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Abstract Background Boron (B) is an essential micronutrient for plants. Inappropriate B supply detrimentally affects the productivity of numerous crops. Understanding of the molecular responses of plants to different B supply levels would be of significance in crop improvement and cultivation practices to deal with the problem. Results We conducted a comprehensive analysis of the transcriptome and proteome of tobacco seedlings to investigate the expression changes of genes/proteins in response to different B supply levels, with a particular focus on B deficiency. The global gene and protein expression profiles revealed the potential mechanisms involved in the responses of tobacco to B deficiency, including up-regulation of the NIP5;1-BORs module, complex regulation of genes/proteins related to cell wall metabolism, and up-regulation of the antioxidant machinery. Conclusion Our results demonstrated that B deficiency caused severe morphological and physiological disorders in tobacco seedlings, and revealed dynamic expression changes of tobacco genes/proteins in response to different B supply levels, especially to B deficiency, thus offering valuable insights into the molecular responses of tobacco to B deficiency.

Abstract Large‐scale topographies affect global extreme weather and climate though dynamic and thermal forcing. However, the impacts of typical topographies on heat waves in different drylands globally remain unclear. In this study, we find that heat waves are mainly occurred in global drylands during 1940–2022. The frequencies and intensities of heat waves in global drylands have significantly increased after 1980s. Multiple numerical model simulations reveal that the impact range of Asian topography on heat waves in global drylands is widest, not only in East Asia, Central, and west Asia locally, but also can reach as far as North Africa and North America. While the impact ranges of topographies in Africa, Arabian Peninsula, North America, and South America on heat waves in drylands are relatively narrow, which are concentrated in localized and their surroundings. These conclusions could provide clues to understand the influence of topography on global weather and climate extremes.

Smooth cordgrass (Spartina alterniflora) is a native salt marsh plant along the Atlantic coast but has become an invasive species in coastal regions in China, as well as other areas. Utilizing it for resources has become a control measure in reducing the spread of S. alterniflora. This study assesses the nutritional and phytochemical properties of the edible stems of S. alterniflora collected from three locations in Fujian province, China. The tender stems of S. alterniflora exhibit a rich nutritional profile, with high levels of protein, carbohydrates, and fats, and significant amounts of essential vitamins, minerals, and antioxidants, indicating their potential as a nutritious addition to the diet or forage. In addition, the levels of potential contaminants, including nitrate, nitrite, cadmium, lead, and chromium, are below the established safe thresholds for consumption. Our results provide valuable information for the sustainable utilization of S. alterniflora resources and will contribute to the integrated control of S. alterniflora.

Abstract Background The Glucan synthase-like (GSL) genes are indispensable for some important highly-specialized developmental and cellular processes involving callose synthesis and deposition in plants. At present, the best-characterized reproductive functions of GSL genes are those for pollen formation and ovary expansion, but their role in seed initiation remains unknown. Results We identified a rice seed mutant, watery seed 1-1 (ws1-1), which contained a mutation in the OsGSL2 gene. The mutant produced seeds lacking embryo and endosperm but filled with transparent and sucrose-rich liquid. In a ws1-1 spikelet, the ovule development was normal, but the microsporogenesis and male gametophyte development were compromised, resulting in the reduction of fertile pollen. After fertilization, while the seed coat normally developed, the embryo failed to differentiate normally. In addition, the divided endosperm-free nuclei did not migrate to the periphery of the embryo sac but aggregated so that their proliferation and cellularization were arrested. Moreover, the degeneration of nucellus cells was delayed in ws1-1. OsGSL2 is highly expressed in reproductive organs and developing seeds. Disrupting OsGSL2 reduced callose deposition on the outer walls of the microspores and impaired the formation of the annular callose sheath in developing caryopsis, leading to pollen defect and seed abortion. Conclusions Our findings revealed that OsGSL2 is essential for rice fertility and is required for embryo differentiation and endosperm-free nucleus positioning, indicating a distinct role of OsGSL2, a callose synthase gene, in seed initiation, which provides new insight into the regulation of seed development in cereals.

IntroductionHarmful algal blooms (HABs) have been increasing in frequency and expanding their ranges on coastlines worldwide in recent decades. Algicidal bacteria play a pivotal role in eliminating HABs, yet the characteristics of bacterial communities and their algicidal activity during a Karenia longicanalis bloom remain poorly understood.MethodsIn this study, we investigated bacterial communities using 16S rRNA sequencing during a K. longicanalis bloom to identify bacteria with high algicidal activity that could be isolated. Five sampling sites in Tongxin Bay, located in Lianjiang County, China, including TX1 to TX5, were selected based on the concentration of K. longicanalis cells.ResultsOur 16S rRNA sequencing results revealed that the TX4 site was enriched with genera known to contain algicidal bacteria, such as Pseudoalteromonas and Alteromonas, which are members of the Gammaproteobacteria class, while Sulfitobacter, a member of the Alphaproteobacteria class, was enriched in the TX5 site. Among the 100 cultivable bacteria isolated from the 5 sampling sites, 6 exhibited an algicidal rate of over 80%, with FDHY-MQ5, isolated from the TX4 site, exhibiting an algicidal rate of approximately 100% against Karenia mikimotoi after 48 hours of challenge with 2% (v/v) bacterial volume (OD600=4.5) concentration. Our 16S rRNA sequencing result showed FDHY-MQ5 was a member of the Pseudoalteromonas genus, and this bacterium also demonstrated high algicidal activity against Heterosigma akashiwo and Alexandrium tamarense.DiscussionOur findings shed light on the changes in bacterial community structure and the algicidal behavior of bacteria towards algae during a K. longicanalis bloom, providing a research basis for a better understanding of HAB management.

Folic acid (FA) is an important water-soluble vitamin and plays an important role as a cofactor and coenzyme in animal growth and development, and regulation of gene expression and methylation. A total of 270 female broiler chickens (1-day-old) were randomly allotted to three dietary treatments supplemented with 0 mg/kg (control group), 5 mg/kg, and 10 mg/kg FA in basal diets for 42 days, respectively. Each treatment had six replicate cages with 15 birds per cage. Dietary supplementation of 5 mg/kg FA significantly enhanced average body weight and average daily gain of 21-day-old broilers (P < 0.05), but significantly reduced subcutaneous fat thickness and widths of an intermuscular fat band of 42-day-old broilers by dietary FA treatments (P < 0.05). Also, a diet with 10 mg/kg FA supplementation significantly increased the relative heart weight of 42-day-old chickens (P < 0.05). Furthermore, dietary FA supplementation significantly improved the serum insulin-like growth factor 2 (IGF2) concentrations (P < 0.01) and IGF2 mRNA expression in the abdominal fat (P < 0.05), but no statistical differences were found in the methylation of IGF2 promoter (P > 0.05). The present study demonstrated that dietary FA supplementation may have positive effects on chicken growth through increased IGF2 gene expression.

Flammulina filiformis, as one of the most popular edible fungi in East Asia, is produced in an industrialized and standardized way. However, its monotonous variety and product convergence have seriously restricted the development of the industry. In this study, 11 cultivated strains and 13 wild strains of F. filiformis were collected from multiple regions of China and Japan and were performed genome sequencing. Together with genome data of six strains previously released, in total 23 dikaryons (formed by two monokaryons mating, can making fruiting body), 35 monokaryons (formed by protoplast-regenerating of dikaryon and isolating) were used for genetic diversity and population structure analysis based on the high-throughput genotyping. Firstly, a set of SNP markers with intrapopulation polymorphism including 849,987 bi-allelic SNPs were developed and basically covered all of 11 chromosomes with a high distribution density of 24.16 SNP markers per kb. The cultivated dikaryotic strains were divided into three subgroups, and their breeding history was made inferences, which is consistent with the available pedigree records. The wild dikaryotic strains were divided into two subgroups and showed varied contributions of genetic components with high genetic diversity. All the investigated dikaryons have a symmetric distribution pattern with their two constituent monokaryons in principal component analysis. Finally, we summarized the pedigree relationship diagram of F. filiformis main strains including six modules, and the genotypes of hybrids can be directly phased by the known parental allele according to it. This study provides a method to distinguish two sets of monokaryon haplotypes, and several valuable genetic resources of wild F. filiformis, and an effective strategy for guiding F. filiformis breeding based on the population structure and pedigree relationship in future.

The evening complex (EC) plays a critical role in photoperiod flowering in Arabidopsis. Nevertheless, the underlying functions of individual components and coordinate regulation mechanism of EC genes in rice flowering remain to be elucidated. Here, we characterized the critical role of LUX ARRHYTHMO (LUX) in photoperiod perception and coordinating vegetative growth and flowering in rice. Non-functional alleles of OsLUX extremely extended vegetative phase, leading to photoperiod-insensitive late flowering and great increase of grain yield. OsLUX displayed an obvious diurnal rhythm expression with the peak at dusk and promoted rice flowering via coordinating the expression of genes associated with the circadian clock and the output integrators of photoperiodic flowering. OsLUX combined with OsELF4a and OsELF3a or OsELF3b to form two ECs, of which the OsLUX-OsELF3a-OsELF4a was likely the dominant promoter for photoperiodic flowering. In addition, OsELF4a was also essential for promoting rice flowering. Unlike OsLUX, loss OsELF4a displayed a marginal influence under short-day (SD) condition, but markedly delayed flowering time under long-day (LD) condition. These results suggest that rice EC genes share the function of promoting flowering. This is agreement with the orthologs of SD plant, but opposite to the counterparts of LD species. Taken together, rice EC genes display similar but not identical function in photoperiodic flowering, probably through regulating gene expression cooperative and independent. These findings facilitate our understanding of photoperiodic flowering in plants, especially the SD crops.

Abstract Background Plant transcription factors (TFs) are key transcriptional regulators to manipulate the regulatory network of host immunity. However, the globally transcriptional reprogramming of plant TF families in response to pathogens, especially between the resistant and susceptible host plants, remains largely unknown. Results Here, we performed time-series RNA-seq from a resistant pepper line CM334 and a susceptible pepper line EC01 upon challenged with Phytophthora capsici, and enrichment analysis indicated that WRKY family most significantly enriched in both CM334 and EC01. Interestingly, we found that nearly half of the WRKY family members were significantly up-regulated, whereas none of them were down-regulated in the two lines. These induced WRKY genes were greatly overlapped between CM334 and EC01. More strikingly, most of these induced WRKY genes were expressed in time-order patterns, and could be mainly divided into three subgroups: early response (3 h-up), mid response (24 h-up) and mid-late response (ML-up) genes. Moreover, it was found that the responses of these ML-up genes were several hours delayed in EC01. Furthermore, a total of 19 induced WRKY genes were selected for functional identification by virus-induced gene silencing. The result revealed that silencing of CaWRKY03–6, CaWRKY03–7, CaWRKY06–5 or CaWRKY10–4 significantly increase the susceptibility to P. capsici both in CM334 and EC01, indicating that they might contribute to pepper’s basal defense against P. capsici; while silencing of CaWRKY08–4 and CaWRKY01–10 significantly impaired the disease resistance in CM334 but not in EC01, suggesting that these two WRKY genes are prominent modulators specifically in the resistant pepper plants. Conclusions These results considerably extend our understanding of WRKY gene family in pepper’s resistance against P. capsici and provide potential applications for genetic improvement against phytophthora blight.

AbstractDeep sequencing-based bulked segregant analysis (BSA-seq) has become a popular approach for quantitative trait loci (QTL) mapping in recent years. Effective statistical methods for BSA-seq have been developed, but how to design a suitable experiment for BSA-seq remains unclear. In this paper, we show in theory how the major experimental factors (including population size, pool proportion, pool balance, and generation) and the intrinsic factors of a QTL (including heritability and degree of dominance) affect the power of QTL detection and the precision of QTL mapping in BSA-seq. Increasing population size can improve the power and precision, depending on the QTL heritability. The best proportion of each pool in the population is around 0.25. So, 0.25 is generally applicable in BSA-seq. Small pool proportion can greatly reduce the power and precision. Imbalance of pool pair in size also causes decrease of the power and precision. Additive effect is more important than dominance effect for QTL mapping. Increasing the generation of filial population produced by selfing can significantly increase the power and precision, especially from F23http://124.71.74.135/BSA-seqDesignTool/https://github.com/huanglikun/BSA-seqDesignTool

High-density genetic maps can significantly improve the resolution of QTL mapping. We constructed a high-density recombination bin-based genetic map of eggplant based on 200 F2 plants from an interspecific cross (Solanum melongena × S. incanum) using the whole genome resequencing strategy. The map was 2022.8 cM long, covering near 99% of the eggplant genome. The map contained 3776 bins, with 3644 (96.5%) being effective (position non-redundant) ones, giving a nominal average distance of 0.54 cM and an effective average distance of 0.56 cM between adjacent bins, respectively. Using this map and 172 F2:3 lines, a major QTL with pleiotropic effects on two anthocyanin pigmentation-related traits, leaf vein color (LVC) and fruit pericarp color (FPC), was steadily detected in a bin interval of 2.28 cM (or 1.68 Mb) on chromosome E10 in two cropping seasons, explaining ~65% and 55% of the phenotypic variation in LVC and FPC, respectively. Genome-wide association analysis in this population validated the QTL and demonstrated the correctness of mapping two bins of chromosome E02 onto E10. Bioinformatics analysis suggested that a WDR protein gene inside the bin interval with reliable effective variation between the two parents could be a possible candidate gene of the QTL.

In this study, we investigate the most severe East Asian dust storm in the past decade that occurred on 14–16 March 2021 based on the Weather Research and Forecasting model coupled with chemistry (WRF-Chem) and a variety of site measurements and satellite retrievals. The dust emissions from the Gobi Desert, especially over Mongolia on March 14, are the dominant sources of this intense dust event. The maximal hourly accumulated dust emissions over Mongolian and Chinese areas reached 1490.18 kt at 07:00 UTC on 14 March and 821.70 kt at 2:00 UTC on 15 March, respectively. During this dust event, the accumulated dust emissions in coarse modes (i.e., bin 4 and bin 5) account for 64.1% of the total dust emission mass, and the accumulated dust emissions in fine modes (i.e., bin 1) are the least, accounting for 7.6% of the total dust emission mass. Because the coarse mode bins of dust dominate the emissions, the downwind transported coarse mode particles can affect the North China Plain, while the fine particles can only affect the desert source and its surrounding regions such as the Gansu and Ningxia provinces. Due to the dust emissions and the dust transport path, the high AOD areas are located in the Gobi Desert and Northwest China and the vertical spatial distributions of aerosol extinction coefficients have the same characteristics. We also found the model drawback of overestimating simulated wind speeds, which leads to the overestimations of dust emissions and concentrations, indicating the urgency of improving the simulated wind field.

The heterosis in hybrid rice is highly affected by the environment and hybrid weakness occurs frequently depending on the genotypes of the hybrid and its parents. Hybrid weakness was also observed in our field experiments on nine rice hybrids produced by 3 × 3 incomplete diallel crosses. Among the nine hybrids, five displayed mid-parent heterosis (MPH) for grain yield per plant, while four showed mid-parent hybrid weakness (MPHW). A sequencing analysis of transcriptomes in panicles at the seed-filling stage revealed a significant association between enhanced non-additive gene expression (NAE) and allele-specific gene expression (ASE) with hybrid weakness. High proportions of ASE genes, with most being of mono-allele expression, were detected in the four MPHW hybrids, ranging from 22.65% to 45.97%; whereas only 4.80% to 5.69% of ASE genes were found in the five MPH hybrids. Moreover, an independence test indicated that the enhancements of NAE and ASE in the MPHW hybrids were significantly correlated. Based on the results of our study, we speculated that an unfavorable environment might cause hybrid weakness by enhancing ASE and NAE at the transcriptome level.

Hyperglycemia-induced endothelial endoplasmic reticulum (ER) stress is implicated in the pathophysiology of diabetes and its vascular complications. Procyanidins are enriched in many plant foods and have been demonstrated to exert several beneficial effects on diabetes, cardiovascular and other metabolic diseases. In the present study, we investigated the effect of procyanidin B2 (PCB2), the most widely distributed natural procyanidin, on ER stress evoked by high glucose in endothelial cells (ECs) and the underlying mechanisms. We showed that PCB2 mitigated the high glucose-activated ER stress pathways (PERK, IRE1α and ATF6) in human vascular ECs. In addition, we found that PCB2 attenuated endothelial ER stress via the activation of peroxisome proliferator-activated receptor δ (PPARδ). We demonstrated that PCB2 directly bound to and activated PPARδ. Conversely, GSK0660, a selective PPARδ antagonist, attenuated the suppressive effect of PCB2 on the ER stress signal pathway. Functionally, PCB2 ameliorated the high glucose-impaired endothelium-dependent relaxation in mouse aortas. The protective effect of PCB2 on vasodilation was abolished in the aortas pretreated with GSK0660 or those from the EC-specific PPARδ knockout mice. Moreover, the protective effects of PCB2 on ER stress and endothelial dysfunction required the inter-dependent actions of PPARδ and AMPK. Collectively, we demonstrated that PCB2 mitigated ER stress and ameliorated vasodilation via a PPARδ-mediated mechanism beyond its classic action as a scavenger of free radicals. These findings further highlighted the novel roles of procyanidins in intervening the ER stress and metabolic disorders related to endothelial dysfunction.

Schizochytrium sp. is the best natural resource for omega-3 long-chain polyunsaturated fatty acids. We report a high-quality genome sequence of Schizochytrium limacinum SR21, which has a 63 Mb genome size, with a contig N50 of 2.67 Mb and 6,838 protein-coding genes. Phylogenomic and comparative genomic analyses revealed that DHA-producing Schizochytrium and Aurantiochytrium strains were highly similar and possessed similar genes. Analysis of the fatty acid synthase (FAS) for LC-PUFAs production results in the annotation of all genes in map00062 and map01212. A gene cluster and 10 ORFs related to PKS pathway were found in the genome. 1,402 differentially expressed genes (DEGs) of the treated groups (0.5 g/L yeast extract) were identified by comparing with the control groups (1.0 g/L yeast extract) at 36 h. A weighted gene coexpression network analysis revealed that 2 of 7 modules correlated highly with the fatty acid and DHA contents. The DEGs and transcription factors were significantly correlated with fatty acid biosynthesis, including MYB, Zinc Finger and ACOX. The results showed that these hub genes are regulated by genes involved in fatty acid biosynthesis pathways. The results providing an important reference for further research on promoting fatty acid and DHA accumulation in S. limacinum SR21.

Abstract Background Host-plant resistance is the most desirable and economic way to overcome BPH damage to rice. As single-gene resistance is easily lost due to the evolution of new BPH biotypes, it is urgent to explore and identify new BPH resistance genes. Results In this study, using F2:3 populations and near-isogenic lines (NILs) derived from crosses between two BPH-resistant Sri Lankan rice cultivars (KOLAYAL and POLIYAL) and a BPH-susceptible cultivar 9311, a new resistance gene Bph33 was fine mapped to a 60-kb region ranging 0.91–0.97 Mb on the short arm of chromosome 4 (4S), which was at least 4 Mb distant from those genes/QTLs (Bph12, Bph15, Bph3, Bph20, QBph4 and QBph4.2) reported before. Seven genes were predicted in this region. Based on sequence and expression analyses, a Leucine Rich Repeat (LRR) family gene (LOC_Os04g02520) was identified as the most possible candidate of Bph33. The gene exhibited continuous and stable resistance from seedling stage to tillering stage, showing both antixenosis and antibiosis effects on BPH. Conclusion The results of this study will facilitate map-based cloning and marker-assisted selection of the gene.

OsWUS has recently been shown to be a transcription factor gene critical for tiller development and fertility in rice. The OsWUS protein consists of three conserved structural domains, but their biological functions are still unclear. We discovered a new rice mutant resulting from tissue culture, which hardly produced tillers and exhibited complete female sterility. The male and female floral organs of the mutant were morphologically indistinguishable from those of the wild type. We named the mutant srt1 for completely sterile and reduced tillering 1. Map-based cloning revealed that the mutant phenotypes were caused by a mutation in OsWUS. Compared with the two previously reported null allelic mutants of OsWUS (tab1-1 and moc3-1), which could produce partial N-terminal peptides of OsWUS, the srt1 protein contained a deletion of only seven amino acids within the conserved homeobox domain of OsWUS. However, the mutant phenotypes (monoculm and female sterility) displayed in srt1 were as typical and severe as those in tab1-1 and moc3-1. This indicates that the homeobox domain of SRT1 is essential for the regulation of tillering and sterility in rice. In addition, srt1 showed an opposite effect on panicle development to that of the two null allelic mutants, implying that the srt1 protein might still have partial or even new functions on panicle development. The results of this study suggest that the homeobox domain is pivotal for OsWUS function.

A two-stage light gas gun driven by gaseous detonation was newly constructed, which can make up for the disadvantages of the insufficient driving capability of high-pressure gas and the constraints of gunpowder. The performance of the gas gun was investigated through experiments and a quasi-one-dimensional modeling of it was also developed and described in detail. The model accounts for the friction and heat transfer to the tube wall for gases by adding a source term. An improved model has been established to consider the inertial loads in the piston or projectile and model the friction force with the tube wall. Besides, the effects of pump tube pressure on the performance of the gas gun are also investigated numerically. Simulations of the pressure histories in the pump tube and the piston and projectile velocities were conducted. A good agreement was observed between the computational predictions and experimental results. The results showed that the friction between the piston and wall had only small influence on the piston velocity. The proposed numerical approach is suitable for the development of two-stage light gas guns and tests of the operating conditions.

Basal or partial resistance has been considered race-non-specific and broad-spectrum. Therefore, the identification of genes or quantitative trait loci (QTLs) conferring basal resistance and germplasm containing them is of significance in breeding crops with durable resistance. In this study, we performed a bulked segregant analysis coupled with whole-genome sequencing (BSA-seq) to identify QTLs controlling basal resistance to blast disease in an F2 population derived from two rice varieties, 02428 and LiXinGeng (LXG), which differ significantly in basal resistance to rice blast. Four candidate QTLs, qBBR-4, qBBR-7, qBBR-8, and qBBR-11, were mapped on chromosomes 4, 7, 8, and 11, respectively. Allelic and genotypic association analyses identified a novel haplotype of the durable blast resistance gene pi21 carrying double deletions of 30 bp and 33 bp in 02428 (pi21-2428) as a candidate gene of qBBR-4. We further assessed haplotypes of Pi21 in 325 rice accessions, and identified 11 haplotypes among the accessions, of which eight were novel types. While the resistant pi21 gene was found only in japonica before, three Chinese indica varieties, ShuHui881, Yong4, and ZhengDa4Hao, were detected carrying the resistant pi21-2428 allele. The pi21-2428 allele and pi21-2428-containing rice germplasm, thus, provide valuable resources for breeding rice varieties, especially indica rice varieties, with durable resistance to blast disease. Our results also lay the foundation for further identification and functional characterization of the other three QTLs to better understand the molecular mechanisms underlying rice basal resistance to blast disease.

CoCrFeCuNi high-entropy alloys (HEAs) prepared by arc melting were irradiated with a 100 keV He+ ion beam. Volume swelling and hardening induced by irradiation were evaluated. When the dose reached 5.0 × 1017 ions/cm2, the Cu-rich phases exhibited more severe volume swelling compared with the matrix phases. This result indicated that the Cu-rich phases were favorable sites for the nucleation and gathering of He bubbles. X-ray diffraction indicated that all diffraction peak intensities decreased regularly. This reduction suggested loosening of the irradiated layer, thereby reducing crystallinity, under He+ ion irradiation. The Nix-Gao model was used to fit the measured hardness in order to obtain a hardness value H0 that excludes the indentation size effect. At ion doses of 2.5 × 1017 ions/cm2 and 5.0 × 1017 ions/cm2, the HEAs showed obvious hardening, which could be attributed to the formation of large amounts of irradiation defects. At the ion dose of 1.0 × 1018 ions/cm2, hardening was reduced, owing to the exfoliation of the original irradiation layer, combined with recovery induced by long-term thermal spike. This study is important to explore the potential uses of HEAs under extreme irradiation conditions.

Low temperature is a major limiting factor in rice growth and development. Mapping of quantitative trait loci (QTLs) controlling cold tolerance is important for rice breeding. Recent studies have suggested that bulked segregant analysis (BSA) combined with next-generation sequencing (NGS) can be an efficient and cost-effective way for QTL mapping. In this study, we employed NGS-assisted BSA to map QTLs conferring cold tolerance at the seedling stage in rice. By deep sequencing of a pair of large DNA pools acquired from a very large F3 population (10,800 individuals), we obtained ∼450,000 single nucleotide polymorphisms (SNPs) after strict screening. We employed two statistical methods for QTL analysis based on these SNPs, which yielded consistent results. Six QTLs were mapped on chromosomes 1, 2, 5, 8 and 10. The three most significant QTLs on chromosomes 1, 2 and 8 were validated by comparison with previous studies. Two QTLs on chromosomes 2 and 5 were also identified previously, but at the booting stage rather than the seedling stage, suggesting that some QTLs may function at different developmental stages, which would be useful for cold tolerance breeding in rice. Compared with previously reported QTL mapping studies for cold tolerance in rice based on the traditional approaches, the results of this study demonstrated the advantages of NGS-assisted BSA in both efficiency and statistical power.