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(Phaseolus Vulgaris L.) GERMPLASM from ETHIOPIA a THESIS SUBMITTED to the SCHOOL of GRADUATE STUDIES COLLEGE of NATURAL SCIENCES ADDIS ABABA UNIVERSITY

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(Phaseolus Vulgaris L.) GERMPLASM from ETHIOPIA a THESIS SUBMITTED to the SCHOOL of GRADUATE STUDIES COLLEGE of NATURAL SCIENCES ADDIS ABABA UNIVERSITY GENETIC DIVERSITY AND POPULATION STRUCTURE OF COMMON BEAN (Phaseolus vulgaris L.) GERMPLASM FROM ETHIOPIA A THESIS SUBMITTED TO THE SCHOOL OF GRADUATE STUDIES COLLEGE OF NATURAL SCIENCES ADDIS ABABA UNIVERSITY BY ZELALEM FISSEHA GEBREEGZIABHER IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY (PhD) IN BIOLOGY (APPLIED GENETICS) ADDIS ABABA JULY 2015 Addis Ababa University School of Graduate Studies This is to certify that the thesis prepared by Zelalem Fisseha Gebreegziabher, entitled: ‗Genetic Diversity and Population Structure of Common Bean (Phaseolus vulgaris L.) Germplasm from Ethiopia‘ and submitted in fulfillment for the requirements for the Degree of Doctor of Philosophy (Biology: Applied Genetics) complies with the regulations of the University and meets the accepted standards with respect to the originality and quality. Signed by the Examining Committee: Examiner: ------------------------- Signature___________ Date__________ Examiner:------------------------- Signature___________ Date__________ Advisor: Dr. Kassahun Tesfaye Signature___________ Date__________ Advisor: Prof. Paul Gepts Signature___________ Date_________ Advisor: Dr. Kifle Dagne Signature ___________ Date_________ _______________________________________________ Chair of Department or Graduate Program Coordinator Genetic Diversity and Population Structure of Common Bean (Phaseolus vulgaris L.) Germplasm from Ethiopia Zelalem Fisseha Gebreegziabher (MSc) PhD Thesis Addis Ababa University College of Natural Sciences Department of Microbial, Cellular, and Molecular Biology 2015 Abstract The common bean is one of the most important diet components and cash crops in Ethiopia and Africa. However, despite having such major benefits, the production and productivity of the crop has been highly constrained by: inadequacy of improved varieties; complex myriads of biotic and abiotic constraints, and narrow genetic base of germplasm used in breeding. The multiple centers of domestication accompanied by heterogenous farming practices adopted by farmers, ever since its introduction to Ethiopia, and recent variety development projects have resulted in a range of morphologically diverse landraces via the preservation and exploitation of useful alleles. To this end, assessing the morphological and molecular diversity; and population structure of common bean landrace accessions in Ethiopia is sine qua non vis-à- vis establishing an efficient breeding and conservation scheme, nationally. Genetic diversity and population structure of 125 common bean accessions were studied using agro- morphological and SSR DNA markers. The morphological diversity assessment was conducted in the main rainy season of 2013 at Melkassa Agricultural Research Center, main research station, Ethiopia. The molecular diversity study was conducted from August 2012 to February 2013 at the lab of the BecA-ILRI hub, Nairobi, Kenya, using 24 fluorescent SSR markers. Higher and significant genetic variability among accessions was evidenced, with respect to 8 quantitative and 11 qualitative agro-morphological traits. On the other hand, results of the association analyses revealed that 100-seed weight and seed diameter had positively-significant correlation and higher positive direct effects on the seed yield of the common bean accessions. Hence, these two traits, 100-seed weight and seed diameter, were recommended, as important traits to be used in the indirect selection of high- yielding common bean cultivars, in conjunction with seed yield. On the other hand, results of the morphological (phenotypic) diversity analyses revealed that both the Tocher and Neighbor-joining clustering methods identified similar five clusters with almost identical members in each. Of these, three and two clusters were predominated by Andean and Mesoamerican accessions, respectively. However, a comparable proportion of accessions had intermediate features between the two gene pools, suggesting the significant presence of inter-gene pool introgressions. The Mahalanobis (D2) analysis of the five clusters indicated mostly significant differences between all the combinations of cluster pairs. This indicated there may be a great opportunity to obtain transgressive segregants and maximum heterosis in future common bean breeding programs. With regards to the molecular genetic diversity using SSR markers among the common bean accessions sampled from different geographical regions, it indicated that most allelic parameters had values comparable to previous similar study results. Specifically, the geographical populations, ‗Amhara‘ and SNNP had the highest number of effective alleles; Shannon‘s diversity index (I); and heterozygosity values. Furthermore, results of Analysis of Molecular Variance (AMoVA) indicated that the greater proportion of the genetic variation was explained between individuals from different populations (58%) and between individuals within the same population (40%). iii In comparison, only 2% of the genetic variation was due the variation among the populations themselves. In addition, the calculated Fst value was small (Fst=0.015), associated with a high gene flow value (Nm=16.282), indicating lower differentiation of the populations, which, in turn, implied significant exchange of planting materials among farmers in the studied populations. Finally, Neighbor-Joining (NJ) cluster and Principal Coordinate analyses (PCoA) revealed that accessions from different collection sites tended to cluster together, probably owing to the high gene flow among the populations. Moreover, five groups of clusters were identified in the NJ dendrogram. In addition to the aforementioned, analysis of structure of genetic diversity of the Ethiopian common bean accessions was undertaken with respect to the Andean and Mesoamerican gene pools of origin. Results indicated at cluster K=2, accessions separated into the Mesoamerican and Andean gene pools. In view of this, the number of accessions from the Mesoamerican gene pool was higher than that of their Andean counterparts. Moreover, STRUCTURE identified K=5, as the optimum cluster number. At K=5, five clusters: three from the Andean gene pool; and two from the Mesoamerican gene pool were identified. On the other hand, based on calculations of hybrid/non-hybrid accessions (from membership coefficient values), 72 out of the 125 accessions were found to be inter-gene pool introgressions. Moreover, results of NJ cluster analysis and PCOA done with the remaining 55 non-hybrid accessions identified at K=5 revealed common bean genetic diversity in Ethiopia, as organized into the Andean and Mesoamerican gene pools. This was exhibited by the clustering of accessions with either of the Andean or Mesoamerican control genotypes. The other peculiar event was mixed membership of Andean/Mesoamerican accessions in some of the clusters. Finally, yet equally importantly, Principal Component Analysis (PCA); stepwise discriminant and canonical correlation analyses; and data recorded in some agro-morphological traits distinguishing the Andean and Mesoamerican gene pools were used in conjunction, in order to determine the identities of the five cluster groups, identified at K=5 of the molecular structure analysis into the ecogeographic races of the Andean or Mesoamerican gene pools. To this end, PCA, done with the 125 accessions identified at K=2 of the molecular analysis, revealed that Mesoamerican and Andean groups of accessions separated along the first Principal Component (PC) axis. Nonetheless, several accessions occupied intermediate positions between the Andean and Mesoamerican control genotypes, which supported the presence of inter-gene pool introgressions detected in both the morphological and molecular analyses. Furthermore, stepwise discriminant and canonical correlation analyses, using the non- hybrid accessions at the molecular analysis STRUCTURE preset K=5, displayed that there was fair level of separation among the Andean and Mesoamerican cluster groups, with some overlaps. Ultimately, the identities of the five cluster groups identified in the molecular structure analysis were determined. In line with this, it was concluded that Ethiopian common bean accessions from the Andean gene pool had a broader base, belonging to ecogeographic races in the gene pool. In other words, the three Andean cluster groups were found to be from two of the three races in the Andean gene pool-‗Nueva Granada‘ and ‗Peru‘, whereas the Mesoamerican accessions had a narrower genetic base, belonging only to race ‗Mesoamerica‘ in the same gene pool. In summary, results of the present study showed that common bean landrace genotypes in Ethiopia had adequate genetic diversity, organized in to the Andean and Mesoamerican gene pools. Importantly, this rich genetic diversity should be harnessed in order to maximize genetic variability in future common bean improvement programs in Ethiopia. On the other hand, the significant presence of inter-gene pool introgressions, by itself, can be used as opportunity, for such introgressions were previously reported to be rich in many adaptation and nutritional genes. This, in turn, can be used as an advantage in prospective common bean breeding works. On the other hand, the higher/significant variations among accessions vis- à-vis qualitative and quantitative agro-morphological traits should be directed towards broadening genetic variability and developing transgressive genotypes, excelling their parents in relation to these traits. Key words: AMoVA,
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