Integrating linkage mapping and comparative transcriptome analysis for discovering candidate genes associated with salt tolerance in rice.

Salinity is one of the most widespread abiotic stresses affecting rice productivity worldwide. Understanding the genetic basis of salt tolerance is key for breeding salt-tolerant rice varieties. Numerous QTLs have been identified to help dissect rice salt-tolerance genetic mechanisms, yet only rare genes located in significant QTLs have been thoroughly studied or fine-mapped. Here, a combination of linkage mapping and transcriptome profiling analysis was used to identify salt tolerance-related functional candidate genes underlying stable QTLs. A recombinant inbred line (RIL) population derived from a cross between Jileng 1 (salt-sensitive) and Milyang 23 (salt-tolerant) was constructed. Subsequently, a high-density genetic map was constructed by using 2921 recombination bin markers developed from whole genome resequencing. A total of twelve QTLs controlling the standard evaluation score under salt stress were identified by linkage analysis and distributed on chromosomes 2, 3, 4, 6, 8 and 11. Notably, five QTL intervals were detected as environmentally stable QTLs in this study, and their functions were verified by comparative transcriptome analysis. By comparing the transcriptome profiles of the two parents and two bulks, we found 551 salt stress-specific differentially expressed genes. Among them, fifteen DEGs located in stable QTL intervals were considered promising candidate genes for salt tolerance. According to gene annotations, the gene OsRCI2-8(Os06g0184800) was the most promising, as it is known to be associated with salt stress, and its differential expression between the tolerant and sensitive RIL bulks highlights its important role in salt stress response pathways. Our findings provide five stable salt tolerance-related QTLs and one promising candidate gene, which will facilitate breeding for improved salt tolerance in rice varieties and promote the exploration of salt stress tolerance mechanisms in rice.

Candidate gene discovery for salt tolerance in rice (Oryza sativa L.) at the germination stage based on genome-wide association study.

Salt stress affects rice seed germination and seedling formation, seriously restricting rice production. Screening salt-tolerant rice varieties and analyzing the genetic mechanisms underlying salt tolerance are therefore very important to ensure rice production. In this study, 313 Oryza sativa ssp. japonica germplasm were used to conduct a genome-wide association study (GWAS) using 1% NaCl as a salt stress treatment during germination stage. The germination potential (GP) on different days and the germination index (GI) under salt stress were used as salt tolerance indicators. The results of population structure analysis showed that the 313 germplasm studied could be divided into two subpopulations, consistent with the geographical origins of the materials. There were 52 loci significantly related to salt tolerance during germination, and the phenotypic contribution rate of 29 loci was > 10%. A region on chromosome 11 (17049672-17249672 bp) was repeatedly located, and the candidate gene LOC_Os11g29490, which encodes a plasma membrane ATPase, was identified in this locus. Further haplotype analysis showed the GP of germplasm with different haplotypes at that locus significantly differed under salt stress (p < 0.05), and germplasm carrying Hap2 displayed strong salt tolerance during the germination stage. Two other promising candidate genes for salt tolerance were identified: LOC_Os01g27170 (OsHAK3), which encodes a potassium transporter, and LOC_Os10g42550 (OsITPK5), which encodes an inositol 1, 3, 4-trisphosphate 5/6-kinase. The results of this study provide a theoretical basis for salt-tolerant gene cloning and molecular design breeding in rice.

Parallel reaction monitoring revealed tolerance to drought proteins in weedy rice (Oryza sativa f. spontanea).

Drought is a complicated abiotic stress factor with severe effects on rice growth and production. Weedy rice is a valuable genetic resource that possesses a strong capacity for drought tolerance, cold tolerance, and salt tolerance, and is an excellent material for studying rice tolerance. Here, according to comprehensive tolerance to drought index D, accession WR16 was selected based on strong drought tolerance among 133 studied weedy red rice germplasms. WR16 was compared with Oryza sativa ssp. Japonica. cv. IAPAR-9, a reference genotype originating from Brazil. In addition, accession WR24 was classified as moderately tolerant to drought accessions. Transcriptomic and proteomic analyses were combined to identify 38 co-upregulated proteins related to drought tolerance, and targeted parallel reaction monitoring (PRM) was used to precisely quantify and verify nine proteins in the complex backgrounds. Result showed that six proteins were significantly (Fisher's exact P value < 0.05) related to drought tolerance in accessions WR16 and WR24. Among them, OS09T0478300-01, OS09T0530300-01, and OS01T0800500-01 formed a combined defense system to respond to drought stress in weedy rice. Results of these studies provide comprehensive information for precisely identifying and verifying tolerance to drought proteins and lay a solid theoretical foundation for research on drought tolerance mechanisms.

Comprehensive Evaluation and Analysis of the Mechanism of Cold Tolerance Based on the Transcriptome of Weedy Rice Seedlings.

In this study, the cold-tolerance capacity of 133 varieties of weedy rice was evaluated based on the comprehensive evaluation index D, with Kongyu 131 used as a cold-tolerant control. A total of 39.8% of the 133 varieties were considered 'strong', indicating that weedy rice populations indeed have relatively strong cold-tolerance capacity as a whole, and the robust cold-tolerant varieties WR29 and WR157 were identified. Regression analysis showed that the metrics including the nitrogen recovery index, superoxide dismutase (SOD) content and malondialdehyde (MDA) content correlated significantly (P < 0.05) with cold tolerance and could be used as indicators of cold tolerance. On the basis of a transcriptome analysis of WR157, a robust cold-tolerant variety identified in this study, a total of 4645 putative DEGs were identified in treated groups compared to the control groups, with 2123 upregulated DEGs and 2522 downregulated DEGs. All upregulated DEGs were enriched on 1388 terms, all downregulated DEGs were enriched on 1566 terms; 911 of the 2123 upregulated DEGs fell into 98 KEGG categories and 1103 of the 2522 downregulated DEGs were in 115 categories. Further analysis showed that GO:0019740 and GO:0006808 are involved in nitrogen utilization; GO:0009269 and GO:0009414 are related to the stress response; and GO:0016491 and GO:0016614 are related to oxidoreductase activity. BACKGROUND: Weedy rice (Oryza) is a related pest species of cultivated rice (Oryza sativa L.) that has strong abiotic stress resistance; however, the comprehensive mechanism governing its cold tolerance is poorly understood. CONCLUSION: Our comprehensive evaluation based on five morphological indices and nine physiological indicators revealed outstanding levels of cold-tolerance capacity among weedy rice varieties from different regions and revealed some terms related to cold tolerance via transcriptome analysis. Our results underscored the reliable evaluation methods for additional cold tolerance studies and revealed several genes related to cold tolerance, which will help researchers breed cultivated rice varieties to increase their cold-tolerance capacity. These traits have the ability to increase seedling survival rate and growth, as well as future yields.

Analysis of evolutionary relationships provides new clues to the origins of weedy rice.

Weedy rice (WR) (Oryza sativa f. spontanea) is considered to be a pest in modern rice production systems because it competes for resources, has poor yield characteristics, and subsequently has a negative effect on rice grain yield. The evolutionary relationships among WR, landrace rice (LR), improved rice (IR) cultivars, and wild rice are largely unknown. In this study, we conducted a population genetic analysis based on neutral markers and gene haplotypes in 524 rice accessions and a comparative transcriptomic analysis using 15 representative samples. The results showed that WR populations have the highest level of genetic diversity (H e = 0.8386) and can be divided into two groups (japonica-type and indica-type). The japonica-type WR accessions from Heilongjiang province (HLJ), Jilin province (JL), Liaoning province (LN), and NX provinces clustered with the landraces grown in these same provinces. The indica-types from Jiangsu province (JS) also clustered with the indica-type landraces from JS province. Comparative transcriptome analysis of WR' IR and LR from HLJ, JL, and LN provinces showed that the WR still clustered with the LR, and that the IR lines comprise a single population. Thirty-two differentially expressed genes were shared by the IR and LR groups as well as between the IR and WR groups. Using Gene ontology (GO) analysis, we identified 19 shared GO terms in the IR and LR groups as well as between the IR and WR groups. Our results suggest that WR populations in China have diverse origins, and comparative transcriptome analysis of different types of rice from HLJ, JL, and LN provinces suggests that IR populations have become a end point in the evolution of WR, which provides a new perspective for the study of WR origins and lays a solid foundation for rice breeding.

Construction of chromosome segment substitution lines of Dongxiang common wild rice (Oryza rufipogon Griff.) in the background of the japonica rice cultivar Nipponbare (Oryza sativa L.).

Dongxiang common wild rice (Oryza rufipogon Griff.) is believed to have the northernmost distribution of any wild rice species. Many favorable genes were lost during rice domestication, and Dongxiang common wild rice is a potential source of many genes related to biological and abiotic stress resistance and high grain yield. Despite its importance, japonica rice has not yet been used as a background material for the construction of introgression lines carrying Dongxiang common wild rice chromosome segments. In this study, we used Dongxiang common wild rice as the donor parent and Nipponbare (Oryza sativa L), a reference-sequence japonica cultivar, as the recurrent parent to develop a set of 104 chromosome segment substitution lines (CSSLs) through crossing and backcrossing with marker-assisted selection based on 203 polymorphic molecular markers evenly distributed across 12 chromosomes. The 104 CSSLs covered 87.94% of the genome of Dongxiang common wild rice. Each CSSL contained an average of four introgressed segments, and the average segment length was 3.3 Mb. Quantitative trait locus (QTL) analysis of the panicle-related traits of the CSSLs at two sites (Beijing and Nanchang, Jiangxi Province) revealed 18 QTLs for eight traits. Among these 18 QTLs were two known grain length- and width-related genes and four novel QTLs. In addition, two QTLs were verified, and two novel QTLs were identified, for panicle neck length, a domestication-related trait. The developed CSSLs potentially represent a valuable population for detecting favorable genes in common wild rice and creating germplasm resources for japonica rice breeding.