Isozyme genetic markers are efficient tools to study genetic variations within and betweenpopulations of less known wild species as well as for studies on spatial distribution of geneticvariation. &nbsp;A study &nbsp;was conducted with &nbsp;four &nbsp;important &nbsp;isozyme markers namely, peroxidase,polyphenol oxidase, esterase and superoxide dismutase in Garcinia gummigutta &nbsp;populationcollected from Western Ghats in South India. The cluster analysis of the marker bands showedthat most of the population from similar geographic locations was the first one to groupthemselves, though a significant pattern was not noticed. The mean percentage of polymorphicloci was 52.5%. Total heterozygocity was 0.97 which is consistent with the average of tropicaltree species.
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P A Mathew, Utpala Parthasarathy, G R Asish, K Jayarajan, R Aravind, B Krishnamoorthy

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29Isozyme diversity of Garcinia gummigutta (L.) N. Robson in Western Ghat region,South IndiaUtpala Parthasarathy, G R Asish, K Jayarajan, R Aravind, B Krishnamoorthy & P A MathewIndian Institute of Spices ResearchCalicut – 673 012, Kerala, India.E-mail: utpala@spices.res.inReceived 8 July 2009; Revised 23 June 2010; Accepted 7 August 2010AbstractIsozyme genetic markers are efficient tools to study genetic variations within and betweenpopulations of less known wild species as well as for studies on spatial distribution of geneticvariation.  A study  was conducted with  four  important  isozyme markers namely, peroxidase,polyphenol oxidase, esterase and superoxide dismutase in Garcinia gummigutta populationcollected from Western Ghats in South India. The cluster analysis of the marker bands showedthat most of the population from similar geographic locations was the first one to groupthemselves, though a significant pattern was not noticed. The mean percentage of polymorphicloci was 52.5%. Total heterozygocity was 0.97 which is consistent with the average of tropicaltree species.Keywords: Garcinia gummigutta, geographic distance, isozyme markers.Abbreviations: Em value (Electrophoretic mobility value), PRX (Peroxidase), PPO (Polyphenoloxidase), SOD (Superoxide dismutase), NTSYS (Numerical taxonomy system).IntroductionAbout 30 species of the genus Garcinia,including G. gummigutta (L.) N. Robson areindigenous to Western Ghats and areextensively distributed in the Western Ghatrange of Kerala and Karnataka. Western Ghatswith its varied environmental conditions isone of the important hotspots of biologicaldiversity including plant species.Isozyme genetic markers have been used formany decades and are still one of the mostreliable and efficient tools in estimation ofheterozygosity and prediction of diversity inplant species (Lewontin & Hubby 1966). Inforestry, isozymes have been used to studygenetic variation within and betweenpopulations. Isozymes are of great use in theelucidation of genetic information forrelatively unknown species and it has beenused to determine species delimitation (Rajora1989). Li (1999) found that isozyme variationin Eucalyptus microtheca F. Muell has significantpattern related to geographic distance. Thisenvironmental variation might also beexpected to favour genetic heterogeneity. Theaim of the present study was to detect isozymepolymorphism in G. gummigutta collected fromJournal of Spices and Aromatic CropsVol. 19 (1 & 2) : 29–33 (2010)         Indian Society for Spices30different geographical locations in Kerala andKarnataka and provide information ongeographical variation that may beresponsible for intra-species genetic variation.This information would help in thedevelopment of conservation and utilizationstrategies of genetic resources of G.gummigutta.Materials and methodsTwenty four natural populations of G.gummigutta were studied. The plants (about 8years old) were collected from various districtsof Kerala and Karnataka located in theWestern Ghats range. The youngest maturedleaf from the plants were used for the study.The leaves were macerated in 1000 μl ofsodium phosphate extraction buffer of pH 7.2in porcelain mortar over ice (Sadasivam &Manikam 1992). The vertical gel bufferelectrophoresis (ATTO) system was used forthe separation of four isozymes namely, PRX,PPO, esterase and SOD. The running gel(10%) and stacking gel (5%) were of polyacrylamide gel. The loci were numbered fromanode to cathode. The cluster analysis wasdone to construct the polygenetic tree usingNTSYS PC2 (Rohlf 1993) software.Collection sites were plotted on the map withthe help of ArcGIS software to verify thesimilarity among clusters with distance(Fig. 1). Using DIVA GIS bioclim model, thealtitude and rainfall grids of the collectionsites were prepared to find out their effect onthe enzymes if any.Genetic parameters such as mean number ofalleles per locus, percentage of polymorphicloci, observed heterozygocities, geneticdistance and similarities were calculated forthe studied natural populations. Among thepopulation, heterogeneity tests were donebased on allele frequencies and polymorphicindex obtained from isozyme analysis(Ashburner et al. 1997).Results and discussionThe number of bands in PPO and PRX variedfrom 2 to 4.  The Em values were almost samefor both the enzymes (Table 1).  For esteraseand SOD, uniform 2 and 3 bands were noticedin all the samples under study. For esterase,the Em values were 0.717 and 0.871 and forSOD, the values were 0.167, 0.667 and 0.807.The polymorphic index calculated for PRXwas 0.0053 and for PPO it was 0.0037. Thisindicates that the intra-population variationof  both the enzymes was not much and themean percentage of polymorphic loci was52.5%. The heterogeneity was worked outbased on allelic frequency and polymorphicindex and was 0.97. Cluster analysis basedon NTSYS pc2 revealed low level of geneticseparation within the population (Fig. 2).The coefficient values were within 0.85 to1.00. The resulting dendrogram showeddifferentiation into three main clusters andFig. 1.  Map of Kerala and part of Karnataka showingthe collection sitesParthasarathy et al.31seven sub-clusters. The altitudinal andprecipitation study of the collection sitesthrew some light on the differences in theenvironment and segregation but furtherstudy is required for better understanding.The mean percentage of polymorphic lociwhich was 52.5%, indicated that geneticvariability within the population of G.gummigutta was not much higher than outcrossing in wind pollinated woody plantswhich is 53%, and the result is consistentwith the average of tropical tree species(60.9%) (Li 1999).Cluster analysis revealed low level of geneticseparation between the studied populations,the coefficient of cluster varied between 0.85to 1.00. The resulting dendrogram showeddifferentiation into three main clusters. Nosignificant pattern related to geographicdistance could be found. The dendrogramshowed that most of populations from similargeographic locations were the first oneswhich group themselves, but the clusteringof Calicut population did not follow anycriterion. The Wayanad collections were inseparate groups (the distance betweenTable 1.  Isozyme band numbers and Em values for polyphenol oxidase and peroxidaseSample Longitude Latitude No. of Em value (poly- Em valuebands (poly- phenol oxidase) (peroxidase)phenoloxidase)/(peroxidase)Thrissur 1 76 030 ’82 ” 10 053 ’56 ” 2 0.5, 0.571 0.421, 0.521Thrissur 2 76 028 ’82 ” 10 043 ’56 ” 2 0.5, 0.571 0.521, 0.62Thrissur 3 76 029 ’82 ” 10 043 ’56 ” 2 0.5, 0.571 0.521, 0.62Thrissur 4 76 027 ’93 ” 10 053 ’56 ” 2 0.5, 0.571 0.421, 0.521Thrissur 5 76 030 ’82 ” 10 053 ’56 ” 2/1 0.5, 0.571 0.521Thrissur 6 76 029 ’23 ” 10 056 ’56 ” 3 0.5, 0.571, 0.642 0.421, 0.521, 0.62Calicut 1 75 078 ’39 ” 11 069 ’22 ” 2 0.5, 0.571 0.421, 0.521Calicut 2 75 080 ’29 ” 11 064 ’56 ” 2 0.5, 0.571 0.421, 0.521Calicut 3 75 077 ’29 ” 11 065 ’49 ” 3 0.5, 0.571, 0.642 0.421, 0.521, 0.62Calicut 4 75 078 ’29 ” 11 063 ’49 ” 3 0.5, 0.571, 0.642 0.421, 0.521, 0.62Calicut 5 75 077 ’29 ” 11 065 ’49 ” 3 0.5, 0.571, 0.642 0.421, 0.521, 0.62Appangala 1 75 077 ’21 ” 12 026 ’21 ” 2 0.5, 0.571 0.421, 0.521Appangala 2 76 000 ’71 ” 12 006 121 ” 3 0.321, 0.5, 0.571 0.295, 0.421, 0.52Appangala 3 75 081 ’45 ” 12 013 ’68 ” 3 0.321, 0.5, 0.571 0.295, 0.521, 0.62Kannur 1 75 044 ’36 ” 12 007 ’53 ” 2 0.5, 0.571 0.521, 0.62Kannur 2 75 049 ’36 ” 12 004 ’53 ” 3 0.5, 0.571, 0.642 0.421, 0.521, 0.62Kannur 3 75 042 ’36 ” 12 008 ’53 ” 3 0.5, 0.571, 0.642 0.421, 0.521, 0.62Kannur 4 75 059 ’36 ” 12 002 ’53 ” 3 0.5, 0.571, 0.642 0.421, 0.521, 0.62Kannur 5 75 049 ’36 ” 12 004 ’53 ” 3 0.5, 0.571, 0.642 0.421, 0.521, 0.62Wayanad 1 76 012 ’04 ” 11 057 ’79 ” 2 0.5, 0.571 0.421, 0.521Wayanad 2 76 008 ’86 ” 11 076 ’22 ” 3 0.5,  0.571, 0.642 0.421, 0.521, 0.62Wayanad 3 76 018 ’39 ” 11 057 ’79 ” 3 0.5, 0.571, 0.642 0.421, 0.521, 0.62Kottayam 1 76 063 ’12 ” 9064 ’25” 4 0.321, 0.5,0.571, 0.64 0.295, 0.421, 0.521, 0.62Kottayam 2 76 076 ’14 ” 9064 ’25” 4 0.321, 0.5, 0.571, 0.64 0.295, 0.421, 0.521, 0.62Isozyme diversity in Garcinia32Fig. 2. Similarity clusters based on NTSYS pc2 software (the accessions are named based on their collectionsites).Sugandhagiri and Pulppally is 55 km), whileThrissur collections are in same group exceptone (separated by10 km). Kottayam (77°6312/9°6425 & 77°7614/9°6425) and Appangala(Kodagu) (75°7721/12°2621, 76°0071/12°0621& 75°8145/12°1368) collections clusteredtogether, but in different sub-groups, thoughthey were collected from nearby geographiclocations.This pattern has also been observed in mostof the studied populations of forest trees(Elenarossello & Cabrera 1996). It isimportant to mention that the clusteranalysis and the resulted dendrogram werebased only on the information of the allelicfrequencies (of four isozymes) of eachpopulation but not on any specialmorphological character in the population.Two sites of Appangala (Kodagu) have thesame altitude and rainfall (800-1000 m andabove 3000 mm) respectively, and in thecluster they showed segregation.In forestry, isozymes have been used to studygenetic variation within and betweenpopulations, population structure,phylogeny, and to elucidate mating patternsamong natural populations as well asexperimental populations (Mitton 1983;Hamrick & Godt 1989). When a littleinformation about genetic diversity withinor between populations is known, isozymescan be an effective genetic marker. In “wild”species like G. gummigutta with scantyinformation on genetic diversity, the need forbasic data such as the levels of heterozygosityand per cent of polymorphic markers withinpopulation are important.  In this study,isozyme data was generated for thecomparison of genetic variation withinpopulations three and seven sub-clusterswere found within 1000 km distance ofWestern Ghats.  Total polymorphicpercentage was 52.5%, which indicated lowheterogenicity. Garcinia being a crosspollinated species, it is expected to observemuch higher diversity but the result isconsistent with the average reported fortropical tree species.Parthasarathy et al.33ReferencesAshburner G R & Thompson W K 1997 RAPDanalysis of South Pacific coconut palmpopulations. Crop Sci. 37: 992-997.Elenarossello J A & Cabrera E 1996 Isozymevariation in natural populations ofcork-oak (Quercus suber L.). Populationstructure, diversity, differentiation andgene flow. Solvae Genetica 245: 229-235.Hamrick & Godt 1989 Effects of life history traitson genetic diversity in plant species.Philosophical Trans.: Biol. Sci. 351: 1291-1298.Lewontin R C & Hubby J L 1966 A molecularapproach to the study of genic heterozy-gosis in natural populations. Genetics54: 595-609.Li C 1999 Isozyme variation in natural popula-tions of Eucalyptus micvotheca. Hereditas130: 117-123.Mitton J B 1983. Conifers. In: Tanksley S D &Ortom T J (eds.) Isozymes in Plant Ge-netics and Breeding, Part B (pp. 443-472). Elsevier Science Publishers B V,Amsterdam.Rajora O P 1989 Genetic structure and identifi-cation of Populus deltoides clones basedon allozymes. Genome 32: 440-448.Rohlf F J 1993 NTSYS pc-Numerical Taxonomyand Multivariate Analysis System,Exeler Software, Version 1.80.  NewYork.Sadasivam S & Manikam A 1992 BiochemicalMethods for Agricultural Sciences.Willey Eastern Limited, New Delhi.Isozyme diversity in Garcinia

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Isozyme diversity of Garcinia gummigutta (L.) N. Robson in Western Ghat region, South India

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Isozyme diversity of Garcinia gummigutta (L.) N. Robson in Western Ghat region, South India

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