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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 Hap2 LOC_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., (© 2024. The Author(s).)

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., (© 2024. The Author(s).)

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 F 4:5 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., (© 2024. The Author(s).)

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., (© 2024. The Author(s).)

Background: Direct seeding is an efficient rice cultivation practice. However, its application is often limited due to O 2 deficiency following submergence, leading to poor seed germination, seedling establishment, and consequently yield loss. Identification of genes associated with tolerance to submergence and understanding their regulatory mechanisms is the fundamental way to address this problem. Unfortunately, the molecular mechanism of rice response to submergence stress is still not well understood., Results: Here, we have performed a genome-wide identification of FCS-like zinc finger (FLZ) proteins and assessed their involvement in submergence response in rice. We identified 29 FLZ genes in rice, and the expression analysis revealed that several genes actively responded to submergence stress. Eight OsFLZ proteins interact with SnRK1A. As a case study, we demonstrated that OsFLZ18 interacted with SnRK1A and inhibited the transcriptional activation activity of SnRK1A in modulating the expression of its target gene αAmy3, a positive regulator in rice flooding tolerance. In line with this, OsFLZ18-overexpression lines displayed retarded early seedling growth and shorter coleoptile following submergence., Conclusions: These data provide the most comprehensive information of OsFLZ genes in rice, and highlight their roles in rice submergence response., (© 2021. The Author(s).)

Background: Bacterial blight (BB), caused by Xanthomonas oryzae pv. oryzae (Xoo), is one of the most devastating diseases in rice all over the world. Due to the diversity and rapid evolution of Xoo, identification and use of the non-race specific quantitative resistance QTLs has been considered the preferred strategy for effective control of this disease. Although numerous QTLs for BB resistance have been identified, they haven't been effectively used for improvement of BB resistance in rice due to their small effects and lack of knowledge on the function of genes underlying the QTLs., Results: In the present study, a genome-wide association study of BB resistance was performed in a rice core collection from South China. A total of 17 QTLs were identified to be associated with BB resistance. Among them, 13 QTLs were newly identified in the present study and the other 4 QTLs were co-localized with the previously reported QTLs or Xa genes that confer qualitative resistance to Xoo strains. Particularly, the qBBR11-4 on chromosome 11 explained the largest phenotypic variation in this study and was co-localized with the previously identified QTLs for BB and bacterial leaf streak (BLS) resistance against diverse strains in three studies, suggesting its broad-spectrum resistance and potential value in rice breeding. Through combined analysis of differential expression and annotations of the predicted genes within qBBR11-4 between two sets of rice accessions selected based on haplotypes and disease phenotypes, we identified the transcription factor OsMYB21 as the candidate gene for qBBR11-4. The OsMYB21 overexpressing plants exhibited decreased resistance to bacterial blight, accompanied with down-regulation of several defense-related genes compared with the wild-type plants., Conclusion: The results suggest that OsMYB21 negatively regulates bacterial blight resistance in rice, and this gene can be a promising target in rice breeding by using the gene editing method. In addition, the potential candidate genes for the 13 novel QTLs for BB resistance were also analyzed in this study, providing a new source for cloning of genes associated with BB resistance and molecular breeding in rice.

Background: Although panicle blast is more destructive to yield loss than leaf blast in rice, the cloned genes that function in panicle blast resistance are still very limited and the molecular mechanisms underlying panicle blast resistance remain largely unknown., Results: In the present study, we have confirmed that the three Oxalate oxidase (OXO) genes, OsOXO2, OsOXO3 and OsOXO4 from a blast-resistant cultivar BC10 function in panicle blast resistance in rice. The expression of OsOXO2, OsOXO3 and OsOXO4 were induced by panicle blast inoculation. Subcellular localization analysis revealed that the three OXO proteins are all localized in the nucleus and cytoplasm. Simultaneous silencing of OsOXO2, OsOXO3 and OsOXO4 decreased rice resistance to panicle blast, whereas the OsOXO2, OsOXO3 and OsOXO4 overexpression rice plants individually showed enhanced panicle blast resistance. More H 2 O 2 and higher expression levels of PR genes were observed in the overexpressing plants than in the control plants, while the silencing plants exhibited less H 2 O 2 and lower expression levels of PR genes compared to the control plants. Moreover, phytohormone treatment and the phytohormone signaling related gene expression analysis showed that panicle blast resistance mediated by the three OXO genes was associated with the activation of JA and ABA signaling pathways but suppression of SA signaling pathway., Conclusion: OsOXO2, OsOXO3 and OsOXO4 positively regulate panicle blast resistance in rice. The OXO genes could modulate the accumulation of H 2 O 2 and expression levels of PR gene in plants. Moreover, the OXO genes mediated panicle blast resistance could be regulated by ABA, SA and JA, and may be associated with the activation of JA and ABA signaling pathways but suppression of the SA signaling pathway.

Background: Direct seeding is an efficient cultivation technique in rice. However, poor low temperature germinability (LTG) of modern rice cultivars limits its application. Identifying the genes associated with LTG and performing molecular breeding is the fundamental way to address this issue. However, few LTG QTLs have been fine mapped and cloned so far., Results: In the present study, the LTG evaluation of 375 rice accessions selected from the Rice Diversity Panel 2 showed that there were large LTG variations within the population, and the LTG of Indica group was significantly higher than that of Japonica and Aus groups (p < 0.01). In total, eleven QTLs for LTG were identified through genome-wide association study (GWAS). Among them, qLTG&#95;sRDP2-3/qLTG&#95;JAP-3, qLTG&#95;AUS-3 and qLTG&#95;sRDP2-12 are first reported in the present study. The QTL on chromosome 10, qLTG&#95;sRDP2-10a had the largest contribution to LTG variations in 375 rice accessions, and was further validated using single segment substitution line (SSSL). The presence of qLTG&#95;sRDP2-10a could result in 59.8% increase in LTG under 15 °C low temperature. The expression analysis of the genes within qLTG&#95;sRDP2-10a region indicated that LOC&#95;Os10g22520 and LOC&#95;Os10g22484 exhibited differential expression between the high and low LTG lines. Further sequence comparisons revealed that there were insertion and deletion sequence differences in the promoter and intron region of LOC&#95;Os10g22520, and an about 6 kb variation at the 3' end of LOC&#95;Os10g22484 between the high and low LTG lines, suggesting that the sequence variations of the two genes could be the cause for their differential expression in high and low LTG lines., Conclusion: Among the 11 QTLs identified in this study, qLTG&#95;sRDP2-10a could also be detected in other three studies using different germplasm under different cold environments. Its large effect and stable expression make qLTG&#95;sRDP2-10a particularly valuable in rice breeding. The two genes, LOC&#95;Os10g22484 and LOC&#95;Os10g22520, were considered as the candidate genes underlying qLTG&#95;sRDP2-10a. Our results suggest that integrating GWAS and SSSL can facilitate identification of QTL for complex traits in rice. The identification of qLTG&#95;sRDP2-10a and its candidate genes provide a promising source for gene cloning of LTG and molecular breeding for LTG in rice.

Background: Seed dormancy directly affects the phenotype of pre-harvest sprouting, and ultimately affects the quality and yield of rice seeds. Although many genes controlling seed dormancy have been cloned from cereals, the regulatory mechanisms controlling this process are complex, and much remains unknown. The MAPK cascade is involved in many signal transduction pathways. Recently, MKK3 has been reported to be involved in the regulation of seed dormancy, but its mechanism of action is unclear., Results: We found that MKKK62-overexpressing rice lines (OE) lost seed dormancy. Further analyses showed that the abscisic acid (ABA) sensitivity of OE lines was decreased. In yeast two-hybrid experiments, MKKK62 interacted with MKK3, and MKK3 interacted with MAPK7 and MAPK14. Knock-out experiments confirmed that MKK3, MAPK7, and MAPK14 were involved in the regulation of seed dormancy. The OE lines showed decreased transcript levels of OsMFT, a homolog of a gene that controls seed dormancy in wheat. The up-regulation of OsMFT in MKK3-knockout lines (OE/mkk3) and MAPK7/14-knockout lines (OE/mapk7/mapk14) indicated that the MKKK62-MKK3-MAPK7/MAPK14 system controlled seed dormancy by regulating the transcription of OsMFT., Conclusion: Our results showed that MKKK62 negatively controls seed dormancy in rice, and that during the germination stage and the late stage of seed maturation, ABA sensitivity and OsMFT transcription are negatively controlled by MKKK62. Our results have clarified the entire MAPK cascade controlling seed dormancy in rice. Together, these results indicate that protein modification by phosphorylation plays a key role in controlling seed dormancy.

Background: Cadmium (Cd) accumulation in rice followed by transfer to the food chain causes severe health problems in humans. Breeding of low Cd accumulation varieties is one of the most economical ways to solve the problem. However, information on the identity of rice germplasm with low Cd accumulation is limited, particularly in indica, and the genetic basis of Cd accumulation in rice is not well understood., Results: Screening of 312 diverse rice accessions revealed that the grain Cd concentrations of these rice accessions ranged from 0.12 to 1.23 mg/kg, with 24 accessions less than 0.20 mg/kg. Three of the 24 accessions belong to indica. Japonica accumulated significantly less Cd than indica (p < 0.001), while tropical japonica accumulated significantly less Cd than temperate japonica (p < 0.01). GWAS in all accessions identified 14 QTLs for Cd accumulation, with 7 identified in indica and 7 identified in japonica subpopulations. No common QTL was identified between indica and japonica. The previously identified genes (OsHMA3, OsNRAMP1, and OsNRAMP5) from japonica were colocalized with QTLs identified in japonica instead of indica. Expression analysis of OsNRAMP2, the candidate gene of the novel QTL (qCd3-2) identified in the present study, demonstrated that OsNRAMP2 was mainly induced in the shoots of high Cd accumulation accessions after Cd treatment. Four amino acid differences were found in the open reading frame of OsNRAMP2 between high and low Cd accumulation accessions. The allele from low Cd accumulation accessions significantly increased the Cd sensitivity and accumulation in yeast. Subcellular localization analysis demonstrated OsNRAMP2 expressed in the tonoplast of rice protoplast., Conclusion: The results suggest that grain Cd concentrations are significantly different among subgroups, with Cd concentrations decreasing from indica to temperate japonica to tropical japonica. However, considerable variations exist within subgroups. The fact that no common QTL was identified between indica and japonica implies that there is a different genetic basis for determining Cd accumulation between indica and japonica, or that some QTLs for Cd accumulation in rice are subspecies-specific. Through further integrated analysis, it is speculated that OsNRAMP2 could be a novel functional gene associated with Cd accumulation in rice.

Background: Harvest index (HI) in rice is defined as the ratio of grain yield (GY) to biomass (BM). Although it has been demonstrated that HI is significantly related to yield and is considered as one of the most important traits in high-yielding rice breeding, HI-based high-yielding rice breeding is difficult due to its polygenic nature and insufficient knowledge on the genetic basis of HI. Therefore, searching for rice varieties with high HI and mapping genes associated with high HI can facilitate marker-assisted breeding for high HI in rice., Results: Yuexiangzhan, a popular indica cultivar with good reputation of high HI was crossed with Shengbasimiao, an indica cultivar with lower HI to develop a recombinant inbred line population, and QTL mapping for HI and its component traits was conducted. In total, five QTLs for HI, three QTLs for GY, and six QTLs for BM were detected in two-year experiments. Among the three GY QTLs, one co-located with the HI QTL on chromosome 8, while the other two co-located with the two tightly-linked BM QTLs on chromosome 3. The co-located QTLs in each of the chromosomal regions produced additive effects in the same direction. Particularly, the HI QTL on chromosome 8, qHI-8, could be detected across two years and explained 42.8% and 44.5% of the phenotypic variation, respectively. The existence of qHI-8 was confirmed by the evaluation of the near isogenic lines derived from a residual heterozygous line, and this QTL was delimitated to a 1070 kb interval by substitution mapping., Conclusion: In the present study, the detected GY QTLs overlapped with both HI QTL and BM QTL, suggesting a positive relationship between GY and HI or BM, respectively. With an understanding of the genetic basis for grain yield, harvest index and biomass, it is possible to achieve higher yield through enhancing HI and BM by pyramiding the favorable alleles for the two traits via marker-assisted selection (MAS). As qHI-8 has a large phenotypic effect on HI and expresses stably in different environments, it provides a promising target for further genetic characterization of HI and MAS of high HI in rice breeding.