The Effect of Ultraviolet Radiation on Physiological and Molecular Characterization on the Capsicum Plants (Solanales: Solanaceae)

Background: Chilli or Pepper (Capsicum species) is an essential and critical plant shown as vegetable cum spice crop that grown for the ornamental, medicinal and pharmaceutical applications. An enormous source of vitamins A and C constituted in the Capsicum species which is an essential and critical plant shown as vegetable and spice crops that grown worldwide for the ornamental, medicinal and pharmaceutical applications. The present paper deals with the relative changes in biochemical composition of the plants because of impacts of the induced mutation and assessment of the changes in the molecular characterization using AFLP innovation. Furthermore, AFLP investigation can be utilized to assess how the mutagenic specialists alter the structure of DNA in living beings and furthermore with the changes in the quantication of physiological and biochemical amounts. Results: Minimum amount of proteins (799.997 µg/ml), carbohydrates (39.13 µg/ml) and free fatty acids (673.2 µg/ml) were found in very high intervening mutated plants. Maximum percentage of inhibition by the peroxidase activity (0.1685 μg/ml), catalase activity (0.078 μg/ml), Diphenyl-1-picrylhydrazyl (68.57 μg/ml) activity and 2, 2’- azino-bis (3-ethylbenzoithioazoline-6-sulphonic acid) (97.88 μg/ml) activities were found in profoundly exposed mutated plants compared to the normal Capsicum plant species. A dendrogram representing the similarity in DNA bands of Capsicum plants in different time intervals of UV-C radiations showed signicant negative anities. Conclusions: This paper encourages us in understanding an induced mutation by UV-radiation on the Chilli plant leaves and its impact on plant cell creation and DNA structure, and acquiring sound and safe plants for mutagenesis. Therefore, this study brings mutation prompts diverse sorts of basic variations from the norm in cell organelles and its structure of the plant sources.

the growth and development of Chilli, speaking to 45% of the world, its e ciency is very low (1 t ha − 1 dry Chilli) when contrasted with USA, China, South Korea, Taiwan, and so on (3-4 t ha − 1 dry Chilli) [6].
A few examinations utilizing morphological, cytogenetical and molecular markers have explored the evolutionary connections among and within the developed species [7][8][9][10][11][12][13]. An imperative connection between the preservation and use of the plant genetic resources is a germplasm portrayal. Utilizing tens to a small number (200) of low throughput, the mostly molecular markers or different DNA markers, for example, restriction fragment length polymorphism (RFLP) [14][15], random ampli ed polymorphic DNA (RAPD) [16], ampli ed fragment length polymorphism (AFLP), simple sequence repeat (SSR) [17][18][19][20], inter simple sequence repeats-PCR (ISSR-PCR) [21]; cleavage ampli ed polymorphic sequence [22] and single nucleotide polymorphism (SNP), the molecular studies had been evaluated the general assorted variety of C. annuum rearing germplasm [23]. For both marker disclosure and measures for known SNP polymorphisms, DNA microarrays at rst was utilized for the expression analysis had been broadly utilized. AFLP investigation was performed for legitimate and an appropriate molecular technique among these all markers study [24].
Mutation has been depicted as a sudden change in the hereditary material of the living cells. Since mutation deals with hereditary material, mutations can be found in every single cell constituent that convey DNA, i.e. the nucleus, the mitochondria and (for plants) plastids. It is the sudden changes in the morphological, cytogenetical and physiological traits of a living beings (including plants and animals) because of the ecological conditions (natural or induced) for example, light, carbon dioxide, oxygen and water, UV radiation, and so forth.
The physical means that origin harm to the DNA molecules of a living being are stated to as physical mutagens or mutagenic radiations. Mutation also is caused by the methods for the ionizing and nonionizing (UV and gamma rays) radiations. Moreover, UV radiation is the portion of the non-ionizing region of the electromagnetic spectrum that contains roughly 8-9% of total solar radiation [25]. UV is customarily separated into three wavelengths, UV-A, UV-B and UV-C. UV-C (200-280 nm) is a great degree hurtful to living creatures, yet not signi cant under natural conditions of solar radioactivity. UV-B (280-320 nm) is speci cally noteworthy because although this wavelength characterizes only roughly 1.5% of the total spectrum, it can have a variability of detrimental effects in plants. UV-A (320-400 nm) denotes about 6.3% of the incoming solar radiation and is the minimum lethal part of UV radiation [26]. Plant reactions to any given dose of radiation are extraordinary, UV-A is less powerful and effective than UV-B and UV-C [26].
Exceedingly lively UV-C (200-280 nm) is emphatically consumed by oxygen and ozone in the stratosphere with the end goal that none of this cleaning radiation is available in global sunlight. Possibly hurtful UV-B (280-320 nm) is emphatically consumed by barometrical ozone, however around 4% of global radiation is UV-B from 290 to 315 nm. UV-A (315-400 nm) is not weakened by air ozone, and this less harming radiation is a vital photomorphogenic signal in plant development.
One of the potential utilizations of the AFLP technique is the identi cation of UV-induced genetic damage. This is a theme of speci c concern since exhaustion in the globe's upper atmosphere and the related larger amounts of UV radiation, has the potential to antagonistically in uence global biota. A research on the impacts of upgraded UV radiation on vegetation (plants -herbs, shrubs and trees) are of incredible signi cance, as these energetic main producers form the foundation of terrestrial food webs. Despite the fact that the impacts of UV irradiance on the genetic material, physiological parameters and diminished development rates have been examined independently, as far as anyone is concerned, there has been no endeavour to elucidate the potential consequences of induced DNA damage on the physiology and Darwinian tness parameters of uncovered populaces.
The seeds of Pusajwala Chilli are exceedingly nutritious, superb, virtue, freshness, simple to utilize and unequivocally handled. These are hot and abandon from light green to red when developed. The pod appears like nger-shaped, curved and with 4.5 inches long. In differentiating, the seeds of Surajmukhi is also a white in shading and blossom consistently, freshness, and simple to development. These plants require sunshine, the more sunlight the fruits receive the warmer they will develop. It will nurture well in a well-drained in 20-35 ºC and productive soil. On the off chance that planting in pots make certain to utilize a decent natural fertilizer that will hold dampness. Therefore, Pusajwala and Surajmukhi was the two main varieties utilized for the present investigation for the experimentation of both pre-and postmutational examinations.
No comprehensive work related to the AFLP-PCR investigation of the varieties of C. annum species with reference to impact of both pre-and post-mutational examinations. Henceforth, the present study on the C. annum species by molecular characterization and genetic relationship before and after the exposure of UV radiations was carried out by concentrating mainly on the following goals.
The present paper goals to emphasis on (i) study the comparative changes in the biochemical composition because of impacts of the mutation in the varieties of the Capsicum species and, (ii) to evaluate the changes in the molecular characterization of the varieties of the Capsicum species by using AFLP technique.

Analysis of Growth Parameter and Biomolecule Analysis
The seed sample of Capsicum species were collected from the "Lalbagh Botanical Garden'', Bengaluru city, India. The seeds of Capsicum species were collected in a container and taken to the laboratory for further studies (Fig. 1). The plants of Capsicum species were grown for several (maximum -four to ve) weeks in controlled environment room and also in vermiculite medium using pots.
The measurement of growth parameters was done on the plants which are grown in the small pits. The plants were transfer to other pits to study the uniform growth of the plants. The parameters such as plant height, leaf width and number of lea ets per plant were measured and it is observed that the growth of all plants was uniform (Table 1 and Fig. 2) and the same plants were exposed to UV radiations in UV chambers at different time intervals (Fig. 3). Effect of Induced Stress

Estimation of Proteins
The amount of protein in control was calculated on the basis of the standard graph (y = 0.0017x + 0.0118 R² = 0.9921) obtained in Capsicum species. Similarly, the least protein content was found in 60 min mutated plant and had 799.997 µg/ml of protein that showed decrease contents compared to the other plants ( Fig. 4). This might be because of damage existed in peptide bonds formed between the a-amino group of one amino acid and the a-carboxyl group of other amino acid corrosive that avoided in yielding a dipeptide and then polypeptide. Thus, polypeptide chain unfurls from various conformations in the protein giving distinctive basic structures.

Estimation of Carbohydrates
The amount of carbohydrate in control was calculated on the basis of the standard graph (y = 0.0404x + 0.0142 R² = 0.9847) obtained in Capsicum species. Similarly, the least carbohydrate content was found in 60 min mutated plant and had 39.13 µg/ml of carbohydrate that showed decrease contents compared to the other plants (Fig. 5). This might be because of harm existed in both straight and spread polymer of homopolysaccharides f framed inside the layer bound cell organelles that counteracted in keeping up trademark appearance and shape. Additionally, harms happened in cellulose by detaching together by b (1®b) linkages. These chains are straightforwardly separated to each other by intermolecular and intramolecular hydrogen bonds and Vander Waals force.

Estimation of Lipids
Lipids or free fatty acids activity was calculated by the formula given for each of the sample in the Capsicum species. Minimum free fatty acid percentage was seen in 673.2 µg/ml concentration in 60 min post mutated samples. Hence, the present study revealed that 60 min of the post mutated samples is required for normal growth of the plant with the minimum lipid content (Fig. 6). This might be because of destruction existed in covalent bonds formed in the long-chain saturated and unsaturated fatty acids that capacities as damage to plant from water gain and from rough harm. Along these lines long chain unfurls from various compliances in the lipids giving distinctive straightforward structures.

Estimation of Enzyme Activity
Estimation of Stress Enzymes by Peroxidase activity The highest percentage of inhibition by the peroxidase activity (0.1685 μg/ml) was found in 50 µl mutated plants of Capsicum species that revealed increase activity compared to the other samples (Fig.  7). In this manner, Ultraviolet-C radiation initiated oxidative worry in Chilli by expanding lipid peroxidation and layer porousness which demonstrates that points of con nement of resistance are substantially less than wounded caused by UV radiation [27]. In this way, the peroxidase activity is additionally a vital part of the

Estimation of Stress Enzymes by Catalase activity
The highest percentage of inhibition by the catalase activity (0.078 μg/ml) was found in 60 µl mutated plants of Capsicum species that showed increased activity compared to the other samples (Fig. 8).
Oxidative stress is supplemented by the amalgamation of hydrogen peroxide, which is typically detoxi ed by catalase activity in the peroxisomes and by ascorbate peroxidase in the cytosol, mitochondria, and chloroplasts of the Capsicum species [29].
Estimation of free radical scavenging activity of Diphenyl-1picrylhydrazyl (DPPH) assay The highest percentage of inhibition by the DPPH activity (68.57 μg/ml) was found in 50 µl mutated plants of Capsicum species that indicated increased activity compared to the other samples ( Fig. 9). After UV exposure, the capacity of marginally higher DPPH radical scavenging capacity was found in UV-C treated plants. UV-C treatments did not cause checked modi cations in DPPH radical scavenging capacity. UV-C treated peppers kept up somewhat higher DPPH radical scavenging capacity mainly when the appearance of the disorder was already advanced. In this way, the UV-C treatment expanded aggregate cell antioxidants [30][31].
Likewise, a decrease in biochemical synthesis of the peppers treated with a UV-C radiation was accounted for by Vicente et al. [32]. UV-C treatments diminished the occurrence of Chilling damage and seriousness. UV-C treated leaves also exhibited lower electrolyte discharge, respiration rate and phenolic compound substance recommending lower injury in response to low temperature stockpiling. The UV-C treatments could be a valuable method for decreasing decay and maintaining bell pepper fruit quality. Moreover, Chilling damage occurrence and seriousness could be decreased by short UV-C treatments. They initially evaluated if a higher dose might be even more bene cial. On analysing the DPPH radical scavenging capacity, immediately after UV-C treatment, distinctions were recognized between control and treated plants [32].
Estimation of free radical scavenging activity of 2, 2'-azino-bis (3ethylbenzoithioazoline-6-sulphonic acid) (ABTS) assay The highest percentage of inhibition by the ABTS activity (97.88 μg/ml) was found in 50 µl mutated plants of Capsicum species that exhibited increased activity compared to the normal plants (Fig. 12).
Enzymes for example, polyphenol oxidase, ascorbate peroxidase, and glutathione reductase in addition to the peroxidase and catalase demonstrated upgraded action in UV-C treated plants and these enzymes proteins may ll in acclimatization systems to scavenge the harmful free radicals of oxygen created under pressure condition. The results of the present work illustrate that in Capsicum species, UV-C radiation generates antioxidant substances that provide protection against UV-C radiation. Along these lines it very well may be presumed that UV radiation induced both enzymatic and nonenzymatic activities that secure plants against UV radiation in a given dose in Capsicum plant species.
Besides, Ultraviolet-C radiation is a possibly harmful, physical mutagenic agent and shape an important component of terrestrial radiation to which plants have been uncovered since attacking area [27]. From that point forward, plants have advanced components to maintain a strategic distance from and x from UV radiation injury. Most of the compounds accumulated are directly involved in UV-C protection; they are either e cient in ltering excess radiation or in scavenging radicals. The substance of chlorophyll a, b and carotenoids of pepper leaves were decreased signi cantly in those plants which were presented to UV-C radiation and contrasted with control plants. Thus, Ultraviolet-C radiation induced oxidative stress in Chilli by increasing lipid peroxidation and membrane permeability which indicates that limits of tolerance are much less than damaged caused by UV-C radiation [27]. Likewise, the impact of UV-C radiation on chlorophyll, avonoids, anthocyanin, proline, membrane permeability, lipid peroxidation and UV-absorbing compound was analysed with reference to the Capsicum species. The seeds were treated with different concentration of gamma rays and the effect of gamma rays on phytochemical constituents in Chilli was considered on M2 generation. The phytochemical constituents induced chlorophyll, Capsaicin, Oleoresin, Capsanthin and Ascorbic acid [33].
The impacts on the structure and ultrastructure of Chilli plants when presented to UV radiation under greenhouse conditions. Changes in shoot development demonstrated the critical decline in UV-C exposed plants. Leaf area also diminished altogether in UV-C-exposed plants. Stomata expanded in number and size in UV-C exposed plants. Chloroplast thylakoids were enlarged and starch decrease was seen at the ultra-basic dimension. UV treatment resulted in the formation of crystals in the peroxisomes of mesophyll cells. The development of these crystals was due to an increase in catalase activity, which is an antioxidant enzyme. The Chilli plants were delicate to UV and the discoveries gave the knowledge into the physiological changes during UV exposure, and showed this plant was progressively touchy to UV-C radiation [34].

Genomic DNA Isolation
In AFLP strategy, a total genomic DNA is processed with two restriction enzymes and adaptors of known sequence are then ligated to the DNA fragments. Primers complementary to the adaptors, with extra 1-3 selective nucleotides on the 3' end are utilized to intensify the restriction fragments. The PCR ampli ed parts would then be able to be isolated by gel electrophoresis and the banding patterns visualized. AFLP pro les require no earlier DNA sequence information and the number and nature of ampli ed fragments are adjusted by the decision of primer pair.
The DNA of Capsicum species is isolated from cells in stepwise manner. DNA was released from the cells and nucleus (Fig. 11). All the chemicals and buffers aids for the release and puri cation of DNA. After extraction of pure DNA from samples, DNA is quanti ed using agarose gel electrophoresis and the DNA fragments/ bands are observed under UV light (Fig. 12). The puri ed DNA fragments are double digestion using the enzyme ECORI and MSEI and the bands are observed under UV light (Fig. 13).
This enzymatic can be used for cleaving DNA molecule at speci c sites, ensuring that all DNA fragments that contain a particular sequence at a particular location have same size, furthermore each fragment that contain the desired sequence has the sequence located at exactly the same position within the fragment it). The resulting digested DNA is very selectively ampli ed using PCR reampli cation (Fig. 14).
A dendrogram showing similarity in the DNA bands of Capsicum species based on the scoring of unexposed and exposed plants in six different time intervals of UV-C radiations with three major clusters showed signi cant negative a nities. 50-60 minutes of UV-C radiations exposed plants accounted for low scoring of DNA bands belonging to the rst cluster; 10 and 40 minutes of UV-C radiations exposed plants of DNA bands in addition to the 20-30 minutes of UV-C radiations exposed plants of DNA bands accounted for moderate scoring in the second cluster, whereas normal plants without exposure of UV-C radiations accounted alone for the third cluster with a high scoring of DNA bands (Fig. 15).
Henceforth, there is a little variety in the occurrence of DNA bands of Capsicum plant mutated at 50-60 minutes intervals after the UV-C radiation exposure when contrasted with the other plant mutated samples. Our outcomes uncovered that the perception of incited UV-C mutation in of Capsicum species was maybe recorded by these divided DNA banding designs. Still more than one hour of UV exposure is required to make/create the fake change in the chose plant source.
DNA from plants presented to UV radiation displayed polymorphic bands which were not detectable in DNA of unexposed or controlled plants of Capsicum species. The upgraded development of AFLP polymorphisms was additionally seen in DNA of plant presented in situ to a physical source for mutagenesis. The correlation among "unexposed" and "exposed" genomes demonstrate that AFLP analysis can be utilized to assess how the mutagenic agents change the structure of DNA in living life forms.
The AFLP procedure likewise has the advantage of sampling many loci simultaneously and it is more robust than discretionary preparing strategies, for example, RAPD, in light of the fact that increasingly stringent conditions are utilized. Along these lines, AFLP gives a novel and amazing asset for gene tagging technique of any origin or complexity [24,35]. It is ordinarily acquired in Mendelian form and in this way be utilized for typing, identi cation of molecular marker and mapping of genetic loci.
The present study privileges an induced mutation by Ultraviolet-radiation and its impact on the plant cells and DNA structure of Chilli plants (Fig. 16) utilizing the physiological methods and AFLP investigations.

Conclusions
The present study on the Capsicum species affords the informations to understand the molecular changes and genetic relationship for the experimentation of both pre-and post-mutational examinations from the acquaintance of UV radiations. Our results indicated the presence of a positive relationship between the biochemical changes and the molecular characterization changes because of the impact of mutation, anyway further examinations are expected to illustrate this association at the molecular level in Capsicum species. The application of UV-C radiations as mutation sources for valuable instigated mutation for the discovery of biochemical and molecular changes in these experiments. Along these lines, Ultraviolet-C (UV-C) with 200-280 nm is amazingly destructive to living beings contrasted to the other of UV radiations (UV-B with 280-320 nm and UV-A with 320-400 nm).
Moreover, the present examination proposes that the AFLP test connected related to different biomarkers from larger amounts of natural association would demonstrate an amazing eco-toxicological device. This should be possible at the molecular level by normal DNA sequencing in which pre-and postmutational changes can be distinguished utilizing Real-time PCR examination. Now most advanced next generation sequencing and genotyping technologies have also been creating more genomic assets in the Capsicum species which must be utilized pro ciently for product enhancement in future.

Materials And Methods
The present investigation was carried out on the evaluation of mutation and its molecular characterization on the Capsicum species (Angiosperms: Solanales: Solanaceae) throughout the experiment. At rst the seeds were sowed to develop the plants which were chosen for the present investigation.

Sowing of seeds
The seeds of Capsicum species for the present study were gathered from the Lalbagh Botanical Garden, Bengaluru, India. The dry seeds of Capsicum species were sown independently in small plastic pots at Azyme Biosciences Laboratory Private Limited, Jayanagar, Bengaluru 560 069, packed with organicmanure (vermicompost). Consistently showering the water for their development of Capsicum species and the development parameter was estimated utilizing standard estimating tape/scale for each substitute days. Fourteen replicates of two rows were used for normal plant development of Capsicum species (see Fig. 1).

Morphometric analysis of growing plants
The morphometric examination on seeding and developing plant of Capsicum species were done from the day of sowing to the 10th day of the developing plant (see Fig. 2). The characteristics of seedling investigated were plant height, number of lea ets per length and diameter of the leaf. The plant height and leaf size of Capsicum species was estimated in centimeter.

Effect of mutation on Capsicum plant in different time intervals
The Chilli plant seed was taken and sown in the pits (eight seeds) in the laboratory providing favourable environmental condition for its appropriate development. The growth of the plant of Capsicum species was detected every week until the point that it came to 15 cm height. After the developing was seen to the 15 cm, the plant was exposed under UV-C radiations (wavelengths 280 nm) for different time intervals with ten minutes gap, (such as 10 min, 20min, 30 min, 40 min, 50 min and 60 min) (Fig. 3).
Leaves from plant of Capsicum species is taken and weighed up to 1 g. Phosphate buffer was prepared by addition of Potassium dihydrogen orthophosphate (100 mM) and Di-potassium hydrogen phosphate (100 mM) and the pH was set to 7. The leaves were homogenized in 10 ml of phosphate buffer. The mixture was transferred into cleaned centrifuge tubes for each sample and subjected to centrifugation at 10,000 rpm for ten minutes. The supernatant was transferred into clean tubes and refrigerated for the further analyses. The leaves of Capsicum species (Chilli) from two different varieties (such as Pusajwala and Surajmukhi) were utilized as material for DNA extraction.

Estimation of biomolecules and stress enzyme
The estimation of biomolecules and the stress enzymes for both the normal Chilli plant and the mutated plant exposed under the UV rays was conducted. The extraction is usually carried out with buffer used for the analysis of enzyme assay. Weigh 1 g of the sample of Capsicum species from both the normal and mutated plant with different time intervals and grind well with a pestle and mortar in 5-10 ml of buffer.
Then centrifuge it and use the supernatant for the estimation of biomolecules and the stress enzymes.

Estimation of Protein by Lowry's method
The proteins were estimated using the method followed by the Lowry et al. [36]. The absorbance of the protein sample was plotted on the standard graph and extrapolated to get the concentration of protein.

Estimation of Carbohydrate using DNS Method
The carbohydrates were adapted by the Miller`s method [37]. The absorbance of the carbohydrate sample was plotted on the standard graph and extrapolated to get the concentration of carbohydrate.

Estimation of Lipids using titration method
The lipids were estimated by the Cox and Pearson [38] and used the following formula:

Enzyme extract
Homogenize plant tissue of Capsicum species in a blender with M/150 phosphate buffer (assay buffer diluted 10 times) at 1-4 ºC and centrifuge stir the sediment with cold phosphate buffer, allow to stand in the cold with occasional shaking and the repeat the extraction once or twice. The extraction should not take longer than 24 h. Use the combined supernatants (sometimes opalescent) for the assay. The enzyme activity can change considerably on storage of the tissue for the comparative studies, therefore, always use the same conditions of extraction, storage and temperature.

Estimation of Peroxidase activity
The peroxidase activity was estimated using the method followed by Malik and Singh [39]. The activity of peroxidase in the sample was calculated by the formula mentioned below:

Agarose Gel Electrophoresis
This was done by the pouring, running and imaging of the gel using the standard protocol.
Puri cation of DNA Take 0.3 ml crude DNA sample in separate Eppendorf tubes and add equal volume of phenol: chloroform: isoamyl alcohol mixture (ratio -25:24:1). The above mixture is vortexed and centrifuged at 10,000 rpm for 10 minutes at 4 °C. Carefully pipette out aqueous layer (top most layer) into another Eppendorf and add equal volume of 24:1 chloroform: isoamyl alcohol mixture. Gently vortex the mixture and centrifuge at 10,000 rpm for 10 minutes at 4° C. Again, transfer the aqueous layer carefully to another Eppendorf tube and add double the volume of chilled ethanol and incubate the sample mixture for 30 minutes in ice cold condition (4 ºC). Centrifuge at 10,000 rpm for 20 minutes at 4° C. The pellet obtained is air dried to remove excess ethanol and dissolve in 0.1 ml of TE buffer and stored at 4 ºC until future use. Agarose gel electrophoresis is once performed again.

Ampli ed Fragment Length Polymorphism (ALFP) Analysis AFLP template preparation
This was done by double digestion and ligation of adapters using the standard protocol.

Ampli cation of AFLP templates
This was done by the preampli cation and re-ampli cation using the standard protocol.

Agarose Gel electrophoresis
After PCR ampli ed product was run agarose gel electrophoresis.
Moreover, Euclidean distances method of cluster analysis of Capsicum species was carried out to create a dendrogram to assess the scoring of DNA bands from UV-C radiations exposed and unexposed plants in six different time intervals using PAST version 1.60 software [44].

Declarations
Ethics approval and consent to participate Not applicable.