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Highlights in BioScience             

ISSN: 2682-4043 

DOI:10.36462/H.BioSci.20202                                                                                                          

 

 

Highlights in BioScience                                                                                                                                                                           April 2020| Volume 3 

http://bioscience.highlightsin.org/ 

Page 1 of 5 

                                               

Research Article 

 

Open Access 

 
 

 

 

 

 

1 Department of Nucleic Acid and Protein 

Structure, Agricultural Genetic Engineering 

Research Institute (AGERI), ARC, Giza, 

Egypt. 
2 Department of Microbiology, Faculty of 

Science, Ain Shams University, 11566, 

Abbassia , Cairo, Egypt. 

 

Contacts of Authors  

 

 

* To whom correspondence should be 

addressed: Ghada Abu El-Heba 

 

Citation: Hassan A.H., A-bouzeid M., and 

Abu El-Heba G. (2020). DNA Variations 

between Medicago truncatula Symbiotic 

Mutant Line and Native Variant Using 

Fluorescence-Based AFLP Marker, Volume 3. 

Article ID 20202, 

dio:10.36462/H.BioSci.20202 

Received:  March 3, 2020 

 

Accepted:  March 20, 2020 

 

Published: April 1, 2020 

 
Copyright: © 2020 Hassan et al. This is an 

open access article distributed under the terms 

of the Creative Commons Attribution License, 

which permits unrestricted use, distribution, 

and reproduction in any medium, provided the 

original author and source are credited. 

 

Data Availability Statement: All relevant data 

are within the paper and supplementary 

materials 

 

Funding: The authors have no support or 

funding to report. 

 

Competing interests: The authors declare that 

they have no competing interests. 

 

 

 

 

 

 

DNA Variations between Medicago truncatula Symbiotic 

Mutant Line and Native Variant Using Fluorescence-

Based AFLP Marker 

 
Asmaa H. Hassan 

1
, Mohamed A-bouzeid 

2
, and Ghada Abu El-Heba *

1
 

 

Abstract 

      Genetic mutagenesis is a very efficient tool in studying genes 

function. Because of great benefits of legumes as human food and animal 

feed worldwide, we used a model plant Medicago truncatula for 

identification gene function related to nitrogen fixation process. Our 

mutant is a Medicago mutant line contains a tobacco Tnt1 retro-

transposon mobile element with the two Long Terminal Repeats (LTR) 

inserted within the genome. Our mutant is predicted to contain a mutation 

in gene/s belonging to symbiotic interaction between legume and 

rhizobia. A novel technique was used based on using fluorescent 

oligonucleotide primers against oligonucleotide primers for Tnt1-LTRs 

of our mutant. This novel protocol was very successful in detection the 

polymorphism between our mutant line and the wild variant R108 using 

Biosystems 310 Genetic Analyzer. Electropherograms of the mutant line 

and wild type gave a total of 561 well- resolved AFLP peaks, 357of 

which were polymorphic peaks and 204 were monomorphic peaks. This 

novel technique enables the calculation percentage of polymorphism 

between the mutant line and the wild type. Additionally primers 

combinations amplified more bands from others to detect polymorphism 

between the plants.   

 

Keywords: Medicago truncatula, AFLP, Polymorphism, Symbiotic mutant, 

Nitrogen fixation.  

 

Introduction  

Legumes played vital roles in improving agriculture and considered as one-

third of the world’s major crop production. They represent about 27% of the 

world’s crop production. Human consumes approximately 33% of the dietary 

protein needs in the form grains. Many food industries based on legumes like 

treated vegetable, oil production, and chicken industry. Animals depend on 

forage legume plants as major source of food like Medicago stavia and Trifolium 

spp (1). 

This group of plants has unique character as they have ability to make 

symbiotic relationship with soil bacteria called rhizobia. Rhizobia are beneficial 

soil bacteria that belong to gram negative group (2)
 
. In this relation legumes can 

fix nitrogen in specific organ called root nodule (1,3). Nitrogen fixation process 

occurs naturally during growing this family of plants and so we can decrease our 

usage of industrial nitrogen fertilizers and related ecosystem pollution (1).  

Many studies focused on genes responsible for nitrogen fixation in legumes 

and rhizobia (4-9). Lotus japonicus and Medicago truncatula are considered as 

more suitable two plants for studying genes responsible for symbiotic relationship 

between both organisms. 



 

 

Hassan et al., 2020                                                                                                                                              Medicago truncatula symbiotic mutant analysis 

 

 

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Numerous technological platforms like; ethyl 

methanesulfonate (EMS) mutagenesis (10), fast neutron 

bombardment and g-rays (11)
 
were developed and used for 

model plants genome studying. DNA-insertion 

mutantgenesis populations save a rapid method for isolating 

genes responsible for symbiotic nitrogen fixation through 

both reverse and forward genetics (12-14). 

Retrotransposon is a type of mobile elements (class I) 

that transpose to new sites in the genome via an RNA 

intermediate. Their primary transcripts are retro-transcribed 

into DNA copies by a reverse transcriptase (15). Tobacco 

retrotransposon Tnt1 is an effective mutagen in M. 

truncatula that is used for molecular studies worldwide (16). 

This transposone element (Tnt1) belongs to retrotransposon 

elements family (17) , its transposition was induced during 

tissue culture of tobacco protoplast(18) and during in vitro 

transformation of A. thaliana. Tnt1 insertions of  are stable 

in the plant and transfer to the progeny because of the 

replication cycle of LTR retrotransposons (19). 

Samuel Roberts Noble Foundation has the largest 

collection of DNA-insertion mutants of all legumes specially 

Tnt1-insertion mutant populations in Medicago truncatula. 

Numerous mutant phenotypes have been screened such as 

mutant loss the ability to form nodules, mutants produce 

dysfunctional nodules, mutants produce large number of 

nodules and mutants with delayed and decreased nodulation 

but efficient in nitrogen fixation (9). 

Among molecular technique, AFLP protocol provides 

a very powerful DNA finger printing method for nucleic 

acid of any organism (20). Fluorescence-labeled PCR 

products were sensed on an automated DNA sequencer 

instead of the standard polyacrylamide gel electrophoresis 

tool (21-22). Capillary electrophoresis has more advanced 

characters from slab gels in speed, resolution and 

availability of numerical information in an electronic format 

following the end of a reaction. In this study, the genetic 

polymorphism between Medicago truncatula symbiotic 

mutant line and the wild type of the same plant has been 

estimated using amplified fragment length polymorphism 

(AFLP) marker analyses. This mutant contains Tnt1 

reterotrasposon element that have mutation in the gene/s 

involving in nitrogen fixation process. 

Materials and Methods 

Plant materials 

The mutant line of Medicago truncatula was provided 

from Noble Foundation Tnt1 mutant collection that 

transformed from M. truncatula R108-1 ecotype (23). The 

wild type plant R108has been used in this study as a 

reference.  

Seed sterilization and germination  
Seeds of Medicago truncatula wild type and mutant 

line were scratched with sand paper and surface sterilize 

with 6.25% (v/v) sodium hypochlorite for 10 minutes. After 

seven washes using sterilize water, seeds were put on 

Murashige and Skoog (MS) basal medium (Murashige and 

Skoog 1962) supplemented with 0.8% agar (Grade J3, 

Gelita) in dark over night at 29° C. Seedlings were 

transferred to growth chamber with this condition at stable 

21° C during a 16-h and 8-h night photon flux density of 

100 ϻ mol m
-2

s
-1

(24). 

 DNA isolation and quantification 

Total genomic DNA was extracted from mutant plants 

and control for using in AFLP analysis. Healthy leaves from 

both line were used for DNA isolation using Qiagen 

Genomic DNA isolation kit, (Qiagen Science,Valence ,  CA 

, USA). The quality and quantity of Genomic DNA were 

estimated using spectrophotometric measurement of ultra 

violet absorbance at wave length 260 and 280 nm in thermo 

scientific Nanodrop 2000™) and were electrophoresed at 

0.5% agarose gel electrophoresis. 

AFLP protocol 

a) Genomic DNA digestion and ligation 

Amount of genomic DNA from mutant line and wild 

type were digested   with 10u EcoR1 and 10u Mfe1 (New 

England Biolabs, Ipswich, MA, USA). The total volume was 

incubated for three hrs at 37°C then the enzymes were 

inactivated by incubation for 20 mins at 65°C. To assess the 

digestion, 5 μl from each sample were electrophoresed on a 

1.5 % agarose gel. The digested fragments DNA were 

ligated by ECO and ASE adaptor ligation using T4 DNA 

ligase (New England biolabs) to produce modified digested 

fragments that were used as a template DNA for 

amplification. 

c) Pre-selective amplification reaction  

The reaction was performed on ligated DNA using two 

AFLP oligonucleotide primers using TakaRa LA Taq kit. 

Then the product was run on 1.5% agarose gel. 

d) Selective amplification reaction 

Selective amplification was performed using two 

AFLP oligonucleotide primers. The first one was 

fluorescence-labeled primer: Eco (Dye-primer-AXX) and 

unlabeled primer (LTR4&LTR6). Three primer 

combinations were used: E-ACC (Ned)/LTR4, E-ACC 

(Ned)/LTR6, E-AAG (6-Fam)/LTR4, E-AAG (6-Fam)/ 

LTR6, E-AAC (Hex)/LTR4 and E-AAC (Hex)/LTR4. AFLP 

procedure was applied according to (25) with a modification 

which is using three florescence labeled oligonucleotide 

primers (E-AAC, E-AAG, and E-ACC) (20). 1ul of selective 

amplified product was added to 12ul Hi-Di formamide and 

0.5 microliter of GeneScan500 ROX internal size standard 

(Applied Biosystems, Foster City, California, USA). The 

mixture of these components was denatured and inoculated 

to the single capillary of Applied Biosystems 310 Genetic 

Analyzer. Gene mapper analysis software version 4.1 

(Applied Biosystems) was used for analyzing the generated 

electropherograms for mutant and R108 from the raw date 

according to AFLP system analysis getting started guide. 



 

 

Hassan et al., 2020                                                                                                                                              Medicago truncatula symbiotic mutant analysis 

 

 

Highlights in BioScience                                                                                                                                                                           April 2020| Volume 3 

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Results 

Pre-selective amplification was achieved using pre-

selective oligonucleotide primer pair and the product was 

run on 1.5% agarose gel. Successful pre-selective 

amplification was confirmed as a smear product that was 

interrupted by a number of bands and the polymorphism 

cannot be detectable at this stage. AFLP electropherograms 

of mutant line and wild type selectively amplified using 

primer combination as illustrated in (Table 1). 

Oligonucleotide primer combinations examined for selective 

amplification of DNA fragments of mutant line and wild 

type gave a total of 561 well- resolved AFLP peaks of which 

357 were polymorphic peaks and 204 were monomorphic 

peaks. The highest number of amplified DNA fragments was 

218 peaks were obtained with primer combination (E-

AGG/LTR4); however the lowest number of amplified DNA 

fragments was 31 peaks from combination (E-ACC/LTR6). 

AFLP electropherograms of both lines were produced using 

the illustrated primer combination E-ACG/LTR4 (as an 

example) was shown in (Figure 1). 

 

Table 1: Showing that the total number of peaks (TNP), 

number of polymorphic peaks (NPP) and number of 

monomorphic peaks (NMP) were produced by each primer 

combination (PC). 

 

 

Discussion 

Legumes have a fundamental role in sustainable 

agriculture for decades. They are able to reduce the 

atmospheric N2 to ammonia through symbiosis interaction 

with rhizobia which is nitrogen-fixing bacteria. This 

criterion enables legumes to propagate well in soils poor 

with nitrogen without any fertilizer addition. Symbiotic 

Nitrogen Fixation (SNF) in legumes produces nitrogen 

about fifty million tons into the agricultural systems every 

year.  Both legumes and non-legumes production is 

sustained by such nitrogenous compound in the soil (21). 

Legumes are essential source for food and feed (1).  

In our research we used Medicago truncatula which is 

a model plant for studying SNF because it has a small 

diploid genome, short life cycle and it performs symbioses 

with (Sinorhizobium meliloti and Sinorhizobium medicae). 

Mutant collections production was necessary to identified 

genes function.  

Large scale mutagenesis was generated in M. 

truncatula via Tnt1 retrotransposon (23). Tnt1 is a Ty1-

copia type retrotransposon with long terminal repeat (LTR), 

isolated from tobacco (26). Tnt1 transpose via copy & paste 

mechanism 
.
Each mutant line has from four to fifty 

insertions per genome (23). Retrotransposon mediated 

mutagenesis was used in the other plant species like rice 

(27), lettuce (19)
,
 and soybean (28). Samuel Roberts Noble 

Foundation has produced about 21,000 Tnt1 mutant lines 

(23). We used AFLP-type protocol in combination with ABI 

310 Genetic Analyzer to determine the genetic 

polymorphism between our mutant line which is putatively 

defects in nodule development and R108 wild type.

 

Figure 1: AFLP electropherograms of R108 and mutant line amplified selectively by the primer combination E-AAG/lTR6. 

Red row indicates a polymorphic peak example that is present in mutant line and absent in R108.

  



 

 

Hassan et al., 2020                                                                                                                                              Medicago truncatula symbiotic mutant analysis 

 

 

Highlights in BioScience                                                                                                                                                                           April 2020| Volume 3 

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AFLP electropherograms of both lines produced a total 

of 561 AFLP peaks were recorded, 357 of which were 

polymorphic and 204 were monomorphic .This corresponds 

to a level of polymorphism of 63.7%. [The percentage of 

polymorphism was calculated as [(the total No. of 

polymorphic peaks / the total No. of peaks) x 100].   

AFLP has the capability to discover the 

polymorphisms between these types of plants without 

knowing the sequence of genome. So, this is one of the best 

advantages of using AFLP technique. The established 

website (http://bioinfo4.noble.org/mutant/) encloses 

information about 11,000 Tnt1 mutant lines with images of 

diverse phenotypes. Our perspective work will include 

AFLP-type PCR to isolate all the Tnt1-flanking sequence 

tags (FTS) for more mutant characterization and 

identification. All fragments will be analyzed and the 

mutated gene/s will be recognized.  

Acknowledgement 

The authors appreciate Dr. Pascal Ratet for providing 

mutant line & R108 wild type. 

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