The following six previously described strains of Dictyostelium giganteum were used to make chromosome preparations: 46a3, 46c6, F4, F5, F15 and F16. 46a3 and 46c6 are soil isolates from a 50-ha plot of undisturbed forest in the Mudumalai nature reserve 12. F4, F5, F15 and F16 are derived from different spores in a single fruiting body isolated from elephant dung, also from the same reserve 211. Following their isolation from the wild, the strains were sub-cultured and maintained either in the form of fruiting bodies on non-nutrient agar plates or stored as spores in glycerol at -80^oC12. Dictyostelium can be grown in suspension or in culture dishes and either axenically or in the presence of bacteria. Media and buffers required for culturing were prepared according to published guidelines and available in the Dictyostelium Web resource 13. Dictyostelium giganteum was grown with Klebsiella aerogenes on SM agar plates using a modified protocol. A lawn of bacteria was grown first on an SM agar plate by overnight incubation at 37°C. A single spore head taken from a D. giganteum fruiting body was picked with a sterile wire loop and transferred to 10µl of autoclaved MilliQ water, and the suspension was dropped in the centre of an agar plate that had a previously grown lawn of K. aerogenes on it. The plate was incubated at 22°C for 40 hrs. Amoebae that emerged from the deposited spores grew outwards from the centre of the plate. The method ensures that the cells in the centre enter starvation and begin the phase of multicellular development, while cells in the periphery are always in a vegetative stage, therefore are mitotically active and readily available for making chromosome preparations.

Chromosome preparations were carried out for all the six stains by doing independent sampling of each of them (same stock revived from -80°C) at least 3 times to avoid misinterpretation of results caused due to possible cross contamination of the strains.

The plate with D. giganteum cells was observed under microscope and the central part of growth area which contained cells in developmental phase was cut and removed. The remaining peripheral area with growth which contained mitotically active cells in vegetative phase was treated with 5ml of 400µg/ml colcemid solution (stock solution of 10mg/ml) prepared in 1xKK₂ buffer. The culture plate was incubated for 2 hours with colcemid at 22°C and at very low rpm so that growth of the cells on the SM agar plate would not be disturbed or minimally disturbed while simultaneously getting treated with the colchicine.

The cells were collected by purging the cells with 1ml pipetman in a 10ml falcon tube. The washing was done for four times in 1xKK₂ buffer for 10 minutes at 1000rpm. Washing with 1xKK₂ buffer ensured the removal of the bacterial cells from the suspension. This was followed by incubation of the cells with water at 22°C for 10 minutes (this worked better than the standard 0.56% KCl hypotonic treatment).

Amoebae were fixed at least 4 times with a slight modification of the standard protocol (Brody and Williams, 1974) by using a 6:1 ratio of methanol and glacial acetic acid. After fixative washes the pellet was re-suspended in 1 ml of fresh fixative.

Slides for the karyotype study of six strains of D. giganteum were prepared by dropping 120 µl of cell suspension on the slide and then warming the slide on a 37°C warmer for 30min. Giemsa staining (using a stock solution of 2%) was carried out for 4min using Sørensen's phosphate buffer and rinsing the slides 15-20 times in autoclaved MilliQ water.

Microscopy: Metaphase chromosomes from the six strains of Dictyostelium giganteum were analysed and imaged using OLYMPUS BX51 microscope at 100x magnification and software from Applied Spectral Imaging (ASI)

Karyotyping: An attempt was made to arrange the chromosomes in the form of a karyotype based on the size of the chromosomes.

Twenty well spread metaphases for each of the six strains were selected. Areas of the individual chromosomes in a metaphase were calculated by using the software Image J 1415. Sum of area of all chromosomes in a metaphase was calculated. The proportion of the area of the individual chromosome with the sum of area of all the chromosomes in the metaphase was considered as genome proportion of the individual chromosome. This was done for all the 20 metaphases each of the six strains.

FISH was carried out on metaphase chromosome preparation of 46a3 strain of Dictyostelium giganteumbwith the help of a standard protocol used for human chromosomes, with slight modification 19.

In-house hybridization buffer (50% (v/v) formamide, 2× SSC, 10% Denhardt's solution, 0.1 M NaPO4 buffer) as described by 20 without SDS and slight variation of remaining solution componentswas used for preparing probe mix. Post- hybridization washing was carried out at 45°C. Hybridization signals were amplified using Fluorescent Antibody Enhancer set for DIG Detection using the protocol described by the manufacturer (Roche Applied Science, Germany). Figure 1

The FISH signals on the metaphase chromosomes, indicating the possible location of centromeres, were captured and analysed using OLYMPUS BX61 fluorescent microscope at 100x magnification and Applied Spectral Imaging software.

A minimum of 60 metaphases were analysed for each of the six strains of Dictyostelium giganteum (46a3, 46c6, F4, F5, F15 and F16); representative figures are shown in Figure 2. In all six, the metaphase chromosome number ranged from 4 to 6 with a frequency that was almost the same but differed slightly between them (Table 3). The modal number in each case was five. It appears reasonable to conclude that the haploid chromosome number in these strains of Dictyostelium giganteum, and probably in the species as a whole, is 5. No meiotic figures were seen in any preparation.

In all the six strains of D. giganteum analysed in this study, the chromosomes can be classified into 3 groups – two large chromosomes, one medium chromosome and two small chromosomes (Figure 3).

Assuming the modal number to be 5 based on the data obtained, 20 well spread metaphases with modal number 5 were selected from each strain. By making use of the software Image J, the area of each chromosome and the proportionate area with respect to the total area of all 5 chromosomes in a metaphase were calculated for each of the 20 metaphases for all the six strains of D.giganteum. This data was used to arrive at a rough estimate of proportion of genomic content of each of the 5 chromosomes in all the six strains (Table 4).

Based on the above data it can be concluded that the genomic content of chromosomes 1, 2, 3, 4 and 5 in all strains of D. giganteum is approximately the same, and amounts to 35%, 30%, 17%, 10% and 8% respectively. However, the small differences may be meaningful, and what they might imply will be addressed elsewhere. Figure 4

Fluorescence in situ hybridization of probes representing conserved centromeric sequences of D. discoideum on the metaphase chromosomes of 46a3 strain of D. giganteum, indicated that two of the large chromosomes are sub metacentric, the medium chromosome is telocentric, one of the small chromosomes is metacentric and the other small chromosome is telocentric.

To the best of our knowledge, apart from D. discoideum, chromosome-based studies have not been carried out on any other species of Dictyostelium except the simple karyotyping in Dictyostelium caveatumn21. Our study is also first inter-strain study. For a long time it was believed that D. discoideum had 7 haploid chromosomes 22. Subsequently, pulse-field gel electrophoresis revealed 5 chromosomes 23; the currently accepted number is 6 242526. The related species Polysphondyliumviolaceum is said to have 11 or 12 chromosomes 27.

Our study involved chromosome analysis of six distinct strains of D.giganteumand 3 independent samples for each strain, leaving little room for any ambiguity regarding the accuracy of chromosome number arrived at. The haploid chromosome number in all six strains of D. giganteum analysed in our study is found to be 5. The chromosomes can be classified into 3 groups, namely group 1 with two large-sized chromosomes, group 2 with one medium-sized chromosome and group 3 with two small-sized chromosomes. The genome size of D. discoideum is 34.042MB and the genome of an axenic strain has been completely sequenced 25. Preliminary results from work in progress indicate that the size of the D. giganteum nuclear genome is about 32 Mb (average of 6 strains; unpublished data). Based on that, and from the relative sizes of the various chromosomes in D. giganteum, we estimate their genomic contents as 11.07Mb, 9.73Mb, 5.40Mb, 3.11Mb and 2.66 Mb for chromosome Nos. 1,2,3,4, and 5 respectively.

FISH has been used in the past to locate the position of the centromere in human chromosomes 2829. Several experiments in past relating to FISH as 3031 has shown that DIRS-1 sequences are characteristic of D. discoideum centromeres. But application of FISH to identify centromere has been done by 32 who used this technique to show that Dictyostelium centromeres contain DIRS-1, but their FISH has not been performed in proper metaphase cells. We find some studies on centromere which give the information that chromosomes appear to be acrocentric 3334, one class of complex repeat in genome of D. discoideum serve as centromere 25 and sequences which compose the functional chromosomal elements like centromere are not conserved and appear to have underwent several modifications 3035. Our study is the first of its kind where we have localised the centromere in the chromosome of D. giganteum using the probe from D. discoideum. Data obtained from our FISH studies has indicated that two of the large chromosomes are sub metacentric, the medium chromosome is telocentric, one of the small chromosomes is metacentric and other small chromosome is telocentric.We have not carried out FISH with other D. giganteum chromosomes, but given the similarity in morphologies, assume that their centromeres will be similarly located.