Identification of Increased Grain Length 1 (IGL1), a novel gene encoded by a major QTL for modulating grain length in rice.

KEY MESSAGE: A novel quantitative trait locus qIGL1, which performed a positive function in regulating grain length in rice, was cloned by the map-based cloning approach; further studies revealed that it corresponded to LOC_Os03g30530, and the IGL1 appeared to contribute to lengthening and widening of the cells on the surface of grain hulls. Grain length is a prominent determinant for grain weight and appearance quality of rice. In this study, we conducted quantitative trait locus mapping to determine a genomic interval responsible for a long-grain phenotype observed in a japonica cultivar HD385. This led to the identification of a novel QTL for grain length on chromosome 3, named qIGL1 (for Increased Grain Length 1); the HD385 (Handao 385)-derived allele showed enhancement effects on grain length, and such an allele as well as NIP (Nipponbare)-derived allele was designated qigl1 HD385 and qIGL1NIP, respectively. Genetic analysis revealed that the qigl1HD385 allele displayed semidominant effects on grain length. Fine mapping further narrowed down the qIGL1 to an ~ 70.8-kb region containing 9 open reading frames (ORFs). A comprehensive analysis indicated that LOC_Os03g30530, which corresponded to ORF6 and carried base substitutions and deletions in HD385 relative to NIP, thereby causing changes or losses of amino-acid residues, was the true gene for qIGL1. Comparison of grain traits between a pair of near-isogenic lines (NILs), termed NIL-igl1HD385 and NIL-IGL1NIP, discovered that introduction of the igl1HD385 into the NIP background significantly resulted in the elevations of grain length and 1000-grain weight. Closer inspection of grain surfaces revealed that the cell length and width in the longitudinal direction were significantly longer and greater, respectively, in NIL-igl1HD385 line compared with in NIL-IGL1NIP line. Hence, our studies identified a new semidominant natural allele contributing to the increase of grain length and further shed light on the regulatory mechanisms of grain length.

The Golgi-localized transporter OsPML4 contributes to manganese homeostasis in rice.

Manganese (Mn), an indispensable plant micronutrient, functions as a vital enzyme co-factor in numerous biochemical reactions. In rice, the Golgi-localized PHOTOSYNTHESIS-AFFECTED MUTANT 71-LIKE 3 (OsPML3), a member of the UNCHARACTERIZED PROTEIN FAMILY (UPF0016), plays a pivotal role in Mn homeostasis, particularly in rapidly developing tissues. This study focused on the functional characterization of another UPF0016 family member in rice, OsPML4, to elucidate its involvement in Mn homeostasis. OsPML4 had a 73% sequence identity with OsPML3 and exhibited expression in both shoots and roots, albeit at a lower transcriptional level than OsPML3. Furthermore, subcellular localization studies confirmed that OsPML4 localizes in the Golgi apparatus. Notably, heterologous expression of OsPML4 restored growth in the Mn uptake-deficient yeast strain Δsmf1 under Mn-limited conditions. Under Mn-deficient conditions, OsPML4 knockout exacerbated the decline in shoot dry weight and intensified necrosis in young leaves of OsPML3 knockout lines, which displayed stunted growth. The Mn concentration in OsPML3PML4 double knockout lines was lower than in wild-type (WT) and OsPML3 knockout lines. At the reproductive phase, OsPML3PML4 double knockout lines exhibited reduced fertility and grain yield compared to WT and OsPML3 knockout lines. Notably, reductions were observed in the deposition of cell wall polysaccharides and the content of Lea (Lewis A structure)-containing N-glycans in the young leaves of OsPML3PML4 double knockout lines, surpassing the reductions in WT and OsPML3 knockout lines. These findings underscore the significance of OsPML4 in Mn homeostasis in the Golgi apparatus, where it co-functions with OsPML3 to regulate cell wall polysaccharide deposition and late-stage Golgi N-glycosylation.

Rice Grain Detection and Counting Method Based on TCLE-YOLO Model.

Thousand-grain weight is the main parameter for accurately estimating rice yields, and it is an important indicator for variety breeding and cultivation management. The accurate detection and counting of rice grains is an important prerequisite for thousand-grain weight measurements. However, because rice grains are small targets with high overall similarity and different degrees of adhesion, there are still considerable challenges preventing the accurate detection and counting of rice grains during thousand-grain weight measurements. A deep learning model based on a transformer encoder and coordinate attention module was, therefore, designed for detecting and counting rice grains, and named TCLE-YOLO in which YOLOv5 was used as the backbone network. Specifically, to improve the feature representation of the model for small target regions, a coordinate attention (CA) module was introduced into the backbone module of YOLOv5. In addition, another detection head for small targets was designed based on a low-level, high-resolution feature map, and the transformer encoder was applied to the neck module to expand the receptive field of the network and enhance the extraction of key feature of detected targets. This enabled our additional detection head to be more sensitive to rice grains, especially heavily adhesive grains. Finally, EIoU loss was used to further improve accuracy. The experimental results show that, when applied to the self-built rice grain dataset, the precision, recall, and mAP@0.5 of the TCLE-YOLO model were 99.20%, 99.10%, and 99.20%, respectively. Compared with several state-of-the-art models, the proposed TCLE-YOLO model achieves better detection performance. In summary, the rice grain detection method built in this study is suitable for rice grain recognition and counting, and it can provide guidance for accurate thousand-grain weight measurements and the effective evaluation of rice breeding.

OsPML2, a chloroplast envelope localized transporter is involved in manganese homeostasis in rice.

Manganese (Mn), a vital element, plays crucial roles in various biochemical and physiological processes by serving as an essential cofactor for numerous enzymes and acting as a catalytically active metal within biological clusters. In this study, we investigate the role of PHOTOSYNTHESIS-AFFECTED MUTANT 71-LIKE 2 (OsPML2), a member of the UNCHARACTERIZED PROTEIN FAMILY 0016 (UPF0016) family, in regulating Mn homeostasis in rice. OsPML2 was highly expressed in young leaves, ovaries, and stigmas. Cross sections from young leaves revealed that OsPML2 was mainly expressed in the phloem region and mesophyll cells. Furthermore, heterologous expression of OsPML2 restored the growth of Mn uptake-defective yeast strain Δsmf1 under Mn-limited conditions. Subcellular localization analysis demonstrated that OsPML2 was specifically localized in the chloroplast envelope. Knockdown of OsPML2 resulted in reduced chloroplast Mn content, significantly affecting plant growth under Mn deficiency. Furthermore, analysis of isolated thylakoid membranes using blue native gels indicated a compromised accumulation of photosystem II (PSII) complexes in OsPML2 knockdown lines. Additionally, grain yield, grain length, and width were significantly reduced in OsPML2 knockdown plants. Collectively, our findings provide insights into the transport function of OsPML2, which facilitates Mn transport from the cytosol to chloroplast stroma and influences the accumulation of PSII complexes in rice.

An intracellular transporter OsNRAMP7 is required for distribution and accumulation of iron into rice grains.

Iron (Fe) is an essential micronutrient for plant growth and human health. Plants have evolved an efficient transport system for absorbing and redistributing Fe from the soil to other organs; however, the molecular mechanisms underlying Fe loading into grains are poorly understood. Our study shows that OsNRAMP7, a member of the natural resistance-associated macrophage protein (NRAMP) family, is a rice Fe transporter that localizes to the Golgi and trans-Golgi network (TGN). OsNRAMP7 was highly expressed in leaf blade, node I, pollen, and vascular tissues of almost tissues at the rice flowering stage. OsNRAMP7 knockdown by RNA interference (RNAi) increased Fe accumulation in the flag leaf blade, but decreased the Fe concentration in node I and rice grains. In addition, the knockdown of OsNRAMP7 also reduced grain fertility, pollen viability, and grain Fe concentration in the paddy fields; OsNRAMP7 overexpression significantly promoted Fe accumulation in the grains. Thus, our results suggest that OsNRAMP7 is required for the distribution and accumulation of Fe in rice grains and its overexpression could be a novel strategy for Fe biofortification in staple food crops.

The Golgi-localized transporter OsPML3 is involved in manganese homeostasis and complex N-glycan synthesis in rice.

Manganese (Mn) is involved in many biochemical pathways as an enzyme cofactor, and is essential for maintaining metabolic processes in various plant cell compartments. Here, we determined the function of a rice (Oryza sativa) Mn transporter, PHOTOSYNTHESIS-AFFECTED MUTANT 71-LIKE 3 (OsPML3), belonging to the UNCHARACTERIZED PROTEIN FAMILY 0016 (UPF0016), in regulating Mn homeostasis and late-stage Golgi N-glycosylation. OsPML3 was highly expressed in rapidly developing tissues such as young leaves, root caps, lateral root primordia, and young anthers. Heterologous expression of OsPML3 restored the growth of Mn uptake-defective yeast strain Δsmf1 under Mn-limited conditions. Sub-cellular localization analysis revealed that OsPML3 localizes in the Golgi apparatus. At the vegetative stage, we observed necrotic root tips and lateral root primordia, and chlorotic young leaves in OsPML3 knockout lines under Mn-deficient conditions. Knocking out OsPML3 reduced the Mn content in the young leaves but did not affect the older leaves. Additionally, knocking out OsPML3 reduced the deposition of cell wall polysaccharides and the content of Lea (Lewis A structure)-containing N-glycan in roots and young leaves. OsPML3 knockout lines grown in the paddy field had reduced pollen fertility. Moreover, we found that the Lewis A structure was reduced in young anthers of OsPML3 knockout lines. Collectively, our results indicate that OsPML3 maintains Mn homeostasis in the Golgi apparatus of the rapidly developing rice tissues, and regulates the deposition of cell wall polysaccharides and late-stage Golgi N-glycosylation, especially biosynthesis of the Lewis A structure.

The tonoplast-localized transporter OsNRAMP2 is involved in iron homeostasis and affects seed germination in rice.

Vacuolar storage of iron (Fe) is important for Fe homeostasis in plants. When sufficient, excess Fe could be stored in vacuoles for remobilization in the case of Fe deficiency. Although the mechanism of Fe remobilization from vacuoles is critical for crop development under low Fe stress, the transporters that mediate vacuolar Fe translocation into the cytosol in rice remains unknown. Here, we showed that under high Fe2+ concentrations, the Δccc1 yeast mutant transformed with the rice natural resistance-associated macrophage protein 2 gene (OsNRAMP2) became more sensitive to Fe toxicity. In rice protoplasts and transgenic plants expressing Pro35S:OsNRAMP2-GFP, OsNRAMP2 was localized to the tonoplast. Vacuolar Fe content in osnramp2 knockdown lines was higher than in the wild type, while the growth of osnramp2 knockdown plants was significantly influenced by Fe deficiency. Furthermore, the germination of osnramp2 knockdown plants was arrested. Conversely, the vacuolar Fe content of Pro35S:OsNRAMP2-GFP lines was significantly lower than in the wild type, and overexpression of OsNRAMP2 increased shoot biomass under Fe deficiency. Taken together, we propose that OsNRAMP2 transports Fe from the vacuole to the cytosol and plays a pivotal role in seed germination.

Analysis of QTLs for yield-related traits in Yuanjiang common wild rice (Oryza rufipogon Griff.).

Using an accession of common wild rice (Oryza rufipogon Griff.) collected from Yuanjiang County, Yunnan Province, China, as the donor and an elite cultivar 93-11, widely used in two-line indica hybrid rice production in China, as the recurrent parent, an advanced backcross populations were developed. Through genotyping of 187 SSR markers and investigation of six yield-related traits of two generations (BC(4)F(2) and BC(4)F(4)), a total of 26 QTLs were detected by employing single point analysis and interval mapping in both generations. Of the 26 QTLs, the alleles of 10 (38.5%) QTLs originating from O. rufipogon had shown a beneficial effect for yield-related traits in the 93-11 genetic background. In addition, five QTLs controlling yield and its components were newly identified, indicating that there are potentially novel alleles in Yuanjiang common wild rice. Three regions underling significant QTLs for several yield-related traits were detected on chromosome 1, 7 and 12. The QTL clusters were founded and corresponding agronomic traits of those QTLs showed highly significant correlation, suggesting the pleiotropism or tight linkage. Fine-mapping and cloning of these yield-related QTLs from wild rice would be helpful to elucidating molecular mechanism of rice domestication and rice breeding in the future.

Additive and over-dominant effects resulting from epistatic loci are the primary genetic basis of heterosis in rice.

A set of 148 F9 recombinant inbred lines (RILs) was developed from the cross of an indica cultivar 93-11 and japonica cultivar DT713, showing strong F1 heterosis. Subsequently, two backcross F1 (BCF1) populations were constructed by backcrossing these 148 RILs to two parents, 93-11 and DT713. These three related populations (281BCF1 lines, 148 RILs) were phenotyped for six yield-related traits in two locations. Significant inbreeding depression was detected in the population of RILS and a high level of heterosis was observed in the two BCF1 populations. A total of 42 main-effect quantitative trait loci (M-QTLs) and 109 epistatic effect QTL pairs (E-QTLs) were detected in the three related populations using the mixed model approach. By comparing the genetic effects of these QTLs detected in the RILs, BCF1 performance and mid-parental heterosis (HMP), we found that, in both BCF1 populations, the QTLs detected could be classified into two predominant types: additive and over-dominant loci, which indicated that the additive and over-dominant effect were more important than complete or partially dominance for M-QTLs and E-QTLs. Further, we found that the E-QTLs detected collectively explained a larger portion of the total phenotypic variation than the M-QTLs in both RILs and BCF1 populations. All of these results suggest that additive and over-dominance resulting from epistatic loci might be the primary genetic basis of heterosis in rice.

Quantitative trait loci underlying domestication- and yield-related traits in an Oryza sativa x Oryza rufipogon advanced backcross population.

To understand the genetic characteristics of the traits related to differentiation between cultivated rice and its wild progenitor, genetic factors controlling domestication- and yield-related traits were identified using a BC3F2 population derived from an accession of common wild rice (donor, Oryza rufipogon Griff.) collected from Yuanjiang, Yunnan province, China, and an indica cultivar, Teqing (recipient, Oryza sativa L.). A genetic linkage map consisting of 125 simple sequence repeat (SSR) markers was constructed. Based on the phenotypes of the 383 BC3F2 families evaluated in two environments, two domestication-related morphological traits, panicle shape and growth habit, were found to be controlled by single Mendelian factors. This implies that the recessive mutations of single genes controlling some morphological traits could have been easily selected during early domestication. By single-point analysis and interval mapping, 59 putative quantitative trait loci (QTLs) that influence 11 quantitative traits were detected at two sites, and 37.5% of the QTL alleles originating from O. rufipogon had a beneficial effect for yield-related traits in the Teqing background. Regions with significant QTLs for domestication- and yield-related traits were detected on chromosomes 1, 4, 5, 7, 8, and 12. Fine mapping and cloning of these domestication-related genes and QTLs will be useful in elucidating the origin and differentiation of Asian cultivated rice in the future.

[Mapping QTLs controlling vascular bundle and panicle-related traits from Yuanjiang common wild rice (Oryza rufipogon Griff.)].

An advanced backcross population (BC3F2), derived from the cross between Yuanjiang common wild rice as the donor parent and Teqing as recurrent parent, was used to map QTLs controlling the number of vascular bundle and panicle-related traits from Yuanjiang common wild rice. Seven QTLs controlling the number of large vascular bundles in peduncle were detected on chromosome 1, 2, 3, 4, 7 and 10. Five QTLs for the number of small vascular bundles in peduncle were mapped on chromosome 1, 2, 3, 4 and 8. A total of 15 QTLs for the number of primary and secondary rachis branches and spikelets per panicle were identified on all chromosomes except chromosome 11 and 12. Most of alleles derived from O. rufipogon Griff. showed negative effect that reduced significantly vascular bundle, rachis branches and spikelets per panicle, suggesting that during the evolution from common wild rice to cultivated rice, favorable alleles were retained while removed undesirable ones. Most of QTLs controlling vascular bundle, rachis branches and spikelets per panicle showed cluster form or close linkage on chromosomes. The directions of their additive effects were consistent, which explained the genetic basis of significant correlation of their phenotypic characters. All the information suggested parallel evolution relationship among these traits.

[Identification of quantitative trait loci controlling plant height and days to heading from Yuanjiang common wild rice (Oryza rufipogon Griff.)].

An advanced backcross strategy was used to identify quantitative trait loci (QTLs) associated with plant height and days to heading in the BC3F2 population derived from an accession of common wild rice (Oryza rufipogon Griff.) from Yuanjiang in Yunnan Province of China, as the donor, and an elite Indica cultivar "Teqing", as the recipient. Based on analyses of 116 SSR markers distributed throughout the rice genome and by using single-point analysis, four putative QTLs derived from O. rufipogon were detected for plant height on chromosome 1, and 6 QTLs for days to heading on chromosome 1, 3, 7, 8 and 11. All Alleles of QTLs for plant height from O. rufipogon could increase plant height of the backcross population. One QTL near RM104 on chromosome 1, explaining 27% and 28% of phenotypic variance and additive value reached 26.24 cm and 26.28 cm respectively in Beijing and Hefei, was detected. The location of this QTL is corresponding to sd-1. One QTL near the marker RM25 on chromosome 1, explaining 13% and 15% of phenotypic variance was identified. The additive value of this QTL reached 4.60 days and 3.65 days respectively in Beijing and Hefei, and the allele from O. rufipogon could delay the backcross population's days to heading.