ESTIMATION OF GENETIC DIVERGENCE IN CHILLI PEPPER (CAPSICUM ANNUUM L.) GENOTYPES FOR MORPHOLOGICAL AND FRUIT TRAITS UNDER HOT CLIMATE OF UMERKOT, SINDH

Thirty-two genotypes of chilli from three different sources (local dandicuts, AVRDC and PGRI PARC) were evaluated for the presence of genetic divergence among them for different agro-economic traits i.e., plant height, stem girth, fruit length, fruit girth, number of fruits per plant, number of seed per fruit, fresh fruit weight and dry fruit weight at Arid Zone Agricultural Research Institute, Umerkot, Sindh during 2018. All these genotypes were found to be significantly different from each other for all the studied traits. For further diversity analysis, multivariate analyses like principal component analysis (PCA) and Cluster analysis were also performed to figure out the traits responsible for maximum variability and grouping of genotypes according to their similarities and differences for their future utilization in chilli breeding programs. PCA analysis showed that first four PCs exhibited 82.79% of the total variability among these 32 chilli genotypes. Cluster analysis showed four different sub-clusters and the genotypes from every single source merge in each sub-cluster, thus showing that chilli genotypes are from different geographical backgrounds. Based on these results, the chilli genotypes can be further utilized in future chilli improvement programs in Pakistan. Genotypes AZRI-DS-14, AZRI-DS-01 and AVPP-9804 showed better performance.


INTRODUCTION 1
Chilli pepper or hot pepper (Capsicum annuum L.), a diploid species having chromosome 2n=24 is an important vegetable-cum-condiment crop of family Solanaceae and genus capsicum (Zulaikha, 2019). Genus capsicum has five cultivated and twenty five wild species (Moscone et al., 2007). It is an important cash crop, used both as fresh vegetable and also processed and used as a condiment (Votava et al., 2005). Due to its pungency this crop is considered as a chief ingredient in culinary preparation in Asian countries like Pakistan, India, Malaysia and Bangladesh (Farhad et al., 2008). Besides its culinary usage chilli is also used as fresh food, condiments in dry form, pickle, sausages and as a guacamole in Mexico (Orellana-Escobedo et al., 2013). Chilli pepper is nutritionally rich in * Corresponding author: rafiqahmad.uaar@gmail.com carbohydrates, lipids, proteins, mineral salts and fibers (Wahyuni et al., 2013;El-Ghorab et al., 2013). It is also an important source of vitamins (A, B, C and K) and phytochemical compounds like carotenoids, flavonoids, ascorbic acid and capsacianoids (El-Ghorab et al., 2013). These vitamins and phytochemicals are useful for cure of chronic disorders like asthma, cancer, used as immune system stimulator, antioxidant and antimicrobial (do Nascimento et al., 2013). Fresh green chillies have more vitamin C than citrus and fresh red chilli pepper have more vitamin A than carrot (Osuna-García et al., 1998;Marin et al., 2004).
Total area of the world under chilli cultivation is about 1856.641 thousand hectares with a production of 4625.833 thousand tones (FAOSTAT, 2017). In Pakistan it was cultivated on 47.349 thousand hectares with a production of 126.943 thousand tones (MNFS and R, 2018-19). Sindh contributes 36.067 thousand hectares in area and 108.578 thousand tones in production followed by Punjab 6.842 thousand hectares in area and 11.698 thousand tones in production, Baluchistan 4.112 thousand hectares in area and 6.285 thousand tones in production and KPK contributes 0.328 thousand hectares in area and 0.382 thousand tones in production (Agriculture Statistics of Pakistan, 2019). Chilli shows good response to warm climatic situations but vulnerable to extreme temperature regimes (Dahal et al., 2015). Kunri is the major area of chilli production in Sindh Province that is known as Asia's biggest market (Wahocho et al., 2016). Presence of genetic diversity is a pre-requisite for improvement in any crop. However, varieties developed lose their genetic potential over the period of time due to continuous cultivation and genetic erosion, therefore, leading to decrease in per acre yield of the available genotypes. However, because of the popularity of the crop and extended history of cultivation at different parts of the world, sufficient genetic variability is present that can be utilized for generation of new and improved varieties (Pujar et al., 2017). For varietal improvement, exploring the potential of base population and selection efficiency are the key factors. Therefore, current study was carried out to investigate thirty-two genotypes of chillies for different agronomic and fruit traits. This study will be helpful in designing a breeding program for new varieties development that should sustain and produce higher yield than existing varieties.

Experimental site
Current study was carried out at field area of Arid Zone Research Institute (AZRI), Pakistan Agricultural Research Council, Umerkot, Sindh, Pakistan. Experimental site was situated at 25.15°N and 69.32°E.

Experimental material and design
Current experiment was carried out by using 32 genotypes; out of which sixteen genotypes were local dundicut (DS) selections, eight genotypes from Asian Vegetable Research and Development Center (AVRDC) and seven genotypes were from Plant Genetic Resources Institute (PGRI, PARC, Islamabad). Nursery was raised on raised bed at field area of Arid Zone Research Institute (AZRI) Umerkot, Pakistan Agricultural Research Council. Seedlings were then transplanted in field in mid of April, 2018 in a randomized complete block design (RCBD) having three replications. Row to row distance was maintained at 75 cm and plant to plant distance of 30 cm (Marame et al., 2009). Four rows were transplanted for each genotype with a row length of 4 m. All agronomic and crop management practices were performed at different crop stages.

Agronomic and fruit characterization of studied chilli genotypes
Data regarding vegetative and fruit traits from five randomly selected plants for all genotypes were collected from all three replications. Data for plant height (cm) was computed with the help of meter rod, number of main branches was counted manually, stem girth (mm), fruit length (mm) and fruit width (mm) were computed with the help of vernier caliper and number of fruits per plant, number of seeds per fruit, fruit fresh weight (g) and fruit dry weight (g) were also determined.

Statistical analysis
Analysis of variance (ANOVA), descriptive statistics, cluster analysis and principal component analysis was performed by using R-Studio Version 1.2.1335.

Morphological variation
Knowledge about the genetic divergence is not only helpful in crop improvement program but also helps in understanding the availability of germplasm diversity to certain area. Analysis of variance showed that considerable diversity is available among studied genotypes (Table 2). All studied traits showed highly significant genotypic mean square values. There was a great variability observed for quantitative traits that includes agronomic and fruit traits (Table 3). Plant height was ranged from 41.5 to 82.5 cm. Number of main fruiting branches varied from 6 to 10 branches per plant. Stem girth varied from 10 to 32 mm. There was great variability observed for number of fruits per plant that varied from 31 to 259 fruits per plant. There was also considerable variability for fresh and dry fruit weight. Fresh fruit weight ranges from 54 to 382 g per plant while dry fruit weight ranges from 15 to 100 g per plant. There was also variability for fruit length and fruit width. Fruit length varied from 4.3 to 7 mm, while fruit width varied from 6.3 to 23.7 mm (Table 3).

Principal component analysis
The principal component analysis (PCA) is useful statistical attribute from multivariate analysis that can prove useful for future breeding and population development program. First four PCs with eigen value > 1, explained 82.79% variability out of the total variation existed in studied chilli genotypes (Figure 1).
Here it can be seen in this figure that maximum variability can be explained by first four principal components. Scree plot explained variance percentage obtained by each component. Maximum variance is accounted for first four PCs after that semi curve line obtained which shows little variance existed in remaining principal components (Figure 2). First eigen vector alone showed variation of about 39.6% out of total explained variation. Principal components results showed all the traits studied had a positive contribution except fruit length. Fruit length has a negative correlation with fruit width, plant height and number of seeds per plant (Figure 2). PC Biplot diagram illustrates the diversity among studied chilli genotypes and different traits studied in current research ( Figure 2). Based on nine morphological and fruit attributes, studied genotypes scattered in four quadrants. Genotypes that are close to each other farming small angle are closely related and having most of the features common to each other. While, genotypes on bi-plot that make an angle more than ninety are diverse genotypes.

Cluster analysis
Cluster heat map was generated through Euclidean distance matrix computed by UPGMA analysis for nine morphological and fruit traits of thirty two chilli genotypes, to study the diversity among these genotypes acquired from different sources in 2018. Heat map diagram generated from the morphological and fruit data of chilli genotypes studied during cropping season 2018, divided these genotypes into four different clusters (Figure 3). First cluster contains 10 genotypes that can further be divided into two sub-clusters, in sub-cluster I, 6 genotypes fall that are from local dundicut selection source from AZRI i.e., AZRI-DS-02, AZRI-DS-03, AZRI-DS-04, AZRI-DS-05, AZRI-DS-06 and AZRI-DS-08 while in sub-cluster II, genotypes AVPP-0701, AVPP-0506, Pr-16162 and AVPP-9704

DISCUSSION
Chilli is an important vegetable cum cash crop of Sindh province, as it produces high quality red (dry) chilli with its unique export quality. Limited research work has been carried out in vegetables seed sectors, especially in chillies. It is the need of time to study the diversity in this crop especially due to ever changing climatic situations. Diversity study is imperative in any crop improvement and population development program. Enhancement of gene pool diversity and its characterization is a foremost goal of a breeder to strengthen breeding program (Bianchi et al., 2020). Current study was aimed to study diversity in hot pepper (red chilli) genotypes collected from three different sources. Significant diversity was observed for different morphological and fruit related traits.
There exists a great variability between different traits like fruit length and weight. Similar findings were also observed by (Castro and D ávila, 2008;Chowdhury et al., 2015;Naegele et al., 2016;Arumingtyas et al., 2017;Moreira et al.,2018). Baba et al. (2016) while studying on Capsicum chinense also found great diversity for morphological and fruit parameters. They also study different molecular markers to study genetic diversity in available germplasm. Principal component analysis and cluster analyses are the two important multivariate analysis approaches. This analysis can be of great help for breeders to select genotypes for launching breeding and improvement program. As it can be seen there was considerable variability existed by analyzing our collected germplasm by PCA and cluster analysis. It was observed that genotypes from different sources were placed in different clusters and showed different relationships by principal components on the basis of studied traits. Genotypic selection can be made on these approaches to strengthen our germplasm and to improve our breeding program. Diversity of chilli was also studied by Hasan et al. (2014) by cluster analysis through D 2 statistics and Principal component analysis. They also studied that broad spectrum variability can be achieved by selecting genotypes from diverse clusters for different morphological and fruit quality traits. Utilizing distantly related parents can be helpful for population improvement and future breeding program as this approach can provides higher heterosis or transgressive segregants. On the basis of results of current study, it can be established that characterization of available germplasm is important and basic criteria to understand the genetic variability of existing population and also for the development of effective strategies germplasm conservation, improvement and breeding purpose.

CONCLUSION
Present study provides us the considerable information for selection of diverse parents to improve our existing cultivars and breed new cultivars with traits of our interests. Some of the study genotypes like AZRI-DS-14, AZRI-DS-01and AVPP-9804 showed better performance for different fruit traits and showed diversity from other genotypes. These genotypes can further have potential to use in future breeding programs for improving fruit yield and fruit quality traits for enhancement of ultimate yield of the chilli crop.

ACKNOWLEDGEMENT
Author is thankful to PSDP for providing funds to conduct Research under strengthening project for AZRI, Umerkot, Sindh.

AUTHOR'S CONTRIBUTION
A. Memon: Performed the experiments and data collected R. Ahmad: Analyzed data and wrote the manuscript M. S. Depar: Design the study and review manuscript A. K. Pathan: Design the study and Review manuscript D. Ibrar: Analyzed data and wrote the manuscript