OsAAP8 mutation leads to significant improvement in the nutritional quality and appearance of rice grains.

Members of the permease gene family are responsible for important biological functions in the growth and development of rice. Here, we show that OsAAP8 is a constitutive expression gene, and its translated protein is localized on the cell membrane. Mutation of the OsAAP8 can promote the expression of genes related to protein and amylopectin synthesis, and also promote the enlargement of protein bodies in its endosperm, leading to an increase in the protein, amylopectin, and total amino acid content of grains in OsAAP8 mutants. Seeds produced by the OsAAP8 mutant were larger, and the chalkiness traits of the OsAAP8 mutants were significantly reduced, thereby improving the nutritional quality and appearance of rice grains. The OsAAP8 protein is involved in the transport of various amino acids; OsAAP8 mutation significantly enhanced the root absorption of a range of amino acids and might affect the distribution of various amino acids. Therefore, OsAAP8 is an important quality trait gene with multiple biological functions, which provides important clues for the molecular design of breeding strategies for developing new high-quality varieties of rice. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-024-01473-w.

CRISPR/Cas9-mediated enhancement of semi-dwarf glutinous traits in elite Xiangdaowan rice (Oryza sativa L.): targeting SD1 and Wx genes for yield and quality improvement.

In rice cultivation, the traits of semi-dwarfism and glutinous texture are pivotal for optimizing yield potential and grain quality, respectively. Xiangdaowan (XDW) rice, renowned for its exceptional aromatic properties, has faced challenges due to its tall stature and high amylose content, resulting in poor lodging resistance and suboptimal culinary attributes. To address these issues, we employed CRISPR/Cas9 technology to precisely edit the SD1 and Wx genes in XDW rice, leading to the development of stable genetically homozygous lines with desired semi-dwarf and glutinous characteristics. The sd1-wx mutant lines exhibited reduced gibberellin content, plant height, and amylose content, while maintaining hardly changed germination rate and other key agronomic traits. Importantly, our study demonstrated that exogenous GA3 application effectively promoted growth by compensating for the deficiency of endogenous gibberellin. Based on this, a semi-dwarf glutinous elite rice (Oryza sativa L.) Lines was developed without too much effect on most agronomic traits. Furthermore, a comparative transcriptome analysis unveiled that differentially expressed genes (DEGs) were primarily associated with the anchored component of the membrane, hydrogen peroxide catabolic process, peroxidase activity, terpene synthase activity, and apoplast. Additionally, terpene synthase genes involved in catalyzing the biosynthesis of diterpenoids to gibberellins were enriched and significantly down-regulated. This comprehensive study provides an efficient method for simultaneously enhancing rice plant height and quality, paving the way for the development of lodging-resistant and high-quality rice varieties.

OsNAC74 affects grain protein content and various biological traits by regulating OsAAP6 expression in rice.

The grain protein content is an important quality trait in cereals, and the expression level of the OsAAP6 can significantly affect the grain protein content in rice. Through site-directed mutagenesis, we found that the position from -7 to -12 bp upstream of the transcription start site of the OsAAP6 was the functional variation site. By using the yeast single hybrid test, point-to-point in yeast, and the local surface plasmon resonance test, the OsNAC74 was screened and verified to be a regulator upstream of OsAAP6. The OsNAC74 is a constitutively expressed gene whose product is located on the cell membrane. The OsAAP6 and the genes related to the seed storage in the Osnac74 mutants were downregulated, and grain protein content was significantly reduced. In addition, OsNAC74 had a significant impact on quality traits such as grain chalkiness and gel consistency in rice. Although the Osnac74 mutant seeds were relatively small, the individual plant yield was not decreased. Therefore, OsNAC74 is an important regulatory factor with multiple biological functions. This study provides important information for the later use of OsNAC74 gene for molecular design and breeding in rice. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-023-01433-w.

Physiological and transcriptomic analysis of OsLHCB3 knockdown lines in rice.

The photosystem II (PSII) outer antenna LHCB3 protein plays critical roles in distributing the excitation energy and modulating the rate of state transition for photosynthesis. Here, OsLHCB3 knockdown mutants were produced using the RNAi system. Phenotypic analyses showed that OsLHCB3 knockdown led to pale green leaves and lower chlorophyll contents at both tillering and heading stages. In addition, mutant lines exhibited decreased non-photochemical quenching (NPQ) capacity and net photosynthetic rate (Pn) by downregulating the expression of PSII-related genes. Moreover, RNA-seq experiments were performed at both tillering and heading stages. The differentially expressed genes (DEGs) mainly involved in chlorophyll binding response to abscisic acid, photosystem II, response to chitin, and DNA-binding transcription factor. Besides, our transcriptomic and physiological data indicated that OsLHCB3 was essential for binding chlorophyll, but not for the metabolism of chlorophyll in rice. OsLHCB3 RNAi knockdown plants affected the expression of PS II-related genes, but not PS I-related genes. Overall, these results suggest that OsLHCB3 also plays vital roles in regulating photosynthesis and antenna proteins in rice as well as responses to environment stresses. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-023-01387-z.

Genome-Wide Association Studies Reveal the Genetic Basis of Fertility Restoration of CMS-WA and CMS-HL in xian/indica and aus Accessions of Rice (Oryza sativa L.).

BACKGROUND: Wild-abortive cytoplasmic male sterility (CMS-WA) and Honglian CMS (CMS-HL) are the two main CMS types utilized in production of three-line hybrid rice in xian/indica (XI) rice. Dissection of the genetic basis of fertility restoration of CMS-WA and CMS-HL in the core germplasm population would provide valuable gene and material resources for development of three-line hybrid combinations. RESULTS: In this study, two F1 populations with CMS-WA and CMS-HL background respectively were developed using 337 XI and aus accessions being paternal parents. Genome-wide association studies on three fertility-related traits of the two populations for two consecutive years revealed that both fertility restoration of CMS-WA and CMS-HL were controlled by a major locus and several minor loci respectively. The major locus for fertility restoration of CMS-WA was co-located with Rf4, and that for fertility restoration of CMS-HL was co-located with Rf5, which are cloned major restorer of fertility (Rf) genes. Furthermore, haplotype analysis of Rf4, Rf5 and Rf6, the three cloned major Rf genes, were conducted using the 337 paternal accessions. Four main haplotypes were identified for Rf4, and displayed different subgroup preferences. Two main haplotypes were identified for Rf5, and the functional type was carried by the majority of paternal accessions. In addition, eight haplotypes were identified for Rf6. CONCLUSIONS: Haplotype analysis of three Rf genes, Rf4, Rf5 and Rf6, could provide valuable sequence variations that can be utilized in marker-aided selection of corresponding genes in rice breeding. Meanwhile, fertility evaluation of 337 accessions under the background of CMS could provide material resources for development of maintainer lines and restorers.

Genetic Architecture of Natural Variation in Rice Nonphotochemical Quenching Capacity Revealed by Genome-Wide Association Study.

The photoprotective processes conferred by nonphotochemical quenching (NPQ) serve fundamental roles in maintaining plant fitness and sustainable yield. So far, few loci have been reported to be involved in natural variation of NPQ capacity in rice (Oryza sativa), and the extents of variation explored are very limited. Here we conducted a genome-wide association study (GWAS) for NPQ capacity using a diverse worldwide collection of 529 O. sativa accessions. A total of 33 significant association loci were identified. To check the validity of the GWAS signals, three F2 mapping populations with parents selected from the association panel were constructed and assayed. All QTLs detected in mapping populations could correspond to at least one GWAS signal, indicating the GWAS results were quite reliable. OsPsbS1 was repeatedly detected and explained more than 40% of the variation in the whole association population in two years, and demonstrated to be a common major QTL in all three mapping populations derived from inter-group crosses. We revealed 43 single nucleotide polymorphisms (SNPs) and 7 insertions and deletions (InDels) within a 6,997-bp DNA fragment of OsPsbS1, but found no non-synonymous SNPs or InDels in the coding region, indicating the PsbS1 protein sequence is highly conserved. Haplotypes with the 2,674-bp insertion in the promoter region exhibited significantly higher NPQ values and higher expression levels of OsPsbS1. The OsPsbS1 RNAi plants and CRISPR/Cas9 mutants exhibited drastically decreased NPQ values. OsPsbS1 had specific and high-level expression in green tissues of rice. However, we didn't find significant function for OsPsbS2, the other rice PsbS homologue. Manipulation of the significant loci or candidate genes identified may enhance photoprotection and improve photosynthesis and yield in rice.

Breeding signatures of rice improvement revealed by a genomic variation map from a large germplasm collection.

Intensive rice breeding over the past 50 y has dramatically increased productivity especially in the indica subspecies, but our knowledge of the genomic changes associated with such improvement has been limited. In this study, we analyzed low-coverage sequencing data of 1,479 rice accessions from 73 countries, including landraces and modern cultivars. We identified two major subpopulations, indica I (IndI) and indica II (IndII), in the indica subspecies, which corresponded to the two putative heterotic groups resulting from independent breeding efforts. We detected 200 regions spanning 7.8% of the rice genome that had been differentially selected between IndI and IndII, and thus referred to as breeding signatures. These regions included large numbers of known functional genes and loci associated with important agronomic traits revealed by genome-wide association studies. Grain yield was positively correlated with the number of breeding signatures in a variety, suggesting that the number of breeding signatures in a line may be useful for predicting agronomic potential and the selected loci may provide targets for rice improvement.

Genetic Architecture of Natural Variation in Rice Chlorophyll Content Revealed by a Genome-Wide Association Study.

Chlorophyll content is one of the most important physiological traits as it is closely related to leaf photosynthesis and crop yield potential. So far, few genes have been reported to be involved in natural variation of chlorophyll content in rice (Oryza sativa) and the extent of variations explored is very limited. We conducted a genome-wide association study (GWAS) using a diverse worldwide collection of 529 O. sativa accessions. A total of 46 significant association loci were identified. Three F2 mapping populations with parents selected from the association panel were tested for validation of GWAS signals. We clearly demonstrated that Grain number, plant height, and heading date7 (Ghd7) was a major locus for natural variation of chlorophyll content at the heading stage by combining evidence from near-isogenic lines and transgenic plants. The enhanced expression of Ghd7 decreased the chlorophyll content, mainly through down-regulating the expression of genes involved in the biosynthesis of chlorophyll and chloroplast. In addition, Narrow leaf1 (NAL1) corresponded to one significant association region repeatedly detected over two years. We revealed a high degree of polymorphism in the 5' UTR and four non-synonymous SNPs in the coding region of NAL1, and observed diverse effects of the major haplotypes. The loci or candidate genes identified would help to fine-tune and optimize the antenna size of canopies in rice breeding.