Microsatellite analysis in the genome of Acanthaceae: An in silico approach
Priyadharsini Kaliswamy, Srividhya Vellingiri, Bharathi Nathan, Saravanakumar Selvaraj
Department of Plant Molecular Biology and Bioinformatics, Centre for Plant Molecular Biology and Biotechnology, Tamil Nadu Agricultural University, Coimbatore, Tamil Nadu, India
|Date of Submission||09-Jan-2014|
|Date of Acceptance||19-May-2014|
|Date of Web Publication||21-Jan-2015|
Department of Plant Molecular Biology and Bioinformatics, Centre for Plant Molecular Biology and Biotechnology, Tamil Nadu Agricultural University, Coimbatore - 641 003, Tamil Nadu
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Background: Acanthaceae is one of the advanced and specialized families with conventionally used medicinal plants. Simple sequence repeats (SSRs) play a major role as molecular markers for genome analysis and plant breeding. The microsatellites existing in the complete genome sequences would help to attain a direct role in the genome organization, recombination, gene regulation, quantitative genetic variation, and evolution of genes. Objective: The current study reports the frequency of microsatellites and appropriate markers for the Acanthaceae family genome sequences. Materials and Methods: The whole nucleotide sequences of Acanthaceae species were obtained from National Center for Biotechnology Information database and screened for the presence of SSRs. SSR Locator tool was used to predict the microsatellites and inbuilt Primer3 module was used for primer designing. Results: Totally 110 repeats from 108 sequences of Acanthaceae family plant genomes were identified, and the occurrence of dinucleotide repeats was found to be abundant in the genome sequences. The essential amino acid isoleucine was found rich in all the sequences. We also designed the SSR-based primers/markers for 59 sequences of this family that contains microsatellite repeats in their genome. Conclusion: The identified microsatellites and primers might be useful for breeding and genetic studies of plants that belong to Acanthaceae family in the future.
Keywords: Acanthaceae, microsatellites, molecular markers
|How to cite this article:|
Kaliswamy P, Vellingiri S, Nathan B, Selvaraj S. Microsatellite analysis in the genome of Acanthaceae: An in silico approach. Phcog Mag 2015;11:152-6
|How to cite this URL:|
Kaliswamy P, Vellingiri S, Nathan B, Selvaraj S. Microsatellite analysis in the genome of Acanthaceae: An in silico approach. Phcog Mag [serial online] 2015 [cited 2021 Jun 20];11:152-6. Available from: http://www.phcog.com/text.asp?2015/11/41/152/149731
Acanthaceae is one of the major groups in angiosperms and the plants, which belong to this family are mostly tropical herbs, shrubs, and rarely trees.  Acanthaceae family has about 202 genera and 3520 species, and most of this family members are well-known medicinal plants (e.g. Acanthus ilicifolius, Andrographis paniculata, Asteracantha longifolia, Hemigraphis hirta, Justicia adhatoda, Nelsonia canescens, etc.) used in traditional medicine as they possess biologically active phytochemicals. ,, The medicinal plants of this family are enriched with secondary metabolites such as alkaloids, flavanoids, glycosides, steroids, saponins, phenols, and tannins. The phytocompounds obtained from these plants are used to treat diarrhea, snake bite, fever, inflammation, hypertension, diabetes, malaria, jaundice, and rheumatism. ,,
Simple sequence repeats (SSRs) or microsatellites are the short DNA sequences with 1-6 base pairs of length. [ 5 ] Several studies suggest that the abundance of SSRs were present in noncoding regions of the genome sequences.  These repeats have a wide application in the field of plant genetics and breeding as they have multi-allelic, reproducible and co-dominant inheritance properties.  The microsatellites were identified in the genomes of many medicinal plants such as Humulus lupulus (hop),  Ocimum basilicum (basil)  and also for some economically important plants including Oryza sativa (rice),  Glycine max (soybean),  Hordeum vulgare L. (barley), Triticum aestivum (wheat), Solanum lycopersicum (tomato), Vitis vinifera (grape), and Helianthus annuus (sunflower).  The SSRs also provide basis for the development of SSR-based markers that have a wide range of application in genetic research including studies of genetic variation, linkage mapping, gene tagging, and evolution. 
The objective of this study includes identification of microsatellites and its associated primers in the genome of Acanthaceae family. In the current study, the different types of SSRs and its distribution from 108 sequences and
59 associated primers were identified. We also reported that the amino acid isoleucine was enriched in all the above SSRs in the genome sequences of Acanthaceae family.
| Materials and methods|| |
Nucleotide sequence retrieval
Totally 4077 nucleotide sequences were retrieved from National Center for Biotechnology Information for Acanthaceae family plants. 
Identification of simple sequence repeat motifs
In order to identify the microsatellites from the genome of Acanthaceae family, the SSR Locator computing tool was used.  It is integrated with the functions such as primer design and PCR simulation.  The sequences were searched for mono-, di-, tri-, tetra-, penta- and hexa-types of SSR motifs with number of repeats 20, 10, 7, 5, 4, and 4, respectively. The enriched amino acids in the predicted SSRs were also identified using the above mentioned SSR Locator tool.
Simple sequence repeat-based primer designing
The in silico based primers for each microsatellite containing nucleotide sequences were designed using Primer3, the interface module in the SSR Locator.  The default parameters such as length of the primer (15-25 base pairs), melting temperature (45-55°C) and GC content (45%) were specified for primer designing.
| Results|| |
Simple sequence repeat identification and distribution in the genome
The SSR motifs were identified from 4077 nucleotide sequences of the Acanthaceae family plants with 31,41,420 base pairs. Totally we found 110 SSR motifs from 108 nucleotide sequences using SSR Locator tool. The microsatellites such as mononucleotide (32%), dinucleotide (50%) and tetranucleotide (11%) were found abundant in number in Acanthaceae family plants. The trimer, pentamer and hexamer motifs occurred comparatively less (about 2%) in the nucleotides. A summary of the predicted SSRs in the sequences was shown in [Table 1]. The distribution of SSR motifs in the Acanthaceae genome was shown in [Figure 1].
|Figure 1: Frequency of simple sequence repeat motifs in the nucleotide sequences of Acanthaceae family plant genomes|
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|Table 1: Summary of the screened microsatellites in the genome of Acanthaceae |
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The mononucleotides, including A and T were found abundant in the Acanthaceae family genomes. We also found eight different dinucleotide repeats (TG, AC, CA, AG, GT, TC, GA, and CT), which were found in about 50% of this genome. TCA, ATC, CAT were identified as trinucleotide repeats and the ATC repeat showed a higher frequency. There were nine tetranucleotide repeats such as AGAC, TATG, ATAC, ACAT, GTAT, TAGA, TATT, CTAT and ATAG with the frequencies of 5, 12, 9, 11, 8, 5, 5, 7, and 5, respectively. The occurrences of pentanucleotide and hexanucleotide are comparatively less in number. TTGAT, a pentanucleotide and two hexanucleotides TTTCTT and TATATC were found in the Acanthaceae family genomes with frequencies of 8, 4, and 5, respectively. The distribution of above mentioned repeats (from mono- to tetra-) were shown in [Figure 2].
|Figure 2: Frequency distribution of (a) mononucleotide, (b) dinucleotide, (c) trinucleotide, and (d) tetranucleotide repeats in the genome of Acanthaceae|
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Frequency of amino acids in the microsatellites
The combination of three nucleotides/triplet codon generally codes for a specific amino acid type. In this study, we found that the trinucleotide repeat ATC was enriched in this genome and it predominantly codes for the essential amino acid isoleucine. The other SSR motifs encode the amino acids including histidine, serine, tyrosine, phenylalanine, leucine, arginine, and lysine that occupied the next levels of this genome. The types of SSRs along with codon and its specific amino acids were shown in [Table 2].
|Table 2: Microsatellites and its corresponding amino acids in the genome sequences of Acanthaceae family plants |
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Simple sequence repeat-based primers/markers
The designing of SSR-based primers/markers through experiments are costly and time consuming as well. Hence, using in silico primer designing methods, 59 out of 108 nucleotide sequences exhibited the primers. The designed forward and reverse primers of this genome also have adequate annealing temperature and GC content as well. The nucleotide accession numbers, forward and reverse primer sequences, melting temperature and product size of the designed primers were given in [Table 3].
| Discussion|| |
The genome sequences of Acanthaceae family plants were retrieved and examined to know the occurrence of microsatellite types, characteristics and distribution. The length of microsatellites 1-6 base pairs were taken into consideration. Totally, we screened 110 SSRs from 108 out of 4077 nucleotide sequences. The occurrence of dinucleotide repeats was comparatively higher than all other repeat types including mono-, tri-, tetra-, penta-, and hexa-repeats and the same repeats pattern was reported in Arabidopsis thaliana., Among mononucleotides, A/T repeat was found to be abundant than C/T repeat in which repeat A (94%) is higher than T repeat. The dinucleotide repeat AG showed a higher frequency (28%) among eight other dinucleotide repeats such as TG (17%), TC (12%), GT (11%), GA (9%), AC (8%), CA (8%), and CT (7%) in the Acanthaceae family plants. The studies on medicinal plant O. basilicum and other plants, including Arabidopsis thaliana, T. aestivum, H. vulgare L., O. sativa, Zea mays, and Prunus dulcis also proposed that the frequency of AG repeats was higher than other dinucleotide repeats.  The trinucleotide repeat ATC (52%) was observed high in number than other trinucleotide repeats TCA (14%) and CAT (14%). The same trinucleotide repeat pattern was reported in Gossypium (cotton) (Gossypium arboretum, Gossypium raimondii, and Gossypium hirsutum) and Arabidopsis thaliana genomes. , The AT rich repeats were abundantly found in tetranucleotides of this family genome. The penta- and hexa-repeats were found in less frequency compared with other repeat types. We also identified that the repeat ATC, which codes for the essential amino acid isoleucine were found to be enriched in trinucleotide to hexanucleotide repeats and the amino acids serine and histidine were seen in less frequency in this genome. It was reported that the same amino acid distribution pattern was seen in the genome of H. lupulus.  The amino acid isoleucine plays a major role in maintaining blood sugar level. Therefore the Acanthaceae species such as Adhatoda vasica, A. paniculata, A. longifolia, Barleria cristata, Barleria noctiflora, Barleria prionotis, Dipteracanthus prostrates, Jacobinia suberecta, and Strobilanthes crispus have been used to treat diabetes in the traditional system of medicine as they contain enriched isoleucine in their genomes.  Moreover, the plants such as Hygrophila auriculata, N. canescens, and Peristrophe bicalyculata were also used to treat various infectious diseases such as diarrheal diseases, cholera, typhoid fever, and tuberculosis.  The SSR-based primers/markers have an extensive application in plant genetics and breeding. Hence, in our study, the potential SSR-based forward and reverse primers were designed for 59 out of 108 nucleotide sequences of the Acanthaceae species including Avicennia germinans, Avicennia alba, Blepharis subvolubilis, Ruellia ciliatiflora, Ruellia nitida, Ruellia eurycodon, Ruellia pedunculosa, Aphanosperma sinaloensis, Kalbreyeriella rostellata, Aphelandra verticillata, A. paniculata, and A. ilicifolius.
| Conclusion|| |
Microsatellites or SSRs play a major role in polymorphism analysis and in marker assisted selection. In silico approach for predicting SSRs in the whole genome, was found to be both cost and time effective and also helps to develop a new generation of molecular markers as well. In our study, the microsatellites and its associated primers were identified for the publically available Acanthaceae family genomes using computational methods. The identified microsatellites and markers might pave the way for further studies in the aspect of breeding and genetic studies of the plants that belong to the family Acanthaceae.
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[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3]