Exploring genetic variation based on drought-induced phenotypic alterations during reproductive stages in Desi and Kabuli types of chickpea

Background: Identification of appropriate varieties adapted to the global warming and enhanced drought changes is imperative. In this study two sets of thirty elite genotypes of Desi and Kabuli chickpea types were used to investigate the effects of water scarcity at the phenotypic level in reproductive stages and surveying different drought-induced responses for them. Results: Alterations in GY and its components, FT, PT, MT, FM, and SDM as well as number of drought tolerance indices were measured in field conditions. The estimated genotypic effects were detected significant at both limited and full irrigation conditions for GY, GN, GW, and SDM; however, these effects had smaller values for environmental effects except in GW. The SDM and GW in water-limited conditions showed significant positive relationship with those of full irrigated for both chickpea types. GMP index provided the most positive correlations with GY for both type either of two conditions. The highest direct effect on GY was represented by SDM for Kabuli at both conditions as well as Desi chickpeas in limited water condition, while GN was the most one in full-irrigated Desi chickpeas. The ideal genotypes, 25 and 321, as Kabuli and Desi chickpeas, respectively, were detected with high stable and high GY. Conclusions: Results of this study showed that tested chickpea genotypes responded differently under different water treatments, suggesting the importance of assessment of genotypes under these conditions in order to identify the best genotype make up for each particular condition. As water stress severity was applied equally, therefore it was thought to be more serious in genotypes with a greater life cycle. However, it seems that chickpea plants have been adapted to the terminal drought stress, which could be due to the same time of vegetative growth with filling pods and transfer capability of photosynthesis assimilates towards more grain yield in tolerant genotypes. It seems to change in plant phenology due to the terminal drought stress more affected GN and GW in Desi and Kabuli chickpeas, respectively. These differences could be clear points for the leadership of breeding programs towards more adaptation of both Desi and Kabuli chickpea types to terminal water stress, respectively. Moderate to a high proportion of G × E effects were observed in combined analysis for GY, GN, and SDM compared to genotypic effects, suggesting that G × E effects played a greater role than genotypic effects. The ideal genotype of Kabuli type i.e. genotype 25 had greater GY as well as SDM in water-limited condition, while genotype 321 as ideal Desi genotype showed acceptable GY and SDM, but could compensate with higher GN.


Introduction
The average of global temperatures has shown an increase of 1.2 ˚C over the past century and it is estimated to rise up to 3 ˚C for 2100 because of global warming [28]. So is widely predicted to increase the frequency and intensity of drought, accompanied by the higher temperatures and higher CO2 concentration in semi-arid and sub-tropical regions out of the climate changes [37]. These alterations could lead to an increase in the rate of evaporation and decrease water availability for crop plants and resulting in yield losses, potentially as a threat to human food security. Thus, multiple improvement strategies are necessary for sustainable crop production, especially since the production of major crop plants could be reduced over 50% under drought stress conditions [9].
Chickpeas with black or brown-colored grain coat are categorized by Desi type and with creamy or beige-colored are named Kabuli. The Desi type is more prominent by accounts up to 80% globally and has a more small grain size as well as thicker grain coat than Kabuli. Chickpea grain provides necessary amino acids (lysine, methionine, threonine, valine, isoleucine, and leucine) to the human diet and health because of up to 70% carbohydrates, 10% fat, 23% fiber, and 31% protein -higher than any other pulse crop.
That of the root, in addition, enriches soil fertility by atmospheric nitrogen fixation [33,16] As a cool-season grain legume, chickpea is predominantly grown in semi-arid regions and its reproductive stages are usually faced with the lack of rain [32]. These regions globally are classified in two major types, i.e. stored soil moisture in subtropical environments with summer-dominant rainfall and rainfall in winter-dominant Mediterranean-type environments that suffer yield losses due to terminal drought stress as rain-fed farming systems of chickpeas growing conditions [40,34]. Nevertheless, water scarcity is an important scourge, which can be addressed by three aspects including severity, timing, and duration. Iran, the fourth major global chickpea producer after India, Pakistan, and Turkey, is a region with the Mediterranean precipitation and has been composed of arid and semi-arid lands with about 250 mm of annual rainfall, equivalent to one-third of average rainfall in the world, in which water scarcity arises owing to its shortage at the end of spring. A large number of studies have been represented in chickpea plants under drought stress and without drought stress, which some of them in phenotypic levels of agronomic traits are mentioned. Behboudian et al (2001) presented that although shoot dry matter and GY of a Desi chickpea cultivar "Sona" were decreased by terminal drought stress, it had not to effect on GW [4]. In addition, Pushpavalli et al (2014) found that drought at the reproductive stage of chickpea can reduce GY and GN but could not influence GW [22]. Kabuli chickpeas with early flowering and early maturity as well as with high HI were maintained as ones, which presented more GY under terminal drought stress [26]. In a comparative study, Kabuli chickpeas have shown a fewer reduction in SDM, while more decreased in GY, GN, HI, and GW have been shown than Desi under terminal drought stress [20]. GY and GW reduced by 53% and 4.5%, respectively, and MT was earlier 2 days 5 for chickpeas grown under the dry-land condition as compared to irrigated condition [36].
Path analysis on chickpea plants has showed that GN per plant and GW have most direct effect contributions for GY under non-drought stress condition [14,27], while SDM and HI were those of in drought stress condition [2,21].
Drought tolerance selection has been made as a complicated procedure because of the

Field location and experimental materials
The field experiment was conducted in the research field of the Department of Agronomy

Experimental Design implementation and data collection
The experiment was laid out in a randomized complete block Design with two replications.
Each plot included 1-meter single row by 50 cm distances and 10 cm plant-to-plant spacing. Blocks were considered as environments, where the genotypes were randomly distributed in each of those, that the experiment had two environments include waterlimited and full irrigation conditions. The cultivation was carried out in the period of February to August 2014, and differential in plant furrow irrigation was started at 50% of the flowering stage, which stopped in the water-limited conditions (rain-fed condition), while continued through crop maturity followed a common irrigation regime of the region in the full water irrigation conditions. The measured phenological traits included days to 50% of flowering, days to 50% of podding, days from flowering to crop maturity, and days to maturity. Eight number of plants, excluding border plants, were harvested individually after the maturity to measure of the shoot dry matter (g plant-1), grain yield (g plant-1), 100-grain weight (g plant-1), and a number of grains (plant-1) under both conditions.
Harvest index (plant-1) was calculated from the formula: HI = grain weight/shoot dry matter. These harvested plants left out for shade drying about a week in separate paper bags before weighting. Drought tolerance indices were calculated based on grain yield in full irrigation and limited water irrigation treatments, represented in Table 1. The degree of stress intensity (SI) applied on plants under water-limited conditions achieved using the formula: SI = (1 -(Yp/Ys)), where, YP is the mean grain yields in full irrigated treatment and YS is the mean grain yields under limited water irrigation treatment.

Statistical model and data analysis
A visualized analysis of genotype main effect and genotype by environment interaction effects (GGE) was worked using GGEbiplot software version 6.0 to evaluate the yield stability in interaction by an environment of the tested chickpea genotypes followed Yan et al (200). Path analysis, a standardized partial regression coefficient was conducted to decompose correlation coefficients into components of direct and indirect effects as well as to examine the strength of the contribution of the various measured traits on grain yield. This purpose was followed using SmartPLS software (version 3.0, SmartPLS GmbH, Boenningstedt, Germany) with partial least square structural equation modeling (PLS-SEM) method developed by Wold (1982). To estimate variance components, minimum norm quadratic unbiased estimation (MINQUE) as a linear mixed model approach (Rao, 1971, Zhu, 189) was deployed in R software version 3.5.1 (R Development Core Team, 2018; www.R-project.org). As deviations from the population mean, in addition, genotypic effects in limited water conditions and full irrigation conditions as well as in a combined analysis were predicted separately by adjusted unbiased prediction method (Zhu, 1993). A significant test of interesting parameters (components of variance and genetic effects) was done according to a randomized 10-group Jackknife method to estimate standard errors (Wu, 2012). An R package named MINQUE performed the estimation of components of variance and prediction of genotypic effects by Wu (2012). These estimations were calculated using a linear mixed model for environmental, chickpea type, genotype, and the interaction of genotype by environment effects followed by equation (1): where Yijk is an observation; µ is a population mean; E i is an environmental effect; T k is a type of chickpea effect; G j is a genotypic effect; GE ij is a genotype by environment interaction effects; B l(i) is a block effects within environment; and e ijk is a random error.
In addition, each environments was analyzed independently in a completely randomized block Design with the linear mixed model followed by equation (2): where Y ij is an observation; µ is a population mean; B i is a block effect; G j is a genotypic effect; and e ij is a random error.

Variance components
Estimated variance components, which expressed as the proportions of the phenotypic variances for limited and full irrigation conditions are summarized in Tables 5 and 6, respectively. The estimated genotypic effect variances were detected highly significant (P ≤ 0.001) for GY, GN, GW, and SDM in Kabuli chickpea genotypes under water-limited treatment. Nevertheless, there were highly significant (P ≤ 0.001) effects of genotypic variances in those of GN, GW, and SDM as well as a significant effect (P ≤ 0.05) for GY in Desi chickpea genotypes at the limited water condition. A moderate significant (P ≤ 0.01) effect of genotypic variance was observed for GY in full irrigation treatment; also, the genotypic variances had highly significant (P ≤ 0.001) effects for GN, GW, and SDM in Kabuli chickpeas. The Desi type of chickpea genotypes showed highly significant effects (P ≤ 0.001) for variances genotypic effects for all the aforementioned traits in full irrigation treatment. The estimated variances of genotypic effects for both chickpea types were larger than residual effects except for that of GY in limited and full irrigation conditions. In a combined model analysis, the estimated environmental variance had the largest values 9 for GY, GN, and SDM with highly significant effects (P ≤ 0.001). The estimated variances for chickpea types have highly significant effects (P ≤ 0.001) which were observed for GY, GW, and SDM, respectively. The estimated genotypic variances for GN, GW, and SDM were highly significant (P ≤ 0.001). Nevertheless, highly significant effects (P ≤ 0.001) for GY, GN, and SDM as well as moderate significant effect (P ≤ 0.01) for GW were estimated as genotype by environment variances represented in Table 7.

Predicted genotypic effects
The predicted genotypic effects of chickpea genotypes tested under limited water and full irrigation treatments for GY, GN, GW, and SDM are given in Tables 8 and 9 The phenotypic correlations calculated between traits and drought tolerance indices are presented in Tables 3 and 4  Terminal water stress decreased GW of Kabuli genotypes by 2.82%, while, an increase of 2.22% was observed for GW in Desi chickpeas. Behboudian et al. (2001) found that GW in Desi chickpea had been not decreased under terminal water stress, which reason of this result was assumed as an accumulation of soluble sugars, amino acids, and proteins in chickpea grains. However, it seems that indirect selection for more drought-tolerant chickpeas could be performed through GW because of the greater correlation between drought and non-drought conditions than other attributes (Figure 1). The results of the variance component analysis also confirmed that GW has not been influenced by environmental factors (Table 7). Noor et al. (2003) found additive gene effects on GW of chickpea-based on high heritability with a high genetic advance in rain-fed conditions. Despite observations in Desi type, three indices including GMP, HM, and SNPI showed a highly positive significant correlation with GW in Kabuli type under drought stress condition. The GMP and HM also had a high correlation with GW at the non-stress conditions ( Table 3). Being additive in effect of genes on GW is a sign of additively gene controlling, hence it could be used through simple and direct selection, which in turn, these indices could provide a promising direction while genetically improvement in Kabuli chickpea yield is followed under rain-fed conditions.
A relationship significant in 0.01 probability level confirmed that 33% of GN in Desi type yielded under water stress condition could be explained by the inherent potential of the crop ( Figure 1). However, this relationship was not observed in the Kabuli genotypes. In Desi and Kabuli chickpea genotypes, terminal drought decreased up to 44.71% and up to 50.10% of GN, respectively ( Table 2). GN of tested genotypes was influenced by water limitation more than other attributes (Table 7). STI and HM indices were the best indicators to select genotypes having more GN in Kabuli and Desi chickpeas, respectively (Tables 3 and 4). GN had a positive effect on GY at both chickpea types and conditions, which was in agreement with Pushpavalli et al. (2014). The HI of Kabuli and Desi chickpeas were reduced by 12.25% and 9.74%, respectively, due to the terminal water stress ( Table 2). Nayyar et al. (2006) also found greater decreasing of HI in Kabuli than Desi chickpeas under terminal drought and stated that presence of relatively higher vegetative dry matter at maturity in Kabuli than Desi genotypes may be a sign of its lower ability to remobilize the assimilates towards developing pods and grains. Plants with higher HI shown better partitioning of photosynthetic assimilate to grain development 19 under water stress conditions . This ability should help the crop in improving its stability of performance under different climatic conditions compared to selection based solely on grain yield (Rehman et al, 2011). The research conducted on Kabuli chickpea showed that precipitation in the last two months of plant growth time has been caused an increasing HI from 0.5% to 0.34% as compared to lack of rain at the same time in sensitive genotype (ILC 3279), whereas non-significant difference was observed in tolerant genotype (ILC 588) (Rehman et al, 2011). The most contribution of HI belonged to GN in both chickpea types either in stress and non-stress conditions (Figures 3, 4, 5, and 6). Plants that combine high values of canopy biomass with greater mobilization of photosynthates to grains have been assumed as drought tolerant. Although HI in Kabuli genotypes did not affect directly by SDM, in Desi genotypes the SDM had a positive effect on HI, which could be evidence of the photosynthates mobilization to grains. On the other, at both chickpea types, there was not any correlation between HI and the drought tolerance indices in water-limited conditions. However, some of the indices such as GMP, MP, ATI, TOL, and K1STI in Kabuli type as well as TOL and ATI in Desi showed positive and significant correlations with HI under full irrigation conditions. According to these results, it seems that the improvement of HI in chickpeas grown under optimal water condition is a straighter approach than selection under terminal water stress.
Considering non-significant estimated genotypic variance of GY in the combined analysis, which could be due to the complexity of involved mechanisms, it seems that indirect selection through each of GW, SDM, and GN could result to more repeatable outcomes (Table 7). Breeding for drought tolerance by selection based on GY solely is difficult, because low heritability of GY under drought conditions, which is due to small genotypic variance or large genotype by environment interaction variances (Ludlow and Muchow, 1990). Environmental factors highly influenced the genetic structure and phenotypic expression of a quantitative trait such as GY, thus genotype by environment interactions is a major barrier for understanding that of inheritance (Breese, 1969). The contribution of genotypic variances as equivalent to the heritability of GY, GN, GW, and SDM in Desi chickpeas was greater in full irrigation condition than water-limited condition. Hence, it could be said that selection without terminal drought conditions will lead to more repeatable results than selection under terminal water stress. In Kabuli chickpeas, however, the greater genotypic variances were detected for GN and SDM in full irrigation condition and for GY and GW were observed under limited-water condition. Therefore, according to the objectives of the selection, doing this selection under conditions with greater genotypic variances dedicated to each trait is better. Hence, as Desi chickpea genotypes 8, 10, 47, and 321 showed significant positive predicted genotypic effects under optimal conditions for the selection, involving these genotypes in multi-parent recombination crosses could be resulting in increased efficient performance. In Kabuli chickpeas, the genotypes 101, 21, 15, 25, and 166 were detected as those of better ones with significant positive predicted genotypic effects.
According to Yan and Kang (2002), an ideal genotype should have a high yield mean among stress and non-stress environments as well as show high stable performance. Rad et al. (2013) stated that the ideal genotype could be found in the center of the concentric circles of AEC method analysis. As AEC abscissa direction towards a more stable grain yield, as shown in Figure 2, the ideal chickpea genotypes have been presented closely to the location of a limited water environment as well as the average environment. As a result, which found consistent with Golabadi et al. (2006) in durum wheat concluded that for high stable grain yield, selection of chickpea in moister-stress environments as well as based on the average of drought stress and non-drought stress conditions could be more advantageous compared with indirect selection only at the non-drought stress conditions.

Conclusion
Results of this study showed that tested chickpea genotypes responded differently under different water treatments, suggesting the importance of assessment of genotypes under these conditions in order to identify the best genotype make up for each particular condition. As water stress severity was applied equally, therefore it was thought to be more serious in genotypes with a greater life cycle. However, it seems that chickpea plants have been adapted to the terminal drought stress, which could be due to the same time of vegetative growth with filling pods and transfer capability of photosynthesis assimilates towards more grain yield in tolerant genotypes. It seems to change in plant phenology due to the terminal drought stress more affected GN and GW in Desi and Kabuli chickpeas, respectively. These differences could be clear points for the leadership of breeding programs towards more adaptation of both Desi and Kabuli chickpea types to terminal water stress, respectively. Moderate to a high proportion of G × E effects were observed in combined analysis for GY, GN, and SDM compared to genotypic effects, suggesting that G × E effects played a greater role than genotypic effects. The ideal genotype of Kabuli type i.e. genotype 25 had greater GY as well as SDM in water-limited condition, while genotype 321 as ideal Desi genotype showed acceptable GY and SDM, but could compensate with higher GN.