4.

4 Genetic Diversity
The binary data matrix generated by the amplified fragments of the 43 A. hypogaea accessions in
the ISSR-PCR analyses was used for the computations of Nei’s genetic distances and Shanon’s
indices for every pairwise comparison of the accessions for the analysis of ISSR data. The Nei’s
gene diversity (H) ranged from 0.11 between GOG-6 (Gursum/Oda-3) and GOB-10
(Babile/Medigana-1) to 0.38 between GOBG-1 (Bale/Ginir) and GOB-14 (Babile/ Tofic-2) with
an average value of 0.245 (Table 3). The Shannon’s indices (I) ranged from 0.24 in GOB-7
(Babile/Ifa-gendi-1) and GOB-16 (Babile/Gende) to 0.41 in GOBG-1 (Bale/Ginir) and GOB-14
(Babile/Tofic-2) with an average value of 0.325. Total gene diversity (Ht) was 0.231, indicating
the existence of low to moderate genetic diversity found in the entire accessions. In the case of
binary data the maximum value of the Ht indices equals 0·5, revealing maximum genetic
diversity (Nei, 1978).

The different levels of genetic diversity for A. hypogaea may be explained, in part by the use of
different ISSR molecular markers and in part by the different sampling strategies, such as the
geographic distance between sampled accessions. The results revealed that GOG-6
(Gursum/Oda-3) and GOB-10 (Babile/Medigana-1) accessions were closely related, having the
lowest genetic distance of 0.11, which could be attributed to the levels of management
implemented and focus on limited accessions disseminated by different stakeholders.
Additionally, the observed low genetic diversity between accessions could be associated with
usage of large sample size representing a few parts of A. hypogaea growing regions
(Grsum/Babile). Further, this low genetic diversity might be explained by the lack in access of
wild relatives, existence of uniformity or monoculture possibly due to vegetative propagation
prevailed in the area, homogeneity due to single or few seed sources during its introduction to
these area.

The low geneticdiversity observed in this study on A. hypogaea indicate that there is high gene flow
through either seeds or seedlings exchange between this different very closely situated areas. The one
reason for the present study, could be due to floral biology and pollination nature of A. hypogaea. The
gene flow could be enhanced via birds, insects, wild and domestic animals by facilitating exchange of
pollens and seeds.
Moreover, insect pollinators including bees and birds might be attracted due to its colorful flower that
facilitate gene flow among populations. Not only the pollination nature of safflower, but also the
existence of seed exchange between nearby districts among local farmers the objective of improving
productivity of A. hypogaea could also be the additional factor.

High genetic exchange or gene flows, which actually have a more homogenizing effect on the genetic
variation among populations by the dispersal of the seeds, can likely explain higher within population
genetic variation. Some seeds may be harvested as weeds together with those of crops and distributed
with the crop seeds via market channels.

Moreover, the presence of long distance marketing of safflower within and among different regions by
farmers and local traders have their own effect on the observed variation of cultivated of safflower.

The highest genetic distance 0.3154 ± 0.209 values belonged to GOBG-1 (Bale/Ginir) and GOB-
14 (Babile/Tofic-2) accessions, which were the most distant accessions and it would
be expected due to its geographical isolation which can be explained by topographic barriers
preventing the dispersal of seeds and pollen..

In addition, McDonald et al. (2003) reported a positive relationship between geographic distance and
genetic divergence among populations in E. cladocalyx. The genetic differentiation among populations
could be the result of an adaptation to environmental gradients. In particular, E. cladocalyx is distributed
across differential environmental conditions with respect to soil composition, altitude and annual
rainfall (MCDONALD et al., 2003; CLARKE et al., 2009), which could drive the acquisition of local
adaptations. Moreover, the environmental conditions could influence flowering times and restrict the
gene flow through pollination and seeds dispersion among populations. In fact, according to the findings
of Contreras-Soto et al. (2011), Mora et al. (2009) and Cané-Retamales et al. (2011), there are significant
differences in flowering times among natural provenances of E. cladocalyx.

These values can be employed in a breeding program such that the accessions with the lowest
genetic divergence could be selected as parents to improve the A. hypogaea accessions.
In contrary to the above, as Balemi (2007) reported it could also be speculated that the high genetic
diversity observed within populations of the Coffea arabica might be due to preferential adaptive gene
complexes adapted to environmental changes being evolved during long evolutionary period in a given
region. In this case, Coffea arabica population uses selfing as mechanisms to prevent influx of the gene
from another portion of the populations that might reduce diversity through disrupting adaptive
genes Lowe et al. (2004).060

Non-genetic factors, such as physical barriers and the geographical isolation of

populations could, block to gene flow among populations

This could be due to high seed exchange among different regions and markets which could lead to
intermix of populations between regions. Unlike other landraces of cultivated plants, Lepidiumsativum
in Ethiopia is not restricted to a given area rather it is wildly exchanged among local community and
markets. This shows that there is very high gene flow between populations and regions.

Hamrick and Godt (1989), stated that, the biological characteristics of a species, population structure,
mating system and multiple evolutionary process (genetic drift: bottleneck and founder effect) are
determine the level and pattern of genetic variation observed in natural population.

The higher within populations’ genetic diversity might be accounted to two contrary reasons. Coffea
arabica is affected by multiple evolutionary forces which operate within historical and biological context
of the plant species. This includes the mating types, gene flow, mode of reproduction and natural
selection etc. Hamrik and Godt (1989). For this reason, it could be speculated from this
result that Coffea arabica might have mixed mating system (partial out-crossing by pollen and seed and
partial selfing) for which some extent of gene flow is expected as reported by Meyer (1965) which could
result in high within population genetic diversity (Loveless and Hamrik 1 984; Aga et al., 2003; Tesfaye,
2006; Oljira, 2006; Balemi, 2007).

Breeding systems of Eucalyptus spp. show a high probability of self-pollination (HARDNER; POTTS, 1995).
In fact, E. cladocalyx has post-zygotic self-incompatibility systems (ELLIS; SEDGLEY, 1992). Nevertheless,
McDonald et al. (2003) reported a high level of inbreeding and low genetic diversity within populations,
which is consistent with the present study. Therefore, other factors are responsible for the low genetic
diversity of E. cladocalyx. The genetic structure can break HardyWeinberg equilibrium due to genetic
and nongenetic factors.. Migration among populations tends to homogenize their allele frequencies, and
therefore, the absence of significant
genetic differentiation among populations indicates that the populations maintain continuous gene
flow. These three sets of authors reported that Cowell hasthe higher flowering intensity and precocity,
and trees from Flinders Chase were the worst for both traits. In the present study, Cowell and Flinders
Chase had the highest genetic differentiation. The provenances of Eyre Peninsula were most genetically
separated from the Kangaroo Island provenance.424

(Table 5). High genetic variation within populations indicated that high genetic dissimilarities among the
individual plants sampled from a single population. The vast majority of diversity studies across
members of the Trifolieae have shown a generally high level of diversity within populations, even among
other inbreeding species such as T. subterraneum (Pecetti and Piano, 2002; Piluzza et al., 2005). The
AMOVA results obtained in the present study do not contradict with the above findings. It is a prevalent
view that self-pollinated species maintain higher genetic variation among populations than within
populations. Though T. quartinianum is self-pollinated plant, higher within populations genetic variation
than among populations was obtained in the present study, contrary to this prevalent view. Moreover,
the pods and mature calyx of T. quartinianum tend to have a coarse surface and points which may
attach to passing animals and be transported to other places.6943-

AMOVA analysis resulted in high genetic diversity within population (94%) and very low genetic diversity
among population (6%). Jiang et al. (2012) who studied on the genetic diversity of
Chimonanthusgrammatus populations by using ISSR marker showed that there was 73.6% within
population variation whereas the rest 26.4% was due to among population variation. As compared to
the present study, there was less gene flow. Jiang et al., (2012) recommended that gene flow, genetic
drift and evolutionary history might have important influence on genetic structure and diversity of a
given population.

AMOVA analysis indicated that majority (>95%) of genetic variation observed in germplasm is due to
variation in individuals instead of from a specific group. In general, genotypes of South America and
South Asia showed higher level of diversity as compared to other geographical regions.

The high value of differentiation reflects the interactions of various factors including: a reduced
geographic range, the saharan and arid interior climates, their breeding system (entomogame), and
genetic drift or genetic isolation of populations. The estimated number of migrants per generation (Nm)
was 0.70, which suggested that the gene flow in A. spinosa was restricted. Gene flow is generally
considered as the main factor that could homogenize the genetic structure of populations in their
distribution area. According to Wright (1931), Nm¼ 1 is sufficient to overcome the effects of genetic
drift. Also, species with low gene flow have higher genetic differentiation than species with high gene
flow. These results can be explained by topographic barriers preventing the dispersal of seeds and
pollen. Also, insect pollination is often associated with a reduced gene flow among populations. On the
other hand, itwas suggested that endemism and limited distributions of populations within a species favor
high genetic differentiation (Hamrick et al., 1990).
Analysis of Molecular Variance (AMOV) and Genetic
Partitioning
When data are available from more than one population, it is usually of interest to evaluate the degree
to which the total gene diversity partitions into within and between population components (Lynch and
Milligan, 1994). In this study, analysis of molecular variance revealed that higher percentage of variation
is attributed to variation within populations (98.9%) and only 1.1% of the variation was attributed to
difference among populations (Table 5). An understanding of the partitioning of genetic variation within
crop gene pools can provide insight into the evolution of crop lineages. In addition to its implication for
conservation, partitioning the genetic variation in to its components has significant impact in the future
breeding and conservation plan. In this investigation, the AMOVA analysis revealed a significant
difference between and within four groups of safflower genotypes. Yang et al. (2007) has reported that
AMOVA revealed a nonsignificant difference between two groups of genotypes (native vs. exotic). The
one reason for high significant genetic variation within safflower population in the present study, could
be due to floral biology and pollination nature of safflower. Moreover, insect pollinators including bees
and birds might be attracted due to its colorful flower that facilitate gene flow among populations. Not
only the pollination nature of safflower, but also the existence of seed exchange among local farmers
could also be the additional factor. Moreover, the presence of long distance marketing of safflower
within and among different regions by farmers and local traders have their own effect on the observed
variation of cultivated of safflower. Hamrick and Godt (1989), stated that, the biological characteristics
of a species, population structure, mating system and multiple evolutionary process (genetic drift:
bottleneck and founder effect) are determine the level and pattern of genetic variation observed in
natural population.

Though a limited number of ISSR markers were used in the study, the results confirm that ISSR markers
are efficient in detecting polymorphism within and among populations of white lupine found in close
geographic proximity. Until the present day information available on the reproductive biology of L. albus
L., suggested that it is a predominantly self-pollinating plant. However, the result of this study might be
attributed to two reasons: one against and the other in favor of the self-pollinating nature of the L. albus
plant. In the former case, the result obtained could be accounted to mixed type of mating, typical of
plant species, in which there is a gene flow, and thus there might be moderate gene flow among the
local populations by effectors such as wind, insect, human (seedling movement) and birds. The other is
that they might have preferential or diverse adaptive genes that are not fixed through self-pollination
until the present day. However, this study used a small sample size, geographic range and limited
primers. Therefore, to find clear patterns of diversity for the whole country and reach a sound
conclusion, further studies should be conducted with large sample sizes and geographic range using
many ISSR primers.

Lepidiumsativum is both self and cross pollinated plant (Quirós and Cárdenas, 1998). Hence, the
proportion of genetic variation is dependent on the type of pollination that the species undergoes. If the
species has large proportion of cross pollination, then we expect high genetic variation within
population and less divergence among populations. In addition to pollination, behavior of insects;
market exchange could facilitate gene flow among regions which could result in higher percent variation
within population and less genetic structure.

This is also supported with the spread of individual accessions on UPGMA, NJ and PCO graphs.

In general, high level of genetic diversity is not expected with strictly limited distribution and a small
population size. Nevertheless, ISSR markers used in this study generated higher level of polymorphism in
24 accessions of from limited geographical T. quartinianum location in North West Ethiopia. This shows
that small populations or individuals are not always associated with a lack or low level of genetic
variation (Yingjuan and Ting , 2009).

The high within population variation observed in this and other study on Ethiopian arabica coffee
indicate that there is high gene flow through either seeds or seedlings exchange. The gene flow could be
enhanced via birds, insects, wild and domestic animals by facilitating exchange of pollens and seeds.
Moreover, coffee farmers could also contribute to gene flow by exchanging seeds and seedlings of
enhanced landraces among nearby districts with the objective of improving productivity of coffee.
The higher within populations’ genetic diversity might be accounted to two contrary reasons. Coffea
arabica is affected by multiple evolutionary forces which operate within historical and biological context
of the plant species. This includes the mating types, gene flow, mode of reproduction and natural
selection etc. Hamrik and Godt (1989). For this reason, it could be speculated from this
result that Coffea arabica might have mixed mating system (partial out-crossing by pollen and seed and
partial selfing) for which some extent of gene flow is expected as reported by Meyer (1965) which could
result in high within population genetic diversity (Loveless and Hamrik 1 984; Aga et al., 2003; Tesfaye,
2006; Oljira, 2006; Balemi, 2007).
In contrary to the above, as Balemi (2007) reported it could also be speculated that the high genetic
diversity observed within populations of the Coffea arabica might be due to preferential adaptive gene
complexes adapted to environmental changes being evolved during long evolutionary period in a given
region. In this case, Coffea arabica population uses selfing as mechanisms to prevent influx of the gene
from another portion of the populations that might reduce diversity through disrupting adaptive
genes Lowe et al. (2004).060

The one reason for high significant genetic variation within safflower population in the present study,
could be due to floral biology and pollination nature of safflower. Moreover, insect pollinators including
bees and birds might be attracted due to its colorful flower that facilitate gene flow among populations.
Not only the pollination nature of safflower, but also the existence of seed exchange among local
farmers could also be the additional factor. Moreover, the presence of long distance marketing of
safflower within and among different regions by farmers and local traders have their own effect on the
observed variation of cultivated of safflower. Hamrick and Godt (1989), stated that, the biological
characteristics of a species, population structure, mating system and multiple evolutionary process
(genetic drift: bottleneck and founder effect) are determine the level and pattern of genetic variation
observed in natural population.049
Breeding systems of Eucalyptus spp. show a high probability of self-pollination (HARDNER; POTTS, 1995).
In fact, E. cladocalyx has post-zygotic self-incompatibility systems (ELLIS; SEDGLEY, 1992). Nevertheless,
McDonald et al. (2003) reported a high level of inbreeding and low genetic diversity within populations,
which is consistent with the present study. Therefore, other factors are responsible for the low genetic
diversity of E. cladocalyx. The genetic structure can break HardyWeinberg equilibrium due to genetic
and nongenetic factors. Non-genetic factors, such as physical barriers and the geographical isolation of
populations (FRANCOIS; DURAND, 2010) could, block to gene flow among populations. Migration among
populations tends to homogenize their allele frequencies, and therefore, the absence of significant
genetic differentiation among populations indicates that the populations maintain continuous gene
flow. The results of the present study indicated that Kangaroo Island is the most genetically distant
region, which is according to McDonald et al. (2003) and Bush and Thumma (2013), and it would
be expected due to its geographical isolation. In addition, McDonald et al. (2003) reported a positive
relationship between geographic distance and genetic divergence among populations in E. cladocalyx.
The genetic differentiation among populations could be the result of an adaptation to environmental
gradients. In particular, E. cladocalyx is distributed across differential environmental conditions with
respect to soil composition, altitude and annual rainfall (MCDONALD et al., 2003; CLARKE et al., 2009),
which could drive the acquisition of local adaptations. Moreover, the environmental conditions could
influence flowering times and restrict the gene flow through pollination and seeds dispersion among
populations. In fact, according to the findings of Contreras-Soto et al. (2011), Mora et al. (2009) and
Cané-Retamales et al. (2011), there are significant differences in flowering times among natural
provenances of E. cladocalyx. These three sets of authors reported that Cowell hasthe higher flowering
intensity and precocity, and trees from Flinders Chase were the worst for both traits. In the present
study, Cowell and Flinders Chase had the highest genetic differentiation. The provenances of Eyre
Peninsula were most genetically separated from the Kangaroo Island provenance.424

In the present study, ISSRs revealed higher genetic diversity ( T H = 0.175) in Ethiopian lentil landraces.
This was greater than the within species diversity ( T H = 0.049) of the cultivated lentil reported by
Ferguson et al. (1998) from RAPD data of 100 lentil landrace accessions from 10 countries. In fact, a
number of literatures indicate that RAPD detects low level of diversity (Abo-elwafa et al., 1995; Ahmad
et al., 1996; Ford et al., 1997; Sonnante and Pignone, 2001), while ISSRs reveal a higher degree of
variation (Sonnante and Pignone, 2001) in lentil. These differences could be explained mainly by the
difference in sensitivity of different molecular markers. The genetic structure of plant populations
reflects the interactions of various factors, including the long-term evolutionary history of the species
(shifts in distribution, habitat fragmentation, and population isolation), genetic drift, mating system,
gene flow and selection (Schaal et al., 1998). In the present study, there was high level of genetic
differentiation ( ST G = 0.455) among populations. This could be attributed to mutation of SSR loci
(annealing site for ISSR primers), random genetic drift and differential selection pressure (by the
environment) on the loci assessed. For most markers, the contribution of mutation to differentiation has
generally been ignored because mutation rates of neutral (traditional isozyme) markers are negligible
when compared to migration rates. This may not be the case when dealing with ISSR, which uses SSRs
(with high mutation rate) as primer annealing sites. Estimates of genetic differentiation between
populations of inbred species based on AMOVA derived by analyzing RAPD markers have usually been >
70%

(Nybom and Bartish, 2000). However, this might not hold in some cases (of food crops, for instance)
where there is high gene flow represented by seed movement through human involvement. In
accordance with this argument, AMOVA analysis in the present study revealed lower among population
genetic variation (43.72%), than within population genetic variation (56.28%). Nei’s diversity statistics
revealed similar result. The values obtained from Nm show the approximate number of individuals
migrating from one population to the other, in a typical island model. Generally, if Nm < 1, then local
differentiation of populations will result, and if Nm > 1, then there will be little differentiation among
populations (Wright, 1951). The fact that estimates of Nm was < 1 suggests that gene flow between
populations is insufficient to counter the effects of random drift (Real, 1994). Genetic differentiation
results from genetic drift only if Nm< 1, but not if Nm > 1 (Slatkin, 1987). According to Slatkin (1981,
1985), Caccone (1985) and Waples (1987) Nm values can be grouped into three categories: high (Nm ≥
1.000), intermediate, (0.250 – 0.990), and low (0.000 – 0.249). In this study, the relatively high genetic
differentiation and intermediate level of gene flow (Nm = 0.600) detected strongly indicate that genetic
drift has greatly affected the genetic composition of individual populations. The lower gene flow among
populations could be attributed to geographical, social or cultural isolation/barrier and limited use of
lentil seeds purchased from one AR for cultivation in another AR. The wide range of interpopulation
genetic distance shows the potential for landrace improvement program.

In general, populations with high intra-population genetic diversity were genetically distantly related to
populations with low intra-population diversity. Similar finding was reported by de la Cruz et al. (2004) in
5 wild populations of Phaseeolus vulgaris L. using ISSR marker.article

The ISSR survey of three populations of T. quartinianum revealed a high level of genetic variation at the
species level (PPL = 100%; h = 0.29; I = 0.44). The least polymorphic and genetically unique population
was Gonder (PPL = 57.14%; h = 0.18; I = 0.27), while Gojam was the most polymorphic and diverse (PPL =
82.14%; h = 0.28; I = 0.41) (Table 4). Previous assessments of genetic diversity in 34 T. quartinianum
accessions of Ethiopia based on eight morphologic and agronomic traits have reported that most of the
accessions showed similarity in morphological characteristics (Basweti and Hanson, 2012). This finding
contradicted with the present study, mainly because morphological features have a number of
limitations including low polymorphism, lowheritability, late expression, and vulnerability to
environmental influences (Smith and Smith, 1992; Konarev, 2000; Muthusamy et al., 2008), which, in
turn limits their utility for assessing real genetic diversity. Up to now, there are no published reports
concerning ISSR method for analyzing the genetic diversity of T. quartinianum. However, ISSR marker
was used to assess the level and pattern of genetic diversity in four Trifolium species represented by two
varieties, one breeding sample and two wild population of T. pratense, four wild populations of T.
medium, two varieties and one population of T. resupinatum, and two varieties and three wild
populations of T. repense. The study showed 69.5% polymorphism in Trifolium medium, 68.9% in T.
resupinatum, 76.2% in T. pretense and 73.6% in Trifolium repense (Dabkevičienė et al., 2011). Recently,
ISSR marker also used for the genetic diversity study of 14 accessions of three species of Trifolium and
60% polymorphism in Trifolium fragiferum, 58.67% in Trifolium hybridum and 77.32% in T. pratense was
found (Aryanegad et al., 2013). The genetic diversity investigated in the present study was higher than
the ones reported by Dabkevičienė et al. (2011) and Aryanegad et al. (2013). Generally, the higher level
of genetic variation found in this study may be due to the fact that geographically isolated populations in
certain geographic locations could accumulate genetic differences and evolve unique genotypes as they
adapt to different environment (Souframanien and Gopalakrishn, 2004). In general, high level of genetic
diversity is not expected with strictly limited distribution and a small population size. Nevertheless, ISSR
markers used in this study generated higher level of polymorphism in 24 accessions of T. quartinianum
from limited geographical location in North West Ethiopia. This shows that small populations or
individuals are not always associated with a lack or low level of genetic variation (Yingjuan and Ting,
2009).

Analysis of molecular variance (AMOVA)

The AMOVA without grouping indicated that most of the total genetic variation in T. quartinianum
populations exists within populations (83.13%), while among population variation (16.87%) was
observed to be low

(Table 5). High genetic variation within populations indicated that high genetic dissimilarities among the
individual plants sampled from a single population. The vast majority of diversity studies across
members of the Trifolieae have shown a generally high level of diversity within populations, even among
other inbreeding species such as T. subterraneum (Pecetti and Piano, 2002; Piluzza et al., 2005). The
AMOVA results obtained in the present study do not contradict with the above findings. It is a prevalent
view that self-pollinated species maintain higher genetic variation among populations than within
populations. Though T. quartinianum is self-pollinated plant, higher within populations genetic variation
than among populations was obtained in the present study, contrary to this prevalent view. High genetic
exchange or gene flows, which actually have a more homogenizing effect on the genetic variation among
populations by the dispersal of the seeds, can likely explain higher within population genetic variation.
Some seeds may be harvested as weeds together with those of crops and distributed with the crop
seeds via market channels. Moreover, the pods and mature calyx of T. quartinianum tend to have a
coarse surface and points which may attach to passing animals and be transported to other places.6943-

AMOVA analysis resulted in high genetic diversity within population (94%) and very low genetic diversity
among population (6%). This could be due to high seed exchange among different regions and markets
which could lead to intermix of populations between regions. Unlike other landraces of cultivated
plants, Lepidiumsativum in Ethiopia is not restricted to a given area rather it is wildly exchanged among
local community and markets. This shows that there is very high gene flow between populations and
regions. Jiang et al. (2012) who studied on the genetic diversity of Chimonanthusgrammatus
populations by using ISSR marker showed that there was 73.6% within population variation whereas the
rest 26.4% was due to among population variation. As compared to the present study, there was less
gene flow. Jiang et al., (2012) recommended that gene flow, genetic drift and evolutionary history might
have important influence on genetic structure and diversity of a given population.

AMOVA analysis indicated that majority (>95%) of genetic variation observed in germplasm is due to
variation in individuals instead of from a specific group. In general, genotypes of South America and
South Asia showed higher level of diversity as compared to other geographical regions.

The high value of differentiation reflects the interactions of various factors including: a reduced
geographic range, the saharan and arid interior climates, their breeding system (entomogame), and
genetic drift or genetic isolation of populations. The estimated number of migrants per generation (Nm)
was 0.70, which suggested that the gene flow in A. spinosa was restricted. Gene flow is generally
considered as the main factor that could homogenize the genetic structure of populations in their
distribution area. According to Wright (1931), Nm¼ 1 is sufficient to overcome the effects of genetic
drift. Also, species with low gene flow have higher genetic differentiation than species with high gene
flow. These results can be explained by topographic barriers preventing the dispersal of seeds and
pollen. Also, insect pollination is often associated with a reduced gene flow among populations. On the
other hand, itwas suggested that endemism and limited distributions of populations within a species favor
high genetic differentiation (Hamrick et al., 1990).