RL is a phenomenon commonly found in grasses and quite commonly described for field crops, but it has not been studied in rye. As leaf morphology is an important agronomic trait in the breeding of rice , the mechanism of RL was of interest, especially to rice researchers. There is no information about the genetic base of molecular mechanisms involved in leaf shape in rye, except one report regarding QTLs controlling leaf area .
A study similar to ours has been conducted on tetraploid wheat ; however, we did not study RL as the reaction to water deficit. The research on drought resistance in durum wheat × wild emmer wheat recombinant inbred line population allowed to detect 11 significant QTLs associated with flag RL, mapped on chromosomes: 1A, 2A, 2B, 4B, 5A, 5B, 6A, 6B, 7A, and 7B . Three of these QTLs were found to be environment responsive.
We detected 43 QTLs, grouped into 28 intervals, which confirms the multigenic base of the trait stated for wheat and rice. Studies on rice showed that no fewer than 70 genes/QTLs for RL have been mapped or cloned till now . Four QTLs for RL were stable in different environments. Due to many QTLs for RL were detected once, their genotype×environment interaction (GEI) could be inferred. GEI is a common characteristic for quantitative traits. For possible breeding purposes (like marker Assisted Selection - MAS), QTLs that are more environment-specific should be treated with caution and more attention should be paid to the repetitive QTLs. However, QTLs of varying manifestation, dependent on the environmental influence are also interesting for cognitive purposes.
In this study, we focused on the analysis of RL per se, without linking this trait with the response to drought. However, co-localization of two QTLs for RL with QTLs for drought index of grain number per spike (4R) and spike number per plant (7R) revealed in other experiments  indicates the relationship of detected loci with adaptive mechanisms to drought-related stress conditions.
Furthermore, 5 out of 11 QTLs for RL mapped on tetraploid wheat were co-localized with QTLs associated with plant productivity . RL was also found to be associated with plant height in four regions (2B, 4B, 6A, and 7A) and with heading earliness (days from planting to heading) in two intervals on chromosomes 4B and 5A . We also found nine QTLs co-localized with QTLs for other agronomic traits mapped in the same population , such as grain number and weight, spike number per plant, compactness of spike and plant height. QTLs for RL were also found to be co-localized with two QTLs for heading earliness (2R and 7R), one with α-amylase activity QTL (7R), and three with preharvest sprouting QTLs (1R, 4R, and 7R) .
Additional confirmation of the association between loci engaged in controlling RL and QTLs responsible for different agronomic traits is the result of comparing markers linked with RL and markers for nine features studied in the other, unrelated rye RILs’ mapping population 541 × Ot1–3 . There were loci for plant height, stem thickness, spike length, awn length, heading date, thousand grain weight, grain length, leaf area, and chlorophyll content localized on the DArT-based high-density map of this population.
Each of the nine traits was characterized by some markers common with these, linked to RL in our population. There were 45 such markers distributed throughout the five chromosomes (3R, 4R, 5R, 6R, and 7R). Among them three DArTs from 7R were common for RL and leaf size (XrPt505931, XrPt389959, and XrPt400276). Seven DArTs were linked to RL and chlorophyll content: three from 5R (XrPt389759, XrPt346583, and XrPt505721) and four from 7R chromosome (XrPt402607, XrPt398519, XrPt347574, and XrPt401795). All these relationships suggest very strong linkages and/or pleiotropic effects of many genes, which remains in agreement with previous results for rye [16, 17, 21], wheat , and rice .
All DArTs significantly linked to RL were subjected to screening the DArT sequences database, followed by NCBI database blasting, in order to find the homologs. A total of 12 records with a known identity were found; majority of rye DArTs sequences were most similar to Aegilops tauschii mRNAs. Only one of them was matched to Secale cereale cds of resistant gene analog (putative disease resistance gene), and one to Triticum aestivum genomic sequence—also connected with resistance, in this case to fusarium head blight (FHB).
Sequence of DArT XrPt506905 from 3R showed similarity to predicted gene, namely, subtilisin-like protease. Subtilisin-like proteases (subtilases) are serine proteases and constitute the largest group of peptidases. Although several subtilases have been identified in plants (e.g., about 60 subtilase genes are known in Oryza sativa and Arabidopsis thaliana), most of their functions in plants remain unknown  (and bibliography therein). It is likely that subtilases contribute significantly to the developmental processes and signaling cascades in plants (Rautengarten et al. 2005, after ). For instance, the loss-of-function mutation in ALE1 leads to abnormal leaf shape . Marker XrPt506905, which is a predicted gene for subtilisin-like protein, in addition to the linkage with RL also showed a relationship with awn and grain length .
DArT XrPt507717, nearest to the peak of a QTL for RL from 3R, revealed similarity to protein-like COV2 mRNA. The role of COV2, inferred from the sequence or structural similarity to COV1 is stem vascular tissue pattern formation. COV1 is predicted to be an integral membrane protein that may be involved in the perception or transport of a signaling molecule that negatively regulates the differentiation of vascular tissue in the developing stem of Arabidopsis . Marker XrPt507717, in addition to the linkage with RL also showed a relationship with awn length .
Two DArTs, XrPt401081 and XrPt398502 from 3R, has sequences homologous to jasmonate O-methyltransferase. This enzyme catalyzes the methylation of jasmonate into methyljasmonate, a plant volatile that acts as an important cellular regulator mediating diverse developmental processes and defense responses (http://www.uniprot.org/uniprot/Q9AR07). It is involved in the pathway of oxylipin biosynthesis, which is a part of lipid metabolism. To this end, 28 differentially expressed proteins related to rolled leaf traits were isolated and identified. Some of the proteins and genes detected are involved in lipid metabolism, which is related to the development of bulliform cells, such as phosphoinositide phospholipase C, Mgll, and At4g26790 .
Sequence of next DArT from 3R, XrPt507473, proved to be similar to Fhb1, a major FHB-resistant gene. Fhb1 was fine mapped on the distal segment of chromosome 3BS of spring wheat (Triticum aestivum L.). One of the recent studies has reported that wheat Fhb1 encodes a chimeric lectin with agglutinin domains and a pore-forming toxin-like domain conferring resistance to FHB . Marker XrPt507473, in addition to the linkage with RL, also showed a relationship with awn length and stem thickness .
XrPt401454 from 5R showed homology to LRR receptor-like serine/threonine-protein kinase FEI11 mRNA. FEI1 is involved in the signaling pathway that regulates cell wall function, including cellulose biosynthesis, likely via an 1-aminocyclopropane-1-carboxylic acid (ACC)-mediated signal; a precursor of ethylene (http://www.uniprot.org/uniprot/C0LGF4). To date, 13 genes associated with rice RL have been isolated or cloned. The cytological mechanism of RL has been found to be largely related to the abnormal development of bulliform cells. NRL1 encodes cellulose synthase and plays a positive role in the regulation of bulliform cell development. In mutant rice plants that lack this gene, shrinkage is found in the area of the bulliform cells, thereby causing inward rolling of rice leaves .
Sequence of XrPt402531 from 5R demonstrated high similarity to predicted A. tauschii transcription factor bHLH79 (basic helix-loop-helix protein 79). The function of this factor is unknown; however, several transcription factors are known to be engaged in the establishment of abaxial/adaxial leaf polarity. For example, mutation in SLL1/RL9, a member of the KANADI family, encoding a transcription factor , leads to the failure of programmed cell death of abaxial mesophyll cells and the suppression of the differentiation of the abaxial cells, and finally to generate adaxially rolled leaves. ROC5 encodes a protein containing a leucine zipper domain, homologous to GLABRA2 in Arabidopsis which results in the negative regulation in the development of the bulliform cells. The number and size of the bulliform cells increased when ROC5 was knocked out, thereby leading to the generation of adaxially rolled leaves, whereas co-suppression of ROC5 resulted in abaxial RL .
Overexpression of a rice gene OsLBD3–7 that encodes a LBD family transcription factor promoted narrow and adaxially rolled leaves by decreasing the size and number of bulliform cells. OsLBD3–7 also upregulated the expression of negative regulators of bulliform cells, which implies that OsLBD3–7 acts as a suppressor of bulliform cell development .
The other rice gene ACL1 encodes an unknown protein with a conserved functional domain; OsZHD1 encodes a domain transcription factor with homologous zinc finger structure. These genes also play a positive role in the regulation of bulliform cell development, and overexpression of these two genes results in an increased number of bulliform cells, thereby causing outward rolling of rice leaves [12, 29].
XrPt402607 from 7R, marker nearest to the peak of QTL for RL and also marker linked with grain length and chlorophyll content , showed homology to predicted polyadenylate-binding protein-interacting protein 7-like gene. The poly(A) binding proteins (PABP) play an important role in the regulation of translation; however, the role of this particular factor is unknown.
Next marker from 7R, the peak of QTL for RL, DArT XrPt401480, seems to be homologous to the predicted gene encoding tubby-like F-box protein 12. Plants include a large number of tubby-like proteins (TLPs/TULPs). For example, there are 11 members of the tubby gene family in Arabidopsis , 14 in rice , 11 in poplar , 4 in wheat , and 8 in sorghum (http://www.ncbi.nlm.nih.gov). The existence of multiple TLPs implies their vital function in plants. F-box proteins regulate diverse cellular processes, including cell cycle transition, transcriptional regulation, and signal transduction. Lai et al.  have demonstrated that AtTLP9 interacts with ASK1 (Arabidopsis Skp1-like 1). According to them, F-box domain containing plant TLPs acting as transcription regulators should have cellular function activities of F-box proteins in signal transduction. AtTLP9 might participate in the abscisic acid signaling pathway . The other example of F-box protein regulating plant growth and development include TIR1 acting in response to auxin .
The function of tubby-like F-box protein 12 encoded by genes of Aegilops tauschii and Brachypodium distachyon, homologous to XrPt401480, is unknown. However, some rice TLPs, especially OsTLP12, were probably involved in the abscisic acid and gibberellin signaling processes. This role might also be attributed to rye TLP12, because the same DArT was pointed as a marker linked to plant height .
XrPt390593 sequence from 7R was similar to vegetative cell wall protein gp1-like mRNA. The nature of cell wall proteins is as varied as the many functions of plant cell walls. Majority of the cell wall proteins are cross-linked into the cell wall and probably have structural functions. If this protein was associated with bulliform and/or hypodermis cells it might have an effect on RL, because these two types of cells are involved in RL in higher plants .
Although the roles described for the aforementioned markers as the potential genes that control RL are likely, their functions and association with RL and other traits should be verified in expression tests and will be studied during further research.