*3.3. Cluster Analysis*

All the genotypes were clustered using pollen germination, pollen viability, cell membrane thermostability, chlorophyll content, boll weight, and boll retention at hightemperature stress as variables. The dendrogram showed three clusters with a minimum of 33.33% similarity level. The highest Euclidean distance was found between clusters 2 and 3 (57.76) and lowest between clusters 1 and 2 (27.95) as presented in Table 6.

**Table 6.** The distance among the various cluster centroid of cotton genotypes under high temperature.


The clusters were divided into two groups Group Y and Group Z. Group Y included two clusters named cluster 1 and cluster 2 while Group Z included only one cluster named cluster 3. Cluster 1 included seven genotypes named CRS-2, MNH-1026, N-777, MNH-1016, VH-377, FH-215, and CIM-343 which represent 53.85% of total genotypes. Cluster 2 includes three genotypes named CIM-602, Cyto-608 and AA-933 represent 23.07% of total genotypes. In cluster 3, three genotypes are included named NIBGE-2, Cyto-124, and N-1048 representing 23.07% of the total genotype (Figure 6). The genotypes within each cluster exhibited similar behavior based on six traits used in this study. The genotypes in cluster 1 showed good performance based on boll weight. The genotypes grouped in cluster 2 are characterized by high pollen germination, pollen viability, chlorophyll content, and CMT. This indicated that the genotypes grouped in cluster 2 could be selected for the breeding program. The genotypes of cluster 3 were grouped by the lowest value of pollen germination, pollen viability, CMT, and boll weight (Table 7).

**Figure 6.** Cluster analysis of thirteen accessions of upland cotton evaluated for high-temperature regimes.


**Table 7.** Means of clusters of 13 cotton genotypes of all observed tr under high-temperature stress.

#### **4. Discussion**

Higher pollen germination and viability percentages from flowers under the canopy of the plant as compared to flowers in direct sunlight were observed in this study. As the temperature in the experimental region in Pakistan rises to 47 ◦C during the time of cotton flowering, this damages the lipid as well as protein parts of the pollen membrane, thus resulting in decreases in pollen viability [12]. Pollen viability was determined by analyzing dehydrogenase enzyme activity in the pollen grains—if the enzyme is active, viable pollen grains change to a red color after TTC staining. However, there may be damage to the pollen grains that reduces germination despite this enzyme activity. It has been reported that the distribution of cell organelles such as mitochondria, vacuoles, and endoplasmic reticulum of pollen cells become disturbed under high temperatures. Lipid and starch granules are also reduced in pollen cells during heat stress [8].

In our study, lower pollen germination was observed as compared to pollen viability. Most pollen could not develop the pollen tube required for germination, likely due to metabolic or structural abnormalities of pollen grains [31]. Drought or heat stress significantly lowers carbohydrate metabolism in the pistil, resulting in a lower energy supply to the pollen tube in the style, thus leading to a failure of fertilization [32]. Under excessively high temperatures, heat shock proteins (HSPs) work to stabilize proteins that were damaged when exposed to stressful conditions. As the expression of HSPs varies between genotypes, some genotypes showed good pollen germination even in the top portion of the plant [33].

Genotypic variability for CMT has been previously reported [3,34]. Here, we have observed CMT differences between plant positions. The significant differences among cultivars are due to several factors including cuticle thickness, secondary metabolites, and heat shock proteins [35–37]. Lower CMT estimates in top leaves were due to sunlight exposure. The membranes of leaves facing direct sunlight in high-temperature conditions were more prone to damage. Sun rays cause oxidative damages to both lipid and protein parts of the cellular membranes and cause the leakage of electrolytes [38]. UV radiation from the sun causes irreversible damage to plant pigments [39]. It causes conformational changes in the structure of nucleic acids, proteins, and macromolecules in the cell and degrades the chlorophyll pigment [40,41]. Heat stress directly affects the flow of fluid through the cell membrane as relative electrical conductivity increases with temperature [42,43].

Since the chlorophyll contents under the canopy were higher as compared to the top position, it has been assumed that chlorophyll loses its integrity under direct sunlight. In addition to direct sunlight, the higher temperature in the top portion of the plant also causes chlorophyll damage [44]. Heat stress that denatures thylakoid membranes results in a loss of chlorophyll [45]. Moreover, the enzymes required for the synthesis of chlorophyll and its normal activity were also denatured under high-temperature conditions [46]. As a result, photosynthetic activity was reduced in the top portion of plants. On average, the genotypes AA-933 and CYTO-608 had good heat tolerance features in bottom positions; therefore, these genotypes would be useful as parents in a breeding program

Ascorbic acid has the potential to mitigate the negative effects of stress. It acts in ROS scavenging and maintains the integrity of membranes, including the thylakoid membrane [47]. So, ascorbic acid could be used to overcome the heat stress problem. The cell membrane thermostability of cotton crops can be improved significantly by applying the foliar application of 40 mg L−<sup>1</sup> ascorbic acid [48].

Boll weight is positively associated with seed cotton yield. It is a complex polygenic trait that depends upon numerous factors namely, the weight of seed, seed size, protein and oil content within the seed, and cellulose deposition during fiber development and maturity [49]. It is one of the most important characters linked to improved yield, and significant variation for this trait has been reported in germplasm [50,51]. Although the genotypes used in this experiment were significantly different for boll weight, no significant differences for this trait were recorded between the top and bottom portions of the same genotypes. Retention of bolls during the developmental period varied significantly between the top and bottom branches. The bottom branches tend to hold more bolls as compared to the top branches. It was noted that the genotypes with higher pollen viability and germination also retained more bolls. This study revealed that the heat tolerance ability of the genotypes was associated with boll retention while heat stress has been considered one of the major factors in bolls dropping before maturity [52,53]. Thus, high temperature in the top portion of a plant due to direct exposure to sunlight can explain retaining a lower number of bolls in this portion of the plant.

The variability was found between the genotypes as shown in Table 3. Cluster analysis has revealed that CIM-602, Cyto-608, and AA-933 grouped in cluster 2 performed well and these genotypes could be used further in any breeding program. Since all genotypes are grown in the Punjab region of Pakistan, these are therefore acclimatized to this environment. These genotypes share some common, as well as different phenotypes, which showed variability based on six traits used in this study. The variability was also observed in the cotton genotypes cultivated in the Punjab region of Pakistan by khan [54].

This study provides an understanding of the role of flowering in the top and bottom portions of the cotton plant in response to high-temperature stress because high temperature is a major factor in reducing yield. It is assumed that by increasing the vegetative growth and leaf surface area, the shading effect can be increased. The spreading-type behavior of the cotton plant could be able to produce more shading. The shading effect will reduce canopy temperature and hence yield could be increased. Likewise, screening for early maturing cultivars and for having more branches on the bottom part of the plant

could be beneficial because the bottom branches have shown more productivity than top branches. Keeping in view the importance of the study, another study may be conducted to assess the temperature of the microenvironment i.e., the temperature of leaf, bud, and/or boll at the top and bottom regions of each genotype, followed by correlation analysis with each trait to understand the relationship of various traits during heat stress.
