'Star Cactus' (Astrophytum) Through Chromosome and RAPD Markers

Assessment of Genetic Diversity and Phylogenetic analysis of ‘Star Cactus’ (Astrophytum) Through Chromosome and RAPD Markers

Anath Bandhu Das*

Cytogenetics Laboratory, Regional Plant Resource Centre, Nayapalli, Bhubaneswar 751015, Orissa, India

Received April 7, 2008; Accepted June 17, 2008

Summary Interspecific variation and molecular phylogenetic analysis among the four species of Astrophytum i.e. A. asterias, A. capricorne, A. myriostigma and A. ornatum of the family Cactaceae were studied using cytological and molecular markers. In all the four species, the somatic chromosomes found were 2n22. Though they showed a high level of similarity in the number of chromosomes, karyotype analysis revealed species-specific chromosomal characteristics. The total chromosome length varied from 53.05 mm in A. aterias to 60.12 mm in A. capricorne. The chromosome volume varied from 41.82 mm3 in A. ornatum to 61.65 mm3 in A. myriostigma. The centromeric index (TF %) among the four species varied from 35.93% in A. myriostigma to 45.25% in A. capricorne. The symmetrical karyotype with same number of secondary constricted chromosomes in A. asterias & A. myriostigma suggests their close affinity between them. The 4C DNA contents were varied significantly from 8.95 pg in A. aterias to 10.23 pg in A. capricorne. The amplification products in PCR generated RAPD analysis of the four species, revealed a total of 315 bands with fragment size ranging from 0.21 kb to 3.1 kb that showed high correlation of A. asterias with A. aterias and A. capricorne with A. ornatum that were also confirmed from the karyotype analysis. Analysis of the nuclear DNA content showed significant variation in the amount of 4C DNA between the three species. The correlation coefficients showed the various chromosomal and nuclear parameters were interdepen- dent to some extent suggesting interspecific relationship between structural and molecular changes of the genome architecture during evolution of speciation.

Key words Cactus, Astrophytum, Chromosome number, Karyotype, Nuclear DNA, RAPD analysis.

Astrophytum, a small group of cacti from Texas and the Eastern part of Mexico with only four species, are two with spines and two without (Schuster 1990). They are with moderate sized spheri- cal to slightly elongated succulent plant body most easily distinguished by the presence of white flakes on the body surface and their relatively few, sharply defined ribs. Their perfect symmetry along with their ornamental appearance is responsible for their popularity in every succulent collection. Astrophytums are lovingly called Bishop’s cap or star cactus due to the prominent wings and ribs, which give a star like appearance. Their striking forms are further enhanced by the most beau- tiful flowers, borne singly or in masses in various shades of yellow, sometimes with a red center during the entire summer season. Interspecific variations were also prominent in number and structure of aerioles, flower colour and size. Propagation of these plants through seeds or cuttings has been carried out extensively in our centre to cope with the increased demand for the production of large number of plants of this horti- cultural important species of Astrophytum. But the production of these plants through seed germination is not suitable mainly due to the low percentage of germination and long germination time.

* Corresponding author, e-mail: [email protected]. 180 Das A. B. Cytologia 73(2)

Growth of these plants through rooted cuttings or shoot budding is highly variable and the main ob- jective was to analyze and evaluate its variability and collection of these genotypes to enlarge the genetic basis of this plant. The level of polymorphism for morphological characteristics in genotypes is inadequate to allow varietals identification. Therefore, chromosome and DNA markers were involved to characterize its phenotypic variability. The present study deals the karyotype analysis and nuclear DNA content to discriminate species specific differences in chromosomes and their ancestry during the process of evolution and to ascertain precisely the importance of DNA in genetic diversity and phylogeny in four species of Astrophytum. Randomly Amplified polymorphic DNA analysis was carried out in these four species using 19 random primers to find out the variability in the amplified products of genomic DNA to establish phylogenetic relationships and affinities and to identify the molecular markers, if any. The morphological parameters of the plant, their spine and flower characteristics has also studied to de- lineate characters for identification and their comparative analysis.

Materials and methods Plant samples for this study were collected from the experimental cactus garden of Regional Plant Resource Centre, Bhubaneswar where above 1000 varieties were maintained collected from different parts of the world.

Morphological analysis Morphological characteristics were evaluated through plant descriptors including number of ribs, presence or absence of areoles and spines, spine characteristics and colour of the ﬂower.

Karyotype analysis For the chromosome study, fresh healthy root-tips were pretreated in 0.02 M hydroxyquinoline for 3 h at 14°C followed by overnight ﬁxation in 1 : 3 propionic alcohol. The root-tips were stained in 2% propionic orcein after cold hydrolysis in 5 N HCl for 5 min and were squashed in 45% propionic acid. Ten well-scattered metaphase plates were selected for karyotype analysis of each species. Form percentage (F%) of individual chromosomes was calculated and the total form percentage (F%) was the average of sum total F% of a karyotype (Das and Mallick 1993). Mean values of total genomic chromosome length and total chromosome volume with standard errors were calculated.

4C nuclear DNA content For Feulgen cytophotometric estimation of 4C DNA content, ﬁxed root-tips from each species were stained in Schiff’s reagent for 2h at 14°C and scorings were made from each slide using Nikon Optiphot microscope ﬁtted with a microspectrophotometer using monochromatic light at 550 nm following the method of Sharma and Sharma (1980). In situ DNA were obtained on the basis of optical density, which was converted to picograms (pg) using Vant Hof’s (1965) 4C nuclear DNA values (67.1 pg) for Allium cepa var. Deshi as a standard.

RAPD analysis Genomic DNA was isolated from young plants using the method of Saghai-Maroof et al. (1994) using extraction buffer containing EDTA (20 mM), Tris-HCl (100 mM), NaCl (1.5 M), CTAB (2%), b-mercaptoethanol (1%) and the DNA concentration was estimated using Versafluor TM Fluorometer (Bio-Rad, USA) using Hoechst 33258 as the fluorimetric dye. The DNA was dilut- 1 ed to a final concentration of 25 ng ml using T10E1 buffer, using as template for RAPD analysis. Polymerase chain reaction (PCR) following the standard protocol of Williams et al. (1990) was performed in a PCR amplification reaction mixture containing 25 ng of template DNA, 200 mM 2008 Assessment of Genetic Diversity and Phylogenetic analysis 181

Figs. 5–8. Diploid somatic chromosome counts in the four species of Astrophytum; A. asterias (2n 22), A. capricorne (2n22), A. myriostigma (2n22) and A. ornatum (2n22) respectively.

each dNTP, 25ng primer, 0.5 unit Taq DNA Polymerase (Bangalore Genei Pvt. Ltd., Ban- galore, India) in a GeneAmp PCR 2400 ther- mal cycler (Perkin Elmer, USA) programmed Figs 1–4. Four species of Astrophytum as distinguished from their morphological features; 1, A. aster- for 45 cycles. The ampliﬁed products were ias; 2, A. capricorne; 3, A. myriostigma; 4, A. separated by electrophoresis in 1.5% agarose ornatum respectively. gel in 1 TAE buffer at 60 V using Gene Ruler 100 bp DNA ladder plus (MBI Ferman- tas, Lithuania) as the marker DNA. The image with the DNA fragments was captured in an image analyzer Gel Doc-G 700 (Bio Rad, USA) for documentation and data analysis.

Statistical analysis chromosome and nuclear DNA analysis Mean values of total genomic chromosome length and volume with standard error were calculated. The correlation coefﬁcient analysis between different chromosomal parameters was done to ﬁnd out the genomic characteristics. The ANOVAs were performed (Sokal and Rohlf 1973) among the nuclear DNA values following Duncan’s multiple range test (Harter 1960).

RAPD data analysis In RAPD analysis, the presence or absence of the bands was taken into consideration and the difference in the intensity of the band was ignored. From RAPD data a similarity matrix was obtained using the Jaccard’s coefﬁcient. From this matrix a phylogenetic dendrogram was obtained by cluster analysis following the neighbourjoining method using NTSYS version 1.7, Exeter Software, New York, USA.

Results Morphological characteristics Phenotypic observation of the 4 species indicated variations among them in the shape of the plants, margin of the ribs, colour and texture of the plant surface, spine and flower characteristics as compared to the original. In general, A. aterias (Zucc.) Lemaire is the smallest sized globular cactus with 8–9 flattened ribs of dull gray-green colour with varying amount of white flakes all over the plants body (Fig. 1). In the middle surface of each rib present a vertical row of circular, raised, gray-white wooly areoles. Its flowers are yellow with a red centre, start appearing on plants only 182 Das A. B. Cytologia 73(2) 0.11 0.15 0.23 0.12 SE) SE) (% 0.98 41.87 2.131.09 45.25 35.93 4.25 36.91 3 m

ome volume; INV: Interphase nuclear vol- INV: ome volume; Fig. 5a–8a. Idiograms representing comparative kary- otypes in the species of Astrophytum; 5a, A. asterias; 6a, A. capricorne; 7a, A. myriostig-

SE) ( ma and 8a, A. ornatum respectively. 0.008 781 0.0060.012 625 862 0.005 598 Astrophytum SE) content (pg 0.181 9.98 0.340.23 10.23 9.12 0.96 8.95 Fig. 9. Standard types of chromosomes found in Astro-

3 phytum species. m

when 2–3 yr. old throughout the summer. 1.06 48.12 1.091.93 50.51 61.65 2.15 57.52 SE) ( A.capricorne (A. Dietr.) Britt et Rose. is TCL DNA INV TCV 4C TF% m globular becoming elongated with age, having m 6–7 narrowly pointed ribs densely covered with white scales giving a mealy appearance (Fig. 2). On the margin of the ribs present a

NSC vertical row of areoles being covered with

Comparative cytological parameters in four species of cytological Comparative large number of ﬂexible spines bent upward.

4D 4 60.76 Flowering is profuse and throughout summer. A. myriostigma Lemaire. is ﬁve-ribbed 4D 6 54.65 2D 6 53.05 2D 4 60.12 14C Table 1.

with tufted areoles and no spines (Fig. 3). On 2B 12C 14C 16C formula ( the margins of the ribs present a vertical row Karyotype of suppressed gray-white wooly areoles without having any spine. A. ornatum (DC.) F. A. C. Weber is the n 22 6A 2222 6A 4B giant of the genus reaching height of up to 3 m with a diameter of 30 cm. The stem of this species is cylindrical with prominent 8 acute

nudum ribs either straight or spiral (Fig. 4). The dark green body is covered with white ﬂecks form- var. ing patterns on the ribs coalescing together at (Zucc.) 22 2A

Species 2 times to form thick white bands. Spines are yellow to dark brown, 5–11 in number per are- : Diploid chromosome number; NSC: Number of secondary constricted chromosomes; TCL: Total chromosome length; TCV: Total chromos Total chromosome length; TCV: : Diploid chromosome number; NSC: Number of secondary constricted chromosomes; TCL: Total n

2 percentage; SE: Standard error. ume; F%: Form ole, all are radials, 3–4 cm long, very sharp (Dietr.) Britt et Rose (Dietr.) A. myriostigma Backb. (R. Mey) A. ornatum A. asterias (Zucc.) Lem. A. capricorne and straight. 2008 Assessment of Genetic Diversity and Phylogenetic analysis 183

Chromosome characteristics The diploid somatic chromosome number 2n22 was observed in all the four species of As- trophytum (Fig. 5a–8a). Though they have same number of chromosomes in their genome, they differed from each other in the minute details of the karyotype. On the basis of the size of the chromosomes and position of the constrictions, four types of chromosomes were recognized (Fig. 9): Type A – Chromosomes with two constrictions in median and sub median positions. Type B – Chromosomes with two constrictions in submedian and sub terminal positions. Type C – Chromosomes with nearly median to median primary constriction. Type D – Chromosomes with nearly submedian primary constriction. The karyotype formula of all the species revealed definite differences in the chromosome structure (Table 1, Fig. 5a–8a). All the types of chromosomes i.e. A, B, C and D were found in the karyotype of only A. ornatum, Type A chromosome was found in all the species except in A. capricorne, where maximum number of B type chromosomes were found. Type B chromosome was ab- sent in A. aterias and A. myriostigma. The minimum of two D type chromosomes were found in A. aterias and A. capricorne which varied from 4–6 in other two species. The number of C type chromosomes was found to be more as compared to D type of chromosomes. Furthermore, dose differences in the median and sub median constricted type C and D chromosomes were noted in all the species. The number of secondary constricted chromosomes were 4 in A. capricorne and A. ornatum and 6 in A. aterias and A. myriostigma. The total chromosome length ranged from 53.05 mm in A. aterias to 60.76 mm in A. ornatum and the total chromosome volume ranged from 48.12 mm3 in A. ornatum to 61.65 mm3 in A. myriostigma (Table 1). The form percentage (TF%) significantly varied from 35.93% in A. myriostigma to 45.25% in A. capricorne. Significant variations in chromosome length, volume and TF% were observed among the studied species of Astrophytum.

INV and 4C nuclear DNA amount Interphase nuclear volume (INV) differed significantly in the four studied species. The maximum value 862.82 mm3 was recorded in A. myriostigma. The data on nuclear DNA amount and other cytological parameters have been presented in Table 1. The 4C DNA content varied significantly from 8.95 pg in A. asterias to 10.23 pg in A. capricorne. The average nuclear DNA content varied from 0.40 pg to 0.46 pg. Total chromosome length was highly correlated with chromosome volume (0.89). Chromosome volume showed significant correlation with INV (0.58). The highest correlation was observed between the 4C DNA content and INV (0.59). The ANOVA and Duncan’s multiple range test showed significant variations in the nuclear DNA values in Astrophytum at 1% level.

RAPD analysis The RAPD profiles were reproducible showing variations in the intra- and interspecific banding patterns within the different plants that were used for further analysis. A total of 64 polymorphic RAPD markers were amplified with random primers. The number of bands for each primer which produced a polymorphic pattern varied from one (OPA11, OPN8 in A. asterias) to 13 (OPA13, OPN5 in A. capricorne and A. ornatum) and varied in size from about 0.5 to 3.1 kb. Some plants showed clear differentiation among the individuals of the same species (Fig. 10). In all the four species, we obtained a total of 315 amplified bands from 19 different random primers (Table 2). Of these, the different primers respond differently and the maximum number of amplified bands in all the four species were observed in primer OPA7 and least number of bands in OPN8. The highest 13 number of bands were observed in A. capricorne in primer OPA13 and the lowest single bands were found in A. asterias amplified with primer OPA14 and OPN8. From the Jaccard’s coefficient similarity matrix, the results showed a lower genetic similarity when compar- isons were made between spine and spineless species. In general, when spineless A. asterias and A. 184 Das A. B. Cytologia 73(2)

Fig. 10. RAPD ampliﬁcation proﬁles of Astrophytum using primers OPD-02, OPA-04 and OPA-11 from left to right. M-Marker DNA (DNA Lad- Fig. 11. Dendogram representing clustering of Astrophy- der plus, MBI, Fermentas), AA, A. asterias; tum species based on the Jaccard’s similarity in- AC, A. capricorne; AM, A. myriostigma; AO, A. dices from RAPD analysis using random ornatum. primers.

Table 2. PCR generated total ampliﬁed bands and polymorphic bands in the respective primers in the RAPD analysis of the 4 species of Astrophytum

Bands ampliﬁed Polymorphic bands Nucleotide Sl. No. Primer sequence AA AC AM AO AA AC AM AO

1OPA-02 TGCCGAGCTG 46703560 2OPA-04 AATCGGGCTG 12 9 10 10 5 6 7 8 3OPA-07 GAAACGGGTG 9 12 12 11 4 7 4 8 4OPA-08 GTGACGTAGG 119997556 5OPA-09 GGGTAACGCC 89786646 6OPA-11 CAATCGCCGT 65263414 7OPA-12 TCGGCGATAG 67364524 8OPA-13 CAGCACCCAC 9 13 8 11 7 11 5 8 9OPA-14 TCTGTGCTGG 15351313 10 OPD-02 GGACCCAACC 11 11 9 9 7 5 5 6 11 OPD-03 GTCGCCGTCA 9 12 10 9 8 9 6 7 12 OPD-04 TCTGGTGAGG 82437121 13 OPD-08 GTGTGCCCCA 8 10 9 9 5 6 6 7 14 OPN-04 GACCGACCCA 11 10 9 11 7 9 7 9 15 OPN-05 ACTGAACGCC 9 11 8 12 7 8 6 11 16 OPN-06 GAGACGCACA 88996555 17 OPN-08 ACCTCAGCTC 12441243 18 OPN-10 ACAACTGGGG 8 11 8 9 5 6 4 7 19 OPN-18 GGTGAGGTCA 56843462

AA: Astrophytum asterias,AC: A. capricorne, AM: A. myriostigma, AO: A. ornatum. 2008 Assessment of Genetic Diversity and Phylogenetic analysis 185 myriostigma were compared with the spined A. capricorne and A. ornatum, the genetic similarity averaged 40.66% to 44.97% compared to 49.42% between species of same spine type. Interestingly, A. capricorne and A. ornatum had a closer relationship with average genetic similarity of 49.42% and A. asterias and A. myriostigma had 45.78%. The percentage of polymorphism varies from 20.2% to 85.0% in the 4 studied species (Table 2). The average percentage of polymorphism was highest between A. aterias and A. ornatum (59.34%) and the lowest percentage of polymorphism was observed between A. capricorne and A. ornatum (50.58%). The degree of similarity in banding pattern was highest in the case of A. capricorne and A. ornatum (49.42%) followed by A. asterias and A. myriostigma (45.78%) and the lowest in A. asterias and A. ornatum (40.66%). The banding pattern obtained from the primers OPA02 revealed 100% similarity between A. capricorne and A. ornatum.

Cluster analysis The cluster analysis of the RAPD banding patterns of all the primers following the method of unweighted pair group with arithmetic mean (UPGMA) revealed that the dendogram (Fig. 11) formed two branches with A. asterias and A. myriostigma clustered together with a similarity coefﬁ- cient of 0.45 and A. capricorne and A. ornatum with similarity coefﬁcient 0.57.

Discussion Karyotype, genome length and nuclear DNA amount Detailed karyotype analysis of four species of Astrophytum revealed the same number of diploid chromosomes, 2n22 in all the studied four species. Although the chromosome numbers were same they differ in their type and number of secondary constricted chromosomes in their karyotype. Among the species studied, the two types of chromosome C, D were common in all the four species. The metaphase chromosome number 2n22 was noted in all the 4 species studied with the number of secondary constricted chromosomes varied from 4 to 6 (Table 1). Numerical variation in the types of chromosomes were noticed in Astrophytum. Type A chromosomes were present in A. aterias, A. myriostigma and A. ornatum. However, dose variation in type A, B, C and D chromosomes was the most important feature in different Astrophytum species. In both A. asterias and A. myriostigma number of the secondary constricted chromosomes were six in number all are A types and A. capricorne and A. ornatum had both 4 secondary constricted chromosomes out of which A. ornatum with two types i.e. A and B type. The median constricted chromosomes varied from 12–16 in all the species studied. The number of C type chromosomes was more compared to the D type in all the species. Comparatively larger chromosomes with a maximum number of secondary constricted chromosomes suggest highly dynamic genome structure in A. ornatum. Evident- ly, structural changes as well as changes in the amount of heterochromatin may have played a vital part in inducing differences at species level (Das et al. 1999a, b, Basak et al. 1998, Jena et al. 2002). The interspecific variation of TF% values from 35.93% to 45.25%, from sub-metacentric to metacentric chromosomes among the four species might be due to structural alternations in the genome during speciation as a result of duplication of parts of some chromosomes, or due to translocations between them, with or without involvement of secondary constrictions (Das et al. 1999a, b). In fact, out of four species, F% was about 35% in A. asterias and A. myriostigma, where- as it was 41.12% in A. ornatum and 45.25% in A. capricorne. The gradual increase in the number of sub-metacentric chromosomes in the karyotype from A. asterias and A. capricorne to A. myriostigma and A. ornatum suggest that the median constricted chromosomes might have evolved from the species with sub median type of chromosomes in the process of evolution and gradual selection of the species. Furthermore, the symmetrical karyotype with same number of secondary 186 Das A. B. Cytologia 73(2) constricted chromosomes in A. asterias and A. myriostigma and A. capricorne and A. ornatum suggests their closer affinity between them. The structural alterations in the morphology as well as variations in the secondary constricted chromosomes of different species might be due to duplication of chromosomes or translocation between the chromosomes with or without secondary constrictions at a very early stage of evolution (Das and Mallick 1993, Das et al. 1998a, b, 1999a, b). The correlation coefficient between average chromosome length and average DNA content did not show significant relationship (r0.135). Chromosome volume showed significant correlation (0.889) with chromosome length. Evidently, the differences in length may be attributed to the differential con- densation and spiralization of chromosome arm. The average DNA content showed a high degree of correlation with average chromosome volume. However, in the eukaryotic system chromosome volume is determined not only by its DNA, but also by its basic and non-basic proteins. The maximum DNA content was found in A. capricorne having maximum number of B type chromosomes and the minimum 35.93 pg was found in A. myriostigma which lacks type B chromosomes. The diversity of DNA amount has often been attributed to loss or addition of highly repetitive DNA sequences rather than the AT- or GC-rich sequences in a genome (Martel et al. 1997). These reach a certain level and become stabilized during micro-evolution and gradual selection (Price et al. 1980). Such interspecific variation in DNA amount is not exceptional in angiosperm species (Rayburn et al. 1989).

Nuclear DNA amount in relation to genomic chromosome volume and INV A detail analysis revealed significant variations in the total chromosome volume per chromosome ranging from 1.9 mm3 in A. ornatum (2n22) to 2.8 mm3 in A. myriostigma (2n22). The DNA content showed no significant correlation with average chromosome length (0.135) but significant or average chromosome volume (r0.331); since in eukaryotic system chromosome volume is determined not directly by DNA but by the basic and non basic proteins as well. The species specific compactness of DNA threads along with nucleosomes or the additional gene sequences (Das and Mallick 1993) with altered non histone proteins in the chromosome played a significant role for chromosomal architecture of a species. INV showing significant correlation with the average DNA content (r0.950) suggest an interdependence of 4C DNA with INV of the species. Perhaps, the differential interaction of genomic characteristics lead to genomic DNA variation (Das et al. 1999a, b).

Diversification in DNA amount Significant differences of 4C DNA were recorded at interspecific level; such variations were noticed in other groups of cactus like Mammillaria (Das et al. 1997, 1998a, b, 1999a, b, Mohanty et al. 1997); Melocactus (Das et al. 1998b) and Ferocactus (Das et al. 1999b). The constancy in the DNA amount at the species level in repeated experiments revealed a stable 4C DNA content in each species. The DNA amount, though, differed significantly at species level, the differences in the DNA content, however, greatly depended on the repetitive sequences in the genome. The maximum (10.23 pg) 4C DNA content was noted in A. capricorne with only B, C and D type of chromosomes and minimum (8.95 pg) in A. asterias (2n22). Perhaps, the C and D types were basic chromosomes and the A and B types (secondary constricted chromosomes) were derived in the evolution process. The 4C DNA, however, showed a high degree of correlation with INV (r0.950). The variability in the stable DNA content in different species might be attributed to the loss or addition of many repeats in the genomes due to the alterations in the micro-and macro-environment during evolution of species (Price et al. 1980). The variability of DNA amount was often attributed to loss or addition of highly repetitive DNA sequences in a genome which reached a certain level and got stabilized during micro-evolution and gradual selection. 2008 Assessment of Genetic Diversity and Phylogenetic analysis 187

Polymorphism in RAPD marker A DNA based molecular technique like RAPD could identify genotypes directly and help to mitigate complications arising from earlier cytological and morphological studies. In the present study of the RAPD analysis of the four species of Astrophytum, the maximum average percentage of polymorphism (59.34%) was noticed between A. asterias and A. ornatum and the minimum (50.58%) in A. capricorne and A. ornatum, therefore suggesting a greater similarity in genetic con- stitution between A. capricorne and A. ornatum as compared with A. asterias. From the similarity matrix index the percentage of similarity varied from 15.0% between A. asterias and A. ornatum to 79.8% between A. capricorne and A. myriostigma (Table 3). The average highest percentage of similarity between A. capricorne and A.. ornatum (49.42%) followed by A. asterias and A. myriostigma (45.78%) and the lowest between A. asterias and A. ornatum (40.66%) suggest a close relationship among the two spined species and a higher percentage of polymorphism among spine and spineless species. The percentage of polymorphism ranged from 20.2% between A. capricorne and A. myriostigma to 85.0% between A. asterias and A. ornatum. The UPGMA cluster analysis of four species shows high coefficient of interspecific similarity between them forming two distinctly separate groups. i.e. A. asterias and A. myriostigma (0.45) and A. capricorne and A. ornatum (0.57) that are also confirmed from the karyotype, 4C DNA content studies. It was clearly found from the dendogram that 2 distinct groups were present, each consist- ing of 2 species. The groups with closely related species consisted of A. asterias paired with A. myriostigma and A. capricorne with A. ornatum. The observations on the RAPD analysis of Astro- phytum are in accord with the other cytological and cytochemical studies. The OPA02 primer yield- ed maximum similarity in all the species at a level of 100% between A. capricorne and A. ornatum. A. asterias showed significant marker bands at 3000 and 330 bp in the primer OPD2, at 530 bp in OPA11. A. ornatum showed marker bands at 700 and 480 bp in the primer OPA2, which was the most prominent of all. A. capricorne and A. ornatum produced 1732 bp, 700 bp, 500 bp DNA marker in the primer OPN8 which differentiates these two species from the other two. A. asterias and A. myriostigma also produced similar marker bands of 1450 and 1050 bp. The distinct RAPD bands could be cloned and used as markers for the species identification. In conclusion, these findings provide an effective tool for species identification and to access the existing interspecific genetic polymorphism to study their phylogeny.

Acknowledgement The authors are thankful to the Ministry of Forest and Environment, Govt of Orissa and India, for providing necessary facilities and Financial Assistance.

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