GWAS Combined with WGCNA of Transcriptome and Metabolome to Excavate Key Candidate Genes for Rice Anaerobic Germination.

Direct seeding of rice is a lightweight and simple cultivation method, which can effectively promote rice production. Anaerobic germination tolerance is one of the main traits of rice adaptability to direct seeding. The mining of related genetic loci, analysis of anaerobic traits and screening of tolerance genes provided valuable genetic resources for improving the anaerobic germination ability of direct seeding rice. This study conducted a dynamic genome-wide association study (GWAS) based on coleoptile-related traits of 591 rice natural populations, and a total of 317 SNP sites were detected. Integrated dynamic widely targeted metabolomics analysis, we found that xanthine, L-alanine and GABA may be key biomarkers that are sensitive and respond strongly to hypoxic stress perception. By WGCNA analysis of targeted metabolomics and transcriptomics, a total of 3 modules were obtained that were significantly correlated with the above three marker metabolites, namely dark green, dark gray and light green modules, respectively, and several key structural genes of OsAlaAT1, OsGAD4, OsAAH and Os09g0424600 that may affect hypoxic germination were screened from the 3 modules. Among them, OsAlaAT1 (Os10g0390500), located in Chr10-12877840, which is within the GWAS location range of CVAN3d, is considered to be a more reliable candidate gene. Overall, in addition to providing new insight into the metabolic regulation of L-alanine, GABA and xanthine during hypoxic germination of rice. This study also provided a reference for the basic theoretical research and breeding application research on the related traits of anaerobic germination in direct-seeding rice.

Small Auxin Up RNA 56 (SAUR56) regulates heading date in rice.

Heading date is a critical agronomic trait that determines crop yield. Although numerous genes associated with heading date have been identified in rice, the mechanisms involving Small Auxin Up RNA (SAUR) family have not been elucidated. In this study, the biological function of several SAUR genes was initially investigated using the CRISPR-Cas9 technology in the Japonica cultivar Zhonghua11 (ZH11) background. Further analysis revealed that the loss-of-function of OsSAUR56 affected heading date in both NLD (natural long-day) and ASD (artificial short-day). OsSAUR56 exhibited predominant expression in the anther, with its protein localized in both the cytoplasm and nucleus. OsSAUR56 regulated flowering time and heading date by modulating the expression of the clock gene OsGI, as well as two repressors Ghd7 and DTH8. Furthermore, haplotype-phenotype association analysis revealed a strong correlation between OsSAUR56 and heading date, suggesting its role in selection during the domestication of rice. In summary, these findings highlights the importance of OsSAUR56 in the regulation of heading date for further potential facilitating genetic engineering for flowering time during rice breeding. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-023-01409-w.

A new method for mutation inducing in rice by using DC electrophoresis bath and its mutagenic effects.

Mutation breeding is a significant means of increasing breeding efficiency and accelerating breeding process. In present study, we explored a new method for mutations inducing in rice (Oryza sativa L.) by using direct current electrophoresis bath (DCEB). The results showed that 20 mM NaCl solution is the optimal buffer, and the mortality of rice seeds followed an upward trend with increasing voltage and processing time of DCEB. By exploring the mutagenic effects of γ-irradiation and DCEB on seed vigor and physiological damages, we found that the physiological damages induced by DCEB on seed vigor were significant compared with that by γ-irradiation. We screened two mutants with low filled grain percentage and one mutant with abnormal hull from the M2 generations. These three mutants were confirmed to be authentic mutants based on 48 SSR markers followed by the protocol NY/T 1433-2014. Whole-genome resequencing detected a total of 503 and 537 polymorphisms in the two mutants, respectively, and the DCEB mutagenesis induced mainly InDel variants, while the exon region of mutant genes occupied a large proportion, especially the SNP variants, which occupied about 20% of the mutation sites in the exon region.

Fine Mapping of the Affecting Tillering and Plant Height Gene CHA-1 in Rice.

The plant architecture of rice is an important factor affecting yield. Strigolactones (SLs) are newly discovered carotenoid-derived plant hormones that play an important role in rice plant architecture. In this study, a high-tillering dwarf mutant, CHA-1, was identified by spatial mutagenesis. CHA-1 was located in the region of 31.52-31.55 MB on chromosome 1 by map-based cloning. Compared with the wild-type THZ, the CHA-1 mutant showed that ACCAC replaced TGGT in the coding region of the candidate gene LOC_Os01g54810, leading to premature termination of expression. Genetic complementation experiments proved that LOC_Os01g54810 was CHA-1, which encodes a putative member of Class III lipase. Expression analysis showed that CHA-1 was constitutively expressed in various organs of rice. Compared with those in THZ, the expression levels of the D17 and D10 genes were significantly downregulated in the CHA-1 mutant. In addition, the concentrations of ent-2'-epi-5-deoxystrigol (epi-5DS) in the root exudates of the CHA-1 mutant was significantly reduced compared with that of THZ, and exogenous application of GR24 inhibited the tillering of the CHA-1 mutant. These results suggest that CHA-1 influences rice architecture by affecting SL biosynthesis.

The DnaJ domain-containing heat-shock protein NAL11 determines plant architecture by mediating gibberellin homeostasis in rice (Oryza sativa).

Ideal Plant Architecture 1 (IPA1) is a key regulator of plant architecture. However, knowledge of downstream genes applicable for improving rice plant architecture is very limited. We identified the plant architecture regulatory gene NARROW LEAF 11 (NAL11), which encodes a heat-shock protein (HSP) containing a DnaJ domain. A promising rare allele of NAL11 (NAL11-923del-1552 ) positively selected in Aus cultivars was identified; this allele exhibited increased expression and generated relatively few tillers, thick stems, and large panicles, components of the ideal plant architecture (IPA). NAL11 is involved in regulating the cell cycle and cell proliferation. NAL11 loss-of-function mutants present impaired chloroplast development and gibberellin (GA) defects. Biochemical analyses show that IPA1 directly binds to elements in the missing fragment of the NAL11-923del-1552 promoter and negatively regulates NAL11 expression. Genetic analyses support the hypothesis that NAL11 acts downstream of IPA1 to regulate IPA by modulating GA homeostasis, and NAL11 may be an essential complement for IPA1. Our work revealed that avoidance of the inhibition of NAL11-923del-1552 caused by IPA1 represents a positive strategy for rescuing GA defects accompanied by the upregulation of IPA1 in breeding high-yield rice.

A Long Noncoding RNA Derived from lncRNA-mRNA Networks Modulates Seed Vigor.

The discovery of long noncoding RNAs (lncRNAs) has filled a great gap in our understanding of posttranscriptional gene regulation in a variety of biological processes related to plant stress responses. However, systematic analyses of the lncRNAs expressed in rice seeds that germinate under cold stress have been elusive. In this study, we performed strand-specific whole transcriptome sequencing in germinated rice seeds under cold stress and normal temperature. A total of 6258 putative lncRNAs were identified and expressed in a stage-specific manner compared to mRNA. By investigating the targets of differentially expressed (DE) lncRNAs of LT-I (phase I of low temperature)/NT-I (phase I of normal temperature), it was shown that the auxin-activated signaling pathway was significantly enriched, and twenty-three protein-coding genes with most of the members of the SAUR family located in chromosome 9 were identified as the candidate target genes that may interact with five lncRNAs. A seed vigor-related lncRNA, SVR, which interplays with the members of the SAUR gene family in cis was eventually identified. The CRISPR/Cas 9 engineered mutations in SVR cause delay of germination. The findings provided new insights into the connection between lncRNAs and the auxin-activated signaling pathway in the regulation of rice seed vigor.

Characteristics of Fertility Transition Response to the Cumulative Effective Low Temperature in a Two-Line Male Sterile Rice Cultivar.

BACKGROUND: Photo-thermo-sensitive genic male sterile (PTGMS) rice (Oryza sativa L.) is usually considered two-line male sterile rice because of its dual-purpose in two-line hybrid rice system: under short days and low temperatures, it is fertile and used for self-propagation, but under long days and high temperatures, it is sterile and used for hybrid seed production. Therefore, photoperiod and temperature conditions are extremely important for the fertility transition of two-line male sterile rice. In recent years, there have been frequent occurrences of abnormally low-temperature (ALT) resulting in failure of two-line hybrid rice seed production. The daily average temperature (DAT) during ALT events is sometimes higher than the critical sterility-inducing temperature (CSIT) of two-line male sterile rice, of which the night temperature is lower than the CSIT. DAT has been traditionally used as the single indicator of pollen fertility transition, but it is unknown why the fertility of two-line male sterile rice in seed production restored fertility under ALT conditions. RESULTS: For Hang93S (H93S), a newly released PTGMS line, we hypothesized fertility transition is determined mainly by the cumulative effective low temperature (ELT) and only a certain duration of low temperature is required every day during the fertility-sensitive period. This study simulated ALTs where the DAT was higher than the CSIT while some segments of night temperature were lower than the CSIT. The results showed H93S exhibited a fertility transition to varying degrees. Moreover, fertility was restored under simulated ALT conditions and pollen fertility increased with increasing cumulative ELT, indicating that the fertility transition was affected primarily by the cumulative ELT. Results also indicated that pollen fertility increased as the number of treatment days increased. CONCLUSIONS: The fertility transition is caused mainly by the cumulative ELT. In two-line male sterile rice breeding, the effects of day length, ALT at night, and continuous response days should be considered together. The present study provides new insight into fertility transition so breeders can more effectively utilize the two-line male sterile rice, H93S, in breeding programs.

Improvement of rice blast resistance by developing monogenic lines, two-gene pyramids and three-gene pyramid through MAS.

BACKGROUND: Rice blast caused by Magnaporthe oryzae (M. oryzae) is one of the most destructive diseases in rice production. Development of resistant varieties through pyramiding of resistant (R) genes is considered as an effective strategy to cope with the disease. However, is it really essential to pyramid more R genes in a specific ecological regions? To answer this question, a set of rice improved lines were developed in this study. Afterwards, the blast disease resistance and agronomic traits of the recurrent parent (RP), donor parents (DPs) and improved lines were investigated. RESULTS: We developed seven improved lines, comprising three monogenic lines, three two-gene pyramids and one three-gene pyramid, by introgression of R gene(s) into a common genetic background using marker-assisted backcross breeding (MABB). Based on 302 SSR markers, the recurrent genome of the seven improved lines reached a range of 89.1 to 95.5%, with the average genome recovery of 92.9%. The pathogenicity assays inoculated with 32 different blast isolates under artificial conditions showed that the resistance spectrum of all the improved lines was significantly broadened. The assays further showed that the two-gene pyramids and the three-gene pyramid exhibited wider resistance spectrum than the monogenic lines. At natural nurseries, the three monogenic lines still showed high ratios of infected panicles, whereas the two-gene pyramids and the three-gene pyramid showed high level of panicle blast resistance. However, the two-gene pyramid R504 reached the similar resistance effect of the three-gene pyramid R507 considering resistance spectrum under artificial conditions and panicle blast resistance under field conditions. Generally, the improved lines showed comparable agronomic traits compared with the recurrent parent (RP), but the three-gene pyramid showed reduced grain yield per plant. CONCLUSIONS: All the improved lines conferred wider resistance spectrum compared with the RP. Yet, the three monogenic lines did not work under field conditions of the two nurseries. Given the similar performances on the main agronomic traits as the RP, the two-gene pyramids have achieved the breeding goals of broad resistance spectrum and effective panicle blast resistance. Whereas, the three-gene pyramid harboring Pi2, Pi46 and Pita seems superfluous considering its reduced yield, although it also showed displayed high level of blast resistance. Thus, rational use of R genes rather than stacking more R genes is recommended to control the disease.

NBS-LRR Protein Pik-H4 Interacts with OsBIHD1 to Balance Rice Blast Resistance and Growth by Coordinating Ethylene-Brassinosteroid Pathway.

The regulation of innate immunity and plant growth, along with the trade-off between them, affects the defense and recovery mechanisms of the plant after it is attacked by pathogens. Although it is known that hormonal crosstalk plays a major role in regulating interaction of plant growth and PAMP-triggered immunity, the relationship between plant growth and effector-triggered immunity (ETI) remains unclear. In a large-scale yeast two-hybrid screening for Pik-H4-interacting proteins, a homeodomain transcription factor OsBIHD1 was identified, which is previously known to function in biotic and abiotic stress responses. The knockout of OsBIHD1 in rice lines carrying Pik-H4 largely compromised the resistance of the rice lines to Magnaporthe oryzae, the fungus that causes rice blast. While overexpression of OsBIHD1 resulted in enhanced expression of the pathogenesis-related (PR) and ethylene (ET) synthesis genes. Moreover, OsBIHD1 was also found to directly bind to the promoter region of ethylene-synthesis enzyme OsACO3. In addition, OsBIHD1 overexpression or deficiency provoked dwarfism and reduced brassinosteroid (BR) insensitivity through repressing the expression of several critical genes involved in BR biosynthesis and BR signaling. During M. oryzae infection, transcript levels of the crucial BR catabolic genes (CYP734A2, CYP734A4, and CYP734A6) were significantly up-regulated in OsBIHD1-OX plants. Furthermore, OsBIHD1 was found to be capable of binding to the sequence-specific cis-elements on the promoters of CYP734A2 to suppress the plant growth under fungal invasion. Our results collectively suggest a model that OsBIHD1 is required for Pik-H4-mediated blast resistance through modulating the trade-off between resistance and growth by coordinating brassinosteroid-ethylene pathway.

Chromosome mapping, molecular cloning and expression analysis of a novel gene response for leaf width in rice.

Genetic analysis revealed that narrow leaf, small panicle, thin and slender stems as well as low fertility rate of an Indica rice variety were recessive traits and controlled by a single gene. Applying map-based cloning strategy, a novel narrow leaf gene, which was named nal11 was delimited to an interval of 58.3 kb between the InDel markers N10 and InD5016. There are 9 genes in the mapping interval, and only a heat shock DNAJ protein encode gene (Os07g09450) has a specific G to T SNP, which was occurred at the last base of the second exon of Os07g09450 in ZYX. 5' and 3' RACE result shown that there were two transcripts in NAL11, and the SNP in nal11 leads to a variable shear of mRNA. In addition, this type of mRNA alternative splicing together with a stop codon closely followed the SNP which caused termination of translation destroyed the DNAJ domain of nal11's product. These results suggested that the heat shock DNAJ gene was most likely to be the candidate gene of nal11. The results of RT-PCR and real-time PCR further verified that the SNP in the ZYX-nal11 gene affects mRNA splicing pattern. Phenotype of ZYX may be caused by a statistically significant reduction in the total number of small veins in leaf, size and number of small vascular bundles and cells in stems, similar to several previous reported mutations. The basic molecular information we provide here will be useful for further investigations of the physiological function of the heat shock DNAJ gene, which will be helpful in better understanding the role of the DNAJ family in regulation of plant type traits such as leaf width of rice.

Quantitative trait loci identification and meta-analysis for rice panicle-related traits.

Rice yield is a complex trait controlled by quantitative trait loci (QTLs). In the past three decades, thousands of QTLs for rice yield traits have been detected, but only a very small percentage has been cloned to date, as identifying the QTL genes requires a substantial investment of time and money. Meta-analysis provides a simple, reliable, and economical method for integrating information from multiple QTL studies across various environmental and genetic backgrounds, detecting consistent QTLs powerfully and estimating their genetic positions precisely. In this study, we aimed to locate consistent QTL regions associated with rice panicle traits by applying a genome-wide QTL meta-analysis approach. We first conducted a QTL analysis of 5 rice panicle traits using 172 plants in 2011 and 138 plants in 2012 from an F2 population derived from a cross between Nipponbare and H71D rice cultivators. A total of 54 QTLs were detected, and these were combined with 1085 QTLs collected from 82 previous studies to perform a meta-analysis using BioMercator v4.2. The integration of 82 maps resulted in a consensus map with 6970 markers and a total map length of 1823.1 centimorgan (cM), on which 837 QTLs were projected. These QTLs were then integrated into 87 meta-quantitative trait loci (MQTLs) by meta-analysis, and the 95 % confidence intervals (CI) of them were smaller than the mean value of the original QTLs. Also, 30 MQTLs covered 47 of the 54 QTLs detected from the cross between Nipponbare and H71D in this study. Among them, the two major and stable QTLs, spp10.1 and sd10.1, were found to be included in MQTL10.4. The three other major QTLs, pl3.1, sb2.1, and sb10.1, were included in MQTL3.3, MQTL2.2, and MQTL10.3, respectively. A total of 21 of the 87 MQTLs' phenotypic variation were >20 %. In total, 24 candidate genes were found in 15 MQTLs that spanned physical intervals <0.2 Mb, including genes that have been cloned previously, e.g., EP3, LP, MIP1, HTD1, DSH1, and OsPNH1. However, it would be beneficial to identify a greater number of candidate genes from these MQTLs. Mining new genes that modulate yield and its related traits would assist researchers to better understand the relevant molecular mechanisms. The MQTLs found in this study that have small physical and genetic intervals are useful not only for marker-assisted selection and pyramiding, but they also provide important information of rice yield and related gene mining for future research.

4-Coumarate-CoA Ligase-Like Gene OsAAE3 Negatively Mediates the Rice Blast Resistance, Floret Development and Lignin Biosynthesis.

Although adenosine monophosphate (AMP) binding domain is widely distributed in multiple plant species, detailed molecular functions of AMP binding proteins (AMPBPs) in plant development and plant-pathogen interaction remain unclear. In the present study, we identified an AMPBP OsAAE3 from a previous analysis of early responsive genes in rice during Magnaporthe oryzae infection. OsAAE3 is a homolog of Arabidopsis AAE3 in rice, which encodes a 4-coumarate-Co-A ligase (4CL) like protein. A phylogenetic analysis showed that OsAAE3 was most likely 4CL-like 10 in an independent group. OsAAE3 was localized to cytoplasm, and it could be expressed in various tissues. Histochemical staining of transgenic plants carrying OsAAE3 promoter-driven GUS (ß-glucuronidase) reporter gene suggested that OsAAE3 was expressed in all tissues of rice. Furthermore, OsAAE3-OX plants showed increased susceptibility to M. Oryzae, and this finding was attributable to decreased expression of pathogen-related 1a (PR1) and low level of peroxidase (POD) activity. Moreover, OsAAE3 over-expression resulted in increased content of H2O2, leading to programmed cell-death induced by reactive oxygen species (ROS). In addition, OsAAE3 over-expression repressed the floret development, exhibiting dramatically twisted glume and decreased fertility rate of anther. Meanwhile, the expressions of lignin biosynthesis genes were significantly decreased in OsAAE3-OX plants, thereby leading to reduced lignin content. Taken together, OsAAE3 functioned as a negative regulator in rice blast resistance, floret development, and lignin biosynthesis. Our findings further expanded the knowledge in functions of AMBPs in plant floret development and the regulation of rice-fungus interaction.