The Journal of Agricultural Sciences - Sri Lanka Vol. 14, No. 1, January 2019. Pp 8-16 http://dx.doi.org/10.4038/jas.v14i1.8452 Open Access Article The use of Microsatellite Markers in Genetic Variation Analysis of Some Introduced Rubber Tree (Hevea brasiliensis) Clones Cultivated in Ghana A. Antwi-Wiredu1*, S. Amiteye2, A. T. Asare2, R. Kusi-Adjei2, R. G. Diawuoh2, C. O. Aryee3, and G. Y. P. Klu4 Received: 28th March 2018 / Accepted: 25th April 2018 ABSTRACT Purpose: Genetic makers are indispensable in the genetic quantifi cation and characterization of plant species of which rubber tree is no exception. Markers have been used severally in genetic identifi cation and diversity analysis of rubber tree species. However, in Ghana genetic diversity and relatedness among introduced rubber tree species are limited. The study is aimed to use microsatellite markers to assess the genetic variability and evaluate genetic relationships among clones of Hevea brasiliensis cultivated in Ghana. Research Method: Genomic DNA was extracted from fresh rubber leaves following the Cetyltrimethyl Ammonium Bromide (CTAB) protocol. The fi ve (5) diff erent SSR primers used for the analysis were hmct1, hmac4, hmct5, gSSR212 and gSSR194. Findings: The fi ve SSR markers showed some degree of relatedness among the rubber clones which suggested genetic similarity (IRCA317-5, IRCA41-2, IRCA331-6, IRCA230-4, IRCA109-3, and B8-23) and diversity (K2-18, IRCA840-7, PB217-8, PB217-10, and IRCA317-16) among the clones irrespective of their geographical area of collection. Research Limitation: The unavailability of polivinylpolipyrolidon (PVPP) and polyacrylamide gel (PAGE). Original Value: The results from the study depict that relatively some rubber clones of interest could be selected for future breeding and propagation programmes in Ghana. Keywords: Microsallites, Diversity, Similarity, Markers, Rubber Clones INTRODUCTION In order to ensure a successful breeding This is particularly important in rubber tree programme, information about the germplasm where there is lack of useful morphological diversity and genetic relatedness is very traits for clonal distinction (Besse et al., 1993). necessary (Mukhtar et al., 2002). Information of such nature can be obtained from pedigree 1* Forestry Research Institute of Ghana, P. O. Box UP 63, analysis, morphological traits characterization KNUST-Kumasi, Ghana. or molecular marker analysis (Mohammadi tonysnas@gmail.com and Prasanna, 2003). There is a limitation in ORCID http://orcid.org/0000-0002-1473-09662 Biotechnology and Nuclear Agriculture Research Institute, genetic diff erentiation in plant species based Ghana Atomic Energy Commission, P. O. Box LG 80, Legon- on morphological characterization. Thus, the Accra. Ghana. use of molecular markers presents a more 3 Cocoa Research Institute of Ghana, P. O. Box 8, New Tafo- promising technique for easy resolution of Akim, Ghana. genetic diff erentiation. 4 School of Nuclear and Allied Sciences, University of Ghana, P. O. Box AE1, Atomic Energy-Accra. Ghana. 8 The Journal of Agricultural Sciences - Sri Lanka , 2019, Vol.14, No. 1 Genetic markers are useful in labeling and species (Lorieux et al., 2000). Between the amplifying DNA and highlighting its variations two types of Simple Sequence Repeats (SSRs), among individual organisms. According to Expressed Sequences Tags (EST-SSR) have Karp et al., (1997), molecular markers provide a stronger transferability among genera and genetic information on genetic distances to species and this has successfully been applied identify particular divergent sub-populations in Hevea brasiliensis than genomic SSR (gSSR) harbouring valuable genetic traits, identifi cation (Lindqvist et al., 2006; Feng et al., 2009). of duplicate accessions, monitor changes in Microsatellites are more effi cient in genetic genetic structure; and characterization with diversity analysis (Laborda et al., 2005; Saha respect to genetic diversity within collections. et al., 2005). Markers may extend and complement characterization based on morphological or In Ghana, genetic studies using molecular biochemical descriptions with more detailed markers to diagnose, quantify and characterize passport data and accuracy than classical genetic variations/diversities in rubber clones phenotypic data (Hodgkin et al., 1995). have received little attention and, therefore, hinders breeding eff orts of rubber tree Investigations into the genetic diversity of improvement. Since the introduction of rubber the rubber tree using of molecular markers in Ghana in 1898, there has not been any have been reported extensively (Lekawipat et critical research into genetic variation analysis al., 2003; Saha et al., 2005; Lam et al., 2009; and relatedness among various clones grown Gouvéa et al., 2010; Oktavia et al., 2011; Feng in the country. It is, therefore, necessary to et al., 2012). In recent past, genetic markers use SSR markers to study the genetic diversity have served as powerful aids in understanding and similarity of the various local Hevea the genetics of Hevea brasiliensis as well as clones being grown by out growers in the assisted in clonal origin and identifi cation. country. The study will be useful in parent-tree selection for Hevea breeding and propagation Among all the markers, microsatellite markers programme, germplasm evaluation and are deemed the most suitable for genetic studies genetic characterization of traits of agronomic because they combine co-dominance and high interest. The objective of this study is to use polymorphism with abundance, locus specifi city microsatellite markers to identify and assess and uniform dispersion in plant genomes (Kalia genetic variability and also evaluate the genetic et al., 2011). A combination of four microsatellite relationships among some introduced clones of markers was used to discriminate uniquely 27 Hevea brasiliensis cultivated in Ghana. Hevea clones and also to generate some clone- specifi c allelic profi les (Saha et al., 2005). The rubber breeding objective is to provide superior and mature budded clones for latex production, MATERIALS AND METHODS rubber-wood production, quality rubber Twenty-three (23) H. brasiliensis clones, products and environmental protection through collected from Plantations Socfi naf Ghana Ltd carbon sequestration (Venkatachalam et al., (PSG), Takoradi (GT1, PB 217, RRIM 703, 2013). Hevea breeding is time-consuming and RRIC 100, IRCA 230, IRCA 41, IRCA 317, expensive. Thus, molecular markers such as IRCA 331); Ghana Rubber Estate Ltd (GREL), microsatellites can be used to reduce the time Western Region (GT1, IRCA 41, IRCA 109, required for breeding the tree crop (Mantello et IRCA 230, IRCA 317, IRCA 331, IRCA 840, PB al., 2012). Similar microsatellite priming sites 217); Forest and Horticultural Crops Research are more likely to be shared by closely related Centre (FOHCREC), Kade, University of species but often, transfer of these functional Ghana (K1, K2, K3); Plant Genetic Resource markers also occur among distantly related Research Institute (PGRRI), Bunso (B1, B5, 9 A. Antwi-Wiredu, S. Amiteye, A. T. Asare, R. Kusi-Adjei, R. G. Diawuoh, C. O. Aryee, and G. Y. P. Klu B7, B8) were used for this study. Fresh leaves for seven cycles, and fi nally 72˚C for 1 minute. were collected from each clone and stored at It was further followed by a normal cycling of -20°C after thoroughly washing them. 94˚C for 30 sec, 56˚C for 1 minute, 72˚C for 1 minute for 23 cycles and a fi nal extension at DNA Extraction 72˚C for 10 minutes. The touch-down protocol Total genomic DNA was extracted from fresh is used to eliminate stuttering and artifact bands. rubber leaves following the Cetyltrimethyl PCR amplicons were ran on 3% agarose gel Ammonium Bromide (CTAB) protocol of Doyle matrix stained with ethidium bromide to a fi nal and Doyle (1990) with some modifi cations. concentration of 0.5μg/ml. Eight (8μl) PCR The DNA pellet was washed with 70% ethanol products were loaded into the wells and ran at two times and dried at room temperature. The 100V for 1hour and were visualized using a UV quality of DNA was confi rmed by agarose gel transilluminator. electrophoresis (0.8% agarose) with ethidium bromide in TAE buff er (40 mM Tris-acetate pH 8.0, 1 mM EDTA) and exposed to UV light RESULTS and photographed. Samples were loaded into agarose gel with 0.25% bromophenol blue, All the fi ve diff erent microsatellites produced 0.25% Xylene cyanol FF and 30% glycerol in bands for the evaluation of genetic diversity/ water as loading buff er. Thereafter, 20μl of similarity among the 23 rubber clones from four each DNA was diluted with sterilize distilled (4) diff erent geographical areas of Ghana. The water to 100μl before PCR amplifi cation. primers provided monomorphic patterns and the number of bands for each primer varied PCR Amplifi cation of DNA Sample from 11 to 16 with an average of 13.75 fragments per primer. The amplifi ed products PCR amplifi cation of DNA was performed ranged from 100 to 650 base pairs in size which following a protocol described by Saha et al., was in consonant with the fi ndings of Sirisom (2005). The amplifi cation reaction was carried and Te-chato (2014). out in 25μl fi nal volume containing 20 ng of genomic DNA (1μl), 25 mM (50μl), 10x Taq Genetic Diversity/Similarity and Cluster buff er (50μl), 0.2 μM (25μl) each of the forward Analysis and reverse primers, 200 μM dNTPs (5μl) and 0.7 units of Taq polymerase (5μl). Three out Figure 02 shows a dendrogram generated using of the four most applied SSR marker pairs in Unweighted Pair Group Method with Arithmetic H. brasiliensis genetic variability analysis Mean (UPGMA) from NYSYSpc version 2.02i. identifi ed from the genomic library of H. The bands generated from SSR (Figure 01) were brasiliensis (Roy et al., 2004; Saha et al., 2005) used to plot the dendrogram/cluster to study together with two other primer pairs reported the genetic similarity and diversity or variation by Pootakham et al., (2012) were used for the among the clones collected. A dendrogram study. These fi ve (5) diff erent SSR primers are generated from the UPGMA cluster analysis hmct1, hmac4, hmct5, gSSR212 and gSSR194. distinguished 23 clones of H. brasiliensis from four (4) diff erent geographical areas of The temperature profi le involved an initial Ghana into two clusters (A and B) at genetic denaturation step of 5 minutes at 95˚C followed distance of 36%. Cluster A was further divided by a touch-down PCR programme. Temperature into two sub-clusters (AI and AII) at genetic profi les of touch-down PCR for seven cycles distance of 53.6%. Sub-cluster AI consisted of were as follows: 94˚C for 30 sec, annealing at H. brasiliensis clones K2-18 (Kade) and AII of (63˚C for 1 minute and 53°C for 30sec each clones GT1-9 (PSG), IRCA317-5 and IRCA41- depends on primer pairs), and decreased 1˚C 2 (GREL) 10 The Journal of Agricultural Sciences - Sri Lanka , 2019, Vol.14, No. 1 Figure 01A: The banding pattern of marker hmct5 on rubber clones numbered 1-23. Forward primer: GTTTTCCTCCGCAGACTCAG, Reverse primer: ATCCACCAAATAAGGCATGA. GREL (GT1-1, IRCA 41-2, IRCA 109-3, IRCA 230-4, IRCA 317-5, IRCA 331-6, IRCA 840-7, PB 217-8); PSG (GT1-9, PB 217-10, RRIM 703-11, RRIC 100-12, IRCA 230-13, IRCA 41-14, IRCA 317-15, IRCA 331-16); Kade (K1-17, K2-18, K3-19); and Bunso (B1-20, B5-21, B7-22, B8-23) and M-1kb ladder. Figure 01B: The banding pattern of marker gSSR212 on rubber clones numbered 1-23. Forward primer: GTTTTCCTCCGCAGACTCAG, Reverse primer: ATCCACCAAATAAGGCATGA. Figure 02: A dendrogram showing genetic diversity/similarity revealed by 5 simple sequence repeat primers among 23 H. brasiliensis clones cultivated in Ghana 11 A. Antwi-Wiredu, S. Amiteye, A. T. Asare, R. Kusi-Adjei, R. G. Diawuoh, C. O. Aryee, and G. Y. P. Klu Contrarily, cluster B was further divided into It was observed that some rubber clones three sub-clusters at 44.8% genetic distance. were genetically similar irrespective of their With this, the fi rst sub-cluster B (III-IV) was geographical location of growth which could made up of RRIC100-12 and RRIM703- be as a result of their development from 11 (PSG). The second sub-cluster B (V-VII) budding of clones with scions of similar genetic comprised K1-17, K3-19 (Kade), B1-20, B5- constitutions. These possible similarities were 21, B7-22, B8-23 (Bunso), IRCA317-15, revealed in rubber clones IRCA317-5, 1RCA41- IRCA230-12, IRCA41-14 (PSG), IRCA840-7 2, IRCA331-6, IRCA230-4, IRCA109-3 and IRCA331-6 (GREL). The fi nal sub-cluster (GREL) and B8-23 (Bunso) which were linked B (VIII-XI) consisted of such rubber clones at 100.0% genetic similarity level. This supports as PB217-8, IRCA230-4, IRCA109-3, GT1-1 a controversial result reported by Cesar et (GREL), PB217-10 and IRCA317-16 (PSG) al., (2006) and Lekawipat et al., (2003) to (Figure 02). express that some relationship existed between geographical areas and sample collection. At genetic distance of 100%; IRCA317- Introduction of planting materials into Ghana 5, IRCA41-2, IRCA331-6, IRCA230-4, from other rubber-producing countries could IRCA109-3 (GREL) and B8-23 (Bunso) were have facilitated the distribution of genes in genetically similar. Moreover, rubber clones various clones irrespective of the country where consisting of K1-17, K3-19 (Kade), IRCA317- they were initially bred. 15, IRCA230-12 (PSG), B1-20, B5-21 and B7- 22 (Bunso) were genetically similar at genetic At a genetic distance of 70.4%, H. brasiliensis distance of 70.4%. Also, at genetic distance clones K1-17, K3-19 from Kade; B1-20, of 80.0% rubber clones GT1-9, RRIC100-12, B5-21, B7-22 from Bunso and IRCA317- RRIM703-11, IRCA41-14 (PSG) and GT1-1 15, IRCA230-12 from PSG showed genetic (GREL) were similar genetically (Figure 02). similarity. Furthermore, at 78.4% level of similarity, H. brasiliensis clones; GT1-9, In contrast, H. brasiliensis clones including RRIC100-12, RRIM703-11, IRCA41-14 from K2-18 (Kade), IRCA840-7, PB217-8 (GREL), the same growing area, PSG and GT1-1 from PB217-10 and IRCA317-16 (PSG) at diff erent GREL showed to be genetically similar. High genetic distances of 53.6%, 73.6%, 64%, genetic similarity among clones could be as 65.6% and 66.8% respectively were genetically a result of the same parent plants selected for dissimilar (Figure 02). The farthest genetic breeding programme over the years. The high distance was shown between Hevea brasiliensis genetic similarity suggests a narrow genetic clones K2-18 (Kade) and GT1-1 (GREL) diversity among the clones and this may be (Figure 02). explained by clonal propagation employed in establishing plantations (Nakkanong et al., 2008). Genetic similarity could occur because DISCUSSION generally vegetative propagation is known to maintain traits of parental lines (Varghese et There was genetic diversity and similarity al., 1997). Genetic similarity assessment might present in Hevea brasiliensis clones from the be useful in selecting the parents for a rubber four diff erent geographical areas of rubber breeding and propagation programme aimed at production in Ghana which has been shown by obtaining heterosis eff ects and high productivity. the dendrogram (Figure 02) . The dendrogram Microsatellite primers provide a large quantity generated a linkage among the Hevea of polymorphic information, for this reason brasiliensis clones at genetic distance of 36.0% they are ideal for diff erentiating genotypes that where two clusters were formed. are genetically very similar (Nakkanong et al., 2008). This was very much shown in this current study. 12 The Journal of Agricultural Sciences - Sri Lanka , 2019, Vol.14, No. 1 Contrarily, H. brasiliensis clones K2-18, IRCA showed genetic similarity even from diverse 840-7, PB217-8, PB217-10 and IRCA317- geographical areas. On the other hand, rubber 16 from diff erent geographical zones did not clones such as K2-18, IRCA 840-7, PB217- show any genetic similarity with other clones 8, PB217-10 and IRCA317-16 also with indicating genetic variability among the regardless to their geographical origins showed genotypes. Genetic diversity observed could some level of genetic dissimilarities. Therefore, be attributed to cross-pollination between the for the purposes of propagation and breeding genotypes since rubber tree is out-crossing. programme of H. brasiliensis, some local Controlled pollinated species produce legitimate clones could be selected based on their genetic genotypes whilst open pollination results into diff erences and relatedness. illegitimate genotypes even if similar parents are from diff erent groups (Gouvéa et al., 2010). Genetic diff erences emanate from gene fl ow RECOMMENDATIONS among populations through seed and pollen dispersal. The genetic variability may have DNA extraction protocol of H. brasiliensis been infl uenced by gene mutation, although it should be modifi ed, especially by adding has low incidence rate in H. brasiliensis species polivinylpolipyrolidon (PVPP) to obtain (Nakkanong et al., 2008). Varghese et al., high quality and pure DNA for amplifi cation. (1997) reported that it could happen because Also, PCR amplicons could be ran using the rubber tree is a cross pollinated plant and polyacrylamide gel (PAGE) to fi nd out if quality very heterozygous. Also, segregation causes bands would be obtainable. proportion of hybrid alleles from parents to vary. Independent assortment and segregation Other types of markers such as SNPs and a do occur in highly heterozygous clones, thus the combination of diff erent markers could be proportion of primer alleles in the F1 hybrids taken into consideration to genetically analyse can greatly diff er. Consequently, true nature of H. brasiliensis clones in Ghana. genetic relatedness may not be shown in highly heterozygous species of similar pedigree and Also apart from trying other markers, the ancestry (Gouvéa et al., 2010). number of microsatellite markers could be increased in future research into the genetic diff erentiation of the Hevea brasiliensis clones grown in Ghana. CONCLUSION A dendrogram was constructed with the un- weighted pair group method with arithmetic ACKNOWLEDGEMENTS averages (UPGMA) on the basis of the diversity/similarity coeffi cients. The band The authors are most grateful to the late size ranged from 100 to 650 base pairs. With Elder Charles K. Hayford who supported and the number of primers employed for the study sponsored this research. We are also thankful some rubber clones (IRCA317-5, 1RCA41- to Prof. Harry Amoatey and Prof. Kenneth E. 2, IRCA331-6, IRCA230-4, IRCA109-3, Danso and all the scientists and technicians B8-23, K1-17, K3-19, B1-20, B5-21, B7-22, of the Biotechnology and Nuclear Agriculture IRCA317-15, IRCA230-12, GT1-9, RRIC100- Research Institute of Ghana Atomic Energy 12, RRIM703-11, IRCA41-14 and GT1-1) Commission for the unrestricted accessibility to their resources. 13 A. Antwi-Wiredu, S. Amiteye, A. T. Asare, R. Kusi-Adjei, R. G. Diawuoh, C. O. Aryee, and G. Y. P. 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