Screening the FIGS Set of Lentil (Lens culinaris Medikus) Germplasm for Tolerance to Terminal Heat and Combined Drought-Heat Stress

Lentil (Lens culinaris Medikus) is one of the most important cool season food legume crops grown in many countries. Seeds are typically rich in protein, fiber, prebiotic carbohydrates and minerals, such as iron and zinc. With changing climate and variability, the lentil crop faces frequent droughts and heat stress of varying intensity in its major production zones. In the present study, a set of 162 lentil accessions selected through the Focused Identification of Germplasm Strategy (FIGS) were screened for tolerance to heat stress and combined heat-drought stresses under field conditions at two contrasting locations, namely Marchouch and Tessaout in Morocco. The results showed a significant genotypic variation for heat tolerance and combined heat-drought tolerance among the accessions at both locations. Based on the heat tolerance index (HTI), accessions, namely ILL 7833, ILL 6338 and ILL 6104, were selected as potential sources of heat tolerance at Marchouch, and ILL 7814 and ILL 8029 at Tessaout. Using the stress tolerance index (STI), ILL 7835, ILL 6075 and ILL 6362 were identified as the most tolerant lines (STI > 1) at Marchouch, and ILL 7814, ILL 7835 and ILL 7804 (STI > 1) at Tessaout, under the combined heat-drought stress conditions. Accession ILL 7835 was identified as a good source of stable tolerance to heat stress and combined heat-drought stress at both locations.


Introduction
Lentil (Lens culinaris Medikus) is an annual, diploid (2n = 14) and self-pollinated crop. Its seeds are rich in protein (22-35%), fiber, prebiotic carbohydrates and minerals, such as iron and zinc [1]. It plays a major role in alleviating malnutrition and micronutrient deficiencies of people living in Central

Plant Material and Study Area
A FIGS set comprising 162 germplasm accessions of lentil developed by the ICARDA gene bank in 2013 was evaluated. These accessions were originated from Pakistan (66), Nepal (68), Ethiopia (13), India (4), Yemen (3), Russia (3), Sudan (2) and Iran (2). The FIGS set, along with the Moroccan cultivar, Bakria as a local check, was evaluated in an alpha lattice design, with two replicates at two locations: Tessaout (31.42  In each row, seeds were sown by hand at a 2 cm depth maintaining a 10 cm space between plants, and the total plot size maintained was 0.6 m 2 . In general, the Tessaout research station represents a typical Mediterranean semi-arid environment, characterized by a hot dry summer with an annual rainfall of 266 mm [48]. The Marchouch station also represents a Mediterranean semi-arid environment, but with higher annual precipitation (400 mm) [49]. The experimental site in Tessaout is silty-clay soil, whereas in Marchouch, it is vertisol.

Treatments
At each of the two locations, three experiments involving the same set of FIGS germplasm were conducted by manipulating the planting date and water supply, in order to impose heat and water stress at the reproductive phase of the plant growth. These three experiments were considered to represent three treatments, namely the normal date of planting (treatment A), late planting with irrigation at field capacity throughout the crop period (treatment B) and late planting without irrigation during the reproductive phase (treatment C), which were referred to as treatments. Treatment A resulted in optimal growing conditions (>150 mm well-distributed rainfall and below 27 • C temperature) without any heat and water stress to the plants. Treatment B (planted 50 days after normal planting date with irrigation at field capacity throughout the crop duration) imposed heat stress, as the plants were exposed under field conditions to a temperature above 32 • C during the reproductive phase (Table A1), while regular irrigation at field capacity avoided any water stress to the plants. Treatment C (planted 50 days after normal planting date without irrigation during the reproductive phase) imposed a combined heat and water stress. Treatment A was sown on 20 December, and no irrigation was applied during the crop period as the crop received well distributed enough rainfall at Tessaout (157.9 mm) and Marchouch (167.8 mm). Treatments B and C were planted on 8 February. Irrigation was applied to maintain water supply at field capacity using sprinkler system throughout the crop duration in treatment B, whereas irrigation was stopped from the flowering initiation stage onward in treatment C, to impose water stress in addition to the heat stress. All the three treatments were kept weed-free throughout the growing season. A wide temperature variation was recorded between normal and late planted treatments at the Tessaout and Marchouch research stations. During the reproductive stage, the averages of the maximum and minimum temperatures were 26.33 • C and 12.72 • C in Treatment A. However, the maximum temperatures during the flowering stage reached the threshold level of 42 • C in treatments B and C at Tessaout and 34 • C at Marchouch (Figure 1). Therefore, late planting with irrigation at field capacity was successful in imposing heat stress during the reproductive phase of test genotypes in treatment B, and late planting without irrigation in imposing drought and heat stress in treatment C.
Agronomy 2020, 10, x 4 of 28 during the flowering stage reached the threshold level of 42 °C in treatments B and C at Tessaout and 34 °C at Marchouch (Figure 1). Therefore, late planting with irrigation at field capacity was successful in imposing heat stress during the reproductive phase of test genotypes in treatment B, and late planting without irrigation in imposing drought and heat stress in treatment C.

Investigation and Calculation of Agronomic Traits
Data were recorded for the phenological traits (days to 50% flowering and maturity) on a plot basis, whereas five plants were selected randomly from each plot for the assessment of the morphological (numbers of primary, secondary and tertiary branches, and plant height) and yield (total numbers of filled and unfilled pods, biological yield, grain yield and 100-seed weight) traits, following the lentil ontology [50]. The heat tolerance index (HTI) was calculated for each genotype, following the multiple regression approach, as suggested by Bidinger et al. [51], and as used in chickpea [52]. Grain yield under stressed and non-stressed conditions was used to assess the tolerance of genotypes against the stress. This approach considers grain yield under heat stress conditions (Ys) to be a function of yield potential (Yp), days to 50% flowering (F) and heat tolerance index (HTI), such that the yield of a genotype can be expressed as Ysi = a + bYp + cFi + HTIi + E, where E is the random error with zero mean and variance σ, and a, b, c are regression parameters estimated by least

Investigation and Calculation of Agronomic Traits
Data were recorded for the phenological traits (days to 50% flowering and maturity) on a plot basis, whereas five plants were selected randomly from each plot for the assessment of the morphological (numbers of primary, secondary and tertiary branches, and plant height) and yield (total numbers of filled and unfilled pods, biological yield, grain yield and 100-seed weight) traits, following the lentil ontology [50]. The heat tolerance index (HTI) was calculated for each genotype, following the multiple regression approach, as suggested by Bidinger et al. [51], and as used in chickpea [52]. Grain yield under stressed and non-stressed conditions was used to assess the tolerance of genotypes against the stress. This approach considers grain yield under heat stress conditions (Ys) to be a function of yield potential (Yp), days to 50% flowering (F) and heat tolerance index (HTI), such that the yield of a genotype can be expressed as Ysi = a + bYp + cFi + HTIi + E, where E is the random error with zero mean and variance σ, and a, b, c are regression parameters estimated by least square methods. The heat tolerance index (HTI) was calculated for each accession as the difference between the estimated late-season grain yield and the estimated optimal-season grain yield plus standardized residuals from regression.
On the basis of grain yield under stress (YS) and normal conditions (YP), the following quantitative tolerance indices were estimated to assess the combined effect of drought and heat stresses: [54]; geometric mean productivity GMP = Y pi * Y si [53]; mean productivity MP = Y pi + Y si 2 [54]; and harmonic [55] where, Ysi = yield of a genotype under stress condition, Ypi = yield of a genotype under normal sown condition, Ys = overall genotypic mean under stress condition, and Yp = overall genotypic mean under normal condition.

Statistical Analysis
Analysis of variance was performed using the general linear model (GLM) using IBM SPSS statistics 23. Treatment means were compared by least significant difference (LSD). Correlation coefficients (Pearson's) were calculated by multivariate analysis for heat stress condition, while Spearman's correlation coefficient was used for the combined heat-drought stress. Hierarchical cluster analysis using Ward's squared Euclidean distance method was performed for genotype grouping.

Effects of Heat Stress on Morphological, Phenological, and Yield Contributing Traits
The analysis of variance (ANOVA) revealed significant differences among genotypes for all traits under stressed and non-stressed conditions at both the locations, Tessaout (Table 1) and Marchouch (Table 2). Furthermore, a highly significant variation (p < 0.001%) was observed for all traits among treatments in Tessaout, as well as at Marchouch. The analysis also showed significant genotype x treatment interactions for all the traits at both locations, except for plant height, number of primary branches per plant, number of tertiary branches per plant and biomass yield per plant at Tessaout.
There existed a wide range of variability among lentil genotypes for phenological traits at both Tessaout (Table 1) and Marchouch (Table 2). Days to 50% flowering in normal planting conditions ranged from 61 to 78 days at Tessaout, and from 79 to 98 days at Marchouch. The range of days to 95% maturity varied from 100 to 119 days at Tessaout, and from 113 to 126 days at Marchouch under normal planting conditions. Heat stress decreased the crop duration by 23% at Tessaout, and 26% at Marchouch, while the combined heat-drought stress caused 28% reduction in crop duration at Tessaout and 27% at Marchouch. At each location, days to 50% of flowering was almost similar under both stress conditions: 46 days at Tessaout and 55 days at Marchouch (Table A1).
Under normal planting, plant height ranged from 17 to 37 cm, with the overall mean of 25 cm at Tessaout and from 14 to 52 cm, with a mean of 31.51 cm at Marchouch. Heat stress reduced plant height by 18% at Tessaout and by 31% at Marchouch. However, the combined heat-drought stress reduced plant height more than the heat stress: 29% at Tessaout and 35% at Marchouch (Table A1).
Heat stress and combined heat-drought stress reduced the number of primary, secondary and tertiary branches per plant significantly. The reduction was more pronounced under combined stress conditions at both locations, particularly at Marchouch. The number of primary branches per plant reduced by 24% at Tessaout and 30% at Marchouch. Likewise, the number of secondary and tertiary branches per plant were reduced by 38% and 63% at Tessaout, whereas in Marchouch, there was an 80% reduction in the number of secondary branches per plant, and a 91% reduction in the number of tertiary branches per plant (Table A1).
Under normal planting, the number of total pods per plant ranged from six to 160 at Tessaout, which decreased by 47% under heat stress and 62% under combined heat-drought stress conditions. In contrast, the number of total pods per plant ranged from three to 230 at Marchouch but declined by 72% due to heat stress and 91% as a result of combined heat-drought stress. Likewise, heat stress and combined heat-drought stresses reduced number of filled pods by 58% and 65% at Tessaout, while it was 69% and 91% at Marchouch, compared to normal planting. The mean numbers of total pods and filled pods per plant were higher under stress conditions at Tessaout than at Marchouch (Table A1).
Biomass per plant ranged from 1.4 to 39.6 g at Tessaout and from 1.07 to 36.2 g at Marchouch under normal planting conditions. The heat stress reduced biomass by 69% at Tessaout and 77% at Marchouch. However, the reduction in biomass was higher under combined stress at both locations: it was recorded 71% at Tessaout and 85% at Marchouch. Similarly, the combined heat-drought stress decreased the seed yield by 76% at Tessaout and 90% at Marchouch, more than the heat stress alone (68% at Tessaout; 71% at Marchouch). The mean grain yields under heat stress and combined heat-drought stress were more at Tessaout than at Marchouch. Under the normal planting, the hundred-seed weight ranged from 0.50 to 3.70 g at Tessaout and 0.50 to 3.85 g at Marchouch. The combined heat-drought stress increased the hundred-seed weight by 21% at Tessaout and 10% at Marchouch. However, the increase was higher under heat stress by 38% at Tessaout and 27% at Marchouch (Table A1).

Correlations among the Traits under Heat Stress and Combined Heat-Drought Stress
The correlations between the variables under normal planting showed highly significant positive correlation at 0.01 level of grain yield with the number of the secondary and tertiary branches per plant and number of filled pods per plant at Tessaout and Marchouch (Tables A2 and A3). A highly positive correlation (p < 0.01%) was also noticed between the grain yield and biomass at Marchouch. However, grain yield was negatively correlated with days to 50% flowering at Marchouch. Days to 50% flowering were positively correlated with plant height, days to 95% maturity, number of secondary and tertiary branches and hundred-seed weight at Tessaout (Table A2). Nevertheless, it was positively correlated only with plant height and days of 95% of maturity at Marchouch under normal planting (Table A3).
Under heat stress, grain yield was positively correlated (p < 0.01%) with the number of the secondary and tertiary branches per plant, the number of total pods and biomass at both stations, Tessaout and Marchouch. Furthermore, grain yield had a positive correlation with days to 95% maturity at only Tessaout. A highly positive correlation of 0.01% was identified among plant height, days to 50% flowering, days to 95% maturity, biomass and hundred-seed weight in heat stress conditions at both stations. In combined heat-drought stress conditions, grain yield was positively correlated with all variables at both stations, except with days to 50% flowering at Marchouch. Additionally, positive associations (p < 0.01%) existed among days to 50% flowering, days to 95% maturity and hundred-seed weight at Tessaout and Marchouch (Tables A2 and A3).

Classification of Genotypes Based on Heat Tolerance Index
Germplasm accessions were classified into representative groups based on the heat tolerance index (HTI). Hierarchical cluster analysis using Ward's incremental squared Euclidean distance method resulted in five clusters. These genotypic clusters differed significantly in HTI and defined as highly heat tolerant (HTI >1), heat tolerant (HTI means 0.68 in Marchouch and 0.66 in Tessaout), moderately heat tolerant (0.25 and 0.10), heat sensitive (−0.08 and −0.25) and highly heat sensitive (−0.37 and −0.55).
Based on the HTI, four accessions (ILL 7833, ILL 6338, ILL 7835 and ILL 6104) showed good performance under heat stress at Marchouch and three accessions (ILL 7835, ILL 7814 and ILL 8029) as highly heat tolerant at Tessaout. ILL 7835 emerged as heat tolerant at both the locations, showing its stable performance under heat stress. These accessions were characterized by a short phenological cycle with 53.5 days to achieve 50% flowering and about 86 days to 95% maturity at Marchouch, however, the duration was less at Tessaout with 45 days to 50% of flowering and 82 days to 95% maturity (Table 3). Additionally, 14 and 26 accessions were categorized as heat tolerant at Marchouch and Tessaout (Table A4), respectively. The moderately-heat tolerant group comprised of 26 accessions at Marchouch and 60 accessions at Tessaout. In this study, heat sensitive and highly heat-sensitive clusters were comprised of 76 and 42 accessions, respectively, at Marchouch. Whereas, it was 39 and 34 accessions at Tessaout (Table A5).

Response to Combined Heat-Drought Stress
Based on the stress tolerance index (STI) and the geometric mean productivity (GMP), genotype ILL 7835 was identified as tolerant to heat and drought at Marchouch and three accessions, ILL 7814, ILL 7835 and ILL 7804 at Tessaout. Genotype ILL 7835 showed tolerance to drought and heat stresses at both locations. The three highly tolerant genotypes at Tessaout achieved 50% flowering in around 43 days, while 50% flowering was achieved in about 60 days for the ILL 7835 at Marchouch. Furthermore, these genotypes reached 95% maturity in 83 days at Tessaout and in 90 days at Marchouch (Table 4). Using Spearman's correlation coefficient, the GMP was strongly correlated to STI (r = 1; p < 0.01) in Tessaout and Marchouch. Furthermore, highly positive correlation (p < 0.01) was recorded between yield potential (Yp) and yield under stress condition (Ys) (p < 0.05) at both stations. The Ys was positively correlated with the Yp, STI, GMP, MP and HARM indices, and negatively correlated with stress tolerance (TOL) at Tessaout and Marchouch (Table 5). Additionally, a non-significant correlation was observed between Ys and TOL under combined heat-drought stress in Marchouch (Table 5).

Discussion
The present study demonstrated delayed sowing as an effective approach to screening lentil germplasm for heat tolerance. A similar methodology was used to assess heat tolerance responses in chickpea [30,31], lentil [19,56] and mung bean [57,58] successfully. High temperature and low vapor pressure deficits (VPD) decrease soil moisture and increase transpiration rate, resulting in combined heat and drought stress [59]. Hence, frequent irrigation must be provided to remove the confounding effects of drought stress, to assess the effect of heat stress. Even the photoperiod may change during late planting, and previous studies by Summerfield et al. [60] and Erskine et al. [61] reported that temperature had a much bigger effect on the flowering time than the photoperiod in lentil.
Our findings showed that heat stress adversely affected plant height, number of branches and pods, grain yield and biomass, which agrees with several investigations in chickpea [62,63], lentil [12] and faba bean [64]. However, the influence of the combined heat-drought stress was more severe when compared to heat stress only, due to the reduction in water use efficiency. Heat and combined heat-drought stresses shortened vegetative and reproductive periods by accelerating the rate of plant growth and development. Similar findings were reported in previous studies [65,66]. The combined heat-drought stress largely decreased the number of pods more than the individual heat stress, leading to severe yield losses and biomass. This is in agreement with the previous research of Sehgal et al. [18,19], which has also shown the impact of combined heat-drought stress in lentil.
The days to 50% flowering under stressed conditions have shown quite similar results in both locations: approximately 46 days at Tessaout and 54 days at Marchouch. Lentil responded in the same way to heat stress and combined heat-drought stress by forcing early flowering (Figure 1) in both locations. Awasthi et al. [22] reported a very similar response in days to 50% flowering eventually, accelerated markedly in chickpea genotypes under heat and combined heat-drought stresses environment.
Overall, grain yield decreased under stress conditions, with a more pronounced effect under the combined heat-drought stress condition. High temperature stress led to yield loss by altering pollen and stigma development, pollination and pod set, while drought directly influenced the seed filling stage [19,67,68]. Moreover, yield is always influenced by photosynthetic ability and the performance of reproductive organs under stressed conditions [69]. In this study, the number of primary, secondary and tertiary branches were significantly positively correlated with seed yield (p < 0.01) under stressed environments, at both stations, except heat stress of Tessaout, which was not correlated with seed yield (Tables A2 and A3). Previous studies agreed with our findings and demonstrated a positive direct effect of primary branches on seed yield in lentil [70,71]. Recently, Ahmadi et al. [72] suggested that if lentil genotypes have a greater number of branches-mainly secondary branches-the final potential would increase the plant yield under stress conditions. It appears that plant biomass was low, mostly due to combined heat-drought stress that demands high water use efficiency. Further, heat stress, in combination with drought, increases leaf temperature and decreased net photosynthetic rate and stomatal conductance, resulting in a more damaging impact under the combined stress condition [73]. Overall, biomass reduction largely affected the number of total pods and seed yield per plant [74]. It was also positively correlated with number of total pods per plant, the numbers of primary, secondary and tertiary branches, and seed yield under heat stress and combined heat-drought stress at Tessaout and Marchouch (Table 5). The present study showed increased 100-seed weight under heat stress and the combined heat-drought stress condition, due to the reduction of seed numbers per plant. Similar results were reported by Chakherchaman et al. [75] in lentil under drought stress.
In the present study, three accessions, namely, ILL7833, ILL6338 and ILL6104, were selected as potential sources of heat tolerance at Marchouch and two accessions (ILL7814 an ILL8029) at Tessaout. Tolerant accessions produced significantly more pods under heat stress, as compared to heat sensitive genotypes. Under the combined stress of heat and drought, the most tolerant lines were ILL 7835, ILL 6075 and ILL6362 (STI >1) at Marchouch and ILL7814, ILL7835 and 7804 (STI >1) at Tessaout. Altogether, ILL7835 was identified as a good source of tolerance under heat stress and combined heat-drought stress at Tessaout, as well as at Marchouch, while ILL7814 was identified as promising accession under both stresses at Tessaout. These selected lines are characterized by early flowering, which helped them to escape from heat and drought stress and shorter crop cycles (Tables 3 and 4). Usually, early flowering and maturity are the stress escape mechanism that can help lentil to perform well under stress conditions, and the development of genotypes with short duration is one of the major strategies used in breeding programs [76]. Although, early duration is considered to be the best adapted trait for chickpea genotypes to Mediterranean (spring sown) and south Indian environments [77,78]. Furthermore, the identified lines had a greater number of filled pods per plant, representing an excellent source for the lentil breeding program, and can be directly used in the crossing program to transfer heat and drought tolerance in a high yielding genetic background.
The selection of superior germplasm against combined stress was carried out by using a stress tolerance index (STI) and geometric mean productivity (GMP). STI and GMP have been used earlier for screening genotypes of chickpea [37,79,80], lentil [81,82], bread wheat [83], barley [84,85], fenugreek [86], oat [87] and maize [88,89] against abiotic stresses. Higher GMP and STI values indicate more tolerance to drought stress [90]. The stress tolerance index (STI) showed a highly positive correlation with both yield under stress conditions (Ys) and yield potential (Yp), GMP, MP and HARM at both locations. These results are in conformity with those of Ganjali et al. [91] in chickpea under stress and non-stress conditions. However, stress tolerance (TOL) was negatively correlated with Ys at Tessaout and non-significantly correlated at Marchouch, while it was positively correlated with potential yield at both locations. Similar results were reported also by Chakherchaman et al. [75] and Rad et al. [81] for lentil under drought stress conditions. Majidi et al. [92] also reported non-significant correlation between TOL and Ys, and a highly significant correlation between TOL and Yp, confirming that selection based on TOL should decrease yield in the moisture stress environment and increase grain yield under normal conditions. The limitations of using the TOL index have been described previously in several studies [35,93]. In general, our findings confirmed the effectiveness of using STI and GMP as reliable selection criteria for terminal heat or drought tolerance, as reported earlier by Fernandez [53], Farshadfar et al. [94] and Talebi et al. [95].

Conclusions
The present study shows that heat, as well as combined heat-drought stress, severely affects flower production and pod set, leading to a substantial loss in grain yield in lentil. The field screening technique has been demonstrated as an effective way for evaluating and selecting promising lines under heat or drought stress. The adaptation by selecting the robust genotypes can be a strong approach to mitigating the impact of climate change. Our study suggests that the FIGS approach in identifying heat tolerant germplasm in lentil is a successful approach under heat and combined heat-drought stress. This research identified a group of highly heat tolerant genotypes using HTI based on flowering time, and grain yield under stress and non-stress conditions. Our findings confirmed that STI, GMP and MP are the most suitable criteria, not only for selecting the high yielding genotypes under individual heat and drought stresses, but also in combined heat-drought stress. These lines would be an excellent source of stress tolerance to increase the adaptation of lentil under climate change, a major issue that currently affects agriculture.