Biotechnology

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Mapping of Novel Quantitative Trait Loci (QTL) for Fusarium Wilt Resistance in Chickpea (Cicer Arietinum L.) and Analysis of the Genomic Diversity of Fusarium Oxysporum F. Sp. Ciceris in Ethiopia
(Addis Ababa University, 2021-05-01) Bekele, Dagnachew; Tesfaye, Kassahun (PhD); Fikre, Asnake (PhD)
Chickpea (Cicer arietinum L.) is one of the most economically important food legumes cultivated in different parts of the world. Ethiopia is the largest producer, consumer and exporter of chickpea in Africa. However, several biotic and abiotic stresses restrict its potential productivity. Among the biotic stresses, fungal diseases are the major yield limiting factors throughout chickpea producing countries. Fusarium wilt, caused by Fusarium oxysporum f. sp. ciceris (Foc), is one of the most dominant and destructive pathogen threatening chickpea production in Ethiopia. Breeding for host plant resistance is the most cost efficient and eco-friendly strategy to control the disease. Nevertheless, chickpea breeding for Fusarium wilt resistance is regularly challenged with high pathogenic variability and limited availability of good resistance sources. So far only few efforts have been made to investigate the genetic diversity and geographic distribution of Foc pathogen in Ethiopia for designing effective breeding and integrated disease management strategies. For this disease, no report is available that encompass the breadth of major and minor chickpea producing areas of the country. In this study four sets of experiments were executed with the main objectives to: investigate the intensity and association of Fusarium wilt/root rot disease of chickpea under diverse biophysical factors in Ethiopia; identify new resistance sources and map a novel wilt resistant quantitative trait loci (QTL) in chickpea; analyses the genomic diversity, pathogenic variability and geographic distribution of Foc pathogen in the country; and develop rapid and reliable disease diagnostic assay for accurate disease diagnosis. In 2015 and 2016 cropping seasons, geo-referenced field surveys were conducted covering a total of 62 major chickpea growing districts located in 19 diverse agro-ecological zones of Ethiopia, and a total of 217 diseased plant samples were collected for pathogen identification and genomics study. Among these, from 51 representative farmers’ fields, three 1 x 1 meter quadrat were surveyed along a diagonal transect to investigate the intensity and association of Fusarium wilt/root rot disease of chickpea under diverse biophysical factors in Ethiopia. Data on major biophysical factors were recorded, and pathogen was isolated based on the established morphological and cultural characteristics. For identification of new Fusarium wilt resistance sources, a total of 315 wild introgression lines and 47 recombinant inbred lines (RILs) were evaluated for Fusarium wilt resistance in sick plot at Debre Zeit Agricultural Research Center. To map Fusarium wilt resistance QTL in chickpea, total of 108 F2 hybrids were generated by crossing Fusarium wilt resistant variety Dera and Fusarium wilt susceptible genotype JG 62, and genotyping-by-sequencing identified 1,659 single nucleotide polymorphisms (SNPs) that distinguish the two parental lines. A total of 166 representative Fusarium isolates collected from different part of the country were sequenced using whole genome sequencing (WGS) with Illumina HiSeq 4000 platform to investigate the genomic diversity, pathogenic variability and geographic distribution of Foc pathogen. For rapid and accurate detection of Foc pathogen directly from symptomatic chickpea plants, broad specificity PCR primers were designed based on the alignment of selected Benchmarking Universal Single Copy Orthologs (BUSCO) genes present and highly conserved in the genomes of a set of 66 Fusarium isolates. Moreover, a cultureindependent broad-range18S amplicon survey was conducted to characterize chickpea-associated eukaryotic communities. The result indicated that Fusarium wilt disease was widely distributed in all growing areas of the country. Across all surveyed sites, Foc was the predominant species encountered among fungi cultured from plant tissue, representing 69.4 % of total isolates. Diseases pressure was significantly (P < 0.05) associated with heavy black soils, Desi type chickpea, early planting, flowering and plant maturity. The highest mean percent diseases incidence per m2 (45.65%) was recorded in the Amhara region, West Gojam zone, where heavy clay soils predominate. Wild introgression lines and advanced recombinant inbred lines showed significant genetic diversity for Fusarium wilt resistance and yield related traits that can be exploited to improve the agronomic value of the chickpea crop. In the present study 20 Fusarium wilt resistant RILs with high yield and desirable agro morphological traits were identified. For Fusarium wilt resistance QTL mapping, a total 836 high quality SNP markers were assigned to six genetic linkage groups, each corresponding to separate chromosomes, with a total map size of 274.9 cM and 3.12 cM average distance between mapped markers. Major QTL explaining 55.28 % of the observed phenotypic variation was identified on chromosome 4 at 44.29 cM with a logarithm of odds (LOD) score of 13.8. Interestingly, Nei’s genetic diversity analysis based on 196, 495 SNPs split test isolates into 20 distinct clusters irrespective of their regions of origin and geographical location. Among these,16 distinct clusters were Fusarium oxysporium ciceris (Foc) isolates. Phylogenetic analysis based on 1,052 highly conserved BUSCO genes also divided test isolates into six distinct Fusarium species, and 16 sub-groups (Foc isolates). Consistent with these results, pairwise average nucleotide identity (ANI) analysis based on 3,695 highly conserved BUSCO genes split test isolates in to six distinct Fusarium species, using 95 % ANI (ANI95) as the lower species boundary. Besides, dendrogram built based on virulence data split Foc isolates into four distinct virulence groups confirming the existence of high pathogenic variability between Foc isolates in Ethiopia irrespective of their geographical origin. Mantel correlation estimate showed very weak correlation between geographical distance and genetic distances of Fusarium isolates in Ethiopia with P = 0.280 and R2 = 0.0006. The results the PCR diagnostic assay showed that, on test with Fusarium specific PCR primer (EOG09331-PTT), 97.5 % of diseased plants with typical symptoms (39 out of 40 plants) gave uniformly strong amplification with the identity of amplicons confirmed by Sanger sequencing. However, some symptomatic plants yielded inconsistent results as PCR based disease diagnosis using organism-specific DNA amplification is unable to assess the presence of all other microbes that might better inform diagnosis. To address these issues, microbial community composition were surveyed using 18S amplicon sequencing. The result nominated Phytophthora medicaginis as alternative pathogens in some fields where Fusarium wilt was suspected. Such analyses represent a potentially powerful alternative to traditional plant disease diagnostics. Without the constraints of culturability and the bias of endpoint PCR, amplicon sequencing can provide powerful insights into disease dynamics. In conclusion, the novel major QTL and associated genetic markers identified in the present study offer molecular tools for breeding wilt resistant against Ethiopian Foc isolates. This study indicated the presence of high genetic diversity and pathogenetic variability between Fusarium isolates in Ethiopia. Therefore, designing effective country wide breeding and integrated disease management strategies against Foc pathogens is key to break the recurrent disease cycle in the country. The results of the present study provide detailed information and appropriate framework to develop effective breeding and integrated disease management strategies to combat Fusarium wilt disease of chickpea in Ethiopia.
Genetic Diversity and Population Structure Analysis of Released and Landrace Sorghum (Sorghum Bicolor (L.) Moench) Genotypes of Northern and Eastern Ethiopia as Revealed by SSR Markers
(Addis Ababa University, 2021-05-01) Misganaw, Abebaw; Feyissa, Tileye (Professor)
Sorghum (Sorghum bicolor (L.) Moench) is the most stable and important food security crop in Ethiopia accounting for nearly 40% of human calorie intake. Knowledge of the natural genetic composition of the crop provides the option to further exploit its genetic potential through breeding. However, there are limited reports on the genetic variability of Ethiopian sorghum using a medium-throughput marker system. Hence, the current study was designed to evaluate the genetic variability of released and landrace Ethiopian sorghum genotypes using polymorphic microsatellite markers. A 92 sorghum accessions collected from five Ethiopian ecological zones were targeted using 12 SSR markers. The study resulted in 77 alleles across the entire loci and populations. All the used microsatellite loci were highly polymorphic with PIC ranging from 0.66 to 0.82 and an overall mean of 0.76. The analysis confirmed the presence of high gene diversity ranging from 0.71 to 0.84 with overall mean of 0.79. There was a higher genetic differentiation (FST=0.21) showing the presence of moderate gene flow. The analyzed molecular variances indicated the existence of large genetic differentiation (FST=0.21) where 90% of the total variation was accounted for within populations genetic variability, leaving only 10% for the among populations variation. The PCoA, clustering, and population structure did not cluster the studied populations into a separate groups according to their geographical areas of sampling due to the presence of considerable gene flow (Nm= 2.13). In conclusion, based on the overall evaluated loci the highest intra-population diversity was observed among populations of North Gondar (Het= 0.75) and South Tigray (Het= 0.74), and hence these areas can be considered as hot spots for the identification of genotypes for breeding program. Therefore, the present study generated valuable information for sorghum breeding programs, and for conservation measures.
Characterization of Wild Indigenous Yeasts from Molasses and other Sugary Substrates and their Potential for Bioethanol Production
(Addis Ababa University, 2021-05-09) Degu, Sisay; Muleta, Diriba (PhD); Tesfaye, Anteneh (PhD)
The increasing demand of energy has been supplied through the combustion of petroleum throughout the world. Due to escalating cost of petroleum, its contribution to global warming and non-renewable nature of this oil, there is collectively a need for renewable and ecofriendly energy sources such as bioethanol. Thus, the objective of this study was aimed at isolating, characterizing and evaluating the potent wild yeasts under different stress conditions from locally available resources for bioethanol production to minimize the utility of fossil fuel. In this study, a total of 35 samples of sugary substrates were collected from Metehara Sugar Factory for wild yeasts isolation following the standard protocols. A total of 305 yeast isolates were retrieved and screened using physiological and osmotic stress tolerance tests. Fermentative and potent wild yeasts were identified to species level using morphological and biolog-based biochemical methods. Out of 305 yeast isolates, 20 (6.56%) and 7 (2.29%) of them were found to be tolerant to 18 and 20% of ethanol, respectively. Out of these 20 ethanol tolerant yeast isolates, 17 tolerated the temperature of 45°C for 48 hrs. From 17 ethanol-thermotolerant isolates, 5 (29.41%), 5 (29.41%) and 7 (41.18%) were found producing gas from glucose at 24, 48 and 72 hrs, respectively. Out of the 17 ethanol-thermotolerant yeast isolates, 12 yeast isolates were able to tolerate 35% of glucose. Out of these 12 ethanol-thermo-and-sugar tolerant yeasts, the 7 were found tolerant to pH 2. From the 7 acidic tolerant yeast isolates, 5 yeast isolates were shown tolerant to 7% of NaCl and identified as K. lodderae, P. guilliemodii B, S. boulardii, Z. rouxii and T. globosa, respectively. During fermentation, K. lodderae, P. guilliemodii B, S. boulardii, Z. rouxii and T. globosa were able to produce bioethanol with the values of (% v/v) 12.62, 11.61, 10.58, 10.82 and 10.44, respectively at 48 hrs and 12.56, 12.52, 12.08, 11.11 and 11.48, respectively at 72 hrs. The initial inoculum cell density was increased from (1.83 to 3.08, 0.59 to 1.81, 0.54 to 1.36, 0.48 to 1.69 and 0.45 to 1.56) x108 cells/ml for K. lodderae, P. guilliemodii B, S. boulardii, Z. rouxii and T. globosa, respectively 0 hr to72 hrs. The fermentation efficiency (%) for K. lodderae, P. guilliemodii B, S. boulardii, T. globosa and Z. rouxii was shown as 98.0, 97.8, 93.1, 90.8 and 88.1, respectively. Thus, on the basis of highest stress tolerant and higher fermentation efficiency features, K. lodderae and P. guilliemodii B wild yeasts were considered to be the best bioethanol producers. Molecular characterization and optimization of fermentation parameters is recommended to utilized these potent yeasts.
Detection of Selection Signatures Breed-Specific SNPs and Linkage Disequilibrium Analysis in Ethiopian Indigenous and European Dairy Cattle Breeds
(Addis Ababa University, 2021-11-20) Dejene, Genet; Sisay, Tesfaye (Professor)
Ethiopia ranks first in Africa with the largest number of cattle populations adapted to diverse environments. Detection of selection signatures and assessment of linkage disequilibrium enable us to asses genetic diversity and genomic region under positive selections. However, in Ethiopian indigenous cattle there is few studies regarding their origin, divergency and genetic adaptability. This study investigated detection of selection signatures, breed-specific SNPs and linkage disequilibrium in Ethiopian cattle populations and European dairy breeds. A total 135 animals representing four Ethiopian indigenous cattle populations: Arsi (n = 29), Begait (n = 40), Boran (n = 40) and Sheko (n = 26) were genotyped with 80K SNP chip. Two European dairy breeds (Holstein, n = 60 and Jersey, n = 38) were used for comparison. The mean of minor allele frequency (MAF) 0.32 ± 0.12, 0.32 ± 0.12, 0.31 ± 0.13, 0.30 ± 0.13,0.19 ± 0.17 and 0.18 ± 0.17 for Arsi, Begait, Boran, Sheko, Holstein and Jersey, respectively. The common variant MAF (≥0.10 and 0.5) distribution across Ethiopian cattle populations and European cattle breeds were 89% and 57%, respectively. Ethiopian cattle specific SNPs were located in genes(CSN2, ABCA7, PDE4B, ABCA1, JAK2, B4GALT4, FOXO3, GHR, ADCY8, ACACB) associated with milk production traits, fertility and growth traits (PGR, GHR, XKR4, ADCY5, POU2F1, IGF1, ABCC2, XKR4) thermo-tolerant, coat color (HSF1, HSPH1, HSPA4, KDR, RAD50, WNT1, KIT), feed intake (XKR4, ACCN1, ACAD11) and fat thickness (XKR4, IGFBP-3 and POU2F1).The top 1% fixation index (Fst) values representing positive selection harbored candidate genes (ABCG2, ABCA7, B4GALNT1, GHR, ITGAV) involved in milk traits such as, milk protein, milk fat and mastitis, milk production and (HSPH1, HSPA4, SOD1, MATR3, RAD50, KDR) for tropical adaptation. The estimated observed heterozygosity (Ho) and expected heterozygosity (He) for Ethiopian cattle populations were found to be, 0.40 and 0.39 respectively. These values were 0.24 and 0.25 for European dairy cattle breeds respectively. Principal component analysis (PCA)clearly separated Sheko from Ethiopian zebu populations with the value of 4.34%(PCA1) and 3.33% (PCA2). Similarly, PCA1 and PCA2 accounted for 65.97 % and 9.50 % of the variation and differentiated bos indicus from European taurine. Additionally, result of the phylogenetic tree analysis supporting PCA revealed that with the exception of Sheko, Ethiopian cattle population were closely clustered and two European dairy breeds (Holstein and Jersey) were located in a clade with Sheko. The overall mean of r2 values were 0.22, 0.23, 0.23, 0.22, 0.24, 0.16, 0.16 in Arsi, Begait, Boran, Sheko, Holstein and Jersey, respectively. In broad, this study revealed that there are genomic regions under strong divergent selection harboring genes involved in milk production traits and in adaptation to tropical environment.
Genetic Diversity Analysis of Sorghum Sorghum Bicolor (L.) Moench in Ethiopia Based on Quantitative Traits and Microsatellite Markers
(Addis Ababa University, 2021-07-01) Mamo, Wubshet; Feyissa, Tileye (PhD)
Sorghum [Sorghum bicolor (L.) Moench] is a very important food security crop in Ethiopia and in the semi-arid and tropical parts of Africa. Genetic analysis based on DNA markers coupled with morphological traits could provide important information on the genetic structure of the crop. Therefore, the present study was targeted to investigate the extent and patterns of genetic variation in 324 sorghum accessions using six quantitative traits measured at three locations in two replications. Among the studied traits, plant height showed the highest genetic advance as percent of mean (38.99%) followed by biomass yield (18.68%). Moreover, the genetic structure of 100 sorghum accessions were further assessed using 15 microsatellite markers. The results revealed the presence of significant differences among the genotypes for all the studied quantitative traits, suggesting the possibility to improve through selection. The polymorphic information content of the loci ranged from 0.68 to 0.89 with overall mean of 0.80, indicating their higher resolution power for genetic analysis. Structure analysis confirmed the three sub-groups with greater degree of genetic admixture. We would like to confirm that the combined application of morphological and marker technology had successfully elucidated the genetic structure of sorghum accessions in Ethiopia. The information will be useful for sorghum improvement through selection, conservation, and wise and sustainable use.
Genetic Diversity and Population Structure Analysis of Zymoseptoria Tritici in Ethiopia at Single Field Scale as Revealed by Simple Sequence Repeat Markers
(Addis Ababa University, 2021-07-12) Mebratu, Taye; Mekonnen, Tilahun (PhD)
Zymoseptoria tritici the causative agent of Septoria tritici blotch (STB), is one of the most economically damaging disease of wheat worldwide. Genetic resistance is a suitable, economical and environmentally safe strategy to control the disease. Knowledge of the genetic structure of the pathogen is vital for designing best management strategy against STB. The present study aimed to investigate the genetic diversity of Z. tritici in Ethiopia at a single field scale using simple sequence repeat (SSR) markers. A total of 200 naturally STB infected wheat leaves were collected from 10 plots of unsprayed single field at Holetta Agricultural Research Center. A total of 147 single-spore derived Z. tritici isolates were subjected to genetic diversity analysis using ten SSR loci. All the tested loci were polymorphic, and highly informative. Different diversity parameters were executed, and highlighted with the number of alleles, gene diversity (0.82), and polymorphic information content ranged from 5-11, 0.71- 0.88 and 0.67 -0.87 with overall mean of 9 , 0.82, and 0.80, respectively. Hierarchical analysis of molecular variance revealed moderate (PhiPT= 0.13) genetic differentiation where the within population genetic variation accounted for 87% of the total variation 4.18. The variation among population accounted only for 13% of the total genetic variation, likely due to the presence of high (Nm = 3.48) gene flow. The neighbor joining, UPGMA and PCoA failed in sharply grouping the populations into their corresponding sampled plots, confirming the presence of high gene flow. Moreover, the Bayesian-model based structure analysis weakly inferred four sub-groups (K= 4) confirming the weak population structuring with high degree of genetic admixture. Therefore, the study confirmed that Z. tritici population shows high genetic variation even in a single field, suggesting need to use integrated disease management strategies to control the disease and also give especial focus for resistance breading to involve pyramiding of several genes that can provide broad spectrum resistance.
Metagenomic Analysis of Microorganisms in Hyper Saline Aquatic and Acidic Habitats of Danakil Depression of the Afar Region of Ethiopia and their Biotechnological Potential
(Addis Ababa University, 2021-12-04) Tilahun, Lulit; Simachew, Addis (PhD); Asrat, Asfawossen (Professor); Wessel, Gary M. (PhD)
The Danakil Depression is one of the hottest place on Earth and is considered as an exceptional place where different extreme environments are found. Five locations in the Danakil Depression were studied for the presence of extremophiles and their biotechnological potentials. The sample sites for this study are the Hyper-saline Acid Ponds [HsAP] (Dallol [DAL], Black Water [BLA], Gaet’ale [GAL]) as well as the Hyper-saline Aquatic Systems [HsAqS] (coastal parts of Lake Asale [LA, LADP5, LADP6], Mud’ara pond [MUP]). All the sites were located South to Mount Dallol at less than -100 m below sea level. In our investigation, we tested for the presence of DNAbased life in the studied sites using Whole Metagenome Shotgun (WMS) DNA sequencing approaches. Bioinformatic tools that adopt techniques of assembly free (Bowtie, MetaPhlAn2) and assembly (MetaSpades, DIAMOND and MEGAN 6-CE) were used to identify the microbial communities and predict the major nutrient metabolic networks. Accordingly, the HsAqS were found to be mainly dominated by archaea Operational Taxonomic Units (OTUs), while the HsAP were dominated by bacteria OTUs. More than 95% of the total OTUs of HsAqS was grouped under phylum Euryarchaea, while more than 90% of the OTUs of HsAP was classified in the phyla Proteobacteria, Actinobacteria and Firmicutus. Halonotius sp J07HN6 and unclassified Salinibacter ruber were the two abundant archaeal and bacterial OTUs in the HsAqS, respectively. On the other hand, unclassified strain of Paraburkholderia fungorum was profiled as the most abundant OTU for DAL and BLA, while unclassified Escherichia and unclassified Burkholderia were top two abundant OTUs for GAL. Insilico prediction of gene functions and metabolic pathways using SEED and KEGG indicated presence of carbon fixation, sulfate oxidation, assimilative nitrogen and sulfate reduction reactions in DAL and BLA samples. Carbon fixation was also predicted in the HsAqS while methanogenesis was exclusively predicted in GAL. Based on the investigation of the sources of the genes of interest, it is clear that syntrophic metabolism is the main strategy adapted to utilize the nutrients in the studied sampling sites. Moreover, the predicted genes from DAL’s and BLA’s nitrogen metabolism helped to discover new protein sequences of cysteine desulfurase and [4Fe-4S] ferredoxin with less than 80% similarity to the homologous reference protein sequence. Images of DAL extremophiles obtained from Transmission Electron Microscopy (TEM) showed high production of nano sized Extracellular Membrane Vesicles (EMV). Further investigation on resistance of DAL’s extremophiles to phenolic compounds under lysing conditions indirectly signified their bio-remediation capability. In general, this preliminary investigation indicates great biotechnological and bioremediation potentials of extremophiles of Danakil Depression.
Genetic and Virulence Variability of Zymoseptoria Tritici Populations and Genome Wide Association Studies for Septoria Tritici Bloch Resistance and Yield Potential in Wheat (Triticum Aestivum L.) in Ethiopia
(Addis Ababa University, 2021-08-25) Mekonnen, Tilahun; Tesfaye, Kassahun (PhD); Hailesilassie, Teklehaimanot (PhD); Abeyo, Bekele (PhD)
Septoria tritici blotch (STB) caused by the fungus Zymoseptoria titici is the major bottleneck to wheat production in Ethiopia and worldwide. The present study was targated to investigate genetic and virulence variability of Zymoseptoria tritici populations; and Genome-Wide association studies for STB resistance and yield potential in bread wheat in Ethiopia. Genetic structure analysis of 182 Z. tritici isolates representing eight populations from major wheat growing areas of Ethiopia using 14 polymorphic microsatellite markers confirmed the presence of high genetic diversity (h = 0.45) where 92% of the total genetic variation resides within populations. Cluster (UPGMA), PCoA and STRUCTURE analyses did not group the Z. tritici populations into sharply genetically distinct clusters according to their geographical areas of sampling. All individual samples shared alleles from two subgroups (K=2). Pathogenicity assay conducted with eight Z. tritici isolates on a set of 45 bread wheat genotypes showed highly significant differences for isolate-by-genotype interaction on both percentage of necrosis leaf area (NLA) and pycnidia coverage (PC). The isolates showed significantly higher differences in virulence patterns. Isolate I1 sourced from Bale was found to be the most virulent (93%) and aggressive (NLA = 58%) isolate. Among tested Stb genes, Stb16 conferred broad-spectrum resistance to 75% of the isolates. Three genotypes (MURGA, Km7 and Hidase) conferred the greatest level of seedling resistance to the tested isolates. Molecular screening for major Stb genes in 180 genotypes using 16 tightly linked markers identified a number of Stb genes ranging from 6.67% for Stb12 to 96.1% for Stb13 gene. Field evaluation conducted on 180 wheat genotypes across six environments revealed a very highly significant difference among the wheat genotypes for most traits. Top 5% selected genotypes showed 10.8 - 21.99% and 52.25- 74.25% superior grain yield performance and STB resistance to standard check (King-bird), respectively. Moreover, population structure, linkage disequilibrium (LD), and GWAS for adult-plant STB resistance, yield, yield- related and phenological traits were conducted for 178 wheat genotypes using 7,776 SNPs resulted from Illumina HiSeq2500 Genotype-by-sequencing. STRUCTURE and PCA confirmed two sub-populations with greater degree of genetic admixture. In all chromosomes, LD between pairs of SNPs declined at r2 = 0.2 within physical distance of 2.26 -105.62 Mbp, with an overall mean of 31.44 Mbp.The association analysis identified 53 loci significantly associated with STB resistance pointing to 33 putative QTLs. Most of these shared similar chromosome locations with already published Septoria resistance genes and QTLs, and were distributed across chromosomes 1B, 1D, 2A, 2B, 2D, 3A, 3B, 3D, 4A, 5A, 5B, 6A, 7A, 7B and 7D. However, five of the putative QTLs identified on chromosomes 1A, 5D and 6B appeared to be novel. Predicting the possible functions of the detected loci from the IWGSC RefSeq Annotation v2.1 revealed the existence of disease resistance-associated genes in the identified QTL regions which are involved in plant defense responses. Seven of the QTLs for STB resistance also co-localized with MTAs for agronomic traits. In conclusion, the study provides new insights into the genetic structure and virulence variability of Z. tritici in Ethiopia to design effective and durable management strategies. The identified Stb genes, QTLs and resistance source materials could be used in wheat genome-assisted resistance breeding against STB. Therefore, the information generated in the present study is useful to improve STB resistance and grain yield performance in wheat which ultimately leads to increased and stable wheat production to ensure food security in Ethiopia and the region at large.
QTL Analysis Molecular Genetics Diversity and Agro-physiological Characterizations of Heat Tolerance in Chickpea (Cicer Arietinum L.)
(Addis Ababa University, 2021-12-08) Getahun, Tsegaye; Tesfaye, Kassahun (PhD)
Chickpea is a cheap source of protein and rich in minerals for people living in developing countries. A major agronomic challenge for chickpea production is temperatures above 35°C, which reduced growth, fertility, development, production and productivity of cultivated chickpea. Besides, domesticated chickpea changes in the nature and intensity of selection during domestication and breeding that constrain the ability to expand the cultivation of crops into arid and semi-arid environments. Therefore, the objectives of the present study were to introgress wild genes into the cultivated gene pool via genomics assisted breeding, assess the molecular genetic diversity and determine the population structures of large collections of chickpea germpalsms; screen heat tolerant genotypes and assess phenotypic variations from all sources of chickpea germplam accessions under heat stress environments; and understand the mechanisms of heat tolerance. A total of 480 chickpea accessions that comprised of 440 F3 lines of C. arietinum x C. reticulatum (17 AR families) and 33 F3 lines of C. arietinum x C. echinospermum (3 AE families), and seven standard heat tolerant and sensitive checks which were grown in two replications at ICRISAT, Hyderabad, India under heat stress conditions. GBS SNPs of 1445 and 1541 were used in AR and AE families, respectively for linkage map construction and QTL mapping. Among the 20 families, 9xKalkan_064 was consistently found to be the most heat tolerant pre-breeding family, while 9xKarabache_081A and 9xDerei_072 were in the ranges of heat sensitive to moderately heat tolerant families. A total of 194 QTLs were detected for 16 traits out of which 38 QTLs had pleotropic effects. The associated SNP markers with pleotropic loci are important for simultaneous selection of genotypes having more than two related traits for heat stress breeding in chickpea. In order to assess the existing molecular genetic diversity and to determine population structure in selected Ethiopian chickpea germplasm accessions (118), a set of 46 simple sequence repeat (SSR) markers equally distributed on chickpea genome were genotyped. A total of 572 alleles were detected from 46 SSR markers and the number of alleles per locus varied from 2 (ICCM0289) to 28 (TA22). The average numbers of alleles per locus, polymorphism information content and expected heterozygosity were 12, 0.684 and 0.699, respectively. Phylogenetic analysis grouped the 118 chickpea genotypes from diverse sources into three evolutionary and/or biological groups (improved desi, improved kabuli and landraces). Population structure analysis revealed six sub-populations from 118 chickpea genotypes used in this study. AMOVA revealed that 57%, 29% and 14% of the total genetic variations were observed among individuals, within populations and among populations. A total of 121 chickpea genotypes were assessed at three field sites in Ethiopia, under heat stressed and non-stressed conditions and 57 genotypes were assessed under high heat stressed conditions at ICRISAT, Hyderabad, India. The results showed that the chickpea genotypes were severely impacted by excessive heat at Hyderabad as compared to those planted under non-heat stressed conditions in Debre Zeit, Ethiopia. At extremely high temperatures, chickpea plants exhibited reduced floral initiation, arrested seed and pod development, shortened life cycles, and reduced plant height, seed size, grain yield and yield related traits. Across stressed and non-stressed environments, there were highly significant differences among the genotypes for most of the traits (ANOVA, P≤0.001). Under heat stressed environments, DZ-Cr-0034 was found to be a highly tolerant, while DZ-Cr-0026 was found to be a highly sensitive genotype. Genetic diversity among the genotypes were determined using 5,722 SNPs, revealing a single group of Ethiopian genotypes with small number of cultivars showing introgression from Middle Eastern germplasm. At the reproductive developmental stages, a total of 13 physiological parameters were measured for 47 chickpea accessions that were grown from February to May, 2019 under a heat stressed environment, Werer (Ethiopia), using a high throughput phenotyping tool (MultispeQ). Based on the physiological responses of chickpea accessions under heat stressed environment, it was confirmed that DZ-2012-ck-0034 and JG 14 were found to be the most heat tolerant, while DZ-2012-ck-0026 and ICC 4567 were the most heat sensitive genotypes. Unlike heat sensitive genotypes, heat tolerant genotypes exhibited high mean values for effective quantum yield of photosystem II (Phi2), chlorophyll fluorescence decline ratio (RFd), coefficients for photochemical quenching (qP and qL), chlorophyll fluorescence (Fv/Fm), pod set and low mean values for regulated (PhiNPQ) and non-regulated heat dissipations (PhiNO). Reproductive traits were studied under heat stressed conditions with 17 and 47 chickpea accessions that were grown at ICRISAT (Hyderbad, India) and Werer (Ethiopia), respectively. The pollen viability study was carried out by using 2% acetocarmine stain. The quantity of pollens per flower was counted with haemocytometer. In vitro pollen germination was determined by using a modified Brewbaker and Kwack liquid medium. Under heat stressed condition, at ICRISAT (Hyderabad, India), the F3 individuals of 9xCudiB_022C family had the highest average percentage of pollen viability (94%) which was associated with high pod set (80%). However, 9xKarabche_081A (9%) and ICC 4567 (10%) had low average percentage of pollen viability which was also associated with low pod set (0-10%). The mean values of the pollen cells per flower, in vitro pollen germination and pod set of the segregating progenies were higher than the landraces, lines and varieties which implied that the segregating wild progenies resisted the heat stress i.e. linked traits can be introgressed into cultivars in order to enhance thermotolerance. The present study revealed that the Ethiopian germplasm accessions have genetic variability for heat tolerance that can be harnessed to meet expected shifts towards warmer climate. The physiological and reproductive traits might enhance the efficiency and accuracy of breeding in chickpea for screening and identifying of tolerant genotypes and help to understand the mechanisms of heat tolerance under heat stress conditions and contribuite to climate resilient agriculture.
Genome-wide Signature of Positive Selection Breed-Specific SNPs and Linkage Disequilibrium in Ethiopian Indigenous and European Beef Cattle Breeds
(Addis Ababa University, 2021-09-05) Mengistie, Dejenie; Sisay, Tesfaye (Professor); Dadi, Hailu (PhD); Edea, Zewdu (PhD)
The detection of selection signatures in Ethiopian indigenous breeds can contribute to the identification of regions of the genome that are, or have been, functionally important and, as a consequence, have been targeted by selection. Despite the availability of genomic methods for determining the origin and divergence of domestic cattle in East Africa, particularly Ethiopia, knowledge regarding their genetic adaptability and divergence remain limited. To investigate signatures of selection, breed-specific SNPs, and linkage disequilibrium (LD), Ethiopian indigenous cattle populations, three Ethiopian cattle populations (Begait n = 40; Boran n = 40; and Fogera n = 43; were genotyped with a high-density 80K SNP array. Three European beef cattle breeds (Angus, n = 42), Hereford (n = 35), and Charolais (n = 37) were also used for comparison purposes. The average minor allele frequency was 0.19 ± 0.17, 0.20 ± 0.17, 0.21± 0.17, 0.31 ± 0.13, 0.32 ± 0.12, 0.32 ± 0.13 for Angus, Herford, Charolais, Boran, Fogera, and Begait cattle, respectively. Across the Ethiopian and European cattle populations, a common variant minor allele frequency (≥0.10 and ≤ 0.5) accounted for 94% and 62%, of the total SNPs, respectively. A total of 7759 and 48 SNPs were identified as breed-specific in the Ethiopian cattle populations and European beef cattle, respectively. These SNPs were associated with 3364 and 17 genes, respectively. Across the sampled Ethiopian cattle populations, the mean observed and expected heterozygosity were 0.403 and 0.400, respectively. Similarly, for European cattle, the observed and expected heterozygosity were 0.25 and 0.26 respectively. Principle component analysis (PCA), and neighbor-joining (NJ)-tree revealed a separation of Ethiopian cattle breeds from European beef breeds. Similarly, the NJ-tree grouped the study cattle according to their breed group with close clustering of the three Ethiopian cattle populations. The average r2 values were 0.22 ± 0.25, 0.23 ± 0.25, and 0.22 ± 0.25 in Boran, Begait, and Fogera, respectively. For Angus, Herford and Charolais it was 0.17 ± 0.28, 0.17 ± 0.28 and 0.18 ± 0.29, respectively. It was observed that average r2 values decreased as physical distance increased. The top 1% FST values were considered to delimit genomic regions under positive selection. Some of the candidate genes are involved in biological processes and pathways linked to meat quality attributes. The candidate genes are involved in meat tenderness (CAPZB, DCTN2, KIF5A, DCTN2, RGS20, STIM1, KLHL3, and ITGB1BP1), IMF (DGAT1, LPCAT1, PLCL2, CTNNA1, and B4GALNT1) meat color (ASAP3, ADAP1, APBB1IP, RTKN2, and STAP2). Furthermore, some of the candidate genes are associated with tropical adaptation — heat tolerance, and resistance to disease (SOD1, DCTN2, GNAS, IGF1, TLN1, RRAS, GNAS, and ASAP3). The significant degree of SNP variation found in this study suggests that these markers could be useful for genetic research in Ethiopian cattle breeds. The high genetic variability observed in Ethiopian cattle breeds can be used as a potential source for future breeding programs. The identified candidate genes have to be further confirmed through GWAS, sequencing, and omics studies.
Roduction of Specific Antisera Against Selected Mammals and Identification of Blood Meals of Phiembotamine Sandjlies Transmitting Visceral Leishmaniasis (VL) in Ethiopia
(Addis Ababa, Ethiopia, 1999) Mamo, Hassen; Hailu, Asrat
In an auempt to know the feeding preferences of phlebotomine sandflies that transmit visceral leishmaniasis (VL) in Eth iopia, Phlebotomus mani"i and P. orielllaiis. and thus to poilll to the possible reservoir host(s) of the disease in the country, blood meals of the vectors were identified using the counter current immunoelectrophoresis (CCIE) technique. First, genus/species-specific antisera were raised against the immunoglobulin G (lgG) of ten potential hosts of the sand fly vectors: human, sheep, goat, donkey , horse, camel, cattle, squirrel, mongoose and hyrax. The IgG was fractionated from the whole sera of the mammals by the ammonium sulphate precipitation method followed by the ion-exchange chromatography technique. The antisera were developed by prim ing (subcutaneously) rabbits, in triplicates, with water-in-oil emulsion of equal volumes of IgG and Freund's Complete Adjuvant and then boosting with Freund 's Incomplete Adjuvant. The potency of the ant isera was determined by assay ing each lest bleed from each rabbit againstlhe homologous IgG. Cross-reactiv ities were checked by screening each test bleed against heterologous IgGs. Human whole serum and IgG were cross-reacted with anti-cattle antisera and vice versa. All other antisera were specific. The titre of each test bleed was qualitatively determined by visualizing the conspicuousness of the precipitin line. Hyperimmune antisera were used in blood meal analysis. Eight blood meal samples (5 SergelJlomyia spp. and 3 P. manilll) were collected , by sticky and CDC light traps, from Aba-Roba in a total of 42 night catches. One hundred and six blood meal samples (94 P. orielJlalis and 12 P. bergerofl) collected, by CDC, from the Middle Awash va lley and stored for about 4 years were used. Each blood meal sample was tested against cortlmercial anti-dog and anti-rat IgGs vi in addition to the 10 ami-host sera raised in the present study. Of 114 blood meals processed, hosts were identified for the 93 (8 1.6%): 2 P. martilli, 79 P. oriellfalis and all P. bergeroli. None of Sergelllomyia blood meals were identified. 37.6% of the blood meals detected were from single host sources: 20 from cattle, 10 camel, 2 squirrel , I donkey, I human and I from mongoose. The rest 62.4% was from mixed sources. Altogether, 80.6% of the meals identified were from caule origin and 59% from camel, singly or in combination with OIher hosts. Thus. P. oriellfatis appears to be an opportunistic feeder with a preference for cattle and camel at least in the Middle Awash where these hosts occur in large numbers. The CCIE technique is specific and sensi tive enough not only to detect mixed blood meals from close ly related hosts but halfdigested meals. The assay is technically simple and inexpensive in terms of reagents required. Thus, CCIE is a robust technique for identification of blood meals of haemalophagous insect vectors in general and smaller nies such as phlebotomine sandfl ies and of biting midges in particular.
Prevalence and Antimicrobial Resistance of Salmonella Enterica in Cow Milk and Cottage Cheese in Major Milk Shades of Oromia Region Ethiopia
(Addis Ababa University, 2021-03-14) Bedassa, Abdi; Sisay, Tesfaye (Professor)
Foods of animal origin are major vehicles of Salmonella infections and a serious public health problem with increasing concern in the world, particularly for developing countries. There has been a median of 6% in raw milk and dairy products but most prior works in this area have focused on biochemical confirmation from dairy isolates. Almost all report of the prevalence of Salmonella spp. in dairy products as well as raw milk was highly varied. To address the limitations of earlier research, molecular methods and the country's milk and dairy value chain were tested. Risk factors related to pathogen prevalence, antibiotic resistance testing, and PCR-based pathogen serotyping were performed. A cross-sectional study was conducted on milk and cottage cheese in major milk shades of the Oromia region of Ethiopia from December to March 2020 to determine the prevalence and of S. enterica. A total of 480 samples (384 milk and 96 cottage cheese) were collected using simple random techniques from producers, collectors, processors, and retailer value chains. The samples were tested for Salmonella using Iso 6579-1: 2008 methods. The risk factor for contamination of these dairy products across the dairy value chain was done using pre-tested questionnaires. Selective plating reveals a higher prevalence of S. enterica among 480 samples, with 67.5% (324/480) for Hektoen enteric agar and 40% (192/480) for Xylose lysine desoxycholate agar while 14.8% (71/480) molecularly by detecting the presence of a highly conserved region of the invA gene. Raw milk accounted for 21.3% (41/192) of the overall prevalence, whereas pasteurized milk accounted for 12.5% (24/192), and cottage cheese accounted for 6.25% (6/96). The collector value chain received 20.8% (20/96), whereas Bishoftu received 23.3% (28/120). Salmonella infection was found to have a statistically significant relationship with access to training and the water temperature with which udder was washed in producers; gender and milk filtration in collectors; sieving in milk processors; and production system in cottage cheese retailer value chain respectively The greatest serotype was S. Heidelberg (59.1%), and clindamycin showed full resistance among 10 antimicrobials, whereas ceftriaxone showed 81.7% susceptibility. The results of this investigation show that there were problems relating to the safety of milk and cottage cheese in the area. Therefore, strict hygienic approaches and quality control measures should be applied to improve the safety of the products. Awareness creation should be required for producers, retailers, processors, and consumers regarding the quality and safety of milk and other dairy products.
Strategies for Improvement of Indigenous Ethiopian Cattle Breeds: Breeding Plans and Sequence Polymorphisms of Selected Candidate Genes (BoLA-DRB3 and Leptin) as a Potential Molecular Markers
(Addis Ababa University, 2021-05-01) Mandefro, Ayele; Sisay, Tesfaye (Professor); Dadi, Hailu (PhD)
Agriculture is the major pillar of Ethiopia’s economy. The livestock sector is one part of agriculture, accounting for about 45% of the total value of agricultural production. Simulation method and genomic selection approaches were implemented to select superior animals for the aim of enhancing production and productivity. Two indigenous cattle breeds of Ethiopia (Boran and Horro) were simulated with the objective of evolving applicable breeding programs. Boran was evaluated for beef, milk, and both beef and meat (dual purpose) while Horro was evaluated for milk and dual purposes. Yearling weight, milk yield, age at first calving, calving interval, and calf survival to yearling were defined as selection criteria for milk production. Moreover, yearling weight, age at first calving, calving interval, and calf survival to yearling were used to improve beef production in the Boran breed. For all goal traits examined, more genetic gains were found from bull selection groups than cow selection groups. The genetic polymorphisms of the BoLA-DRB3 and leptin gene in Ethiopian and Asian zebu and taurine cattle breeds were evaluated by sequence-based typing (SBT). For the BoLA-DRB3 exon2 locus we detected 59 total alleles and 16 alleles were newly identified from this study. The Hardy Weinberg genetic equilibrium (HWE) of all the breeds investigated revealed non-significant excess of heterozygosis that could show over-dominant selection. The results of pairwise FST values directed low genetic differentiation of the Ethiopian breeds with Asian Zebu and taurine, rooted from the evolutionary history of cattle breeds. Exon2 and exon3 were evaluated for leptin gene IV | P a g e polymorphism. Five SNPs at positions 73 (C>T), 143 (C>A), 399 (T>C), 411 (T>C), and 495 (C>T) were identified and only the second SNP (C143A) was new to this study. Mutations detected on exon2 region were missense that causes a change of amino acids from arginine to cysteine (R25C) and from threonine to lysine (T48K). The evolutionary divergence showed that large-sized cattle breeds were closely clustered together. Simulation study could be used to improve the performance of cattle production. Also, selection of superior animals based on selected candidate genes (BoLA-DRB3 and leptin) could be important to improve cattle genetics.
Multiplex Polymerase Chain Reaction Based Serotyping of Diarrheagenic Escherichia Coli Isolates from Calves and Children in Ethiopia
(Addis Ababa University, 2021-03-03) Alefew, Bewekete; Sisay, Tesfaye (Professor)
Diarrheagenic E. coli strains (DEC) are associated with many disease outbreaks and sporadic cases mostly in children and calves. Traditionally serotyping of the pathogen is based upon the conventional serotyping using their surface O and H antigens. However, this method has its own drawbacks and as a result cost- effective, rapid and more sensitive detection method is required. Thus, the objective of this study was to identify the serotypes of diarrheagenic E. coli isolated from diarrheic calves and children from different parts of Ethiopia using three multiplex PCR assays. In the present study a total of 280 DEC isolates from diarrheic children (n=116) and calves (n=164) were distinguished based on their O-antigen gene clusters particularly, wzx (O-antigen filppase) and wzy (O- antigen polymerase). Results revealed that 12 (10.34 %) isolates from diarrheic children and 18 (10.97%) isolates from calves were found to be positive for one of the targeted O-serogroup specific primers used. A total of 12 O-serogroups were identified among the 21 O-serogroups targeted based on their importance in clinical cases. Of the identifiable serotype in diarrheic children O15, 8(6.89%), was the most frequently detected serotype. While in diarrheic calves the most frequently detected serotype was O55, 5(3.04%). Moreover, O55 and O128 serotypes were identified in both diarrheic calves and children. Furthermore, O15 was more distributed across three sampling sites, such as in Jimma 3(2.59%), Addis Ababa sub cities 3(2.59%) and Hawassa 2(1.72%) from diarrheic children. In diarrheic calves O55 was identified from Fiche 3(1.82%), Bahirdar 1(0.60%) and South Wollo 1(0.60%). Out of 12 O-serogroup samples detected from diarrheic Children 25% belonged to Enteropathogenic E.coli (EPEC) and while 8.33% were Enterohemorhagic E.coli (EHEC) and Shiga toxin producing E.coli. The 18 O-serotypes detected from diarrheic calves were classified to STEC, EPEC, and EHEC strains at 27.77%, 16.66%, and 16.7% respectively. The common serogroups O15, O118, O128, and O55 were identified in children and in calves O55, O123, O128, O145 and O26 were detected. From these, O128 and O55 are potentially involved in zoonotic transmission. The finding clearly indicates the potential of multiplex PCR to serotype diarrheagenic E. coli isolates. Thus, further use of Multiplex PCR for serotyping of diarrheagenic isolates in Ethiopia and elsewhere is highly recommended.
Profiles of Physicochemical Characteristics, Antibiotic Residues, Antibiotic Resistance Genes and Bacteria Community Structure of Batu Tannery Wastewater Released to Little Akaki River
(Addis Ababa University, 2020-06-10) Admassu, Tesfaye; Assefa, Fasil (PhD); Feleke, Adey (PhD); G. Love, Nancy (Professor)
Tanneries produce one of the highest COD wastewaters afterusing large volume of water and different chemicals to produce semi-finished and finished leather products. In Ethiopia, most of the tannery wastewater is discharged directly into water bodies with little or no treatment. These days, different treatment systems are constructed to treat tannery wastewaters that necessitate research to evaluate the efficiency. To this end, a research was conducted on Batu Tannery wastewater treatment plant to evaluate the physicochemical characteristics, bacteria community dynamics, antibiotic residues and antibiotic resistance genes in the different stages of the tannery wastewater treatment plant. The raw tannery wastewater showed variable strength from time to time, andthe highest mean COD (7204+8) mg/l, ammonia-N (190±3.6) mg/l, total nitrogen (308±2)mg/l, and total chromium (22±2.7)mg/lwere observed in April samples. The range of pH remains between 8.0 and 9.0 in raw tannery wastewater and 7.2 and 8.6 in the final effluent. The tannery effluent affected the physicochemical characteristics of the downstream river water of Little Akaki, except temperature and pH. The tannery effluent released to the Little Akaki River caused an increase in the physicochemical values by an average of 3.8%, 9% and 9.5% for COD; 23%, 10.4% and 12.5% for TN; 13.5%, 18.4% and 14.8% forammonia_N; and 31.5%, 28% and 52.8% for total chromium in the November, February, and April samples, respectively. However, only the COD, sulfate and total chromium cause significant (p < 0.05) difference in the downstream river water after tannery effluent was released. 16S rRNA amplicon-based bacterial community analysis from the treatment plant revealed that Firmicutes (48.5%), Bacteroidetes (32.6%) and Proteobacteria (11.5%) were the most represented bacterial phyla across the treatment plant. At the genus level, the dominant genera were Clostridium (15%), Synergistes (5%), Psychrobacter (4%), Acinetobacter (2.5%), Bacteroidetes (1.8%), Anaerovorax (1.3%), Arcobacter (1.3%) and Shewanella (1%). Most of the core bacteria communities in the tannery wastewater were common signatures of gastrointestinal bacteria of ruminants. Some of the core bacteria arepotential pathogens tohuman including the Clostridium, Acinetobacter, Arcobacter and Shewanella. Antibiotic residues and antibiotic resistance genes were detected in the tannery wastewater. The antibiotic residues of tetracycline, oxytetracycline, erythromycin, penicillin G, trimethoprim, sulfadiazine, sulfadoxine, sulfamethoxazole and ciprofloxacin were present in all samples of the tannery wastewater, but penicillin V, amoxicillin, streptomycin, ampicillin and vancomycin were not detected in the same samples. The antibiotic resistance genes tet(A), tet(O), tet(M), erm(B), Sul(I), Sul(II) and Otr(A) were also detected in the tannery wastewater samples. An assessment on the use trend of antibiotics in livestock indicated that antibiotic contamination to tannery wastewater was highly likely to come from the misuse ofantibiotics in livestock disease treatment.
Genetic Diversity Study of Napier Grass (Cenchrus Purpureus L.) Collections from Different Part of the World and Progeny Plants
(Addis Ababa University, 2021-02-21) Bizuneh, Tadelech; Shehabu, Meki (PhD); Feyissa, Tileye (PhD); Habte, Ermias (PhD)
There are limited studies on characterization of Napier grass forage through the application of currently available advanced molecular techniques. Furthermore, ILRI genebank held collection of this forage grass from different part of the world with little information. Therefore, the objective of this study was to evaluate the extent of the genetic diversity of Napier grass accessions collected from different parts of the world and progeny plants raised from naturally produced seeds. The genomic DNA was extracted from leaves of 347 Napier grass genotypes and genotyped by the DArTseq platform. A total of 96,454 Silico DArT and 96,321 SNP markers were generated, of which highly informative 1001 SNP markers were selected for diversity analysis after screening. The average polymorphic information content (PIC) values of Silico DArT and SNP markers were 0.21 and 0.15 and Average heterozygosity of 0.26 and 0.18 respectively. Two major groups and ten sub-clusters were identified from STRUCTURE, Hierarchal clustering population stratification and diversity analysis. The sub-clusters obtained by cluster analysis were also confirmed through discriminant analysis of principal component (DAPC). Analysis of molecular variance (AMOVA was highly significant (P<0.001) with 60% variation among the populations. The mean values of fixation index (Fst) per cluster ranged from 0.3398 in cluster VI that consist progeny plants to 0.7621 in cluster VII that consist most of the ILRI collections and the largest divergence (0.3777) was also between sub-cluster IV and VII. All these parameters showed the presence of high diversity and genetic differentiation among the assayed genotypes. This result indicates the potential of progenies and introduced genotypes for being used as parental plant in future hetrotic breeding and suitability of the studied population in Molecular genetic studies.
Genetic Diversity and Population Structure of Zymoseptoria Tritici Populations of Southern Ethiopia Using Ssr Markers
(Addis Ababa University, 2020-09-28) Molla, Messele; Hailesilassie, Teklehaimanot (PhD)
Septoria Tritici Blotch Caused By The Fungal Pathogen Zymoseptoria Tritici Is Considered To Be One The Major Threats To Wheat Production In Ethiopia And Elsewhere In The World. Understanding The Genetic Structure Of The Pathogen Has Great Importance In Designing And Implementing Suitable Management Strategies. Therefore, The Present Study Was Targeted To Explore The Genetic Structure Of 51 Z. Tritici Isolates Collected From Four Wheat Producing Zones Of South And Southwesten Parts Of Ethiopia Using Nine Microsatellite Marker Systems. Different Diversity Indices Were Computed. A Z. Tritici Specific Diagnostic Marker That Targets The ITS Rdna Had Amplified An Expected Fragment Size Of 345bp In All The Tested Isolates. All The Markers Were Found To Be Highly Polymorphic And Thus Useful Tools To Depict The Genetic Structure Of The Pathogen Populations. The Analysis Revealed A High Degree Of Genetic Diversity Within Populations With Number Of Alleles, Gene Diversity And Polymorphic Information Content Per Locus Ranging From 9 - 14, 0.80 - 0.88 And 0.70 - 0.87, Respectively. Analysis Of Molecular Variance (AMOVA) Confirmed A Moderate (0.14) Genetic Differentiation Where 86% Of The Total Genetic Variability (3.93) Resides Within Populations, And Only 14% Was Due To Among Populations‘ Difference. Dendrogram Produced UPGMA And Pcoa Also Showed A Moderate Population Clustering In Which The Populations Were Not Distinctly Clustered According To Their Areas Of Sampling Due To Presence Of High Gene Flow. Moreover, Bayesian Model-Based Population Structure Analysis Weakly Clustered The Population Into Five (ΔK) Sub-Groups With High Level Of Genetic Admixture. Among The Considered Populations, Populations Of Kembata -Tembaro Zone And Hadiya Zone Showed Relatively Higher Genetic Variability, And Thus Can Be Considered As STB Hot Spots For Further Studies On The Dynamics Of The Pathogen And Germplasm Screening And Host-Pathogen Interactions. Therefore, The Present Study Had Generated Useful Baseline Information For Wheat Breeders And Pathologists For Designing And Implementing Durable And Effective STB Control Strategies.
Genetic Diversity and Population Structure Analysis of Noug (Guizotia Abyssinica) Accessions Collected from Ethiopia Using Microsatellite Markers
(Addis Ababa University, 2021-06) Terefe, Motbaynor; Birmeta, Genet (PhD)
Abstract Noug (Guizotia abyssinica) is an Ethiopian indigenous oilseed crop cultivated primarily for its oil and various economic importances. Its production and productivity is very low due to different constraints. The crop has receiveda littleattention in implementing improvement programs and until very recently remained as underutilized oilseed crop. Crop improvement requires the availability and understanding of the genetic resource in terms of genetic variation and population structure.However, estimationofgenetic diversity of Noug with a wider representation of Ethiopia’s Noug growing regions using reliable molecular marker such as, microsatellite markers are rare. Thus, this study was aimed to uncover the extent of genetic diversity and population structure of 161 Noug genotypes collected from Ethiopia using 13 microsatellite markers. The analysis revealed a total of 158 alleles generated with a mean of 12.15 per locus. All the 13 microsatellite markers were polymorphic and highly informative with a mean polymorphic information content (PIC) value of 0.82. The genetic diversity indices across the 14 collection regionsindicated the presence of higher genetic variability among genotypes; including the Shannon’s information index (I) which ranged from 0.89 to 1.26 with a mean value of 1.57. Observed heterozygosity (Ho), expected heterozygosity or gene diversity (He), unbiased expected heterozygosity (uHe) was0.17, 0.74, and 0.79, respectively. Genotypes from Shewa, Wollo, Gojjam, Tigray and Benishangul Gumuz (B/G), shared the highest in both expected hetrozygosity or gene diversity and unbiased expected heterozygosity. Individuals in the populations had a fixation index (F) ranging from 0.67 to 0.80, with a mean of 0.77. The percentage of polymorphic loci (%Pl) in all the populations showed the highest (100%) and lowest (94.33%) with a mean of 99.19%.The pairwise Nei’s genetic distance showed the highest between Eritrea and Bale (0.892) followed by Eritreaand Illubabor (0.875).AMOVA revealed that 96.06% of the total genetic variation was attributed to within populations while only 3.94% was attributed to among populations, indicatingthat the highest variation was between genotypes rather than variation due to geographic distances. The NJ or UPGMA cluster and PCOA poorly grouped the genotypes based on their origin indicating high gene flow or seed exchange across geographic regions.Furthermore, the model-based population structureanalysis weakly inferred the predefinedpopulations into K=2 withconsiderable genetic admixture among tested populations.In conclusion,the microsatellites used in the present study are highly informative and could be used in future marker assisted breeding. Genotypes from Shewa, Wollo, Gojjam, Tigray and B/G are identified as hotspot areas for their high genetic diversity. They also showed the highest number of private alleles in which special focus should be given in these areas to implement breeding and future improvement programs. Key words/Phrases; Cluster analysis, Gene flow, Heterozygosity, hotspots, SSR Markers
Ethnobotanical Knowledge of Lablab (Lablab Purpureus (L.) Sweet - Fabaceae) in Konso Zone and Genetic Diversity of Collections from Ethiopia Using SSR Markers
(Addis Ababa University, 2020-10-23) Tamiru, Solomon; Feyisa, Tileye (PhD); Asfaw, Zemede (Professor); Disasa, Tesfaye (PhD)
Lablab is an important multipurpose legume crop used for human consumption, animal feed and soil conservation. In spite of these qualities, the potential value of this crop has not been fully utilized, and little research attention has been given to this crop. The main objective of the study was to build the knowledge base from farmers’ perspectives and molecular genetic diversity analysis of Lablab collections using 15 SSR markers. The field study was conducted in December 2018 in six kebeles distributed in Konso zone by interviewing a total of 84 informants containing 72 randomly selected general informants and 12 purposively selected key informants (42 men and 42 women) of above 18 years age. The data were analyzed by entering the data into the excel spreadsheet version 2007 and summarized using descriptive statistics. A total of six Lablab farmers’ varieties were identified and the majority of the farmers give names to their varieties based on seed color. The main cultivation practices in the study area were intercropping Lablab with sorghum, maize and finger millet, and sole crop in the margin of terracing and fence line. In Konso zone, Lablab is mainly used for human food in the form of boiled grain (NIFRO), animal feed and soil conservation purpose. The molecular genetic diversity study of 91 lablab collections from the entire country revealed a total of 225 alleles with an average of 14.80 alleles per locus. All markers across the entire populations were found to be highly polymorphic and informative with PIC values ranging from 0.92 to 0.78 with a mean value of 0.85. The average expected heterozygosity and gene diversity was 0.75 and 0.86 respectively, indicating a high level of genetic diversity. Analysis of molecular variance showed that 94% of the total genetic variation was attributed to within populations while only 6% was attributed to among populations. The smaller Fixation Index value (0.061) recorded indicates the presence of moderate population differentiation as a result of higher gene flow (Nm =3.820) among populations. Cluster, PCoA and Structure analysis revealed a weak association between geographical origin and genetic diversity confirming the presence of population admixtures due to seed exchange and sharing. The observed higher genetic diversity in Konso and West Wellega zones indicates hot spot area for genetic diversity and germplasm evaluation. Generally, ethnobotanical knowledge and genetic diversity obtained from this study provides inputs for Lablab conservation and improvement in Ethiopia.
Production and Characterization of Xylanolytic Enzyme from White Rot Fungi Isolated from Decayed Woods
(Addis Ababa University, 2021-02-22) Abena, Tariku; Simachew, Addis (PhD)
Utilization of enzymes like xylanase, in different industrial processes across the globe gained more attention than the chemical catalyst because they are highly selective, easy to control and have a negligible environmental impact since they produce very small amount of byproducts. Therefore, this study was undertaken with the objective of production, purification and characterization of xylanase from white rot fungi. In the present investigation, 68 white rot fungi (WRF) isolates were screened for xylanolytic potential using qualitative (plate assay) and quantitative (ferementation) methods. The optimum growth conditions for xylanase production were determined by growing fungal isolates at different: - pH values, incubation temprature and carbon and nitrogen sources. The feremetation was conducted under SSF and SmF condition using wheat straw as sole carbon source. The enzyme was assayed using 3, 5-dinitrosalysalic acid (DNS) method by measuring the amount of xylose released in μmol/ml/min at 540nm. Partial purification of the enzyme was conducted by ammonium sulfate precipitation method and protein concentration determined. Partial purified xylanas was characteriazed under different physico-chemical condtions to determine the optimum pH, temperature and the effect of metal ions. Among 68 WRF qualitatively screened, 17 isolates with ≥4.55 cm hydrolysis zone on mineral salt medium (MSM) or basal medium were selected and subjected to qualitative screenng. Out of quantitatively screened isolates, five WRF isolates with high xylanase yield (73.63±0.0283–63.6±0.01247 U/ml) were selected and used for further study. It was found that, the optimum xylanse production was at pH 5.0 and 28 oC temperature. Among carbon and nitrogen sources, xylan and yeast extract respectively, enhanced xylanase production. Solid state fermentation (SSF) favored xylanase production than that of submerged fermentation (SmF). The partial purified xylanase showed higher specific activity than the crude extract. Highest xylanase activity (80.9–61.274 U/ml) was recorded in the pH range of 5.0–6.5 and at 50 oC. Among the metal ions tested, Mg2+, Ca2+ and Mn2+ enhanced relative activity of xylanse (127.28–110.06%) while Cu2+, Fe2+ and K+ showed inhibitive effect with 43.4–17% loss of relative activity. In this study, kinetics parameters (Km and Vmax) were determined from Lineweaver-Burk plot. Xylanase from the isolates showed 0.32–0.545 mg/ml Km and 86.95–113.63 μmol min-1mg-1 Vmax. Xylanase from white rote fungal isolates exhibited properties suitable for applications in animal feed and different food industries like bakery, fruit juice and wine industries.

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