The TaSnRK1-TabHLH489 module integrates brassinosteroid and sugar signalling to regulate the grain length in bread wheat.

Regulation of grain size is a crucial strategy for improving the crop yield and is also a fundamental aspect of developmental biology. However, the underlying molecular mechanisms governing grain development in wheat remain largely unknown. In this study, we identified a wheat atypical basic helix-loop-helix (bHLH) transcription factor, TabHLH489, which is tightly associated with grain length through genome-wide association study and map-based cloning. Knockout of TabHLH489 and its homologous genes resulted in increased grain length and weight, whereas the overexpression led to decreased grain length and weight. TaSnRK1α1, the α-catalytic subunit of plant energy sensor SnRK1, interacted with and phosphorylated TabHLH489 to induce its degradation, thereby promoting wheat grain development. Sugar treatment induced TaSnRK1α1 protein accumulation while reducing TabHLH489 protein levels. Moreover, brassinosteroid (BR) promotes grain development by decreasing TabHLH489 expression through the transcription factor BRASSINAZOLE RESISTANT1 (BZR1). Importantly, natural variations in the promoter region of TabHLH489 affect the TaBZR1 binding ability, thereby influencing TabHLH489 expression. Taken together, our findings reveal that the TaSnRK1α1-TabHLH489 regulatory module integrates BR and sugar signalling to regulate grain length, presenting potential targets for enhancing grain size in wheat.

The OsMAPK5-OsWRKY72 module negatively regulates grain length and grain weight in rice.

Grain size and grain weight are important determinants for grain yield. In this study, we identify a novel OsMAPK5-OsWRKY72 module that negatively regulates grain length and grain weight in rice. We found that loss-of-function of OsMAPK5 leads to larger cell size of the rice spikelet hulls and a significant increase in both grain length and grain weight in an indica variety Minghui 86 (MH86). OsMAPK5 interacts with OsMAPKK3/4/5 and OsWRKY72 and phosphorylates OsWRKY72 at T86 and S88. Similar to the osmapk5 MH86 mutants, the oswrky72 knockout MH86 mutants exhibited larger size of spikelet hull cells and increased grain length and grain weight, whereas the OsWRKY72-overexpression MH86 plants showed opposite phenotypes. OsWRKY72 targets the W-box motifs in the promoter of OsARF6, an auxin response factor involved in auxin signaling. Dual-luciferase reporter assays demonstrated that OsWRKY72 activates OsARF6 expression. The activation effect of the phosphorylation-mimicking OsWRKY72T86D/S88D on OsARF6 expression was significantly enhanced, whereas the effects of the OsWRKY72 phosphorylation-null mutants were significantly reduced. In addition, auxin levels in young panicles of the osmapk5 and oswrky72 mutants were significantly higher than that in the wild-type MH86. Collectively, our study uncovered novel connections of the OsMAPKK3/4/5-OsMAPK5-mediated MAPK signaling, OsWRKY72-mediated transcription regulation, and OsARF6-mediated auxin signaling pathways in regulating grain length and grain weight in an indica-type rice, providing promising targets for molecular breeding of rice varieties with high yield and quality.

Identification of TaGL1-B1 gene controlling grain length through regulation of jasmonic acid in common wheat.

Grain length is one of the most important factors in determining wheat yield. Here, a stable QTL for grain length was mapped on chromosome 1B in a F10 recombinant inbred lines (RIL) population, and the gene TaGL1-B1 encoding carotenoid isomerase was identified in a secondary large population through multiple strategies. The genome-wide association study (GWAS) in 243 wheat accessions revealed that the marker for TaGL1-B1 was the most significant among all chromosomes. EMS mutants of TaGL1 possessed significantly reduced grain length, whereas TaGL1-B1-overexpressed lines possessed significantly increased grain length. Moreover, TaGL1-B1 strongly interacted with TaPAP6. TaPAP6-overexpressed lines had significantly increased grain length. Transcriptome analysis suggested that TaPAP6 was possibly involved in the accumulation of JA (jasmonic acid). Consistently, JA content was significantly increased in the TaGL1-B1 and TaPAP6 overexpression lines. Additionally, the role of TaGL1-B1 in regulating carotenoids was verified through QTL mapping, GWAS, EMS mutants and overexpression lines. Notably, overexpression of TaGL1-B1 significantly increased wheat yield in multiple locations. Taken together, overexpression of TaGL1-B1 enhanced grain length, probably through interaction with TaPAP6 to cause the accumulation of JA that improved carotenoid content and photosynthesis, thereby resulted in increased wheat yield. This study provided valuable genes controlling grain length to improve yield and a potential insight into the molecular mechanism of modulating JA-mediated grain size in wheat.

OsBAK2/OsSERK2 expression is repressed by OsBZR1 to modulate brassinosteroid response and grain length in rice.

Brassinosteroids (BRs) are a class of polyhydroxylated steroidal phytohormones that are essential for plant growth and development. Rice BRASSINOSTEROID-INSENSITIVE1 (BRI1)-ASSOCIATED RECEPTOR KINASES (OsBAKs) are plasma membrane-localized receptor kinases belonging to the subfamily of leucine-rich repeat receptor kinases. It has been found that in Arabidopsis, BRs induce the formation of a BRI1-BAK1 heterodimer complex and transmit the cascade signal to BRASSINAZOLE RESISTANT1/bri1-EMS-SUPPRESSOR1 (BZR1/BES1) to regulate BR signaling. Here, in rice (Oryza sativa ssp. japonica), we found that OsBZR1 binds directly to the promoter of OsBAK2, but not OsBAK1, and represses the expression of OsBAK2 to form a BR feedback inhibition loop. Additionally, the phosphorylation of OsBZR1 by OsGSK3 reduced its binding to the OsBAK2 promoter. The osbak2 mutant displays a typical BR-deficiency phenotype and negative modulates the accumulation of OsBZR1. Interestingly, the grain length of the osbak2 mutant was increased whereas in the cr-osbak2/cr-osbzr1 double mutant, the reduced grain length of the cr-osbzr1 mutant was restored, implying that the increased grain length of osbak2 may be due to the rice somatic embryogenesis receptor kinase-dependent pathway. Our study reveals a novel mechanism by which OsBAK2 and OsBZR1 engage in a negative feedback loop to maintain rice BR homeostasis, facilitating a deeper understanding of the BR signaling network and grain length regulation in rice.

A Pseudo-near isogenic F2 population strategy for rapid QTL cloning.

Near isogenic F2 (NIF2) population frequently developed by conventional backcross has dramatically contributed to QTL identification in plants. Developing such a NIF2 population is time-consuming. Thus, it is urgent to rapidly produce a NIF2 population for QTL cloning. Here, we proposed a rapid QTL cloning strategy by generating a Pseudo-near isogenic F2 population (Pseudo-NIF2), which segregates at the target QTL but is fixed at other QTLs for the target trait. Nineteen QTLs for GL, GW, and TGW were detected in the F2 population from the cross between Zhenshan 97 and Egy316. To verify the efficiency of Pseudo-NIF2 in QTL quick cloning, the novel moderate QTL qGL10.1 which explained 9.1% and 5.6% of grain length variation in F2 and F2:3 populations was taken as an example. An F2 plant (F2-120), which segregated at qGL10.1 but fixed at other 8 QTLs for grain length, was screened to generate a Pseudo-NIF2 population by selfing cross. In the Pseudo-NIF2 population, the segregation ratio of plants with long grains to short grains fits 3:1, indicating that one gene controlled the variation of grain length. Based on the Pseudo-NIF2 and its progeny, qGL10.1 was fine mapped to a 19.3-kb region, where a gene OsMADS56 was verified as the candidate by functional polymorphism between parental alleles. Pseudo-NIF2 strategy is a rapid way for QTL cloning, which saves 3 to 4 cropping seasons compared to the conventional way. Applying the method for cloning QTL with moderate or major effects is promising. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-023-01408-x.

Rapid Identification by Resequencing-Based QTL Mapping of a Novel Allele RGA1-FH Decreasing Grain Length in a Rice Restorer Line 'Fuhui212'.

Grain size is one of the most frequently selected traits during domestication and modern breeding. The continued discovery and characterization of new genes and alleles in controlling grain size are important in safeguarding the food supply for the world's growing population. Previously, a small grain size was observed in a rice restorer line 'Fuhui212', while the underlying genetic factors controlling this trait were unknown. In this study, by combining QTL mapping, variant effect prediction, and complementation experiments, we recovered a novel allele RGA1-FH that explains most of the phenotypic changes. The RGA1-FH allele contains an A-to-T splicing site variant that disrupts the normal function of RGA1. While population analysis suggests extremely strong artificial selection in maintaining a functional allele of RGA1, our study is the first, to the best of our knowledge, to prove that a dysfunctional RGA1 allele can also be beneficial in real agricultural production. Future breeding programs would benefit from paying more attention to the rational utilization of those overlooked 'unfavored' alleles.

Fine Mapping and Candidate Gene Analysis of Rice Grain Length QTL qGL9.1.

Grain length (GL) is one of the crucial determinants of rice yield and quality. However, there is still a shortage of knowledge on the major genes controlling the inheritance of GL in japonica rice, which severely limits the improvement of japonica rice yields. Here, we systemically measured the GL of 667 F2 and 1570 BC3F3 individuals derived from two cultivated rice cultivars, Pin20 and Songjing15, in order to identify the major genomic regions associated with GL. A novel major QTL, qGL9.1, was mapped on chromosome 9, which is associated with the GL, using whole-genome re-sequencing with bulked segregant analysis. Local QTL linkage analysis with F2 and fine mapping with the recombinant plant revealed a 93-kb core region on qGL9.1 encoding 15 protein-coding genes. Only the expression level of LOC_Os09g26970 was significantly different between the two parents at different stages of grain development. Moreover, haplotype analysis revealed that the alleles of Pin20 contribute to the optimal GL (9.36 mm) and GL/W (3.31), suggesting that Pin20 is a cultivated species carrying the optimal GL variation of LOC_Os09g26970. Furthermore, a functional-type mutation (16398989-bp, G>A) located on an exon of LOC_Os09g26970 could be used as a molecular marker to distinguish between long and short grains. Our experiments identified LOC_Os09g26970 as a novel gene associated with GL in japonica rice. This result is expected to further the exploration of the genetic mechanism of rice GL and improve GL in rice japonica varieties by marker-assisted selection.

Grain Size Associated Genes and the Molecular Regulatory Mechanism in Rice.

Grain size is a quantitative trait that is controlled by multiple genes. It is not only a yield trait, but also an important appearance quality of rice. In addition, grain size is easy to be selected in evolution, which is also a significant trait for studying rice evolution. In recent years, many quantitative trait loci (QTL)/genes for rice grain size were isolated by map-based cloning or genome-wide association studies, which revealed the genetic and molecular mechanism of grain size regulation in part. Here, we summarized the QTL/genes cloned for grain size and the regulation mechanism with a view to provide the theoretical basis for improving rice yield and breeding superior varieties.

Natural variations in the promoter of Awn Length Inhibitor 1 (ALI-1) are associated with awn elongation and grain length in common wheat.

Wheat awn plays a vital role in photosynthesis, grain production, and drought tolerance. However, the systematic identification or cloning of genes controlling wheat awn development is seldom reported. Here, we conducted a genome-wide association study (GWAS) with 364 wheat accessions and identified 26 loci involved in awn length development, including previously characterized B1, B2, Hd, and several rice homologs. The dominant awn suppressor B1 was fine mapped to a 125-kb physical interval, and a C2 H2 zinc finger protein Awn Length Inhibitor 1 (ALI-1) was confirmed to be the underlying gene of the B1 locus through the functional complimentary test with native awnless allele. ALI-1 expresses predominantly in the developing spike of awnless individuals, transcriptionally suppressing downstream genes. ALI-1 reduces cytokinin content and simultaneously restrains cytokinin signal transduction, leading to a stagnation of cell proliferation and reduction of cell numbers during awn development. Polymorphisms of four single nucleotide polymorphisms (SNPs) located in ALI-1 promoter region are diagnostic for the B1/b1 genotypes, and these SNPs are associated with awn length (AL), grain length (GL) and thousand-grain weight (TGW). More importantly, ali-1 was observed to increase grain length in wheat, which is a valuable attribute of awn on grain weight, aside from photosynthesis. Therefore, ALI-1 pleiotropically regulates awn and grain development, providing an alternative for grain yield improvement and addressing future climate changes.

The basic helix-loop-helix transcription factor OsBLR1 regulates leaf angle in rice via brassinosteroid signalling.

Leaf angle is a key factor in plant architecture and crop yield. Brassinosteroids (BRs) regulate many developmental processes, especially the leaf angle in monocots. However, the BR signalling pathway is complex and includes many unknown members. Here, we propose that Oryza sativa BRASSINOSTEROID-RESPONSIVE LEAF ANGLE REGULATOR 1 (OsBLR1) encodes a bHLH transcription factor, and positively regulates BR signalling to increase the leaf angle and grain length in rice (Oryza sativa L.). Lines overexpressing OsBLR1 (blr1-D and BLR1-OE-1/2/3) had similar traits, with increased leaf angle and grain length. Conversely, OsBLR1-knockout mutants (blr1-1/2/3) had erect leaves and shorter grains. Lamina joint inclination, coleoptile elongation, and root elongation assay results indicated that these overexpression lines were more sensitive to BR, while the knockout mutants were less sensitive. There was no significant difference in the endogenous BR contents of blr1-1/2 and wild-type plants. These results suggest that OsBLR1 is involved in BR signal transduction. The blr1-D mutant, with increased cell growth in the lamina joint and smaller leaf midrib, showed significant changes in gene expression related to the cell wall and leaf development compared with wild-type plants; furthermore, the cellulose and protopectin contents in blr1-D were reduced, which resulted in the increased leaf angle and bent leaves. As the potential downstream target gene of OsBLR1, the REGULATOR OF LEAF INCLINATION1 (OsRLI1) gene expression was up-regulated in OsBLR1-overexpression lines and down-regulated in OsBLR1-knockout mutants. Moreover, we screened OsRACK1A as an interaction protein of OsBLR1 using a yeast two-hybrid assay and glutathione-S-transferase pull-down.

Identification of qLG2, qLG8, and qWG2 as novel quantitative trait loci for grain shape and the allelic analysis in cultivated rice.

MAIN CONCLUSION: Three novel QTLs for grain shape were genetically fine mapped, with two of which to a 250-kb target interval on rice chromosome 2 that contains fourteen candidate genes. Grain shape (grain length, width, and thickness) determines crop yield and grain quality. However, the trait is regulated by numerous naturally occurring quantitative trait loci (QTLs) and the underlying mechanism remains largely unknown. Here, we report the genetic mapping of three new QTLs, qLG2, qWG2, and qLG8 that each exerts a semi-dominant effect on grain shape in cultivated rice. These QTLs were validated using populations derived from the corresponding chromosome segment substitution lines (CSSLs), and were further delimited to small genomic intervals in progeny testing experiments. Especially, qLG2/qWG2 was placed into an about 250-kb genomic candidate region, and 14 predicted ORFs localized within the interval. We also evaluated the individual and pyramiding genetic effect(s) of these QTL(s) using the corresponding nearly isogenic lines, and found that they have additive effects on the traits. Collectively, these findings provided useful information as a tool to improve grain shape in crop breeding programs and established foundations for future QTL cloning.

Analysis of the functions of TaGW2 homoeologs in wheat grain weight and protein content traits.

GW2 is emerging as a key genetic determinant of grain weight in cereal crops; it has three homoeologs (TaGW2-A1, -B1 and -D1) in hexaploid common wheat (Triticum aestivum L.). Here, by analyzing the gene editing mutants that lack one (B1 or D1), two (B1 and D1) or all three (A1, B1 and D1) homoeologs of TaGW2, several insights are gained into the functions of TaGW2-B1 and -D1 in common wheat grain traits. First, both TaGW2-B1 and -D1 affect thousand-grain weight (TGW) by influencing grain width and length, but the effect conferred by TaGW2-B1 is stronger than that of TaGW2-D1. Second, there exists functional interaction between TaGW2 homoeologs because the TGW increase shown by a double mutant (lacking B1 and D1) was substantially larger than that of their single mutants. Third, both TaGW2-B1 and -D1 modulate cell number and length in the outer pericarp of developing grains, with TaGW2-B1 being more potent. Finally, TaGW2 homoeologs also affect grain protein content as this parameter was generally increased in the mutants, especially in the lines lacking two or three homoeologs. Consistent with this finding, two wheat end-use quality-related parameters, flour protein content and gluten strength, were considerably elevated in the mutants. Collectively, our data shed light on functional difference between and additive interaction of TaGW2 homoeologs in the genetic control of grain weight and protein content traits in common wheat, which may accelerate further research on this important gene and its application in wheat improvement.

Identification of the quantitative trait loci controlling spike-related traits in hexaploid wheat (Triticum aestivum L.).

MAIN CONCLUSION: Totally, 48 loci responsible for six spike-related traits were identified in wheat, and a major locus QGl-4A for grain length was mapped and validated for marker-assisted selection in breeding. Wheat yield is determined by the number of spikes, number of grains per spike (GN), and one-thousand kernel weight (TKW), among which GN and TKW are greatly related to the spike development and thus the spike-related traits, including spike length (SL), number of spikelet per spike (SN), grain length (GL) and grain width (GW). To identify the key loci governing the spike-related traits (SL, SN, GN, TKW, GL and GW), we conducted the quantitative trait loci (QTL) analysis combined with wheat 660K SNP chip and Kompetitive allele-specific PCR (KASP) assay, using the F2 and F2:3 populations derived from Luohan6 (LH6) with big spike and grain and Zhengmai366 with small spike and grain, and identified a total of 48 QTLs on 18 chromosomes. Moreover, a major stable QTL for GL on chromosome 4A, designated as QGl-4A, was mapped into a 0.37 cM interval between KASP markers Xib4A-10 and Xib4A-12, corresponding to 20 Mb physical region in the Chinese Spring genome. This QTL explained 17.30% and 5.12% of the phenotypic variation for GL in the F2 and F2:3 populations. Further association analysis of flanking markers Xib4A-10 and Xib4A-12 in 192 wheat varieties showed that these two markers could be used for marker-assisted selection in breeding. These results provide valuable information for map-based cloning of the target genes involved in the regulation of spike-related traits in common wheat.

Natural alleles of GLA for grain length and awn development were differently domesticated in rice subspecies japonica and indica.

Rice (Oryza sativa L.) cultivars harbour morphological and physiological traits different from those of wild rice (O. rufipogon Griff.), but the molecular mechanisms underlying domestication remain controversial. Here, we show that awn and long grain traits in the near-isogenic NIL-GLA are separately controlled by variations within the GLA (Grain Length and Awn Development) gene, a new allele of GAD1/RAE2, which encodes one member of the EFPL (epidermal patterning factor-like protein) family. Haplotype analyses and transgenic studies revealed that InDel1 (variation for grain length, VGL) in the promoter region of GLA (GLAVGL ) increases grain length by promoting transcription of GLA. Absence of InDel3 (variation for awn formation, VA) in the coding region (CDS) of GLA (GLAva ) results in short awn or no awn phenotypes. Analyses of minimum spanning trees and introgression regions demonstrated that An-1, an important gene for awn formation, was preferentially domesticated and its mutation to an-1 was followed by GLA and An-2. Gene flow then occurred between the evolved japonica and indica populations. Quality analysis showed that GLA causes poor grain quality. During genetic improvement, awnlessness was selected in ssp. indica, whereas short-grained and awnless phenotypes with good quality were selected in japonica. Our findings facilitate an understanding of rice domestication and provide a favourable allele for rice breeding.

Alternative splicing of OsLG3b controls grain length and yield in japonica rice.

Grain size, one of the important components determining grain yield in rice, is controlled by the multiple quantitative trait loci (QTLs). Intensive artificial selection for grain size during domestication is evidenced in modern cultivars compared to their wild relatives. Here, we report the molecular cloning and characterization of OsLG3b, a QTL for grain length in tropical japonica rice that encodes MADS-box transcription factor 1 (OsMADS1). Six SNPs in the OsLG3b region led to alternative splicing, which were associated with grain length in an association analysis of candidate region. Quantitative PCR analysis indicated that OsLG3b expression was higher during the panicle and seed development stages. Analysis of haplotypes and introgression regions revealed that the long-grain allele of OsLG3b might have arisen after domestication of tropical japonica and spread to subspecies indica or temperate japonica by natural crossing and artificial selection. OsLG3b is therefore a target of human selection for adaptation to tropical regions during domestication and/or improvement of rice. Phylogenetic analysis and pedigree records showed that OsLG3b had been employed by breeders, but the gene still has much breeding potential for increasing grain length in indica. These findings will not only aid efforts to elucidate the molecular basis of grain development and domestication, but also facilitate the genetic improvement of rice yield.

OsLG3 contributing to rice grain length and yield was mined by Ho-LAMap.

BACKGROUND: Most agronomic traits in rice are complex and polygenic. The identification of quantitative trait loci (QTL) for grain length is an important objective of rice genetic research and breeding programs. RESULTS: Herein, we identified 99 QTL for grain length by GWAS based on approximately 10 million single nucleotide polymorphisms from 504 cultivated rice accessions (Oryza sativa L.), 13 of which were validated by four linkage populations and 92 were new loci for grain length. We scanned the Ho (observed heterozygosity per locus) index of coupled-parents of crosses mapping the same QTL, based on linkage and association mapping, and identified two new genes for grain length. We named this approach as Ho-LAMap. A simulation study of six known genes showed that Ho-LAMap could mine genes rapidly across a wide range of experimental variables using deep-sequencing data. We used Ho-LAMap to clone a new gene, OsLG3, as a positive regulator of grain length, which could improve rice yield without influencing grain quality. Sequencing of the promoter region in 283 rice accessions from a wide geographic range identified four haplotypes that seem to be associated with grain length. Further analysis showed that OsLG3 alleles in the indica and japonica evolved independently from distinct ancestors and low nucleotide diversity of OsLG3 in indica indicated artificial selection. Phylogenetic analysis showed that OsLG3 might have much potential value for improvement of grain length in japonica breeding. CONCLUSIONS: The results demonstrated that Ho-LAMap is a potential approach for gene discovery and OsLG3 is a promising gene to be utilized in genomic assisted breeding for rice cultivar improvement.

Increased pericarp cell length underlies a major quantitative trait locus for grain weight in hexaploid wheat.

Crop yields must increase to address food insecurity. Grain weight, determined by grain length and width, is an important yield component, but our understanding of the underlying genes and mechanisms is limited. We used genetic mapping and near isogenic lines (NILs) to identify, validate and fine-map a major quantitative trait locus (QTL) on wheat chromosome 5A associated with grain weight. Detailed phenotypic characterisation of developing and mature grains from the NILs was performed. We identified a stable and robust QTL associated with a 6.9% increase in grain weight. The positive interval leads to 4.0% longer grains, with differences first visible 12 d after fertilization. This grain length effect was fine-mapped to a 4.3 cM interval. The locus also has a pleiotropic effect on grain width (1.5%) during late grain development that determines the relative magnitude of the grain weight increase. Positive NILs have increased maternal pericarp cell length, an effect which is independent of absolute grain length. These results provide direct genetic evidence that pericarp cell length affects final grain size and weight in polyploid wheat. We propose that combining genes that control distinct biological mechanisms, such as cell expansion and proliferation, will enhance crop yields.

Identification of quantitative trait loci for rice grain quality and yield-related traits in two closely related Oryza sativa L. subsp. japonica cultivars grown near the northernmost limit for rice paddy cultivation.

Quantitative trait loci (QTLs) associated with eating quality, grain appearance quality and yield-related traits were mapped in recombinant inbred lines (RILs) derived from closely related rice (Oryza sativa L. subsp. japonica) cultivars, Yukihikari (good eating quality) and Joiku462 (superior eating quality and high grain appearance quality). Apparent amylose content (AAC), protein content (PC), brown grain length (BGL), brown grain width (BGWI), brown grain thickness (BGT), brown grain weight per plant (BGW) and nine yield-related traits were evaluated in 133 RILs grown in four different environments in Hokkaido, near the northernmost limit for rice paddy cultivation. Using 178 molecular markers, a total of 72 QTLs were detected, including three for AAC, eight for PC, two for BGL, four for BGWI, seven for BGT, and six for BGW, on chromosomes 1, 2, 3, 4, 6, 7, 8, 9, 11 and 12. Fifteen intervals were found to harbor multiple QTLs affecting these different traits, with most of these QTL clusters located on chromosomes 4, 6, 8, 9 and 12. These QTL findings should facilitate gene isolation and breeding application for improvement of eating quality, grain appearance quality and yield of rice cultivars.