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Abstract 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.

Abstract 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.

Abstract 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.

Abstract 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.

Cadmium (Cd) accumulation in rice grain is of health concern. Identifying genes involved in grain Cd accumulation and performing molecular breeding may reduce it. In this study, knockout of OsNRAMP2, a member of the NRAMP family, reduced grain Cd concentrations by more than 38%, and overexpressing OsNRAMP2 increased grain Cd concentrations by more than 50%. Physiological experiments showed that OsNRAMP2 facilitated Cd translocation from root to shoot by positively regulating Cd efflux from the vacuoles. At filling stage, OsNRAMP2 was highly expressed in all tissues except for husk, suggesting its role in Cd remobilization. Changes in OsNRAMP2 expression affected the concentrations of Fe, Mn, Zn, and Cu in grain and also affected rice growth. Phylogenetic analysis showed that the distribution of OsNRAMP2 haplotypes between japonica and indica was different. Among the four haplotypes of OsNRAMP2, Hap 1, with a 6-bp nucleotide insertion in exon 1, had grain Cd concentration at least 45.3% lower than any of the other three haplotypes. Almost all (99.3%) japonica accessions but rare indica accessions (4.44%) from the 3K sequenced rice genomes carry Hap 1 of OsNRAMP2. Our study sheds light on the molecular mechanism of grain Cd accumulation and provides a promising target for low-Cd rice breeding.

Objective To predict sonographic features of malignancy in thyroid nodules by using convolutional neural networks (CNNs) ResNet50 model. Methods A cohort of 461 patients having sonographic thyroid nodules with histology diagnosis were randomly split into training set (70%), validation set (15%) and testing set (15%). Labeled sonographic patterns of thyroid nodules were used to train CNNs ResNet50 in training set, where algorithm pipelines were used to explicitly delineate structures of interest using segmentation algorithms to measure predefined characteristics of these structures as to be predictive and to use these features to train models that predict the malignancy in thyroid nodules. The prognostic accuracy of ResNet50 model was evaluated on validation set and compared with the individual sonographic specialists in testing set. Accuracy, sensitivity, specificity, and efficiency of ResNet50 model was measured using receiver operating characteristic (ROC) curve. Results Measurements showed the evaluation indexes of ResNet50 model were as follows: accuracy: 94.39%, sensitivity: 92.45%, specificity: 96.30%, efficiency: 96.08%, F1 value: 94.23%, and AUC: 93.40%. The prognostic accuracy and other indexes of ResNet50 model was not subordinate compared to sonographic specialists (P < 0.05). Conclusion These results highlight the emerging role of deep learning techniques including CNNs in precision medicine and suggest an expanding utility for computational analysis of sonographic images in the future practice. This study showed a computational approach can be used for learning sonographic features of thyroid nodules using ResNet50 model to combine the power of adaptive machine learning and algorithms with traditional sonographic assessment.

Abstract 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.

【Objective】Accurate differentiation of indica-japonica nature in rice is of great importance in the aspects of advantage utilization and evolutionary studies of indica-japonica subspecies hybrids. Molecular markers designed based on nucleotide sequence differences between indica rice and japonica rice have been widely used to discriminate indica-japonica in rice. However, it remains unclear whether these published molecular markers for indica-japonica discrimination still show indica-japonica specificity in experimental materials with diverse genetic backgrounds; moreover, the current indica-japonica discrimination is mostly based on the comparison between the line to be tested and two control varieties, which cannot reflect the indica-japonica similarity between the line to be tested and the indica (japonica) subspecies population. Therefore, it is necessary to screen a set of molecular markers that can show indica-japonica specificity in all diverse genetic materials and to establish an indica-japonica group discrimination system to objectively discriminate indica-japonica in rice.【Method】In the Rice Diversity Germplasm Platform 2 (RDP2), which represents the genetic diversity of rice in the world, 92 rice varieties (lines) we selected that retained population genetic diversity based on the genotypes of 70000 SNP molecular markers, and screened 51 pairs of previously reported molecular markers for indica-japonica discrimination; and selected 5 indica rice and 5 japonica rice to form indica and japonica discrimination groups based on the clustering results of indica-japonica-specific molecular markers, and used indica discrimination values to quantify rice indica-japonica.【Result】Twenty-four pairs of indica-japonicaspecific molecular markers were screened out of 51 pairs (both indica/japonica rice clusters showed specific band types with a frequency of higher than 69.5%), which were evenly distributed on 12 chromosomes of rice. Based on the banding results of the indica-japonica-specific molecular markers, a clustering analysis of indica-japonica nature was performed. Ninety-two varieties (lines) could be divided into 2 clusters of indica and japonica rice; and the clustering results were in perfect consitant with the known indica-japonica nature of these varieties (lines). Based on the clustering results, a cluster discrimination system was constructed and indica discrimination values were calculated for 10 randomly selected varieties (lines). Among them, there were 1 indica-leaning line, 1 japonica-leaning line, 4 indica lines and 4 japonica lines, and the quantitative discrimination of their indica-japonica nature was accurately achieved.【Conclusion】In this study, we screened a set of molecular markers for indica-japonica identification in materials with rich genetic background, and established an indica-japonica discrimination system based on indica-japonica clusters, which can be used to discriminate indica-japonica of rice efficiently and accurately.

Grain chalkiness is the main factor determining the market value of rice. Reducing chalkiness is an important breeding goal for genetic improvement of high quality rice. Identification of QTLs or genes controlling chalkiness is the prerequisite for molecular breeding in rice. Here, we conducted a genome-wide association study to identify QTLs associated with grain chalkiness including percentage of grains with chalkiness (PGWC) and degree of endosperm chalkiness (DEC) in 450 rice accessions consisting of 300 indica and 150 japonica rice in two environments. A total of 34 QTLs were identified, including 14 QTLs for PGWC and 20 QTLs for DEC. Among them, seven QTLs were commonly identified in two environments, and eight QTLs were simultaneously related to two traits. Based on the haplotype analysis, LD decay analysis, RNA-sequencing, qRT-PCR confirmation and haplotype comparisons, four genes (LOC_Os10g36170, LOC_Os10g36260, LOC_Os10g36340 and LOC_Os10g36610) were considered as the candidate genes for qDEC-10c1w,2wj, which could be identified in both environments and had the most significant p-value among the newly identified QTLs. These results provided new insight into the genetic basis of grain chalkiness and gene resources for improving quality by molecular breeding 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.

High seedling vigor can improve the ability to compete against weeds and flooding at the seedling stage and is essential for the direct seeding of rice. Early shoot length is an important performance index in seedling vigor evaluation. However, information on the identity of rice germplasm with high seedling vigor, and the genetic basis of seedling vigor are not well understood. In this study, we have conducted a genome-wide association study using 302 international diverse rice accessions from the Rice Diversity Panel 2. Six quantitative trait loci (QTLs) were found to associate with shoot length (SL). The locus qSL2 was further analyzed for candidate gene characterization. We identified OsCPS1, which encodes CDP synthase and functions in GA (Gibberellins) biosynthesis in rice, exhibits differential expression between long and short SL accessions. Using the Nipponbare genome sequence as the reference, we identified a 36 bp deletion in the 5’ UTR of OsCPS1 in long SL accessions, which is absent in short SL accessions. GA content analysis showed that the levels of bioactive GA1 and GA4 are considerably higher in long SL accessions than in short SL accessions. Genome-wide gene expression analysis indicated the expression of some photosynthesis genes is higher in long SL accessions than in short SL accessions. In contrast, genes involved in ABA (Abscisic Acid)-activated signal pathway showed lower expression in long SL accessions. Population analysis across wild rice, indica and japonica, suggested that OsCPS1 may be under selection in japonica during domestication. The results suggest that OsCPS1 is a candidate gene underlying qSL2. These data provide a promising source for candidate genetic variation associated with seedling vigor, with practical applications in rice breeding.

Abstract Background Direct seeding is an efficient rice cultivation practice. However, its application is often limited due to O2 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.

Abstract 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 H2O2 and higher expression levels of PR genes were observed in the overexpressing plants than in the control plants, while the silencing plants exhibited less H2O2 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 H2O2 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.

Abstract 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.

High cadmium (Cd) accumulation in rice is a serious threat to human health. The genetic mechanism of Cd accumulation in rice is highly complicated. To identify the low Cd accumulation in rice germplasm, investigate the genetic mechanism underlying Cd accumulation, and mine the elite genes of significant importance for rice breeding of low Cd accumulation varieties, we performed a genome-wide association study (GWAS) for rice Cd concentration in the shoot. The rice accessions were 315 diverse indica rice accessions selected from the 1568 rice accessions with 700,000 SNPs. Within the high rate of linkage disequilibrium (LD) decay, eight QTLs related to rice Cd accumulation were identified. Transcriptomic analysis showed there were 799 differentially expressed genes (DEGs) in the root and 857 DEGs in the shoot, which are probably considered to be the cause of the significant difference in Cd accumulation between high and low Cd accumulation varieties. In qCd11-1, we detected a crucial candidate gene, LOC_Os11g11050, which encodes an initiation factor, expressed differently in the root between the high and low Cd accumulation varieties. Furthermore, under Cd treatment, the expression levels of LOC_Os11g11050 significantly decreased in both the high and low Cd accumulation varieties. Sequence comparison and qRT-PCR revealed that there were indel sequences and base substitutions in the promoter region of LOC_Os11g11050 correlated with the LOC_Os11g11050 expression level, as well as the phenotype of Cd concentration differences in shoot between the high and low Cd accumulation accessions. LOC_Os11g11050 might play important roles in Cd accumulation. The results of our study provide valuable resources for low Cd accumulation in indica varieties and the candidate functional gene, as well as molecular mechanisms for Cd accumulation in indica rice. The genetic architecture underlying Cd accumulation in indica can be used for further applying the low Cd gene existing in indica for decreasing Cd accumulation in rice.

Pericarp colors (PC) in rice are determined by the types and content of flavonoids in the pericarp. The flavonoid compounds have strong antioxidant activities and are beneficial to human health. However, the genetic basis of PC in rice is still not well-understood. In this study, a genome-wide association study (GWAS) of PC was performed in a diverse rice collection consisting of 442 accessions using different phenotyping methods in two locations over 2 years. In the whole population consisting of white and colored pericarp rice, a total of 11 quantitative trait loci (QTLs) were identified using two phenotyping methods. Among these QTLs, nine were identified using the phenotypes represented by the presence and absence of pigmentation in pericarp, while 10 were identified using phenotypes of the degree of PC (DPC), in which eight are common QTLs identified using the two phenotyping methods. Using colored rice accessions and phenotypes based on DPC, four QTLs were identified, and they were totally different from the QTLs identified using the whole population, suggesting the masking effects of major genes on minor genes. Compared with the previous studies, 10 out of the 15 QTLs are first reported in this study. Based on the differential expression analysis of the predicted genes within the QTL region by both RNA-seq and real-time PCR (RT-PCR) and the gene functions in previous studies, LOC_Os01g49830, encoding a RAV transcription factor was considered as the candidate gene underlying qPC-1, a novel QTL with a large effect in this study. Our results provide a new insight into the genetic basis of PC in rice and contribute to developing the value-added rice with optimized flavonoid content through molecular breeding.

Abstract 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

Leaf rolling and discoloration are two chilling-injury symptoms that are widely used as indicators for the evaluation of cold tolerance at the seedling stage in rice. However, the difference in cold-response mechanisms underlying these two traits remains unknown. In the present study, a cold-tolerant rice cultivar, Lijiangxintuanheigu, and a cold-sensitive cultivar, Sanhuangzhan-2, were subjected to low-temperature treatments and physiological and genome-wide gene expression analyses were conducted. Leaf rolling occurred at temperatures lower than 11 °C, whereas discoloration appeared at moderately low temperatures such as 13 °C. Chlorophyll contents in both cultivars were significantly decreased at 13 °C, but not altered at 11 °C. In contrast, the relative water content and relative electrolyte leakage of both cultivars decreased significantly at 11 °C, but did not change at 13 °C. Expression of genes associated with calcium signaling and abscisic acid (ABA) degradation was significantly altered at 11 °C in comparison with 25 °C and 13 °C. Numerous genes in the DREB, MYB, bZIP, NAC, Zinc finger, bHLH, and WRKY gene families were differentially expressed. Many aquaporin genes and the key genes in trehalose and starch synthesis were down regulated at 11 °C in comparison with 25 °C and 13 °C. These results suggest that the two chilling injury symptoms are temperature-specific and are controlled by different mechanisms. Cold-induced leaf rolling is associated with calcium and ABA signaling pathways and is regulated by multiple transcriptional regulators. The suppression of aquaporin genes and reduced accumulation of soluble sugars under cold stress results in a reduction in cellular water potential and consequently leaf rolling. Keywords: Rice (Oryza sativa L.), Cold-induced leaf yellowing, Cold-induced leaf rolling, Physiological analysis, Genome-wide expression profiling

Abstract 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.

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.

Abstract 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.

Abstract 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.

Abstract 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.

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.

With rising living standards, there is an increasing demand for high-quality rice. Rice quality is mainly defined by milling quality, appearance quality, cooking and eating quality, and nutrition quality. Among them, chalkiness is a key trait for appearance quality, which adversely affects cooking and eating quality, head rice yield, and commercial value. Therefore, chalkiness is undesirable, and reducing chalkiness is a major goal in rice quality improvement. However, chalkiness is a complex trait jointly influenced by genetic and environmental factors, making its genetic study and precision improvement a huge challenge. With the rapid development of molecular techniques, much knowledge has been gained about the genes and molecular networks involved in chalkiness formation. The present review describes the major environmental factors affecting chalkiness and summarizes the quantitative trait loci (QTL) associated with chalkiness. More than 150 genes related to chalkiness formation have been reported. The functions of the genes regulating chalkiness, primarily those involved in starch synthesis, storage protein synthesis, transcription regulation, organelle development, grain shape regulation, and high-temperature response, are described. Finally, we identify the challenges associated with genetic improvement of chalkiness and suggest potential strategies. Thus, the review offers insight into the molecular dynamics of chalkiness and provides a strong basis for the future breeding of high-quality rice varieties. [ABSTRACT FROM AUTHOR]