Molecular divergence analysis of chilli (Capsicum annuum L.) germplasm using SSR markers

Introduction

Among the 25 species of the genus capsicum of chilli crop only five species are being grown worldwide (Bosland and Botava, 2000; Costa et al., 2009) [2,4] . It is cultivated for its fruits which are utilized in various forms such as vegetable, condiment, spice, and in processed forms like powders, sauce, paste, pickle, flakes, etc. (Timmarao et al., 2025) [19] . It is mainly valued for its pungency, which is due to crystalline acrid volatile alkaloid capsaicin, present in the placenta of fruits. Due to the numerous benefits offered by capsaicin, it is used widely in various pharmaceutical products. It is used for industrial purpose by extraction of oleoresin. In food processing factories, oleoresin extracted from chilli is used for better colour distribution in food. From global perspective, chilli crop is an major spice cum vegetable crop (Saisupriya et al., 2024) [17] . The diverse utilization of chilli from culinary, to health and industrial uses has driven its cultivation worldwide (Lozada et al., 2022) [12] . For optimal growth, chilli plants thrive in daytime temperatures between 20 °C and 30 °C and prefer well-drained loamy soils with a pH between 5.5-6.8 (Khan et al. 2025) [10] . Genetic diversity is the total variety of genes present in a single population and it estimates the inherent genetic potential of a cross in subsequent advanced generations. It can be evaluated at various levels such as genotypic, molecular and phenotypic level. Such studies facilitate understanding of genetic relationships between accessions, identification of germplasm excesses and admixtures, and identification of genitor pairs with sufficient genetic distance (Delfini et al., 2021) [6] . DNA markers are prominent tools for identification of genotypes and diverse parents for hybridization. Given that these molecular markers are not influenced by environmental variability, they are crucial tools for genetic mapping and assessing the diversity. The markers play a very important role in determining the position of genes and that gene can be used for targeted traits and novel varieties can be developed by using this genetic makeup (Bashir et al., 2022) [1] . With the development of molecular biology technique, many DNA markers have been emerged that are useful in identification of polymorphism at genetic level. Various DNA markers advented for chilli include random amplified polymorphic DNAs (RAPDs), restriction fragment length polymorphisms (RFLPs), simple sequence repeats (SSRs) and amplified fragment length polymorphisms (AFLPs). SSR have numerous benefits among genetic markers such as high reproducibility and useful in analysis of genetic diversity in various spp. (Jamir et al., 2022) [9] . In cultivated species with low levels of variation, simple sequence repeats are the most widely used for analysis of molecular diversity (Geleta et al., 2004) [7] . They are highly polymorphic in nature and used to predict heterosis. They are powerful tools for estimating genetic similarities and diversity (Sharmin et al., 2018) [18] . Thus, SSR markers preferred to perform the molecular characterization research due to its adaptation to automation and in particular, a quite reliable system, as the results cannot change in the repetition of the reactions (Polat et al., 2025) [13] . They are used for gene tagging, marker-assisted selection and gene or population mapping (Hasan et al., 2021) [8] . Profiling DNA sequences using molecular markers can reveal genetic differences among accessions. Successful characterization supports the development of improved cultivars and sustains crop diversity, particularly under diverse environmental conditions (Khan et al., 2025) [10] . Hence the present experiment was conducted with an aim to capture the potential genetic diversity of different chilli genotypes at molecular level by using SSR markers.

Materials and Methods

The present experiment was carried out with 35 chilli accessions as treatments at College of Horticulture, SKLTSHU, Rajendranagar, Hyderabad. Forty (40) SSRs primers were selected from the map of chilli genome for the amplification of genomic DNA. From each genotype, genomic DNA was isolated was performed by cetyl trimethyl ammonium bromide (CTAB) method, (Clarke 2009) [3] with specific changes. DNA was quantified by using agarose gel electrophoresis (Dean et al., 1999) [5] . DNA was amplified in 0.2ml PCR tubes holding reaction mixture of 25 μl. PCR products were separated on 3% agarose gel electrophoresis.

Scoring of Bands

Ultra violet transilluminator was used for studying the SSR-PCR bands. They were later photographed in gel documentation unit. Scoring of SSR bands was done by with presence scored as 1 and absence as 0. Standard marker of 100bp is used for assessment of sizes of the bands. This data is used to generate Bi-nomial data for all the genotypes using excel sheet. Against each primer the banding patterns of all genotypes were differentiated. Bands were considered variable if they are present in one genotype and absent in another. Number of alleles and polymorphic information content were calculated to check the informativeness level and discriminatory efficiency of SSR primers used in this study. PIC value was estimated based on the method proposed by Roldan-Ruiz et al. (2000) [15] .

Diversity Analysis

Similarity matrix and cluster analysis of 35 genotypes was done using the collected data. The cluster analysis and a dendrogram were constructed with the help of NTSYS software based on unweighted paired group of arithmetic mean average (UPGMA). Linkage distance was calculated among these 35 genotypes and they were divided into various clusters, sub-cluster and sub-sub clusters.

Results and Discussion Molecular Diversity of Chilli Accessions

Genetic diversity analysis of 35 chill genotypes was carried out using 40 SSR primers. PCR analysis of the genotypes using 40 SSRs ensured polymorphism with 39 markers and monomorphic with 1 marker. Using the allelic data of 39 polymorphic markers, binary matrix data was prepared. A total of 203 alleles were observed and the number of alleles varied from 1 (monomorphic) to 11 with an average of 5.07. Highest number of alleles were observed with HpmsE 074 and the same marker was used by Nanda et al., (2016) for tagging genomic regions controlling anthracnose resistance and reported polymorphic to F2 mapping population parents (PBC 80 and SB 1) in Capsicum baccatum.

Polymorphism Information Content (PIC)

Genetic diversity among the genotypes was assessed on the basis of Polymorphic information content (PIC) value. PIC values among SSRs varied widely from 0.09 to 0.89 with an average of 0.53 (Table 1). The observation of the present study suggests the polymorphism in the genotypes which is reflected by the PIC values. Highest PIC value of 0.89 was observed with HpmsE140, HpmsE066 followed by HpmsE071 (0.85), Epms331 (0.80) implying their further use in genetic variability studies of chilli germplasm. Whereas lowest PIC value of 0.09 was observed with Gpms 178 marker. The observed average PIC value is similar to the PIC value outlined by Yong sham et al., 2005 [20] (0.53).

Cluster Analysis

The binary matrix data derived from the 40 SSR markers analysis was used for diversity assessment. Jaccard's similarity coefficient values were calculated for 35 chilli accessions using NTSYS-pc version 2.02e (Rohlf 1998) [14] and the dendrogram was constructed by UPGMA The genotypes IC-214965 and IC-410423 were also found to be very closely related (85%), which belong to cluster III. Out of 35 genotypes, two genotypes IC-570408 and IC-561648 were found to be the most diverse, grouping separately with least similarity. The diversity of these two genotypes was also seen in phenotypical cluster analysis by Tocher's method. Similarly other 2 genotypes IC-610383 and IC-399581 were also most diverse with less similarity. Thus, from the present study two diverse genotypes (IC-570408 and IC-561648) and two highly similar genotypes (IC-215012 and IC-572498) were identified which can be deployed in chilli breeding and crop improvement programmes. The results confirmed that the rich diversity existing in chilli genotypes could be used as a genetic resource in designing breeding programs.

Conclusion

Detection of genetic diversity provides valuable information in selecting superior genotypes for plant breeding. Among the 35 chilli genotypes, SSR markers showed genetic variability. These markers are important tools for calculating the genetic similarities and diversity. The genetic relationships presented among the studied genotypes are useful for selection of parents for future breeding programs.

Based on cluster analysis done using 40 SSRs diverse genotypes have been identified for developing mapping populations, crop improvement and heterosis breeding.

The present study revealed that SSR analysis is quick and reliable procedure for genetic diversity analysis as they show sufficient polymorphism within the experimental population. The polymorphic markers with more PIC values are highly informative SSR markers for genetic studies. Markers with high PIC value identified in the present investigation can be used for varietal identification, germplasm conservation, DUS testing, DNA fingerprinting and analysis of genetic purity. Therefore, the information yielded from this study will be useful for further chilli breeding studies.

Declaration

There is no competing of interest amongst the authors.

Acknowledgement

AcknowledgmentAuthors acknowledge SKLTGHU for the invaluable support and provision of resources during the research.

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