Analyzing weather data during the two growing seasons indicates that January and February were the coldest months (Supplementary Table S1). During this time, all field trials were exposed to temperatures less than 5 ℃ indicating that the tested genotypes were exposed to vernalization conditions. For rainfall amounts, RB station received ~10% (33.5 mm) less precipitation than the long-term rainfall average during the first growing season, while JU and JUST stations received 11.5% (58 mm) and 40% (86.5 mm) more, respectively (Supplementary Table S1). During the second growing season, all locations received more rainfall than the long-term average where RB received 3% (11 mm), JU 6% (28 mm) and JUST 10% (20.6 mm) above the long-term average. The rainy season in 2012 was terminated in April in all stations and was accompanied by high temperatures at the end of the season (Supplementary Table S1). In 2013, the rainy season was terminated earlier than expected in RB in March, while only 7.8 mm were received only in JUST that resulted in severe terminal drought conditions. The amounts of water provided by supplementary irrigation for irrigated plots are given in Supplementary Table S1.
The combined ANOVA for the studied traits showed highly significant differences (P<0.001) between genotypes, locations, water regimes, sowing dates, and their interactions (Supplementary Table S2). The mean values for the studied traits across the six environments and their combinations with the sowing date and water regime are given in Supplementary Table S3. For correlation analysis, positive high significant correlations (P<0.001) were found between all traits where GY showed a strong correlation with yield component-related traits and with HD, MD and GFP (Figure S1).
For GY, the percentage of variance explained by main factors varied where genotypic effect attributed 12.95%, environment (location-year) attributed 12.54%, water regime attributed 42.75% and sowing date attributed 23.78% from the total variance. Mean values of GY differed significantly between genotypes and ranged from 585 g/m2 in Rum to 367 g/m2 in Steptoe. For the sowing date, GY mean values differed significantly with December produced the highest GY mean value (605 g/m2) compared to February (341 g/m2). The GY mean values of water regime treatment differed significantly with the irrigation treatment produced 650 g/m2 compared to rainfed 296 g/m2. In addition, the interaction between sowing date, water regime and genotype on GY was highly significant (P<0.001; Supplementary Table S2). Rum produced the highest mean value of GY (889 g/m2) under irrigated conditions and December sowing date, while Steptoe produced the lowest mean value of GY (73 g/m2) under rainfed conditions and February sowing date where Rum produced 188 g/m2 (LSD (0.05) = 13.9). The interaction effect between environments, water regime, sowing date and genotypes on the GY showed highly significant differences (P<0.001) (Supplementary Table S1; Supplementary Table S3; Figure 1A). For instance, Steptoe produced the lowest mean value (50 g/m2) for GY under rainfed conditions in JUST-2013 for the December sowing date, while Rum under the same conditions produced 388 g/m2 (LSD (0.05) = 42.2) (Supplementary Table S3). In JUST-2013, no significant differences were found between GY mean values of all tested genotypes under rainfed treatment for the February sowing date, however, Steptoe produced significantly the lowest GY mean values in all remaining environments (Supplementary Table S3). Under irrigated conditions, mean values of GY differed significantly between genotypes where Rum and Acsad176 produced the highest mean values for GY across all environments irrespective of sowing date treatment except when compared to Steptoe sown in December in JU2012 (Supplementary Table S3).
For HD, the percentage of variance explained by main factors varied where genotypic effect attributed 9.03%, environment (location-year) attributed 5.05%, water regime attributed 0.47% and sowing date attributed 82.31% from the total variance. Heading date mean values of tested genotypes ranged from 90.2 days in Rum (significantly the earliest genotype to flower) to the significantly latest mean value (107.6 days) recorded in Steptoe. For the main effect of the sowing date, December produced significantly the highest HD mean value (116.7 days) when compared to February (75.7 days). For the water regime effect, the irrigation treatment produced significantly the highest HD mean value (97.8 days) when compared to rainfed (94.7 days). The interaction effect between sowing date and water regime on HD was significant (P<0.05), and this indicates that the effect of water availability depended on the sowing date (Supplementary Table S2). The interaction effect between environments, water regime, sowing date and genotype on the HD showed a significant effect (P<0.001) (Supplementary Table S2; Supplementary Table S3; Figure 1B). Steptoe produced the highest mean values for HD in any given environment × water regime combinations for December sowing date treatment (Figure 1B; Supplementary Table S3). For February sowing date treatment, clear significant differences were identified only in 2013 environments (JU-2013, RB-2013 and JUST-2013) between the HD mean values of Steptoe and other genotypes irrespective of water regime treatments (Supplementary Table S3).
To analyze genotype-specific adaptation to specific environments for the GY trait, the GGE biplot was used. For this purpose, environments were reclassified based on the combination of each location, year, sowing date and water regime treatments to produce 24 distinct environmental groups. The relationships between the GY of tested genotypes and the 24 environmental groups and the degree by which each group is represented are shown in Figure 2A. The two principal components (PC1 and PC2) together captured 98.32% of the interaction effects and the variations due to GGE. Three irrigated environments from the 2013 season fell in the sector in which Acsad176 cultivar was the vertex genotype, which means that Acsad176 was the best genotype in these environments (Figure 2A). On the other hand, Rum was the best cultivar in the rest of the environments (21 out of 24) and specifically in all rainfed environments irrespective of location, sowing date, or season. By contrast, Morex and Steptoe did not perform well in all testing environments and the two genotypes were considered the poorest across all tested environments (Figure 2A). Interestingly, both genotypes were placed on different sectors indicating different responses to tested environments. For stability as measured by projection to the Average-Tester Axis y-axis, Rum was the most stable followed by Acsad176, while Steptoe and Morex were considered the least stable genotypes (Figure 2B).
Based on the results above and to analyze the relationship between HD and agronomic performance, a new correlation analysis was performed using the field data of Rum and Steptoe alone after reclassifying the environments into four different groups based on the combination of sowing dates and water regime treatments (December-irrigated, December-rainfed, February-irrigated and February-rainfed). As shown in Figure S2, negative significant correlations were found across the four environmental groups between HD with GY and SPW. For December-irrigated conditions, the negative significant correlations with HD included HI and TKW, while for December-rainfed conditions the negative significant correlations with HD included TW and STW. For the February sowing date and irrespective of water regime treatment, negative significant correlations were detected between HD with TW, SN, GN, GS, TKW and HI (Figure S2).
Controlled Conditions Experiment
In this study, the transition to the reproductive stage under LD was significantly more advanced in Rum genotype when compared to Steptoe irrespective of photoperiod, vernalization and water regime combinations (Figure 3; Figure S3). After four weeks under LD and well-watered conditions, a difference was observed between non-vernalized and vernalized Rum plants. On the other hand, Steptoe reproductive meristem was in an advanced developmental stage under LD and well-watered conditions when compared to stressed plants irrespective of vernalization treatment (Figure 3A; Figure S3). After eight weeks of LD incubation under well-watered conditions, non-vernalized Steptoe reproductive meristem was in an advanced developmental stage when compared to well-watered and vernalized Steptoe plants.
Under SD and irrespective of the vernalization and water regime treatments, the progression of reproductive meristem development in both genotypes was less advanced when compared to LD conditions (Figure 3; Figure S3). Significant differences were observed between the tested genotypes after eight weeks of incubation where stressed Rum plants subjected to vernalization showed a clear advanced developmental stage. Under the same conditions, stressed Steptoe plants showed a clear less advanced reproductive meristem when compared to other treated plants. After eight weeks of SD conditions and irrespective of water regime treatment, non-vernalized Rum plants showed a clear advanced developmental stage when compared to Steptoe (Figure 3B; Figure S3).
At the end of the experiment, the mean values of HD of Rum plants grown under LD conditions were significantly lower than the mean values of Steptoe (Figure S4). On the other hand, vernalized Rum plants showed a significantly earlier HD under LD conditions when compared to non-vernalized Rum plants. Under LD conditions and irrespective of vernalization treatment, a significant delay in heading was observed in stressed Steptoe plants when compared to well-watered plants (Figure S4). Under SD and vernalization conditions, stressed Rum plants were significantly the earliest to flower when compared to well-watered plants.
The effects of water stress on RWC, stomatal resistance, maximum quantum efficiency (Fv/Fm) of photosystem II was analyzed in treated plants as an index for drought tolerance in the two tested genotypes. Significant differences between well-watered and stressed in the two genotypes were observed for the tested physiological parameters under different photoperiod and vernalization conditions (Figure S5). For instance, the mean values of RWC of the tested genotypes were lower in stressed plants when compared with well-watered (Figure S5). Starting the 4th week, the mean value of RWC of non-vernalized Steptoe plants grown under LD and drought conditions was significantly the lowest. Clear effects of drought stress on stomatal resistance and maximum quantum efficiency (Fv/Fm) of photosystem II were also observed in both genotypes (Figure S5).
The expression patterns of Ppd-H1, Vrn-H1, Vrn-H3 and HVA22 genes were investigated in both tested genotypes in response to drought and vernalization treatments after two and four weeks for LD conditions and after four and six weeks for SD conditions. After two weeks of LD conditions and irrespective of water regime treatment, the Ppd-H1 expression in vernalized Rum plants showed the highest levels when compared to other treated plants (Figure 4). After four weeks of LD incubation and irrespective of water regime treatment, the expression levels of Ppd-H1 in vernalized Rum plants were lower compared to non-vernalized Steptoe and Rum plants (Figure 4). For SD conditions, no major changes in Ppd-H1 expression levels were observed between the treated genotypes (Figure 4).
After two weeks of LD conditions, the relative expression levels of Vrn-H1 were higher in vernalized plants when compared to non-vernalized for both tested genotypes (Figure 4). Irrespective of vernalization treatment, Rum showed higher Vrn-H1 expression after two weeks of LD incubation when compared to Steptoe. Irrespective of water regime treatment, Vrn-H1 expression levels after two weeks of LD incubation were significantly lower in non-vernalized Steptoe plants compared to vernalized plants (Figure 4). After four weeks of LD incubation, the same trend of high expression of Vrn-H1 was observed in vernalized plants when compared to non-vernalized plants (Figure 4). However, the expression levels of Vrn-H1 in vernalized and stressed Steptoe plants were significantly lower when compared to stressed Rum plants and when compared to well-watered Steptoe (Figure 4). After six weeks of SD conditions, the expression of Vrn-H1 was significantly higher in stressed and vernalized Rum plants when compared with other plants subjected to different treatment combinations. After eight weeks of SD conditions, Rum showed higher Vrn-H1 expression levels when compared to Steptoe (Figure 4)
After two weeks of LD conditions, the expression levels of Vrn-H3 were significantly higher in vernalized Rum plants when compared to other plants subjected to different treatment combinations (Figure 4). Under LD conditions, non-vernalized Rum plants showed significant differences of Vrn-H3 expression levels when compared to non-vernalized Steptoe. The relative expression levels of Vrn-H3 under LD conditions were higher in vernalized Steptoe plants when compared to non-vernalized plants (Figure 4). After four weeks of LD conditions, non-vernalized Rum plant showed higher Vrn-H3 expression when compared to other treated plants. On the other hand, well-watered and vernalized-Steptoe plants showed higher Vrn-H3 expression when compared to stressed and vernalized-Steptoe plants (Figure 4). Under SD conditions, stressed and vernalized Rum plants showed higher Vrn-H3 expression levels when compared to other plants subjected to other treatment combinations (Figure 4). Furthermore, the expression levels of Vrn-H3 in vernalized and well-watered Rum plants were also significantly higher when compared to Steptoe plants, irrespective of treatment combinations.
For HVA22 gene expression, no major changes were observed between the different growth conditions and treatments for both Rum and Steptoe where a clear increment in its expression level was observed under stress conditions (Figure 4).