The present study reports on the genetic dissection of PC and WS by using 301 BC2DH lines of a cross between barley cultivar Scarlett and wild accession ISR42-8. An advanced backcross population was utilized for a straightforward detection and introgression of favorable exotic alleles in the Scarlett background according to Tanksley and Nelson
. Our data showed a significant variation of PC and WS between parents as well as among the BC2DH lines. This population showed skewed frequency distributions for both traits because normal distribution is not expected in a BC2DH population. Mather and Jinks
 proposed a bias to one of the parent when two parents bear difference for a trait because of successive backcrossing of the recurrent parent. The present work reports on high resolution QTL mapping data by using the biggest double haploid population in barley. It is accepted that the strength of a QTL analysis primarily depends upon the size of mapping population and the density of markers on the genetic map
QTL analysis identified four QTL for PC where at three QTL the elite alleles from Scarlett were associated to heighten PC. These findings agree with higher PC in the susceptible parent Scarlett which suggests that higher proline levels may define its drought sensitivity. Proline level was used as a metabolic measure to screen drought stress tolerance in barley and higher PC were found in drought susceptible genotypes
[30, 31] which agrees with the response of Scarlett in the present study. It has been reported that proline levels vary considerably in different plant organs. It was found higher in reproductive organs than vegetative as well as in the organs bearing endogenously controlled dehydration, e.g. in seeds or pollen
[38–40]. However, in barley leaves the preferential accumulation of proline in epidermis and vascular bundles was only observed under stress conditions
, suggesting proline induction a reliable marker for the measurement of drought stress response in barley. Our data accounted for the preeminence of Scarlett's alleles at QTL effect QPC.S42.3H, QPC.S42.4H and QPC.S42.6H to enhance PC as compared to respective exotic alleles. Interestingly, these effects revealed both M and M x T interaction effects thus indicating their role under control and drought stress conditions. The knowledge gained in the model plant Arabidopsis suggests that proline biosynthesis occurs both in normal and stress conditions. Under normal conditions, it is synthesized to maintain the housekeeping function of the cell. Altogether, three enzyme coding genes, P5CS1 and P5CS2 and P5CR have been described to regulate proline biosynthesis in Arabidopsis. Under drought, the expression of P5CS1 and P5CR increased which result in higher proline synthesis in the chloroplast whereas P5CS2 is primarily linked to housekeeping proline synthesis in the cytosole
[23, 24]. The map position of P5CS1 and P5CS2 and P5CR are not known in barley and hence the identification of QPC.S42.3H, QPC.S42.4H and QPC.S42.6H on chromosomes 3H, 4H and 6H may provide an initial knowledge of proline biosynthetic loci in barley.
The strongest QTL effect was detected on chromosome 5H, where an exotic allele enhanced PC by 54%. This exotic allele showed an M x T interaction effect only which suggests that QPC.S42.5H may underlie a drought inducible gene of wild origin possibly similar to transcriptional activity of P5CS1 or P5CR which induced under drought stress conditions
. However, it is still an open question whether QPC.S42.5H underlies an exotic variant of P5CS1 or P5CR or a new component of proline biosynthesis in barley. To address this question candidate gene approach will be adapted to test DNA sequence polymorphism and expression differences of barley orthologs in Scarlett and ISR42-8. Alternatively, QTL bearing ILs are available for positional cloning of QPC.S42.5H. A single report was found on the genetic mapping of PC in barley where Siahsar and Narouei
 reported two QTL for salt stress related to proline accumulation on chromosome 5H by using 72 double haploid lines of a cross between Steptoe and Morex. To see the effect of PC on plant performance we measured plant biomass which revealed major shoot weight QTL were associated to Scarlett's alleles suggesting a possible linkage drag of the exotic alleles on shoot biomass as expected from wild barley accession ISR42-8 being inferior in shoot development with respect to Scarlett (data not shown). Therefore, the effect of PC variation on biomass production may not be visible in such mixed background but candidate IL can be used to test its effect on plant performance. Although, the role of proline is still a debate in crop plants, its utility has been validated in the process of drought stress tolerance in Arabidopsis and rice via transgenic approaches
[43–45]. Therefore, more efforts are needed to generalize these effects in crops like barley.
The extent of leaf WS is a straightforward criterion to measure a plant's ability to tolerate water limiting conditions which may be linked to drought inducible metabolites like proline. Our data revealed highly significant but weak correlation of PC and WS. Stewart
 studied proline accumulation in wilted barley leaves and found that wilting caused 40 fold stimulation of proline biosynthesis in non-starved leaves when compared to starved leaves. He suggested the role of carbohydrates which appeared to supply precursors for proline bio-synthesis. These data indicate that although leaf wilting may stimulate proline biosynthesis, it was unable to modulate the process of its biosynthesis which agrees with the partial dependence of PC and WS. No co-localization of QTL was found which also suggest an independent genetic inheritance of both traits. Our data showed four QTL for WS where at QWS.S42.1H and QWS.S42.4H the introgression of exotic alleles was associated to reduce WS under drought stress conditions. Although these QTL are genetically independent to PC, they may still have significance in modulating proline metabolism which requires lower wilting environment. These findings support the idea of introgressing favorable exotic alleles from a tolerant wild accession ISR42-8 into Scarlett where higher PC may be ceased because of precocious leaf wilting and death under drought stress condition. Abscisic acid (ABA) is one major factor implicated for leaf drying (senescence) under severe drought
. Quarrie et al.
 made a QTL analysis to dissect the genetics behind ABA accumulation and found ten QTL for low and high ABA accumulation in rice under drought stress conditions. In a previous study, they found a QTL for ABA accumulation on 5A of wheat in the region of vernalization gene Vrn1 . Thus, the localization of QWS.S42.4H at marker locus VrnH2 may indicate a putative orthologous region of Vrn1 variant in barley. QWS.S42.1H was linked to HvABAIP where Tondelli et al.
 described QTL for drought stress tolerance in barley. HvA1 (group 3 LEA protein), which is known as ABA induced barley effector gene, was mapped at the similar region on chromosome 1H whose overexpression confers dehydration tolerance in transgenic rice plants
[50, 51]. QTL effects QWS.S42.2H and QWS.S42.3H were associated to superior performance of elite alleles for decrease in WS. These findings suggest that the susceptible parent Scarlett also harbors useful alleles for WS that may underlie valuable components of shoot development and their replacement with detrimental exotic alleles might be a reason behind such variation. Strikingly, all four QTL for WS showed no M x T interaction effect suggesting significant and stable QTL effects across control and drought stress blocks. A possible reason may lie in the genetic control of WS where the associated QTL alleles are active across both treatments. Alternatively, it is possible that QTL were unable to pass the criteria of M x T interaction effects due to error created by different environmental (E) conditions across years as phenotypic evaluations were made in each year in single replication. It is worthwhile to mention that QTL x E (years) was not the focus of the present study because the experiments were carried out inside a tunnel and not under real field conditions. Furthermore, two epistatic interactions were detected where exotic alleles presented an additive role in the development lower WS. Here, the drought sensitive elite allele seems epistatic to drought tolerant exotic alleles which overshadowed the performance of exotic alleles in the determination of drought tolerance. A tolerant WS is evident in the donor parent ISR42-8 of these exotic alleles. However, lower wilting due to the interaction of a flowering gene specific marker HvFT2 on chromosome 3H is an interesting outcome which suggests a putative role of flowering as a regulator in drought stress tolerance. Wang et al.
 identified DNA polymorphism in the 3'UTR of HvFT2 between Scarlett and ISR42-8. Von Korff et al.
 studied heading date variation in population S42 and found at most of the ten QTL identified that the introgression of the wild allele was associated with enhanced flowering. They indicated early and heterogeneous flowering habits in wild barley were presumably linked to adaptation in a water limiting environment. These data also highlight the necessity of digenic interactions effect for a high resolution QTL analyses. The role of epistatic interactions has been accepted crucial for the determination of a final phenotype of quantitative traits