Molecular Genetic Diversity
In this study, the overall of 122.43 alleles were detected across the genome of 81 genotypes as revealed by 14 microsatellite loci. The number of alleles noticed ranged from 6.11 (CNLTS5) to 12.4 (CNLTS463) with a mean number of 8.75 alleles per locus through the genome of the entire populations. On the other hand, from all loci there was high inbreeding coefficient (f>0.5) except loci CNLTS 463 (0.3021) (Fig 1). In addition, the heterozygosity of the studied materials ranged from 0.to 0.69 with overall mean of 0.1457.
Relatively the markers CNLTS463, CNLTS22, CNLTS27, and CNLTS20 have high heterozygosity and Shannon´s index. The germplasm showed the highest genetic differentiation (Fst = 0.27) and the lowest value of gene flow (Nm =0.69) from CNLTS458 (Fig 1).
Four of the microsatellite loci (CNLTS22, CNLTS25, CNLTS27 and CNLTS463) did not significantly deviated from the HW-equilibrium over the entire populations.And the overall polymorphic information, from all the microsatellite loci utilized was found profoundly instructive (0.96-0.98) (Fig.1)
Analysis of Molecular Variance
The analysis of molecular variance (AMOVA) showed that 1% of the total genetic variation and low genetic differentiation (FST = 0.014) for the variation among populations, 86% among genotypes within populations and 13% variation among genotypes across all populations (Table 4). This study confirmed genetic differentiation (FIS =0.870) of genotypes within population and (FIT=0.872) among genotypes across all population (From 81 genotypes=one large grouped) with a small value of overall genetic differentiation coefficient (p = 0.001) (Table 4).
On the other side, released varieties and core germplasm collection of overall genetic differentiation coefficient among the population (p = 0.832), Fst=-0.004 and gene flow (Nm: 0) were observed (Table 4). A total genetic variation of 87% and 13% were explained by within populations and among genotypes across all populations respectively (Table 4).
Table 4: AMOVA results of 81 tef genotypes evaluated using 14 SSRs
Source
|
df
|
SS
|
MS
|
Est. Var.
|
%
|
Fstat
|
P-value
|
Among Pops
|
9
|
114.3
|
14.29
|
0.199
|
1%
|
Fst=0.014
|
0.002
|
Among Indiv
|
71
|
909.07
|
12.63
|
5.885
|
86%
|
Fis=0.87
|
0.001
|
Within Indiv
|
81
|
71.00
|
0.85
|
0.88
|
13%
|
Fit=0.87
|
0.001
|
Total
|
161
|
1094.37
|
|
6.85
|
100%
|
|
|
Gene flow among population (Nm= 17.02)
|
Among Pops
|
1
|
12.01
|
12.01
|
0.00
|
0%
|
Fst=0.004
|
0.832
|
Among Indiv
|
74
|
944.61
|
12.775
|
5.92
|
87%
|
Fis=0.865
|
0.001
|
Within Indiv
|
76
|
70.00
|
0.92
|
0.92
|
13%
|
Fit=0.865
|
0.001
|
Total
|
151
|
1026.62
|
|
6.84
|
100%
|
|
|
Gene flow among core germplasm and released varieties (Nm=0)
|
Where, df = degree of freedom, SS=sum of squares, MS=mean squares and Nm=gene flow, FST=inbreeding coefficient among population, FIS=inbreeding coefficient with population, FIT=inbreeding coefficient of within groups (across all population=one)
Genetic Distance and Gene Flow Among Populations
The pairwise population genetic distance and gene flow for both core germplasm and released varieties of tef genotypes are presented in (Table 5). The pairwise Fst standard genetic differentiation among populations varied from (0.000-0.036) which means 0,000 in between Wollo and Wellega, Gojam and Wollo ,Gojam and Wellega, Wollo and released varieties, Wellega and released varieties and 0.036 shows in between Arsi and Wellega.
Table 5: Fst values (below diagonal) and gene flow (above the diagonal) among tef genotypes originating from nine sources
Origins
|
JIM
|
TG
|
GOJ
|
WO
|
WEL
|
E. S
|
W. S
|
AR
|
R. Var
|
JIM
|
0.000
|
7.435
|
29.00
|
63.830
|
20.547
|
32.239
|
17.402
|
8.045
|
99.402
|
TG
|
0.027
|
0.000
|
24.846
|
12.423
|
7.022
|
10.686
|
8.448
|
7.042
|
13.039
|
GOJ
|
0.009
|
0.010
|
0.000
|
0.000
|
0.000
|
31.000
|
8.674
|
8.161
|
100.00
|
WO
|
0.005
|
0.020
|
0.000
|
0.000
|
0.000
|
20,000
|
11.691
|
7.393
|
0.000
|
WEL
|
0.013
|
0.034
|
0.000
|
0.000
|
0.000
|
29.000
|
12.631
|
6.605
|
0.000
|
ES
|
0.008
|
0.027
|
0.008
|
0.012
|
0.010
|
0.000
|
21.196
|
10.788
|
73.356
|
WS
|
0.014
|
0.020
|
0.026
|
0.019
|
0.020
|
0.020
|
0.000
|
79.772
|
20.52
|
AR
|
0.029
|
0.031
|
0.030
|
0.035
|
0.036
|
0.030
|
0.003
|
0.000
|
13.073
|
RV
|
0.003
|
0.020
|
0.002
|
0.000
|
0.000
|
0.003
|
0.012
|
0.022
|
0.000
|
JIM=Jimma, TG =Tigray, GOJ= Gojam, WO= Wollo, WEL=Wellega, ES= East Shoa, WS= West Shoa, AR=Arsi and R. Var=Released Variety
Allelic Diversity Among Populations
The number of observed alleles (Na) was higher for accessions collected from East Shoa, West Shoa and Wollo with values of 18.857, 12.57 and 10.714, respectively (Fig 2). Similarly, the germplasms from East Shoa, West Shoa and Wollo in that order have the highest numbers of effective alleles with 16.756, 11.592 and 9.732 and Shannon’s information indices with 2.855, 2.436 and2.306, and numbers of private alleles with 13.071, 8.500 and6.357. (Fig 2)
Small number of observed heterozygosity were observed for all of the populations. However, relatively high number of heterozygosity were noted for the lines from Jimma (0.193), Released varieties (0.19) Wellega (0.16) Tigray (1.5) and E.shoa (1.5) and the (Fig 2). And all core germplasm tef collection of populations used in this study showed similar value in percentage of polymorphism (100), except the materials originated from Arsi. (Fig 2)
Population Structure Analysis
Cluster Analysis
Cluster analysis based on UPGMA method clustered the 81 tef genotypes into three major groups consisting of 52%, 44% and 4% of the genotypes in clusters 1, 2 and 3, respectively (Figure.1A). It was observed that the genotypes in cluster 1 comprised in total of genotypes per population:16/25 from East Shoa, 4/7 from released varieties, 6/11 from Wollo, 3/4 from Wellega, 3/4 from Gojam, 1/5 from Arsi, 1/16 from Jimma, 3/4 from Tigray and 5/15 from West Shoa.
The genotypes in second cluster included 10 from West Shoa, 4 from Arsi, 7 from East Shoa, 2 from Wollo, 3 from the 7 released varieties 1 of the 4 from Tigray, and 4 from Jimma. On the other hand, only 3 of the germplasm lines from Wollo are grouped in cluster 3
Principal Coordinate Analysis
PCoA showed that the first three coordinates count in anti clockwise direction explained 93.34% of the gross genetic variation. The gross variation explained was 65.45%, 22.93% and 4.96% by the first, second, and third principal coordinates, respectively (Fig. 3).
Population Structure
The most function of structure in population hereditary qualities is the recognizable proof of hereditarily homogeneous bunches of individuals based on modular value of ΔK. These are determined by Bayesian algorithm implemented in the software STRUCTURE based on true number of cluster (K) suggested by Evanno et al. (2005). In this study the algorithm allows estimating the true number of clusters at (K=4) (Figure 3a) in the sample of 81 individuals included in the study. Which means that these samples (81 individuals) come from four ancestors. But the bar plot appeared that there is no clear geographic origin-based organizing of population this is what indicates there is admixture of tef genes.