V.N.Mutharaian?R.Kamalakannan?A.Mayavel?S.Makesh?S.H.Kwon?K.-S.Kang

Introduction

The taxonomically complex genus Acacia is mostly represented by trees that are of central importance to the rural economy of many of the world’s arid and semi-arid zones(Allen and Allen 1981).In view of the great diversity and potential of Acacia species,it is perhaps surprising that only a few species have been widely promoted for use on farms or in afforestation programs and even fewer have been studied in detail(Fagg and Stewart 1994).The genus of Indian Acacia encompasses a wide variety of species of which Acacia leucophloea(Roxb.)Willd.is the most common.It is a small to medium-sized,deciduous thorny tree that can attain a height of more than 20 m.In fact,among other nations where the species is native,India has a wider distribution area of the species.

Commonly known as white babul tree or safe babul,it grows over a wide latitude,from central to southern India.Woodlands of A.leucophloea have played an important socioeconomic role for local rural populations by providing small-scale uses such as fodder,fuel wood,smaller timber and shade for livestock pasture over centuries.The species is also extremely tolerant to heat and drought;however,the nature of its tolerance to salinity or alkalinity remains conflicting.Besides,it is also reported to have certain medicinal properties(Parotta 2001).Though the breeding system for A.leucophloea has not been investigated in detail,outcrossing may be favored as reported for a majority of tropical Acacias(Moran et al.1989;Mandal and Ennos 1995;Tandon and Shivanna 2001).In line with other acacias,the species has the potential to enrich degraded,vulnerable ecosystems such as arid and semi-arid lands.Despite its crucial role as a promising livelihood species,information available on the genetic diversity as well as germplasm collections of the species is not satisfactory and is a major obstacle to developing conservation measures.

In general,species with wide geographical ranges develop local adaptations and exhibit differences in morphological features.These variations are attributed to evolutionary adaptation of the species to that particular environment and can be determined genetically.For these reasons,selection and establishment of provenance trials have gained importance in recent decades(O’Brien et al.2007).But in case of A.leucophloea,based on morphological attributes,so far it has not been categorized into varieties or subspecies as reported for several other acacias(Brenan 1983;Rutishauser and Sattler 1986).However,in the present study,we were able to identify five provenances of the species that had been selected in the 1980s for evaluating biomass production and tannin content.We included this collection for the present genetic diversity analysis.

Characterizing plant genotypes provides valuable information for any kind of breeding program.Recent developments in molecular biology,in particular,random amplified polymorphic DNA(RAPD)markers,have provided numerous DNA-based markers for characterizing plants.The present study used the RAPD technique(Williams et al.1990)to determine the genetic diversity among provenances and between species of Acacia.Sources of polymorphism in RAPD assays may include a base change within a priming site,sequence deletion of a priming site,and an insertion that renders priming sites too distant to support amplifications and deletions or insertions that change the size of DNA without preventing its amplification(Williams et al.1990).In addition,the polymorphisms in RAPD markers were observed as a difference in DNA fragment size.Therefore,difference in markers in different genotypes may be the result of DNA recombination,mutation,or random segregation of chromosome in meiosis processing during hybridization and subsequent selection(Darnell et al.1990;Huchett and Botha 1995).Though RAPD markers are dominant in nature,a large number of available random primers can generate immense genetic data across the entire genome(Kremer et al.2005),which has made them useful for species where the DNA sequence is not available,as for A.leucophloea.

The present study was designed to determine the genetic diversity among five provenances of A.leucophloea distributed in natural stands of southern India and how the species is clustered with other relatives of Acacias in the region.RAPD markers were used to characterize polymorphisms among the five provenances of A.leucophloea and to detect genetic relatedness of the species with six other Acacias.

Materials and methods

Plant materials and DNA extraction

Plant material consisted of five provenances of A.leucophloea,namely,Dharapuram (10°45′N,77°34′E,245 m), Thirumangalam (13°1′N, 80°2′E, 160 m),Pudukottai(10°23′N,78°49′E,90 m),Sendurai(11°2′N,79°18E,80 m)and Dharmapuri(12°08′N,78°13′E,460 m)from southern India and seven species of Acacias viz.A.holosericea,A.auriculiformis,A.mangium,A.dealbata,A.ferruginea,A.nilotica and A.leucophloea.Ten individuals representing each provenance were selected,and fresh leaves were harvested from their germplasm assembled near Neyveli,Tamil Nadu,India.During our study,these were the only available genotypes representing various provenances of A.leucophloea in Tamil Nadu.This was an earlier collection for estimating the variation of tannin content among different provenances.To date,no new provenances or germplasm collection have been made.Leaf samples of different species of Acacia were also collected from localities in and around Coimbatore(11°N,77°E),Tamil Nadu,India.

Genomic DNA was extracted from approximately 100 mg of young leaves by following the protocol as mentioned in GeNei(Genei Laboratories Pvt.Ltd.,Bangalore,India)Plant Genomic DNA Purification Kit(Cat#612115700021730).The quality and quantity of the extracted genomic DNA was determined using gel electrophoresis(0.7%agarose gel).DNA concentration for PCR amplification was estimated by comparing the band intensity of a sample with the band intensities of known dilutions of Lambda DNA/EcoRI+Hind III Marker(Fermentas,#SM0191).On the basis of band intensity,the DNA was further fractionized to the required concentration(25–50 ng)using double distilled water.About 25–50 ng of an individual DNA sample from 10 individuals of each provenance were pooled together and from this bulk DNA 25–50 ng was used for PCR amplification.

DNA amplification

After an initial screening of 50 primers,fifteen 10-mer primers(B2,B7,B9,B10,B12,B14,B17,B19,B20,B21,B23,F3,F4,F5,D25 supplied by Bangalore Genei)that produced clear and visible bands were used for RAPD reactions.Almost all the selected primers had a high content of GC rather than AT.We used the protocol of Williams et al.(1990)with slight modifications in the temperature profile.The reaction mixture(25 μL)consisted of 25 ng of template DNA,2.5 μL of 10× buffer with 1.5 mM MgCl2,0.5 μL of 10 mM each of dATP,dTTP,dGTP and dCTP(Fermentas Cat#R0181),0.5 μL 20 pMol primer(Genei),1 unit Taq polymerase(Genei)and sterile,deionized water to make up the final volume.

The amplification reactions were carried out in Bio-Rad(MyCycler thermal cycler)programmed for an initial denaturation at 94°C for 5 min;40 cycles of 1 min denaturation at 94 °C,1 min annealing at 35 °C and 2 min extension at 72 °C;and a final extension at 72 °C for 10 min.The amplified PCR products were separated electrophoretically in 1×TBE buffer in a 2%agarose gel at 200 V/h.Two DNA ladders,Stepup 100 bp DNA ladder(Cat#612652670501730)and Stepup 500 bp DNA ladder(Cat#612651970501730)were also used.Gels were stained with ethidium bromide and viewed under UV light.Gels that produced ambiguous products were repeated to confirm the reproducibility.Photographs were taken and documented.

Data analysis

Data from unambiguous,intensely stained and clear bands among different genotypes were used for statistical analysis.At each locus,bands were scored for their presence as‘1’or absence as ‘0’for matrix analysis.The pooled data from the RAPD profiles of the primers were subjected to cluster analysis.A similarity matrix was constructed using Jaccard’s coefficient,and the similarity values were used for cluster analysis.Sequential agglomerative hierarchical non-overlapping(SAHN)clustering was done using the neighbor joining algorithm.Data analysis was done using NTSYSpc version 2.02(Rohlf 1997).The cluster analysis was performed for provenances and species based on Jaccard’s similarity coefficient matrix calculated from the RAPD marker to generate a dendrogram using neighbor joining.

Results

Though a variable number of bands were amplified among the provenances and among species,all primers used were known to provide reproducible RAPD profiles in multiple reactions.The 15 primers produced 194 scorable markers across the five provenances,of which 57 were polymorphic and accounted for 29.38%of the polymorphism.The products ranged from 210 to 2000 bp(Table 1),and 9–18 markers were produced per primer.Primers B23 and F3 produced only 1 polymorphic locus,while primer B7 produced a maximum of 8 polymorphic loci(also more monomorphic loci).Thus,the average number of markers produced per primer was 12.93.

Amplification by the same set of primers for the 7 species of Acacia including A.leucophloea,yielded 326 markers.The size range of product was 150–2800 bp.Individual primer pairs yielded 16–27 markers.Primer B14 scored 6 polymorphic loci,whereas three primers B7,B10 and B17 produced 17 polymorphic loci,respectively,and produced the most markers(Table 1).The average number of marker produced per primer was 21.73.Of the 326 markers generated in the species studies,182 were polymorphic,accounting for 55.82%of the polymorphisms.The patterns of amplification generated by primers B7 and B21 were shown in Fig.1.

The genetic similarity coefficient values for the provenances are listed in Table 2.They indicate a close relationship(1.00)between the Thirumangalam and Dharmapuri provenances.Among the provenances,Pudukottai and Sendurai were most distantly placed(0.545).However,all provenances shared 50%similarity.To validate the correctness of the similarity,A.holosericea as an outgroup and showed a greater genetic distance(46.7–73.3).

Table 1 Primer sequences for RAPDs,loci details and product size generated for five provenances of Acacia leucophloea and seven species of Acacia

In the cluster analysis and dendrogram based on the genetic similarity coefficient,the five provenances separated into two major clusters;i.e.,four provenances Dharapuram,Thirumangalam,Pudukottai and Dharmapuri into one cluster and the Sendurai provenance into the other(Fig.2).The outgroup species A.holosericea was distantly placed as a separate entity in the tree.The present study generated 36 provenance-specific markers(PSMs)with various molecular weights,of which Sendurai had 9 specific markers,and Dharmapuri had 3.The PSMs discriminating the provenances are listed in Table 3.

Between species,based on the cluster analysis,the genetic similarity coefficients with RAPD markers ranged from 0.214(A.nilotica and A.dealbata)to 1.000(A.holosericea and A.ferruginea)(Table 4).The dendrogram obtained by the neighbor joining algorithm separated the 7 species of Acacia into 3 major groups(A,B and C;Fig.3).Group A was further divided into three subgroups;i.e.,A.holosericea and A.ferruginea into the same subgroup,and A.auriculiformis and A.nilotica each into a separate subgroup.Group B comprised two subgroups:A.mangium and A.leucophloea.Interestingly,group C had only one species,A.dealbata,which was distantly related to all the other acacias in the cluster(Fig.3 and Table 4).A total of 162 species-specific markers(SSMs)were generated by the 15 primers in interspecific relatedness.A.dealbata had a maximum of 38 SSMs,while A.auriculiformis had a minimum of 12 SSMs(Table 5).

Discussion

With the advent of DNA fingerprinting,the field of molecular genetics has generated a numerous insights on Acacian diversity and phylogeny(Harrier et al.1997;Shrestha et al.2002;Nanda et al.2004;George et al.2006;Omondi et al.2010).In the present study,we demonstrated the reliability of RAPD markers for discriminating the provenances of A.leucophloea and their relatedness with other Acacia species.This marker analysis revealed that considerable variation among individuals of the different provenances.

If A.leucophloea is considered to be an outcrosser,as reported for other Acacia species(Philp and Sherry 1946;Moffett 1956;Moran et al.1989;Mandal and Ennos 1995;Tandon and Shivanna 2001),the results of the similarity matrix in the present study provide evidence that the progenies collected from different regions differ at the DNA level;i.e.,at a relatedness level.To validate this difference,the provenance bulk was divided further for the presence of specific markers among individuals.Though the level of genetic relatedness was high,as already reported among individuals of Acacia species(Nanda et al.2004),there was still considerable variation among provenances at the genomic level,as evidenced by the presence of 36 provenance-specific RAPD markers.

Fig.1 RAPD patterns generated by primers B7 and B21 for five provenances of Acacia leucophloea and seven different Acacia species

In our study,the provenance Sendurai was distantly placed in genetic relatedness from other provenances,and the provenance Dharmapuri could record a minimum of 3 provenance-specific markers.These results were enough to justify the genetic variation between the selected provenances even though they are representatives of dry zones.Hence,there is a strong possibility that of geographical isolation,and the outcrossing behaviour of A.leucophloea has led to high genetic diversity among individuals as well as between various provenances.Such variations have been previously documented in the provenances of Azadirachta indica(neem;Ranade and Farooqui 2002)and A.auriculiformis and A.mangium(Nguyen et al.2004).

Fig.2 Dendrogram of five provenances of Acacia leucophloea based on 15 RAPD markers and the SAHN clustering algorithm

The RAPD profiles produced from the species-specificmarkers clearly distinguished the seven different Acacia species.A total of 326 markers were generated across 7 species of Acacia using fifteen 10-mer primers in this study.Earlier,Nanda et al.(2004)who used the same RAPD technique to study genetic relationship of the 6 species of Acacia scored 249 markers using seventeen 10-mer primers.They also indicated that RAPD pattern of genetic diversity wasadequate to analyze the similarity and distances between Acacia species.Previous studies on interspecific variations employing RAPD markers in different species of Acacia(Harrier et al.1997),Pinus(Nkongolo et al.2002),Prosopis(Landeras et al.2006)and Jatropha(Ganesh Ram et al.2008)have proved to be more informative.

The genetic cluster analysis resolved the 7 species of Acacia into 3 groups,of which group A is the major one consisting of A.holosericea,A.auriculiformis,A.ferruginea and A.nilotica.While dissecting the origin of these species,we inferred that A.holosericea and A.auriculiformis were from Australia,and A.ferruginea and A.nilotica were from Africa.All these species are trees except for the shrub A.holosericea.Group B included A.mangium and A.leucophloea.A.mangium is of Australian origin,while A.leucophloea is of Asian origin,and their morphology is dissimilar.Thus,the two species in the group B had apparent morphological differences,but they might have co-evolved in their habitat.This view is supported by Bukhari et al.(1999)who suggested that A.nilotica and A.farnesiana are sister species in spite of their different origins.

Table 2 Similarity matrix computed among five provenances of Acacia leucophloea based on Jaccard’s coefficient(A.holosericea was included as an outgroup species)

Table 3 Provenance-specific markers(PSMs)generated by RAPD polymorphisms using three primer sets

Table 4 Similarity matrix between seven Acacia species based on Jaccard’s coefficient

Fig.3 Dendrogram produced by a neighbor-joining cluster analysis of seven Acacia species

In the cluster analysis,A.dealbata alone formed a separate group C and was distant from all other relatives.It would be more logical to consider this in a separate cluster as it has consistent morphological and adaptive differences from the other species of Acacia in this study.Though the different Acacia species do not seem to cluster in accordance with geographical distances,the dendrogram represents their relatedness according to numerical taxonomy as suggested by Sneath and Sokal(1973).

A.leucophloea remains under-utilized because the various germplasm collections and the evaluation of traits are highly fragmented.Results of marker studies have confirmed considerable variation among provenances.Though drought adaptation by the species is unquestionable,its salt tolerance is ambiguous.Under these circumstances,the study of trait-specific markers in this species would be worthwhile.RAPD markers for salt tolerance in A.auriculiformis and A.mangium have been reported(Nguyen et al.2004).The PSMs and SSMs generated in the present study can now be compared with those reported in A.auriculiformis and A.mangium.Such studies should greatly bene fit coastal land management because most of the Indian coast is dominated by exotics acacias.

The present investigation has shown that RAPD patterns can be analyzed to correlate genetic similarity and distance between Acacia species,then predict their origin.The 162 species-specific markers scored in this study will also be helpful to identify species hybrid progeny.Alternatively,these markers can be used to generate SCARs(sequence characterized amplified regions)with higher sensitivity and reproducibility,which could be useful for developing eff icient tree breeding programs in Acacia species to increase the production of biomass(i.e.,fuel wood)and tannins.

Table 5 Species-specific markers(SSMs)generated by RAPD polymorphism using three primer sets

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