Biotechnology

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    Comparative in Silico Study of Lodging-Resistant Genes of Tef (Eragrostis Tef (Zucc.) Trotter) Against Wheat, Barley and Rice
    (Addis Ababa University, 2024-07) Tigist Shewafera; Abiy Zegeye
    Tef (Eragrostis tef), a cereal crop indigenous to Ethiopia, is essential in ensuring food and nutrition security. It is well-known for its gluten-free properties, rendering it suitable for individuals with celiac disease. However, tef is highly susceptible to lodging that negatively impacts its productivity. Efforts to develop varieties that are lodging-resistant have had limited success; particularly, because there have been a dearth of targeted genetic interventions. The aim of this study is to computationally predict and characterize six lodging-resistant genes (Rht1, BRI1, COMT1, CAD8C, CESA1 and CESA4) and the corresponding polypeptides of tef vis-à-vis three economically important cereal crops. The study began by selecting three economically important crops, namely: wheat, barley and rice and their six lodging-resistant genes. Using in silico probes, six homologous candidate lodging-resistant genes were retrieved from tef subgenomes. The candidate tef lodging-resistant nucleotide sequences were analyzed to computationally predict their gene structures and then comparatively analyzed against their homologues in the selected three crops. Putative polypeptide sequences of the six lodging-resistant genes, derived from their obtained predicted tef genes, were subjected to further amino acid level analysis to identify variations unique to tef, map the variations to specific functional or structural domains, predict the potential effects of the said variations and examine the possible impact of significant variations on the 3 dimensional structure of the proteins. Multiple sequence alignment of polypeptide sequences revealed distinct amino acid differences unique to tef in key functional and structural domains encoded by these lodging-resistant genes, with potential implications for tef's lodging susceptibility attributes. Variant effect predictions of counterfactual amino acid variations point to a subset of the unique tef variations having significant likelihood of causing damage in four of the six proteins: Rht1, BRI1, COMT1 and CESA1. Further examination of significant variation effects on the 3D structures of the four proteins revealed possible effects on the secondary structure of the polypeptides. Overall, the findings provide insights into several of tef's genetic variations related to lodging resistance features. Further experimental validation studies are needed to confirm these findings and elucidate the precise roles of these genes in tef's lodging susceptibility.
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    Isolation and Characterization of Lytic Bacteriophages from Various Sources in Addis Ababa Against Antimicrobial Resistant Diarrheagenic E. Coli Strains and Evaluate their Therapeutic Potentials
    (Addis Ababa University, 2023-10) Tamirat Salile; Tesfaye Sisay
    Escherichia coli is a common fecal coliform, facultative aerobic, gram-negative bacterium. Pathogenic strains of such microbe have evolved to cause diarrhea, urinary tract infection and septicemias. The emergences of antibiotic-resistance urged to find an alternative strategy. The use of lytic bacteriophages against the control of pathogenic E. coli in the clinics and different environmental setups (waste and drink water management) become an alternative therapy to antibiotic therapy. Thus, this study aimed to isolate and characterize the lytic bacteriophage from various sources in Addis Ababa and tested against antimicrobial resistant diahrrgenic E. coli strains and evaluates their therapeutic potentials under invitro conditions. A total of 14 samples were processed against six different diahrrgenic E. coli strains. Conventional culture and plaque analysis agar overlay method was conducted to recover lytic bacteriophage isolates. The phage isolates were characterized to determine their lytic effect, growth characteristics, host range activity and stability under different temperature and pH conditions. Phage isolates were identified by Scanning Electron Microscope (SEM), molecular techniques (PCR), and whole genome sequencing. Totally, 17 phages were recovered from 84 tested plates. Totally, 17 phages were recovered from 84 tested plates. Of the 17 phage isolates, 11(65%) were Myoviridae-like phages, 6 (35%) phage isolates were Podoviridae and Siphoviridae by morphology and PCR identification. Bacteriophage genome sequencing revealed that each bacteriophage has a linear double-stranded DNA genome. The GC content of phage genomes ranged from 43 to 54%, while their sizes ranged from 40,427 to 143,710 bp. The whole genome sequence analysis of 7 potent coliphages showed that phage isolates were taxonomically classified as 4 (57%) of Myoviridae phages and 3 (43%) of Siphoviridae phages. Based on the host range test, growth characteristics and stability test 7 potent phages were selected. These phages demonstrated better growth characteristics, including short latent periods, highest burst sizes, and wider host ranges, as well as thermal stability and the ability to survive in a wide range of pH levels. These phages' promising effect against AMR pathogens has raised the possibility of their use in biological control of bacterial infections.
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    The Effect of Urine-Derived Fertilizers on Soil Microbiota and Nutritional Contents of Selected Vegetables
    (Addis Ababa University, 2023-02) Tseganesh Darsema; Adey Feleke
    There is now a growing trend to explore environmentally friendly, low cost and effective soil fertility techniques in substitute of chemicals that applied to the soil. Application of human urine is being used as organic fertilizer because of its nutrient rich and pathogen free nature. The aim of this study is to explore the effect of human urine fertilizers on soil microbial community and nutritional values of the plant by comparing it with widely used chemical fertilizer. On this specific study four different vegetables: Cabbage, Ethiopian kale, Carrot and Tomato were used as experimental subjects in green house condition. Five different treatments (Stored urine, struvite urine, chemical fertilizer and unfertilized and control) with twice replication of each were used for this experiment. The plants grown by these different treatments in soil were measured for their nutritional content values and physiological characters. Soil sample obtained from each of experimental pots were also used to elaborate the effects of chemical and organic fertilizers (urine) on the microbial community of soil. Shotgun metagenomic analysis was used to assess the distribution of soil microbial community at phylum and genus level upon the application of various fertilizers. In all selected plants, the distribution of the identified bacterial groups was relatively better in struvite urine fertilized soil than unfertilized ones. This was the same both in Phylum and genus levels. In carrot, struvite urine fertilized soil showed better composition of identified bacteria compared to chemical treated soil both at phylum and genus levels. In Ethiopian kale, the soil bacterial community distributed better in stored urine fertilized soil than both struvite urine treated soil and unfertilized soil. The nutritional analysis section of this study concluded that there was no significant difference in nutritional values of the plants treated with different fertilizers. However, the phosphorous, nitrogen and protein contents were slightly higher in plants fertilized with organic fertilizers while moisture contents were slightly higher on those plants treated with chemical fertilizers.
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    Morphological Characterizations, Genetic Diversity and Population Structure of Date Palms (Phoenix Dactylifera L.) in Ethiopia
    (Addis Ababa University, 2024-10) Workia Ahmed; Tileye Feyissa
    Date palm tree is aperennial plant that belongs to Arecaceae family and it significantly contributes to agricultural sustainability and socio-economic values for many countries including Ethiopia. Scientific evidences from molecular and morphological genetic data of date palms are the baseline for establishment of gene banks, conservation and to develop breeding programs. In Ethiopia, there is lack of genetic information about date palm trees. Therefore, the objective of this dissertation is to evauate the genetic diversity and population structure of date palms in Ethiopia. In this study, 45 morphological traits for 11 individual date palms, 10 Inter Simple Sequence Repeat (ISSR) markers for 113 date palm genotypes, 10 Simple Sequence Repeat (SSR) markers for 124 date palm genotypes and 4 Single Nucleotide Polymorphism (SNPs) markers for 15 date palm genortypes were used. The morphological diversity analysis was based on principal component analysis using the total 45 traits showed 37% variations exist among 11 individuals. The morphological data analysis using 25 vegetative traits alone as well as the data from 20 reproductive traits alone 29% and 32% of variations was observed respectively. Fourty three morphological traits exhibited significant differences at p < 0.05 in analysis of variance and 23 traits showed significant variances at p < 0.05 in homogeneity of variance analysis among cultivars. Dendrograms indicated clear genetic relationships of cultivars. From ISSR analysis, a total of 241 DNA fragments were generated by all primers and each primer showed 100% of polymorphism. The highest (37) and the lowest (10) number of bands were produced by (AGG)6 and (AG)10C primers, respectively. AMOVA result showed 49% and 51% within and among population diversity, respectively, and also, the first three PCoA accounted for 10.55%, 9.17% and 7.68% variations. The genotyes were also clustered according to their geographical location. From SSR analysis: a total of 112 of alleles were detected, Neighbour-joining clustering analysis based on dissimilarity coefficient values the date palm popuatons grouped into five major categories. Population structure analysis at the highest K value grouped the genotypes into three groups. Principal coordinate analysis explained a total variation of 17.33%. The discriminant analyses of principal components also separated date palm genotypes into eight clusters. From SNPs data, genetic variations were shown in phylogenetic tree, nucleotide content and pairwise distance analysis of date palm genotypes. Generally, evaluation of genetic diversity and phylogenetic structure of date palms using molecular and morphological markers is very important. The overall findings of this study are helpfull to design genetic improvement, conservation and germplasm introduction programs of date palms in Ethiopia and elsewhere.
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    Morphological Characterizations, Genetic Diversity and Population Structure of Date Palms (Phoenix Dactylifera L.) in Ethiopia
    (Addis Ababa University, 2024-10) Workia Ahmed; Tileye Feyissa
    Date palm tree is aperennial plant that belongs to Arecaceae family and it significantly contributes to agricultural sustainability and socio-economic values for many countries including Ethiopia. Scientific evidences from molecular and morphological genetic data of date palms are the baseline for establishment of gene banks, conservation and to develop breeding programs. In Ethiopia, there is lack of genetic information about date palm trees. Therefore, the objective of this dissertation is to evauate the genetic diversity and population structure of date palms in Ethiopia. In this study, 45 morphological traits for 11 individual date palms, 10 Inter Simple Sequence Repeat (ISSR) markers for 113 date palm genotypes, 10 Simple Sequence Repeat (SSR) markers for 124 date palm genotypes and 4 Single Nucleotide Polymorphism (SNPs) markers for 15 date palm genortypes were used. The morphological diversity analysis was based on principal component analysis using the total 45 traits showed 37% variations exist among 11 individuals. The morphological data analysis using 25 vegetative traits alone as well as the data from 20 reproductive traits alone 29% and 32% of variations was observed respectively. Fourty three morphological traits exhibited significant differences at p < 0.05 in analysis of variance and 23 traits showed significant variances at p < 0.05 in homogeneity of variance analysis among cultivars. Dendrograms indicated clear genetic relationships of cultivars. From ISSR analysis, a total of 241 DNA fragments were generated by all primers and each primer showed 100% of polymorphism. The highest (37) and the lowest (10) number of bands were produced by (AGG)6 and (AG)10C primers, respectively. AMOVA result showed 49% and 51% within and among population diversity, respectively, and also, the first three PCoA accounted for 10.55%, 9.17% and 7.68% variations. The genotyes were also clustered according to their geographical location. From SSR analysis: a total of 112 of alleles were detected, Neighbour-joining clustering analysis based on dissimilarity coefficient values the date palm popuatons grouped into five major categories. Population structure analysis at the highest K value grouped the genotypes into three groups. Principal coordinate analysis explained a total variation of 17.33%. The discriminant analyses of principal components also separated date palm genotypes into eight clusters. From SNPs data, genetic variations were shown in phylogenetic tree, nucleotide content and pairwise distance analysis of date palm genotypes. Generally, evaluation of genetic diversity and phylogenetic structure of date palms using molecular and morphological markers is very important. The overall findings of this study are helpfull to design genetic improvement, conservation and germplasm introduction programs of date palms in Ethiopia and elsewhere.
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    Plasmodium Vivax Duffy Binding Protein Copy Number Variation and their Effects on Reticulocyte Invasion in Selected Parts of Ethiopia
    (Addis Ababa University, 2024-01) Yasin Nasir; Alemayehu Godana; Fitsum Girma
    The connection between the Duffy binding protein and the Duffy antigen receptor for chemokine is required for Plasmodium vivax penetration into human reticulocytes. A previous analysis of Plasmodium vivax samples in Ethiopia determined that Duffy binding protein duplications are more prevalent than in any other Plasmodium vivax location. However, its prevalence and importance in large samples remain unclear. Duffy blood group genotyping was done by amplifying the GATA1 transcription factor-binding region of DARC geneamong 349 Plasmodium vivax isolates were determined by a real-time PCR technology. In addition,duplications of Duffy binding protein and their relationship with Duffy-negativity and parasite densitywere examined. Duffy binding protein duplications and Duffy-negative antigens were detected in 74% and 3.2% isolates respectively. Most of the Duffy negative participants were found in Gondar Zuria district (72.7%). To know whether Duffy binding protein amplification contributes to the takeover of Duffy-negative reticulocytes, the relationship between Duffy binding protein copy numbers and Duffy status was investigated using Fisher's exact test and post-hoc tests. Fisher's exact test reveals a significant association (p = 0.000313). In addition, a post-hoc test indicates a significant association between the Duffy binding protein copy numbers (single and 2-3 copy) and the DARC status (p = 0.000281). However, the number of Duffy-negative patients infected by multiple Duffy binding protein copy is small. Therefore, we cannot decide that multiple Duffy binding protein copy number increase parasite's invading ability against Duffy-negative patients. The mean parasite burden differs between Duffy negative and positive in a statistically significant way (P <0.0001). Duffy-negative patients aren't resistant to Plasmodium vivax however; the detailed mechanisms of the infection in Duffy-negative patients remain unclear. In reference to the high rate of Duffy-binding protein duplication, further investigation should be explored by extending study site across Ethiopia by using the most sensitive molecular detection tools known as digital PCR. It is also useful to look into additional parasite ligands related to the invasion of Duffy-negative reticulocytes.
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    Chloroquine Resistance Transporter (Pfcrt) and Multidrug Resistance 1 (Pfmdr1) Genes Mutation in Plasmodium Falciparum Population Under Varying Level of Endemicity with Plasmodium Vivax in Selected Parts of Ethiopia
    (Addis Ababa University, 2023-02) Tajudin Abdurahman; Alemayehu Godana
    The global controlling of Plasmodium falciparum infections faces significant challenges due to the spread of parasites resistant to antimalarial drugs. In Ethiopia, where both P. vivax and P. falciparum coexist, the treatment for uncomplicated falciparum malaria shifted from chloroquine (CQ) to sulfadoxine-pyrimethamine (SP) in 1998 and then to Coartem (artemether-lumefantrine (AL)) in 2004. AL has been the standard treatment for over two decades for P. falciparum, while P. vivax is still treated with CQ. The coexistence of these two species and the accessibility of CQ for P. vivax treatment raise questions about whether switching from CQ to AL for P. falciparum treatment might lead to the resurgence of CQ -susceptible P. falciparum strains due to reversal mutations or the efficacy of AL. The sudy aimed to assess the prevalence of pfcrt-76 and pfmdr1-86 gene mutations in the P. falciparum population under varying levels of endemicity with P. vivax in selected parts of Ethiopia. In this study the frequency of gene mutations of P. falciparum chloroquine resistance transporter 76 (pfcrt-76) and P. falciparum multidrug resistance 1-86 (pfmdr1-86) in P. falciparum collected from malaria-infected patients in Abobo, Dera, Fentale, and Metema districts, Ethiopia, were examined. Confirmed P. falciparum samples (n = 258) with microscope were collected through health facility-targeted cross-sectional surveys in areas with varying levels of P. falciparum and P.vivax prevalence. Genomic DNA was extracted from dried blood spots by using MagMAX DNA Multi-Sample Kit, operated by the Kingfisher Flex Automated Extractor. An 18S rRNA gene-based multiplex real-time PCR assay was employed to confirm P. falciparum mono-infection samples (n = 258). The study analyzed 258 P. falciparum-infected patients using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), examining Pfmdr1-86 and Pfcrt-76 gene mutations. Fisher's exact test determined marker distribution significance. Out of 250 genotyped for Pfcrt K76T, 68.8% had mutant 76T, and 31.2% were wild-type K76. For 257 genotyped for Pfmdr1-N86Y, 98.44% was N86 wild-type, and 1.56% was 86Y mutants. The mutant Pfcrt-76T was more prevalent in areas with higher P. vivax endemicity, including 93.33% (Fentale), 84.71% (Dera), 57.5% (Metema), and 43.75% (Abobo). Pfcrt-76 gene single nucleotide polymorphisms (SNPs) significantly correlated with P. vivax endemicity (P = 0.000). Pfmdr1-N86 was predominantly 100% except in Abobo, where 95.18% were N86, and 4.81% were 86Y. Pfmdr1 gene SNPs were significantly associated with P. vivax endemicity (P = 0.025). Despite CQ discontinuation for over two decades in Ethiopia, a substantial proportion of P. falciparum isolates still carry mutant 76T genotypes, indicating latent CQ pressure. The use of AL for uncomplicated P. falciparum malaria may lead to the return of Pfmdr1 N86 wild-type genes. Further molecular epidemiological investigations in varied endemic regions with different CQ usage histories are recommended to understand chloroquine (CQ) susceptibility recovery and AL therapy efficacy.
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    Isolation and Molecular Characterization of Campylobacter Jejuni and Campylobacter Coli Isolated from Ethiopian Dairy Supply Chain and Evaluation of their Associated Risk Factors and Antimicrobial Resistance
    (Addis Ababa University, 2024-03) Abera Admasie; Tesfaye Sisay
    Campylobacter is among the leading bacterial foodborne pathogens, causing a high foodborne disease burden worldwide. There is a limited information on the prevalence, risk factor, and whole genome sequencing of Campylobacter in Ethiopian milk and milk products in major milk sheds in Ethiopia. To this end, a cross-sectional study was carried out to isolate and characterize the genomic diversity,antimicrobial resistance patterns and associated risk factors of Campylobacter species from milk and dairy products collected from representative regional sites (Oromia, Amhara, and SNNP). in Ethiopia. A total of 1140 dairy food samples were collected in the dry and wet seasons of which 456 samples were used for seasonal comparison. Samples were tested for Campylobacter by following the ISO 10272-1:2017 standard and confirmed by PCR with Illumina MiSeq instrument (v3 600-cycle cartridge) for the paired-end sequencing run. Amrfinderplus_db NCBI was used to detect gyrA and 50S_L22_A103V gene mutations. NCBI Pathogen Detection database was used for the genomic similarity . A total of 141 Campylobacter isolates were tested for susceptibility to three antibiotics using a disk diffusion method.. The result indicated that Campylobacter was detected in 12% of tested food samples. The highest prevalence of Campylobacter jejuni and Campylobacter coli was found in raw milk (19%), followed by pasteurized milk (10 %) and cottage cheese (3%) (P<0.001). The prevalence did not differ significantly between the wet (20%) and dry (16%) seasons (P=0.27). However, there was a five times more chance of finding Campylobacter species in milk and milk products during the wet season than the dry season (COR = 4.5 (1.8-12), P = 0.002). in the Oromia region, Besides, 89% of the samples were contaminated with C. jejuni, and 11% with C. coli. Two different C. jejuni MLST sequence types, namely, ST 51 (clonal complex ST-443) and ST 2084 (clonal complex 353) were detected; they were clustered in different clades (B and C), respectively. Two ST 1628 and 2 ST 830 C. coli from clonal complex 828 were grouped into a single clade (C). Phenotypically, 89 %, 74%, and 57% of Campylobacter species were resistant to tetracycline, erythromycin, and ciprofloxacin respectively. Moreover, 43% of the tested isolates were resistant to more than two drugs. Genomically, ten isolates of 8 C. jejuni ST 2084 and 2 C. coli ST 1628 had a T86I mutation in the gryA gene, which is associated with resistance to Quinolone (ciprofloxacin), and all 14 C. jejuni carry 50S_L22_A103V gene, associated with resistance against Macrolide (erythromycin). Of these, all Campylobacter species carried CTD genes, chemotaxis-related genes (cheA, cheB, cheR, and cheY), and invasive genes (flaC, ciaB, and ciaC). We can conclude that 12% of Campylobacter species were present during the dry and wet seasons. The data also showed that , 43% of the isolates acquired more than two antibiotic resistance genes and a mutation was present in the 50S_L22_A103V and gryA genes in C. jejuni. The risk factor analysis showed that using warm water and soap for cleaning cow udders and teats on farms (AOR=0.3, P=0.023), filtering milk with a cloth, or plastic filter (AOR=0.065, P=0.005), and storing milk in an aluminum container (AOR=0.23, P=0.027) reduced the likelihood of detecting Campylobacter in raw milk. In contrast, Campylobacter detection was significantly higher in milk samples collected at collection centers with concrete floors (AOR=5.2, P=0.004). The odds of detecting Campylobacter in milk were 17 times greater (AOR=17, P=0.007) in milk processing facilities that did not calibrate a pasteurizer on an annual basis. Likewise,, having a separate refrigerator for milk storage reduced the occurrence of Campylobacter in retail (AOR=0.29, P=0.021). In conclusion, Compared to samples of pasteurized and cottage cheese, the raw milk was more contaminated. Additionally, a mutation was found in the 50S_L22_A103V and gryA genes of C. jejuni, and 43% of the isolates that were studied possessed more than two antibiotic resistance genes. Thus, understanding the genetic composition and prevalence of Campylobacter in the dairy supply chain may help identify potential contamination sources and create effective management plans that ensure the safety and caliber of dairy products.
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    Evaluation of Microbes from Teff (Eragrostis Tef (Zucc. ) Trotter) Ersho for Injera Starter Culture Development
    (Addis Ababa University, 2024-06) Hagere Hailemariam; Anteneh Tesfaye
    Injera is fermented Ethiopian ethnic traditional staple food prepared usually form tef (Eragrostis tef (Zucc.) Trotter). The teff injera is prepared of households by mixing teff flour with water and using a starter (ersho) which is left over of the last fermentation. The batter is fermented using the back-slopping technique which involves adding ersho. The microbial composition of ersho is not defined.Therefore, this study was undertaken to isolate, screen, and identify fermentative microbes from ersho and their potential for starter culture development. A ninety-one ersho samples were collected from Debrebirhan, Fiche, Chacha, Sebata, Teji, Holeta, Adama, Bishoftu, and Dukam. Lactic acid bacteria, aerobic mesophilic bacteria, yeasts, and molds were isolated from the ersho samples collected. Isolated microbes were screened for their sugar utilization stress test, temperature stress test, pH tolerance test and hydrogen sulfide production test. The best performing isolates were used to formulate the starter culture for injera fermentation by combining the four microbial groups. The sensory acceptability of the selected injera scored highest mean of other, was produced from a combination of T4 (L1+L2+Y1+Y2+AMB+M) and fermented for 28 hrs. Finally, based on DNA sequencing, bacteria identified as Bacillus subtilis, Lacticaseibacillus paracasei and bacterium strain AGE YJ E2. The yeast and mold belong to the Pichia fermentans strain and the Aspergillus niger strain, respectively. The selected starter culture requires further analysis of its profile, performance, and shelf life.
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    Deciphering the Conserved Cis-Regulatory Elements of Major Milk Protein Genes by Computational Analysis
    (Addis Ababa University, 2024-04) Addis Tekaw; Abiy Zegeye
    Milk genes are exclusively expressed in MECs during lactation, regulated by lactogenic hormones like prolactin that act through specific TFs. The recruitment of TF is determined by cis-regulatory motifs in their gene promoters; but there is limited evidence of shared motifs among the major milk gene across species. In this study, Jalview, Meme-Suite, TomTom, and GOMo software were utilized to construct phylogenetic trees, discover motifs, identify TFs, and determine GO terms, respectively, in the 2kb upstream putative promoter region of CSN1S1, CSN1S2, CSN2, CSN3, LALBA and BLG milk genes among twenty-three mammalian species. The analysis revealed three common TFBSs for STAT1, STAT5a, and STAT5b TFs in all milk genes across the species studied, except for BLG, within the region -390 to -80bp from the canonical TSS, with a few shared TFBSs located in the distal promoter region upstream of -600bp. STAT3 was also detected in CSN1S1, CSN2, CSN3, and LALBA genes, sharing binding sites with STAT1, STAT5a, and STAT5b. Furthermore, TFBSs for Sox9 and Sox6 TFs were found to be shared between the LALBA and BLG putative promoters within -400 to -100bp. Moreover, GO analysis linked GPCR with regulatory motifs in all six milk genes across 23 species, essential for enhancing STAT5 phosphorylation through Gαq activation in the JAK-STAT pathway. The totality of the result suggests that the relative abundance of STAT5 and STAT3 proteins may play a role in regulating the expression level of caseins and LALBA commensurate with the stage of lactation. Together, these results offer valuable insights into the potential of the milk genes’ promoter for constructing eukaryotic expression vectors and provide essential information for transgenic studies.
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    Plantlets and Micro-Rhizome Regeneration, Genetic Diversity and Evaluation of Ginger (Zingiber Officinale Roscoe) for Bacterial Wilt in Ethiopia
    (Addis Ababa University, 2024-06) Genene Gezahegn; Tileye Feyissa; Yeyise Rezene
    Ginger (Zingiber Officinale Roscoe) is an important crop used for many purposes and is a major spice across Ethiopia since 13th century. Ginger production, productivity, and product quality were limited due to different biotic and abiotic factors. Among the biotic factors, it was challenged primarily due to bacterial wilt disease eruption in the last decade. The use of clean tissue culture generated seed rhizome as part of integrated management with other cultural practices was an option to reduce the disease pressure. However, the disease still poses a threat to ginger cultivation in the country. In the absence of efficient strategies to reduce the disease impact, the use of tolerant cultivars appears to be the best disease control strategy. Hence, this study aimed to enhance disease-free planting materials generation and ginger diversity analysis. The first and second experiments targeted in vitro disease-free plantlets generations and micro-rhizome induction respectively. Experiments three, and four are with the objectives of genetic diversity assessment among ginger accessions using morphological and molecular markers consecutively. The last objective of the study focused on evaluating the study materials for resistance against the pathogen. In the course of in vitro regeneration, alternative nitrogen source salts replaced 1.65 g/l ammonium nitrate in Murashige and Skoog medium. xvi Ammonium chloride at 1.0 g/l, potassium nitrate (3.8 g/l), and urea (3.0 g/l) are the best alternatives. Micro-rhizome induction resulted in viable in vitro generated micro-rhizomes that are planted directly in the soil. Murashige and Skoog medium supplied with elevated levels of sucrose (80 g/l) and benzyl amino purine (6.0 mg/l) was the best treatment among thirteen treatment combinations for the micro-rhizome induction. For genetic diversity analysis, both by morphological traits and SSR markers, 100 ginger accessions including two released varieties (Boziab and Yali) and wild species (mango ginger) were collected from different agro-ecologies of south, central, and southwestern parts of Ethiopia. The morphological trait-based analysis was done by using 24 quantitative and qualitative traits. The analysis has revealed the availability of genetic diversity via high values of genetic coefficient of variation, heritability, and genetic advance as mean. Cluster analysis also grouped the 100 accessions into four clusters. The study materials assigned to four sub populations; Southern, Central, Southwest and Oromia based on the area of the collection were also assessed for genetic diversity using twelve polymorphic SSR markers. The SSR based molecular diversity population structure anlysis has grouped the 100 accessions into three clusters unlike morphological-based analysis which resulted in four clusters. AMOVA has showed 96% among individuals and 4% variation was among the four populations. Sprouted buds were found better for quality DNA extraction in ginger, which is the first of its kind. In both morphological and SSR-based diversity analysis, the wild type (JW89) was unique due to some traits like rhizome size and unique fragment bands. The diversity analysis based on morphological traits and SSR markers, in general, revealed that there is high genetic diversity among ginger accessions. Evaluation of the 100 study materials against bacterial wilt disease pathogen under the protected conditions in the screen house and further tested in the field also revealed that there is response variation among the accessions. Disease severity, disease incidence, days to severe disease symptoms, fresh and dry yield losses showed high variations among accessions. the evaluation experiment revealed that (12) accessions were grouped as very tolerant accessions. The majority, 88 accessions also showed different response levels from tolerant to very susceptible based on disease scores and yield loss analysis results. Four accessions (BASP19, BSSB47, BSSB49, and OKW63) and wild type (JW89) have shown consistent variations being grouped to very tolerant in disease screening, in similar clusters during morphological and molecular genetic diversity analysis. The study results of the three separate experiments' morphological traits, SSR markers, and screening against pathogen gave clue that there might be potential markers linked to disease tolerance and high rhizome yield, which needs further verification.
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    Heavy Metal Removal Capability of Bacterial Isolates from Awash Tannery Effluents and Wastewater Sources of Selected Rivers in Addis Ababa
    (Addis Ababa University, 2024-08) Smegnew Melese; Addis Simachew
    Heavy metals from various industrial processes for different purposes pollute soil and groundwater worldwide. They have toxic, mutagenic, and carcinogenic effects, which pose health risks to the natural biota. Microbe-based biological treatment methods are an environmentally benign and more efficient heavy metal removal technology than conventional ones. The objective of this study was to evaluate the heavy metal removal capability of naturally occurring heavy metal-resistant bacteria both in NAM-SHM and in the actual wastewater, which was collected from ATE and selected tributaries of AARW. The result showed that four of the 130 heavy metal-resistance isolates were identified based on their morphological and molecular characteristics. Isolates S. xylosusT14Cd and B. cereusT4Zn were obtained from the Cd and Zn metal-supplemented NAM of the ATE, while S. xylosysW1Cr and S. xylosusW12Pb were obtained from the Cr and Pb of the selected tributaries of AARW. For all those isolates, 30°C and pH 8 were the optimum growth conditions. Isolates S. xylosusW1Cr, S. xylosusT14Cd, S. xylosusW12Pb, and B. cereusT4Zn were shown to have MICs of 3500, 4000, 5000, and 7000 ppm against Cr6+, Cd2+, Pb2+, and Zn2+, respectively. Isolates’ resistance order to a mixture of heavy metals Pb2+, Zn2+, Cr2+, and Cd2+ in 3:3:1:1 (50-800 ppm), respectively, was MCC>S. xylosusT14Cd>S. xylosusW1Cr>S. xylosusW12Pb>B. cereusT4Zn. On the NAM-SHM inoculation, isolates S. xylosusT14Cd, S. xylosusW12Pb, and B. cereusT4Zn gave the maximum removal efficiency for Cd2+ (82.19%), Pb2+ (84.24%), and Zn2+ (80.94%) metals in 8 days, respectively, and Cr6+ (69.9%) was recorded for S. xylosusW1Cr in 6 days of incubation. Whereas, for MCC Cr6+ (79.36%), Cd2+ (88.66%), and Zn2+ (90.22%) in 8 days, and Pb2+ (93.44%) in 6 days, removal efficiency was recorded. The maximum removal efficiency of S. xylosusW1Cr and S. xylosusW12Pb was 83.00 and 79.60% in 6 days, while S. xylosusT14Cd, B. cereusT4Zn, and MCC was 84.88, 76.05, and 87.98% of Cr6+ in 8 days of incubation on the ATE. Moreover, the maximum removal efficiency of S. xylosusW1Cr was 89.75, 98.33, 97.57, and 99.46% of Cr6+, Cd2+, Pb2+, and Zn2+, respectively, on the selected tributaries of AARW in 6 days of incubation. After 6 days of incubation, S. xylosusT14Cd was able to remove the maximum concentration of metals from AARW, with values of 89.76, 99.89, and 99.08% of Cr6+, Cd2+, and Pb2+, respectively, and 99.12% of Zn2+ after 8 days. S. xylosusW12Pb was able to remove up to 99.03% of Zn2+ on same medium in 8 days of incubation and also 88.70, 98.93, and 97.77% of Cr6+, Cd2+, and Pb2+, respectively, in 6 days. Similarly, the maximum removal efficiency of B. cereusT4Zn was also 80.48 and 97.25% of Cr6+ and Zn2+, respectively, in 8 days of incubation, while 98.35 and 83.28% of Cd2+ and Pb2+, respectively, in 6 days. Whereas, for MCC Cr6+ (90.29%) and Zn2+ (99.99%), in 8 days, while Cd2+ (99.97%) and Pb2+ (99.37%), in 6 days of incubation, removal efficiency was recorded. In conclusion, the four tested isolates are highly efficient for the removal of heavy metals from the NAMSHM and actual wastewater samples. Hence, using these identified and tested bacteria for heavy metal bioremediation is recommended for tannery factory effluents and other industries.
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    Antimicrobial Resistance Profile of Escherichia Coli Isolated from Hospital Sewage and Polluted River: the Cases of Adama Hospital Medical College in Adama and Yerer River in Dukem
    (Addis Ababa University, 2024-11) Kajelcha Fikadu; Alemayehu Godana
    The antimicrobial resistance profile of Escherichia coli is dramatically increasing across the world, particularly in low- and middle-income countries where untreated wastewater can disseminate resistant bacteria into the environment. Untreated wastewater discharged from hospitals and industries is the most known vehicle for the emergence and spread of antimicrobial resistance genes. The study aimed to evaluate the antimicrobial resistance profile of E. coli isolates from hospital sewage and polluted river samples in Adama and Dukem, respectively. The hospital sewage and polluted water samples were collected from Adama Hospital Medical College and two rivers in Dukem. The samples were transported to Addis Ababa University, Institute of Biotechnology Laboratory. For the purpose of isolating E. coli, the samples were cultivated on Eosin Methylene Blue agar, MacKonkey agar, and Nutrient agar media. Biochemical tests, including Gram staining, IMViC, TSI, and SCA, were used for the identification of E. coli isolates. The researchers evaluated the antimicrobial susceptibility patterns of the E. coli isolates using the Kirby-Bauer’s disc diffusion method. The identified E. coli isolates were then cultured on Mueller Hinton agar media at 37°C for 24 hours, after which the inhibition zone was measured by a digital ruler. Polymerase chain reaction (PCR) was used to confirm the presence of resistance-associated genes in the E. coli isolates. A total of 75 presumed E. coli isolates were obtained via culture. Among these isolates, 50 isolates were subjected to AST. The isolates from Yerer River before the industrial waste entry site (YRBI) did not show multiple antimicrobial resistance (MAR), while the isolates from Xadacha River (XR) and Yerer River After Industrial waste entry site (YRAI) showed resistance profiles of 66.67% and 90%, respectively. This study also revealed that 86.67% of hospital sewage isolates and 65% of river water isolates showed MAR. PCR amplification confirmed the presence of tetA and blaTEM genes in 83.33% and 57.14% of the AMR isolates, respectively. In conclusion, the study showed that untreated hospital sewage and pollute river water are considered major reservoirs for the emergence of antimicrobial-resistant E. coli among humans, animals, and the environment. The study advocates for improved wastewater treatment, stringent regulations on antimicrobial use, and regular monitoring to curb the emergence and spread of antimicrobial resistance.
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    Isolation of Yeast and Lactic Acid Bacteria from Selected Ethiopian Fermented Foods and Evaluation of their Leavening Capacity
    (Addis Ababa University, 2024-10) Martha Yishak; Diriba Muleta
    There is a high demand for baker’s yeast for various Ethiopian foods and bioprocess industries. Currently, the baker’s yeast for the country’s demand is fully imported from abroad, and a huge amount of foreign currency has to be spent for this purpose, which necessitates the need for alternative import substitution products. Therefore, the main objective of the current study was to screen indigenous yeasts and lactic acid bacteria isolates from Borde, Tella, and Teff dough having superior bread leavening abilities and evaluating their impact on bread’s sensory quality and shelf life investigation. A total of 230 yeasts and 42 lactic acid bacteria were isolated and purified. Out of which, 47 yeast and 20 lactic acid bacteria isolates were selected for further analysis based on their baking ability; carbon dioxide production, growth rate, hydrogen sulfide (H2S) production, temperature tolerance, flocculation, and ethanol tolerance, within 24 hours of incubation. Four yeasts and five lactic acid bacteria isolates were chosen to develop starter cultures for bread making. The combined effect of two selected yeast isolates was also tested. The findings demonstrated that the five most potent yeast isolates (Y3, Y1, Y2,Y4 and Y5) tolerated different values of temperature that ranged up to 45°C and alcohol concentrations up to 16%. The isolate from Tella (Y3), had a maximum leavening capacity of (251.67 ±7.6 ml) at 4 hours of fermentation, but the commercial strain had a maximum leavening capacity of (245.67 ± 5.9 ml) at 3 and 4 hours. There was no significant difference (p>0.05) between Tella and the commercial strain. Effect of the three co-inoculated isolates (Y3 + Y1 + Y2) was found the highest at 275 ml at 120 min, compared co-inoculation of two combinations (Y3+Y1) 261 ml at 120 min. The five screened lactic acid bacteria isolates (L1, L2, L5, L4 and L3) revealed very poor leavening capacity; none of the isolates were selected for further testing. A panel of judges assessed the bread's organoleptic quality based on its appearance, color, texture, flavor, and overall acceptability. A nine-point "Hedonic scale" was used to gauge consumer acceptability of the items, and the commercial strain scored the highest (6.18 ± 0.17), isolates from Tella (Y3) scored (5.70 ± 0.57) although there was no statistically significant difference (p>0.05) between the two isolates. The bread made with Tella isolates (BY3) had the highest shelf life of 5 days, compared the bread made with commercial yeast (BCY) 4 days. The results of the present study indicated that mixed cultures showed superior leavening potential than that of single cultures and commercial yeast, and the wild yeasts isolated from Ethiopian traditional fermented products can be used for the development of bakery yeast.
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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.
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    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.
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    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.
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    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.
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    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.
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    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.