Evaluation of genetic diversity in germplasm of paprika (Capsicum spp.) using random amplified polymorphic DNA (RAPD) markers

Capsicum spp. is one of the most important economical horticulture crops due to its high consumption either by fresh vegetable or dried spice. Molecular genetic markers offer a number of applications in the genetic improvement of crop plants, which plays an important role in the areas of plant classification and breeding programs. The polygenetic characters of rare species, which are difficult to analyze by traditional methods can, be analyzed easily and classified by using molecular markers. In our study, genetic relationships of twenty-two paprika species were examined to estimate their genetic variations/similarities and to detect the polymorphism present within and among the paprika species by using RAPD-PCR markers. The results revealed that the maximum similarities among the 16 ICBD lines were 100%. The ICBD 03 had 76% similarity compared with other ICBD lines. The CC01 had comparatively low similarity with ICBD forms (30%), followed by EC01 (28%), EC02 (33%), CC02 (35%), and Kt.Pl-19 (60%). The similarity between EC01 and EC02 were 54%. Kt.Pl-19 showed different similarities compared to CC01 (41%), CC02, EC01 (38%), EC02 (29%) and ICBD 03 (40%). The different combinations were tried to optimize the RAPD-PCR profile, which helped to assessing the polymorphism/similarities within and among the Paprika germoplasms were studied. Research Article Evaluation of genetic diversity in germplasm of paprika (Capsicum spp.) using random amplifi ed polymorphic DNA (RAPD) markers Renganathan P1, Ruíz-Alvarado C2, Hernández-Montiel LG3, Prasath Duraisamy4 and Rueda-Puente EO5* 1Department of Plant Biotechnology, School of Life Science, Bharathidasan University, Trichy, Tamilnadu, India 2Instituto de Ciencias Agrícolas de la Universidad Autónoma de Baja California, México 3Centro de Investigaciones Biológicas del Noroeste. CIBNOR. La Paz, B.C.S. México 4Indian Institute of Spices Research, Indian Council of Agriculture Research, Calicut, Kerala, India 5Departamento de Agricultura y Ganadería, Universidad de Sonora, Hermosillo, Sonora, México *Address for Correspondence: Rueda-Puente EO, Departamento de Agricultura y Ganadería, Universidad de Sonora, Hermosillo, Sonora, México. E-mail: erueda04@santana.uson.mx Submitted: 16 September 2017 Approved: 26 September 2017 Published: 27 September 2017 Copyright: 2017 Renganathan P, et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


Introduction
Peppers and pepper fruits (Capsicum spp.) represent an important part of the fresh vegetable market in India, and are also signi icant worldwide in the segment of condiments, spices and salt preserves [1][2][3]. Among them, ive domesticated species have been globally recognized which includes C. annuum L., C. frutescens L., C. chinense Jacq., C. baccatum Jacq., and C. pubescens (4). C. annuum continues to be the most popular and important species, the next being C. frutescens [5,6]. Some varieties are famous for red color because of the pigment capsanthin, others are known for biting pungency attributed by capsaicin [7][8][9]. Paprika (Capsicum spp.) is the ground product from the mild or sweet varieties of Capsicum, whereas red and chili peppers are blends of different varieties of more pungent peppers [10,11]. Consideration and assessment of genetic variability existing in a population is the basic requirement for any effective crop improvement program. Germplasm characterization is an important link between the conservation and utilization of plant genetic resources. Molecular DNA techniques allow researchers to identify accessions at the taxonomic level and assess the relative diversity within and among the species [12]. Molecular markers are important tool for breeding selection, genotype identi ication and studying the organization and evolution of plant genomes [13]. Randomly Ampli ied Polymorphic DNA (RAPD) is attractive for less-known species and widely used for describing levels and patterns of diversity and genetic structure in a number of plant species [12,13]. RAPD analysis has been found to be effective in varietal identi ication of several crops including lily, Capsicum, eggplant, Chrysanthemum, mulberry, Jatropha, castor bean and Cymbopogon [14][15][16]. Our present investigation was undertaken to assess the genetic variability among the paprika germplasms conserved at Indian Institute of Spices Research, Calicut using RAPD markers with the following objectives: [1] to estimate genetic relationship among the paprika accessions; and [2] to detect the polymorphism present within and among the accessions.

Experimental site
All experiments were conducted at Laboratory of Plant Breeding, Division of Crop Improvement and Biotechnology, Indian Institute of Spices Research (IISR), Kozhikode, Kerala, India.

Genomic DNA extraction
Total genomic DNA was extracted from fresh young and disease free leaves of 22 paprika accessions by using a modi ied version of CTAB method described by Doyle (18). 10-15 ml of CTAB buffer containing 2% CTAB (trimethylammonium bromide), 5 M NaCl, 2-mercaptoethanol, 0.5 EDTA, 1M Tris-HCl (pH 8.0), 2% PVP was added to 50 ml Oak Ridge tube and preheated for 20 min at 65°C in a water bath. Ground 2 g of fresh leaf tissue into ine powder with the help of mortar and pestle after freezing in liquid nitrogen, and it was transferred to the Oak Ridge tube containing preheated CTAB buffer solution. Incubate the sample at 65°C for 15-60 min with optional occasional gentle swirling. After incubation, add equal volume of chloroform:isoamyl alcohol (24:1), mix gently but thoroughly for 15 min. The tubes were centrifuged at 10,000 rpm for 15 min at 4°C. Removed the aqueous phase with wide bore pipette, transferred to clean Oak Ridge tube, added 2/3 volumes of ice-cold isopropanol, and mix gently to precipitate the DNA. The tubes were centrifuged at 10,000 rpm for 15 min at 4°C and discard the supernatant. DNA pellet was washed with 70% ethanol and allowed the DNA pellet to air dry at room temperature. Finally, the pellet was dissolved with 500 μl of nuclease free distilled water and was transferred to micro centrifuge tubes.

DNA purifi cation
Two microlitre of RNase enzyme was added to micro centrifuge tubes containing dissolved extracted total genomic DNA, mixed gently and incubated for 1 h at 37°C in a water bath with intermittent shaking. After incubation, equal volume of phenol:chloroform:isoamyl alcohol (25:24:1) was added. Mixed thoroughly and centrifuged at 10,000 rpm for 10 min at 4°C. And the supernatant was transferred to a fresh micro centrifuge tube. Equal volume of chloroform:isoamyl alcohol (25:1) was added to the supernatant, mixed well and centrifuged at 10,000 rpm for 10 min at 4°C.
Supernatant was collected to a fresh tube and add 100% ice-cold alcohol and stored at 4°C for 1 h to precipitate DNA. Precipitated DNA was collected by centrifugation at 10,000 rpm for 10 min at 4°C and washed with 70% ethanol and dried. The dried pellet was dissolved with 100 μl of nuclease free water, the DNA was dissolved and stored at -20°C. The quality of the extracted DNA was determined by taking the ratio of absorbance at 260 nm and 280 nm.

DNA quantifi cation
The DNA was quanti ied on 1% agarose gel in 1x TAE buffer was prepared with 1.5 μl of ethidium bromide. 2 μl of λ DNA ladder was loaded as marker. 5 μl of DNA samples were loaded with 3 μl of gel loading dye. Gel electrophoresis was carried out for 90 min at 60-80 volts. The DNA bands were visualized on a UV Trans illuminator and the gel was documented by gel documentation system (Alpha Image 1220).

RAPD-PCR amplifi cation
The primer´s used for RAPD-PCR were listed in table 1. The PCR reactions were set up in a 25 μl reaction containing 45 ng genomic DNA, 1X PCR reaction buffer containing 25 mM MgCl 2 , 5 pmols primer, 10 mM each of dCTP, dGTP, dTTP and dATP and 1U of Taq DNA polymerase (Perkin Elmer). Ampli ication was performed on a thermal cycler (Model 2 400, Perkin Elmer) with a programme of initial denaturation cycle at 94ºC for 3 min, denaturation at 94ºC for 1 min, annealing at 37ºC for 1 min, extension at 72ºC for 2 min, and inal extension at 72ºC for 7 min with 35 cycle repeats. The ampli ication products were electrophoresed on a (Owl Electrophoresis Unit) 2% agarose gel (Genei Q Biogene) with a standard DNA marker (1kb DNA ladder) at 67V for 2 h stained by ethidium bromide (1.5 μl/ml) and photographed by using gel documentation system (Alpha Image 1220).

Data analysis
The electrophoretic pattern was visually analyzed and DNA bands were scored as present [1] or absent (0). The matrix obtained was entered into NTSYS-pc program [19] and Jaccard's Similarity Index (JSI) was calculated to each pair of samples. A UPGMA dendrogram was constructed based on JSI.

Results and Discussion
We determined the genetic variations/similarities among the twenty-two paprika accessions of various types. The results are conformity with [20,21]. The application of molecular markers in the improvement of Capsicum species for characterization of the genotypes, genetic ingerprinting, identi ication and cloning of important genes, marker assisted selection and in understanding of inter-relationships at molecular level. RAPD markers have been extensively used in assessing genetic variation and taxonomic relatedness of both wild and cultivated species [22][23][24]. RAPD markers can be ef icient in the routine assessment of variety identi ication and of hybrid seed purity in pepper [25,26].
The evolutionary relationships among and within the cultivated Capsicum species have been investigated in several studies, using morphological, cytogenetically and molecular markers [20,27]. RAPD markers have also been used to determine evolutionary relationships and levels of genetic variation among wild and cultivated populations of a number of crop species, including Capsicum species [20,21]. In the present study, the total genomic DNA was extracted from the young and fresh leaves of twenty-two paprika accessions by using modi ied version of CTAB method. The quality of extracted DNA samples were tested on 1% agarose gels along with standard λ DNA. The DNA yield ranged from 25-250 ng/μl. Though comparison with standard λ DNA marker indicated high molecular weight DNA, a considerable amount of RNA contamination was found in some of the extracted DNA samples of paprika. In order to eliminate or reduced the RNA contamination. The DNA samples were treated with RNase enzyme followed by phenol:chloroform:isoamyl alcohol extraction. Different combinations and concentrations of Taq DNA Polymerase (1U, 2U and 3U per 25μl reaction volume) and MgCl2 (25mM and 50mM per 25μl reaction volume) were tried for good RAPD-PCR ampli ication. Among the different combinations, 1U of Taq DNA Polymerase and 25 mM MgCl2 concentration offered ampli ication with clear bands without non-speci ic banding. Hence, 45ng of DNA, 10 X Assay buffer, 25 mM of MgCl2, 10mM of dNTP, 1U of Taq Polymerase were optimized for generating a consistent RAPD-PCR ampli ication.
The two temperature pro iles were tested for optimizing the PCR conditions with the initial denaturation cycle at 94ºC for 3 min, denaturation at 94ºC for 1 min, annealing at 40ºC for 1 min, extension at 72ºC for 2 min and inal extension at 72ºC for 7 min with 38 cycle repeats (reaction I) and initial denaturation cycle at 94ºC for 3 min, denaturation at 94ºC for 1 min, annealing at 37ºC for 1 min, extension at 72ºC for 2 min and inal extension at 72ºC for 7 min with 35 cycle repeats (reaction II). Among the two reaction pro iles, the ampli ication products and number of ampli ied bands were generated more under reaction II than reaction I. In the second reaction pro ile, the primer annealing time was with 1 min at 37ºC. i.e., the stringency of the reaction was reduced from 40ºC to 37ºC. Also the cycle repeats were reduced from 38 to 35. Twenty two Operon decamer primers were used for assessing the polymorphism/similarities within and among the Paprika accessions. The primer OPB-07 showed a consistent band around 600 bp in almost all accessions. Similarly, OPD-02 showed a consistent band around 1 kb and as well as OPA-06 around 500 bp. These common bands may represent the genetic characters of Capsicum sp. An example of the ampli ied pattern obtained with one RAPD primer is presented in igue 1. Seven polymorphic bands were observed with OPA-04 with the amplicon size ranged from 250 to 2000 bp ( Figure  1). The bands from 400 to 1300 bp were found to be polymorphic. Similarities in the banding pattern were noticed between the ICBD accessions around 800 bp. On the other hand, ive polymorphic bands were observed at other ive accessions (CC 01, CC 02, Kt.Pl-19, EC 01 and EC 02). Seven polymorphic bands were observed with OPA-05. The amplicon size ranged from 250 to 2500 bp. The bands from 600 to 1500 bp were found to be polymorphic. The results are conformity with [20,21]. Ampli ication with quality was observed and the maximum numbers of ampli ied bands were nine with OPA-11. Similarities in patterns were noticed in the ICBD accessions. The patterns were also similar with minor differences in ICBD03 around 800 bp with the amplicon size ranged from 250 to 2500 bp. The bands from 300 to 1600 bp were found to be polymorphic. On the other hand, nine polymorphic bands were observed at other ive accessions (CC 01, CC 02, Kt.Pl-19, EC 01 and EC 02).
The UPGMA cluster dendrogram constructed based on the Jaccard's Similarity Index (JSI) showed a single cluster with seven groups (Figure 2

Conclusions
The modi ied protocol used for the isolation of DNA was found to be suitable for Paprika, which showed quality DNA from the leaves. Among the different combinations tried to optimize the PCR pro ile, which helped to give ampli ication with quality and clear bands without non-speci ic banding. Cluster dendrogram revealed maximum similarities/polymorphism were observed within the 16 ICBD accessions except ICBD 03. Apart from ICBD line, other accessions like EC 01, EC 02, CC 01, CC 02, and Kt.Pl had comparatively low similarity with ICBD forms.