A newly evolved rice-specific gene JAUP1 regulates jasmonate biosynthesis and signalling to promote root development and multi-stress tolerance.

Root architecture and function are critical for plants to secure water and nutrient supply from the soil, but environmental stresses alter root development. The phytohormone jasmonic acid (JA) regulates plant growth and responses to wounding and other stresses, but its role in root development for adaptation to environmental challenges had not been well investigated. We discovered a novel JA Upregulated Protein 1 gene (JAUP1) that has recently evolved in rice and is specific to modern rice accessions. JAUP1 regulates a self-perpetuating feed-forward loop to activate the expression of genes involved in JA biosynthesis and signalling that confers tolerance to abiotic stresses and regulates auxin-dependent root development. Ectopic expression of JAUP1 alleviates abscisic acid- and salt-mediated suppression of lateral root (LR) growth. JAUP1 is primarily expressed in the root cap and epidermal cells (EPCs) that protect the meristematic stem cells and emerging LRs. Wound-activated JA/JAUP1 signalling promotes crosstalk between the root cap of LR and parental root EPCs, as well as induces cell wall remodelling in EPCs overlaying the emerging LR, thereby facilitating LR emergence even under ABA-suppressive conditions. Elevated expression of JAUP1 in transgenic rice or natural rice accessions enhances abiotic stress tolerance and reduces grain yield loss under a limited water supply. We reveal a hitherto unappreciated role for wound-induced JA in LR development under abiotic stress and suggest that JAUP1 can be used in biotechnology and as a molecular marker for breeding rice adapted to extreme environmental challenges and for the conservation of water resources.

A Large-Scale Hydroponic Evaluation of Rice Mutants for Pythium Resistance.

Rice is a major staple crop worldwide. However, the occurrence of rice diseases during cultivation poses a significant challenge to achieving optimal yields. Among the major pathogens, Pythium species, which cause seedling blight, are of particular concern. Pythium infects rice roots through zoospores, mycelia, or oospores, leading to root rot, stunting, yellowing, and ultimately seedling damping-off. While many disease resistance-related genes have been reported in rice, only very limited research has been associated with resistance to Pythium infection. In this study, we aimed to establish a rapid screening system to identify rice lines that are resistant or susceptible to Pythium pathogen in rice nurseries. We conducted evaluations on important factors, including virulence, inoculation method, seed soaking period, and the measurement of disease severity. As a result, we successfully developed a screening system that allows for high-throughput and rapid screening of the Taiwan Rice Insertional Mutant (TRIM) library for mutant lines exhibiting resistance to P. arrhenomanes. Furthermore, we identified a slightly resistant TRIM line and explored potential genes encoding endglucanase-1 precursor and malonyl-CoA decarboxylase that may be involved in conferring resistance to P. arrhenomanes.

A single promoter-TALE system for tissue-specific and tuneable expression of multiple genes in rice.

In biological discovery and engineering research, there is a need to spatially and/or temporally regulate transgene expression. However, the limited availability of promoter sequences that are uniquely active in specific tissue-types and/or at specific times often precludes co-expression of multiple transgenes in precisely controlled developmental contexts. Here, we developed a system for use in rice that comprises synthetic designer transcription activator-like effectors (dTALEs) and cognate synthetic TALE-activated promoters (STAPs). The system allows multiple transgenes to be expressed from different STAPs, with the spatial and temporal context determined by a single promoter that drives expression of the dTALE. We show that two different systems-dTALE1-STAP1 and dTALE2-STAP2-can activate STAP-driven reporter gene expression in stable transgenic rice lines, with transgene transcript levels dependent on both dTALE and STAP sequence identities. The relative strength of individual STAP sequences is consistent between dTALE1 and dTALE2 systems but differs between cell-types, requiring empirical evaluation in each case. dTALE expression leads to off-target activation of endogenous genes but the number of genes affected is substantially less than the number impacted by the somaclonal variation that occurs during the regeneration of transformed plants. With the potential to design fully orthogonal dTALEs for any genome of interest, the dTALE-STAP system thus provides a powerful approach to fine-tune the expression of multiple transgenes, and to simultaneously introduce different synthetic circuits into distinct developmental contexts.

Closer vein spacing by ectopic expression of nucleotide-binding and leucine-rich repeat proteins in rice leaves.

KEY MESSAGE: Elevated expression of nucleotide-binding and leucine-rich repeat proteins led to closer vein spacing and higher vein density in rice leaves. To feed the growing global population and mitigate the negative effects of climate change, there is a need to improve the photosynthetic capacity and efficiency of major crops such as rice to enhance grain yield potential. Alterations in internal leaf morphology and cellular architecture are needed to underpin some of these improvements. One of the targets is to generate a "Kranz-like" anatomy in leaves that includes decreased interveinal spacing close to that in C4 plant species. As C4 photosynthesis has evolved from C3 photosynthesis independently in multiple lineages, the genes required to facilitate C4 may already be present in the rice genome. The Taiwan Rice Insertional Mutants (TRIM) population offers the advantage of gain-of-function phenotype trapping, which accelerates the identification of rice gene function. In the present study, we screened the TRIM population to determine the extent to which genetic plasticity can alter vein density (VD) in rice. Close vein spacing mutant 1 (CVS1), identified from a VD screening of approximately 17,000 TRIM lines, conferred heritable high leaf VD. Increased vein number in CVS1 was confirmed to be associated with activated expression of two nucleotide-binding and leucine-rich repeat (NB-LRR) proteins. Overexpression of the two NB-LRR genes individually in rice recapitulates the high VD phenotype, due mainly to reduced interveinal mesophyll cell (M cell) number, length, bulliform cell size and thus interveinal distance. Our studies demonstrate that the trait of high VD in rice can be achieved by elevated expression of NB-LRR proteins limited to no yield penalty.

How does rice cope with too little oxygen during its early life?

Most crops cannot germinate underwater. Rice exhibits certain degrees of tolerance to oxygen deficiency for anaerobic germination (AG) and anaerobic seedling development (ASD). Direct rice seeding, whereby seeds are sown into soil rather than transplanting seedlings from the nursery, becomes an increasingly popular cultivation method due to labor shortages and opportunities for sustainable cultivation. Flooding is common under direct seeding, but most rice varieties have poor capability of AG/ASD, which is a major obstacle to broad adoption of direct seeding. A better understanding of the physiological basis and molecular mechanisms regulating AG/ASD should facilitate rice breeding for enhanced seedling vigor under flooding. This review highlights recent advances on molecular and physiological mechanisms and future breeding strategies of rice AG/ASD.

Rice Big Grain 1 promotes cell division to enhance organ development, stress tolerance and grain yield.

Grain/seed yield and plant stress tolerance are two major traits that determine the yield potential of many crops. In cereals, grain size is one of the key factors affecting grain yield. Here, we identify and characterize a newly discovered gene Rice Big Grain 1 (RBG1) that regulates grain and organ development, as well as abiotic stress tolerance. Ectopic expression of RBG1 leads to significant increases in the size of not only grains but also other major organs such as roots, shoots and panicles. Increased grain size is primarily due to elevated cell numbers rather than cell enlargement. RBG1 is preferentially expressed in meristematic and proliferating tissues. Ectopic expression of RBG1 promotes cell division, and RBG1 co-localizes with microtubules known to be involved in cell division, which may account for the increase in organ size. Ectopic expression of RBG1 also increases auxin accumulation and sensitivity, which facilitates root development, particularly crown roots. Moreover, overexpression of RBG1 up-regulated a large number of heat-shock proteins, leading to enhanced tolerance to heat, osmotic and salt stresses, as well as rapid recovery from water-deficit stress. Ectopic expression of RBG1 regulated by a specific constitutive promoter, GOS2, enhanced harvest index and grain yield in rice. Taken together, we have discovered that RBG1 regulates two distinct and important traits in rice, namely grain yield and stress tolerance, via its effects on cell division, auxin and stress protein induction.

EAT-Rice: A predictive model for flanking gene expression of T-DNA insertion activation-tagged rice mutants by machine learning approaches.

T-DNA activation-tagging technology is widely used to study rice gene functions. When T-DNA inserts into genome, the flanking gene expression may be altered using CaMV 35S enhancer, but the affected genes still need to be validated by biological experiment. We have developed the EAT-Rice platform to predict the flanking gene expression of T-DNA insertion site in rice mutants. The three kinds of DNA sequences including UPS1K, DISTANCE, and MIDDLE were retrieved to encode and build a forecast model of two-layer machine learning. In the first-layer models, the features nucleotide context (N-gram), cis-regulatory elements (Motif), nucleotide physicochemical properties (NPC), and CG-island (CGI) were used to build SVM models by analysing the concealed information embedded within the three kinds of sequences. Logistic regression was used to estimate the probability of gene activation which as feature-encoding weighting within first-layer model. In the second-layer models, the NaiveBayesUpdateable algorithm was used to integrate these first layer-models, and the system performance was 88.33% on 5-fold cross-validation, and 79.17% on independent-testing finally. In the three kinds of sequences, the model constructed by Middle had the best contribution to the system for identifying the activated genes. The EAT-Rice system provided better performance and gene expression prediction at further distances when compared to the TRIM database. An online server based on EAT-rice is available at http://predictor.nchu.edu.tw/EAT-Rice.

The Nucleotide-Dependent Interactome of Rice Heterotrimeric G-Protein α -Subunit.

The rice heterotrimeric G-protein complex, a guanine-nucleotide-dependent on-off switch, mediates vital cellular processes and responses to biotic and abiotic stress. Exchange of bound GDP (resting state) for GTP (active state) is spontaneous in plants including rice and thus there is no need for promoting guanine nucleotide exchange in vivo as a mechanism for regulating the active state of signaling as it is well known for animal G signaling. As such, a master regulator controlling the G-protein activation state is unknown in plants. Therefore, an ab initio approach is taken to discover candidate regulators. The rice Gα subunit (RGA1) is used as bait to screen for nucleotide-dependent protein partners. A total of 264 proteins are identified by tandem mass spectrometry of which 32 were specific to the GDP-bound inactive state and 22 specific to the transition state. Approximately, 10% are validated as previously identified G-protein interactors.

Ectopic Expression of WINDING 1 Leads to Asymmetrical Distribution of Auxin and a Spiral Phenotype in Rice.

Ectopic expression of the rice WINDING 1 (WIN1) gene leads to a spiral phenotype only in shoots but not in roots. Rice WIN1 belongs to a specific class of proteins in cereal plants containing a Bric-a-Brac/Tramtrack/Broad (BTB) complex, a non-phototropic hypocotyl 3 (NPH3) domain and a coiled-coil motif. The WIN1 protein is predominantly localized to the plasma membrane, but is also co-localized to plasmodesmata, where it exhibits a punctate pattern. It is observed that WIN1 is normally expressed in roots and the shoot-root junction, but not in the rest of shoots. In roots, WIN1 is largely localized to the apical and basal sides of cells. However, upon ectopic expression, WIN1 appears on the longitudinal sides of leaf sheath cells, correlated with the appearance of a spiral phenotype in shoots. Despite the spiral phenotype, WIN1-overexpressing plants exhibit a normal phototropic response. Although treatments with exogenous auxins or a polar auxin transport inhibitor do not alter the spiral phenotype, the excurvature side has a higher auxin concentration than the incurvature side. Furthermore, actin filaments are more prominent in the excurvature side than in the incurvature side, which correlates with cell size differences between these two sides. Interestingly, ectopic expression of WIN1 does not cause either unequal auxin distribution or actin filament differences in roots, so a spiral phenotype is not observed in roots. The action of WIN1 appears to be different from that of other proteins causing a spiral phenotype, and it is likely that WIN1 is involved in 1-N-naphthylphthalamic acid-insensitive plasmodesmata-mediated auxin transport.

Ectopic expression of specific GA2 oxidase mutants promotes yield and stress tolerance in rice.

A major challenge of modern agricultural biotechnology is the optimization of plant architecture for enhanced productivity, stress tolerance and water use efficiency (WUE). To optimize plant height and tillering that directly link to grain yield in cereals and are known to be tightly regulated by gibberellins (GAs), we attenuated the endogenous levels of GAs in rice via its degradation. GA 2-oxidase (GA2ox) is a key enzyme that inactivates endogenous GAs and their precursors. We identified three conserved domains in a unique class of C20 GA2ox, GA2ox6, which is known to regulate the architecture and function of rice plants. We mutated nine specific amino acids in these conserved domains and observed a gradient of effects on plant height. Ectopic expression of some of these GA2ox6 mutants moderately lowered GA levels and reprogrammed transcriptional networks, leading to reduced plant height, more productive tillers, expanded root system, higher WUE and photosynthesis rate, and elevated abiotic and biotic stress tolerance in transgenic rice. Combinations of these beneficial traits conferred not only drought and disease tolerance but also increased grain yield by 10-30% in field trials. Our studies hold the promise of manipulating GA levels to substantially improve plant architecture, stress tolerance and grain yield in rice and possibly in other major crops.

Genetic resources offer efficient tools for rice functional genomics research.

Rice is an important crop and major model plant for monocot functional genomics studies. With the establishment of various genetic resources for rice genomics, the next challenge is to systematically assign functions to predicted genes in the rice genome. Compared with the robustness of genome sequencing and bioinformatics techniques, progress in understanding the function of rice genes has lagged, hampering the utilization of rice genes for cereal crop improvement. The use of transfer DNA (T-DNA) insertional mutagenesis offers the advantage of uniform distribution throughout the rice genome, but preferentially in gene-rich regions, resulting in direct gene knockout or activation of genes within 20-30 kb up- and downstream of the T-DNA insertion site and high gene tagging efficiency. Here, we summarize the recent progress in functional genomics using the T-DNA-tagged rice mutant population. We also discuss important features of T-DNA activation- and knockout-tagging and promoter-trapping of the rice genome in relation to mutant and candidate gene characterizations and how to more efficiently utilize rice mutant populations and datasets for high-throughput functional genomics and phenomics studies by forward and reverse genetics approaches. These studies may facilitate the translation of rice functional genomics research to improvements of rice and other cereal crops.

A late embryogenesis abundant protein HVA1 regulated by an inducible promoter enhances root growth and abiotic stress tolerance in rice without yield penalty.

Regulation of root architecture is essential for maintaining plant growth under adverse environment. A synthetic abscisic acid (ABA)/stress-inducible promoter was designed to control the expression of a late embryogenesis abundant protein (HVA1) in transgenic rice. The background of HVA1 is low but highly inducible by ABA, salt, dehydration and cold. HVA1 was highly accumulated in root apical meristem (RAM) and lateral root primordia (LRP) after ABA/stress treatments, leading to enhanced root system expansion. Water-use efficiency (WUE) and biomass also increased in transgenic rice, likely due to the maintenance of normal cell functions and metabolic activities conferred by HVA1 which is capable of stabilizing proteins, under osmotic stress. HVA1 promotes lateral root (LR) initiation, elongation and emergence and primary root (PR) elongation via an auxin-dependent process, particularly by intensifying asymmetrical accumulation of auxin in LRP founder cells and RAM, even under ABA/stress-suppressive conditions. We demonstrate a successful application of an inducible promoter in regulating the spatial and temporal expression of HVA1 for improving root architecture and multiple stress tolerance without yield penalty.

Serotonin accumulation in transgenic rice by over-expressing tryptophan decarboxylase results in a dark brown phenotype and stunted growth.

A mutant M47286 with a stunted growth, low fertility and dark-brown phenotype was identified from a T-DNA-tagged rice mutant library. This mutant contained a copy of the T-DNA tag inserted at the location where the expression of two putative tryptophan decarboxylase genes, TDC-1 and TDC-3, were activated. Enzymatic assays of both recombinant proteins showed tryptophan decarboxylase activities that converted tryptophan to tryptamine, which could be converted to serotonin by a constitutively expressed tryptamine 5' hydroxylase (T5H) in rice plants. Over-expression of TDC-1 and TDC-3 in transgenic rice recapitulated the stunted growth, darkbrown phenotype and resulted in a low fertility similar to M47286. The degree of stunted growth and dark-brown color was proportional to the expression levels of TDC-1 and TDC-3. The levels of tryptamine and serotonin accumulation in these transgenic rice lines were also directly correlated with the expression levels of TDC-1 and TDC-3. A mass spectrometry assay demonstrated that the darkbrown leaves and hulls in the TDC-overexpressing transgenic rice were caused by the accumulation of serotonin dimer and that the stunted growth and low fertility were also caused by the accumulation of serotonin and serotonin dimer, but not tryptamine. These results represent the first evidence that over-expression of TDC results in stunted growth, low fertility and the accumulation of serotonin, which when converted to serotonin dimer, leads to a dark brown plant color.

Using Machine Learning Approaches to Predict Target Gene Expression in Rice T-DNA Insertional Mutants.

To change the expression of the flanking genes by inserting T-DNA into the genome is commonly used in rice functional gene research. However, whether the expression of a gene of interest is enhanced must be validated experimentally. Consequently, to improve the efficiency of screening activated genes, we established a model to predict gene expression in T-DNA mutants through machine learning methods. We gathered experimental datasets consisting of gene expression data in T-DNA mutants and captured the PROMOTER and MIDDLE sequences for encoding. In first-layer models, support vector machine (SVM) models were constructed with nine features consisting of information about biological function and local and global sequences. Feature encoding based on the PROMOTER sequence was weighted by logistic regression. The second-layer models integrated 16 first-layer models with minimum redundancy maximum relevance (mRMR) feature selection and the LADTree algorithm, which were selected from nine feature selection methods and 65 classified methods, respectively. The accuracy of the final two-layer machine learning model, referred to as TIMgo, was 99.3% based on fivefold cross-validation, and 85.6% based on independent testing. We discovered that the information within the local sequence had a greater contribution than the global sequence with respect to classification. TIMgo had a good predictive ability for target genes within 20 kb from the 35S enhancer. Based on the analysis of significant sequences, the G-box regulatory sequence may also play an important role in the activation mechanism of the 35S enhancer.

Comparisons within the Rice GA 2-Oxidase Gene Family Revealed Three Dominant Paralogs and a Functional Attenuated Gene that Led to the Identification of Four Amino Acid Variants Associated with GA Deactivation Capability.

BACKGROUND: GA 2-oxidases (GA2oxs) are involved in regulating GA homeostasis in plants by inactivating bioactive GAs through 2ß-hydroxylation. Rice GA2oxs are encoded by a family of 10 genes; some of them have been characterized, but no comprehensive comparisons for all these genes have been conducted. RESULTS: Rice plants with nine functional GA2oxs were demonstrated in the present study, and these genes not only were differentially expressed but also revealed various capabilities for GA deactivation based on their height-reducing effects in transgenic plants. Compared to that of wild-type plants, the relative plant height (RPH) of transgenic plants was scored to estimate their reducing effects, and 8.3% to 59.5% RPH was observed. Phylogenetic analysis of class I GA2ox genes revealed two functionally distinct clades in the Poaceae. The OsGA2ox3, 4, and 8 genes belonging to clade A showed the most severe effect (8.3% to 8.7% RPH) on plant height reduction, whereas the OsGA2ox7 gene belonging to clade B showed the least severe effect (59.5% RPH). The clade A OsGA2ox3 gene contained two conserved C186/C194 amino acids that were crucial for enzymatic activity. In the present study, these amino acids were replaced with OsGA2ox7-conserved arginine (C186R) and proline (C194P), respectively, or simultaneously (C186R/C194P) to demonstrate their importance in planta. Another two amino acids, Q220 and Y274, conserved in OsGA2ox3 were substituted with glutamic acid (E) and phenylalanine (F), respectively, or simultaneously to show their significance in planta. In addition, through sequence divergence, RNA expression profile and GA deactivation capability analyses, we proposed that OsGA2ox1, OsGA2ox3 and OsGA2ox6 function as the predominant paralogs in each of their respective classes. CONCLUSIONS: This study demonstrates rice has nine functional GA2oxs and the class I GA2ox genes are divided into two functionally distinct clades. Among them, the OsGA2ox7 of clade B is a functional attenuated gene and the OsGA2ox1, OsGA2ox3 and OsGA2ox6 are the three predominant paralogs in the family.

Large-scale phenomics analysis of a T-DNA tagged mutant population.

Rice, Oryza sativa L., is one of the most important crops in the world. With the rising world population, feeding people in a more sustainable and environmentally friendly way becomes increasingly important. Therefore, the rice research community needs to share resources to better understand the functions of rice genes that are the foundation for future agricultural biotechnology development, and one way to achieve this goal is via the extensive study of insertional mutants. We have constructed a large rice insertional mutant population in a japonica rice variety, Tainung 67. The collection contains about 93 000 mutant lines, among them 85% with phenomics data and 65% with flanking sequence data. We screened the phenotypes of 12 individual plants for each line grown under field conditions according to 68 subcategories and 3 quantitative traits. Both phenotypes and integration sites are searchable in the Taiwan Rice Insertional Mutants Database. Detailed analyses of phenomics data, T-DNA flanking sequences, and whole-genome sequencing data for rice insertional mutants can lead to the discovery of novel genes. In addition, studies of mutant phenotypes can reveal relationships among varieties, cultivation locations, and cropping seasons.

Candidate regulators of Early Leaf Development in Maize Perturb Hormone Signalling and Secondary Cell Wall Formation When Constitutively Expressed in Rice.

All grass leaves are strap-shaped with a series of parallel veins running from base to tip, but the distance between each pair of veins, and the cell-types that develop between them, differs depending on whether the plant performs C3 or C4 photosynthesis. As part of a multinational effort to introduce C4 traits into rice to boost crop yield, candidate regulators of C4 leaf anatomy were previously identified through an analysis of maize leaf transcriptomes. Here we tested the potential of 60 of those candidate genes to alter leaf anatomy in rice. In each case, transgenic rice lines were generated in which the maize gene was constitutively expressed. Lines grouped into three phenotypic classes: (1) indistinguishable from wild-type; (2) aberrant shoot and/or root growth indicating possible perturbations to hormone homeostasis; and (3) altered secondary cell wall formation. One of the genes in class 3 defines a novel monocot-specific family. None of the genes were individually sufficient to induce C4-like vein patterning or cell-type differentiation in rice. A better understanding of gene function in C4 plants is now needed to inform more sophisticated engineering attempts to alter leaf anatomy in C3 plants.

Lack of Genotype and Phenotype Correlation in a Rice T-DNA Tagged Line Is Likely Caused by Introgression in the Seed Source.

Rice (Oryza sativa) is one of the most important crops in the world. Several rice insertional mutant libraries are publicly available for systematic analysis of gene functions. However, the tagging efficiency of these mutant resources-the relationship between genotype and phenotype-is very low. We used whole-genome sequencing to analyze a T-DNA-tagged transformant from the Taiwan Rice Insertional Mutants (TRIM) resource. The phenomics records for M0028590, one of the TRIM lines, revealed three phenotypes-wild type, large grains, and tillering dwarf-in the 12 T1 plants. Using the sequencing data for 7 plants from three generations of this specific line, we demonstrate that introgression from an indica rice variety might occur in one generation before the seed was used for callus generation and transformation of this line. In addition, the large-grain trait came from the GS3 gene of the introgressed region and the tillering dwarf phenotype came from a single nucleotide change in the D17 gene that occurred during the callus induction to regeneration of the transformant. As well, another regenerant showed completely heterozygous single-nucleotide polymorphisms across the whole genome. In addition to the known sequence changes such as T-DNA integration, single nucleotide polymorphism, insertion, deletion, chromosome rearrangement and doubling, spontaneous outcrossing occurred in the rice field may also explain some mutated traits in a tagged mutant population. Thus, the co-segregation of an integration event and the phenotype should be checked when using these mutant populations.

Source-Sink Communication: Regulated by Hormone, Nutrient, and Stress Cross-Signaling.

Communication between source organs (exporters of photoassimilates) and sink organs (importers of fixed carbon) has a pivotal role in carbohydrate assimilation and partitioning during plant growth and development. Plant productivity is enhanced by sink strength and source activity, which are regulated by a complex signaling network encompassing sugars, hormones, and environmental factors. However, key components underlying the signaling pathways that regulate source-sink communication are only now beginning to be discovered. Here, we discuss recent advances in our understanding of the molecular mechanisms regulating sugar mobilization during seed development and seedling establishment in cereals, which provide the majority of nutrition for humans. Insights into these mechanisms may provide strategies for improving crop productivity.

Increasing leaf vein density by mutagenesis: laying the foundations for C4 rice.

A high leaf vein density is both an essential feature of C4 photosynthesis and a foundation trait to C4 evolution, ensuring the optimal proportion and proximity of mesophyll and bundle sheath cells for permitting the rapid exchange of photosynthates. Two rice mutant populations, a deletion mutant library with a cv. IR64 background (12,470 lines) and a T-DNA insertion mutant library with a cv. Tainung 67 background (10,830 lines), were screened for increases in vein density. A high throughput method with handheld microscopes was developed and its accuracy was supported by more rigorous microscopy analysis. Eight lines with significantly increased leaf vein densities were identified to be used as genetic stock for the global C4 Rice Consortium. The candidate population was shown to include both shared and independent mutations and so more than one gene controlled the high vein density phenotype. The high vein density trait was found to be linked to a narrow leaf width trait but the linkage was incomplete. The more genetically robust narrow leaf width trait was proposed to be used as a reliable phenotypic marker for finding high vein density variants in rice in future screens.