Mapping and candidate gene analysis of QTLs for grain shape in a rice chromosome segment substitution line Z485 and breeding of SSSLs.

Grain shape is one of the most important factors that affects rice yield. Cloning novel grain shape genes and analyzing their genetic mechanisms are crucial for high yield breeding. In this study, a slender grain CSSL-Z485 with 3-segments substitution in the genetic background of Nipponbare was constructed in rice. Cytological analysis showed that the longer grain length of Z485 was related to the increase in glume cell numbers, while the narrower grain width was associated with the decrease in cell width. Three grain shape-related quantitative trait locus (QTLs), including qGL12, qGW12, and qRLW12, were identified through the F2 population constructed from a cross between Nipponbare and Z485. Furthermore, four single segment substitution lines (SSSLs, S1-S4) carrying the target QTLs were dissected from Z485 by MAS. Finally, three candidate genes of qGL12 for grain length and qGW12 for grain width located in S3 were confirmed by DNA sequencing, RT-qPCR, and protein structure prediction. Specifically, candidate gene 1 encodes a ubiquitin family protein, while candidate genes 2 and 3 encode zinc finger proteins. The results provide valuable germplasm resources for cloning novel grain shape genes and molecular breeding by design. Supplementary information: The online version contains supplementary material available at 10.1007/s11032-024-01480-x.

OsOFP6 Overexpression Alters Plant Architecture, Grain Shape, and Seed Fertility.

OVATE family proteins (OFPs) play important roles in plant growth and development, hormone signaling, and stress response pathways. However, the functions of OsOFPs in rice are largely unknown. In this study, a novel gain-of-function rice mutant, Osofp6-D, was identified. This mutant exhibited decreased plant height, erect leaves, reduced panicle size, short and wide seeds, delayed seed germination time, and reduced fertility. These phenotypic changes were attributed to the increased expression of OsOFP6, which was caused by a T-DNA insertion. Complementation of the Osofp6-D phenotype by knockout of OsOFP6 using the CRISPR/Cas9 system confirmed that the Osofp6-D phenotype was caused by OsOFP6 overexpression. In addition, transgenic plants overexpressing OsOFP6 with the 35S promoter mimicked the Osofp6-D phenotype. Cytological observations of the glumes showed that OsOFP6 overexpression altered the grain shape, mainly by altering the cell shape. Hormone response experiments showed that OsOFP6 was involved in the gibberellin (GA) and brassinolide (BR) signaling responses. Further studies revealed that OsOFP6 interacts with E3BB, which is orthologous to the Arabidopsis central organ size-control protein BIG BROTHER (BB). This study further elucidates the regulation mechanism of the rice OFP family on plant architecture and grain shape.

A Glu209Lys substitution in DRG1/TaACT7, which disturbs F-actin organization, reduces plant height and grain length in bread wheat.

Plant height and grain size are two important agronomic traits that are closely related to crop yield. Numerous dwarf and grain-shape mutants have been studied to identify genes that can be used to increase crop yield and improve breeding programs. In this study, we characterized a dominant mutant, dwarf and round grain 1 (drg1-D), in bread wheat (Triticum aestivum L.). drg1-D plants exhibit multiple phenotypic changes, including dwarfism, round grains, and insensitivity to brassinosteroids (BR). Cell structure observation in drg1-D mutant plants showed that the reduced organ size is due to irregular cell shape. Using map-based cloning and verification in transgenic plants, we found that a Glu209Lys substitution in the DRG1 protein is responsible for the irregular cell size and arrangement in the drg1-D mutant. DRG1/TaACT7 encodes an actin family protein that is essential for polymerization stability and microfilament (MF) formation. In addition, the BR response and vesicular transport were altered by the abnormal actin cytoskeleton in drg1-D mutant plants. Our study demonstrates that DRG1/TaACT7 plays an important role in wheat cell shape determination by modulating actin organization and intracellular material transport, which could in the longer term provide tools to better understand the polymerization of actin and its assembly into filaments and arrays.

Regulation of OsPIL15 on rice quality.

The phytochrome-interacting factor-like gene OsPIL15 negatively regulates grain size and 1000-grain weight, but its regulatory effect on rice quality traits is unknown. Here, knock-down, knock-out, and over-expression of OsPIL15 transgenic rice lines were used to investigate the effects of OsPIL15 on rice yield and quality traits. The results showed that knock-down or knock-out of OsPIL15 increased grain length and width, chalkiness, amylose content, glutenin and globulin content, and total protein content but reduced amylopectin content, total starch content, prolamin and albumin content, and gel consistency. Over-expression of OsPIL15 showed the opposite results, except for the reduction of prolamin content. Although OsPIL15 changed the grain size and weight, it had no effect on grain length/width ratio, brown rice rate, and milled rice rate. KEGG pathway enrichment analysis of differentially expressed genes between transgenic lines and wild type showed that OsPIL15 mainly regulated genes related to ribosome, metabolic pathways, and biosynthesis of secondary metabolites. Gene expression analysis showed that RNAi transgenic lines decreased OsCIN2 and OsSUS1 expression and increased OsGBSSI, OsSSI, OsAPGL2, and OsAPGL3 expression level, while over-expression of OsPIL15 increased OsCIN2, OsSUS1, OsSUS6, and OsSSI and decreased OsSSIIa, OsSSIIc, and OsAPGL2 expression level. These results revealed that OsPIL15 plays an important role in rice grain development. In addition to grain shape, OsPIL15 also regulates chalkiness, starch content, protein content, and gel consistency. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-022-01311-x.

Snow albedo reductions induced by the internal/external mixing of black carbon and mineral dust, and different snow grain shapes across northern China.

Previous studies have indicated that black carbon (BC) potentially induces snow albedo reductions across northern China. However, the effects of other light-absorbing particles (LAPs, e.g., mineral dust, MD), snow grain shape, or BC-snow mixing state on snow albedo have been largely ignored. Here we evaluate the BC- and MD-induced snow albedo reductions and radiative forcings (RFs) using an updated Snow, Ice, and Aerosol Radiation radiative transfer model, considering all of the potential factors that can be derived from the field observations across northern China. The results highlight that the LAP-induced albedo reductions for nonspherical snow grains are 2%-30% less than those for spherical grains. Furthermore, BC-snow internal mixing can significantly enhance albedo reduction by a factor of 1.42-1.48 relative to external mixing, with snow grain radius ranging from 100 to 1000 µm. The mean regional BC + MD-induced snow albedo reductions are amplified by the increase of snow grain radius, ranging from 0.012 to 0.123 for fresh snow to 0.016-0.227 for old snow. Finally, we discuss the relative contributions of BC and MD to the albedo reductions and RFs, highlighting the dominant role of BC in reducing snow albedo across northern China.

UNetGE: A U-Net-Based Software at Automatic Grain Extraction for Image Analysis of the Grain Size and Shape Characteristics.

The shape and the size of grains in sediments and soils have a significant influence on their engineering properties. Image analysis of grain shape and size has been increasingly applied in geotechnical engineering to provide a quantitative statistical description for grain morphologies. The statistic robustness and the era of big data in geotechnical engineering require the quick and efficient acquirement of large data sets of grain morphologies. In the past publications, some semi-automation algorithms in extracting grains from images may cost tens of minutes. With the rapid development of deep learning networks applied to earth sciences, we develop UNetGE software that is based on the U-Net architecture-a fully convolutional network-to recognize and segregate grains from the matrix using the electron and optical microphotographs of rock and soil thin sections or the photographs of their hand specimen and outcrops. Resultantly, it shows that UNetGE can extract approximately 300~1300 grains in a few seconds to a few minutes and provide their morphologic parameters, which will ably assist with analyses on the engineering properties of sediments and soils (e.g., permeability, strength, and expansivity) and their hydraulic characteristics.

Identification and Allele Combination Analysis of Rice Grain Shape-Related Genes by Genome-Wide Association Study.

Grain shape is an important agronomic character of rice, which affects the appearance, processing, and the edible quality. Screening and identifying more new genes associated with grain shape is beneficial to further understanding the genetic basis of grain shape and provides more gene resources for genetic breeding. This study has a natural population containing 623 indica rice cultivars. Genome-wide association studies/GWAS of several traits related to grain shape (grain length/GL, grain width/GW, grain length to width ratio/GLWR, grain circumferences/GC, and grain size/grain area/GS) were conducted by combining phenotypic data from four environments and the second-generation resequencing data, which have identified 39 important Quantitative trait locus/QTLs. We analyzed the 39 QTLs using three methods: gene-based association analysis, haplotype analysis, and functional annotation and identified three cloned genes (GS3, GW5, OsDER1) and seven new candidate genes in the candidate interval. At the same time, to effectively utilize the genes in the grain shape-related gene bank, we have also analyzed the allelic combinations of the three cloned genes. Finally, the extreme allele combination corresponding to each trait was found through statistical analysis. This study's novel candidate genes and allele combinations will provide a valuable reference for future breeding work.

The divergence of brassinosteroid sensitivity between rice subspecies involves natural variation conferring altered internal auto-binding of OsBSK2.

Japonica/geng and indica/xian are two major rice (Oryza sativa) subspecies with multiple divergent traits, but how these traits are related and interact within each subspecies remains elusive. Brassinosteroids (BRs) are a class of steroid phytohormones that modulate many important agronomic traits in rice. Here, using different physiological assays, we revealed that japonica rice exhibits an overall lower BR sensitivity than indica. Extensive screening of BR signaling genes led to the identification of a set of genes distributed throughout the primary BR signaling pathway with divergent polymorphisms. Among these, we demonstrate that the C38/T variant in BR Signaling Kinase2 (OsBSK2), causing the amino acid change P13L, plays a central role in mediating differential BR signaling in japonica and indica rice. OsBSK2L13 in indica plays a greater role in BR signaling than OsBSK2P13 in japonica by affecting the auto-binding and protein accumulation of OsBSK2. Finally, we determined that OsBSK2 is involved in a number of divergent traits in japonica relative to indica rice, including grain shape, tiller number, cold adaptation, and nitrogen-use efficiency. Our study suggests that the natural variation in OsBSK2 plays a key role in the divergence of BR signaling, which underlies multiple divergent traits between japonica and indica.

Identification and allele mining of new candidate genes underlying rice grain weight and grain shape by genome-wide association study.

BACKGROUND: Grain weight and grain shape are important agronomic traits that affect the grain yield potential and grain quality of rice. Both grain weight and grain shape are controlled by multiple genes. The 3,000 Rice Genomes Project (3 K RGP) greatly facilitates the discovery of agriculturally important genetic variants and germplasm resources for grain weight and grain shape. RESULTS: Abundant natural variations and distinct phenotic differentiation among the subgroups in grain weight and grain shape were observed in a large population of 2,453 accessions from the 3 K RGP. A total of 21 stable quantitative trait nucleotides (QTNs) for the four traits were consistently identified in at least two of 3-year trials by genome-wide association study (GWAS), including six new QTNs (qTGW3.1, qTGW9, qTGW11, qGL4/qRLW4, qGL10, and qRLW1) for grain weight and grain shape. We further predicted seven candidate genes (Os03g0186600, Os09g0544400, Os11g0163600, Os04g0580700, Os10g0399700, Os10g0400100 and Os01g0171000) for the six new QTNs by high-density association and gene-based haplotype analyses. The favorable haplotypes of the seven candidate genes and five previously cloned genes in elite accessions with high TGW and RLW are also provided. CONCLUSIONS: Our results deepen the understanding of the genetic basis of grain weight and grain shape in rice and provide valuable information for improving rice grain yield and grain quality through molecular breeding.

Effect of multi-allele combination on rice grain size based on prediction of regression equation model.

Rice yield potential is partially affected by grain size and weight, which associates with a great number of genes and QTLs. However, it is still unclear that how multiple alleles in different genes take a combined effect on grain shape/size. Here, we investigated seven core grain size-related functional genes (GL7, GS3, GW8, GS5, TGW6, WTG1, and An-1) and observed a wide phenotypic variation for five agronomic traits (grain length, grain width, grain length-width ratio, grain thickness and thousand-grain weight) in 521 rice germplasm. The correlation analysis showed a strong association among these grain traits which have distinct impacts on determining the final rice grain size. Genotyping analysis demonstrated that a relatively small number of allele combinations were preserved in the diverse population and these allele combinations were significantly associated with differences in grain size. Furthermore, alleles were regarded as individual variables to develop the multiple regression equation. We found that B and C allelic types of GS3 and conventional type of WTG1 played relevant roles in grain size and thousand-grain weight, separately. The models would conduce to devise instructive approaches by selecting appropriate candidate alleles, which could fuel further research for breeding preferred grain shape and high-yielding crop.

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.

OsOFP19 modulates plant architecture by integrating the cell division pattern and brassinosteroid signaling.

Characterization of OVATE family proteins (OFPs) has revealed that they exert functions by interacting with different types of transcription factor. However, the molecular bases of these processes are poorly understood. Here, we report that OsOFP19 negatively modulates brassinosteroid (BR) response and integrates it with the cell division pattern to affect plant architecture, including grain shape, through interaction with both DWARF AND LOW-TILLERING (DLT) and Oryza sativa homeobox1 (OSH1). Overexpression of OsOFP19 caused a semi-dwarf stature with thicker leaves and stronger culms and roots, which result from an increase in cell layers in the sub-epidermal tissue. Further studies revealed that OsOFP19 interacts with OSH1, and that this interaction mutually enhances the transcriptional activity of these proteins and leads to a transition from anticlinal to periclinal cell division. Furthermore, DLT interacts with both OsOFP19 and OSH1 and acts as an antagonist in the two interactions. Therefore, OsOFP19, OSH1 and DLT form a functional complex which plays a pivotal role in modulating BR signaling and determining the cell division pattern during plant growth and development.

Pleiotropic effects of the wheat domestication gene Q on yield and grain morphology.

MAIN CONCLUSION: Transformation from q to Q during wheat domestication functioned outside the boundary of threshability to increase yield, grains m-2, grain weight and roundness, but to reduce grains per spike/spikelet. Mutation of the Q gene, well-known affecting wheat spike structure, represents a key domestication step in the formation of today's free-threshing, economically important wheats. In a previous study, multiple yield components and spike characteristics were associated with the Q gene interval in the bread wheat 'Forno' × European spelt 'Oberkulmer' recombinant inbred line population. Here, we reported that this interval was also associated with grain yield, grains m-2, grain morphology, and spike dry weight at anthesis. To clarify the roles of Q in agronomic trait performance, a functional marker for the Q gene was developed. Analysis of allelic effects showed that the bread wheat Q allele conferred free-threshing habit, soft glumes, and short and compact spikes compared with q. In addition, the Q allele contributed to higher grain yield, more grains m-2, and higher thousand grain weight, whereas q contributed to more grains per spike/spikelet likely resulting from increased preanthesis spike growth. For grain morphology, the Q allele was associated with reduced ratio of grain length to height, indicating a rounder grain. These results are supported by analysis of four Q mutant lines in the Chinese Spring background. Therefore, the transition from q to Q during wheat domestication had profound effects on grain yield and grain shape evolution as well, being a consequence of pleiotropy.

Genome wide association mapping for grain shape traits in indica rice.

MAIN CONCLUSION: Using genome-wide association mapping, 47 SNPs within 27 significant loci were identified for four grain shape traits, and 424 candidate genes were predicted from public database. Grain shape is a key determinant of grain yield and quality in rice (Oryza sativa L.). However, our knowledge of genes controlling rice grain shape remains limited. Genome-wide association mapping based on linkage disequilibrium (LD) has recently emerged as an effective approach for identifying genes or quantitative trait loci (QTL) underlying complex traits in plants. In this study, association mapping based on 5291 single nucleotide polymorphisms (SNPs) was conducted to identify significant loci associated with grain shape traits in a global collection of 469 diverse rice accessions. A total of 47 SNPs were located in 27 significant loci for four grain traits, and explained ~44.93-65.90 % of the phenotypic variation for each trait. In total, 424 candidate genes within a 200 kb extension region (±100 kb of each locus) of these loci were predicted. Of them, the cloned genes GS3 and qSW5 showed very strong effects on grain length and grain width in our study. Comparing with previously reported QTLs for grain shape traits, we found 11 novel loci, including 3, 3, 2 and 3 loci for grain length, grain width, grain length-width ratio and thousand grain weight, respectively. Validation of these new loci would be performed in the future studies. These results revealed that besides GS3 and qSW5, multiple novel loci and mechanisms were involved in determining rice grain shape. These findings provided valuable information for understanding of the genetic control of grain shape and molecular marker assistant selection (MAS) breeding in rice.

Molecular functions of genes related to grain shape in rice.

Because grain shape is an important component of rice grain yield, the discovery of genes related to rice grain shape has attracted much attention of rice breeding programs. In recent years, some of these genes have been cloned and studied. They have been found not only regulate grain shape by changing the shape of the spikelet hull, but also regulate endosperm development through control of cell division using different molecular mechanisms. In this paper, we review the recent research on genes related to rice grain shape and their possible regulatory mechanisms.

Advanced backcross QTL analysis reveals complicated genetic control of rice grain shape in a japonica × indica cross.

Grain shape is an important trait for improving rice yield. A number of quantitative trait loci (QTLs) for this trait have been identified by using primary F2 mapping populations and recombinant inbred lines, in which QTLs with a small effect are harder to detect than they would be in advanced generations. In this study, we developed two advanced mapping populations (chromosome segment substitution lines [CSSLs] and BC4F2 lines consisting of more than 2000 individuals) in the genetic backgrounds of two improved cultivars: a japonica cultivar (Koshihikari) with short, round grains, and an indica cultivar (IR64) with long, slender grains. We compared the ability of these materials to reveal QTLs for grain shape with that of an F2 population. Only 8 QTLs for grain length or grain width were detected in the F2 population, versus 47 in the CSSL population and 65 in the BC4F2 population. These results strongly suggest that advanced mapping populations can reveal QTLs for agronomic traits under complicated genetic control, and that DNA markers linked with the QTLs are useful for choosing superior allelic combinations to enhance grain shape in the Koshihikari and IR64 genetic backgrounds.

QTL analysis on rice grain appearance quality, as exemplifying the typical events of transgenic or backcrossing breeding.

Rice grain shape and yield are usually controlled by multiple quantitative trait loci (QTL). This study used a set of F9-10 recombinant inbred lines (RILs) derived from a cross of Huahui 3 (Bt/Xa21) and Zhongguoxiangdao, and detected 27 QTLs on ten rice chromosomes. Among them, twelve QTLs responsive for grain shape/ or yield were mostly reproducibly detected and had not yet been reported before. Interestingly, the two known genes involved in the materials, with one insect-resistant Bt gene, and the other disease-resistant Xa21 gene, were found to closely link the QTLs responsive for grain shape and weight. The Bt fragment insertion was firstly mapped on the chromosome 10 in Huahui 3 and may disrupt grain-related QTLs resulting in weaker yield performance in transgenic plants. The introgression of Xa21 gene by backcrossing from donor material into receptor Minghui 63 may also contain a donor linkage drag which included minor-effect QTL alleles positively affecting grain shape and yield. The QTL analysis on rice grain appearance quality exemplified the typical events of transgenic or backcrossing breeding. The QTL findings in this study will in the future facilitate the gene isolation and breeding application for improvement of rice grain shape and yield.

The OsCLV2s-OsCRN1 co-receptor regulates grain shape in rice.

The highly conserved CLV-WUS negative feedback pathway plays a decisive role in regulating stem cell maintenance in shoot and floral meristems in higher plants, including Arabidopsis, rice, maize, and tomato. Here, we find significant natural variations in the OsCLV2c, OsCLV2d, and OsCRN1 loci in a genome-wide association study of grain shape in rice. OsCLV2a, OsCLV2c, OsCLV2d, and OsCRN1 negatively regulate grain length-width ratio and show distinctive geographical distribution, indica-japonica differentiation, and artificial selection signatures. Notably, OsCLV2a and OsCRN1 interact biochemically and genetically, suggesting that the two components function in a complex to regulate grain shape of rice. Furthermore, the genetic contributions of the haplotypes combining OsCLV2a, OsCLV2c, and OsCRN1 are significantly higher than those of each single gene alone in controlling key yield traits. These findings identify two groups of receptor-like kinases that may function as distinct co-receptors to control grain size in rice, thereby revealing a previously unrecognized role of the CLV class genes in regulating seed development and proposing a framework to understand the molecular mechanisms of the CLV-WUS pathway in rice and other crops.

Rice OseIF6.1 encodes a eukaryotic translation initiation factor and is essential for the development of grain and anther.

The eIF6 proteins are distributed extensively in eukaryotes and play diverse and essential roles. The bona fide eIF6 protein in Arabidopsis, At-eIF6;1, is essential for embryogenesis. However, the role of eIF6 proteins in rice growth and development remains elusive and requires further investigation. Here, we characterized the functions of OseIF6.1, which is homologous to At-eIF6;1. OseIF6.1 encodes an eukaryotic translation initiation factor with a conserved eIF6 domain. The knockdown of OseIF6.1 resulted in a decrease in grain length and pollen sterility, whereas the overexpression of OseIF6.1 displayed opposite phenotypes. Further studies revealed that OseIF6.1 regulates grain shape by influencing cell expansion and proliferation. In addition, OseIF6.1 interacts with OsNMD3, which is a nuclear export adaptor for the 60S ribosomal subunit. The knockdown of OsNMD3 in plants exhibited reduced fertility and seed setting. Therefore, our findings have significantly enriched the current understanding of the role of OseIF6.1 in rice growth and development.