The 14-3-3 protein OsGF14f interacts with OsbZIP23 and enhances its activity to confer osmotic stress tolerance in rice.

Drought, which can induce osmotic stress, is the leading environmental constraint on crop productivity. Plants in both agricultural and natural settings have developed various mechanisms to cope with drought stress. The identification of genes associated with drought stress tolerance and understanding the underlying regulatory mechanisms are prerequisites for developing molecular manipulation strategies to address this issue. Here, we reported that the G-BOX FACTOR 14-3-3f (14-3-3 protein OsGF14f) positively modulates osmotic stress tolerance in rice (Oryza sativa). OsGF14f transgenic lines had no obvious change in crucial agronomic traits including yield and plant height. OsGF14f is transcriptionally induced by PEG treatment, and in rice, overexpression or knockout of this gene leads to enhanced or weakened osmotic stress tolerance, respectively. Furthermore, OsGF14f positively regulates abscisic acid (ABA) responses by interacting with the core ABA-responsive transcription factor BASIC LEUCINE ZIPPER 23 (OsbZIP23) to enhance its transcriptional regulation activity toward downstream target genes. Further genetic analysis showed that OsGF14f is required for the full function of OsbZIP23 in rice osmotic response, and OsGF14f-mediated osmotic stress tolerance partially depends on OsbZIP23. Interestingly, OsGF14f is a direct target gene of OsbZIP23. Taken together, our findings reveal a genetic and molecular framework by which the OsGF14f-OsbZIP23 complex modulates rice osmotic response, providing targets for developing drought-tolerant crops.

OsFLZ2 interacts with OsMADS51 to fine-tune rice flowering time.

Flowering time is an important agronomic trait affecting crop yield. FCS-LIKE ZINC FINGER (FLZ) proteins are plant-specific regulatory proteins that are involved in multiple biological processes. However, their roles in plant flowering time control have not been clarified. Here, we report that OsFLZ2 is a negative regulator of rice flowering time. OsFLZ2 delays flowering by repressing the expression of key floral integrator genes. Biochemical assays showed OsFLZ2 physically interacts with OsMADS51, a flowering activator under short-day (SD) conditions. Both OsFLZ2 and OsMADS51 are highly expressed in rice leaves before floral transition under natural SD conditions, and their proteins are colocalized in the nucleus. Co-expression of OsFLZ2 can destabilize OsMADS51 and weaken its transcriptional activation of the downstream target gene Early heading date 1 (Ehd1). Taken together, these results indicate that OsFLZ2 can interfere with the function of OsMADS51 to fine-tune rice flowering time.

Genome-wide association mapping combined with gene-based haplotype analysis identify a novel gene for shoot length in rice (Oryza sativa L.).

KEY MESSAGE: Genome-wide association mapping revealed a novel QTL for shoot length across multiple environments. Its causal gene, LOC_Os01g68500, was identified firstly through gene-based haplotype analysis, gene expression and knockout transgenic verification. Strong seedling vigor is an important breeding target for rice varieties used in direct seeding. Shoot length (SL) is one of the important traits associated with seedling vigor characterized by rapid growth of seedling, which enhance seedling emergence. Therefore, mining genes for SL and conducting molecular breeding help to develop varieties for direct seeding. However, few QTLs for SL have been fine mapped or cloned so far. In this study, a genome-wide association study of SL was performed in a diverse rice collection consisting of 391 accessions in two years, using phenotypes generated by different cultivation methods according to the production practice, and a total of twenty-four QTLs for SL were identified. Among them, the novel QTL qSL-1f on chromosome 1 could be stably detected across all three cultivation methods in the whole population and indica subpopulation. Through gene-based haplotype analysis of the annotated genes within the putative region of qSL-1f, and validated by gene expression and knockout transgenic experiments, LOC_Os01g68500 (i.e., Os01g0913100 in RAP-DB) was identified as the causal gene for SL, which has a single-base variation (C-to-A transversion) in its CDS region, resulting in the significant difference in SL of rice. LOC_Os01g68500 encodes a DUF538 (Domain of unknown function) containing protein, and the function of DUF538 protein gene on rice seedling growth is firstly reported in this study. These results provide a new clue for exploring the molecular mechanism regulating SL, and promising gene source for the molecular breeding in rice.

An aspartic protease 47 causes quantitative recessive resistance to rice black-streaked dwarf virus disease and southern rice black-streaked dwarf virus disease.

Rice black-streaked dwarf virus disease (RBSDVD) and southern rice black-streaked dwarf virus disease (SRBSDVD) are the most destructive viral diseases in rice. Progress is limited in breeding due to lack of resistance resource and inadequate knowledge on the underlying functional gene. Using genome-wide association study (GWAS), linkage disequilibrium (LD) decay analyses, RNA-sequencing, and genome editing, we identified a highly RBSDVD-resistant variety and its first functional gene. A highly RBSDVD-resistant variety W44 was identified through extensive evaluation of a diverse international rice panel. Seventeen quantitative trait loci (QTLs) were identified among which qRBSDV6-1 had the largest phenotypic effect. It was finely mapped to a 0.8-1.2 Mb region on chromosome 6, with 62 annotated genes. Analysis of the candidate genes underlying qRBSDV6-1 showed high expression of aspartic proteinase 47 (OsAP47) in a susceptible variety, W122, and a low resistance variety, W44. OsAP47 overexpressing lines exhibited significantly reduced resistance, while the knockout mutants exhibited significantly reduced SRBSDVD and RBSDVD severity. Furthermore, the resistant allele Hap1 of OsAP47 is almost exclusive to Indica, but rare in Japonica. Results suggest that OsAP47 knockout by editing is effective for improving RBSDVD and SRBSDVD resistance. This study provides genetic information for breeding resistant cultivars.

Overexpression of OsGF14f Enhances Quantitative Leaf Blast and Bacterial Blight Resistance in Rice.

Although it is known that rice 14-3-3 family genes are involved in various defense responses, the functions of OsGF14f in response to diseases have not been reported. Here, we showed that the transcription of OsGF14f was significantly induced by leaf blast infection, and the overexpression of OsGF14f quantitatively enhanced resistance to leaf blast and bacterial blight in rice. Further analysis showed that the expression levels of salicylic acid (SA) pathway-associated genes (PAL1, NH1, PR1a and PR10) in the OsGF14f-overexpressing plants, were higher than those in wild-type plants after inoculation with the blast isolate (Magnaporthe oryzae Barr). In addition, the expression level of OsGF14f was significantly induced after SA treatment, and higher endogenous SA levels were observed in the OsGF14f-overexpressing plants compared with that in wild-type plants, especially after blast challenge. Taken together, these results suggest that OsGF14f positively regulates leaf blast and bacterial blight resistance in rice via the SA-dependent signaling pathway.

NAC transcription factor ONAC066 positively regulates disease resistance by suppressing the ABA signaling pathway in rice.

KEY MESSAGE: This is the first time to dissect the mechanism of NACs-mediated disease resistance in plants using metabolomic approach and discover the involvement of ABA signaling pathway in NACs-mediated disease resistance. NAC transcription factors have been validated as important regulators in stress responses, but their molecular mechanisms in plant disease resistance are still largely unknown. Here we report that the NAC gene ONAC066 (LOC_Os01g09550) is significantly activated by rice blast infection. ONAC066 is ubiquitously expressed and this protein is localized in the nucleus. Overexpression of ONAC066 quantitatively enhances resistance to blast disease and bacterial blight in rice. The transcript levels of PR genes are also dramatically induced in ONAC066 overexpressing plants. Exogenous abscisic acid (ABA) strongly activates the transcription of ONAC066 in rice. Further analysis shows that overexpression of ONAC066 remarkably suppresses the expression of ABA-related genes, whereas there are no obvious differences for salicylic acid (SA) and jasmonic acid (JA)-related genes between wild-type and ONAC066 overexpressing plants. Consistently, lower endogenous ABA levels are identified in ONAC066 overexpressing plants compared with wild-type plants before and after blast inoculation, while no significant differences are observed for the SA and JA levels. Yeast one-hybrid assays demonstrate that ONAC066 directly binds to the promoters of LIP9 and NCED4 to modulate their expression. Moreover, the metabolomic study reveals that the ONAC066 overexpressing plants accumulated higher contents of soluble sugars and amino acids both before and after pathogen attack, when compared to wild-type plants. Taken together, our results suggest that ONAC066 positively regulates rice resistance to blast and bacterial blight, and ONAC066 exerts its functions on disease resistance by modulating of ABA signaling pathway, sugars and amino acids accumulation in rice.

OsWRKY67 positively regulates blast and bacteria blight resistance by direct activation of PR genes in rice.

BACKGROUND: WRKY proteins are one of the largest gene families and are well-known for their regulatory roles in many aspects of plant development, including plant response to both biotic and abiotic stresses. Although the roles of WRKY proteins in leaf blast resistance have been well-documented in rice, their functions in panicle blast, the most destructive type of blast disease, are still largely unknown. RESULTS: Here, we identified that the transcription of OsWRKY67 was strongly activated by leaf and panicle blast infection. OsWRKY67 is ubiquitously expressed and sub-localized in the nucleus. Rice plants overexpressing OsWRKY67 showed quantitatively enhanced resistance to leaf blast, panicle blast and bacterial blight. In contrast, silencing of OsWRKY67 increased the susceptibility to blast and bacterial blight diseases. RNA-seq analysis indicated that OsWRKY67 induces the transcription of a set of defense-related genes including the ones involved in the salicylic acid (SA)-dependent pathway. Consistent with this, the OsWRKY67-overexpressing plants accumulated higher amounts of endogenous SA, whereas lower endogenous SA levels were observed in OsWRKY67-silenced plants relative to wild-type Nipponbare plants before and after pathogen attack. Moreover, we also observed that OsWRKY67 directly binds to the promoters of PR1a and PR10 to activate their expression. CONCLUSIONS: These results together suggest the positive role of OsWRKY67 in regulating rice responses to leaf blast, panicle blast and bacterial blight disease. Furthermore, conferring resistance to two major diseases makes it a good target of molecular breeding for crop improvement in rice.

A novel functional gene associated with cold tolerance at the seedling stage in rice.

Identification and cloning of cold-tolerant genes that can stably express under different cold environments are crucial for molecular rice breeding for cold tolerance. In the previous study, we identified a cold-tolerant QTL at the seedling stage, qCTS-9 which could be detected under different cold environments using a recombinant inbred line (RIL) population derived from a cold-tolerant variety Lijiangxintuanheigu (LTH) and a cold-sensitive variety Shanhuangzhan 2 (SHZ-2). In this study, eight candidate genes within the qCTS-9 interval were identified through integrated analysis of QTL mapping with genomewide differential expression profiling of LTH. The qRT-PCR assay showed that only Os09g0410300 exhibited different expression patterns between LTH and SHZ-2 during cold stress, and significantly positive correlation was found between cold induction of Os09g0410300 and seedling cold tolerance in the RI lines. Five SNPs and one InDel in the promoters of Os09g0410300 were detected between LTH and SHZ-2, and the InDel marker ID410300 designed based on the insertion-deletion polymorphism in the promoter was significantly associated with seedling cold tolerance in RIL population. Further, Os09g0410300 over-expression plants exhibited enhanced cold tolerance at the seedling stage compared with the wild-type plants. Thus, our results suggest that Os09g0410300 is the functional gene underlying qCTS-9. To our knowledge, it is a novel gene contributed to enhance cold tolerance at the seedling stage in rice. Identification of the functional gene underlying qCTS-9 and development of the gene-specific marker will facilitate molecular breeding for cold tolerance at the seedling stage in rice through transgenic approach and marker-assisted selection (MAS).

Small RNAs in regulating temperature stress response in plants.

Due to global climate change, temperature stress has become one of the primary causes of crop losses worldwide. Much progress has been made in unraveling the complex stress response mechanisms in plants, particularly in the identification of temperature stress responsive protein-coding genes. Recently discovered microRNAs (miRNAs) and endogenous small-interfering RNAs (siRN As) have also been demonstrated as important players in plant temperature stress response. Using high-throughput sequencing, many small RNAs, especially miRNAs, have been identified to be triggered by cold or heat. Subsequently, several studies have shown an important functional role for these small RNAs in cold or heat tolerance. These findings greatly broaden our understanding of endogenous small RNAs in plant stress response control. Here, we highlight new findings regarding the roles of miRNAs and siRNAs in plant temperature stress response and acclimation. We also review the current understanding of the regulatory mechanisms of small RNAs in temperature stress response, and explore the outlook for the use of these small RNAs in molecular breeding for improvement of temperature stress tolerance in plants.

OsGF14b Positively Regulates Panicle Blast Resistance but Negatively Regulates Leaf Blast Resistance in Rice.

Although 14-3-3 proteins have been reported to be involved in responses to biotic stresses in plants, their functions in rice blast, the most destructive disease in rice, are largely unknown. Only GF14e has been confirmed to negatively regulate leaf blast. We report that GF14b is highly expressed in seedlings and panicles during blast infection. Rice plants overexpressing GF14b show enhanced resistance to panicle blast but are susceptible to leaf blast. In contrast, GF14b-silenced plants show increased susceptibility to panicle blast but enhanced resistance to leaf blast. Yeast one-hybrid assays demonstrate that WRKY71 binds to the promoter of GF14b and modulates its expression. Overexpression of GF14b induces expression of jasmonic acid (JA) synthesis-related genes but suppresses expression of salicylic acid (SA) synthesis-related genes. In contrast, suppressed GF14b expression causes decreased expression of JA synthesis-related genes but activation of SA synthesis-related genes. These results suggest that GF14b positively regulates panicle blast resistance but negatively regulates leaf blast resistance, and that GF14b-mediated disease resistance is associated with the JA- and SA-dependent pathway. The different functions for 14-3-3 proteins in leaf and panicle blast provide new evidence that leaf and panicle blast resistance are controlled by different mechanisms.

The germin-like protein OsGLP2-1 enhances resistance to fungal blast and bacterial blight in rice.

KEY MESSAGE: This is the first report that GLP gene (OsGLP2-1) is involved in panicle blast and bacterial blight resistance in rice. In addition to its resistance to blast and bacterial blight, OsGLP2-1 has also been reported to co-localize with a QTLs for sheath blight resistance in rice. These suggest that the disease resistance provided by OsGLP2-1 is quantitative and broad spectrum. Its good resistance to these major diseases in rice makes it to be a promising target in rice breeding. Rice (Oryza sativa) blast caused by Magnaporthe oryzae and bacterial blight caused by Xanthomonas oryzae pv. oryzae are the two most destructive rice diseases worldwide. Germin-like protein (GLP) gene family is one of the important defense gene families which have been reported to be involved in disease resistance in plants. Although GLP proteins have been demonstrated to positively regulate leaf blast resistance in rice, their involvement in resistance to panicle blast and bacterial blight, has not been reported. In this study, we reported that one of the rice GLP genes, OsGLP2-1, was significantly induced by blast fungus. Overexpression of OsGLP2-1 quantitatively enhanced resistance to leaf blast, panicle blast and bacterial blight. The temporal and spatial expression analysis revealed that OsGLP2-1is highly expressed in leaves and panicles and sub-localized in the cell wall. Compared with empty vector transformed (control) plants, the OsGLP2-1 overexpressing plants exhibited higher levels of H2O2 both before and after pathogen inoculation. Moreover, OsGLP2-1 was significantly induced by jasmonic acid (JA). Overexpression of OsGLP2-1 induced three well-characterized defense-related genes which are associated in JA-dependent pathway after pathogen infection. Higher endogenous level of JA was also identified in OsGLP2-1 overexpressing plants than in control plants both before and after pathogen inoculation. Together, these results suggest that OsGLP2-1 functions as a positive regulator to modulate disease resistance. Its good quantitative resistance to the two major diseases in rice makes it to be a promising target in rice breeding.

OsGF14e positively regulates panicle blast resistance in rice.

Though GF14e has been reported to negatively regulate bacterial blight and sheath blight resistance in rice, its effect on panicle blast, the most destructive disease in rice is still unknown. In the present study, we identified that GF14e was highly expressed in panicles and was induced in panicles infected by blast pathogen. Overexpression of GF14e enhances resistance to panicle blast whereas silencing GF14e results in increased susceptibility to panicle blast, suggesting that GF14e plays a positive role in quantitative panicle blast resistance in rice. Our results also demonstrate that GF14e is regulated by WRKY71 and GF14e-mediated panicle blast resistance is related to activation of SA-dependent pathway and suppression of JA-dependent pathway. The functional confirmation of GF14e in panicle blast resistance makes it to be a promising target in molecular rice breeding.

Global transcriptional profiling of a cold-tolerant rice variety under moderate cold stress reveals different cold stress response mechanisms.

Gene expression profiling under severe cold stress (4°C) has been conducted in plants including rice. However, rice seedlings are frequently exposed to milder cold stresses under natural environments. To understand the responses of rice to milder cold stress, a moderately low temperature (8°C) was used for cold treatment prior to genome-wide profiling of gene expression in a cold-tolerant japonica variety, Lijiangxintuanheigu (LTH). A total of 5557 differentially expressed genes (DEGs) were found at four time points during moderate cold stress. Both the DEGs and differentially expressed transcription factor genes were clustered into two groups based on their expression, suggesting a two-phase response to cold stress and a determinative role of transcription factors in the regulation of stress response. The induction of OsDREB2A under cold stress is reported for the first time in this study. Among the anti-oxidant enzyme genes, glutathione peroxidase (GPX) and glutathione S-transferase (GST) were upregulated, suggesting that the glutathione system may serve as the main reactive oxygen species (ROS) scavenger in LTH. Changes in expression of genes in signal transduction pathways for auxin, abscisic acid (ABA) and salicylic acid (SA) imply their involvement in cold stress responses. The induction of ABA response genes and detection of enriched cis-elements in DEGs suggest that ABA signaling pathway plays a dominant role in the cold stress response. Our results suggest that rice responses to cold stress vary with the specific temperature imposed and the rice genotype.

Pangenome-Wide Association Study and Transcriptome Analysis Reveal a Novel QTL and Candidate Genes Controlling both Panicle and Leaf Blast Resistance in Rice.

Cultivating rice varieties with robust blast resistance is the most effective and economical way to manage the rice blast disease. However, rice blast disease comprises leaf and panicle blast, which are different in terms of resistance mechanisms. While many blast resistant rice cultivars were bred using genes conferring resistance to only leaf or panicle blast, mining durable and effective quantitative trait loci (QTLs) for both panicle and leaf blast resistance is of paramount importance. In this study, we conducted a pangenome-wide association study (panGWAS) on 9 blast resistance related phenotypes using 414 international diverse rice accessions from an international rice panel. This approach led to the identification of 74 QTLs associated with rice blast resistance. One notable locus, qPBR1, validated in a F4:5 population and fine-mapped in a Heterogeneous Inbred Family (HIF), exhibited broad-spectrum, major and durable blast resistance throughout the growth period. Furthermore, we performed transcriptomic analysis of 3 resistant and 3 sensitive accessions at different time points after infection, revealing 3,311 differentially expressed genes (DEGs) potentially involved in blast resistance. Integration of the above results identified 6 candidate genes within the qPBR1 locus, with no significant negative effect on yield. The results of this study provide valuable germplasm resources, QTLs, blast response genes and candidate functional genes for developing rice varieties with enduring and broad-spectrum blast resistance. The qPBR1, in particular, holds significant potential for breeding new rice varieties with comprehensive and durable resistance throughout their growth period.

The Function of SD1 on Shoot Length and its Pyramiding Effect on Shoot Length and Plant Height in Rice (Oryza sativa L.).

Strong seedling vigor is imperative to achieve stable seedling establishment and enhance the competitiveness against weeds in rice direct seeding. Shoot length (SL) is one of the important traits associated with seedling vigor in rice, but few genes for SL have been cloned so far. In the previous study, we identified two tightly linked and stably expressed QTLs for SL, qSL-1f and qSL-1d by genome-wide association study, and cloned the causal gene (LOC_Os01g68500) underlying qSL-1f. In the present study, we identify LOC_Os01g66100 (i.e. the semidwarf gene SD1), a well-known gene controlling plant height (PH) at the adult-plant stage, as the causal gene underlying qSL-1d through gene-based haplotype analysis and knockout transgenic verification. By measuring the phenotypes (SL and PH) of various haplotypes of the two genes and their knockout lines, we found SD1 and LOC_ Os01g68500 controlled both SL and PH, and worked in the same direction, which provided the directly genetic evidence for a positive correlation between SL and PH combined with the analysis of SL and PH in the diverse natural population. Moreover, the knockout transgenic experiments suggested that SD1 had a greater effect on PH compared with LOC_ Os01g68500, but no significant difference in the effect on SL. Further investigation of the pyramiding effects of SD1 and LOC_Os01g68500 based on their haplotype combinations suggested that SD1 may play a dominant role in controlling SL and PH when the two genes coexist. In this study, the effect of SD1 on SL at the seedling stage is validated. In total, two causal genes, SD1 and LOC_ Os01g68500, for SL are cloned in our studies, which controlled both SL and PH, and the suitable haplotypes of SD1 and LOC_ Os01g68500 are beneficial to achieve the desired SL and PH in different rice breeding objectives. These results provide a new clue to develop rice varieties for direct seeding and provide new genetic resources for molecular breeding of rice with suitable PH and strong seedling vigor.

GWAS and Transcriptomic Analysis Identify OsRING315 as a New Candidate Gene Controlling Amylose Content and Gel Consistency in Rice.

Cooking quality is the main factor determining the market value of rice. Although several major genes and a certain number of QTLs controlling cooking quality have been identified, the genetic complexity and environmental susceptibility limit the further improvement for cooking quality by molecular breeding. This research conducted a genome-wide association study to elucidate the QTLs related to cooking quality including amylose content (AC), gel consistency (GC) and alkali spreading value (ASV) by using 450 rice accessions consisting of 300 indica and 150 japonica accessions in two distinct environments. A total of 54 QTLs were identified, including 25 QTLs for AC, 12 QTLs for GC and 17 QTLs for ASV. Among them, 10 QTLs were consistently observed by the same population in both environments. Six QTLs were co-localized with the reported QTLs or cloned genes. The Wx gene for AC and GC, and the ALK gene for ASV were identified in every population across the two environments. The qAC9-2 for AC and the qGC9-2 for GC were defined to the same interval. The OsRING315 gene, encoding an E3 ubiquitin ligase, was considered as the candidate gene for both qAC9-2 and qGC9-2. The higher expression of OsRING315 corresponded to the lower AC and higher GC. Three haplotypes of OsRING315 were identified. The Hap 1 mainly existed in the japonica accessions and had lower AC. The Hap 2 and Hap 3 were predominantly present in the indica accessions, associated with higher AC. Meanwhile, the GC of accessions harboring Hap 1 was higher than that of accessions harboring Hap 3. In addition, the distribution of the three haplotypes in several rice-growing regions was unbalanced. The three traits of cooking quality are controlled by both major and minor genes and susceptible to environmental factors. The expression level of OsRING315 is related to both AC and GC, and this gene can be a promising target in quality improvement by using the gene editing method. Moreover, the haplotypes of OsRING315 differentiate between indica and japonica, and reveal the differences in GC and AC between indica and japonica rice.

A Novel Function of GW5 on Controlling the Early Growth Vigor and its Haplotype Effect on Shoot Dry Weight and Grain Size in Rice (Oryza sativa L.).

Strong early growth vigor is an essential target in both direct seeded rice breeding and high-yielding rice breeding for rice varieties with relatively short growth duration in the double-cropping region. Shoot dry weight (SDW) is one of the important traits associated with early growth vigor, and breeders have been working to improve this trait. Finding stable QTLs or functional genes for SDW is crucial for improving the early growth vigor by implementing molecular breeding in rice. Here, a genome-wide association analysis revealed that the QTL for SDW, qSDW-5, was stably detected in the three cultivation methods commonly used in production practice. Through gene-based haplotype analysis of the annotated genes within the putative region of qSDW-5, and validated by gene expression and knockout transgenic experiments, LOC_Os05g09520, which is identical to the reported GW5/GSE5 controlling grain width (GW) and thousand grain weight (TGW) was identified as the causal gene for qSDW-5. Five main haplotypes of LOC_Os05g09520 were identified in the diverse international rice collection used in this study and their effects on SDW, GW and TGW were analyzed. Phenotypic comparisons of the major haplotypes of LOC_Os05g09520 in the three subpopulations (indica, japonica and aus) revealed the same patterns of wider GW and higher TGW along with higher SDW. Furtherly, the haplotype analysis of 138 rice varieties/lines widely used in southern China showed that 97.8% of the cultivars/lines carry Hap2LOC_Os05g09520. These results not only provide a promising gene source for the molecular breeding of rice varieties with strong early growth vigor, but also elucidate the effect of the LOC_Os05g09520 haplotypes on SDW, GW, and TGW in rice. Importantly, this study provides direct genetic evidence that these three traits are significantly correlated, and suggests a breeding strategy for developing high-yielding and slender grain-shaped indica cultivars with strong early growth vigor.

Artificially Selected Grain Shape Gene Combinations in Guangdong Simiao Varieties of Rice (Oryza sativa L.).

BACKGROUND: Grain shape is a key trait in rice breeding. Although many QTLs and genes of grain shape have been identified, how different combinations of alleles of these genes affect grain shape is largely unknown. It is important to understand the effects of grain shape gene combinations for breeding by design. In the present study, we performed genetic dissection of the grain shapes in Guangdong Simiao varieties, a popular kind of rice in South China, to identify the effective alleles and their combination for breeding. RESULTS: We selected two hundred nineteen indica accessions with diverse grain shapes and fifty-two Guangdong Simiao varieties with long and slender grain shapes for genome-wide selection analysis. The results showed that four (GS3, GS5, GW5 and GL7) of the twenty grain shape genes fall into the regions selected for in Guangdong Simiao varieties. Allele analysis and frequency distribution of these four genes showed that GS3allele3 and GW5allele2 accounted for 96.2%, and GL7allele2 and GS5allele2 accounted for 76.9% and 74.5% of the Simiao varieties, respectively. Further analysis of the allelic combinations showed that 30 allelic combinations were identified in the whole panel, with 28 allelic combinations found in the international indica accessions and 6 allelic combinations found in Guangdong Simiao varieties. There were mainly three combinations (combinations 17, 18 and 19) in the Guangdong Simiao varieties, with combination 19 (GS3allele3 + GW5allele2 + GL7allele2 + GS5allele2) having the highest percentage (51.9%). All three combinations carried GS3allele3 + GW5allele2, while combinations 17 (GL7allele1) and 19 (GL7allele2) showed significant differences in both grain length and length/width ratio due to differences in GL7 alleles. Pedigree analysis of Guang8B, the maintainer of the first released Simiao male sterile line Guang8A, showed that the parent lines and Guang8B carried GS3allele3 + GW5allele2 + GS5allele2, while the GL7 allele differed, resulting in significant differences in grain size. CONCLUSION: The results suggest that specific alleles of GS3, GS5, GW5 and GL7 are the key grain shape genes used in the Guangdong Simiao varieties and selected for grain shape improvement. Combination 19 is the predominant allelic combination in the Guangdong Simiao varieties. Our current study is the first to dissect the genetics of grain shape in Guangdong Simiao varieties, and the results will facilitate molecular breeding of Guangdong Simiao varieties.

A pangenome analysis pipeline provides insights into functional gene identification in rice.

BACKGROUND: A pangenome aims to capture the complete genetic diversity within a species and reduce bias in genetic analysis inherent in using a single reference genome. However, the current linear format of most plant pangenomes limits the presentation of position information for novel sequences. Graph pangenomes have been developed to overcome this limitation. However, bioinformatics analysis tools for graph format genomes are lacking. RESULTS: To overcome this problem, we develop a novel strategy for pangenome construction and a downstream pangenome analysis pipeline (PSVCP) that captures genetic variants' position information while maintaining a linearized layout. Using PSVCP, we construct a high-quality rice pangenome using 12 representative rice genomes and analyze an international rice panel with 413 diverse accessions using the pangenome as the reference. We show that PSVCP successfully identifies causal structural variations for rice grain weight and plant height. Our results provide insights into rice population structure and genomic diversity. We characterize a new locus (qPH8-1) associated with plant height on chromosome 8 undetected by the SNP-based genome-wide association study (GWAS). CONCLUSIONS: Our results demonstrate that the pangenome constructed by our pipeline combined with a presence and absence variation-based GWAS can provide additional power for genomic and genetic analysis. The pangenome constructed in this study and the associated genome sequence and genetic variants data provide valuable genomic resources for rice genomics research and improvement in future.

Overexpression of OsGF14C enhances salinity tolerance but reduces blast resistance in rice.

High-salinity and blast disease are two major stresses that cause dramatic yield loss in rice production. GF14 (14-3-3) genes have been reported to play important role in biotic and abiotic stresses in plants. However, the roles of OsGF14C remain unknown. To understand the functions and regulatory mechanisms of OsGF14C in regulating salinity tolerance and blast resistance in rice, we have conducted OsGF14C-overexpressing transgenic experiments in the present study. Our results showed that overexpression of OsGF14C enhanced salinity tolerance but reduced blast resistance in rice. The enhanced salinity tolerance is related to the reduction of methylglyoxal and Na+ uptake instead of exclusion or compartmentation and the negative role of OsGF14C in blast resistance is associated with the suppression of OsGF14E, OsGF14F and PR genes. Our results together with the results from the previous studies suggest that the lipoxygenase gene LOX2 which is regulated by OsGF14C may play roles in coordinating salinity tolerance and blast resistance in rice. The current study for the first time revealed the possible roles of OsGF14C in regulating salinity tolerance and blast resistance in rice, and laid down a foundation for further functional study and crosstalk regulation between salinity and blast resistance in rice.