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Genetic Divergence Study in Grewia Optiva through Quantitative and Molecular Markers

Shikha Bhagta, Poonam Thakur and Dushyant Sharma

  • Page No:  029 - 033
  • Published online: 28 Feb 2021
  • DOI: HTTPS://DOI.ORG/10.23910/2/2020.0396

  • Abstract
  •  bhagtashikha@gmail.com

Diversity analysis amongst 10 different families of Grewia optiva was carried out by using RAPD (random amplified polymorphic DNA) and ISSR (inter simple sequence repeats) marker. Grewia optiva families were raised by seed collected from various districts of Himachal Pradesh (India) and selected based on morphological parameters. Using 15 RAPD and 20 ISSR primers and 9 RAPD and 12 ISSR primers show amplification respectively. Nine RAPD primers showed 68.96% polymorphism and 12 ISSR primers showed 71.25% polymorphism. Similarity matrices and Dendrograms were generated using SAHN module of NTSYSpc ver.2.02h. Jaccard’s similarity matrix revealed maximum similarity coefficient 0.88 between ‘SO-7’ and ‘SO-3’ with RAPD primers. For ISSRs, coefficient values ranged from 0.52 to 0.80. Dendrograms also revealed to larger extent similar results and maximum similarity found among the 10 families of Grewia optiva collections was 88% between ‘SO-7’ and ‘SO-3’with RAPD primers and 80% between ‘SH-7’ and ‘SO-4’ with ISSRs. RAPD and ISSR were found effective in revealing polymorphisms among 10 different genotypes of Grewia optiva. UPGMA based dendrograms of both RAPD and ISSR confirmed the placement of different genotypes into different clusters and sub clusters as per geographic distribution and genetic constitution. Family SH-7 came as outliner as revealed by both RAPD and ISSR study.

Keywords :   Fodder tree, genetic diversity, Grewia optiva, ISSR, RAPD

  • Introduction

    The genus Grewia consists of some 150 species in world, out of which 42 species are found in Indian subcontinent whereas five species of genus Grewia are found in Himachal Pradesh. Grewia optiva (local name: Beul; 2n=18) belonging to family Tiliaceae is one of the most important fodder tree species found in Himachal Pradesh. G. optiva is very popular agroforestry tree of low and mid-hills regions in the western and central Himalaya on account of its utility as fodder, fuel and fiber. It is naturally distributed in India, Bhutan, Nepal and Pakistan. In India, it is distributed in areas of Himachal Pradesh, Jammu and Kashmir, Punjab, Sikkim and Uttar Pradesh (Hooker, 1875). It occurs at elevations from 500-2100 meters, where the temperature ranges from 2° to 38°C and rainfall from 1,200 to 2,500 mm in the year (Joshie and Narain, 1992).

    With the increase in demand for fodder, there is a need to select superior genotypes for inclusion in breeding program develop clones of genetically superior trees. Therefore, it is essential to understand the genetic architecture of Grewia optiva, which provides useful guidelines to determine the source population and from which it is possible to derive appropriate genotypes with desired characters. Use of molecular markers facilitate breeding processes, since it can provide means of detecting and resolving complications and accelerate the generation of new varieties and allow association of phenotypic traits with genomic loci.Molecular markers not only help in studying genetic diversity but also allow the easy and reliable identification of breeding lines, hybrids and cultivars. Because F1 hybrids contain DNA from both parents, identification of male and female parent specific markers will allow differentiation of true hybrids. In this regard, in recent years molecular techniques are providing a useful tool for the correct identification of plant species, included hybrid taxa. In particular, DNA markers have been often used for hybrid characterization in tree species (Dumolin et al., 1995). Among these, RAPDs and ISSRS have been the preferred markers for finger printing tree species. RAPDs are fragments of genomic DNA amplified through PCR using a decamer primer of random sequence, where polymorphism depends upon the presence or absence of an amplification product. The use of RAPDs in different organisms began in the late 80’s (Williams et al., 1990), and due to their simplicity and speed they have become a very valuable tool for cultivar identification and genetic similarity studies in plants. On the other hand, ISSR markers share most of the advantageous features of RAPD markers with the addition of a potentially co-dominant pattern of inheritance (Zietckiewicz et al., 1994) and have been considered a promising source of a large number of reliable, highly-polymorphic markers (Salimath et al., 1995).


  • Materials and Methods

    In the present study, top ten best performing families (Table 1) out of forty families of G. optiva evaluated for morphometric and fodder quality parameters were evaluated for molecular diversity through molecular markers. Fresh and disease free leaves were collected from the selected ten trees in seedling seed orchard of Grewia optiva. Fresh, green leaves were separately excised from different plants. Before plucking, the leaves were wiped off the soil with tissue paper and then wrapped in aluminum foil and brought to the laboratory in icebox and stored in deep freezer at -80oC till further use.


    Genomic DNA from the collected leaves of top ten families separately isolated using CTAB method of Doyle and Doyle (1987) with some modifications wherever required.

    2.1.  RAPD amplification

    DNA amplification was carried out for RAPD analysis using fifteen decamer random primers. DNA was amplified by PCR amplification reaction. The 25 μl of reaction mixture contained 4 μl of DNA (5 ng μl-1), 0.25μl of Taq DNA Polymerase (3U μl-1), 2.5 μl of Taq buffer (10X), 1.25 μl of dNTPs (2.5 mM), 2.0μl of Primer (10 ng) and 15 μl of sterile distilled water. PCR condition for RAPD amplification included initial denaturation for 3min at 94°C followed by 45 cycles of amplification (denaturation at 92°C for 45 seconds, annealing of primer at 36°C for 1 min and primer amplification at 72°C for 2 min) and final extension at 72°C for 10 min.

    2.2.  ISSR amplification

    Fifteen ISSR primers synthesized by M/S Banglore Genei, India Limited were used in the current study. DNA was amplified by PCR amplification reaction. The 24 μl of reaction mixture contained 4μl of DNA (5ng μl-1), 0.25 μl of Taq DNA polymerase (3U μl-1), 1.0μl of primer (10 ng), 1.25 μl of dNTPs (2.5 mM), 2.5μl of Taq buffer (10X) and 15μl of Sterile distilled water. PCR condition for ISSR amplification included initial denaturation for 3 min at 94°C followed by 45 cycles of amplification (denaturation at 92°C for 45 seconds, annealing of primer at 55°C for 1min and primer amplification at 72°C for 2min) and final extension at 72°C for 10 min. The amplified DNA was mixed thoroughly with 6X loading dye and then electrophoresed in 2% agarose gel in 1X TAE buffer. The gel was run at constant voltage at the rate of 5V cm-1 under submerged conditions for about two hours. Ethidium bromide at the rate of 0.5 µg ml-1 was incorporated in the gel. Stock solution of ethidium bromide @ 10 mg ml-1 was kept ready. DNA profiles were visualized on UV Transilluminator and photographed on Gel Documentation System (Syngene, Cambridge, UK).

    2.3.  Scoring of bands and data analysis

    The scored bands were analyzed in the form of binary system to prepare the similarity index. The bands with same molecular weight and mobility were treated as identical fragments. Data matrices were prepared in which the presence of a band was coded as one whereas the absence as zero. The data matrices were analyzed by the SIMQUAL Program of NTSYS-PC (Version 2.2) and similarities between Families were estimated using Jaccard similarity coefficient, calculated as J = A ÷ (N-D), where A is the number of positive matches (i.e. presence of band in both samples), D is the number of negative matches (i.e. absence of band in both samples) and N is the total sample size including both the number of matched and unmatched. Dendrogram was produced from the resultant similarity matrices using the UPGMA method.


  • Results and Discussion

    3.1.  RAPD (Random amplified polymorphic DNA) studies

    After initial screening of 15 RAPD, nine RAPD primers producing intense banding pattern and showing polymorphism were used for further study. Total of 29 amplified bands were scored with 9 primers in 10 families of Grewia optiva. Six polymorphic and 4 monomorphic bands were observed. Amplification pattern of the primers maximum number of amplified bands i.e. five were produced by primers ‘P1’and ‘P4’ whereas minimum number of bands i.e. one was produced by primer P9. Out of the total 29 scorable bands, 20 showed polymorphism and 9 bands exhibited monomorphism resulting in 68.96% polymorphism among ten families. Five of nine primers exhibited 100% polymorphism.   

    3.1.1.  RAPD data analysis

    The data matrix so obtained was analyzed with NTSYS- 2.2 software to obtain the Jaccard’s similarity correlation coefficient. The mean coefficient value of any families or accession gave an idea about its overall relatedness with all other families or accession in the study. The coefficient values ranged from 0.413 (among the family HA-2 and SI-14) to 0.875 (among the family SO-7 and SO-3). This indicated a fair range of variability in the similarity coefficient values suggesting a broad genetic base of ten accessions included in the experiment. The above finding are in agreement with Qi et al. (2003), who reported screening of twenty-five primers from 119 random primers in jute, and a total of 329 DNA fragments were amplified ranging from 0.3-3.0 kb, 253 (87.78%), which were polymorphic. Similar results were also revealed by Vaishali et al. (2008) who reported that out of total 145 fragments generated by random decamer primers, 126 (86%) were polymorphic with an average of 10 polymorphic products primer-1. The number of products amplified by the polymorphic primers varied from 8-17. Similarly, Wang et al. (2011) screened 16 decamer primers that showed polymorphisms within five populations of Dalbergia sissoo used, and that generated 101 bands ranging in molecular size 200 to 1700 bp (Table 2).


    In the dendrogram (Figure 1), the 10 families separated into two main clusters, ‘I’ and ‘II’, at 59% similarity. This revealed less similarity between cluster I and cluster ‘II’. Cluster ‘II’ was further subdivided into two clusters i.e. IIa and IIb at similarity value of 70%. It was concluded that ‘S0-3’ and ‘SO-7’ were closely related as they showed 88% similarity.


    3.2.  Inter simple sequence repeat (ISSR) studies

    Initial screening of 20 ISSR primers 12 primers produced ISSR profiles with intense banding pattern, which showed polymorphism between 10 accessions used in the study. ISSR analysis revealed high levels of genetic diversity within the reference set of G. optiva families. Out of the total 74 scorable bands, 57 showed polymorphism and 17 bands exhibited monomorphism. Total number of amplified and polymorphic fragments generated per ISSR primer revealed 71.25% polymorphism among families (Table 3).


    3.2.1.  ISSR data analysis

    The similarity coefficient values ranged from 0.52 to 0.80. This indicated a fair range of variability suggesting a broad genetic base of thirty accessions included in the experiment. The highest value (0.80) was found between SH-7 and SO-4. The lowest value of 0.52 was exhibited between SI-15 and SO-7, SI-15 and HA-2 depicting that the families were more diverse respectively.

    In the dendrogram (Figure 2), the 10 families separated into two main clusters, ‘I’ and ‘II’, at 60% similarity. Cluster ‘I’ contained only one family i.e. SH-7 ‘and Cluster‘II’ accommodated rest nine families. This revealed less similarity between cluster I and cluster ‘II’. Cluster ‘II’ was further subdivided into two clusters i.e. IIa and IIb at similarity value of 63%. Families ‘SO-3’ and ‘SO-7’ were closely related as they showed 80% similarity.

    ISSR markers have been successfully used for varietal identification and assessment of genetic relationships in many plant species (Ajibade et al., 2000). In a similar study by Chatterjee et al. (2004) in Morus alba,ten ISSR primers generated a total of 58 bands, out of which 43 were polymorphic, thus generating 74.13% polymorphism. In contrast, Verma (2012) in the study of Grewia optiva showed that RAPD primers revealed more DNA polymorphism (96.31%) among the genotypes than ISSR primers (91.72%).


  • Conclusion

    RAPD and ISSR were found effective in revealing polymorphisms among 10 different families of Grewia optiva. UPGMA based dendrograms of both RAPD and ISSR confirmed the placement of different genotypes into different clusters and sub clusters as per both RAPD and ISSR study.


    Reference

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    Vaishali, Khan, S., Sharma, V., 2008. RAPD based assessment of genetic diversity of Butea monosperma from different agro-ecological regions of India. Indian Journal of Biotechnology7, 320−327.

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    Wang, B.Y., Shi, L., Ruan, Z.Y., Deng, J., 2011. Genetic diversity and differentiation in Dalbergia sissoo (Fabaceae) as revealed by RAPD. Genetics and Molecular Rresearch 10, 114−120.

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Cite

1.
Bhagta S, Thakur P, Sharma D. Genetic Divergence Study in Grewia Optiva through Quantitative and Molecular Markers IJEP [Internet]. 28Feb.2021[cited 8Feb.2022];8(1):029-033. Available from: http://www.pphouse.org/ijep-article-details.php?art=267

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