Plant Biotechnology
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Item GENETIC DIVERSITY AND GENOME-WIDE ASSOCIATION MAPPING IN TETRAPLOID WHEAT (TRITICUM TURGIDUM SPP.) GERMPLASM OF ETHIOPIA(Hawassa University College of Agriculture, 2024) MIHERETU FUFA GELETAGenetic Diversity and Genome-Wide Association Mapping in Tetraploid Wheat (Triticum turgidum spp.) Germplasm of Ethiopia PhD Dissertation Miheretu Fufa Geleta Hawassa University, 2024 Ethiopia is a center of diversity for Tetraploid wheat (Triticum turgidum spp.) species, which hold ample genetic variation; howevr, the country remains a net importer of wheat due to a huge gap between production and consumption. The present study was aimed to investigate the extent and pattern of diversity based on phenotypic traits, grain quality traits, and molecular markers; and Genome-Wide association studies for phenotypic and grain quality traits. Diversity assessment at the entire genotypes based on the qualitative traits revealed intermediate (0.60±0.01) diversity and high diversity concerning glum color (0.78), spike density (0.61), and seed color (0.86); however, low diversity for awndness (0.15). All qualitative traits showed highly significant (p<0.001) variation across regions of collections and altitudinal classes; however, only spike density was significantly (p<0.05) different at the species level. Within populations diversity was higher than between population diversity. Analysis of variance based on the quantitative traits revealed highly significant variation (p<0.001) among genotypes and for genotype by location interaction in all traits except for the number of effective tillers per plant. The observed mean and range values of the phenotypic traits revealed high variability in the accessions. Phenotypic Coefficient of Variation (PCV) and Genotypic Coefficient of Variation (GCV) values were high for grain yield, biomass yield, and harvest index. The estimates of heritability (H2 ) ranged from 41.78 to 84.62 % respectively for grain yield and the number of seeds per spikelet. High genetic advance as a percentage of mean was observed for the number of seeds per spikelet, the number of effective tillers per plant, grain yield, biomass yield, and harvest index. The number of seeds per spikelet gave a high value of genetic advance and heritability implying high genetic gain from its selection. Grain yield showed a highly significant (p<0.001) negative correlation with days to booting and days to maturity and a positive correlation with the remaining quantitative traits. The combined ANOVA based on the grain quality traits revealed a highly significant variation (p < 0.001) among the genotypes and for genotype by location interaction for all traits. The genotypes showed a wide range in their thousand kernel weight, zeleny index, and vitreousness percentage. Grain yield and protein content, respectively, showed high and low values of PCV and GCV; however, an intermediate value of PCV and GCV was observed for gluten content and thousand kernel weight. Zeleny index and vitreousness percentage showed high PCV and intermediate GCV; however, protein content showed low PCV and GCV. The estimate of broad sense heritability (H2 ) ranged from 33.57 % for vitreousness percentage to 66.36 % for zeleny index. The values of genetic advance and GAM observed were respectively high for grain yield (31.94, 26.91) and zeleny index (31.89, 29.34); intermediate for thousand kernel weight (18.27, 16.59), gluten content (15.70, 13.55), and vitreousness percentage (15.75, 12.97) and low for protein content (9.81, 8.44). Grain yield showed a highly significant positive correlation with thousand kernel weight (0.43***) and vitreousness percentage (0.19**) and positive correlation, but not significant, with protein content, gluten content, and zeleny index. xxi Principal component analysis (PCA) based on phenotypic traits showed that the first and second principal components (PC) respectively accounted for 19.74% and 15.96 of the total variation in the entire genotypes, respectively. The first five principal components combined explained 61.21 % based on the entire genotypes. On the other hand, PCA of grain quality traits showed that the first and second PCs respectively accounted for 34.12 % and 20.65% of the total variation and the three PCs explained 69.11% of the total variation in the entire genotypes. Eleven landraces and eleven improved varieties were top performing based on their grain yield and grain quality traits. Clustering based on phenotypic traits and grain quality traits respectively grouped the whole genotypes into seven and six clusters without clear regional grouping. Furthermore, the genetic diversity and population structure were investigated for the study panel as well as the landraces based on 10349 DArTSeq markers distributed across A and B genomes. The study panel was clustered into two populations: pop1 and pop2 respectively comprising 150 landraces and 3 released varieties and 19 released varieties and 12 landraces pop2 being more diverse than pop1; likewise, landraces were clustered into two subgroups. Analysis of molecular variance (AMOVA) revealed highly significant (P<0.001) variation between populations and within populations the latter being higher than the former implying that more attention should be given to individual accessions within populations to explore the existing genetic diversity. Little to moderate genetic differentiation (Fst) was respectively reported for the landraces (0.028) and the study panel (0.117) implying no significant differentiation among populations. High gene flow (1.297-8.818) was recorded based on DArTSeq markers at all level. PCA based on DArTSeq markers grouped the released varieties and the landraces separately in the study panel; however, PCA of the landraces did not show clear groupings. 127 genotypes were reported to have one or more private alleles at 755 loci, an indication of key adaptive genes at these loci to be exploited in breeding programs. In conclusion, high genetic diversity was detected in Ethiopian tetraploid wheat germplasm, which could be utilized for future wheat breeding programs. The landraces ETW115 and ETW135 and the released varieties (Tesfaye and Bekelcha) could be used in a crossing program owing to their maximum genetic distance. Analysis of genome-wide LD in the whole collection showed that 44.98% of the total marker pairs had a significant LD (p<0.01) with a mean r2 value of 0.129791. 44.59% (18668) and 8.62% (3609) of the significant marker pairs showed r2 values above 0.2 and 0.7, respectively, indicating a higher level of LD in the genome. The number of significant marker pairs observed was higher in the B genome (24.44%) than the A genome (20.54%). GWAS identified 44 SNPs, two of which are pleiotropic, across chromosome 1A, 2A, 2B, 3A, 3B, 4B, 5A, 5B, 6A, 6B, and 7B that were significantly (FDR <0.05) associated with some of the traits studied. Thirty-five QTLs were reported while the remaining 9 QTLs were found to be novel, which need to be validated for further use in the breeding program. We also identified several candidate genes, potentially regulating the traits, and encoding various proteins involved in plant growth and development. Hence, this study highlights the significance of the Ethiopian tetraploid wheat gene pool for improving tetraploid wheat globally; thus, a breeding strategy focusing on accumulating favorable alleles at these loci could improve tetraploid wheat production in Ethiopia and beyondItem Morpho-Agronomical, Physiological, and Molecular Genetic Diversity of Amaranths (Amaranthus species) in Ethiopia(Hawassa University College of Agriculture, 2024) MEKONNEN YESHITILA DEGEFUMorpho-Agronomical, Physiological, and Molecular Genetic Diversity of Amaranths (Amaranthus species) in Ethiopia The genus Amaranthus L. is one of the world's underutilized cosmo-politant and genetically potential orphan crops, with a diverse range of morphological characteristics and geographic distribution. It is one of the few dicotyledonous, non-grass mesophytes that uses specialized C4 annuals or short-lived perennials to produce significant amounts of edible small-seeded pseudo cereals. Amaranths are versatile plants with the potential for high yields. They have great photosynthetic performance as a result of eliminating the rival photorespiration mechanism. A diversified family of food crops known as amaranths is impressively adaptable to new environments despite a variety of biotic and abiotic constraints. Moreover, to maintain a healthy lifestyle and prevent disease, people are becoming more and more concerned with their diet and selective about the foods they eat. People are therefore moving away from the typical cereals and staple meals they have been eating for a long time and toward more nutrient-dense options. Regular cereals lack essential minerals, amino acids, a greater nutritional value, and contain gluten. As a result, pseudo-céréals, particularly amaranth, are excellent alternatives. The potential of Amaranthus spp. is still underexploited in Ethiopia. This is also evident from the limited genetic research and selection for desirable traits in amaranths. Consequently, the number of genotypes being cultivated is small, thus reducing the genetic variability of the elite germplasm and ultimately increasing potential vulnerability to biotic and abiotic stresses. In addition, the amaranth genotypes are poorly represented in gene banks. Moreover, the plant is being neglected due to a lack of extensive research, discrimination, ignorance, and, among other things, the long-term genetic growth of the plant in Ethiopia. As a result, there is a lack of data on its genetic diversity, necessitating this research. Therefore, it is important to broaden the germplasm collection of this crop and assess the genetic diversity existing within the germplasm to support future collection, conservation, and crop improvement programs. The objectives of the current study were to explore the potential of the genetic resources of amaranth genotypes for further conservation and exploitation and make it easier to incorporate them into breeding programs in order to increase productivity. In this study, one hundred twenty amaranth genotypes were evaluated over two years using an alpha lattice design with two replications using agro-morphological markers. The analysis of variance indicated that the mean square due to year and genotype-by-year interaction varied significantly for most measured traits. The estimates of variability, heritability, and genetic advance found in this study indicate high genetic diversity in amaranth genotypes and the strength of selection response for these traits in the population. Furthermore, the potential for amaranth improvement through appropriate selection is revealed by the existence of significant differences between the number of superior and inferior genotypes for the majority of examined traits. This suggests that these traits are governed more by additive gene action and that selection based on these traits might be successful in achieving the desired genetic gains for improvement. Aside from this, selection based on yield alone may not be effective for yield improvement in plant breeding initiatives. So yield should be considered along with potential yield contributing traits to progress the genetic gains during selection. On the other hand, the results revealed significant positive phenotypic and genotypic associations on leaf yield, with leaf area, leaf breadth, branch number, leaf number, plant height at flowering, and grain yield all having positive direct effects. Similar strong positive phenotypic and genotypic relationships were found for grain yield and grain sinking and filling rates. This study consequently shows the need for traits with significant positive indirect impacts via leaf area to be considered indirect selection criteria for improving leaf yield in amaranth genotypes. The grain sink filling rate also significantly improved grain yield indirectly at both the xxiv phenotypic and genotypic levels, mainly via days to flowering and leaf yield. This demonstrated that selection that mainly targeted days to flowering, leaf yield, and grain sink filling rate would ultimately boost the grain yield in amaranth genotypes. Principal component analysis showed that the first six principal components with eigenvalues greater than one contributed 80.41% of the variability. However, the first two principal components explained 52.42% of the total variation. The highest contributing traits in the first component were days to flowering, basal stem diameter, plant height at flowering, plant height at maturity, auxiliary inflorescence length, number of branches, terminal inflorescence lateral length, days to maturity, terminal inflorescence stalk length, leaf number, leaf length, top lateral branch length.. The traits with the greatest weight on the second component were leaf area, basal lateral branch length, leaf length, and leaf width, grain filling period, grain sinking filling rate, and grain yield. Therefore, selection based on these traits would be effective for yield improvement in amaranth genotypes. Additionally, the hierarchical clustering grouped all the genotypes into five clusters. The findings suggest that amaranth genotypes in Ethiopia have a lot of genetic variation, which might be used for future breeding and ought to be conserved. Twenty qualitative descriptors were utilized for a morphological diversity study for 120 amaranth genotypes. The overall mean of the Shannon diversity indices (H') was 0.61. The estimated diversity indices showed more intra-regional diversity (0.66) than inter-regional diversity (0.34), demonstrating the existence of gene flow between growing regions. Shannon-Weaver Diversity Index, ranged from 0.00 for auxiliary inflorescence to 1.94 for leaf coloration, with an overall mean of 19 characters (95%) that were found to have high diversity (>0.76) while auxiliary inflorescence was invariant. The hierarchical clustering grouped all the genotypes into three clusters. The first cluster included the most genotypes (58), followed by the second (47), and the third cluster contained the fewest (15). The study unequivocally demonstrated that, even when the genotypes were grouped into a small number of clusters, there was still enough divergence within the clusters to demonstrate the genotypes of amaranth to have a high genetic diversity. These results indicate that there is substantial genetic diversity among Ethiopian amaranth genotypes, which should be safeguarded and may be utilized in breeding in the future. The results of the analysis of variance showed that all examined physio-morphological parameters, except the rate of photosynthesis and stomata conductance, had mean squares that varied considerably (P < 0.001) owing to genotypes. The estimates of genetic variability, heritability, and expected genetic advance indicated high genetic diversity among amaranth genotypes, with a significant selection pressure for these traits in the population to produce better genotypes for improved amaranth. Selection based on desirable features such as leaf-to-air vapor pressure deficit, transpiration rate, chlorophyll a, chlorophyll b, total chlorophyll, carotenoid, leaf area, plant height, leaf number, and root weight can be useful in achieving the intended genetic gains for improvement since these traits appear to be more controlled by additive gene activity. Thus, selection in amaranth genotypes may consider these desired yield-related features. Moreover, the study showed that certain genotypes (ALE-073) exhibited better intercellular CO2 concentration (Ci), leaf-to-air vapor pressure deficit (VPD), transpiration rate (E), and leaf number (LN), resulting in better grain yield. Understanding the relationship between LA and E can help in selecting crops for high E and may provide an avenue to improve leaf yield. Furthermore, some of the selected genotypes in this study could be used as potential parents for improving the genetic gain in amaranth breeding programs. The study concluded that there was additive gene action present since the Ch a, Ch b, TCh, and Tca markers exhibited 100% heritability. This showed that the use of these characteristics for selection, which indicated a potentially exploitable variation, would be more effective and successful in the long run in breeding programs than the use of other traits for splitting generations. Hierarchical clustering grouped all the genotypes into seven clusters. The sixth cluster had the fewest genotypes (6), while the seventh cluster had the greatest number of genotypes (35) and was followed by Cluster Five (25), Cluster Three (22), Cluster One (14), Cluster Two (11), and Cluster Four (7). High leaf and grain yield-producing genotypes were grouped into the sixth cluster. The second cluster (C2) was characterized by high mean values for LA, LY, and GY. The maximum and significant genetic divergence was found between clusters C4 and C6 (D2 =103.44) and the least non-significant genetic divergence was found between clusters C5 and C7 (D2 =17.00). The first six PCs with eigenvalues greater than one accounted for 79.30% of the total variation in amaranth genotypes. The cumulative variance of 39.40% xxv was explained by 1st two principal components (PC1 and PC2) with eigenvalues greater than 1. Component 1 with a variance of 21.11 had a contribution from root length, root weight, total chlorophyll, and carotenoid while principal component 2 accounting for18.29% total variability had a contribution from transpiration rate, rate of net photosynthesis, chlorophyll b and, stomata conductance. The remaining variability of 13.02%, 11.33%, 9.37%, and, 6.17 % was consolidated in PC3, PC4, PC5, and PC6. These give scope for the selection of parents for breeding programs from these clusters, genetic divergence, and principal components to realize high genetic variation and novel combinations for yield incremen t. The present study was conducted to determine the genetic diversity and population structure of 40 amaranth genotypes using four inter-simple sequence repeat (ISSR) markers. The polymorphic information content (PIC), marker index (MI), resolving power (RP) and effective multiplex ratio (EMR) showed average values of 0.46, 10.41, 4.74, and 4.22 per primer, indicating high polymorphism values. The degree of polymorphism among the genotypes ranged from 0.00% for the Amhara population to 100% for the Southern Nations, Nationalities, and Peoples' Region population, with a grand mean of 70.83+11.34%. The observed number of alleles (Na), effective number of alleles (Ne), expected heterozygosity (He ), and genetic diversity estimated by Shannon’s information index (I) were 1.51, 1.50, 0.28 and 0.42, among collection regions, respectively. The total genetic diversity, Ht (0.400+0.006), and the average intrapopulation genetic diversity, Hs (0.285+0.004). A high level of gene flow (Nm = 2.12) between populations implies the presence of gene flow (Nm > 1) and reflects high genetic differentiation (Gst = 0.281). The analysis of molecular variance showed that the maximum value of genetic variation was found within populations (99%), whereas a low value of genetic variance was observed among populations (1%). The structure analysis, principal coordinate analysis (PcoA), and unweighted pair-group method with arithmetic averages (UPGMA) analysis clustered the 40 amaranths genotypes into four distinct subpopulations. The majority of genotypes (35%) were clustered in Pop2, mainly obtained from Southern Nations, Nationalities, and Peoples' regions constituted dominantly of breeding lines and varieties, implying target selection contributed to the formation of distinct populations. The findings of this study provide important and relevant information for future breeding and conservation efforts of amaranths. Therefore, the use of molecular markers would be valuable for the effective utilization of amaranth in breeding programs. In conclusion, the characterization of the genotypes using the molecular marker, and phenotypic traits compositions indicated the presence of diversity and variation among Ethiopian amaranth genotypes. The results from these studies suggest possibilities for identification of amaranth genotypes superior for leaf and grain yield, indicating the need for the initiation of a planned breeding program. The outcome of this study provided new insights into the genetic diversity and population structure in Ethiopian amaranth genetic resources for designing an effective collection and conservation strategies for efficient utilization in future breeding.Item ISOLATION, CHARACTERIZATION AND EVALUATION OF PLANT GROWTH PROMOTING RHIZOBACTERIA FROM TOMATO RHIZOSPHERE FROM DIFFERENT PARTS OF ETHIOPIA.(Hawassa University College of Agriculture, 2024) DEREJE HAILE SEMERERhizobacteria inhabit and colonize the plant root-zone and play a significant role in soil quality, health, biodiversity, and productivity. Rhizobacteria that possess beneficial traits in plant growth-promotion and disease protection are called plant growth-promoting rhizobacteria (PGPR). Phosphate solubilizing bacteria (PSB) are the known plant growthpromoters with their multidimensional benefits in plant nutrient access, stress alleviation, phytohormone production, and broad host-range interaction, among others. They improve plants’ physiological processes to grow well and produce quality yield. Therefore, exploring and developing potential or competent plant growth-promoter is required to improve soil fertility, farm production, and yield quality with a sustainable and eco-friendly approach. A huge diversity of soil microbes capable of P-solubilization and other plant growth-promoting attributes have been reported so far and remain to be explored. However, local isolation, strain identification, and host-specific evaluation are helpful for better farm production, indigenous competition, and sustainable environmental health. Multistage screening procedures (lab screening, greenhouse trials, and field evaluation) help to select efficient and competent strains. Each stage of evaluation possibly confers the different dimensional attributes of the candidate strain and then empirically strengthens the screening technique. This project aimed to screen efficient PSB strains and develop competent biofertilizers and plant growth-promotors with multidimensional benefits possibly applicable for tomato and other crop production at Koka, Meki, and Ziway Zuria. The project started by assessing the overall tomato production system, agrochemicals, and biofertilizer practices in these specific sites using randomly selected smallholder farmers. Accordingly, smallholder farmers in the study sites produced different crops, including tomatoes, in both seasons under repeated agrochemical practices. Sample soils were collected from different sites, including the tomato rhizosphere at Koka, Meki, and Ziway Zuria, as well as the natural forest soil from Wendogenet and Yirgalem Zuria. Then the experiment proceeded in the laboratory by screening potential PSB strains from collected soil samples using selective media and other preliminary screening techniques. The top 10 PSB isolates were selected and then further evaluated under different conditions. Their P dissolving ability was evaluated in liquid medium from different sources and the amount of dissolved P concentration quantified, plant growth-promoting traits verified, representative biochemical tests conducted, molecular characters (sequence of the 16S rDNA and IGS region between 16S and 23S) analysed and taxonomy identified, antagonistic effects, N2-fixing ability, symbiotic interactions, and host viii range were evaluated accordingly. PSB and possible P-substrates dual inoculation impact on tomato growth and yield were evaluated both at a greenhouse and open field levels, while a host range symbiotic interaction trial was conducted at field conditions using 5 different crops (maize, wheat, faba bean, kidney bean, and onion). From each experimental unit, various parameters were analysed against each PSB strain. During screening in the lab for instance, upon 8 days of incubation on PVK agar plate, Mk-1-25 and K-10-41 strains recorded a higher solubilization index (3.1 and 3.0, respectively), while 5 days of incubation in PVK broth resulted in a growing medium pH change where K-10-41 and K-10-27 significantly lowered the pH to 4.02 and 4.12, respectively. Inoculation in modified PVK broth with different P-substrates (iron phosphate, aluminium phosphate, and bone meal) and incubation for 10 days resulted in a substantial medium pH change and dissolved P. Accordingly, strains from the Koka site dissolved the highest overall mean P concentrations (260.83, 260.38, and 241.91 µg/ml by K-1-29, K-10-41, and K-10-27, respectively) and lower medium pH (4.93 and 4.95 by K-10-27 and K-10-41, respectively). Likewise, Z-12-20 was isolated from Ziway Zuria and found to be a competent strain in dissolving an average of 223.52 µg/ml P and reducing the pH to 4.98 upon 10 days of incubation. Among Psubstrates, the highest dissolved P (253.46 µg/ml) was obtained from TCP; nevertheless, the rest of the substrates recorded comparable concentrations. Furthermore, these potential 10 PSB isolates were characterized (morpho-biochemically and molecularly) and taxonomically identified. As a result, they placed to Bacillus, Priestia and Burkholderia species (i.e., K-1- 29, Mk-20-7, and Mk-20-20 belong to Priestia megaterium, while K-10-27 and Z-12-20 belong to Bacillus subtilis; Mk-1-25 and Z-13-4 Bacillus halotolerance; K-10-41 Bacillus velezensis, Mk-13-16 belongs to Bacillus amyloliquefaciens, and Z-1-16 belongs to Burkholderia cenocepacia). These strains are highly studied and repeatedly cited by different scholars for their plant growth-promoting and biocontrol roles. To evaluate their symbiotic effectiveness and plant assay responses, the current PSB strains were transferred and inoculated to tomato under greenhouse conditions. So as to improve their interaction and responses, all of them were co-inoculated with six possible P sources (TCP, BM, FeP, AlP, DAP fertilizer, and compost) independently. Accordingly, strains inoculation significantly improved plant growth parameters: shortened tomato germination, shoot length (highest 108.19, 106.43, and 101.81 cm by Z-12-20, Mk-1-25, and K-10-41), leaf parameters (substantially improved by Mk-1-25 and K-1-29), branch and node number, floral parameters (number, cluster, bud), fruit parameters (number, weight, marketability) (on average 6.1 and 5.95 fruits per plant were harvested from K-1-29 and K-10-27, respectively, ix 158.7 and 149.2 g fruits collected from Mk-20-20 and Z-1-16 inoculation, respectively), root length, root fresh and dry weight substantially were promoted by Z-12-20, shoot fresh and dry weight were significantly enhanced by Mk-1-25 and Z-12-20. Moreover, the overall highest phosphate-based symbiotic effectiveness was recorded by Mk-1-25 (PBSE%=176), followed by Z-12-20 and K-1-29 (PBSE%=144). Among the added P substrates, compost induced the symbiotic interaction, which then resulted in enhanced tomato vegetative growth, fruit number (6.03), and biomass, while AlP and AmP promoted fruit weight (collected on average of 156.73 and 149.61 g, respectively). Having these encouraging and positive responses/results from laboratory and greenhouse trials, the strains were further evaluated at open field conditions using tomato and other host crops. This is because, the in vitro solubilization and beneficial response of the strains might not be appropriately reflected and related to their effects at field level. Similar to greenhouse experiment, tomato-field trial was conducted under dual-inoculation of PSB and possible external P substrates (BM, compost, DAP fertilizer, and a 50% rate mixture of DAP and compost). This synergetic inoculation significantly improved the tomato’s growth, development, and yield parameters over uninoculated (control) group. For instance, elevated average shoot length (67.2 cm recorded by Mk-20-7), branch and leaf development (Z-12-20 and Mk-1-25), floral development (K-1- 29, Mk-1-25, Mk-20-20, and K-10-41), and fruit parameters (average highest total fruit number: 21.87, 21.82, 21.31, and 20.69 by K-10-41, K-10-27, K-1-29, and Mk-20-20, respectively) and highest fruit weight: 2821.6, 2793.3, and 2780.53 g harvested by K-10-41, K-1-29, and Mk-20-7, inoculation respectively). From the collected total fruits, a greater average number of marketable fruits were obtained from the inoculation of Mk-20-7 (10.44), K-10-41 (10.42), and K-1-29 (10.0 fruits per plant). However, because of strong biological competitors (birds, porcupines, and hyena), blossom end rot and early harvesting, a substantial number of unmarketable fruits were collected from the inoculation of K-10-27 (12.64) and Mk-13-16 (11.44). Compost application improved tomato-bacterial symbiotic interaction and significantly promoted tomato early vegetative growth, while a 50% rate mixture of DAP and compost demonstrated substantial responses at late growth period and resulted in highest tomato shoot length (67.39 cm) and fruit yield (on average 63.06 fruits with a gross weight of 2617.39 g). Similarly, the addition of bone meal enhanced fruit yield (total fruit number (62.82), quality (larger fruit with 10 cm length), and marketability (9.67 healthy fruits per plant)). To strengthen the screening processes and to promote farm practices, the candidate strains were inoculated to different crops to check their symbiotic interaction/response and growth promotion efficiencies at open field. Accordingly, maize, x wheat, onion, faba, and kidney bean were inoculated, and agronomy parameters were evaluated. Because of biological competitors’ attacks, grain yield was excluded from the analysis for most of these crops. Among the 10 PSB inoculants, Mk-20-20, Z-12-20, and Z13-4 significantly induced maize shoot length, whereas leaf number, cob number, and shoot fresh and dry weight were enhanced by inoculation of Z-12-20 and Z-13-4. Similarly, wheat inoculation with Z-1-16 promoted shoot length, tiller, and leaf development; Z-12-20 enhanced wheat flower development; and Mk-20-7 improved wheat biomass yield. Onion inoculation with Mk-20-20 increased shoot length (53.45 cm), shoot fresh weight (44.11 g), Mk-1-25 improved shoot length, leaf number, shoot fresh weight, bulb length, and Z-12-20 induced bulb diameter and bulb weight, while K-10-41 (which was one of the top promotors to tomato and legumes) showed the poorest interaction and limited response against onion. Inoculation of Mk-1-25, Z-1-16, Z-12-20, K-10-41, and K-1-29 promoted faba bean and kidney bean growth and biomass yield. Generally, hierarchical procedures (laboratory-tofield work) were conducted to screen these potential biological growth-promotors. In conclusion, 10 competent PSB stains were selected and evaluated under various conditions including plant beneficial traits, substrate, and host crop preference. Consequently, the current strains have shown positive and encouraging plant-benefiting traits and responses on tomato and other crops especially, K-1-29, K-10-27, K-10-41, Z-12-20, Mk-20-7, and Mk-1- 25, can be developed and recommended as biotechnological farm-inputs as potential biofertilizers and PGPR. Besides screening efficient strains, it is critical to apply other supplementals (possible P-substrates) for fruitful symbiotic effectiveness and improved production. Moreover, the future perspective should focus on conducting site-specific field trials, exploring more potential strains, and studying the detailed molecular and physiological symbiotic interactions and responses of the strains to develop competent bioinoculants that are cheap, affordable, ecofriendly, and sustain production especially for smallholder farmers.Item MORPHO-PHYSIOLOGICAL, SEED OIL COMPOSITIONAL, AND MOLECULAR DIVERSITY IN ETHIOPIAN MUSTARD (Brassica carinata A. Braun) GERMPLASM(Hawassa University College of Agriculture, 2025) YIRSSAW DEMEKE AMBAWMorpho-Physiological, Seed Oil Compositional and Molecular Diversity in Ethiopian Mustard (Brassica carinata A.Braun) Germplasm Ethiopian mustard (Brassica carinata A. Braun) is an economically significant selfpollinating oilseed crop valued for its adaptability and diverse industrial applications. Despite its long cultivation history in Ethiopia and the availability of extensive germplasms, a comprehensive understanding of its diversity is lacking, hindering effective breeding strategies. This study aimed to evaluate the morpho-physiological, seed oil, fatty acid, and genetic diversity of 386 collections of B. carinata accessions. Three independent experiments were conducted. First, 386 accessions were phenotypically characterized for 27 agro-morphological and physiological traits for two successive growing seasons in 2022 and 2023 using an augmented block design, replicated twice. Second, seed oil content and fatty acid profiles were analyzed using Nuclear Magnetic Resonance Spectroscopy (NMRS) and Near-Infrared Reflectance Spectroscopy (NIRS), respectively, to identify significant variation (P < 0.05) and promising accessions for industrial and edible oil applications. Third, genetic diversity was assessed in 188 accessions using 3793 DArTSeqgenerated single nucleotide polymorphism (SNP) markers. Phenotypic analyses revealed significant variation (P < 0.001) within accessions for all measured traits indicates substantial phenotypic diversity. Cluster analysis identified four distinct groups with significant inter-cluster divergence, indicating potential for selection. Promising accessions (n = 19) with high seed yields and oil contents were identified. Seed oil content ranged from 37.88% to 46.98%, with high heritability (85-94%) and genetic advance (22.30-59.29%) observed for all traits. Specific accessions (n = 19) were identified as valuable sources of high oil contents and desirable fatty acid profiles. Genetic diversity and population structure analysis using STRUCTURE, principal coordinate analysis (PCoA), and neighbor-joining trees identified two distinct subpopulations with limited genetic differentiation (PhiPT = 0.02) and high gene flow (Nm = 5.74). PCoA indicated low molecular variation, and genetic diversity indices (HE = 0.21, PIC = 0.13) suggested heterozygote deficiency, likely due to restricted cross-fertilization. Accessions did not cluster strictly according to geographical origin, indicating that other factors influence genetic diversity. Higher genetic variation was observed within populations (65.19%) than between populations (44.81%). In conclusion, this study revealed substantial phenotypic variability and medium genetic diversity in the Ethiopian mustard germplasm. These findings highlight the importance of intra-population diversity in breeding programs and suggest that geographic origin is not the primary determinant of genetic diversity. Promising accessions identified for high seed yield, oil content, and desirable fatty acid profiles provide valuable resources for future improvement and commercialization. This study contributes to a more comprehensive understanding of B. carinata diversity, informing efficient conservation and breeding strategies.Item GENETIC CONSIDERATIONS AND MOLECULAR TOOLS FOR FOREST CONSERVATION AND RESTORATION: EMPHASIZING Afrocarpus gracilior (Pilg.) C. N. Page IN SOUTHERN ETHIOPIA(Hawassa University College of Agriculture, 2025) NIGUSSU BEGASHAW ABATELarge-scale tree planting initiatives and ambitious global forest restoration commitments aim to mitigate the impacts of deforestation. Ensuring the long-term success of these efforts requires integrating genetic principles into restoration practices. This study aims to enhance forest conservation and restoration by generating molecular genetic insights and tools, using Afrocarpus gracilior, a native dioecious conifer, as a case study. Specifically, it evaluates the extent to which genetic principles are considered in species selection and seed procurement, investigates the genetic consequences of population fragmentation, assesses inbreeding depression, and develops molecular markers for early sex identification. To achieve these objectives, the study combined a survey of tree nurseries and seed vendors with genetic analyses using DArTseq-generated SNPs to assess genetic diversity and population differentiation. Additionally, in vitro germination and early progeny growth experiments were conducted to evaluate inbreeding depression, while RAPD markers and bulk-segregant analysis were used for sex identification. Assessment of genetic considerations in forest restoration revealed that crucial guidelines are frequently overlooked in species selection and seed procurement, with exotic species dominating while native species remain underrepresented. Seed collection practices often neglect essential measures for preserving genetic diversity, heightening the risk of inbreeding and reducing adaptive potential. Notably, 84% of seed collectors sourced seeds indiscriminately, 87% of nurseries received seeds without passport data, 97% of seed collectors failed to meet the minimum required number of mother trees per collection event, and 88% ignored recommended spacing between selected mother trees. Genetic diversity analysis of A. gracilior populations showed overall low genetic diversity (Hₑ < 0.1), with progeny cohorts exhibiting even lower diversity than adults. Progeny from isolated or few mother trees had the lowest genetic diversity, indicating heightened genetic drift and inbreeding. In contrast, sacred forests and larger remnant patches harbored relatively higher genetic diversity, indicating their importance for in situ conservation. Further assessment of inbreeding depression in fragmented populations revealed significant reductions in progeny fitness, including 53% lower germination rates, 33% reduced acclimatization, 30% and 41% slower growth in diameter and height, respectively, and a 62% increase in leaf scorch. Screening for sex-linked markers identified OPD-18 (5’-GAGAGCCAAC-3’) as a 600 bp male-specific RAPD marker, providing a foundation for early sex determination in A. gracilior. xix The results highlight the critical role of genetic considerations in successful forest restoration, yet current practices often overlook these principles, increasing the risk of inbreeding and loss of adaptive potential. Very low genetic diversity was recorded across all fragmented populations of A. gracilior, with progeny from isolated or few mother trees exhibiting the lowest genetic diversity and significantly reduced fitness. These findings indicate increased genetic erosion, drift, and inbreeding depression, jeopardizing the species’ long-term survival. Recommendations include establishing and enforcing policies that promote the use of native species and genetic standards in seed procurement; sourcing seeds from diverse, larger populations; prioritizing in situ conservation of sacred sites; and further developing robust molecular markers to improve the reliability of early-stage sex identification in the dioecious A. gracilior. Implementing these measures would enhance the long-term success of restoration initiatives, fostering resilient forest ecosystems that support biodiversity conservation and sustainable land management.Item GENETIC DIVERSITY AND TRAIT VARIABLITY IN SOME COFFEE [Coffea arabica L.] GENOTYPES FROM SOUTH ETHIOPIAN(Hawassa University College of Agriculture, 2025) HABTAMU GEBRESELASSIE ADDODIVERSITY OF SOUTH ETHIOPIAN COFFEE [Coffea arabica L.]: AGROMORPHOLOGICAL, BIOCHEMICAL AND MOLECULAR ASPECTS Ethiopia is the center of origin and genetic diversity for Coffea arabica, a crop that plays a central role in the country's economy, culture, and identity. Coffee provides a livelihood to about 25 million people. Despite Ethiopia's favorable climate, soil, and rich genetic resources, national coffee productivity remains low as compared to leading producer and world average. This is partly due to our incomplete knowledge of the wider adaptability and stability, morphological, biochemical, and molecular diversity among characteristics of the plant and the relevant agricultural traits of the plant in general, and South Ethiopian genotypes, in particular. To address this gap, five integrated studies were conducted to evaluate the morphological, biochemical, and molecular diversity of South Ethiopian Arabica coffee genotypes and their relationship to yield and quality performance. These studies aimed to support genetic improvement efforts through better trait characterization, genotype selection, and understanding of genotype × environment interaction (GEI). The first two studies involved the evaluation of 17 genotypes (14 selections and 3 released varieties) at Awada, Wonago, and Shebedino using a randomized complete block design (RCBD). A total of 26 quantitative and 15 qualitative traits were recorded. Results revealed wide phenotypic variation, with Shannon-Weaver diversity indices ranging from 0.22 to 1.12. Significant differences (p<0.05) were found for most qualitative traits, confirming high morphological diversity. Quantitative trait analysis showed significant variation among genotypes, environments, and their interaction for several yield-related traits. Traits such as number of branches, canopy diameter, fruit size, and seed traits exhibited moderate-to-high heritability and genetic advance, indicating potential for improvement via selection. Principal component and cluster analyses grouped the genotypes based on trait similarities, highlighting key contributors to phenotypic diversity. Genotypes AW7705 and AW105 showed superior yield performance (1.37 t/ha), with AW7705 also demonstrating yield stability across environments. The third study analyzed GEI effects using AMMI and GGE-biplot models across 12 environments. The interaction was significant, accounting for 17.5% of total variation. Genotype AW7705 (G16) was identified as both high-yielding and stable, making it a best candidate for xxii variety development in Sidama, Gedeo, and similar agro-ecologies. Conversely, AW105 (G7) was high-yielding but less stable, suggesting potential for targeted environments. These results emphasize the importance of multi-location trials in coffee breeding to capture GEI effects and ensure stability. In the fourth study, 30 genotypes were assessed for physical traits, cup quality, and biochemical composition using RCBd design. Significant variation was found among genotypes and environments almost all the studied traits. Most genotypes scored above 80%, qualifying them as specialty coffee. Biochemical diversity included a wide caffeine range (0.52%–1.53%), offering opportunities to develop low-caffeine varieties. PCA and cluster analyses identified key traits contributing to quality variation, including caffeine, trigonelline, chlorogenic acid, acidity, and flavor. Genotype AW9648 consistently exhibited high-quality scores, making it suitable for both quality breeding and specialty market promotion. The fifth study, the genetic diversity of 40 accessions from Sidama, Guji, Amaro, and Jinka and 10 improved varieties analyzed using four ISSR markers. A total of 74 bands were generated, with private bands and higher polymorphism observed in improved varieties. Genetic diversity indices showed considerable variation among populations, with AMOVA revealing 67% of total variation occurring among populations. Principal coordinate analysis explained 43% of the genetic variation across the first three axes. UPGMA clustering grouped genotypes into four major clusters, often reflecting geographic origin. These findings confirm the rich genetic base in southern Ethiopian coffee and support its use in breeding and conservation programs. The integrated results of these five studies confirm the presence of significant agromorphological, quality, and molecular diversity among South Ethiopian coffee genotypes. Significant GEI effects further underline the need for multi-environment testing to develop widely adaptable and stable varieties. AW7705 and AW105 were high yielder and found to be promising candidate for yield competitive variety development; whereas, AW9648 was identified as superior quality. These results provide a foundation for selection, hybridization, and conservation strategies in Arabica coffee improvement. Future research should include more accessions and high-resolution molecular markers to enhance the precision of genetic diversity studies and guide sustainable crop improvement.Item MORPHO-AGRONOMIC, BIOCHEMICAL AND MOLECULAR CHARACTERIZATION OF BLACK CUMIN (Nigella sativa L.) IN(Hawassa University College of Agriculture, 2025) BASAZINEW DEGU GEBREMEDINBlack cumin (Nigella sativa L.) is a diploid annual flowering plant native to Southern Europe, North Africa, and Southwest Asia. It is cultivated worldwide for its medicinal and aromatic values. Ethiopia is an important center of black cumin genetic diversity. This study aims to characterize Ethiopian black cumin genotypes using morpho-agronomic, biochemical, and molecular markers (SNPs). A total of 64 genotypes, including 8 improved varieties and 56 genotypes from five Ethiopian regions, were characterized for morpho-agronomic and biochemical traits at Debre Zeit and Kulumsa Agricultural Research Centers. Essential oils and fixed oils were extracted, and their compositions analyzed using GC-MS. Molecular characterization was conducted using DArTseq SNP markers to determine genetic variation and population structure of 94 genotypes. Significant differences were found among genotypes for most morpho-agronomic traits, with substantial variability in essential oil compositions. Biochemical analysis revealed significant differences in fixed oil yield, essential oil content, and yield among genotypes. Molecular analysis indicated high levels of genetic diversity within regions and among genotypes, clustering into two distinct groups. The findings reveal significant diversity and variation among Ethiopian black cumin genotypes, offering valuable insights for conservation and breeding programs. The study emphasizes the need for equal attention to all growing areas for effective crop improvement.Item GENETIC DIVERSITY AND DROUGHT TOLERANCE IN ETHIOPIAN DURUM WHEAT (Triticum turgidum subsp. durum) GENOTYPES(Hawassa University College of Agriculture, 2025) BANTEWALU HAILEKIDAN DUKAMODurum wheat (Triticum turgidum subsp. durum), the second most widely cultivated wheat species after common wheat, plays a crucial role in global food security. Ethiopia, recognized as a secondary center of origin and diversity for durum wheat, harbors a broad and unique genetic reservoir well adapted to diverse agroecological conditions. However, productivity remains low due to limited availability of improved, drought-tolerant varieties and insufficient exploitation of genetic diversity. This study aimed to characterize Ethiopian durum wheat landraces, identify drought-tolerant genotypes, and assess their potential for breeding programs by integrating field, greenhouse, and molecular analyses. The research involved three interlinked components. The first was a field experiment conducted at Dera (1500 masl) and Debrezeit (1920 masl), where 104 genotypes (100 landraces and 4 checks) were evaluated under drought-stressed and non-stressed conditions using an augmented design. Thirteen agronomic traits related to yield, phenology, and canopy status were measured across both environments. Additional data were collected from an extended growing season, and drought tolerance was assessed using various indices. ANOVA, correlation, principal component analysis (PCA), and clustering were used to identify promising genotypes and trait associations. The second component involved a greenhouse experiment at Hawassa University using 20 top-performing landraces and four checks selected from the field study. Genotypes were grown under well-watered (70% field capacity) and drought-stressed (35% field capacity) conditions in a completely randomized design with three replications. Data on morphological, physiological, and biochemical traits were collected, including grain yield, relative leaf water content (RLWC), chlorophyll content, canopy temperature, and proline accumulation. Statistical analyses were employed to evaluate drought responses, including correlation, PCA, cluster, and path coefficient analysis. The third component focused on the molecular characterization of 94 genotypes (86 landraces and 8 improved varieties) using SNP markers generated through DArTSeq technology. Genotyping was performed by SEQART AFRICA, producing 17,092 highquality SNPs. Genetic diversity, population structure, and linkage disequilibrium (LD) were analyzed to assess genome-wide variation and support genotype selection. Field results revealed significant genetic variation among landraces across all measured traits. Genotypes ETDW/15DZ23, 34493, ETDW/15DZ4, 34522, MCD3-14, 34217, and 31831 demonstrated superior grain yield under stress and non-stressed conditions. High heritability (h²b = 32.84–97.87) and genetic advance estimates for traits such as spike length, kernel number per spike, and tiller number indicated strong potential for selection. Drought indices, including stress tolerance index (STI), mean productivity (MP), and yield stability index (YSI), identified ETDW/15DZ23, 34493, and ETDW/15DZ4 as topperforming, drought-resilient genotypes. Strong positive correlations (r = 0.88) between grain yields under stressed and non-stressed conditions further confirmed their stability and xviii adaptability. The greenhouse experiment revealed significant effects of genotype, treatment, and their interaction (P<0.001) for most traits. Under stress, grain yield decreased by up to 68%, RLWC dropped from 93.07% to 44.91%, and proline content increased markedly, indicating drought response. Cluster analysis grouped genotypes based on resilience, with one cluster showing the highest yield (5.99 t/ha) under well-watered conditions, while another showed superior RLWC (65.80%) and yield (2.90 t/ha) under stress. Path analysis underscored the importance of RLWC, proline, and chlorophyll content in drought tolerance. Molecular analysis revealed 14,136 informative SNPs distributed across the A and B genomes, with chromosome 2B having the highest marker density. Population structure analysis indicated considerable variation within and among landraces, with AMOVA showing 51.75% of genetic variation within populations and 48.15% within individuals. The genome-wide LD decay threshold was 4.58 Mbp, with the highest LD values on chromosome 4B. Average polymorphic information content (PIC) and gene diversity values were moderate, indicating a diverse and informative marker set for future breeding applications. This study highlights the significant phenotypic and genotypic diversity within Ethiopian durum wheat and identifies promising genotypes for drought tolerance and yield stability. The integration of field performance, physiological traits, and genomic data provides a robust platform for developing improved durum wheat cultivars. These findings support the use of landraces in breeding programs targeting climate resilience and food security. Future work should focus on multi-environment trials, genome-wide association studies (GWAS), and marker-assisted selection to accelerate genetic improvement and enhance drought tolerance in Ethiopian durum wheat