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African Journal of Agricultural Marketing ISSN: 2375-1061 Vol. 12 (7), pp. 001-007, July, 2024. Available online 
at www.internationalscholarsjournals.org © International Scholars Journals 

 

Author(s) retain the copyright of this article. 
 
 

Full Length Research Paper 

  

Genetic Diversity and Morphological 
Characterization of M7 Soybean Mutant Lines 

Using RAPD Markers 
 
Mehdi- Younessi Hamzekhanlu1, Ali Izadi-Darbandi1*, Nejat Pirvali-Beiranvand2, Mohammad 

Taher-Hallajian2 and Abbas Majdabadi2 
 

1
Deparment of Agronomy and Plant Breeding science, College of Aburaihan, University of Tehran, P. O. Box. 

3391653775, Tehran- Iran. 
2
Agricultural, Medical and Industrial Research School, Nuclear Science and Technology Research Institute, Atomic 

Energy Organization of Iran. 
 

Accepted 28 February, 2024 
 

When genetic variability is diminished using traditional breeding methods, induced mutation is a good 
way to increase genetic diversity in soybean. Hence, genetic diversity amongst 33 M7 generation 
soybean mutant lines with high N2 fixation character and one non-irradiated cultivar (L17) was studied 
by using random amplified polymorphic DNA (RAPD) markers and some morphological traits. RAPDs 
established 104 major amplified products using 10 polymorphic primers. Out of 104 markers, 34 were 
monomorph and the remaining (70) were polymorph. Cluster analysis of studied lines in terms of RAPD 
markers, separated mutant lines group from parent cultivar. Both morphological and RAPD markers 
successfully detected genetic variation within induced mutant lines and the variation between irradiated 
and non irradiated lines which were morphologically indistinguishable also detected by RAPD. 
Therefore RAPD markers with average PIC = 0.80, can be more useful for detecting induced diversity 
among mutant lines. It can be inferred from the results that irradiation did induce significant genetic 
variability with regard to majority of studied traits such as number of nodule per plant and harvest 
index. Sequencing and cloning of band pattern (3 kb) obtained from parent cultivar with OPA09 primer 
and introducing it as a SCAR marker can be used in marker assisted selection. 

 
Key words: diversity, morphological traits, mutation, random amplified polymorphic DNA, soybean. 

 
INTRODUCTION 

 
Mutation breeding in crop plants is an effective tool in 
hands of plant breeders especially in crops having narrow 
genetic base. Many mutants have been identified as 
donors of desirable traits in breeding program. Mutation 
breeding work in soybean crop has yielded in 
identification of many mutant lines with desirable traits 
like high germination and survival percent (Rahman et al., 
1994). Despite the richness of the soybean germplasm 
collection the genetic base of the present day collection 
remains poor (Delannay et al., 1983). Because of this  
 
 
 
*Corresponding author. E-mail: alizadi110@yahoo.com or 
aizady@ut.ac.ir. Tel: 09128024109. 

 
 
 

 
problem, induction of genetic diversity and its use in 
soybean breeding programs is essential to create 
performance mutant lines to meet the worldwide soybean 
demand.  

It can be possible to increase the genetic variability by 
inducing many mutations in plants with ionized radiations 
at the in vivo and in vitro studies of mutation breeding. 
After the creation of mutant lines genetic diversity of 
these lines can be monitored using morphological as well 
as genetic based tools, DNA techniques (Bennici et al., 
2003) and advanced molecular methods (Barazani et al., 
2002; Shiran et al., 2007). The polymerase chain reaction 
(PCR) based method for DNA profiling, random amplified 
polymorphic DNA (RAPD) techniques (Li et al., 2001; 
Fracaro et al., 2005) has been extensively applied in 



2 

 

 
 
 

 

evaluation of genetic diversity of different plant species 
and soybean cultivars. The RAPD assay is efficient for 
screening for nucleotide sequence polymorphism among 
individuals as each primer (on average) will direct the 
amplification of several discrete loci within a genome 
(Samal et al. 2003). As primers modified by even a single 
nucleotide produce different banding profiles, the RAPD 
technique can generate polymorphisms between very 
closely-related genotypes (Deng et al., 1995).  

Present investigation was, therefore, undertaken to 
obtain information on effect of gamma irradiation on 
morphological traits of the M7 generation of soybean 
mutant lines. There are problems about soybean disease 
and pests such as cyst-nematode in Iran and also lack of 
genetic diversity in soybean, traditional breeding 
programs have not been resulted in favorable results. In 
our research the studied lines were previously 
established and has no genetic evaluation was not 
conducted on these lines. The objective of this study was 
mainly to evaluate the rate of genetic diversity amongst 
induced mutant plants and their parent cultivar (L17) and 
their classification using RAPDs and morphological 
markers. We also expected the mutant lines that are 
separated from parent cultivar allow them to create 
resistant varieties against pests and disease such as 
cyst-nematode. We can use these mutant lines in other 
soybean mutation breeding programs. 
 

 
MATERIALS AND METHODS 
 
Plant materials 
 
This experiment containing 33 M7 generation soybean mutant lines 
which previously evolved by γ ray (cobalt - 60 ) from L17 cultivar 
irradiated with doses 150, 200 and 250 Gray (absorbed dose) and 
L17 cultivar. Seeds of mutant lines and parent cultivar were planted 
in pots in greenhouse of Nuclear Research Center for Agriculture 
and Medicine Karaj – Iran. Experiment was conducted in a 
completely randomized design (CRD) with three replications, in this 
research treatments were 33 mutant lines and one parent cultivar 
(L17). 

 

Morphological characteristics 
 
Each line was characterized using 11 traits taken at the different 
stages. Number of leafs per plant at vegetative stage, number of 
pods per plant, number of seed per pod, number of seed per plant, 

number of nodule per plant at R8 stage, and 100-seed weight (g), 
plant dry weight (g), seed yield per plant (g), harvest index, root dry 
weight (g), nodule dry weight (g) after harvesting were measured. 

 

The genomic DNA isolation and PCR 

 
The DNA was extracted from leaves using the procedure described 
by Sharma et al. (2003). Ten random primers were used for PCR 
amplifications. Amplification for RAPD was carried out in 25 µl 
volumes containing 50 ng of template DNA, 400 ng primer, 2.5 U of 
Taq polymerase (Fermentas), 2.5 µl 10X PCR buffer, 300 µM 

dNTPs, 1.4 M MgCl2 and sterile distilled water to 25 µl. The 
amplifications were performed in Biorad thermalcycle. It was 

 
 
 
 

 
programmed for two cycles at 94°C for 2 min, 35°C for 1 min, and 
72°C , two cycles at 94°C for 1 min, 35°C for 30 s and 72°C for 1 
min, and finally 40 cycles at 94°C for 15 s, 35°C for 30 s and 72°C 
for 1 min, and an additional elongation step at 72°C for 5 min. The 
products were held at 4°C until analyzed. Amplified samples were 
run on a 1% agarose gel stained with ethidium bromide at 100 V for 
up to 90 min with 1X TAE as running buffer and gels were 
visualized under UV light and photographed with Polaroid film. 
Duplicate reactions were performed to ensure reproducibility. 

 

Data analysis 
 
Amplification product profiles were scored for the presence (1) or 
absence (0) of bands. Molecular size of the amplified fragments 
was estimated using SM-0403 DNA ladder (Fermentas). RAPD 
assays generating weak or ambiguous amplification products were 
repeated to confirm the consistency of these markers. The NTSYS-
pc software was used to estimate genetic similarities with the 
Jaccard’s similarity coefficient. The generated matrix of similarities 
was analyzed by the complete linkage (CL) or farthest neighbor, 
using the sequential hierarchical agglomerative and nested 
clustering (SHAN) module. Analysis of variance have been carried 
out on the experimental data to evaluate the gamma irradiation 
effect on yield and other traits and their pertinent means were 
separated through the Duncan Multiple Range Test popularly 
known as Duncan’s Test with SAS 9.0 software. Also morphological 
traits were analyzed by using Wards method and Euclidian distance 
coefficient with the SPSS-pc (ver. 16.0). The average polymorphic 
information content (PIC) was calculated for RAPD markers through 
following formula, given by Powell et al. (1996): 
 
He=1-ΣPi

2
 , where He is the expected hetrozygosity or PIC and Pi is 

the frequency of ith allele. 
 

 

RESULTS 

 

Variance analyses (ANOVA) of studied traits are 
presented in Table 1. There were highly significant 
differences (P < 0.01) for number of leaf per plant, 
number of grain per plant, number of pod per plant, plant 
dry weight (shoot dry weight), root dry weight, harvest 
index, number of nodule per plant, nodule dry weight, and 
significant differences (P < 0.05) for 100 seed weight and 
seed yield per plant among mutant lines and parent 
cultivar. Only for number of seed per pod there are no 
significant differences among mutant lines and parent 
cultivar. Means of number of nodule per plant, harvest 
index and shoot dry because of their importance are 
shown in Table 2. Majority of mutant lines such as M13 
mutant line showed significantly (P < 0.05) increased 
number of nodule per plant as compared to parent L17. 
The mutant line M1showed higher harvest index and 
shoot dry weight as compared to parent L17 and other 
mutant lines.  

Cluster analysis of the studied lines using 11 traits was 
carried out. The dendrogram and clustering pattern are 
observed in Figure 1. The ward dendrogram based on 
morphological characterization indicated that all lines 
were clustered into four major groups. According to the 
proximity matrix mutant lines number 24 (M24) and 26 
(M26) were nearest lines to parent cultivar and fourth 



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Table 1.  Analysis of variance of studied traits in M7 generation of soybean mutant lines and parent cultivar M17.      
 

            
 

     MS       
 

Seed yield 100 seed 
Shoot dry weight 

Number of pod per Number of grain Number of grain Number of leaf per 
DF 

Source of   
 

per plant weight plant per plant per pod plant variation 
  

 

     
 

∗∗308/0 ∗∗139/5 ∗∗0003/0  NS  007/0 ∗01/0 ∗063/0 ∗∗0006/0 33 genotype   
 

119/0 74/0 00008/0  005/0 005/0 035/0 0001/0 68 Error   
 

46/17 42/7 17/7  75/17 67/17 58/8 87/10  %CV   
 

    MS        
 

Nodule dry weight Number of nodule per plant Root dry weight  Harvest index DF Source of variation 
 

∗∗0044/0  ∗∗188/0 ∗∗320/0  ∗∗53/1 33 genotype   
 

00008/0   003/0 082/0  44/0 68 Error   
 

89/11   62/9 21/5  08/16  %CV   
 

 
*, ** = significant at the 5 and 1% of probability level, respectively; ns = not significant. 

 

Table 2. Means of number of nodule per plant, harvest index, shoot dry weight in M7 generation of mutant lines (M1-M33) and parent cultivar L17.  
 
 Number of line Number of nodule Harvest index Shoot dry weight Number of line Number of nodule Harvest index Shoot dry weight 
 M1  14.77D  0.333A   8.33A M18 11.76E 0.266ABC 8.3AB 
               

 M2 6.53KIJ 0.31AB 6.67D-J M19 8.55GH 0.316AB 7.46A-F 
 M3 4.55MLN 0.283ABC 7.1C-I M20 9.99GF 0.253ABC 7.56A-D 
 M4  14.89D 0.316AB 6.86D-J M21 8.77GH 0.23BCD 6.43G-J 
 M5 3.44MN 0.283ABC 7.44A-G M22 17.44C 0.303AB 7.46A-F 
 M6  14.5D 0.263ABC 6.45F-G M23 4.99KML 0.23BCD 6.56D-J 
 M7  19.88B  0.3AB 6.7D-J M24 11.22EF 0.253ABC 6.67D-J 
 M8 16.1CD 0.26ABC 7.13C-I M25 10.88EF 0.29AB 7.36A-H 
 M9 4.66MLN 0.28ABC 6.23IJ M26 8.66GH 0.26ABC 6.7D-J 
 M10  19.55B 0.293AB 7.33C-H M27 3.21N 0.3AB 6.36H-J 
 M11  3.11N 0.313AB 6.86D-J M28 5.44KLJ 0.253ABC 6.86D-J 
 M12  7.44HI 0.313AB 7.46A-F M29 6.55KIJ 0.196CD 6.23IJ 
 M13  23.77A 0.29AB  8A-C M30 10.66EF 0.26ABC 6.5F-J 
 M14  17.1C  0.313AB 7.9A-C M31 8.77GH 0.24BCD 6.5E-J 
 M15 3.44MN 0.28ABC 7.23C-I M32 6.77JL 0.303AB 7.1C-I 
 M16 5.88KILJ 0.316AB 7.53A-E M33 11.87E 0.303AB 7.05C-J 
 M17 6.44KIJ 0.26ABC  6.06J P(L17) 7.55HI 0.26ABC 7.23C-I 



4 

 

  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 

Figure 1. Dendrogram of 33 M7 generation mutant lines and parent L17 cultivar (p) of 
soybean based on morphological traits. 

 

 

Table 3. Means of studied traits for each group derived from cluster analysis of 33 M7 generation mutant lines and L17 cultivar of soybean.  
 

Means of traits   
 

Number 
Grain Grain  Pod  

Dry 100 seed Yield per Harvest Root dry 
Nodule Nodule 

 

  per  per  per   per  dry  

Group of leaf 
   

weight(g) weight(g) plant(g) index weight(g) 
  

 

 pod  plant plant  plant weight(g)  

                      
 

1 8.9  2.18  18.03  8.21 7.1  11.73 2.09 0.29 5.32 5.58  0.051 
 

                                   

2  9.21  2.11  15.06  7.2  6.54 11.02 1.64 0.25 5.58 6.72  0.053 
 

3 9.01  2.29  18.16 7.96 7.35 12.12 2.19 0.29 5.61 17.55 0.129 
 

                                

4 8.68  2.26  17.61 7.83 7.29 11.4  1.98 0.24 5.61 11.08 0.085 
 

 
Underlined numbers have highest. 
 

 

groups mutant lines including number 2 (M2), 17 (M17) 
and 30 (M30) were entirely different from parent cultivar. 
Means of each group are shown in Table 3. In terms of 
studied traits mutant lines group (2, 3 and 4) were higher 
as compared with parent cultivar group (1). Amplification 
patterns are performed twice in order to evaluate the 
reproducibility of the RAPD markers. The results showed 
that reproducible RAPD patterns can be obtained under 
the same amplification conditions for two replicates. The 
ten polymorphic primers resulted in 104 storable bands, 
ranging from 200 to 3100 bp in size. Of these, 70 (67%) 
bands were polymorphic (Table 4). The number of bands 
for each primer varied from 6 (OPA - 01) to 12 (OPA - 03, 
OPA - 07) with an average of 10.4 bands per primer. To 
estimate the similarities among studied lines, the Jacard 
coefficient provided similarity values ranging from 0.35 to 
0.79. According to the CL dendrogram (Figure 2) the 
studied lines (mutant lines pulse parent cultivar) were 

 
 

 

clustered into 5 major clusters. According to the similarity 
matrix, the mutant line number 32 (M32) was closer to 
parent cultivar and the mutant line number 9 (M9) was 
entirely different from parent cultivar (L17). For each of 
the 10 RAPD primers, PIC were calculated and PIC 
values ranged 0.73 to 0.86 (Table 4). The mean PIC 
values for all loci were 0.80. 
 

 

DISCUSSION 

 

Morphological variation 

 

In this research induct variation by gamma irradiation was 
assessed using morphological traits in m7 generation of 
soybean mutant lines. It can be inferred from the results 
that irradiation did induce significant genetic variability 
with regard to majority of studied traits such as number of 



5 

 

  
 
 

 

Table 4. Degree of polymorphism and information content for RAPD primers along with their sequences, applied to 33 M7 
generation mutant lines and L17 cultivar of soybean.  

 
 

No. Primers Sequence 
Total (T) Polymorphic (POL) % Polymorphism 

PIC  

 
fragment fragment (POL/T*100)  

     
 

 1 OPA-01 CAGGCCCTTC 6 5 83 0.77 
 

 2 OPA-02 TGCCGAGCTG 11 7 64 0.79 
 

 3 OPA-03 AGTCAGCCAC 12 7 58 0.80 
 

 4 OPA-04 AATCGGGCTG 9 4 44 0.73 
 

 5 OPA-05 AGGGGTCTTG 11 8 73 0.87 
 

 6 OPA-07 GAAACGGGTG 12 10 83 0.83 
 

 7 OPA-08 GTGACGTAGG 11 9 82 0.86 
 

 8 OPA-09 GGGTAACGCC 11 7 64 0.80 
 

 9 OPA-10 GTGATCGCAG 10 6 60 0.74 
 

 10 OPA-11 CAATCGCCGT 11 7 64 0.82 
  

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

Figure 2. Dendrogram of 33 M7 generation mutant lines and parent cultivar (L17) of soybean based on RAPD. 
 
 

 

nodule per plant and harvest index. Also results 
demonstrate that radio mutation induction and selection 
could be successfully used in the soybean cultivars to 

improve the N2 fixation and some of the agronomic 
characters, e.g. number of nodule per plant, harvest 
index and shoot dry weight. Similar results were also 
reported by Padavai and Dhanavel (2004) and Singh and 
Kole (2005). Increase in studied traits in mutant lines may 
be attributed to chromosomal damages. Also separation 
of mutant lines from parent cultivar shows gamma ray 
effect in creating genetic diversity that resulted into 
separate mutant lines from parent cultivar. According to 

 
 
 

 

the means of studied traits in each group (Table 2) 
mutant lines groups (second, third and fourth groups) in 
comparison with parent group (first group) have highest 
value. Since these traits are most important agronomical 
traits such as yield and yield components traits therefore 
relevant mutant lines can be selected from segregated 
group. Using radiation techniques, similar improvements 
in the agronomic characters of soybean and other crops 
were achieved (Rahman et al., 1994; Odeigah et al., 
1998; Dubey et al., 2007; Arulbalachandran et al., 2010). 
The next step will be to verify the disease reaction of 
these materials and to test them for combining ability. 



6 

 

  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 

Figure 3. RAPD pattern of M7 generation mutant lines (M1-M33) and parent cultivar (L17) obtained with primer OPA-09. S.M. demonstrated 
the size marker DNA (SM-0403). According to the figure parent cultivar (L17) have a band which this band deleted in mutant lines. 
 

 

The best lines such as M13 and M1 will be used for 
hybrid production. 
 

 

Molecular markers variation 

 

Changes in DNA caused by mutagens result in genetic 
variation detected by RAPD analysis (Rani et al., 1995; 
Teparkum and Veilleux, 1998). Polymorphic amplification 
products which represent one allele per locus can result 
from changes in either the sequence of the primer binding 
site, such as point mutations, or from changes altering 
the size or preventing successful amplification of a target 
DNA such as insertions, deletions, and inversions (Rani 
et al., 1995). Mutations resulting in polymorphisms are 
those occurring on primer binding sites, leading to an 
increase or decrease in the total number of primer 
binding sites, and consequently the number of amplified 
fragments. In the present study, 10 RAPD primers have 
revealed polymorphic fragments among soybean mutant 
lines that are most likely due to treatment with the 
physical mutagen gamma. These polymorphisms indicate 
presence of genetic differences in soybean mutant lines. 
It is possible that these mutations have occurred in 
different loci, although it is not yet known whether these 
mutations and polymorphism bands have correlated with 
useful traits. RAPD polymorphisms observed in this study 
are likely due to alterations in the number of primer 
binding sites following mutagenesis. Point mutations at 
other regions of the genome that fall within the amplified 
fragments are likely to go undetected. In this study, 
detected out of 70 polymorph fragments indicate a high 
level of mutation 

 
 

 

due to gamma mutagenesis. Thus, RAPD markers are 
useful in detecting polymorphisms in soybean mutant 
lines as they provide sufficient numbers of DNA 
fragments for conducting assays. This approach can then 
be used to rapidly screen mutant lines in efforts to detect 
mutants with desirable agronomic traits.  

This study showed molecular markers such as RAPD is 
more useful for analysis of genetic diversity because 
RAPD also detected variation between the irradiated and 
non irradiated lines, which were morphologically 
indistinguishable therefore RAPD markers can be more 
useful for detecting induced diversity among mutant lines. 
Sequencing of polymorphism bands between mutant 
lines and parent cultivar and additional research are 
needed for screening and detecting of new genes that are 
linked with desirable traits. The sequencing of unique 
band that produced in parent (Figure 3) and introducing it 
as a SCAR marker can be used for marker assisted 
selection. 

 
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