Int. J. Biosci.
2014
International Journal of Biosciences | IJB |
ISSN: 2220-6655 (Print) 2222-5234 (Online)
http://www.innspub.net
Vol. 4, No. 1, p. 75-81, 2014
RESEARCH PAPER
OPEN ACCESS
The study of genetic variation for Lolium perenne using ISSR
molecular markers
Hooshmand Safari1*, Hooman Shirvani2, Ali Ashraf Jafari3, Sara Mahdavi4
1
Agriculture and Natural Resources Research Center, Kermanshah, Iran
2
Department of Agriculture, Payame Noor University, Iran
3
Research Institute of Forests and Rangelands, Tehran, Iran
4
Department of Agriculture, Borujerd Branch, Islamic Azad University, Borujerd, Iran
Key words: Genetic diversity, Lolium perenne, accessions, ISSR molecular markers.
doi: http://dx.doi.org/10.12692/ijb/4.1.75-81
Article published on January 01, 2014
Abstract
Genetic variation for 12 accessions of Lolium perenne were surveyed using the number of 12 ISSR primers, that
the number of 10 primers can be scored. The ISSR primers can be produced the number of 62 bands, which the
polymorphism was showed for the number of 47 bands. The average of bands was 6.2 for each primer. The
primer of IS9 showed the highest number of band (11 bands) and IS 15 showed the lowest number of band (3
bands). A desirable polymorphism between genotype was observed based on ISSR markers, which the primers of
IS9, IS10, IS13 and IS16 were determined for genetic variation study in Lolium perenne as desirable primers.
Average PIC in the used primers was 0.33 that the highest amount of PIC related to primer IS 12 that amount of
PIC was 0.45 in this primers and primers IS3 and IS14 with the lowest value of PIC don’t have ability in the
separation of accessions. Yatsyn (G11) had the most genetic distance with Grenisle (G7). Cluster analysis and
Scatter plot based on first and second axis from principal coordinate analysis for genotypes, showed that genetic
variation did not agreement with the geographical distribution. Therefore the genotypes which had the most
genetic distance based on used ISSR marker can be considered as suitable material in breeding programs for
using hetrosis.
* Corresponding
Author: Hooshmand Safari hooshmand.safari@gmail.com
75 Safari et al.
Int. J. Biosci.
2014
Introduction
of DNA profiling of populations is to determine
Perennial ryegrass (Lolium perenne L.) is the most
diversity among populations in order to develop
important grass species in temperate climates of the
genetically distinct subsets of populations in a
world. Perennial ryegrass is a diploid species
breeding program or to check for duplicates in a gene
(2n=2x=14) with a two-locus self incompatibility
bank. In these cases, it may be possible to determine
system, which ensures a high degree of genetic
diversity among populations by profiling bulked DNA
variation in populations (Bolaric et al., 2005a).
of the individuals (Rouf et al., 2002). Inter Simple
Lolium perenne (Poaceae) was chosen as the model
Sequence Repeat (ISSR) is a dominant molecular
species and small populations of 15 different cultivars
marker revealed in mass. ISSR has recently been
of this species were established in each experimental
developed as an anonymous, RAPD–like approach
community. Lolium perenne is one of most important
that accesses variation in the numerous microsatellite
grass species in central Europe and worldwide used
regions dispersed throughout the various genomes
for the sowing and regeneration of temperate
and circumvents the challenge of characterizing
agricultural grassland. Its productivity is stimulated
individual loci that other molecular approaches
by nitrogen fertilization and it has a high fodder
require. They are characterized by mono-, di- or multi
quality and grazing tolerance. Beside its importance
- nucleotide repeats that have 4-10 repeat units’ side-
as a fodder crop, perennial ryegrass is frequently used
by-side. Extremely high variability combined with
as a turf species in lawns (Beddows, 1967).
greater robustness in repeatability experiments and
less prone to changing band patterns with changes in
Knowledge of genetic variation is a useful tool in
constituent or DNA concentration template make
genebank management, helping in the establishment
them superior to other readily available marker
of core collections, facilitating efficient sampling and
systems in investigations of genetic variation (Fang
utilization
and/or
and Roose, 1997). Genetic diversity based on
eliminating duplicates in the gene stock), and
Molecular markers for within different species of
selecting of desirable genotypes to be used in
Lolium was reported by many researchers (Elazreg et
breeding programs (Elham et al., 2010; Vishwanath
al., 2011; Vieira et al., 2004; Majidi and Mirlohi,
et al., 2011). On the other hands, understanding
2010).
of
germplasm
(identifying
genetic diversity of certain species is not only useful
in addressing questions about evolutionary process
Thus, the present study aimed to determine and
and the development of conservation strategies, but
assess the genetic variability of the Lolium collection
also a prerequisite for efficient use of genetic
and better understand the genetic diversity structure
resources in breeding programs. Interest in the
of the available accessions using ISSR molecular
genetic structure of natural populations of grass
markers.
species has been increased in the last few years due to
the necessity of broadening the knowledge of genetic
Materials and methods
variations in cultivated species (Che and Li, 2007).
Plant materials
Assessment of genetic diversity with molecular
In order to evaluate the genetic variation, 12
markers is a
alternative. Molecular
accessions of Lolium perenne were prepared from
characterization can also be re-applied after years of
gene bank of Research Institute of Forests and
maintenance and new accessions can be related to
Rangelands, Tehran, Iran (Table 1).
existing
promising
collections.
Moreover,
molecular
characterization of genetic diversity provides base
DNA extraction and ISSR method
information, which can be used to select a promising
Total genomic DNA was extracted for young leaves of
range of accessions for different breeding programs
greenhouse-grown plants using a modified CTAB
(Roldan-Ruiz et al., 2001). Often, the initial objective
(Murry and Tompson, 1980) with modification
76 Safari et al.
Int. J. Biosci.
2014
described by De la Rosa et al., (2002). Quality and
information content (PIC) calculated for each primer
quantity of extracted DNA were examined using 0.8%
(Anderson et al., 1993). Similarity matrix computed
agarose gel. The compounds of polymerase chain
based on Dice's coefficient and cluster analysis
reaction were carried out according to Table 2.
performed for grouping accessions based on Dice's
coefficient by UPGMA methods. Principal coordinate
Template DNA was initially denatured at
92oC
for 5
analysis performed to better interpret the genetic
min, followed by 35 cycles of PCR amplification under
variation
the following parameters: denaturation for 30
molecular variance analysis performed for the three
seconds at 95oC, primer annealing for 30 seconds at
groups from cluster analysis. Statistical analyses were
the temperature based on primer temperature
done by Darwin 5 and Gen ALEX 6.2.
between
accessions
and
finally
the
(Temperatures of annealing in this study was 50, 55
and 60 oC) and primer extension for 1 min at 72oC. A
final incubation was performed for 5 min at
72oC
to
Results
ISSR polymorphism
ensure that the primer extension reaction proceeded
Genetic variation for 12 accessions of Lolium perenne
to completion. The PCR amplified products were
were surveyed using the number of 12 ISSR primers,
separated by electrophoresis on a 1.5% agarose gels
that the number of 10 primers can be scored. For all
using TBE buffer. The gels were put in the Ethidium
primers, the number of 62 bands was scored that
bromide for 30-45 min and visualized by gel
polymorphism was observed for 47 of them. The
document.
average of bands was 6.2 for each primer. IS9 primer
with 11 bands had the highest and primer IS15 with 3
Statistical analysis
bands had the lowest number of bands. The average
ISSR bands were treated as binary characters and
of bands for each primer was 5.1 for 12 genotypes that
coded accordingly (presence =1, absence = 0). The
genotype Spelga (G12) had the most and genotype
Number
of
Fontoon (G10) had the lowest band. Band pattern of
of
accessions for IS9 showed in Fig. 1.
of
polymorphic
scored
bands
bands
(NSB),
(NPB),
number
percentage
polymorphism bands (PPB) and polymorphism
Table 1. Gen bank cod and Origin of accessions of Lolium perenne.
Gene bank
Name
code
genotype
1303
Grenisle
1312
Tyrone
Origin
Number Gen bank cod
Southern Ireland
Northern Ireland
G7
G8
1309
1307
Name
genotype
Aubisque
Green Gold
1311
Napoleon
Denmark
G9
1308
Magician
1330
1302
1306
Fontoon
Yatsyn
Spelga
Southern Ireland
New Zealand
Northern Ireland
G10
G11
G12
1305
1313
1301
Carat
Gilford
Moy
Origin
Number
Netherlands
Southern
Ireland
Southern
Ireland
Netherlands
Ireland
Northern
Ireland
G1
G2
Table 2. Compounds of optimized ISSR reaction.
To provide 20 µl
12.6 µl
2 µl
1.5 µl
0.4 µl
1.2 µl
0.3 µl
2 µl
20 µl
77 Safari et al.
Compounds of a sample
Water distillated twice
Buffer PCR (X10)
Colored manyazium (50 mmol)
Nucleotides mixture (10 mmol)
Primer (10 µmol)
Tag polymerase
DNA (10 ng)
Total
G3
G4
G5
G6
Int. J. Biosci.
2014
Average of polymorphism percent was 80.24%. The
Primers sequences, code, number of bands scored,
lowest percent of polymorphism belonged to IS15
number
(50%) and the highest percent of polymorphism was
polymorphic
100% for primers IS3, IS9, IS10, IS13 and IS16. Average
information content (PIC) were showed for ISSR
of PIC for all primers was 0.33 that the highest value
primers in Table 3.
of
polymorphic
bands
bands,
(PPB)
and
percent
of
polymorphism
of PIC related to IS12 and the lowest belonged to IS3
and IS14 (Table 3).
Table 3. ISSR primers used in this study and some summary results.
ISSR code
IS3
Primer sequence
5' GAGAGAGAGAGAGAGAYC 3'
NSB
4
NPB
4
PPB
100%
PIC
0.257
IS5
IS9
5' AG AG AG AG AG AG AG AGC 3'
5' CTCTCTCTCTCTCTCTG 3'
11
4
8
4
72.73%
100%
0.304
0.368
IS10
5' GAGAGAGAGAGAGAGARC 3'
8
8
100%
0.339
IS11
5' ACACACACACACACACC 3'
7
4
57.14%
0.344
IS12
5' TGTGTGTGTGTGTGTGG 3'
5
3
60%
0.458
IS13
5' AGAGAGAGAGAGAGAGYT 3'
3
3
100%
0.324
IS14
5' GACAGACAGACAGACA 3'
8
5
62.50%
0.280
IS15
5' GGATGGATGGATGGAT 3'
8
4
50%
0.336
IS16
5'DBDACACACACACACACA3'
4
4
100%
0.340
Average
6.2
4.7
80.24
0.335
Similarity Matrix
of similarity was 0.70 [Between the accessions of
Similarity matrix based on Dice’s coefficient for
Yatsyn (G11) and Grenisle (G7)] to 0.87 [Between the
accessions
accessions of Aubisque (G1) with Grenisle (G7)].
showed that (Table 4) the average of
Similarity between accessions was 0.79 and the range
Table 4. Similarity matrix for studying accessions based on Dice’s coefficient.
accessions G1
G2
G3
G4
G5
G6
G7
G8
G9
G10
G11
G2
G3
G4
G5
G6
G7
G8
G9
G10
G11
G12
0.787
0.825
0.805
0.732
0.8
0.831
0.814
0.789
0.736
0.8
0.782
0.818
0.809
0.771
0.738
0.839
0.747
0.766
0.804
0.785
0.775
0.767
0.8
0.786
0.764
0.800
0.75
0.84
0.8
0.831
0.86
0.829
0.767
0.778
0.747
0.753
0.814
0.743
0.782
0.778
0.817
0.795
0.857
0.709
0.786
0.815
0.862
0.756
0.8
0.865
0.791
0.809
0.784
0.736
0.812
0.822
0.795
0.714
0.873
0.778
0.836
0.776
0.827
0.787
0.747
0.744
included accessions of Carat (G4), Green Gold (G2)
Cluster Analysis
grouping
and Tyrone (G8), which the average similarity
accessions based on Dice's coefficient (Fig. 2) were
coefficient was 0.81 for this group. The third group
identified the three distinctive groups. The first group
consisted of Aubisque (G1), Gilford (G5), Grenisle
consisted of accessions Yatsyn (G11), Spelga (G12),
(G7), Napoleon (G9) and Fontoon (G10). The average
Magician (G3) and Moy (6G), which the average
of Dice's coefficient was 0.83 for this group.
UPGMA
hierarchical
clustering
for
similarity was 0.79 for this group. The second group
78 Safari et al.
Int. J. Biosci.
2014
Cluster analysis of the similarity matrix for grouping
between groupings of accessions based on cluster
showed, the most similar among Third group and
analysis (Table 6). The results showed a significant
Second group, and the most distance for accession in
(P<0.01) difference between groups and the portion
groups Second and first. (Table5).
of variance percent for between group and within
Table 5. Similar groups from the cluster analysis.
group were 13% and 87%, respectively.
Groups
C1
C2
C3
C1
1
0.767
0.778
C2
C3
Principal Coordinate Analysis
1
0.795
Scatter plot for accessions based on first (33.25) and
1
second (17.26) axis from principal coordinate analysis
Molecular Variance Analysis
(Fig. 3) showed that Genetic variation did not
Analysis of molecular variance was performed for
matching with the geographical distribution of
ISSR bands to determine of significant difference
accessions. These results confirmed by cluster
analysis and similarity matrix.
Table 6. Molecular variance analysis.
S.O.V
Df
SS
MS
Est. Var.
Var%
PhiPT
Between group
2
27.63
13.82
1.323
13%
0.133*
Within group
9
77.70
8.633
8.633
87%
Total
11
105.33
9.957
100%
Est. Var; Calculated variance for Within and between group %Var; Percent of variance of each source to total
variance.
Discussion
determine of genetic diversity between and within
The results revealed that ISSR markers are suitable
different Lolium species (Pivoriene et al., 2008; Hu et
tools for detecting the genetic variation in Lolium
al., 2011; Posselt et al., 2006; Bolaric et al., 2005b).
accessions. According to this study a Significant
The similarity between accessions based on Dice's
variation was observed among accessions. We believe
coefficient was high, therefore can be stated that there
that there needs a molecular markers studies as a
was a low genetic variation among accessions. It was
complementary studies for the morphological traits in
noticed that the majority of the similarity coefficients
the field. It will reduce the amount of materials for
between accessions was close to 0.78. This indicated
study as well as the costs of experiments.
the
This
close
relationships
between
the
evaluated
technique has been used to study a variety of plants.
accessions, though they are collected from different
The results of this study Ghariani et al (2003) were
origin. Grouping of accessions based on cluster
consistent. The Average percentage of polymorphism
analysis and principal coordinate analysis indicated
was 80.24% that showed a good polymorphism
that genetic variations do not in agreement with the
between genotypes. Between 10 used primers, the
geographical distribution of accessions.
primers IS3, IS9, IS10, IS13 and IS16 showed 100%
polymorphism that indicate high ability for survey
molecular variation and high variation among
genotypes. PIC values estimate the discriminatory
power of a marker. The mean PIC values for markers
used in present study were 0.33. Marker with high
PIC values such as IS12 could be effectively used in
genetic diversity studies in Lolium. Efficiency of ISSR
primers were reported by other researchers to
79 Safari et al.
Int. J. Biosci.
2014
high
genetic
variation
between
accessions.
Information about current genetic diversity permits
the classification of our available germplasm into
various/heterotic
groups,
which
is
particularly
important to hybrid/cross-breeding programs in
Lolium. Even though the genetic mechanisms that
explain heterosis are not fully understood, it is well
documented that crosses between unrelated and
genetically distant parents, show greater hybrid vigor
than crosses between closely related parents. The
magnitude and pattern of genetic variation detected
Fig. 1. The band pattern for accessions using IS9
in this study can be useful for more systematic
germplasm management and utilization in breeding
primer.
programs.
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