Molecular and chemical characterization of mutant and nonmutant genotypes of saffron grown in Saudi Arabia

Abstract Saffron (Crocus sativus L.) is an important spice and medicinal plant that is cultivated in Asia, Europe, North Africa, and North America. Its morphological and biochemical parameters, such as the changes in the floral parts (six tepals, three stamens, three stigmata), biomass, and chlorophyll content, are primarily affected by environmental conditions. A polymerase chain reaction–rapid amplified polymorphic DNA (PCR‐RAPD) approach was used to analyze the extent of the polymorphisms between C. sativus genotypes grown in the Saudi climate. In this research study, the DNA fingerprints of the stigmata of C. sativus genotypes [K1 & K2 = C. sativus var. cashmerianus, C1 = C. sativus (nonmutant), T1 = mutant (T0‐2B), T2 = mutant (T1‐2B), T3 = mutant (T4‐2A)] were determined according to the floral parts, and a total of 10 decamer primers were used for PCR‐RAPD analysis. Only three pairs of arbitrary primers showed polymorphisms (33.3%–88.2%) in the total genomic DNA extracted from these genotypes. Jaccard's similarity index (JSI) ranged from 0.88 to 1.0. An unweighted pair group method with arithmetic mean (UPGMA) similarity and dendrogram matrix showed that two genotypes (T1‐2B and T4‐2A) were closely related to each other and to the strain CM‐cashmerianus, while the T0 of C. sativus genotype showed divergence.

of molecular techniques, therefore, helps a taxonomist not only to identify genotypes but also to assess and exploit genetic variability via molecular markers (Whitkus, Doebley, & Wendel, 1994).
Saudi Arabia is one of the countries with the highest levels of consumption of saffron spice. In the year 2009, the price for one kg of saffron spice in the local market reached 18,000 SR (~US$ 5,000). The development of domestic saffron production in Saudi Arabia is therefore important. In this context, Sharaf-Eldin, Fernandez, Al-Khedhairi, and Elsayed (2013) reported, for the first time, the cultivation of saffron in the Kingdom of Saudi Arabia (KSA), in particular within Alkharj Governorate, and recommended the cultivation of saffron corms in the first half of September, with the expectation of harvesting in the fourth week of November.
Polymerase chain reaction-random amplified polymorphic DNA (PCR-RAPD) is an important tool for identifying molecular markers and differentiates plant genomes on the basis of genomic DNA banding (Tivang, Skroch, Nienhuis, & De Vos, 1996;Williams, Kubelik, Livak, Rafalski, & Tingey, 1990). The polymorphisms and variability of genomic DNA can be seen after performing electrophoresis on the DNA bands that result from the primer binding sites. Significant variations in plant genome size occur in different species and differentiate the molecular characteristics of plants, allowing phylogenetic relationships to be established, as recognized by Goodspeed (1954).

| Plant material
Corms (each 10-12 g) of C. sativus L., and C. sativus var. cash-  Figure 1). This is a semiarid region, with an average annual rainfall of 15-25 mm. The main weather information for Alkharj, KSA, concerning temperature (T) is given in Table 1. Physical and chemical analyses of the field experimental soil (Table 2) were carried out before planting followed the method of Chapman and Pratt (1978). Saffron flowers with five or three stigmata were harvested during November and December of both growing seasons (2013 and 2014).

| Experimental design
We set up the experiment in a randomized complete block design with three replicates in each growing season. Plant population is based on the spacing between plants within row (intrarow = 20 cm) as well as the spacing between two adjacent rows (inter-row = 1 m) had been designed. Each plot was 2 m long and had two rows 1 m apart with 20 corms in each. The corms were planted at a depth of 10 cm, and we took care to provide all necessary agricultural support [irrigation (drip method), fertilizer, and weed control] to ensure unstressed conditions (Sharaf-Eldin, Fernandez, Al-Khedhairi, & Elsayed, 2013).

| Flower samples
About 75 days after the corms were planted, we harvested the saffron flowers. This was done by hand early every morning before the tepals started to open. The crop cycle of saffron was estimated as 6 months.
We harvested the first flower at the end of November and the last three weeks after that. The biomass data were assessed based on the floral dry weight, including the stigma, stamen, and tepal.

| Genomic DNA extraction
We isolated genomic DNA from C. sativus L. genotypes in our laboratories using a modified version of Doyle and Doyle's protocol (1990).
The DNA was purified from contaminants such as RNA, proteins, phenols, terpenes, and other secondary metabolites, as well as the colored compounds present in the C. sativus leaves. The purity of the DNA was checked on a 1.0% agarose gel and resolved to appear as fine bands. We then treated the extracted DNA sample in TE with 1 μl RNase (10 mg/ml stock) for 10 min at 37°C, and then added an equal volume of the mixture of chloroform: isoamyl alcohol (24:1). We centrifuged the mixture at 11,180 g for 10 min at 4°C, before extracting the aqueous phase and adding 0.6 volume of isopropanol. We then froze the mixture at −20°C for 10 min before centrifuging it once again at 10,000 rpm for 5 min at 4°C and carefully decanting the supernatant. We washed the resulting pellet twice with 70% ethanol and dried it at 37°C for 10 min. Finally, the DNA pellet was dissolved in 50 μl of 1× TE buffer. The absorbance of the DNA solution was taken at 260 nm using a spectrophotometer (Specgen, Darmstadt, Germany) in order to determine the concentration and purity of the DNA samples.

| Generation of randomly amplified polymorphic DNA (RAPD) markers
We extracted DNA from leaf samples of C. sativus genotypes (CMcashmerianus, T0, T1-2B, T4-2A) and subjected these DNA samples to RAPD analysis. To this end, we undertook PCR amplification with 10 sets of random primers (Table 3). The PCRs were conducted in 100μl Eppendorf tubes in a final volume of 25 μl, which contained the template (15 ng/μl), dNTP mix (2.5 mM each), Taq DNA polymerase, reaction buffer (10×), and 50 ng of random primers, adjusting the final volume to 25 ml with distilled water. The PCR amplification was conducted by using the Applied Biosystems, USA, using the following conditions: initial denaturation at 94°C for 5 min, followed by 40 cycles of denaturation at 94°C for 1 min, annealing at 35°C for 1 min, and extension at 72°C for 2 min; a final extension at 72°C for 5 min; and holding at 4°C. The PCR products were electrophoresed on 1.2% agarose gels and photographed using a gel documentation system (Bio-Rad, Gladesville, Australia). TA B L E 1 The mean temperature (°C) in Alkharj city throughout the experimental period

| Phylogenetic analyses using RAPD fingerprints
We used the ten random primer sets to amplify all possible and reproducible monomorphic and polymorphic bands for the initial screening against the C. sativus genotypes (CM-cashmerianus, T0, T1-2B, T4-2A) so as to identify RAPD markers (Table 3). Each sample was run in triplicate during PCR to verify its reproducibility. The RAPD primers were classified based on the manufacturer's primer code and corresponded to the 10-mer oligo used in this study followed by a four-digit number that indicated the size of the product in base pairs. The phenotype of each RAPD marker was scored as 1 when bands were present and 0 when no bands were observed. We performed a genotype cluster analysis to generate a dendrogram based on Jaccard's similarity coefficients earlier used by Sharaf-Eldin et al. (2015) and unweighted pair group mean analyses (UPGMA) with NTSYS-PC ver. 2.0 to develop the phylogenetic tree (Rohlf, 2001).

| Estimation of chlorophyll content
Leaf chlorophyll content is a recognized indicator of many concerns, such as plant photosynthesis activity, plant nutritional state, presence of mutations, and stresses. We therefore determined the total chlorophyll content of the leaves of the C. sativus genotypes (CM-cashmerianus, T0, T1-2B, T4-2A) using the method of Hiscox and Israelstam (1979). Specifically, we used dimethyl sulfoxide at 65°C for 2.5 hr to isolate the chlorophyll from 100 mg samples of C. sativus leaves that were taken during early December. We then measured the absorbance of the reaction mixture at 645 and 660 nm in a UV-Vis spectrophotometer (Spectroscan 80 DV, USA), and after it was recorded, the total chlorophyll content was calculated (Arnon, 1949).

| Extraction and HPLC analyses of the crocin and safranal content of saffron genotypes
Fifteen milligrams of saffron stigmata dried at 60°C (CM-cashmerianus, T0, T1-2B, T4-2A) was used to extract the metabolites. We suspended the samples in 10 ml of methanol-water (50:50, v/v) and mixed them using a magnetic stirrer for 24 hr at room temperature in the dark according to Alam, Elkholy, Hosokawa, Mahfouz, and Sharaf-Eldin (2016).

| Statistical analyses
The treatments in these experiments were arranged in a randomized complete block design. Each treatment included three sets of replicate data for each season and was statistically analyzed applying the ANOVA test (MS DOS/Costat Exe Program) according to Gomez and Gomez (1984). The least significant difference (at a level of 5%) was used to compare between different means, according to Snedecor and Cochran (1982).

| Generation of RAPD fingerprints from the saffron genotypes
Different RAPD fingerprints were obtained from all the saffron genotypes. Twenty random GCC primers were used for the RAPD analysis. The RAPD profile showed monomorphisms (similar bands) but also polymorphisms that were unique to the selected random GCC primer. The sizes of the bands varied in the range of 100-1,000 bp for both the monomorphic and polymorphic forms (Table 4, Figure 2). The amplification pattern was more pronounced with GCA-01 (CAGGCCCTTC), while the other random primers were unable to amplify uniformly. GCC-01, however, amplified the genomic DNA of all saffron genotypes and generated unique fragments.

| Analysis of the genetic similarity between the saffron genotypes
On the basis of the RAPD fingerprint polymorphisms between the genotypes of saffron bearing five stigmata, a similarity matrix was obtained after multivariate analysis using the "Nei and Li" coefficient (Nei & Li, 1979); this matrix is presented in Table 5. Crocus genotypes, as shown by the numerical taxonomy.
The dendrogram of the Crocus genotypes clearly indicated that the PCR-RAPD correlated with the similarities and distances between the C. sativus genotypes, from which one could to a large extent predict the origin of the species (Figure 3). PCR-RAPD also showed the mutational pattern among the genotypes.

| Biomass
The dry biomass of the stigmata, stamens, and tepals was studied in genotypes of C. sativus. The dry biomass of the stigmata of T0-2B (10 mg/flower), CM (6.8 mg per flower), and T1-2B (5.8 mg per flower) was higher than that of the T0 stigmata (5.2 mg per flower), while the dry biomass of the tepals did not differ in any of the mutant flowers (10 mg per flower) with respect to the control T0 (Table 6).

| Analysis of crocin and safranal
The crocin and safranal concentrations were evaluated in the saffron genotypes. The crocin content in genotypes with five stigmata, CM types, as shown in Table 8.

| D ISCUSS I ON
Saffron (C. sativus L.) is one of the most valuable and expensive spices; it has high medicinal value and is used in the treatment of many diseases. The production of saffron is a very low worldwide because of its growth characteristics and high demand for labor (Sharaf-Eldin et al., 2008).
Crocus sativus is a triploid (2n = 3x = 24) sterile plant; it fails to produce viable seeds and is totally dependent on human support (Rubio-Moraga et al., 2009). In the recent past, it has been produced by breeders, which provides a better platform for maintaining its genetic balance. The quality of saffron mainly depends upon stigma processing and species origin (Sharaf-Eldin et al., 2008). Caiola, Somma, and Lauretti (2000) reported that the phenological evaluation of C. sativus flowers obtained from corms from various regions of the world showed some variation in the color of the flowers as well as their fragrance and tepal lobes, but not in the pollen size (Caiola et al., 2000).

Improvements in DNA biology have provided new information
for taxonomic analysis among accessions (Caiola, Caputo, & Zanier, 2004), and some studies have demonstrated that the relationship between molecular and biochemical characterization is sufficiently robust to classify and clarify the systematics and phylogeny of plants (Frello & Heslop-Harrison, 2000 The advantages of the PCR-RAPD technique include its rapidity; the low concentration of genomic DNA, dNTPs, and primers needed; and the ability to obtain genetic information without the use of radioisotopes (Srivastava et al., 2012;Williams et al., 1990). The reproducibility of PCR-RAPD is affected by the quality of the DNA, the concentration of genomic DNA and the primers, and the source of the DNA polymerase (Ellsworth, Rittenhouse, & Honeycutt, 1993).
The random primers used in RAPD are decamers (Williams et al., 1990) and are mostly designed based on microsatellite/minisatellite regions, which contain highly repetitive sequences. The PCR conditions used in this study for the RAPD analysis of the saffron genomic DNA were 94°C for denaturation, 35°C for annealing, and 72°C for elongation, repeated for 40 cycles. These conditions yielded the most bands. This result could be due to the low annealing temperature (35°C), which could allow maximum primer-DNA annealing and the maximum number of amplicons. These types of parameters have also been used by other investigators in different plant species (Busconi, Sebastiani, & Fogher, 2006;Miller & Bayer, 2001;Srivastava et al., 2012).
The morphological traits (e.g., tepal, stamen, and stigma) of  Table 5. This finding could be due to the effect of the climatic conditions on the different saffron genotypes. This study provides strong proof that RAPD polymorphisms can be used as an important tool to reveal phylogenetic relationships among species and genotypes. Our results are also supported by observations from several investigators (Heikal, Abdel-Razzak, & Hafez, 2008;Li, Fatokun, Ubi, Singh, & Scoles, 2001).
Similarly, the chlorophyll and biomass were also affected in these genotypes. The chlorophyll content of the T0 genotype (with three stigmata) (21.90 mg/g fresh weight basis) was higher than that of  Tables 6 and 7.

| CON CLUS ION
The main goals of this study were to explore the genetic relationships among genotypes or accessions by gaining a better understanding of the genetic differences among them. Furthermore, reproducible DNA markers, such as inter simple sequence repeats (ISSRs) and sequence characterized amplified regions (SCARs), can also support relationships among accessions or genotypes or species of the plants. Thus, PCR-RAPD could be helpful in the identification of commercial saffron lines and could be a useful tool to supplement uniformity, distinctness, and stability analyses for saffron genotypes to maintain their original identity and protect the crop in the future.

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interests.

E TH I C A L S TATEM ENT
This article does not contain any studies with human participants or animals performed by any of the authors.