Application of stress susceptibility index and multivariate analysis of tomato (Solanum lycopersicum L.) to identify thermo-tolerant genotypes


 Climatic parameters have become key mechanisms in controlling crop productivity worldwide. The more frequency of heat waves urges the breeding for thermo-tolerance. The motive of this study was to furnish an accurate and deep understanding on the heat tolerance of reproductive key traits (flowers with exerted stigma, pollen viability, fruits set per cent and number of fruits per cluster), along with earliness (days to first fruit set) and yield traits (average fruit weight, pericarp thickness, number of fruits per plant and yield per plant) in tomato. The study reasoned 35 genotypes employing three no-identical analysis tools, ANOVA of field assessment, stress susceptibility index and multivariate analysis based on genetic diversity. Insights of genetic architecture of the reproductive traits under heat stress might improve our core understanding, and might have applied value. The study substantiated the essence of heat tolerance for the genotypes EC-620395, EC-620401, EC-620406 and EC-620410. Divergence analysis revealed five clusters corresponding to specific characters. Therefore on the ground of the investigation new breeding lines and breeding strategies can be implemented under changing environmental conditions with special reference to elevated temperature.


Introduction
Tomato (Solanum lycopersicum L.) occupies an eminent position among the vegetables and its cultivation has roll out globally. The agro-climatic conditions of tropical, subtropical and temperate zones are well suited for tomato . In traditional agriculture, tomato turns up well in winter months (Ruggieri et al. 2019). During summer months the crop does not grow well due to hostile weather conditions . With rising zeal for its cultivation, the farmers are aptly looking towards upgraded varieties to meet out higher demands of seasonal dissimilarity (Dhillon et al 2019;Zdravkovic et al. 2013). Course of action to take the edge off yield cutback bringing on heat, encompass the production and use of heat tolerant genotypes, and for this a finer perception of the plant response and adaptations to heat stress is a precondition (Jha et al. 2014). A temperature surpassing 30ºC weaken various vital physiological processes viz; respiration and transpiration (Stone 2001), pollen production and viability (Camejo et al. 20105), seedling emergence, photosynthesis (Xu et al. 2017a) and that eventually result in the substantial yield loss (Adam et al. 2001;Pressman et al. 2002;Barnab et al. 2008;Hedly 2008;Zinn et al. 2010). The most commercial cultivars of tomato in Kerala perform well in the wintertime cultivation. Yet they break down in summertime. Kerala relish the classic tropical climate, where the summer temperature obviously crosses 38ºC (Alam et al. 2010).
Different strategies, for instance screening and selection of varieties (Zamir 2001;Srivastava et al. 2016) may provide a resource for evolving resistant and relatively resistant genotypes, since stress tolerance is a developmentally regulated state-specific phenomenon. To differentiate the degree of resistance amidst the screened genotypes, Stress Susceptibility Index (SSI) has been proposed (Fisher and Maurer 1978). It is the ratio of genotypic performance under stress and non-stress conditions (Jha et al. 2014). Safeguarding the crop by protected cultivation was in subject of research team in the previous periods (Jha et al. 2014;Singh et al. 2014).
In this manuscript, 35 tomato genotypes were screened for high temperature tolerance inside rainshelter. Analysis was accomplished with field screening and heat susceptibility indexing. The current evaluations at different temperature regimens, pointed out varying degree of resistance. Therefore the genotypes were further studied in multivariate analysis to discern the genetic relationship among them. The joint analysis could be a powerful tool for thermo stable line identification and would play a vital role in future agriculture.

Materials and methods
The core segment of the current work was the field appraisal of tomato genotypes for tandem season, one summer (from January 2018 to May 2018) and one rainy (July 2018-December 2018) at Vegetable Science Department, College of Agriculture, Vellanikkara, Thrissur at an altitude of 22.25 m above mean sea level, between 10 o 31 ' N latitude and 76 o 13 ' E longitude, with tropical warm humid climate and mean annual rainfall of 2663 mm (sandy loam soil with pH 5.7). The investigation material was 35 tomato genotypes, 29 genotypes from NBPGR, New Delhi, Pusa Ruby, 3 IIHR Bangalore varieties (Arka Abha, Arka Saurabh and Arka Alok), and 2 KAU released varieties (Akshaya and Anagha). The genotypes were raised in nursery pro-trays filled with potting mixture red earth, sand and cow dung in the proportion of 2:1:1 for twenty one days and shifted to the rainshelter (24m × 16m × 3.1m) at spacing of 1 m × 0.5 m on raised beds (23 m × 1 m), enveloped with black and white double shaded polythene mulch of 30-micron thickness, with drip irrigation. The configuration of the experiment was Randomized Block Design (RBD) with two replications and ten plants per replication. The plants were hold up on long pole and regularly pruned. Plant protection has been shouldered as per the ad-hoc guidelines of KAU (Estelitta et al. 2016).

Phenotypic evaluation
The heat tolerance distinctions assessed were flowers with exerted stigma (%), pollen viability (%), fruit set per cent and number of fruits per cluster (number).
For pollen viability the pollen grains from flowers that bloom on the very day were spotted in acetocarmine stain and observed under microscope (Abdul-Baki 1992) ( Figure 1) The earliness of the genotypes was recognized from the days to first fruit set. The yield properties of average fruit weight (g), pericarp thickness (g), number of fruits per plant (number) and yield per plant (g) were rated as well. The mean of all quantitative traits mentioned above were subjected to statistical analysis and mean was compared with the help of ANOVA for each character separately at p=0.05 (Fisher 1957).

Heat Susceptibility Index
To evaluate the heat tolerance of investigated genotypes, the HSI was determined (Fisher and Maurer 1978) for pollen viability, fruit set per cent, average fruit weight and yield per plant as differences in the results obtained for stressed (during summertime) and non-stressed (in the rainy season) by fitting into the equation: A three point scale was used for scoring. The HSI value below 0.5 implies heat tolerance, in the range 0.5 to 1.0 denoted moderate tolerance and exceeding 1.0 hinted the heat susceptibility of the genotype (Singh et al. 2017).

Multivariate cluster analysis
Multivariate cluster analysis was also employed as a selection standard, for understanding divergence of the investigated genotypes in reference to phenotypic characteristics. Divergence was estimated using Euclidean distance with complete genes attachment to grouping. The genotypes were clustered by Tocher's method (Rao 1952). The analysis was performed implementing and using R package (R version 3.6.3 (2020-02-

Result and discussion
The seasonal appraisal delineated notable dissimilarity amidst the genotypes and the differential response of genotypes to unlike weather conditions.

Flowers with exerted stigma (%)
Protrusion of style over the anther cone, the most outstanding reaction of uplifted temperature on female reproductive organ, was observed in the current work as well (Table 1 and Figure 2). During summertime the genotype EC-620382 towered (56.9%), and during the rainy season Arka Abha (36.5%).
Sixteen genotypes displayed additional incidence of stigma protrusion in summertime, and the deviation exceeded 5%.  Gonzalo et al. 2020). The result of current work was consistent with the previous documents (Giorno et al. 2003;Sato et al. 2006;Din et al. 2015;Saeed et al. 2017;Xu et al. 2017b).

Pollen viability (%)
The temperature during the pollen formation is ample to cause major differences in the pollen viability.
The current work noted fifteen genotypes exhibiting more than 5% difference between the seasons, with excessive pollen viability in the rainy season (Table 1). In both the seasons the genotype EC-165395 towered pollen viability (64.4% and 65.4% for summer and rainy season respectively). Two genotypes, EC-165700 and EC-620378, exhibited more pollen viability in the summer season. Two genotypes, EC-620376 and Pusa Ruby, exhibited non significant changes betwixt the season. Continuous exposure of tomato plants to high temperature impart differential accumulation of hexose Vs sucrose in developing pollen grains (Paupiere et al. 2014) and delayed tapetum development (Alsamir et al. 2017;Razzaq et al. 2019), thereupon imparting different levels of tolerance. Metabolic alterations in the thermo-tolerant genotypes leads to the proline accumulation, acting as a heat shock protein (Giorno et al. 2003;Sato et al. 2006;Din et al. 2015;Saeed et al. 2017;Xu et al. 2017b). The differential response in the pollen viability of genotypes to high temperature has remarkably been documented (Zamir 2001;Singh et al. 2017;Xu et al. 2017b;Dhillon et al. 2019).

Fruit set per cent
Stress outcome on the fruit set has more dramatic repercussions on the yield. The current work remarked better fruit set per cent in rainy season for 30 genotypes (Table 1)

Number of fruits per cluster (number)
Number of fruits per cluster is viewed as good prediction for fruit retention capacity at elevated temperature. The investigation noted more number of fruits per cluster in the rainy season for 22 genotypes (Table 1)

Days to first fruit set (number of days)
Days to first fruit set is cardinal in regulating the earliness of the crop. The appraisal noted similar pattern of earliness in the summer season for 29 genotypes for days to first fruit set ( and EC-620410, documented non-significant changes for days to first fruit set for tandem appraisal. Days to first fruit set fabricated a consistent trend (Agele et al. 2002). The high precipitation and relative humidity in the rainy season advocate the production of huge biomass, and the fruiting incline toward the late side (Oladin and Oluwasemire 2018). Late season crop flowering earlier than the rainy season planting was previously been observed (Agele et al. 2002; Oladin and Oluwasemire 2018).

Average fruit weight (g)
Fruit weight is one chief element that contributes to the yield. The investigation recorded (Table 2) average fruit weight in the extend of 4.2 g (EC-165700) and 94.2 g (EC-538153) for summer assessment, and in the radius of 10.9 g (EC-620376) and 95.3 g (EC-538153) for the rainy season assessment. There was increased average fruit weight in the rainy season for 25 genotypes.

Pericarp thickness (cm)
The fruit morphology constitutes reference to robust and stable fruit phenotypes. EC-620406 and EC-

Number of fruits per plant (number)
Number of fruits per plant envisaged an index of yield prediction in tomato. The work noted 17 genotypes with improved number of fruits per plant in the rainy season assessment ( for dissimilar weather conditions. Genotypes exhibited independency in the stress responsive mechanism (Sato et al. 2002). Heat stress correlated fruit number trimming, primarily linked with reduced fruit set and pollen viability, (Sato et al. 2002;Zhou et al. 2017;Driedonks et al. 2018), was consistent in this work as well. Heat tolerant genotypes exhibit increased chlorophyll content, higher number of stomata and bigger stomatal pore size (Sato et al. 2002), thereby maintaining substrate carbon conversion about 25% irrespective of temperature (Panthee et al. 2018). These seasonal and varietal interplay in number of fruits per cluster has been reported by Rajashekar et al. 2006;Alam et al. 2010;Ashrafuzzman et al. 2010;Hossain et al. 2014.

Yield per plant (g)
Any abiotic stress has a direct impact on the yield of tomato. The work noted a rise in the rainy season yield for 12 genotypes (Table 2). EC-620387 noted significantly higher returns in the summer appraisal.
Remaining genotypes exhibited non-significant changes over the seasons.

Heat Susceptibility Index (HSI)
The assessment of reaction of investigated genotypes to heat was further done by estimating Heat Susceptibility Index for pollen viability, fruit set per cent, average fruit weight, and yield per plant. The HSI for yield per plant in Figure 6 plotted values from -9.07 to 7.

Multivariate analysis
The final estimation of genotypes in the selection process involved the use of cluster method classifying different genotypes in similar classes. The analysis distributed genotypes into five clusters based on genetic divergence (Table 3). Cluster II was the largest group containing 15 genotypes. The formation of five clusters indicated the existence of divergence of population as suggested by Ahmad et al. 2003;Basavaraj et al. 2010.
The D 2 technique measures the forces of differentiation at two levels, intra-cluster and inter-cluster.
The inter-cluster distance was greater than intra-cluster distance (Table 4)  The D 2 technique is thus practically advantageous for the selection of genetically divergent parents for the exploitation in the hybrid vigour (Lekshmi and Celin 2015).
The contribution of each trait to the variations was reflected in the cluster mean value (Table 5) was also identified. the results also support the existing distinct screening modules for the identification of stable heat tolerant genotypes. The new insights gained and the wealth of data furnished from this investigation can contribute much to the refinement of current breeding models for stress tolerance. Clustering pattern could be utilized in choosing parents for hybridisation likely to generate the highest possible variability for the economic characters. Heat stress is often connected with drought stress in field conditions, which demand the integrated study of heat and drought stress that boosts the current work. Extra work on chlorophyll content can ameliorate the work as well. since heat tolerance hangs on multiple traits, the strategy cited and available set of genotypes therefore will help in deciphering the genetic layout fundamental in tolerance mechanism and illuminate on the future breeding proposals for stress tolerance.