DNA fingerprinting of cultivated and wild genotypes of Curcuma species from agroclimatic regions of Chhattisgarh

In the present study we had tried to investigate different genotypes of Curcuma isolated from different agroclimatic zones of Chattisgarh based on molecular fingerprinting as they contain varying range of Curcumin. Genetic fingerprints of 25 genotypes of Curcuma caesia, Curcuma longa and Curcuma aromatica by using 13 and 11 SSR primers. These primers were used to determine the diversity in genes for their proper utilization, genotypic conservation and commercial production of curcuma genotypes. Among all the studied varieties, 13 ISSR and 11SSR primers generated 107 and 51 different amplified fragments, of which 97% and 86% showed polymorphism, respectively. Through all the ISSR and SSR primers in all the genotypes amplified 1080 and 567 loci out of which 102 polymorphic and 5 monomorphic alleles were found in ISSR primers. 47 polymorphic and 4 monomorphic alleles were found in SSR. Different ISSR and SSR markers were used to estimate the DNA fingerprinting, genetic similarity and distance among or between the Curcuma genotypes. UPGMA data scoring methods were to analyze the variations between these genotypes of Curcuma . Thus, this study provides a baseline data for conservation of these curcuma genotypes that could be an important research towards optimizing industry level production of Curcumin production.


Introduction
Plants contain secondary metabolites for their protection that are responsible for different pharmacological properties of the medicinal plants. Especially the medicinal herbs of species Curcuma of family Zingiberaceae is highly valued worldwide for their medicinal and economic significance. Curcuma belongs more than 90 accepted species in the world that includes C. longa named as "Indian Saffron" having wide medicinal value. More than 100 components isolated from rhizomes and leave of turmeric, mainly consisting curcumin, bismethoxycurcumin and desmethoxycurcumin. Curcumin exhibits variety of biological activity including anti-inflammatory agent, colic and chest pains, wound healing, antibacterial, antifungal, anticancer, antioxidant. It is also known for its anti-fertility, neuroprotective, human immunodeficiency virus-1 (HIV-1) and HIV-2 protease inhibitor, antialzheimer, anti-hepatotoxic, immunomodulatory activity and in menstrual difficulties (Rajasekaram, 2011;Seo et al., 2017;Ameruoso et al., 2017). C. caesia locally call as Black turmeric is an uncommon endemic as well as ethno medicinally important plant of south East Asia.
Rhizomes of black turmeric have bluish black color and emit a camphorous smell due to presence of essential oil. C. aromatica is commonly known as wild turmeric, kasturimanjal, it is found effective in snake bite, sprain, bruise, inflammation, tonic, carminatives, astringent, antioxidant and for antimicrobial activity (Sikha et al., 2015).
All the three targeted species of turmeric have great demand in increasing demand in the global market due to their medicinal applications. In spite of this no major breakthrough has been noticed in boosting the production of turmeric and increasing the exports. Chhattisgarh, comprising 3 agroclimatic zones, produces 83,470 Mt. of turmeric from an area of 9747 ha with an average productivity of 8.56 Mt/ha which is not satisfactory. The major bottle-neck is the non-availability of requisite high curcumin containing genotypes. Usually turmeric with curcumin content more than 5% has commercial value. Therefore it is imperative to study the extent of variability in curcumin content of different cultivars and accessions of turmeric from different regions of Chhattisgarh so as to select the genotype best suited to each of these regions. Though turmeric displays great morphological and phytochemical diversity, meager information is available on the magnitude of variation in curcumin content among available germplasm of Chhattisgarh. These concerns warrant identification of elite chemotypes of medicinal plant species in order to grown in suitable environments, harvested at appropriate age and time so as to maintain uniformity in the desired chemical constituents. Recently genetic fingerprinting has been proved to be very useful in germplasm characterization i.e. for identification and cloning of important genes and understanding interrelationship at molecular level. It can be used to identify unambiguously a plant variety or clone through different markers.
Thus, the present study was preformed determine genetic proximity between curcuma species collected from three Agro-Climatic Zone of Chhattisgarh which include Bastar Plateau Zone, Chhattisgarh Plain Zone and North Hills Zone, thus originated keeping this in mind the importance of Curcuma species including endemic ones have 25 genotypes from different agroclimatic region of Chhattisgarh using two different sets of molecular markers, viz., SSR, ISSR, respectively 2. Materials And Methods

Collection and Extraction of DNA from curcuma species
The species of curcuma was collected different regions of Chhattisgarh, India as shown in Table 1. In these species 25 genotypes of C. longa L. and C. caesia Roxb. were cultivated varieties. However, C.
aromatica Salibs. varieties were of wild habitant. The protocol for deoxyribonucleic acid (DNA) extraction was as described by Edwards et al. (1991) and Das et al., (2011) with slight modification.
Genomic DNA was isolated from frozen leaf samples by grinding with a mortar, pestle in extraction buffer (100 mM Tris-HCl [pH 8.0], 1.4 M NaCl, 20 mM EDTA, 2% SDS) and incubated at 65°C for 1 h in 500 µl of sodium dodecyl sulphate (SDS) extraction buffer. After extraction with an equal volume of chloroform-isoamylalcohol (24:1) and centrifugation at 5000 rpm for 15 min, the upper phase containing DNA was treated with 5 µl (10 mg/ml) RNase for samples to eliminate RNA by incubation at 37°C for 15 min.Then again treated with Chloroform:Isoamylalcohol to for removal of nonnucleic acid compounds. Genomic DNA was precipitated out of the upper phase by adding 0.6 vol of chilled ethanol and 100 µl of 5M NaCl and keeping at less than 4°C for several hours. After incubation centrifuge at 10000 rpm for 15 min and the precipitate was washed several times with 75% ethanol, and then air-dried and dissolved in appropriate volume of TE buffer (10 mM Tris-HCl [pH 8.0], 1 mM EDTA). The quality and quantity of the DNA was determined with a Thermo Scientific Nano drop and genomic DNA was also quantified by agarose gel electrophoresis with use of standard lambda DNA as a reference.

Primer screening
31 synthesized inter simple sequence repeat (ISSR) primers (Oligos) or 25 simple sequence repeat (SSR) primers (Bioserve Hyderabad) were initially screened to determine the suitability of each primer for the study. Primers were selected for further analysis based on their ability to detect distinct, clearly resolved, and polymorphic amplified products within the genotypes. To ensure reproducibility, the primers generating no, weak, or complex patterns were discarded select only 13 ISSR and 11 SSR primers for further study (Table 2, 3).

Genetic diversity analysis by Molecular markers
Thirteen ISSR primers (Oligos) and Eleven SSR primers (Bioserve, Hyderabad, India) were used for PCR amplification. Based on results, good resolution and reproducibilityability, all ISSR and SSR primers were selected out of several primers utilized during screening. PCRs with a single primer were carried out in a final volume of 20 μl Containing (2.5 µl) 50 ng template DNA, 2 µl 2mM of dNTP mix (Himedia), 2 µl 10mM of oligonucleotides synthesized primer, 2 µl 1X Taq buffer and 0.3 µl 2U Taq DNA polymerase (Thermo fisher). Amplification was performed in a thermal cycler (Eppendorf and Biorad). In ISSR, PCR was performed at an initial temperature of 95ºC for 5 min for complete denaturation. The second step consisted of 45 cycles having three ranges of temperature, i.e., at 94ºC for 1 min for denaturation of template DNA, primer annealing temperature was set at 2ºC lower than the melting temperature for 1 min and at 72ºC for 2 min for primer extension, followed by running the samples at 72ºC for 8 min for final extension. The PCR products obtained from ISSR were analyzed in 2 % agarose gel stained with 3µl ethidium bromide. PCR products from ISSR markers were combined with 3µl of a loading buffer (orange green gel loading dye Himedia and were analyzed directly on 2% agarose gels in 1X TAE buffer. Electrophoresis was done for about 45 min at 120 volts. 100 bp ladder (Himedia) was used to compare the molecular weights of amplified products.
Visualization of the amplified bands was done by gel documentation system BioRad. The detection of microsatellite polymorphism was performed using 5 SSR markers characterized by (Singh, 2015) and 6 different SSR primers. The SSR amplification condition was as follows: an initial hot start and denaturing step at 95ºC for 5 min followed by 40 cycles of a 1 min denaturation at 94ºC, a 1 min annealing based on primer tm, and a 1 min primer elongation at 72ºC. A final extension step at 72ºC for 5 min was performed. The PCR amplified products were resolved in a 2% agarose gel.
Electrophoresis was done for about two and half hours at 60 volts. 100 bp ladder (Himedia) was used to compare the molecular weights of amplified products.

Data scoring and analysis
Only clear, unambiguous and reproducible bands were considered for data analysis. Each band was considered to be a single locus. Data were scored as ''1'' for presence and ''0''for absence. To avoid taxonomic ambiguities, the intensity of bands was not taken into considerations, only the presence of band was taken as indicative. The binary data of the ISSR and SSR fingerprints were used further for population genetic analyses. The numbers of monomorphic and polymorphic bands were derived from the binary data, and their percentages were calculated. The level of similarity between species was established as the percentage of polymorphic bands, and a matrix of genetic similarity was compiled using Jaccard's similarity coefficient (JSI) (Jaccard, 1908). Similarity coefficients were used to construct the dendrogram using the unweighted pair group method with arithmetic average (UPGMA) and the sequential hierarchical and nested clustering routine in the Past 3 software representing

Quality of extracted DNA samples
Extracted genomic DNA of Curcuma genotypes was quantified by Nano drop system anintegrity of DNA on 0.8% agarose gel is shown in Fig. 1.

ISSR polymorphism
The ISSR derived banding pattern is presented in Fig. 2. Thirteen ISSR primers were used to characterize the genetic diversity present among the genotypes of Curcuma species (Table 2).
Thirteen of these primers showed a total of 102 polymorphic reproducible fragments that ranged from 250-2000 bp in size. High percentage of polymorphism was displayed in 10 primers (100%) other all the 3 primers showed IG-10 (78%), IG-14 86% and UBC-812 (71%) of polymorphism among all three species of Curcuma. Total of 102 polymorphic bands and 5 monomorphic bands were detected (Table   4). The percentage of ISSR polymorphism for different Curcuma species studied ranged from 71 to 100%. The PIC values for ISSR primers ranged from 0.44-0.5 with an average of 0.48 of all the primers. The primer UBC-811 and UBC-841 produced maximum number of 11 bands, while the primer IG-2, B17898 and UBC-836 resulted in amplification of only 6 bands. Among these ISSR primers, minimum PIC was found to be 0.44 in IG-03.

SSR polymorphism
Eleven SSR primers will be amplified and showed diversity so we choose only these out of 25 synthetic oligos (Table 3). The primer combinations had amplified 51 loci among which 4 were found to be monomorphic in nature and the rest were polymorphic (Fig 4a, 4b and Table 6). Maximum number of 9 bands was resolved for the primer CIR-343 and the minimum 1 for CIR-375. Maximum number of polymorphism was found in primer CIR-263, CIR-343, Cumisat-22(100%) and minimum in primer Cumisat-27 (50 %). The average PIC of all the primers was 0.43. Bands resolved between 400 to 50 bp were consider for the present investigation.

Data scoring and Cluster analysis
The dendrogram constructed through UPGMA algorithm using Jaccard's similarity coefficient of ISSR and SSR by using Past 3 software. Dendrogram cluster analysis is an explorative analysis that tries to identify structures within the data it is also known as taxonomy analysis. Cluster analysis is used to identify groups of cases if the grouping is not previously known. In ISSR matrix of Curcuma species shown that it has been divided the dendrogram in two main clusters with 25 genotypes. Jaccard's coefficient showed the genotypes and species closeness with a similarity value 0.94 and distance value 0.81. Genetic variation of ISSR primers among the genotypes is presented in Table 5. Constructed dendrogram, is shown in Fig. 3. In another extent the variation in SSR primers among the genotypes and Jaccard's coefficient by UPGMA algorithm showed that the species were most closely related with a similarity value 0.91and distance value 0.77. Genetic variations in SSR primers among all the Curcuma genotypes is presented in Table 7 and dendrogram tree of SSR marker is presented in Fig. 5.

Discussion
Dendrogram constructed on the basis of different ISSR primers with their respective genotypes genotypes was divided into the two groups, first group contained 5 genotypes of Curcuma caesia and second group contained remaining of the genotypes of all Curcuma species. The second cluster was again divided into two sub-clusters and it was divided into again four sub cluster were consisting of Curcuma longa and Curcuma aromatica because these are much similar species but Curcuma caesia and Curcuma aromatica have also show more similary to each other so rest of the subclusters containing genotypes of Curcuma caesia and Curcuma aromatica. As same ISSR primers similarity is clearly seen in dendrogram and it has been divided into 2 main groups with two sub clusters. Second small cluster contain few genotypes due to genetic difference to the other genotypes. Correlation between individual genotypes to each other is significant. Archana Das et al.,(2011) to provide frame work for large scale production and cultivation of best genotypes Mohanty et al., 2014;Ashwani Kumar Singh et al., 2015. In SSR distance matrix of Curcuma species shown that it has been divided the dendrogram in three main clusters then again it has to be divided into number of sub clusters due to the presence of greater polymorphism exhibited in between genotypes of wild and cultivated varieties of Curcuma species due to lots of environmental, soil, agroclimatic and its genetic makeup. SSR primers dendrogram plot shown that Curcuma longa genotypes form again different subcluster due to minor changes in its genetic sequence and one genotype of Curcuma caesia ICC place in single sub cluster in their group because it showed unique bands in most of the SSR primers which have not present at all.

Conclusion
This study provides a baseline data for conservation of these curcuma genotypes that could be an important research towards optimizing industry level production of Curcumin production. Consent for publication -Not applicable.
Availability of data and materials -It can be made available on request.
Competing interests -There is no competing interest.       Figure 1 Integrity of isolated Genomic DNA of Curcuma genotypes on 0.8 % agarose gel Figure 2 Banding patterns of ISSR fragments of 25 genotypes of three targeted Curcuma species