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Applicability of Different Molecular Markers Techniques for Genetic Distinguish Between Two Genera Cressa Linn. and Cuscuta Yunck. Family Convolvulaceae



Asmaa Amer, Hussein Taha, Nagwa Ammar, Maha Salama and Taha El-Alfy
 
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ABSTRACT

Background and Objective: The DNA fingerprinting is used to determine the relationship between species in the same genus or between genera related to the same family. The aim of this study was to determine the relationships between two samples related to the same family Convolvulaceae, representing two genera, Cressa Linn. and Cuscuta Yunck. by RAPD, ISSR and SCoT molecular techniques (PCR based DNA fingerprint). Materials and Methods: The RAPD, ISSR and SCoT based DNA fingerprinting techniques were implemented to identify the fingerprint diversity between two genera, Cressa Linn. and Cuscuta Yunck-belonging to the family Convolvulaceae. Results: Applying of RAPD technique revealed that using OP-A02, OP-A09, OP-A10, OP-C04 and OP-M01 primers recorded 60, 83.33, 100, 50 and 70.66% polymorphism, respectively. On the other hand, ISSR technique recorded 40, 50, 100, 66.67, 33.33 and 37.5% polymorphism with 44B, HB-08, HB-09, HB-10, HB-11 and HB-12 primers, respectively. However, amplification of SCoT technique, SCoT 1, SCoT 2, SCoT 3, SCoT 4, SCoT 6, SCoT 8, SCoT 10 and SCoT 12 primers recorded 33.33, 28.57, 14.28, 66.66, 25, 40, 42.85 and 50%, respectively . The total polymorphism recorded 73.33, 54.58 and 37.7% for RAPD, ISSR and SCoT techniques, respectively. Conclusion: The result of this study indicated that SCoT technique was more efficient and sustainable for distinguish between two genera under investigation.

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  How to cite this article:

Asmaa Amer, Hussein Taha, Nagwa Ammar, Maha Salama and Taha El-Alfy, 2018. Applicability of Different Molecular Markers Techniques for Genetic Distinguish Between Two Genera Cressa Linn. and Cuscuta Yunck. Family Convolvulaceae. Pakistan Journal of Biological Sciences, 21: 179-186.

DOI: 10.3923/pjbs.2018.179.186

URL: https://scialert.net/abstract/?doi=pjbs.2018.179.186
 
Copyright: © 2018. 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.

INTRODUCTION

Deoxyribonucleic acid (DNA) fingerprinting is considered as one of the most important tools for genetic identification in plant breeding and germplasm management of cultivar or varietal identification1,2. It is also used to study and characterize relationships between different plant genera in the same family or different species and to measure genetic diversity among genotypes of the same species, based on the inherent DNA polymorphism present among those genotypes3,4. Hybridization-based fingerprinting and polymerase chain reaction (PCR) were developed by Williams et al.5.

Random amplified polymorphic DNA (RAPD) marker is easy technique, quick and not requires information about the sequence. The method depends on the amplification of random genomic DNA fragments by arbitrarily selected primers. The generated patterns depend on the sequence of the PCR primers and the nature of the template6,7. Amplification product derived from a region of the genome having two short DNA segments with some homology to the primer, must be present on opposite DNA strands and sufficiently close to each other to allow DNA amplification8-10. Polymorphism is then observed and scored as the presence or absence of a fragment and relates to sequence variation due to nucleotide insertion, deletion or substitution11,12. The homozygous presence of fragment is not distinguishable from its heterozygote and hence, RAPDs are dominant markers. RAPD polymorphism results from mutation or rearrangements at or between oligonucleotide primer binding sites in the genome were indicated. Such polymorphism behave as dominant genetic markers13-15. Inter Simple Sequence Repeat (ISSR) amplification is another method, which a marker system now referred to. This makes the usage of anchored primers to amplify simple sequence repeats without the requirement for prior sequence information16,17. This technique is more reliable than the RAPD technique and generates larger numbers of polymorphisms per primer18. Theoretically, polymorphisms should be easier to detect because variable regions in the genome are targeted16,19,20.

Start codon targeted (SCoT) polymorphism is recommended as a new, simple and reliable gene-targeted marker technique based on the translation start codon21,22. Their markers are more reproducible than RAPD and ISSR. It is reported that this primer has been used in genetic diversity analysis in a number of plant species23-27. The SCoT markers were successfully utilized to analyze the genetic diversity of rice21, peanut28, mango29-31, potato22, grape25, Lycoris32 and Dendrobium nobile33 for their highly polymorphism and efficiency.

The genus Cressa is perennial herbaceous plant34-36. Cressa cretica Linn. is a remarkable salt tolerant plant, common in coastal areas and represent the genus37. It is used in folk medicine as tonic, stomachic, anthelmintic and in the treatment of hepatic disorders, urinary discharges, diabetes, asthma, ulcers, constipation and also in aphrodisiac purposes38. Traditionally, the plant is used as expectorant, emetic and antibilious agent in Bahrain and Sudan39.

The genus Cuscuta is a widespread parasitic weeds, occurring in both crop- cultivated and non-crop wild areas40. Cuscuta seeds have been used as a Chinese medicine for many years. However, there has been little scientific investigation into the actual effectiveness of its use41. Among this genus Cuscuta campestris Yunck.42. which is known by its Arabic name Al-hamol34. It is used in traditional Chinese medicine and in popular medicine for the treatment of fresh wounds, hepatic, stomach and urinary tract disorders43.

This study aimed to determine the relationships between two samples related to the same family Convolvulaceae, representing two genera, Cressa Linn. and Cuscuta Yunck. Three molecular techniques (PCR based DNA fingerprint), RAPD (5 primers), ISSR (6 primers) and SCoT (8 primers) were adopted to identify the molecular basis between the two genera under investigation.

MATERIAL AND METHODS

This investigation was carried out in Plant Biotechnology Department, National Research Centre, Cairo, Egypt during the period from January, 2017-March, 2018.

Plant materials: Cressa cretica Linn. aerial parts was collected from the road to Qaron lake, El-Faiyum, Egypt. Cuscuta campestris Yunck. aerial parts was collected in flowering stage as a parasite on Ipomoea cairica L. sweet from Banha, Minia el kamh road, Kaliobeya Governorate, Egypt. These plants were kindly authenticated by Dr. Abd Elhalim Abd El Motgali, Professor of flora and phyto taxonomy researches, Horticultural Research Institute, Agriculture Research Centre, Dokki, Cairo (Egypt). They have voucher specimens numbers 23-2-2015 A and B, respectively. These plants are kept in the herbarium of the Pharmacognosy Department, Faculty of Pharmacy, Cairo University.

DNA extraction: Samples of DNA were prepared, extracted and performed according to the described method by Dellaporta et al.44.

Polymerase chain reaction (PCR)
RAPD-PCR analysis: In this process, various factors were optimized and performed according to Williams et al.5. However, only five primers were succeeded to generate reproducible polymorphic DNA products.

ISSR and SCoT-PCR analysis: The PCR amplification was completed and performed using random 10 mer arbitrary primers synthesized by Operon biotechnologies, Inc. (Germany). In order to obtain clear reproducible amplification products, different preliminary experiments were carried out in which a number of factors were optimized as followed method by Williams et al.5. In PCR reaction, only six and eight primers were succeeded to generate reproducible polymorphic DNA products for ISSR and SCoT techniques, respectively.

Statistical analysis: The DNA bands generated by each primer were counted and their molecular sizes were compared with those of the DNA markers. The bands scored from DNA profiles generated by each primer were pooled together. Then the presence or absence of each DNA band was treated as a binary character in a data matrix (coded 1 and 0, respectively) to calculate genetic similarity among the studied samples45.

RESULTS

The RAPD, ISSR and SCoT banding profiles produced by using primers in the two examined samples of plants, Cressa cretica Linn. and Cuscuta campestris Yunck. were illustrated in Fig. 1 and Table 1-3.

The RAPD banding profiles produced by the five primers OP-A02, OP- A09, OP-A10, OP-C04 and OP-M01. Illustrated data in Table 1 showed that using OP-C04 primer presented 8 bands giving zero monomorphic and 8 polymorphic bands with 100% polymorphism between genera Cressa and Cuscuta. While, OP-A10 showed 6 bands illustrated 1 monomorphic and 5 polymorphic bands producing 83.33% polymorphism. On the other hand, OP-M01 showed 6 bands resulted into 3 monomorphic and 3 polymorphic bands with 50% polymorphism. The total polymorphism after applying the RAPD method was found 73.33% between the two genera under study.

Table 1: Primers sequence, total band, monomorphic and polymorphic percentage of polymorphism revealed by the five 10-mer primers using RAPD technique

Table 2: Percentage of polymorphism revealed by the five 10-mer primers using ISSR technique

Table 3: Percentage of polymorphism revealed by the five 10-mer primers using SCoT technique

Regarding ISSR banding profiles produced by the six primers, 44B, HB-08, HB-09, HB-10, HB-11 and HB-12, in the two examined samples of Cressa Linn. and Cuscuta Yunck were illustrated in Table 2.

From Table 2 primer, HB-09 showed 8 bands giving zero monomorphic and 8 polymorphic bands with 100% polymorphism between genera Cressa and Cuscuta, while HB-10 showed 6 bands giving 2 monomorphic and 4 polymorphic bands resulting in 66.67% polymorphism. However, HB-11 showed 9 bands giving 6 monomorphic and 3 polymorphic bands indicating 33.33% polymorphism. The total polymorphism after applying the ISSR method was found 54.58%.


Fig. 1: RAPD, ISSR and SCoT amplification electrophoretic profile of Cressa cretica Linn. and Cuscuta campestris Yunck
  M: DNA marker (1 Kb ladder) 1: Cressa cretica Linn., 2: Cuscuta campestris Yunck

Table 4: Summary of the generated data by RAPD, ISSR and SCoT techniques

The SCoT amplification Technique resulting in banding profiles produced by the eight primers were illustrated in Table 3.

From Table 3 primers, SCoT 4 showed 9 bands giving 3 monomorphic and 6 polymorphic bands with 66.66% polymorphism between genera Cressa and Cuscuta. While SCoT 1 showed 9 bands giving 6 monomorphic and 3 polymorphic bands with 33.33% polymorphism. On the otherwise, SCoT 6 producing 4 bands with 3 monomorphic and 1 polymorphic bands indicating 25% polymorphism. The summary of the amplification of the three techniques for making DNA analysis to assess the genetic diversity between the two genera under investigation were presented in Table 4.

DISCUSSION

The obtained results of this study revealed that SCoT technique shows 37.7% polymorphism between the two genera under investigation, Cressa Linn. and Cuscuta Yunck. belong to the same family, Convolvulaceae recording more efficient than that of other techniques, RAPD and ISSR (73.33 and 54.58%), respectively. The extracted results were firstly done, since no reports could be traced concerning this study based on molecular markers. These techniques; RAPD, ISSR and ScoT were previously used to investigate the polymorphism between cultivars of other plants.

The RAPD and ISSR markers46 have been used extensively in genetic analysis of prokaryotes and eukaryotes. Identification of many crops including potato can be achieved using RAPD technique47-50. In this respect and in agreement of current extracted results, RAPD, ISSR, Simple Sequence Repeat (SSR) and Amplified Fragment Length Polymorphism (AFLP) were reported to be successfully distinguished between 39 potato cultivars, respectively51.

Moreover, primers of ISSR technique have high efficiency power in fingerprinting and diversity analysis for many crops being easy to generate, inexpensive and powerful in detecting polymorphisms52,53. In close of present obtained results ISSR was reported54,55 to be useful in detecting genetic diversity and population structure of coffee, teff, lentils and barley than RAPD technique.

In similarity, SCoT markers were proved to be useful in evaluating the genetic relationship among different cultivars and showed high level of polymorphism21,22,25,31,33,56,57. Moreover, Genetic diversity had been assessed through molecular data provided by application of SCoT markers as being an efficient and inexpensive way in DNA analysis.

CONCLUSION

This study is the first record to distinguish between the two genera under investigation related to the same family. Furthermore, it assessed the ability of the RAPD, potentially useful ISSR marker and efficiency of SCoT systems to distinguish the studied samples and make comparison based on fragment polymorphism. The obtained results of RAPD technique showed a high percentage of polymorphism (73.33%), while ISSR and SCoT techniques resulted in lower percentage (54.58 and 37.7%) than the other, respectively.

Finally, it can be recommended that SCoT technique is the most efficiency to distinguish between two genera, Cressa Linn. and Cuscuta Yunck. based molecular analysis than ISSR and RAPD techniques, consequently.

SIGNIFICANT STATEMENT

This study discovered the applying of SCoT as molecular technique that can be beneficial for genetic diversity between two genera; Cressa Linn. and Cuscuta Yunk. belong to the same family, Convolvulaceae rather than RADP and ISSR techniques. Further, this study will help the researchers to uncover the critical areas of genetic bands which related to the same family that many researchers were not able to explore. Thus a new theory on genetic distinguish between different genera belong to the same family may be arrived at.

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