Cold and salt stresses are major environmental factors that constrain rice production. Understanding their mechanisms is important to enhance cold and salt stress tolerance in rice. MicroRNAs (miRNAs) are a class of non-coding RNAs with only 21–24 nucleotides that are gene regulators in plants and animals. Previously, miR2871b expression was suppressed by cold stress in Dongxiang wild rice (DXWR, Oryza rufipogon Griff.). However, its biological functions in abiotic stress responses remain elusive. In the present study, miR2871b of DWXR was overexpressed to investigate its function under stress conditions. When miR2871b of DWXR was introduced into rice plants, the transgenic lines were more sensitive to cold and salt stresses, and their tolerance to cold and salt stress decreased. The increased expression of miR2871b in rice plants also increased the levels of reactive oxygen species (ROS) and malondialdehyde (MDA); however, it markedly decreased the activities of peroxidase (POD), superoxide dismutase (SOD), and catalase (CAT) and the contents of proline (Pro) and soluble sugar (SS). These data suggested that miR2871b of DXWR has negative regulatory effects on cold and salt stress tolerance. Meanwhile, 412 differentially expressed genes (DEGs) were found in rice transgenic plants using transcriptome sequencing, among which 266 genes were up-regulated and 146 genes were down-regulated. Furthermore, the upstream cis-acting elements and downstream targets of miR2871b were predicted and analyzed, and several critical acting elements (ABRE and TC-rich repeats) and potential target genes (LOC_Os03g41200, LOC_Os07g47620, and LOC_Os04g30260) were obtained. Collectively, these results generated herein further elucidate the vital roles of miR2871b in regulating cold and salt responses of DXWR.

Dongxiang wild rice (Oryza rufipogon Griff.) (DXWR) has strong seed storability and identifying its elite gene resources may facilitate genetic improvements in rice seed storability. In this study, we developed two backcross inbred lines (BILs) populations, with DXWR as a common donor parent and two rice varieties (F6 and R974) as recipient parents. Bulked segregant analysis via whole genome sequencing (BSA-seq) was used to identify seed storability-related loci in the DXWR and F6 population. Two main genomic regions containing 18,550,000–20,870,000 bp on chromosome 4 and 7,860,000–9,780,000 bp on chromosome 9 were identified as candidate loci of DXWR seed storability; these overlapped partially with seed storability-related quantitative trait loci (QTLs) discovered in previous studies, suggesting that these loci may provide important regions for isolating the responsible genes. In total, 448 annotated genes were predicted within the identified regions, of which 274 and 82 had nonsynonymous and frameshift mutations, respectively. We detected extensive metabolic activities and cellular processes during seed storability and confirmed the effects of the seed storability-related candidate loci using four BILs from DXWR and R974. These results may facilitate the cloning of DXWR seed storability-related genes, thereby elucidating rice seed storability and its improvement potential.

Rice (Oryza sativa) is a staple food for more than half of the world’s population, and its production is critical for global food security. Moreover, rice yield decreases when exposed to abiotic stresses, such as salinity, which is one of the most detrimental factors for rice production. According to recent trends, as global temperatures continue to rise due to climate change, more rice fields may become saltier. Dongxiang wild rice (Oryza rufipogon Griff., DXWR) is a progenitor of cultivated rice and has a high tolerance to salt stress, making it useful for studying the regulatory mechanisms of salt stress tolerance. However, the regulatory mechanism of miRNA-mediated salt stress response in DXWR remains unclear. In this study, miRNA sequencing was performed to identify miRNAs and their putative target genes in response to salt stress in order to better understand the roles of miRNAs in DXWR salt stress tolerance. A total of 874 known and 476 novel miRNAs were identified, and the expression levels of 164 miRNAs were found to be significantly altered under salt stress. The stem-loop quantitative real-time PCR (qRT-PCR) expression levels of randomly selected miRNAs were largely consistent with the miRNA sequencing results, suggesting that the sequencing results were reliable. The gene ontology (GO) analysis indicated that the predicted target genes of salt-responsive miRNAs were involved in diverse biological pathways of stress tolerance. This study contributes to our understanding of DXWR salt tolerance mechanisms regulated by miRNAs and may ultimately improve salt tolerance in cultivated rice breeding using genetic methods in the future.

Dongxiang wild rice (DXWR) (O. rufipogon Griff.), which has the northernmost worldwide distribution of a wild rice species, is a valuable genetic resource with respect to improving stress tolerance in cultivated rice (Oryza sativa L.). In the three-line hybrid rice breeding system, restorer lines play important roles in enhancing the tolerance of hybrid rice. However, restorer lines have yet to be used as a genomic background for development of substitution lines carrying DXWR chromosome segments. We developed a set of 84 chromosome segment substitution lines (CSSLs) from a donor parent DXWR × recurrent parent restorer line R974 (Oryza sativa indica) cross. On average, each CSSL carried 6.27 introgressed homozygous segments, with 93.37% total genome coverage. Using these CSSLs, we identified a single QTL, qDYST-1, associated with salt stress tolerance on chromosome 3. Furthermore, five CSSLs showing strong salt stress tolerance were subjected to whole-genome single-nucleotide polymorphism chip analyses, during which we detected a common substitution segment containing qDYST-1 in all five CSSLs, thereby implying the validity and efficacy of qDYST-1. These novel CSSLs could make a significant contribution to detecting valuable DXWR QTLs, and provide important germplasm resources for breeding novel restorer lines for use in hybrid rice breeding systems.

Grain number per panicle (GNPP) is a major factor influencing rice yield (Oryza sativa L.). However, the molecular mechanisms of GNPP determination are not well understood. A rice GNPP mutant, ngp4a, was isolated from an ethyl methanesulfonate-mutagenized rice library of japonica Nipponbare. ngp4a produced fewer grains than wild-type plants at maturity as the number of secondary branches decreased significantly. The mutant phenotype of ngp4a was controlled by a recessive nuclear gene, which was fine-mapped into a 155.2 kb region on chromosome 4. One GNPP-related gene, Gnp4/LAX2 (LOC_Os04g32510), was found in the mapped region. The deletion of 3-bp nucleotides in the first exon of NGP4A resulted in a threonine residue loss. The mutation in NGP4A was responsible for the mutant phenotype of ngp4a. These results suggest that NGP4A is a new allele for Gnp4 and LAX2, while the mutant phenotype and underlying causation differed. Notably, transcriptome analysis revealed that NGP4A could regulate GNPP determination through the phenylpropanoid biosynthesis and mitogen-activated protein kinase signaling pathways. Our results further elucidated the vital roles of Gnp4/LAX2 in GNPP determination, providing a new genetic resource and theoretical basis to further explore the molecular mechanisms of GNPP in rice.