Rice (Oryza sativa L.) is a widely cultivated food crop around the world, especially in Asia. However, rice seedlings often suffer from cold stress, which affects their growth and yield. Here, RNA-seq analysis and Meta-QTLs mapping were performed to understand the molecular mechanisms underlying cold tolerance in the roots of 14-day-old seedlings of rice (RPY geng, cold-tolerant genotype). A total of 4779 of the differentially expressed genes (DEGs) were identified, including 2457 up-regulated and 2322 down-regulated DEGs. The GO, COG, KEEG, and Mapman enrichment results of DEGs revealed that DEGs are mainly involved in carbohydrate transport and metabolism, signal transduction mechanisms (plant hormone signal transduction), biosynthesis, transport and catabolism of secondary metabolites (phenylpropanoid biosynthesis), defense mechanisms, and large enzyme families mechanisms. Notably, the AP2/ERF-ERF, NAC, WRKY, MYB, C2H2, and bHLH transcription factors participated in rice’s cold–stress response and tolerance. On the other hand, we mapped the identified DEGs to 44 published cold–stress-related genes and 41 cold-tolerant Meta-QTLs regions. Of them, 12 DEGs were the published cold–stress-related genes and 418 DEGs fell into the cold-tolerant Meta-QTLs regions. In this study, the identified DEGs and the putative molecular regulatory network can provide insights for understanding the mechanism of cold stress tolerance in rice. In addition, DEGs in KEGG term-enriched terms or cold-tolerant Meta-QTLs will help to secure key candidate genes for further functional studies on the molecular mechanism of cold stress response in rice.

Salinity is a major abiotic stress that limits plant growth and crop productivity. Indica rice and japonica rice show significant differences in tolerance to abiotic stress, and it is considered a feasible method to breed progeny with stronger tolerance to abiotic stress by crossing indica and japonica rice. We herein developed a high-generation recombinant inbred lines (RILs) from Luohui 9 (indica) X RPY geng (japonica). Based on the high-density bin map of this RILs population, salt tolerance QTLs controlling final survival rates were analyzed by linkage mapping and RTM-GWAS methods. A total of seven QTLs were identified on chromosome 3, 4, 5, 6, and 8. qST-3.1, qST-5.1, qST-6.1, and qST-6.2 were novel salt tolerance QTLs in this study and their function were functionally verified by comparative analysis of parental genotype RILs. The gene aggregation result of these four new QTLs emphasized that the combination of the four QTL synergistic genotypes can significantly improve the salt stress tolerance of rice. By comparing the transcriptomes of the root tissues of the parents’ seedlings, at 3 days and 7 days after salt treatment, we then achieved fine mapping of QTLs based on differentially expressed genes (DEGs) identification and DEGs annotations, namely, LOC_Os06g01250 in qST-6.1, LOC_Os06g37300 in qST-6.2, LOC_Os05g14880 in qST-5.1. The homologous genes of these candidate genes were involved in abiotic stress tolerance in different plants. These results indicated that LOC_Os05g14880, LOC_Os06g01250, and LOC_Os06g37300 were the candidate genes of qST-5.1, qST-6.1, and qST-6.2. Our finding provided novel salt tolerance-related QTLs, candidate genes, and several RILs with better tolerance, which will facilitate breeding for improved salt tolerance of rice varieties and promote the exploration tolerance mechanisms of rice salt stress.

Submerged germination ability is a key trait of rice (Oryza sativa L.) in the success of water direct seeding. Therefore, mining submerged germination‐related loci and seeking high tolerant rice germplasms are important for developing rice direct seeding breeding strategies. Here, we treated 272 recombinant inbred lines (RILs, F15) from a cross from Luohui 9 (indica) and RPY geng (japonica) with simulated submergence for 7 days. We successfully bred several superior homozygous RILs with high submergence resistance, namely, R53, R137, R148, R176, and R180, via indica and japonica hybridization strategies. Then, the QTL mapping of coleoptile length trait was performed by R/qtl and IciMapping softwares based on the high‐density bin marker genetic map and two QTLs responsible for coleoptile length were detected in chromosomes 1 (qCL‐1.1) and 3 (qCL‐3.1). The haplotype results of RILs showed that the aggregation of elite alleles of these two QTLs in an individual was beneficial to improving the rice submergence tolerance. According to contrasting genotype of qCL‐1.1 and qCL‐3.1 with different coleoptile lengths, RIL07 (R07) and R180 were selected to further RNA‐seq analysis for analyzing the possible molecular mechanisms of rice under submergence stress. Finally, LOC_Os01g04430, LOC_Os01g04530, and LOC_Os03g22720 for qCL‐1.1 and qCL‐3.1 were characterized to affect submergence stress by the overlapped analysis of two independent sets of RNA‐seq data, qRT‐PCR verification, and gene sequence alignments. In this study, the submerged germination‐related QTLs/genes and high submergence‐tolerant RILs provided new genetic resources for breeding rice varieties suitable for direct seeding via molecular breeding strategies in the immediate future.

Polyol transporters (PLTs), also called polyol/monosaccharide transporters, is of significance in determining plant development and sugar transportation. However, the diverged evolutionary patterns of the PLT gene family in Gramineae crops are still unclear. Here a micro-evolution analysis was performed among the seven Gramineae representative crops using whole-genome sequences, i.e., Brachypodium distachyon (Bd), Hordeum vulgare (Hv), Oryza rufipogon (Or), Oryza sativa (Os), Sorghum bicolor (Sb), Setaria italica (Si), and Zea mays (Zm), leading to the identification of 12, 11, 12, 15, 20, 24, and 20 PLT genes, respectively. In this study, all PLT genes were divided into nine orthogroups (OGs). However, the number of PLT genes and the distribution of PLT OGs were not the same in these seven Gramineae species, and different OGs were also subject to different purification selection pressures. These results indicated that the PLT OGs of the PLT gene family have been expanded or lost unevenly in all tested species. Then, our results of gene duplication events confirmed that gene duplication events promoted the expansion of the PLT gene family in some Gramineous plants, namely, Bd, Or, Os, Si, Sb, and Zm, but the degree of gene family expansion, the type of PLT gene duplication, and the differentiation time of duplicate gene pairs varied greatly among these species. In addition, the sequence alignment and the internal repeat analysis of all PLTs protein sequences implied that the PLT protein sequences may originate from an internal repeat duplication of an ancestral six transmembrane helical units. Besides that, the protein motifs result highlighted that the PLT protein sequences were highly conserved, whereas the functional differentiation of the PLT genes was characterized by different gene structures, upstream elements, as well as co-expression analysis. The gene expression analysis of rice and maize showed that the PLT genes have a wide range of expression patterns, suggesting diverse biological functions. Taken together, our finding provided a perspective on the evolution differences and the functional characterizations of PLT genes in Gramineae representative crops.

Ammonium (NH4+) is one of the major nitrogen sources for plants. However, excessive ammonium can cause serious harm to the growth and development of plants, i.e., ammonium toxicity. The primary regulatory mechanisms behind ammonium toxicity are still poorly characterized. In this study, we showed that OsCIPK18, a CBL-interacting protein kinase, plays an important role in response to ammonium toxicity by comparative analysis of the physiological and whole transcriptome of the T-DNA insertion mutant (cipk18) and the wild-type (WT). Root biomass and length of cipk18 are less inhibited by excess NH4+ compared with WT, indicating increased resistance to ammonium toxicity. Transcriptome analysis reveals that OsCIPK18 affects the NH4+ uptake by regulating the expression of OsAMT1;2 and other NH4+ transporters, but does not affect ammonium assimilation. Differentially expressed genes induced by excess NH4+ in WT and cipk18 were associated with functions, such as ion transport, metabolism, cell wall formation, and phytohormones signaling, suggesting a fundamental role for OsCIPK18 in ammonium toxicity. We further identified a transcriptional regulatory network downstream of OsCIPK18 under NH4+ stress that is centered on several core transcription factors. Moreover, OsCIPK18 might function as a transmitter in the auxin and abscisic acid (ABA) signaling pathways affected by excess ammonium. These data allowed us to define an OsCIPK18-regulated/dependent transcriptomic network for the response of ammonium toxicity and provide new insights into the mechanisms underlying ammonium toxicity.