Intraspecific variations in cardamom (Elettaria cardamomum Maton): assessment of genomic diversity by flow cytometry, cytological studies and ISSR analysis

Plant materials

Solanum lycopersicum L. Stupicke polni rane (2C DNA content is 1.96 pg), Glycine max Merr. Polanka (2C DNA content is 2.50 pg) and Zea mays L. CE-777 (2C DNA content is 5.43 pg) obtained from Laboratory of Molecular Cytogenetics and Cytometry, Institute of Experimental Botany, Sokolovska, Czech Republic were used as internal reference standards for genome size estimation study (Dolezel et al. 2007).

Genome size determination using flow cytometry

Samples were prepared according to a standard two step procedure (Otto 1990). About 50 mg of young and fresh incompletely expanded leaf tissue of test sample and internal standard were co-chopped with a sharp razor blade in a plastic petridish containing 2 ml of ice-cold Otto I buffer (0.1 M citric acid, 0.5 % Tween 20). This was kept at −4 °C for 40 min for releasing nuclei from cells to buffer. The nuclear suspension was filtered through a 40 µm Nylon mesh and centrifuged at 150g for 5 min. Nuclei were gently resuspended in 100 µl of fresh Otto I buffer after removing the supernatant. 1 ml of Otto II buffer (0.4 M Na₂HPO₄·12H₂O) supplemented with propidium iodide for absolute DNA content determination and RNase (both at a final concentration of 100 µg/ml) was added (Bures et al. 2003; Leong-Skornickova et al. 2007; Lysak et al. 2000; Palomino et al. 2003; Pecinka et al. 2006; Roux et al. 2003). All the sample preparation methods were done on ice. The fluorescence intensity of 5000 particles was recorded using a BD FACSAria III Cell Sorter (BD Biosciences, California, USA). Solanum lycopersicum L. Stupicke polni rane, Glycine max Merr. Polanka and Zea mays L. CE-777 were used as internal reference standards to determine the genome size of cardamom and Z. mays L. CE-777 was found to be appropriate reference standard there exist around a twofold variation between the mean peak intensity of the sample and the standard (Petr et al. 2012). Each sample was analysed twice for getting reliable results.

The number of nuclei and coefficient of variation (CV) for peaks of both the cardamom and maize were procured by gating using the BD FACs Diva software. Rectangular gates were drawn manually around the section which is of interest on the histogram of PI fluorescence-Area against PI fluorescence-width to discard the intervention of debris. The mean peak intensity of nuclei of cardamom and maize in the G1 phase of the cell cycle were compared and 2C DNA content of cardamom was estimated (Wang et al. 2015). The formula used to estimate the nuclear DNA content of cardamom (Gregory et al. 2015) was: 2C DNA nuclear content (pg) of cardamom = (sample peak mean/standard peak mean) × 2C DNA content of the reference standard (pg). One way ANOVA and t Test were used to check the significance of genome size variation among the accessions (n = 30) (Sheidai et al. 2014).

Determination of chromosome count

The root tips of cardamom were collected around 10.30 am which is the peak hour of mitotic activity in cardamom plants. Somatic chromosomes from root tip cells were fixed in Carnoy’s fluid (ethanol and glacial acetic acid in the ratio 3:1). The root tips were pretreated in 0.002 M solution of 8-hydroxyquinoline for 2 h at 4 °C. The smear and squash preparations were stained in 2 % acetocarmine, and well spread chromosome preparations were photomicrographed using Leica DM 100 digital camera attached with Leica DM 2500 trinocular microscope (Ngamriabsakul 2004).

ISSR analysis

The bands obtained with each primer were scored as diallelic characters: 1 denotes present and 0 denotes absent (Lamyai et al. 2014; Mark et al. 2015). The amplified products extends in size from 200 to 2000 bp. A binary qualitative data matrix was formed and estimated the Observed number of alleles (na), Effective number of alleles (ne), Nei’s gene diversity (h), Shannon’s Information index (I) and number of polymorphic loci (p) using the software POPGENE ver 1.31 (Yeh et al. 1999; Amit et al. 2014).

In an attempt to get a deeper understanding of the relationship between genetic variability and genome size, 2C-values of the accessions which are common in the present study and that of our previous research (Anjali et al. 2015) were subjected to correlation analysis with ISSR and EST-SSR based genetic variability parameters such as number of alleles per loci and polymorphic alleles.

2C nuclear DNA content estimation

Flow cytometric study generated histograms with good resolution peaks (Fig. 1). Nuclear DNA content of thirty accessions of cardamom were estimated (Table 3). 2C DNA amount shows low variation among the studied accessions. Maximum genome size was 3.22 pg shown in accession C61 (ICRI-2, a released variety) and minimum was found in C39 (a wild accession from Cheenikkala, Sankhili) as 2.57 pg. It shows a difference of about 1.25 times. ANOVA and t Test analysis did not find out significant differences in nuclear DNA content among the 30 accessions analysed as wild, feral, and cultivar groups. Mean 2C nuclear DNA content of all the thirty accessions of cardamom was calculated as 2.87 pg and the mean genome size is 2.81 × 10⁹ bp. This is the first report on genome size of E. cardamomum.

Acc. no.	Type	2C DNA (pg)	Genome size (bp)
C11	LR	2.82	2.76 × 109
C51	Feral	2.71	2.65 × 109
C55	LR	3.02	2.95 × 109
C61	RV	3.22	3.15 × 109
C62	RV	2.68	2.62 × 109
C63	LR	2.93	2.87 × 109
C65	RV	3.10	3.03 × 109
C96	RV	3.07	3.00 × 109
C97	RV	2.84	2.78 × 109
C16	Feral	2.83	2.77 × 109
C17	Feral	2.89	2.83 × 109
C28	Feral	2.96	2.89 × 109
C31	Feral	2.69	2.63 × 109
C38	Feral	2.78	2.72 × 109
C39	Feral	2.57	2.51 × 109
C40	Feral	2.77	2.71 × 109
C52	Feral	2.95	2.89 × 109
C53	Feral	2.72	2.66 × 109
C70	Feral	2.72	2.66 × 109
C81	Feral	3.04	2.97 × 109
C83	Feral	3.14	3.07 × 109
C84	Feral	2.75	2.69 × 109
C86	Feral	2.85	2.79 × 109
C91	Feral	2.74	2.68 × 109
C26	Wild	2.91	2.85 × 109
C68	Wild	3.14	3.07 × 109
C74	Wild	2.74	2.68 × 109
C75	Wild	2.70	2.64 × 109
C76	Wild	2.97	2.90 × 109
C77	Wild	2.86	2.80 × 109

RV released variety, LR landrace

Chromosome count

The chromosome number was found to be 2n = 48 in the studied accessions by observing the mitotic metaphase stage. The experimental was repeated with different samples, but no variation in chromosome number was observed.

Genetic diversity analysis using ISSR markers

Correlation between genome size and genetic variability

The analysis revealed no correlation between the genetic variability and genome size in the studied accessions (Fig. 2). Further, the thirty accessions were divided into four groups based on their wild/cultivar nature. The one-way ANOVA was estimated from these four groups, viz. feral [n = 16, mean (m) = 2.82, variance (v) = 0.0213], landrace (n = 3, m = 2.92, v = 0.0100), released varieties (n = 5, m = 2.98, v = 0.0474), and wild (n = 6, m = 2.87, v = 0.0257). Since if F(1.4992) < F(2.9752) crit, it was concluded that the four groups did not differ significantly from each other. Further t Tests were also performed to test each of the above pairs of the means. These tests also did not show any significant differences between any of the pairs.

Genome size estimation

Variation in nuclear DNA content was observed among the studied accessions of cardamom which ranged from 2.54 to 3.22 pg. However, this variation was not statistically significant as estimated using one-way ANOVA and t Test. This means that genome size estimates among the 30 accessions or among the various groups (wild, feral, released variety and cultivar) did not differ significantly from each other. According to various reports, intraspecific variation in nuclear DNA content of plants is a disputable subject in the scientific community. Very few studies are reported with remarkable level of intraspecific genome size variation (Bennett and Leitch 2005). As per some of the earlier studies on Collinsia verna, Pisum sativum and Glycine max, it was observed that there was a significant variation. But later Greilhuber (1998) came out with the conclusion that they were merely due to some technical issues. Based on some works which described the variation in genome size within species using authentic controls and standards, Murray (2005) explained that this intraspecific variation has some taxonomic relevance if the former could be related to morphological variation. About 1.19 times variation in genome size was reported for Linum glaucum populations in Iran and difference in nuclear DNA content was found to be associated with altitude and morphological characters (Seyed et al. 2015). Marek et al. (2009) reported a high level intraspecific variation in different populations of Picris hieracioides. Significant level of intraspecific variation was noted in five species of Lathyrus genus (Nandini et al. 1997). The contradiction of having low intraspecific variation and high interspecific variation in diploid Triticeae species proposed that emergence of genome size occurs just before, during or right after the process of speciation (Eilam et al. 2007). 2C nuclear DNA content analysis exhibited a 1.1 fold difference and thus a low level intraspecific variation in Camellia sinesnsis var. assamica (Huang et al. 2013). 1C nuclear DNA content of the populations of Pinus nigra was reported to have around 23 pg (Faruk et al. 2007). Sandra et al. (2015) described that DNA content shows only a small variation within Miscanthus sinensis cultivars. Considerable differences in DNA content was not observed among the five populations of Pinus heldreichii when a t Test was performed (Bogunic et al. 2003).

Genome size in Zingiberaceae members

A search in the C-value database of RBG Kew using the term ‘Zingiberaceae’ resulted in 39 records belonging to 7 genus such as Curcuma, Paracautleya, Hitchenia, Kaempferia, Stahlianthus, Alpinia and Zingiber. Average 2C value of the family is 2.79 pg and the minimum and maximum values are 1.66 and 12.05 respectively. The mean 2C value of cardamom estimated in the present study was 2.87 pg which falls in comparable range of the family.

Use of genome size estimation

In addition to chromosome numbers and ploidy levels, genome size has been estimated in many plants as it is an important attribute in biology and biodiversity (Bennett et al. 2000; Hanson et al. 2001) and pre-requisite for genome sequencing experiments. The co-processed samples with different genome sizes always gave two distinct peaks, which is the most convincing evidence for genuine differences in DNA content (Greilhuber 2005). Genome size variation has significant consequences at cellular, tissue and organismal levels and also influences phenological and ecological behaviour. Comparisons of genome size data within genus and species levels and probable phylogenetic relationships suggest that both increases and decreases of genome size have occurred in evolution (Wendel et al. 2002; Soltis et al. 2003).

Ploidy level and genome size

The occurrence of different chromosome numbers (2n = 48, 52) in cardamom was reported in early cytological studies (Gregory 1936; Sharma and Bhattacharya 1959; Chakravarti 1948). Variations in chromosome numbers were observed in Mysore and Malabar varieties of cardamom indicted that aneuploidy as well as structural alterations in the chromosome have contributed to the varietal differentiation (Chandrasekhar and Sampathkumar 1986). Earlier workers have reported that cardamom is of amphidiploid origin from wild species (Peter et al. 2007). As ploidy level is well correlated with the mode of reproduction, detailed knowledge of genome size in particular species plays an important role in forming hypotheses about evolutionary potential of the species and evolutionary processes in the genus (Chrtek et al. 2007). The present study did not find any ploidy changes among the studied accessions as evident from observations based on cytology and the present flow cytometry analyses. In general, ploidy screening can be done through analysing a test sample with those of the same species having known ploidy using the same instrument settings. This would result in a histogram from which ploidy can be determined from the G1 peak position. The wild cardamom plant collected from Therakkudy, Ernakulam (C75) was screened for chromosome number by root tip squash technique and found to be tetraploid (2n = 4x = 48). While calculating the 2C DNA content of cardamom using maize as internal reference standard, the G1 peak of all the cardamom accessions were positioned around channel 26. So it can be concluded that all the 30 accessions of cardamom that were taken for this particular study are having tetraploid nature.

Genome size and genetic variability

It is well cited that there is no correlation between the amount of DNA per cell and organismal advancement or genetic complexity (Sparrow et al. 1972; Price 1988) which has been historically termed the “C-value paradox” (Thomas 1971). Since the discovery of non-coding DNA and its impact on genome size variation [for example, retrotransposons have increased the size of the maize genome two- to five-fold since the divergence of maize and sorghum from a common ancestor about 16 million years ago (Sanmiguel and Bennetzen 1998)], “paradox” has been replaced by “enigma” in order to address the matter properly (Gregory 2002, 2004). It is now generally agreed that the C-value enigma is due to the differential amplification and proliferation of the repetitive sequences of the genome among organisms (Bennetzen 2000, 2002; Kidwell 2002; Hawkins et al. 2006).

Microsatellites are ubiquitous class of simple repetitive DNA sequences which are abundant in all eukaryotes analyzed and is thought to result from the mutational effects of replication slippage (Tautz and Schlötterer 1994). To find out the relation between genome size and microsatellite occurrence, the microsatellite frequency was analyzed in plant species with a 50-fold range in genome size that is mostly attributable to the recent amplification of repetitive DNA (San Miguel et al. 1998). It was found that the overall frequency of microsatellites was inversely related to genome size and to the proportion of repetitive DNA but remained constant in the transcribed portion of the genome among species (Morgante et al. 2002). This indicates that most microsatellites reside in regions pre-dating the recent genome expansion in many plants. The microsatellite frequency was higher in transcribed regions, especially in the untranslated portions, than in genomic DNA (Morgante et al. 2002). In a more recent study, it was found that EST-SSR density in the coding regions was not associated with genome size in selected genera of woody trees representing gymnosperms (17 species from seven genera) and angiosperms (40 species from eight genera) (Ranade et al. 2014).

In the present study, high genetic variation was observed among different accessions of cardamom using ISSR markers (for example, Shannon’s information index (Table 4) ranged from 0.23 to 0.53, around 130 % increase). Similarly, the variability in genome size was also clearly observed. However, there was no correlation between the increase in the genetic variability parameters and genome size in the studied accessions (Fig. 2) which is at par with a previous finding that EST-SSR density was not associated with genome size in neither angiosperms nor gymnosperms (Ranade et al. 2014).