity; HE, expected heterozygosity; HEQ, heterozygosity values under mutation drift equilibrium; HO, observed heterozygosity; HT, total gene diversity; IAM, infinite allele model; SMM, stepwise mutation model; TPM, two-phase model; LM, lower Midlands; UM, upper midlands; HB, Homabay; SY, Siaya; BU, Busia counties; SSR, simple sequence repeats

Effects of postharvest handling practices on quality of groundnuts and aflatoxin contamination

The increased cases of aflatoxin contamination are exacerbated by poor post-harvest management practices, coupled with adverse climatic conditions at harvest and post-harvest stages. This study therefore was carried out to improve safety and quality of groundnuts from aflatoxin contamination, through use of proper postharvest handling practices. Specifically the study determined the effects of harvesting dates and drying methods on aflatoxin contamination. Field experiments were carried out both at Chitedze and Chitala Agricultural Research Stations in Malawi during 2017/2018 growing season. A randomized complete block design in a split plot arrangement with three harvesting dates as the main plot and four drying methods as the sub-plots replicated three times was used. Groundnut was assessed for kernel infection by Aspergillus flavus, and level of aflatoxin contamination. Significantly low levels of about 0.5μg/ Kg of A. flavus infection and aflatoxin contamination were observed at 90 days after sowing (DAS). Higher aflatoxin contamination of up to 5μg/ Kg was observed at 80 DAS, and 10 days late after physiological maturity (100 DAS). This study also identified Mandela cock, aframe drying rack as effective drying method that can reduce aflatoxin contamination in groundnuts by 75 %. Moreover, Mandela cock drying method was shown as the most effective compared to A-frame and drying rack drying method. Current study therefore recommends for adoption of timely harvesting at physiological maturity, and drying using either Mandela cock or A-frame and drying rack. Further studies need to be carried on biological control of aflatoxin contamination

Improving Drought Tolerance in Sorghum bicolor L. Moench: Marker-Assisted Transfer of the Stay-Green Quantitative Trait Loci (QTL) from a Characterized Donor Source into a Local Farmer Variety

Drought stress is a major constraint to sorghum production in Kenya, especially during flowering stage. This study aimed at developing drought tolerant sorghum varieties by transferring the stay green trait that confers drought tolerance in sorghum from a mapped and characterized donor source into an adapted farmer preferred variety. The drought tolerance donor source, E36-1 originally from Ethiopia was backcrossed into a Kenyan farmer-preferred variety, Ochuti until BC2F1 generation and the stay-green Quantitative Trait Loci (QTL) were transferred through Marker Assisted Breeding (MAB) strategy. Five polymorphic Simple Sequence Repeat (SSR) markers were used to select the 3 stay green QTL of E36-1 found in SBI-01, SBI-07 and SBI-10 linkage groups. In the F1 generation, two of these QTL, were transferred into three genotypes. In the BC1F1 generation, 32 genotypes had at least one QTL incorporated. From a population of 157 BC2F1 progenies, 45 genotypes had incorporated either one or two of the stay-green QTL. Despite a few number of genotypes obtained through the backcrosses, the results showed that stay-green QTL and consequently drought tolerance can be transferred successfully into farmer preferred sorghum varieties through MAB

Molecular characterization of ' Candidatus Liberibacter' species/strains causing huanglongbing disease of citrus in Kenya

This study was undertaken to characterize the alpha subgroup of the proteobacteria causing the huanglongbing (HLB) disease of citrus from three different ecological zones of Kenya namely the Lower highlands (LH2, LH3, 1800-1900 m above sea level); Upper midlands (UM3, UM4, 1390-1475m), Lower midlands (LM5, LM4, LM3 of 1290-1340-1390m), by isolation and sequencing DNA encoding the L10 and L12 ribosomal proteins and the intergenic region. A 7I6-basepair DNA fragment was amplified and sequenced and consisted of 536 basepairs of DNA encoding the L10 protein, 44 basepairs of DNA intergenic region and 136 basepairs of DNA that partially encodes the L12 protein. Sequences of rpL10/L12 protein genes from Kenyan strains were 98% and 81% similar to the South African ' Candidatus Liberibacter africanus strain Nelspruit' and the Asian 'Candidatus Liberibacter asiaticus' strains, respectively. The intergenic rDNA sequence of Kenyan strain from UM and LM showed 84% similarity with 'Candidatus L. africanus strain Nelspruit' and 50% similarity with 'Candidatus L. asiaticus' strain. However, the LH strain had an 11- basepairs deletion, while the LM4 had a 5- basepair deletion in the intergenic region compared to 'Candidatus L. africanus strain Nelspruit'. The L10 amino acid sequence was 100% homologous among HLB bacteria obtained from the agro-ecological zones in Kenya and the L10 protein sequence was also homologus to 'Candidatus L. africanus strain Nelspruit'. Nevertheless, the L10 amino acid sequence of 'Candidatus L. asiaticus' and the 'Candidatus L. africanus subsp. capensis' differed from the Kenyan strains by 18.36% and 11.82%, respectively. Phylogenetic analysis of both the L10/L12 rDNA sequences and the L10 amino acid sequences clustered the Kenyan strains of the 'Candidatus Liberibacter' species with members of alpha subdivision of proteobacteria

Real time PCR mediated determination of the spontaneous occurrence of Sorghum bicolor alleles in wild sorghum populations

The study evaluates the utility of Real Time PCR (RT-PCR) in quantitative and qualitative analysis of alleles in sorghum populations and the spontaneous occurrence of Sorghum bicolor alleles in wild populations of sorghum. Leaf and seed material from wild sorghum accesions were sampled in Homabay, Siaya and Busia counties to represent Western Kenya sorghum producing regions. A second sampling was done on S2 populations of S. bicolor, Sorghum halepense and Sorghum sudanense maintained in the greenhouse. Crop loci were evaluated in all materials using a LightCycler® 2.0 system. Real Time PCR was effective in qualitative and quantitative determination of crop alleles in both crop and weedy backgrounds of S. sudanense, S. halepense and S. verticilliflorum. Crossing point values ranged between 19.7 from 30 ng template to 35.9 from 0.015 pg of template on locus SB1764. Melting peaks analysis ranged between 83.29 to 88°C on locus SB1764 and between 86.01 to 80.88°C on locus SB3420 effectively differentiating the 4 species. RealTime-PCR was successful in quantitative and qualitative analysis of specific crop alleles from loci SB1764 and SB3420 from seed and leaf DNA. Spontaneous occurrence of crop and rare alleles in wild sorghum populations growing in sympatry with crop cultivars showed the presence of crop and rare alleles in wild sorghum populations. Means of wild populations from lower midland1 (LM1), LM2, LM3 and LM4 AEZs were not significantly different. It is therefore vital to test S. bicolor seeds and other plant materials in transit, at entry points and populations of growing plants for foreign genes including transgenes using RT-PCR. Keywords: Real time polymerase chain reaction (PCR), Sorghum bicolor, Sorghum halepense, Sorghum sudanense

Molecular characterization of ' Candidatus Liberibacter' species/strains causing huanglongbing disease of citrus in Kenya

This study was undertaken to characterize the alpha subgroup of the proteobacteria causing the huanglongbing (HLB) disease of citrus from three different ecological zones of Kenya namely the Lower highlands (LH2, LH3, 1800-1900 m above sea level); Upper midlands (UM3, UM4, 1390-1475m), Lower midlands (LM5, LM4, LM3 of 1290-1340-1390m), by isolation and sequencing DNA encoding the L10 and L12 ribosomal proteins and the intergenic region. A 7I6-basepair DNA fragment was amplified and sequenced and consisted of 536 basepairs of DNA encoding the L10 protein, 44 basepairs of DNA intergenic region and 136 basepairs of DNA that partially encodes the L12 protein. Sequences of rpL10/L12 protein genes from Kenyan strains were 98% and 81% similar to the South African ' Candidatus Liberibacter africanus strain Nelspruit' and the Asian 'Candidatus Liberibacter asiaticus' strains, respectively. The intergenic rDNA sequence of Kenyan strain from UM and LM showed 84% similarity with 'Candidatus L. africanus strain Nelspruit' and 50% similarity with 'Candidatus L. asiaticus' strain. However, the LH strain had an 11- basepairs deletion, while the LM4 had a 5- basepair deletion in the intergenic region compared to 'Candidatus L. africanus strain Nelspruit'. The L10 amino acid sequence was 100% homologous among HLB bacteria obtained from the agro-ecological zones in Kenya and the L10 protein sequence was also homologus to 'Candidatus L. africanus strain Nelspruit'. Nevertheless, the L10 amino acid sequence of 'Candidatus L. asiaticus' and the 'Candidatus L. africanus subsp. capensis' differed from the Kenyan strains by 18.36% and 11.82%, respectively. Phylogenetic analysis of both the L10/L12 rDNA sequences and the L10 amino acid sequences clustered the Kenyan strains of the 'Candidatus Liberibacter' species with members of alpha subdivision of proteobacteria

A review of the Amplified Fragment Length Polymorphism (AFLP) technique in genotyping and DNA fingerprinting studies [Review Article]

ABSTRACT Amplified fragment length polymorphism (AFLP) is a marker based on polymerase chain reaction amplification of restricted fragments ligated to synthetic adaptors and amplified using primers which carry selective nucleotides at their 3' ends. The technique generates highly reproducible markers from DNA of any organism and allows high resolution genotyping. AFLP has broad applications and has been used to investigate genomes of different complexity from microbes to higher organisms for purposes of species, strains and varieties identification, systematics, pathotyping, population genetics, simple and complex trait mapping, population genetics, construction of linkage and physical maps. In addition, it is being used in medical diagnostics, forensic analysis and microbial typing. AFLP is superior compared to other markers in that it has time efficiency, generates more information, is highly reproducible and has a wide range of applications. The marker has a drawback in that it generates dominant rather than co-dominant markers and can also be expensive if automated systems are used

Identification of molecular markers linked to a gene conferring resistance to coffee berry disease (Colletotrichum kahawae) in Coffea arabica

Coffee berry disease (CBD) caused by Colletotrichum kahawae is a major constraint to Arabica coffee (Coffea arabica) production in Africa. One source of resistance to the disease is a natural interspecific hybrid between C. arabica and C. canephora and its derivatives. This study is aimed at deciphering the genetic basis of the host resistance and identification of molecular markers associated with it. CBD is a mature stage disease and in the absence of a mature mapping population, early detection of disease reaction phenotypes of mapping individuals is required. Two F-2 populations from crosses of cv. Catimor (resistant) and cv. SL28 (susceptible) were screened for resistance by a two step procedure. First, half of each population was screened 6 weeks after germination by inoculating hypocotyls with the pathogen. The surviving seedlings (G1) were considered to be resistant and were raised in a nursery together with the other unscreened halves (G2). Secondly, after one year, all the seedlings (G1 + G2) were screened by inoculation. Analysis of 57 microsatellites and 31 AFLP markers in 56 and 95 seedlings from G1 and G2, respectively, were performed. Eight AFLP and two microsatellites markers linked tightly to the resistant phenotype were identified and mapped to one unique chromosomal fragment introgressed from C. canephora. The gene conferring the resistance was localized within an 11 cM segment. It is concluded that the locus carries a major resistance gene designated Ck-1, which is likely to be synonymous to the T gene described in previous studies