Pyramiding of gn1a, gs3, and ipa1 Exhibits Complementary and Additive Effects on Rice Yield.

Pyramiding of quantitative trait loci (QTLs) is a powerful approach in breeding super-high-yield varieties. However, the performance of QTLs in improving rice yield varies with specific genetic backgrounds. In a previous study, we employed the CRISPR/Cas9 system to target three yield-related genes, gn1a, gs3, and ipa1 in japonica 'Zhonghua 11', mutants of which featured large panicle, big grain, few sterile tillers, and thicker culm, respectively. In this paper, four pyramided lines, including gn1a-gs3, gn1a-ipa1, gs3-ipa1, and gn1a-gs3-ipa1, were further generated by conventional cross-breeding to be tested. Agronomic traits analysis showed that: (1) the stacking lines carried large panicles with an increased spikelet number in the main panicle or panicle; (2) the grain weight of the stacking lines, especially gs3-ipa1 and gn1a-gs3-ipa1, were heavier than those in single mutants; (3) both gn1a-gs3 and gs3-ipa1 produced more grain yield per plant than single mutant lines; (4) pyramided lines were higher than single mutants and transcriptome analysis found improved expression levels of genes related to lipid, amino acid, and carbohydrate transport and metabolism in lines pyramiding three mutant alleles, possibly as a result of complementary and additive effects. Accordingly, the alteration of gene-expression patterns relating to hormone signaling, plant growth, and seed size control was characterized in pyramided lines. The present study not only investigates the effects of pyramiding genes, but also may provide an efficient strategy for breeding super-high-yield rice by reducing the time cost of developing pyramided lines.

Roles of AGD2a in Plant Development and Microbial Interactions of Lotus japonicus.

Arabidopsis AGD2 (Aberrant Growth and Death2) and its close homolog ALD1 (AGD2-like defense response protein 1) have divergent roles in plant defense. We previously reported that modulation of salicylic acid (SA) contents by ALD1 affects numbers of nodules produced by Lotus japonicus, but AGD2's role in leguminous plants remains unclear. A combination of enzymatic analysis and biological characterization of genetic materials was used to study the function of AGD2 (LjAGD2a and LjAGD2b) in L. japonicus. Both LjAGD2a and LjAGD2b could complement dapD and dapE mutants of Escherichia coli and had aminotransferase activity in vitro. ljagd2 plants, with insertional mutations of LjAGD2, had delayed flowering times and reduced seed weights. In contrast, overexpression of LjAGD2a in L. japonicus induced early flowering, with increases in seed and flower sizes, but reductions in pollen fertility and seed setting rates. Additionally, ljagd2a mutation resulted in increased expression of nodulin genes and corresponding increases in infection threads and nodule numbers following inoculation with Rhizobium. Changes in expression of LjAGD2a in L. japonicus also affected endogenous SA contents and hence resistance to pathogens. Our results indicate that LjAGD2a functions as an LL-DAP aminotransferase and plays important roles in plant development. Moreover, LjAGD2a activates defense signaling via the Lys synthesis pathway, thereby participating in legume-microbe interaction.

Genome-Wide Identification, Expression Patterns and Sugar Transport of the Physic Nut SWEET Gene Family and a Functional Analysis of JcSWEET16 in Arabidopsis.

The Sugars Will Eventually be Exported Transporters (SWEET) family is a class of sugar transporters that play key roles in phloem loading, seed filling, pollen development and the stress response in plants. Here, a total of 18 JcSWEET genes were identified in physic nut (Jatropha curcas L.) and classified into four clades by phylogenetic analysis. These JcSWEET genes share similar gene structures, and alternative splicing of messenger RNAs was observed for five of the JcSWEET genes. Three (JcSWEET1/4/5) of the JcSWEETs were found to possess transport activity for hexose molecules in yeast. Real-time quantitative PCR analysis of JcSWEETs in different tissues under normal growth conditions and abiotic stresses revealed that most are tissue-specifically expressed, and 12 JcSWEETs responded to either drought or salinity. The JcSWEET16 gene responded to drought and salinity stress in leaves, and the protein it encodes is localized in both the plasma membrane and the vacuolar membrane. The overexpression of JcSWEET16 in Arabidopsis thaliana modified the flowering time and saline tolerance levels but not the drought tolerance of the transgenic plants. Together, these results provide insights into the characteristics of SWEET genes in physic nut and could serve as a basis for cloning and further functional analysis of these genes.

Target Lines for in Planta Gene Stacking in Japonica Rice.

The clustering of transgenes at a chromosome location minimizes the number of segregating loci that needs to be introgressed to field cultivars. Transgenes could be efficiently stacked through site-specific recombination and a recombinase-mediated in planta gene stacking process was described previously in tobacco based on the Mycobacteriophage Bxb1 site-specific integration system. Since this process requires a recombination site in the genome, this work describes the generation of target sites in the Japonica rice genome. Agrobacterium-mediated gene transfer yielded ~4000 random-insertion lines. Seven lines met the criteria of being single copy, not close to a centromere, not inserted within or close to a known gene or repetitive DNA, having precise recombination site sequences on both ends, and able to express the reporter gene. Each target line tested was able to accept the site-specific integration of a new gfp-containing plasmid and in three of those lines, we regenerated fertile plants. These target lines could be used as foundation lines for stacking new traits into Japonica rice.

Ectopic Expression of JcCPL1, 2, and 4 Affects Epidermal Cell Differentiation, Anthocyanin Biosynthesis and Leaf Senescence in Arabidopsis thaliana.

The CAPRICE (CPC)-like (CPL) genes belong to a single-repeat R3 MYB family, whose roles in physic nut (Jatropha curcas L.), an important energy plant, remain unclear. In this study, we identified a total of six CPL genes (JcCPL1-6) in physic nut. The JcCPL3, 4, and 6 proteins were localized mainly in the nucleus, while proteins JcCPL1, 2, and 5 were localized in both the nucleus and the cytoplasm. Ectopic overexpression of JcCPL1, 2, and 4 in Arabidopsis thaliana resulted in an increase in root hair number and decrease in trichome number. Consistent with the phenotype of reduced anthocyanin in shoots, the expression levels of anthocyanin biosynthesis genes were down-regulated in the shoots of these three transgenic A. thaliana lines. Moreover, we observed that OeJcCPL1, 2, 4 plants attained earlier leaf senescence, especially at the late developmental stage. Consistent with this, the expression levels of several senescence-associated and photosynthesis-related genes were, respectively, up-regulated and down-regulated in leaves. Taken together, our results indicate functional divergence of the six CPL proteins in physic nut. These findings also provide insight into the underlying roles of CPL transcription factors in leaf senescence.

The Temperature-Dependent Retention of Introns in GPI8 Transcripts Contributes to a Drooping and Fragile Shoot Phenotype in Rice.

Attachment of glycosylphosphatidylinositols (GPIs) to the C-termini of proteins is one of the most common posttranslational modifications in eukaryotic cells. GPI8/PIG-K is the catalytic subunit of the GPI transamidase complex catalyzing the transfer en bloc GPI to proteins. In this study, a T-DNA insertional mutant of rice with temperature-dependent drooping and fragile (df) shoots phenotype was isolated. The insertion site of the T-DNA fragment was 879 bp downstream of the stop codon of the OsGPI8 gene, which caused introns retention in the gene transcripts, especially at higher temperatures. A complementation test confirmed that this change in the OsGPI8 transcripts was responsible for the mutant phenotype. Compared to control plants, internodes of the df mutant showed a thinner shell with a reduced cell number in the transverse direction, and an inhomogeneous secondary wall layer in bundle sheath cells, while many sclerenchyma cells at the tops of the main veins of df leaves were shrunken and their walls were thinner. The df plants also displayed a major reduction in cellulose and lignin content in both culms and leaves. Our data indicate that GPI anchor proteins play important roles in biosynthesis and accumulation of cell wall material, cell shape, and cell division in rice.

Characterization of miRNA profiles in the mammary tissue of dairy cattle in response to heat stress.

BACKGROUND: MicroRNAs (miRNAs) are a class of small noncoding RNAs that play important roles in the regulation of gene expression. However, the role of miRNAs in bovine mammary gland responses to heat stress is not well understood. RESULTS: In the present study, we performed a deep RNA sequencing analysis to identify miRNAs associated with the heat stress potential of the bovine mammary gland. We identified 27 miRNAs that were differentially expressed significantly between the mammary tissue of Holstein cattle heat stress and normal conditions. Twenty miRNAs had higher expression in the mammary tissue of heat-stressed Holstein cattle. The seven highest differentially expressed candidate miRNAs (bta-miR-21-5p, bta-miR-99a-5p, bta-miR-146b, bta-miR-145, bta-miR-2285 t, bta-miR-133a, and bta-miR-29c) identified by deep RNA sequencing were additionally evaluated by stem-loop qPCR. Enrichment analyses for targeted genes revealed that the major differences between miRNAs expression in the mammary gland of heat-stressed versus control were associated with the regulation of Wnt, TGF-ß, MAPK, Notch, and JAK-STAT. CONCLUSIONS: These data indicated that the differentially expressed miRNAs identified in this study may act as dominant regulators during heat stress. We might reduce heat stress damage of Holstein cows by up-regulating or down-regulating these differentially expressed miRNAs.

The Phenylalanine Ammonia Lyase Gene LjPAL1 Is Involved in Plant Defense Responses to Pathogens and Plays Diverse Roles in Lotus japonicus-Rhizobium Symbioses.

Phenylalanine ammonia lyase (PAL) is important in the biosynthesis of plant secondary metabolites that regulate growth responses. Although its function is well-established in various plants, the functional significance of PAL genes in nodulation is poorly understood. Here, we demonstrate that the Lotus japonicus PAL (LjPAL1) gene is induced by Mesorhizobium loti infection and methyl-jasmonate (Me-JA) treatment in roots. LjPAL1 altered PAL activity, leading to changes in lignin contents and thicknesses of cell walls in roots and nodules of transgenic plants and, hence, to structural changes in roots and nodules. LjPAL1-knockdown plants (LjPAL1i) exhibited increased infection thread and nodule numbers and the induced upregulation of nodulin gene expression after M. loti infection. Conversely, LjPAL1 overexpression delayed the infection process and reduced infection thread and nodule numbers after M. loti inoculation. LjPAL1i plants also exhibited reduced endogenous salicylic acid (SA) accumulation and expression of the SA-dependent marker gene. Their infection phenotype could be partially restored by exogenous SA or Me-JA application. Our data demonstrate that LjPAL1 plays diverse roles in L. japonicus-rhizobium symbiosis, affecting rhizobial infection progress and nodule structure, likely by inducing lignin modification, regulating endogenous SA biosynthesis, and modulating SA signaling.

Integrated genome sequence and linkage map of physic nut (Jatropha curcas L.), a biodiesel plant.

The family Euphorbiaceae includes some of the most efficient biomass accumulators. Whole genome sequencing and the development of genetic maps of these species are important components in molecular breeding and genetic improvement. Here we report the draft genome of physic nut (Jatropha curcas L.), a biodiesel plant. The assembled genome has a total length of 320.5 Mbp and contains 27,172 putative protein-coding genes. We established a linkage map containing 1208 markers and anchored the genome assembly (81.7%) to this map to produce 11 pseudochromosomes. After gene family clustering, 15,268 families were identified, of which 13,887 existed in the castor bean genome. Analysis of the genome highlighted specific expansion and contraction of a number of gene families during the evolution of this species, including the ribosome-inactivating proteins and oil biosynthesis pathway enzymes. The genomic sequence and linkage map provide a valuable resource not only for fundamental and applied research on physic nut but also for evolutionary and comparative genomics analysis, particularly in the Euphorbiaceae.

Global analysis of gene expression profiles in physic nut (Jatropha curcas L.) seedlings exposed to drought stress.

BACKGROUND: Physic nut (Jatropha curcas L.) is a small perennial tree or large shrub, which is well-adapted to semi-arid regions and is considered to have potential as a crop for biofuel production. It is now regarded as an excellent model for studying biofuel plants. However, our knowledge about the molecular responses of this species to drought stress is currently limited. RESULTS: In this study, genome-wide transcriptional profiles of roots and leaves of 8-week old physic nut seedlings were analyzed 1, 4 and 7 days after withholding irrigation. We observed a total of 1533 and 2900 differentially expressed genes (DEGs) in roots and leaves, respectively. Gene Ontology analysis showed that the biological processes enriched in droughted plants relative to unstressed plants were related to biosynthesis, transport, nucleobase-containing compounds, and cellular protein modification. The genes found to be up-regulated in roots were related to abscisic acid (ABA) synthesis and ABA signal transduction, and to the synthesis of raffinose. Genes related to ABA signal transduction, and to trehalose and raffinose synthesis, were up-regulated in leaves. Endoplasmic reticulum (ER) stress response genes were significantly up-regulated in leaves under drought stress, while a number of genes related to wax biosynthesis were also up-regulated in leaves. Genes related to unsaturated fatty acid biosynthesis were down-regulated and polyunsaturated fatty acids were significantly reduced in leaves 7 days after withholding irrigation. As drought stress increased, genes related to ethylene synthesis, ethylene signal transduction and chlorophyll degradation were up-regulated, and the chlorophyll content of leaves was significantly reduced by 7 days after withholding irrigation. CONCLUSIONS: This study provides us with new insights to increase our understanding of the response mechanisms deployed by physic nut seedlings under drought stress. The genes and pathways identified in this study also provide much information of potential value for germplasm improvement and breeding for drought resistance.

Knockdown of LjALD1, AGD2-like defense response protein 1, influences plant growth and nodulation in Lotus japonicus.

The discovery of the enzyme L,L-diaminopimelate aminotransferase (LL-DAP-AT, EC 2.6.1.83) uncovered a unique step in the L-lysine biosynthesis pathway in plants. In Arabidopsis thaliana, LL-DAP-AT has been shown to play a key role in plant-pathogen interactions by regulation of the salicylic acid (SA) signaling pathway. Here, a full-length cDNA of LL-DAP-AT named as LjALD1 from Lotus japonicus (Regel) Larsen was isolated. The deduced amino acid sequence shares 67% identity with the Arabidopsis aminotransferase AGD2-LIKE DEFENSE RESPONSE PROTEIN1 (AtALD1) and is predicted to contain the same key elements: a conserved aminotransferase domain and a pyridoxal-5'-phosphate cofactor binding site. Quantitative real-time PCR analysis showed that LjALD1 was expressed in all L. japonicus tissues tested, being strongest in nodules. Expression was induced in roots that had been infected with the symbiotic rhizobium Mesorhizobium loti or treated with SA agonist benzo-(1, 2, 3)-thiadiazole-7-carbothioic acid. LjALD1 Knockdown exhibited a lower SA content, an increased number of infection threads and nodules, and a slight reduction in nodule size. In addition, compared with wild-type, root growth was increased and shoot growth was suppressed in LjALD1 RNAi plant lines. These results indicate that LjALD1 may play important roles in plant development and nodulation via SA signaling in L. japonicus.

RD29A promoter constitutively drives a rice Hd3a expression to promote early-flowering in Saussurea involucrate Kar. et Kir. ex Maxim.

S. involucratae, an endemic and endangered plant, is a valuable and traditional Chinese medicinal herb. In order to control the flowering time of S. involucratae, we used the well-known stress inducible RD29A promoter to drive Hd3a (a FT ortholog from rice) expression in S. involucratae. Unexpectedly, the majority of regenerated buds in RD29A::Hd3a transgenic lines (S-RH) produced flowers in tissue culture stage under normal growth (25 ± 2 °C) condition. Their flowering time was not further influenced by salt treatment. Hd3a in S-RH was strongly expressed in MS media supplemented with or without 50 mM NaCl. RD29A::GUS transgenic experiments further revealed that RD29A constitutively promoted GUS expression in both S. involucrate and halophyte Thellungiella halophile, in contrast to glycophic plants Oryza sativa L. 'Zhonghua 11', in which its expression was up-regulated by cold, salinity, and drought stress. The results supported the hypothesis that RD29A promoter activity is inducible in stress-sensitive plants, but constitutive in stress-tolerant ones. Importantly, S-RH plants produced pollen grains and seeds under normal conditions. Additionally, we found that OsLEA3-1::Hd3a and HSP18.2::Hd3a could not promote S. involucrate to flower under either normal conditions or abiotic stresses. Taken together, we demonstrated the potential of RD29A::Hd3a might be served as a feasible approach in breeding S. involucrate under normal condition.

Highly stretchable, injectable hydrogels with cyclic endurance and shape-stability in dynamic mechanical environments, by microunit reformation.

Traditional injectable hydrogels have so far found it difficult to accommodate resistance to large deformation and shape-stability under cyclic deformation. Polyampholyte (PA) hydrogels exhibit resistance to large deformation, good fatigue-resistance and rapid self-healing under dynamic forces. The limitations of the preparation process result in non-injectability of polyampholyte (PA) hydrogels. Electrostatic interactions as a medium for resistance to large deformation and shape-stability after cyclic deformation in reformed injectable hydrogels has been explored in this study. The prepared hydrogels (as-prepared PA-N) were dried and smashed into microunits and then mixed with 0.9% NaCl solution to transform them into reformed hydrogels (as-reformed PA-N) via a needle to achieve injectability. The as-reformed PA-N could exhibit 913.6% elongation at break and showed shape-stability under cyclic deformation due to the efficient self-healing abilities of the microunits and the inherited structure of the prepared hydrogels, which are superior to those of current tough injectable hydrogels. Potential applications in elbow cyclic bending and frequent movement of mobile wounds have been proved in this study. Overall, the results showed that the as-reformed PA-N achieved convenient injectability with resistance to large deformation and shape-stability under cyclic deformation at the same time.

Microstructure-united heterogeneous sodium alginate doped injectable hydrogel for stable hemostasis in dynamic mechanical environments.

Injectable hydrogels that can withstand compressive and tensile forces hold great promise for preventing rebleeding in dynamic mechanical environments after emergency hemostasis of wounds. However, current injectable hydrogels often lack sufficient compressive or tensile performance. Here, a microstructure-united heterogeneous injectable hydrogel (MH) was constructed. The heterogeneous structure endowed MH with a unique "microstructures consecutive transmission" feature, which allowed it to exhibit high compressive and tensile performance simultaneously. In this work, two types of sodium alginate doped hydrogels with different microstructures were physically smashed into microgels, respectively. By mixing the microgels, MH with one micro-pores featured microstructure and another nano-pores featured microstructure can be formed. The obtained MH can withstand both compressive and tensile forces and showed high mechanical performance (compressive modulus: 345.67 ± 10.12 kPa and tensile modulus: 245.19 ± 7.82 kPa). Furtherly, MH was proven to provide stable and sustained hemostasis in the dynamic mechanical environment. Overall, this work provided an effective strategy for constructing injectable hydrogel with high compressive and tensile performance for hemostasis in dynamic mechanical environments.

Rapid generation of rice mutants via the dominant negative suppression of the mismatch repair protein OsPMS1.

Mismatch repair (MMR) is a conservative pathway for maintaining the genome integrity of different organisms. Although suppression of MMR has resulted in various mutation phenotypes in Arabidopsis, the use of this strategy for mutation breeding in major crops has not been reported. Here, we overexpressed a truncated version of the OsPMS1 protein in rice; this approach is expected to suppress the rice MMR system through a dominant negative mechanism. We observed a wide spectrum of mutation phenotypes in the progeny of the transgenic plants during seed germination and the plant growth stages. Genomic variations were detected with inter-simple sequence repeat (ISSR), and sequencing of the differential ISSR bands revealed that the mutation occurred as a point mutation or as microsatellite instability at high frequencies. Plant lines with agronomically important traits, such as salt and drought tolerance, various tiller number, and early flowering, were obtained. Furthermore, we obtained mutants with important traits that are free of the transgene. Together, these results demonstrate that MMR suppression can be used as an efficient strategy for mutation breeding in rice.

Transcriptional Regulations on the Low-Temperature-Induced Floral Transition in an Orchidaceae Species, Dendrobium nobile: An Expressed Sequence Tags Analysis.

Vernalization-induced flowering is a cold-relevant adaptation in many species, but little is known about the genetic basis behind in Orchidaceae species. Here, we reported a collection of 15017 expressed sequence tags (ESTs) from the vernalized axillary buds of an Orchidaceae species, Dendrobium nobile, which were assembled for 9616 unique gene clusters. Functional enrichment analysis showed that genes in relation to the responses to stresses, especially in the form of low temperatures, and those involving in protein biosynthesis and chromatin assembly were significantly overrepresented during 40 days of vernalization. Additionally, a total of 59 putative flowering-relevant genes were recognized, including those homologous to known key players in vernalization pathways in temperate cereals or Arabidopsis, such as cereal VRN1, FT/VRN3, and Arabidopsis AGL19. Results from this study suggest that the networks regulating vernalization-induced floral transition are conserved, but just in a part, in D. nobile, temperate cereals, and Arabidopsis.

Ectopic expression of WRINKLED1 in rice improves lipid biosynthesis but retards plant growth and development.

WRINKLED1 (WRI1) is a transcription factor which is key to the regulation of seed oil biosynthesis in Arabidopsis. In the study, we identified two WRI1 genes in rice, named OsWRI1a and OsWRI1b, which share over 98% nucleotide similarity and are expressed only at very low levels in leaves and endosperms. The subcellular localization of Arabidopsis protoplasts showed that OsWRI1a encoded a nuclear localized protein. Overexpression of OsWRI1a under the control of the CaMV 35S promoter severely retarded plant growth and development in rice. Expressing the OsWRI1a gene under the control of the P1 promoter of Brittle2 (highly expressed in endosperm but low in leaves and roots) increased the oil content of both leaves and endosperms and upregulated the expression of several genes related to late glycolysis and fatty acid biosynthesis. However, the growth and development of the transgenic plants were also affected, with phenotypes including smaller plant size, later heading time, and fewer and lighter grains. The laminae (especially those of flag leaves) did not turn green and could not unroll normally. Thus, ectopic expression of OsWRI1a in rice enhances oil biosynthesis, but also leads to abnormal plant growth and development.

Ectopic expression of FaDREB2 enhances osmotic tolerance in paper mulberry.

Dehydration-responsive element binding (DREB) proteins are a subfamily of AP2/ERF transcription factors that have been shown to improve tolerance to osmotic stresses in plants. To improve the osmotic stress tolerance of paper mulberry (Broussonetia papyrifera L. Vent), an economically important tree, we transformed it with a plasmid carrying tall fescue (Festuca arundinacea Schreb) FaDREB2 under the control of CaMV 35S. The ectopic expression of FaDREB2 did not cause growth retardation, and the paper mulberry seedlings expressing FaDREB2 showed higher salt and drought tolerance than wild-type plants (WT). After 13 d of withholding water, or 15 d in the presence of 250 mM NaCl, all the WT plants died, while the plants expressing FaDREB2 survived. The FaDREB2 transgenic plants had higher leaf water and chlorophyll contents, accumulated more proline and soluble sugars, and had less membrane damage than the WT plants under high salt and water-deficient conditions. Taken together, the results indicate the feasibility of improving tolerance to multiple environmental stresses in paper mulberry seedlings via genetic engineering, by introducing FaDREB2, which promotes the increased accumulation of osmolytes (soluble sugars and proline), to counter osmotic stresses caused by abiotic factors.

Overexpression of SGR results in oxidative stress and lesion-mimic cell death in rice seedlings.

It is thought that the Stay Green Rice (SGR) gene is involved in the disaggregation of the light harvesting complex and in the subsequent breakdown of chlorophyll and apo-protein during senescence. In this study, we found that overexpression of SGR (Ov-SGR) resulted in the generation of singlet oxygen and other reactive oxygen species and produced a chlorophyll-dependent regional cell death phenotype on leaves of rice seedlings. Transcriptome analyses using Affymetrix Rice GeneChips revealed that Ov-SGR rice seedlings exhibited a number of signs of singlet oxygen response. The genes and their associated biochemical pathways identified provide an insight into how rice plants respond to singlet oxygen at the molecular and physiologic level.

Monitoring homologous recombination in rice (Oryza sativa L.).

Here we describe a system to assay homologous recombination during the complete life cycle of rice (Oryza sativa L.). Rice plants were transformed with two copies of non-functional GUS reporter overlap fragments as recombination substrate. Recombination was observed in all plant organs examined, from the seed stage until the flowering stage of somatic plant development. Embryogenic cells exhibited the highest recombination ability with an average of 3x10(-5) recombination events per genome, which is about 10-fold of that observed in root cells, and two orders of that observed in leaf cells. Histological analysis revealed that recombination events occurred in diverse cell types, but preferentially in cells with small size. Examples of this included embryogenic cells in callus, phloem cells in the leaf vein, and cells located in the root apical meristem. Steady state RNA analysis revealed that the expression levels of rice Rad51 homologs are positively correlated with increased recombination rates in embryogenic calli, roots and anthers. Finally, radiation treatment of plantlets from distinct recombination lines increased the recombination frequency to different extents. These results showed that homologous recombination frequency can be effectively measured in rice using a transgene reporter assay. This system will facilitate the study of DNA damage signaling and homologous recombination in rice, a model monocot.