Field trials
Analyzing weather data during the two growing seasons (2012 and 2013) in the three locations indicates that January and February were the coldest months (Additional file 1). 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 average of rainfall during the first growing season, while JU and JUST stations received 11.5% (58 mm) and 40% (86.5 mm) more than the long term average of rainfall, respectively (Additional file 1). 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 long-term average of rainfall. The rainy season in 2012 was terminated in April in all stations and was accompanied by high temperatures at the end of the season (Additional file 1). In 2013, the rainy season was terminated earlier than expected in Rabbah starting March, while only 7.8 mm were received in JUST that resulted in severe terminal drought conditions. The amounts of water provided by supplementary irrigation for irrigated plots are given in Additional file 1.
The combined ANOVA for the studied traits showed highly significant differences (P < 0.01) between genotypes, locations, water regimes, sowing dates, and their interactions (Additional file 2). The mean values for the studied traits across the six environments and their combinations with the sowing date and water regime are given in Additional file 3. For correlation analysis, positive significant correlations (P < 0.01) were found between all traits where GY showed a strong correlation with yield component-related traits and to less extent with HD, MD and GFP traits (Additional file 4).
The mean values of GY of tested genotypes (main effect of genotype) ranged from 585 g/m2 in Rum (significantly the highest genotype) to the significantly lowest mean value of 367 g/m2 in Steptoe. The main effect of sowing date treatment showed that December produced significantly the highest GY mean value (605 g/m2) compared with February (341 g/m2). The main effect of water regime treatment showed a significantly higher GY mean value for irrigated treatment (650 g/m2) when compared with rainfed (296 g/m2). The combined effect of the sowing date, water regime and genotype on GY showed high significancy level (P < 0.01) (Additional file 2). Rum produced significantly the highest mean value of GY (889 g/m2) under irrigated conditions and December sowing date, while Steptoe produced significantly 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 between environments, water regime, sowing date and genotypes showed a clear significant effect on the GY (Additional file 2; Additional file 3; Fig. 1a). For instance, Steptoe produced significantly the lowest mean value (50 g/m2) for GY under rainfed conditions in JUST-2013 for December sowing date treatment, while Rum produced 388 g/m2 (Additional file 3). In JUST-2013, no significant differences were found between GY mean values of all tested genotypes under rainfed treatment for February sowing date, however, Steptoe produced significantly the lowest GY mean values in all remaining environments (Additional file 3). Under irrigated conditions, Rum and Acsad176 produced significantly the highest mean values for GY across all environments irrespective of sowing date treatment except for Steptoe sown in December in JU2012 where no significant differences were observed with Rum and Acsad176 GY mean values (Additional file 3).
For HD, the mean values of tested genotypes ranged from 90.2 days in Rum (significantly the fastest genotype to flower) to the significantly lowest 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 with February (75.7 days). For the main effect of the water regime, the irrigation treatment produced significantly the highest HD mean value (97.8 days) when compared with rainfed (94.7 days). The effect of interaction between sowing date and water regime treatments on HD indicates the presence of significant differences between tested genotypes (Additional file 2). The interaction between environments, water regime, sowing date and genotype showed a clear significant effect on HD (Additional file 2; Additional file 3; Fig. 1b). For instance, Steptoe produced the highest mean values for HD in any given environment × water regime combinations for December sowing date treatment (Fig. 1b; Additional file 3). For February sowing date treatment, clear significant differences were identified only in 2013 environments (JU-2013, RB-2013 and JUST-2013) between HD mean values of Steptoe and other genotypes irrespective of water regime treatments (Additional file 3).
To analyze genotype-specific adaptation to specific environment for the GY trait, the GGE biplot was used. For this purpose, the environments were reclassified based on the combination of each location, year, sowing date and water regime treatments to produce 24 distinct environments. The relationships between the GY of tested genotypes and the 24 tested environments and the degree by which each environment is represented are shown in Fig. 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 (Fig. 2a). On the other hand, Rum was the winner 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 win in any testing environments and the two genotypes were considered losers and they were the poorest genotypes across all tested environments (Fig. 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 (Fig. 2b).
Based on the results above and to analyze the relationship between heading date and agronomic performance, a new correlation analysis was perfumed using the field data of Rum and Steptoe 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 Additional file 5, negative significant correlations (P < 0.05) were found across the four groups between HD with GW and SPW. For December-irrigated conditions (Additional file 5), 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 (Additional file 5). For the February sowing date and irrespective of water regime treatment, negative significant correlations were detected between HD with TW, SN, GN, G.S. TKW and HI (Additional file 5).
Controlled conditions experiment
In this study, the transition to reproductive stage under LD was significantly faster in Rum genotype when compared with Steptoe irrespective of photoperiod, vernalization and water regime combinations (Fig. 3; Additional file 6). Under LD conditions, Steptoe reproductive meristem was in an advanced developmental stage under well-watered conditions when compared with stressed plants irrespective of vernalization treatment (Fig. 3a; Additional file 6). After eight weeks of LD incubation under well-watered conditions, non-vernalized Steptoe reproductive meristem was in an advanced developmental stage when compared with well-watered and vernalized Steptoe plants. For Rum plants grown under LD conditions, a significant difference was observed only after four weeks of incubation between well-watered and non-vernalzied plants when compared with well-watered and vernzalied plants (Fig. 3a; Additional file 6). Under SD, the progression of reproductive meristem development in both genotypes was slower when compared with LD conditions irrespective of vernalization and/or water regime treatments with significant differences observed between the tested genotypes after eight weeks of incubation (Fig. 3b; Additional file 6). After eight weeks of SD incubation, stressed Rum plants subjected to vernalization showed a clear and significantly different advanced developmental stage, while under the same conditions, stressed Steptoe plants showed a clear slower reproductive meristem development (Fig. 3b; Additional file 6). For non-vernalized plants, Rum showed a clear advanced developmental stage after eight weeks of SD conditions, irrespective of water regime treatment, when compared with Steptoe.
At the end of the experiment, the mean values of HD of Rum plants grown under LD conditions and irrespective of vernalization and water regime treatments were significantly lower than the mean values of Steptoe (Additional file 7). On the other hand, vernalized Rum plants showed a significant earlier HD under LD conditions when compared with 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 with well-watered plants (Additional file 7). Under SD conditions, stress-treated and vernalized Rum plants were significantly the earliest to flower when compared with well-watered and vernalized Rum plants (Additional file 7).
The effects of water stress on RWC, stomatal resistance maximum quantum efficiency (Fv/Fm) of photosystem II have been analyzed in treated plants as an index for drought tolerance of the two tested genotypes. Significant differences between well-watered and stressed plants were observed for the tested parameters in the two tested genotypes under different photoperiod and vernalization conditions (Additional file 8). For instance, the mean values of RWC of the tested genotypes were lower in stressed plants irrespective of photoperiod or vernalization treatments and the effects were more pronounced after four weeks of incubation (Additional file 6). Starting the 4th week, the mean values of RWC of non-vernalized Steptoe plants grown under SD and water deficit conditions were significantly the lowest. Clear effects of water deficit stress on stomatal resistance and maximum quantum efficiency (Fv/Fm) of photosystem II were also observed in both genotypes irrespective of day length conditions or vernalization treatments (Additional file 8).
The expression patterns of Vrn-H1, Ppd-H1 and HVA22 genes were investigated in both tested genotypes in response to water deficit, vernalization and photoperiod treatments at selected time points. After two weeks of LD conditions, the relative expression levels of Vrn-H1 were higher in vernalized plants when compared with non-vernalized for both tested genotypes (Fig. 4). Irrespective of vernalization treatment, Rum always showed higher Vrn-H1 expression after two weeks of LD incubation than that observed in Steptoe. Irrespective of water regime treatment, Vrn-H1 expression levels after two weeks of LD incubation were significantly low in non-vernalized Steptoe plants compared with vernalized plants (Fig. 4). After four weeks of LD incubation, the same trend of high expression of Vrn-H1 was observed in vernalized plants when compared with non-vernalized plants, however, the expression levels were significantly lower in stressed Steptoe plants when compared with stressed Rum plants (Fig. 4). Under SD conditions, Rum showed higher Vrn-H1 expression levels when compared with Steptoe (Fig. 4). Interestingly, the expression of Vrn-H1 was significantly the highest in stressed and vernalized Rum plants when compared with Rum plants subjected to other combinations.
After two weeks of LD incubation and irrespective of water regime treatment, the Ppd-H1 expression in vernalized Rum plants showed the highest levels when compared with non-vernalized Rum (Fig. 4). At the same time, no significant differences were detected between Ppd-H1 levels in Steptoe plants subjected to different combinations of water regime and vernalization. After four weeks of LD incubation and irrespective of water regime treatment, vernalized Rum plants showed lower Ppd-H1 expression levels when compared with non-vernalized Steptoe and non-vernalized Rum plants (Fig. 4). For SD conditions, Ppd-H1 levels were slightly higher in Rum plants when compared with Steptoe with a slight increase under well-watered conditions (Fig. 4). For HVA22 gene expression, no major changes were observed between the different growth conditions under SD and LD for both Rum and Steptoe genotypes where both showed an increment in expression level under stress conditions (Fig. 4).