4.5.1 Phenological and morpho-qualitative traits
As indicated in Figure 4.1, the test genotypes were clustered into three clusters, two of them divided into various subgroups. The largest cluster, Cluster I, composed of five sub-clusters containing 32 genotypes within which were 15, 11 and 6 genotypes from South Wello, North Wello and Oromiya Special Zones, respectively. This Cluster mainly consisted of high yielder sorghum genotypes selected from the previous chapter (Chapter 3) such as E-72457, E-72438, E-72435 and E-72449.
The first sub-cluster of Cluster I, contained five genotypes representing the three collection sites with two genotype except South Wello. The second sub-cluster contained two genotypes only from South Wello. The third, fourth and fifth sub-clusters contained 4, 8, and 13 genotypes, respectively. Cluster II, the second largest cluster comprised four sub-clusters. This cluster contained argonomically better performing genotypes such as E-206214 and E-75460. The four sub-clusters of Cluster II contained 3, 2, 3 and 5 genotypes, respectively. Cluster III comprised of five genotypes of which 80% of them were collected from North Wello. In general, the phenological and morpho-qualitative traits did not distinctly group genotypes according to their geographic origin/area of collection, materials from the different area tended to cluster together within each group, indicating that their geographic origin didn‟t play a role in the selection of germplasm used.
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Figure 4.1 Morphological diversity analysis of 50 medium maturing sorghum genotypes based on Gower‟s distance matrics
Red : South Wello accessions Blue : North Wello accessions Green: Oromiya Special accessions
102 4.5.2 Polymorphism of SSR markers
Summary on genetic parameters are presented in Table 4.2. Numbers, PIC and heterozygosity fragments amplified by each SSR marker are listed in Table 4.2. Results showed that 100% of the markers used in this study were polymorphic and a total of 279 alleles were generated. Majority of the SSRs generated 2 to 9 alleles; six markers generated 12 to 14 alleles and the remaining three markers generated 11 to 18 alleles with a mean of 7.15 alleles per locus. Major allele frequency ranged from 0.174 to 0.880 with a mean of 0.48. The observed allele sizes ranged from 93 (msbCIR238) to 312 base pairs (gpsb123). The two accessions with the lowest PIC values in the loci of mSbCIR246 and mSbCIR300 were E-72437 and E-72444, respectively. The accessions with the highest PIC values in the loci of Xgap206 and Xtxp265 were E-20006 and E-75454, respectively.
Gene diversity is defined as the probability that two randomly chosen alleles from the population are different. It varied from 0.253 (E-75458) to 0.894 (E-210972), with a mean of 0.64. Heterozygosity values of the 39 polymorphic SSR markers ranged from 0.00 (E-72438, E-72444) to 0.98 (E-72439) with a mean of 0.24, suggesting that each detected a single genetic locus and that each of the sorghum genotypes used was reasonably inbred and homogeneous.
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Table 4.2 Genetic parameters estimated using 39 polymorphic SSR loci screened across 50 sorghum genotypes
Marker Motif type MAF NA Size AR GD HE PIC
gpsb067 (GT)10 0.38 7 188-202 1.00 0.74 0.00 0.71
gpsb123 (CA)7+(GA)5 0.38 4 305-312 0 .96 0.67 0.00 0.60
mSbCIR223 (AC)6 0.74 4 127-138 0.94 0.42 0.17 0.38
mSbCIR238 (AC)26 0.28 12 93-135 0.90 0.84 0.20 0.82
mSbCIR240 (TG)9 0.28 15 126-187 0.98 0.86 0.31 0.84
mSbCIR246 (CA)7.5 0.84 2 113-119 0.98 0.27 0.12 0.24
mSbCIR248 (GT)7.5 0.70 3 113-115 0.98 0.45 0.16 0.39
mSbCIR262 (CATG)3.25 0.54 3 231-235 0.94 0.58 0.11 0.51
mSbCIR276 (AC)9 0.58 3 246-250 0.96 0.55 0.19 0.47
mSbCIR283 (CT)8 (GT)8.5 0.37 12 133-166 1.00 0.79 0.42 0.77
mSbCIR286 (AC)9 0.40 8 127-156 0.96 0.76 0.31 0.73
mSbCIR300 (GT)9 0.86 5 122-129 1.00 0.25 0.20 0.24
mSbCIR306 (GT)7 0.70 3 140-144 0.98 0.46 0.02 0.41
mSbCIR329 (AC)8.5 0.38 7 128-134 0.98 0.74 0.27 0.70
Xgap72 (AG)16 0.31 7 205-217 0.98 0.78 0.39 0.75
Xgap206 (AC)13/(AG)20 0.17 18 120-140 0.98 0.89 0.63 0.89
Xgap84 (AG)14 0.44 13 203-225 0.98 0.77 0.22 0.75
Xisep0310 (CCAAT)4 0.52 4 195-227 1.00 0.59 0.24 0.51
SbAGB02 (AG)35 0.50 11 113-145 0.98 0.70 0.27 0.68
Xcup02 (GCA)6 0.39 5 216-218 1.00 0.68 0.26 0.61
Xcup14 (AG)10 0.45 3 226-230 0.96 0.59 0.35 0.51
Xcup53 (TTTA)5 0.42 5 201-219 0.98 0.67 0.18 0.60
Xcup61 (CAG)7 0.54 2 216-218 0.96 0.50 0.13 0.37
Xcup63 (GGATGC)4 0.77 2 158-164 0.98 0.36 0.06 0.29
Xtxp010 (CT)14 0.42 7 155-170 1.00 0.74 0.20 0.71
Xtxp012 (CT)22 0.34 13 184-224 0.98 0.82 0.37 0.80
Xtxp015 (TC)16 0.46 8 233-247 0.70 0.73 0.31 0.70
Xtxp021 (AG)18 0.54 8 185-200 1.00 0.67 0.10 0.65
Xtxp040 (GGA)7 0.52 4 150-160 0.96 0.57 0.15 0.48
Xtxp057 (GT)21 0.41 9 260-283 0.98 0.76 0.20 0.73
Xtxp114 (AGG)8 0.41 4 235-253 0.92 0.64 0.98 0.57
Xtxp136 (GCA)5 0.71 3 255-258 0.96 0.42 0.21 0.34
Xtxp141 (GA)23 0.30 9 169-189 0.98 0.81 0.22 0.78
Xtxp145 (AG)22 0.43 14 231-263 0.96 0.78 0.52 0.77
Xtxp265 (GAA)19 0.24 14 224-273 0.98 0.87 0.29 0.86
Xtxp273 (TTG)20 0.50 9 231-256 0.96 0.67 0.23 0.63
Xtxp278 (TTG)12 0.85 3 264-276 0.96 0.26 0.13 0.24
Xtxp320 (AAG)20 0.33 8 281-305 0.92 0.78 0.09 0.74
Xtxp321 GT)4+(AT)6+(CT)2 0.50 8 212-226 1.00 0.69 0.14 0.65
Mean 0.48 7.15 188-205 0.96 0.64 0.24 0.60
MAF = major alleles frequency; NA=total number of alleles; AR = allelic richness; GD = gene diversity;
HE=heterozygocity and PIC= polymorphic information content
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Group III
Group II
Group I 4.5.3 Genetic diversity revealed through cluster analysis
The cluster analysis based on Neighbor-Joining method grouped and displayed the 50 sorghum genotypes into three main clusters (Figure 4.2). The dendrogram provided a basic overview of diversity analysis among sorghum genotypes. No clustering according to area of collection could be observed in the dendrogram. Group I could be considered as an outlier as it contained only one genotype (E-212636). Group II composed of five sub-groups and mainly contained drought tolerant and high yielding genotypes such as E-72457, E-72438, E-206214, E-75460, E-72449 and E-72435.
Genotypes in Group III exhibited moderate yielding potential under drought condition when compared to Group II genotypes.
Figure 4.2 Dendrogram illustrating the genetic diversity of 50 sorghum genotypes with 39 SSR markers using “Neighbor Joining” method
Red : South Wello Blue : North Wello Green: Oromiya Special
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4.5.4 Genetic diversity through principal component analysis
Factorial analysis of 50 sorghum genotypes was performed based on Rogers-Tanimoto dissimilarity index. The two first principal components (PC) accounted for 38.29% of the total variation with PC1 accounting to 25.08% and PC2 to 13.21%. Three major sets are displayed (Figure 4.3). The samples used in the present study were more diverse in terms of their genetic distinctiveness than that of their area of collection. Set I contained 4, 3 and 5 genotypes collected from North Wello, South Wello and Oromiya Special Zones, respectively. Genotype E-72437, better performing genotype under drought condition was also presented in Set-I. Twenty-two genotypes were presented in Set-II. This set comprised of mainly drought tolerant and high yielding genotypes (E-72457, E75457, E-20006, E- 206100, E-206214, E-72435, E-75458 and E-75460). Set-III composed of 16 genotypes of which four were drought tolerant high yielding genotypes (E72449, E-200070, E-72444 and E-75453).
Red : South Wello Blue : North Wello Green: Oromiya Special
25.08%
13.21%
Set-I Set-II
Set-III
Figure 4.3 B-plot of PC1 (25.58%) and PC2 (13.21%) from principal component analysis illustrating the grouping
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K 4.5.5 Structure Analysis
The natural logarithm of the probability of the data, proportional to the posterior probability of K, showed clear peak for K = 3 and hence the determination of the true number of populations (K) was simple. The rate of change of Napierian logarithm probability relative to the standard deviation (ΔK) as described by Evanno et al. (2005) was estimated. The results showed the highest peak at K = 3 indicating the presence of three major clusters: collections from North Wello, South Wello and Oromiya Special Zones. Structure is considered to be uniform when more than 80% of the accessions in one group have more than 80% of membership in this group.
(a) Mean L (K) over 20 runs for each K value; (b) Maximum delta K (△K) values were used to determine the uppermost level of structure for K ranging from 2 to 5, here K is three and three clusters.
Red zone: South Wello; Green zone: Oromiya Special; Blue zone: North Wello.
0 5 10 15 20
1 2 3 4 5
1 2 3 4 5
Delta K
a b
K=3
Figure 4.4 Three groups of 50 sorghum genotypes inferred from STRUCTURE analysis and the description of detected the optimum value of K by using graphical method
107 4.5.6 Analysis of Molecular Variance
To assess distinctiveness among and within the sub-populations, an Analysis of Molecular Variance was carried out. The AMOVA analysis (Table 4.3) attributes 0.91% of genetic variation to the differentiation among populations, 61.85% due to differentiation among individuals and 37.24% was due to difference within individuals in a population.
Table 4.3 Results of molecular variance analysis with 50 sorghum genotypes
Sources of variation df Sum of
Squares
Variance components
Percentage of variation
Among population 2 39.246 0.09358 0.91
Among individuals within populations 47 779.964 6.37749 61.85
Within individuals 50 192 3.84000 37.24
Total 99 1011.21 10.31106
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