Rolled Leaf Mutant Research Articles

Characterization and Mapping of a Rolling Leaf Mutant Allele rlT73 on Chromosome 1BL of Wheat.

Leaf rolling is regarded as an important morphological trait in wheat breeding. Moderate leaf rolling is helpful to keep leaves upright and improve the photosynthesis of plants, leading to increased yield. However, studies on the identification of genomic regions/genes associated with rolling leaf have been reported less frequently in wheat. In this study, a rolling leaf mutant, T73, which has paired spikelets, dwarfism, and delayed heading traits, was obtained from a common wheat landrace through ethyl methanesulfonate mutagenesis. The rlT73 mutation caused an increase in the number of epidermal cells on the abaxial side and the shrinkage of bulliform cells on the adaxial side, leading to an adaxially rolling leaf phenotype. Genetic analysis showed that the rolling leaf phenotype was controlled by a single recessive gene. Further Wheat55K single nucleotide polymorphism array-based bulked segregant analysis and molecular marker mapping delimited rlT73 to a physical interval of 300.29-318.33 Mb on the chromosome arm 1BL in the Chinese Spring genome. We show that a point mutation at the miRNA165/166 binding site of the HD zipper class III transcription factor on 1BL altered its transcriptional level, which may be responsible for the rolling leaf phenotype. Our results suggest the important role of rlT73 in regulating wheat leaf development and the potential of miRNA-based gene regulation for crop trait improvement.

RLM1, Encoding an R2R3 MYB Transcription Factor, Regulates the Development of Secondary Cell Wall in Rice.

Leaf morphology is an important component of rice ideal plant type. To date, many regulatory genes influencing leaf morphology in rice have been cloned, and their underlying molecular regulatory mechanism has been preliminarily clarified. However, the fine regulation relationship of leaf morphogenesis and plant type remains largely elusive. In this study, a rolling-leaf mutant, named rlm1-D, was obtained and controlled by a pair of dominant nuclear genes. Cytological observations revealed that the rlm1 was mainly caused by abnormal deposition of secondary cell walls. Molecular evidence showed ectopic expression of a MYB-type transcription factor LOC_Os05g46610 was responsible for the phenotype of rlm1-D. A series of experiments, including the transcription factor-centered technology, DNA-binding assay, and electrophoretic mobility shift assay, verified that RLM1 can bind to the promoter of OsCAD2, a key gene responsible for lignin biosynthesis in rice. An interacting partner of RLM1, OsMAPK10, was identified. Multiple biochemical assays confirmed that OsMAPK10 interacted with RLM1. OsMAPK10 positively regulated the lignin content in the leaves and stems of rice. Moreover, OsMAPK10 contributes to RLM1 activation of downstream target genes. In particular, RLM1 is exclusively expressed in the stems at the mature plant stage. The yield of RLM1 knockdown lines increased by over 11% without other adverse agricultural trait penalties, indicating great practical application value. A MAPK-MYB-OsCAD2 genetic regulatory network controlling SCW was proposed, providing a theoretical significance and practical value for shaping the ideal plant type and improving rice yield.

CRISPR/Cas9-Induced Mutagenesis of Semi-Rolled Leaf1,2 Confers Curled Leaf Phenotype and Drought Tolerance by Influencing Protein Expression Patterns and ROS Scavenging in Rice (Oryza sativa L.)

Rice leaf morphology is an essential agronomic trait to develop drought-tolerant genotypes for adequate and stable crop production in drought-prone areas. Here, rolled leaf mutant plants were acquired by CRISPR/Cas9-based mutagenesis of Semi-rolled leaf1,2 (SRL1 and SRL2) genes, and isobaric tags for relative and absolute quantification (iTRAQ) based proteomic analysis was performed to analyze the subsequent proteomic regulation events. Homozygous mutants exhibit decreased chlorophyll content, transpiration rate, stomatal conductance, vascular bundles (VB), stomatal number, and agronomic traits with increased panicle number and bulliform cells (BCs). Under drought stress, mutant plants displayed lower malondialdehyde (MDA) content while higher survival rate, abscisic acid (ABA) content, superoxide dismutase (SOD), catalase (CAT) activities, and grain filling percentage compare with their wild type (WT). Proteomic results revealed that 270 proteins were significantly downregulated, and 107 proteins were upregulated in the mutant line compared with WT. Proteins related to lateral organ boundaries’ (LOB) domain (LBD) were downregulated, whereas abiotic stress-responsive proteins were upregulated in the CRISPR mutant. LBD proteins (Q5KQR7, Q6K713, Q7XGL4, Q8LQH4), probable indole-3-acetic acid-amido synthetase (Q60EJ6), putative auxin transporter-like protein 4 (Q53JG7), Monoculm 1 (Q84MM9) and AP2 (Apetala2) domain-containing protein (Q10A97) were found to be hub-proteins. The hybrids developed from mutant restorers showed a semi-rolled leaf phenotype with increased panicle number, grain number per panicle, and yield per plant. Our findings reveal the intrinsic value of genome editing and expand the knowledge about the network of proteins for leaf rolling and drought avoidance in rice.

Rice Morphogenesis and Chlorophyll Accumulation Is Regulated by the Protein Encoded by NRL3 and Its Interaction With NAL9.

Rice yield is closely related to plant leaf shape and chlorophyll content. In this study, we isolated and identified a narrow and rolled leaf mutant, temporarily named nrl3 with darker green leaves. Histological analysis showed that nrl3 has a reduced number of vascular bundles and undergoes abnormal abaxial sclerenchymatous cell differentiation. The NRL3 mutant phenotype was controlled by a single recessive gene, fine-mapped to a 221 kb interval between Indel3 and RM2322 on Chr3. There are 42 ORF in this interval. Sequencing identified an SNP mutant leading to a premature stop in ORF 18, the candidate gene. Bioinformation analysis indicated that NRL3 encodes a novel protein with unknown function. NRL3 is localized in cytoplasm, membrane and nucleus. Expression analysis of nrl3 showed that genes involved in chlorophyll synthesis were significantly up-regulated while those involved in chlorophyll degradation and programmed cell death (PCD) were significantly down-regulated. The expression levels of photosynthesis genes were also affected. Y2H and BIFC assays indicated that NRL3 interacts directly with NAL9/VYL to regulate leaf morphology in rice. Thus, NRL3 plays an important role in leaf morphogenesis and chlorophyll accumulation, and can be used as a new gene resource for constructing improved rice.

Identification, cloning and sequence analysis of <italic>shallot like1-Fuhui673</italic> from a rolled leaf mutant in rice

Leaves are the important organ for photosynthesis in rice and the strong and weak photosynthesis will be directly influenced with their morphologic structure of leaves. Moderate rolling leaf could keep plants upright, improve light transmittance of population, increase efficiency of light utilization and then increase rice yield. We obtained a rolled leaf mutant, named sll1-fh673 ( shallot like1-Fuhui673 ) derived from the restorer line Fuhui673 ( Oryza sativa L. ssp. indica ) of hybrid rice, which were treated with 60 Co-γ radiation. The leaves of the sll1-fh673 rolled inward like a cylinder during the whole growth period. The characteristics of the sll1-fh673 were distinct from those of the wild-type plants, with shorter on plant height and ear length, malformed spikelets, decreased the grain number per spike, smaller grain shape, worse development of the seed. The result of genetic analysis indicated that the phenotype of rolled leaves of sll1-fh673 was controlled by a single recessive nuclear gene. Meanwhile, the sll1-fh673 gene was mapped to a 52-kb region between inDel marker Ri9-20 and Ri9-7 on the Chromosome 9 of rice with Bulked Segregant Analysis and Recessive-class Analysis methods. But there was a cloned rolled leaf gene SLL1 (LOC_Os09g23200) in the mapped regions, which might be the candidate gene of sll1-fh673 . The sequencing analysis revealed that there was a single-base substitution from C to A and five-base deletion in the first exon in sll1-fh673 comparing with the SLL1 gene, leading to frame-shift mutation after the 148 aa and dysfunction of sll1 protein. So we speculated that the sll1-fh673 gene might be allelic to SLL1 gene. These results will deepen our understanding the mechanisms of the leaf rolling and enriches the allelic resource of the SLL1 gene. We will predict binding sites of the sll1-fh673 gene and find interacting protein of down-regulation, and then carry out the molecular mechanism of the leaf rolling, such as electrophoretic mobility shift assay (EMSA), DNA Chip Technology and Yeast One Hybrid System in the future.

Comparative genomic hybridization and transcriptome sequencing reveal that two genes, OsI_14279 (LOC_Os03g62620) and OsI_10794 (LOC_Os03g14950) regulate the mutation in the γ-rl rice mutant.

We previously established the genetic locus of the rolled-leaf mutant, γ-rl, to chromosome 3. In this study, we performed a comparative genomic hybridization (CGH) analysis to identify the genes responsible for the γ-rl mutant phenotype. This was combined with RNA transcriptome sequencing (RNA-seq) to analyze differences in the mRNA expression in seeds 12h after germination. Using the reference genome of the "indica type" rice from GenBank, we created a chip with 386,000 high density DNA probes designed to target chromosome 3. The genomic DNA from γ-rl and Qinghuazhan (the wild-type) was used for hybridization against the chip to compare signal differences. We uncovered 49 regions with significant differences in hybridization signals including deletions and insertions. RNA-seq analysis between γ-rl and QHZ identified 1060 differentially expressed genes, which potentially regulate numerous biological activities. Moreover, we identified 72 annotated genes in the 49 regions discovered in CGH. Among these, 44 genes showed differential expression in RNA-seq. qRT-PCR validation of the candidate genes confirmed that seven of the 44 genes showed a significant change in their expression levels. Among these, four genes [OsI_10125 (LOC_Os03g06654), OsI_14045 (LOC_Os03g62490), OsI_14279 (LOC_Os03g62620) and OsI_14326 (LOC_Os03g63250)] were down regulated and three genes [(OsI_10794 (LOC_Os03g14950), OsI_11412 (LOC_Os03g21250) and OsI_14152 (LOC_Os03g61360)] were up regulated with a fold change ≥2.0 and a P value≤0.01. Finally, we constructed transgenic plants to study the in vivo functions of these genes. RNAi knock down of LOC_Os03g62620 resulted in rolled-leaf, lower seed-setting and decreased seed growth phenotypes. Transgenic plants with LOC_Os03g14950 over-expression showed dwarf plants with a shortened leaf phenotype. Our results, LOC_Os03g62620 and LOC_Os03g14950 as the essential genes responsible for creating the γ-rl mutant phenotypes suggested that these genes may play crucial roles in regulating rice leaf development and seed growth.

Analysis of the differential gene and protein expression profile of the rolled leaf mutant of transgenic rice (Oryza sativa L.).

The importance of leaf rolling in rice (Oryza sativa L.) has been widely recognized. Although several studies have investigated rice leaf rolling and identified some related genes, knowledge of the molecular mechanism underlying rice leaf rolling, especially outward leaf rolling, is limited. Therefore, in this study, differential proteomics and gene expression profiling were used to analyze rolled leaf mutant of transgenic rice in order to investigate differentially expressed genes and proteins related to rice leaf rolling. To this end, 28 differentially expressed proteins related to rolling leaf traits were isolated and identified. Digital expression profiling detected 10 genes related to rice leaf rolling. 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 gene, and At4g26790 gene. The “omics”-level techniques were useful for simultaneously isolating several proteins and genes related to rice leaf rolling. In addition, the results of the analysis of differentially expressed proteins and genes were closely consistent with those from a corresponding functional analysis of cellular mechanisms; our study findings might form the basis for further research on the molecular mechanisms underlying rice leaf rolling.

Semi-dwarf narrow rolled leaf mutant in rice: Photosynthetic efficiency and physiological response to gibberellic acid (GA3)

Leaf is the major component for fixation of carbondioxide to produce more photoassimilates and maintains source sink relationship in plants. The present investigation was carried out to study the photosynthetic efficiency and physiological response of GA3 of semi-dwraf narrow rolled leaf mutant. Semi-dwarf narrow rolled leaf mutant exhibited reduced plant height, reduced leaf width, reduced panicle length and number of grains per panicle when compared to wild type. Photosynthetic efficiency study revealed that the mutant showed higher photosynthetic efficiency than wild type. Complementation test with GA3 clearly revealed that this mutant is sensitive to GA3 and revert to normal plant height. Scanning Electron Microscope study revealed that mutant had exhibited minimum number of inter-veins between veins, minimum distance between midrib and has slightly larger cell size. Hence, this loss of function mutant is very useful to map the regions associated with panicle length, number of grains per panicle and other traits related to enhancement of yield. The result provides an important clue for further understanding the mechanism of rice leaf development and plant architecture.

Identification of a novel candidate gene for rolled leaf in rice

A semi-narrow and adaxially rolled leaf mutant, rl15(t), was induced from Korean japonica rice cultivar Ilpum by chemical mutagenesis using ethyl methanesulfonate. We characterized the mutant and identified the novel gene causing the mutant phenotype. Cytological analysis of mutant leaves indicated that the adaxial leaf-rolling phenotype is due to the reduced size and number of bulliform cells in the mutant. Genetic analysis showed that the rolled leaf trait is controlled by a single recessive gene, designated rl15(t). Using an F2 mapping population generated from a cross between Milyang23 and the mutant, we mapped the candidate region to a 174 kb interval on the long arm of chromosome 1 near the centromeric region. Through whole genome sequencing in bulk and MutMap analysis, we identified the causal SNP within the candidate region. The results of RT-PCR analysis indicated that a splicing error occurred due to a base change from G to A at the beginning of the fifth intron of LOC_Os01g37837, which encodes a putative seryl-tRNA synthetase, resulting in the mutant phenotype. Further study of the rl15(t) gene will facilitate analysis of leaf architecture and morphogenesis in rice plants.

Characterization and Gene Mapping of Rolled Leaf Mutant 28 ( rl28 ) in Rice ( Oryza sativa L.)

Characterization and Gene Mapping of Rolled Leaf Mutant 28 ( <i>rl28</i> ) in Rice ( <i>Oryza sativa</i> L.)

Genetic Analysis and Scanning Electron Microscopy of an Abaxial Rolled-leaf Mutant in Rice

Genetic Analysis and Scanning Electron Microscopy of an Abaxial Rolled-leaf Mutant in Rice

Cloning, characterization and expression of $\emph{OsFMO}_{{\mathbf{(}\emph{t}\mathbf{)}}}$ in rice encoding a flavin monooxygenase

Flavin monooxygenases (FMO) play a key role in tryptophan (Trp)-dependent indole-acetic acid (IAA) biosynthesis in plants and regulate plant growth and development. In this study, the full-length genomic DNA and cDNA of OsFMO(t), a FMO gene that was originally identified from a rolled-leaf mutant in rice, was isolated and cloned from wild type of the rolled-leaf mutant. OsFMO(t) was found to have four exons and three introns, and encode a protein with 422 amino acid residues that contains two basic conserved motifs, with a 'GxGxxG' characteristic structure. OsFMO(t) showed high amino acid sequence identity with FMO proteins from other plants, in particular with YUCCA from Arabidopsis, FLOOZY from Petunia, and OsYUCCA1 from rice. Our phylogenetic analysis showed that OsFMO(t) and the homologous FMO proteins belong to the same clade in the evolutionary tree. Overexpression of OsFMO(t) in transformed rice calli produced IAA-excessive phenotypes that showed browning and lethal effects when exogenous auxins such as naphthylacetic acid (NAA) were added to the medium. These results suggested that the OsFMO(t) protein is involved in IAA biosynthesis in rice and its overexpression could lead to the malformation of calli. Spatio-temporal expression analysis using RT-PCR and histochemical analysis for GUS activity revealed that expression of OsFMO(t) was totally absent in the rolled-leaf mutant. However, in the wild type variety, this gene was expressed at different levels temporally and spatially, with the highest expression observed in tissues with fast growth and cell division such as shoot apexes, tender leaves and root tips. Our results demonstrated that IAA biosynthesis regulated by OsFMO(t) is likely localized and might play an essential role in shaping local IAA concentrations which, in turn, is critical for regulating normal growth and development in rice.

Photosynthetic characterization of a rolled leaf mutant of rice (Oryza sativa L.)

A new rolling leaf rice mutant was identified which showed an apparently straighter longitudinal shape normal transverse rolling characters at all developing stages. The chlorophyll contents per fresh weight of this mutant leaves were lower than those of wild-type. The electron transfer rate (ETR) and photochemical quenching (qP) were a little higher than those of wild-type. However, because of significant increase of non-photochemical quenching (NPQ), the maximal photosystem II (PSII) photochemistry (Fv/Fm) and the efficiency of excitation energy trapping by open PSII reaction centers in the light–adapted state (Fv’/Fm’) were lower than those of wild-type. Low temperature fluorescence analysis showed that rolling leaf mutant assigned more excited energy to photosystem I (PSI) than to PSII. The superoxide dismutase (SOD) content, soluble sugar content, proline content and malonaldehyde (MDA) content of the rolling leaf mutant were nearly 39.4, 91.2, 96.7 and 143.7% of those of wild-type, respectively. The great increase of MDA content suggests that membrane lipid system was damaged in rolling leaf mutant leaves. These results indicate that rolling leaf mutant decrease the efficiency of light utilization compared to the wild-type. This was because of the reduction of leaf area and chlorophyll contents, and the dissipation of more excitation energy as NPQ as a result of avoiding potential damage of membrane structure.

Functional Analysis for Rolling Leaf of Somaclonal Mutants in Rice (Oryza sativa L.)

This study was carried out to facilitate the functional analysis of rice genes. Some 297 insertion plants (1.7%) of the entire lines with the endogenous retrotransposon Tos17 were produced. Phenotypes of these plants in the S2 genera-tion were observed in the field according to different leaf types. Rolling leaf mutants showed thinner sclerenchyma-tous cells, defective arrangement of vascular bundles, and well-formed bulliform cells as compared to the parental cultivar. Two new copies of Tos17 were detected in the rolling leaf type. In the new leaf type, the copy number and activation of Tos12, 15 did not appear as ‘Ilpum’. Flanking sequence tag (FST) analysis of Tos17 in the rolling leaf mutant indicated that new copies of Tos17 were transposed on chromosomes 11 and 12. Annotated homologues of the tagging genes on chromosome 11 were arabinoxylan rabinofuranohydrolase isoenzyme AXAH-I and II. The tagging gene in chromosome 12 was highly correlated with 6 kinds of genes including a transcript regulated factor and a rough sheath 2-like protein. This rolling leaf and flanking sequence data will stimulate the functional analysis of rice genes.

Genetic analysis and gene fine mapping of a rolling leaf mutant (rl 11(t) ) in rice (Oryza sativa L.)

The exploration of new genes controlling rice leaf shape is an important foundation for rice functional genomics and plant architecture improvement. In the present study, we identified a rolling leaf mutant from indica variety Yuefeng B, named rl11(t), which exhibited reduced plant height, rolling and narrow leaves. Leaves in rl11(t) mutant showed abnormal number and morphology of veins compared with those in wild type plants. In addition, rl11(t) mutant was less sensitive to the inhibitory effect of auxin than the wild type. Genetic analysis suggested that the mutant was controlled by a single recessive gene. Gene rl11(t) was initially mapped between SSR markers RM6089 and RM124 on chromosome 4. Thirty-two new STS markers around the rl11(t) region were developed for fine mapping. A physical map encompassing the rl11(t) locus was constructed and the target gene was finally delimited to a 31.6 kb window between STS4-25 and STS4-26 on BAC AL606645. This provides useful information for cloning of rl11(t) gene.

Genetic Analysis and Gene Mapping of a Novel Rolled Leaf Mutant &lt;I&gt;rl12&lt;/I&gt;(&lt;I&gt;t&lt;/I&gt;) in Rice

Leaf is an important organ in photosynthesis, its moderate rolling could facilitate the improvement of plant population's structure and enhance light-use efficiency, which is significant in ideotype breeding. Recently, more attention has been paid to the identification of rice rolled leaf mutants and related gene cloning by breeders and geneticists. This paper reported a rolled leaf mutant, temporarily named rl12(t), from the restorer line Jinhui 10 treated by EMS. In the mutant, the initializing leaves did not roll, the mature leaves curled the upper 1/3 section of them and the older mature leaves rolled completely, the pigment contents increased significantly. To date, such phenotype has never been reported. One CMS line Xinong 1A with normal leaves was crossed with the rl12 (t) mutant, the F1/F2 populations were used for genetic analysis, suggesting that the mutant trait were controlled completely by one single dominant nuclear gene. rl12 (t) was finally located on the chromosome 10 between SWU-1 and SWU-2 with genetic distances of 1.5 and 0.2 cM, respectively. There has been no rolled leaf gene reported on this chromosome. Therefore, the RL12 (t) should be novel, and also the unique dominant rolled leaf gene mapped by molecular markers up to now. The results provide a basis for RL12 (t) gene cloning and functional analysis, as well as mechanism studies of rolled leaf and the application in plant-type breeding.

Genetic Analysis and Gene Mapping of a Novel Rolled-Leaf Mutant rl12(t) in Rice

Leaf is an important organ for photosynthesis. Moderate leaf rolling could facilitate structure improvement of plant population and enhance light-use efficiency, which is important in breeding for ideotype plants. A rolled leaf mutant temporarily named rl12(t), was obtained from the rice ( Oryza sativa L.) restorer line Jinhui 10 treated with ethyl methyl sulphonate (EMS). In the mutant, the newly developing leaves of the mutant did not roll, the upper 1/3 section of mature leaves was curled, and the older mature leaves were rolled completely. The pigment contents of the mutant increased significantly. The cytoplasmic male sterile (CMS) line Xinong 1A with flat leaves was crossed with the rl12(t) mutant to produce F 1 and F 2 populations. Genetic analysis indicated that the mutant was controlled by a single dominant gene. Gene rl12(t) was finally located on chromosome 10 between SWU-1 and SWU-2 with the genetic distances of 1.5 and 0.2 cM, respectively. Because no genes for rolled leaf trait have been previously located on this chromosome, RL12(t) should be a novel and unique dominant gene for rolled leaf.

Genetic analysis and gene mapping of a new rolled-leaf mutant in rice (Oryza sativa L.)

To understand the development of rice leaf blades, we identified a new rolled-leaf mutant, w32, from indica cultivar IR64 through EMS mutagenesis. The mutant showed a stable rolled-leaf phenotype throughout the life cycle. Two F2 populations were developed by crossing w32 to cultivar IR24 and PA64. Genetic analysis showed that the rolled-leaf phenotype was controlled by a single recessive gene. To determine the location of the gene, bulked segregant analysis was carried out using mutant and wild-type DNA pools and 1846 mutant-type F2 individuals derived from the cross w32/PA64 were genotyped to locate the gene on the short arm of chromosome 7. The rolled-leaf gene, tentatively named rl11(t), is likely a new gene as no other rolled-leaf genes have been identified near the region. By developing new SSR and InDel markers, the gene was delimited to a 52 kb region near the end of the short chromosome arm. Further fine mapping and cloning of the gene are currently underway.

Anatomical and Chemical Characteristics of a Rolling Leaf Mutant of Rice and Its Ecophysiological Properties

The anatomical and chemical characteristics of a rolling leaf mutant ( rlm) of rice ( Oryza sativa L.) and its ecophysiological properties in photosynthesis and apoplastic transport were investigated. Compared with the wild type (WT), the areas of whole vascular bundles and xylem as well as the ratios of xylem area/whole vascular bundles area and xylem area/phloem area were higher in rlm, whereas the area and the width of foliar bulliform cell were lower. The Fourier transform infrared (FTIR) microspectroscopy spectra of foliar cell walls differed greatly between rlm and WT. The rlm exhibited lower protein and polysaccharide contents of foliar cell walls. An obvious reduction of pectin content was also found in rlm by biochemical measurements. Moreover, the rate of photosynthesis was depressed while the conductance of stoma and the intercellular CO 2 concentration were enhanced in rlm. The PTS fluorescence, which represents the ability of apoplastic transport, was 11% higher in rlm than in WT. These results suggest that the changes in anatomical and chemical characteristics of foliar vascular bundles, such as the reduction of proteins, pectins, and other polysaccharides of foliar cell walls, participate in the leaf rolling mutation, and consequently lead to the reduced photosynthetic dynamics and apoplastic transport ability in the mutant.

Genetic analysis and fine mapping of a dynamic rolled leaf gene,RL10(t), in rice (Oryza sativaL.)

A dynamically rolled leaf mutant (rl10) was identified from a spontaneous mutation in an Oryza sativa L. subsp. indica line, II-32B. The leaf chlorophyll content of rl10 is higher than that of the wild type. Genetic analysis using 3 F2 segregating populations derived from crosses between rl10 and the rice lines Mian5B, II-32B, and D62B, respectively, confirmed that the rolled leaf trait of rl10 is controlled by a single recessive gene. Of 719 SSR primer pairs that showed polymorphism between D62B and rl10, 151 were adopted to map the RL10(t) gene using an F2 segregating population of the cross rl10 x D62B, which contained 352 recessive plants. RL10(t) was primarily mapped on the long arm of chromosome 9, 5.09 cM from marker RM105 and 5.13 cM from marker RM3912. Using a novel set of 22 primer pairs between RM105 and RM3912, RL10(t) was further mapped between markers rlc3 (0.72 cM in distance) and rlc12 (0.1 cM in distance) using an F2/F3 population containing 1172 recessive individuals. Mapped position analysis and homology analysis of the 20 genes within the 194-kb region between these 2 markers both indicated that a gene encoding a Myb-like domain transcription factor with homology to Arabidopsis KANADI (annotated in PAC clone AP005904) is the most probable candidate for RL10(t). This study enables further investigation of whether KANADI-like Myb genes are involved in leaf polarity modeling in monocots, as they are in dicots.