Synergizing breeding strategies via combining speed breeding, phenotypic selection, and marker-assisted backcrossing for the introgression of Glu-B1i in wheat.

Wheat is a major food crop that plays a crucial role in the human diet. Various breeding technologies have been developed and refined to meet the increasing global wheat demand. Several studies have suggested breeding strategies that combine generation acceleration systems and molecular breeding methods to maximize breeding efficiency. However, real-world examples demonstrating the effective utilization of these strategies in breeding programs are lacking. In this study, we designed and demonstrated a synergized breeding strategy (SBS) that combines rapid and efficient breeding techniques, including speed breeding, speed vernalization, phenotypic selection, backcrossing, and marker-assisted selection. These breeding techniques were tailored to the specific characteristics of the breeding materials and objectives. Using the SBS approach, from artificial crossing to the initial observed yield trial under field conditions only took 3.5 years, resulting in a 53% reduction in the time required to develop a BC2 near-isogenic line (NIL) and achieving a higher recurrent genome recovery of 91.5% compared to traditional field conditions. We developed a new wheat NIL derived from cv. Jokyoung, a leading cultivar in Korea. Milyang56 exhibited improved protein content, sodium dodecyl sulfate-sedimentation value, and loaf volume compared to Jokyoung, which were attributed to introgression of the Glu-B1i allele from the donor parent, cv. Garnet. SBS represents a flexible breeding model that can be applied by breeders for developing breeding materials and mapping populations, as well as analyzing the environmental effects of specific genes or loci and for trait stacking.

OsLRR-RLP2 Gene Regulates Immunity to Magnaporthe oryzae in Japonica Rice.

Rice is an important cereal crop worldwide, the growth of which is affected by rice blast disease, caused by the fungal pathogen Magnaporthe oryzae. As climate change increases the diversity of pathogens, the disease resistance genes (R genes) in plants must be identified. The major blast-resistance genes have been identified in indica rice varieties; therefore, japonica rice varieties with R genes now need to be identified. Because leucine-rich repeat (LRR) domain proteins possess R-gene properties, we used bioinformatics analysis to identify the rice candidate LRR domain receptor-like proteins (OsLRR-RLPs). OsLRR-RLP2, which contains six LRR domains, showed differences in the DNA sequence, containing 43 single-nucleotide polymorphisms (SNPs) in indica and japonica subpopulations. The results of the M. oryzae inoculation analysis indicated that indica varieties with partial deletion of OsLRR-RLP2 showed susceptibility, whereas japonica varieties with intact OsLRR-RLP2 showed resistance. The oslrr-rlp2 mutant, generated using clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9), showed increased pathogen susceptibility, whereas plants overexpressing this gene showed pathogen resistance. These results indicate that OsLRR-RLP2 confers resistance to rice, and OsLRR-RLP2 may be useful for breeding resistant cultivars.

Acceleration of wheat breeding: enhancing efficiency and practical application of the speed breeding system.

BACKGROUND: Crop breeding should be accelerated to address global warming and climate change. Wheat (Triticum aestivum L.) is a major food crop. Speed breeding (SB) and speed vernalization (SV) techniques for spring and winter wheat have recently been established. However, there are few practical examples of these strategies being used economically and efficiently in breeding programs. We aimed to establish and evaluate the performance of a breeder-friendly and energy-saving generation acceleration system by modifying the SV + SB system. RESULTS: In this study, a four-generation advancement system for wheat (regardless of its growth habits) was established and evaluated using an energy-efficient extended photoperiod treatment. A glasshouse with a 22-hour photoperiod that used 10 h of natural sunlight and 12 h of LED lights, and minimized temperature control during the winter season, was successful in accelerating generation. Even with one or two field tests, modified speed breeding (mSB) combined with a speed vernalization system (SV + mSB) reduced breeding time by more than half compared to traditional field-based methods. When compared to the existing SV + SB system, the SV + mSB system reduced energy use by 80% to maintain a 22-hour photoperiod. Significant correlations were found between the SV + mSB and field conditions in the number of days to heading (DTH) and culm length (CL). Genetic resources, recombinant inbred lines, and breeding materials that exhibited shorter DTH and CL values under SV + mSB conditions showed the same pattern in the field. CONCLUSIONS: The results of our SV + mSB model, as well as its practical application in wheat breeding programs, are expected to help breeders worldwide incorporate generation acceleration systems into their conventional breeding programs.

Tumor budding as a predictor of disease-free survival in patients with cholangiocarcinoma.

Background: Tumor budding is considered a prognostic factor in several solid cancer types. However, we lack comprehensive information on the importance of tumor budding in cholangiocarcinoma. Therefore, we aimed to assess the prognostic value of tumor budding in intrahepatic and extrahepatic cholangiocarcinomas and to evaluate its correlations with other clinicopathological parameters. Methods: We monitored 219 patients who underwent surgery for intrahepatic or extrahepatic cholangiocarcinoma at the Pusan National University Hospital between 2012 and 2021. Tumor budding was evaluated using the International Tumor Budding Consensus Conference scoring system. Tumor budding was classified into low (0-4), intermediate (5-9), and high (≥10). For statistical analysis, tumor budding was divided into two groups based on the cut-off value of 10 (lower: 0-9 vs. higher: ≥10). The correlations between clinicopathological parameters were examined using the chi-square and Fisher's exact test. The prognostic values of the variables were analyzed using the log-rank test and Cox regression analysis. Results: Low, intermediate, and high tumor buddings were identified in 135 (61.6%), 63 (28.8), and 21 (9.6%), patients, respectively. Higher tumor budding was related to the presence of lymphatic invasion (p = 0.017), higher tumor grade (p = 0.001), higher N category (p = 0.034). In the univariable and multivariable analyses, higher tumor budding was associated with shorter disease-free survival in 97 (44.3%) patients who underwent R0 resection (p < 0.001 and p = 0.011). Tumor budding did not significantly correlate with disease-specific survival in entire patients. Conclusion: Tumor budding may serve as a prognostic factor for intrahepatic and extrahepatic cholangiocarcinomas treated with R0 resection.

Proteome profiling highlights mechanisms underlying pigment and tocopherol accumulation in red and black rice seeds.

Rice is a major component of the human diet and feeds more than 50 million people across the globe. We previously developed two pigmented rice cultivars, Super-hongmi (red seeds) and Super-jami (black seeds), that are highly rich in antioxidants and exhibit high levels of radical scavenging activities. However, the molecular mechanism underlying the accumulation of pigments and different antioxidants in these rice cultivars remains largely elusive. Here, we report the proteome profiles of mature Super-hongmi and Super-jami seeds, and compared them with the Hopum (white seeds) using a label-free quantitative proteomics approach. This approach led to the identification of 5127 rice seed proteins of which 1628 showed significant changes in the pigmented rice cultivar(s). The list of significantly modulated proteins included a phytoene desaturase (PDS3) which suggested accumulation of ζ-carotene in red seeds while the black seeds seem to accumulate more of anthocyanins because of the higher abundance of dihydroflavonol 4-reductase. Moreover, proteins associated with lignin and tocopherol biosynthesis were highly increased in both red and black cultivars. Taken together, these data report the seed proteome of three different colored rice seeds and identify novel components associated with pigment accumulation in rice.

Multi-omics analysis reveals the genetic basis of rice fragrance mediated by betaine aldehyde dehydrogenase 2.

INTRODUCTION: Fragrance is an important economic and quality trait in rice. The trait is controlled by the recessive gene betaine aldehyde dehydrogenase 2 (BADH2) via the production of 2-acetyl-1-pyrroline (2AP). OBJECTIVES: Variation in BADH2 was evaluated at the population, genetic, transcriptional, and metabolic levels to obtain insights into fragrance regulation in rice. METHODS: Whole-genome resequencing of the Korean World Rice Collection of 475 rice accessions, including 421 breeding lines and 54 wild accessions, was performed. Transcriptome analyses of a subset of 279 accessions, proteome analyses of 64 accessions, and volatile profiling of 421 breeding lines were also performed. RESULTS: We identified over 3.1 million high-quality single nucleotide polymorphisms (SNPs) in Korean rice collection. Most SNPs were present in intergenic regions (79%), and 190,148 SNPs (6%) were located in the coding sequence, of which 53% were nonsynonymous. In total, 38 haplotypes were identified in the BADH2 coding region, including four novel haplotypes (one in cultivated and three in wild accessions). Tajima's D values suggested that BADH2 was under balancing selection in japonica rice. Furthermore, we identified 316 expression quantitative trait loci (eQTL), including 185 cis-eQTLs and 131 trans-eQTLs, involved in BADH2 regulation. A protein quantitative trait loci (pQTL) analysis revealed the presence of trans-pQTLs; 13 pQTLs were mapped 1 Mbp from the BADH2 region. Based on variable importance in projection (VIP) scores, 15 volatile compounds, including 2AP, discriminated haplotypes and were potential biomarkers for rice fragrance. CONCLUSION: We generated a catalog of haplotypes based on a resequencing analysis of a large number of rice accessions. eQTLs and pQTLs associated with BADH2 gene expression and protein accumulation are likely involved in the regulation of 2AP variation in fragrant rice. These data improve our understanding of fragrance and provide valuable information for rice breeding.

Mutations in OsDET1, OsCOP10, and OsDDB1 confer embryonic lethality and alter flavonoid accumulation in Rice (Oryza sativa L.) seed.

Morphological and biochemical changes accompanying embryogenesis and seed development are crucial for plant survival and crop productivity. Here, we identified a novel yellowish-pericarp embryo lethal (yel) mutant of the japonica rice cultivar Sindongjin (Oryza sativa L.), namely, yel-sdj. Seeds of the yel-sdj mutant showed a yellowish pericarp and black embryo, and were embryonic lethal. Compared with wild-type seeds, the yel-sdj mutant seeds exhibited significantly reduced grain size, grain weight, and embryo weight, and a remarkably lower rate of embryo retention in kernels subjected to milling. However, the volume of air space between embryo and endosperm, density of embryo, and total phenolic content (TPC) and antioxidant activity of mature grains were significantly higher in the yel-sdj mutant than in the wild type. Genetic analysis and mapping revealed that the yel-sdj mutant was non-allelic to the oscop1 null mutants yel-hc, yel-cc, and yel-sk, and its phenotype was controlled by a single recessive gene, LOC_Os01g01484, an ortholog of Arabidopsis thaliana DE-ETIOLATED 1 (DET1). The yel-sdj mutant carried a 7 bp deletion in the second exon of OsDET1. Seeds of the osdet1 knockout mutant, generated via CRISPR/Cas9-based gene editing, displayed the yel mutant phenotype. Consistent with the fact that OsDET1 interacts with CONSTITUTIVE PHOTOMORPHOGENIC 10 (OsCOP10) and UV-DAMAGED DNA BINDING PROTEIN 1 (OsDDB1) to form the COP10-DET1-DDB1 (CDD), seeds of oscop10 and osddb1 knockout mutants also showed the yel phenotype. These findings will enhance our understanding of the functional roles of OsDET1 and the CDD complex in embryogenesis and flavonoid biosynthesis in rice seeds.

TMT-based quantitative membrane proteomics identified PRRs potentially involved in the perception of MSP1 in rice leaves.

Pathogen-associated molecular patterns (PAMPs) play a key role in triggering PAMPs triggered immunity (PTI) in plants. In the case of the rice-Magnaporthe oryzae pathosystem, fewer PAMPs and their pattern recognition receptors (PRRs) have been characterized. Recently, a M. oryzae snodprot1 homolog protein (MSP1) has been identified that functions as PAMP and triggering the PTI responses in rice. However, the molecular mechanism underlying MSP1-induced PTI is currently elusive. Therefore, we generated MSP1 overexpressed transgenic lines of rice, and a tandem mass tag (TMT)-based quantitative membrane proteomic analysis was employed to decipher the potential MSP1-induced signaling in rice using total cytosolic as well as membrane protein fractions. This approach led to the identification of 8033 proteins of which 1826 were differentially modulated in response to overexpression of MSP1 and/or exogenous jasmonic acid treatment. Of these, 20 plasma membrane-localized receptor-like kinases (RLKs) showed increased abundance in MSP1 overexpression lines. Moreover, activation of proteins related to the protein degradation and modification, calcium signaling, redox, and MAPK signaling was observed in transgenic lines expressing MSP1 in the apoplast. Taken together, our results identified potential PRR candidates involved in MSP1 recognition and suggested the overview mechanism of the MSP1-induced PTI signaling in rice leaves. SIGNIFICANCE: In plants, recognition of pathogen pathogen-derived molecules, such as PAMPs, by plant plant-derived PRRs has an essential role for in the activation of PTI against pathogen invasion. Typically, PAMPs are recognized by plasma membrane (PM) localized PRRs, however, identifying the PM-localized PRR proteins is challenging due to their low abundance. In this study, we performed an integrated membrane protein enrichment by microsomal membrane extraction (MME) method and subsequent TMT-labeling-based quantitative proteomic analysis using MSP1 overexpressed rice. Based on these results, we successfully identified various intracellular and membrane membrane-localized proteins that participated in the MSP1-induced immune response and characterized the potential PM-localized PRR candidates in rice.

Speed vernalization to accelerate generation advance in winter cereal crops.

There are many challenges facing the development of high-yielding, nutritious crops for future environments. One limiting factor is generation time, which prolongs research and plant breeding timelines. Recent advances in speed breeding protocols have dramatically reduced generation time for many short-day and long-day species by optimizing light and temperature conditions during plant growth. However, winter crops with a vernalization requirement still require up to 6-10 weeks in low-temperature conditions before the transition to reproductive development. Here, we tested a suite of environmental conditions and protocols to investigate whether the vernalization process can be accelerated. We identified a vernalization method consisting of exposing seeds at the soil surface to an extended photoperiod of 22 h day:2 h night at 10°C with transfer to speed breeding conditions that dramatically reduces generation time in both winter wheat (Triticum aestivum) and winter barley (Hordeum vulgare). Implementation of the speed vernalization protocol followed by speed breeding allowed the completion of up to five generations per year for winter wheat or barley, whereas only two generations can be typically completed under standard vernalization and plant growth conditions. The speed vernalization protocol developed in this study has great potential to accelerate biological research and breeding outcomes for winter crops.

Cytokinin increases vegetative growth period by suppressing florigen expression in rice and maize.

Increasing the vegetative growth period of crops can increase biomass and grain yield. In rice (Oryza sativa), the concentration of trans -zeatin, an active cytokinin, was high in the leaves during vegetative growth and decreased rapidly upon induction of florigen expression, suggesting that this hormone is involved in the regulation of the vegetative phase. To elucidate whether exogenous cytokinin application influences the length of the vegetative phase, we applied 6-benzylaminopurine (BAP) to rice plants at various developmental stages. Our treatment delayed flowering time by 8-9 days when compared with mock-treated rice plants, but only at the transition stage when the flowering signals were produced. Our observations also showed that flowering in the paddy field is delayed by thidiazuron, a stable chemical that mimics the effects of cytokinin. The transcript levels of florigen genes Heading date 3a (Hd3a) and Rice Flowering locus T1 (RFT1) were significantly reduced by the treatment, but the expression of Early heading date 1 (Ehd1), a gene found directly upstream of the florigen genes, was not altered. In maize (Zea mays), similarly, BAP treatment increased the vegetative phage by inhibiting the expression of ZCN8, an ortholog of Hd3a. We showed that cytokinin treatment induced the expression of two type-A response regulators (OsRR1 and OsRR2) which interacted with Ehd1, a type-B response regulator. We also observed that cytokinin did not affect flowering time in ehd1 knockout mutants. Our study indicates that cytokinin application increases the duration of the vegetative phase by delaying the expression of florigen genes in rice and maize by inhibiting Ehd1.

Global Identification and Characterization of C2 Domain-Containing Proteins Associated with Abiotic Stress Response in Rice (Oryza sativa L.).

C2 domain-containing proteins (C2DPs) have been identified in different genomes that contain single or multiple C2 domains in their C- or N-terminal. It possesses higher functional activity in the transmembrane regions. The identification of C2 domains were reported in a previous study, such as multiple C2 domains and transmembrane-region proteins (MCTPs) and N-terminal-TM-C2 domain proteins (NTMC2s) of rice, Arabidopsis thaliana, and cotton, whereas the C2DP gene family in rice has not been comprehensively studied, and the role of the C2DP gene in rice in response to abiotic stress is not yet fully understood. In this study, we identified 82 C2DPs in the rice genome and divided them into seven groups through phylogenetic analysis. The synteny analysis revealed that duplication events were either exhibited within the genome of rice or between the genomes of rice and other species. Through the analysis of cis-acting elements in promoters, expression profiles, and qRT-PCR results, the functions of OsC2DPs were found to be widely distributed in diverse tissues and were extensively involved in phytohormones-related and abiotic stresses response in rice. The prediction of the microRNA (miRNA) targets of OsC2DPs revealed the possibility of regulation by consistent miRNAs. Notably, OsC2DP50/51/52 as a co-tandem duplication exhibited similar expression variations and involved the coincident miRNA-regulation pathway. Moreover, the results of the genotypic variation and haplotype analysis revealed that OsC2DP17, OsC2DP29, and OsC2DP49 were associated with cold stress responses. These findings provided comprehensive insights for characterizations of OsC2DPs in rice as well as for their roles for abiotic stress.

Comparative proteome profiling of susceptible and resistant rice cultivars identified an arginase involved in rice defense against Xanthomonas oryzae pv. oryzae.

Xanthomonas oryzae pv. oryzae (Xoo), the causative agent of bacterial blight, is one of the major threats to rice productivity. Yet, the molecular mechanism of rice-Xoo interaction is elusive. Here, we report comparative proteome profiles of Xoo susceptible (Dongjin) and resistant (Hwayeong) cultivars of rice in response to two-time points (3 and 6 days) of Xoo infection. Low-abundance proteins were enriched using a protamine sulfate (PS) precipitation method and isolated proteins were quantified by a label-free quantitative analysis, leading to the identification of 3846 proteins. Of these, 1128 proteins were significantly changed between mock and Xoo infected plants of Dongjin and Hwayeong cultivars. Based on the abundance pattern and functions of the identified proteins, a total of 23 candidate proteins were shortlisted that potentially participate in plant defense against Xoo in the resistant cultivar. Of these candidate proteins, a mitochondrial arginase-1 showed Hwayeong specific abundance and was significantly accumulated following Xoo inoculation. Overexpression of arginase 1 (OsArg 1) in susceptible rice cultivar (Dongjin) resulted in enhanced tolerance against Xoo as compared to the wild-type. In addition, expression analysis of defense-related genes encoding PR1, glucanase I, and chitinase II by qRT-PCR showed their enhanced expression in the overexpression lines as compared to wild-type. Taken together, our results uncover the proteome changes in the rice cultivars and highlight the functions of OsARG1 in plant defense against Xoo.

Lymphocyte-activating gene-3 expression is associated with tumor-infiltrating lymphocyte levels in HER2-positive breast cancers.

ABSTRACT: Lymphocyte-activating gene-3 (LAG-3, CD223) is the third inhibitory receptor targeted for immunotherapy. Several clinical trials investigating the use of interventions targeting LAG-3 are underway. The exact signaling mechanism downstream of LAG-3 is largely unknown, especially in breast cancer. The prognostic significance of tumor-infiltrating lymphocytes (TILs) in breast cancer has been previously determined.Among 167 human epidermal growth factor receptor 2 (HER2)-positive breast cancer patients, 90 and 78 patients were positive and negative for the hormone receptor, respectively. LAG-3 mRNA and protein expression levels in TILs were evaluated by quantitative real-time polymerase chain reaction and immunohistochemistry, respectively, among 12 and 167 HER2-positive breast cancer samples, respectively.High expression of LAG-3 in TILs was significantly correlated with high levels of TILs (P = .003) and an abundance of tertiary lymphoid structures around invasive components (P = .014). In addition, high expression of LAG3 was significantly associated with positivity for programmed death-ligand 1 (PD-L1) in tumor cells, a high immunostaining score of PD-L1 in TILs, and a high total immunostaining score for PD-L1 in tumor cells and TILs (all, P < .001). High expression levels of LAG-3 mRNA were associated with high levels of TILs (P = .091).LAG-3 protein expression was not a prognostic factor in HER2-positive breast cancers, and LAG-3 expression in TILs was significantly associated with the levels of TILs in HER2-positive breast cancer, although it was not a prognostic factor.

Genome-Wide Association Study Reveals the Genetic Basis of Chilling Tolerance in Rice at the Reproductive Stage.

A genome-wide association study (GWAS) was used to investigate the genetic basis of chilling tolerance in a collection of 117 rice accessions, including 26 Korean landraces and 29 weedy rices, at the reproductive stage. To assess chilling tolerance at the early young microspore stage, plants were treated at 12 °C for 5 days, and tolerance was evaluated using seed set fertility. GWAS, together with principal component analysis and kinship matrix analysis, revealed five quantitative trait loci (QTLs) associated with chilling tolerance on chromosomes 3, 6, and 7. The percentage of phenotypic variation explained by the QTLs was 11-19%. The genomic region underlying the QTL on chromosome 3 overlapped with a previously reported QTL associated with spikelet fertility. Subsequent bioinformatic and haplotype analyses suggested three candidate chilling-tolerance genes within the QTL linkage disequilibrium block: Os03g0305700, encoding a protein similar to peptide chain release factor 2; Os06g0495700, encoding a beta tubulin, autoregulation binding-site-domain-containing protein; and Os07g0137800, encoding a protein kinase, core-domain-containing protein. Further analysis of the detected QTLs and the candidate chilling-tolerance genes will facilitate strategies for developing chilling-tolerant rice cultivars in breeding programs.

Genome-Wide Identification and Genetic Variations of the Starch Synthase Gene Family in Rice.

Starch is a major ingredient in rice, and the amylose content of starch significantly impacts rice quality. OsSS (starch synthase) is a gene family related to the synthesis of amylose and amylopectin, and 10 members have been reported. In the present study, a synteny analysis of a novel family member belonging to the OsSSIV subfamily that contained a starch synthase catalytic domain showed that three segmental duplications and multiple duplications were identified in rice and other species. Expression data showed that the OsSS gene family is involved in diverse expression patterns. The prediction of miRNA targets suggested that OsSS are possibly widely regulated by miRNA functions, with miR156s targeted to OsSSII-3, especially. Haplotype analysis exhibited the relationship between amylose content and diverse genotypes. These results give new insight and a theoretical basis for the improved amylose content and eating quality of rice.

OsGATA16, a GATA Transcription Factor, Confers Cold Tolerance by Repressing OsWRKY45-1 at the Seedling Stage in Rice.

BACKGROUND: Cold stress is the main abiotic stress in rice, which seriously affects the growth and yield of rice. Identification of cold tolerance genes is of great significance for rice to solve these problems. GATA-family transcription factors involve diverse biological functions, however, their role in cold tolerance in rice remains unclear. RESULTS: In this study, a GATA-type zinc finger transcription factor OsGATA16, which can improve cold tolerance, was isolated and characterized from rice. OsGATA16 belongs to OsGATA subfamily-II and contains 11 putative phosphorylation sites, a nuclear localization signal (NLS), and other several conserved domains. OsGATA16 was expressed in all plant tissues, with the strongest in panicles. It was induced by cold and ABA treatments, but was repressed by drought, cytokinin and JA, and acted as a transcriptional suppressor in the nucleus. Overexpression of OsGATA16 improves cold tolerance of rice at seedling stage. Under cold stress treatments, the transcription of four cold-related genes OsWRKY45-1, OsSRFP1, OsCYL4, and OsMYB30 was repressed in OsGATA16-overexpressing (OE) rice compared with wild-type (WT). Interestingly, OsGATA16 bound to the promoter of OsWRKY45-1 and repressed its expression. In addition, haplotype analysis showed that OsGATA16 polarized between the two major rice subspecies japonica and indica, and had a non-synonymous SNP8 (336G) associated with cold tolerance. CONCLUSION: OsGATA16 is a GATA transcription factor, which improves cold tolerance at seedling stage in rice. It acts as a positive regulator of cold tolerance by repressing some cold-related genes such as OsWRKY45-1, OsSRFP1, OsCYL4 and OsMYB30. Additionally, OsGATA16 has a non-synonymous SNP8 (336G) associated with cold tolerance on CDS region. This study provides a theoretical basis for elucidating the mechanism of cold tolerance in rice and new germplasm resources for rice breeding.

Genome-Wide Association Study of Root System Development at Seedling Stage in Rice.

Root network structure plays a crucial role in growth and development processes in rice. Longer, more branched root structures help plants to assimilate water and nutrition from soil, support robust plant growth, and improve resilience to stresses such as disease. Understanding the molecular basis of root development through screening of root-related traits in rice germplasms is critical to future rice breeding programs. This study used a small germplasm collection of 137 rice varieties chosen from the Korean rice core set (KRICE_CORE) to identify loci linked to root development. Two million high-quality single nucleotide polymorphisms (SNPs) were used as the genotype, with maximum root length (MRL) and total root weight (TRW) in seedlings used as the phenotype. Genome-wide association study (GWAS) combined with Principal Components Analysis (PCA) and Kinship matrix analysis identified four quantitative trait loci (QTLs) on chromosomes 3, 6, and 8. Two QTLs were linked to MRL and two were related to TRW. Analysis of Linkage Disequilibrium (LD) decay identified a 230 kb exploratory range for detection of candidate root-related genes. Candidates were filtered using RNA-seq data, gene annotations, and quantitative real-time PCR (qRT-PCR), and five previously characterized genes related to root development were identified, as well as four novel candidate genes. Promoter analysis of candidate genes showed that LOC_Os03g08880 and LOC_Os06g13060 contained SNPs with the potential to impact gene expression in root-related promoter motifs. Haplotype analysis of candidate genes revealed diverse haplotypes that were significantly associated with phenotypic variation. Taken together, these results indicate that LOC_Os03g08880 and LOC_Os06g13060 are strong candidate genes for root development functions. The significant haplotypes identified in this study will be beneficial in future breeding programs for root improvement.

Unveiling the Crucial Role of Type IV Secretion System and Motility of Helicobacter pylori in IL-1ß Production via NLRP3 Inflammasome Activation in Neutrophils.

Helicobacter pylori is a gram-negative, microaerophilic, and spiral-shaped bacterium and causes gastrointestinal diseases in human. IL-1ß is a representative cytokine produced in innate immune cells and is considered to be a key factor in the development of gastrointestinal malignancies. However, the mechanism of IL-1ß production by neutrophils during H. pylori infection is still unknown. We designed this study to identify host and bacterial factors involved in regulation of H. pylori-induced IL-1ß production in neutrophils. We found that H. pylori-induced IL-1ß production is abolished in NLRP3-, ASC-, and caspase-1/11-deficient neutrophils, suggesting essential role for NLRP3 inflammasome in IL-1ß response against H. pylori. Host TLR2, but not TLR4 and Nod2, was also required for transcription of NLRP3 and IL-1ß as well as secretion of IL-1ß. H. pylori lacking cagL, a key component of the type IV secretion system (T4SS), induced less IL-1ß production in neutrophils than did its isogenic WT strain, whereas vacA and ureA were dispensable. Moreover, T4SS was involved in caspase-1 activation and IL-1ß maturation in H. pylori-infected neutrophils. We also found that FlaA is essential for H. pylori-mediated IL-1ß production in neutrophils, but not dendritic cells. TLR5 and NLRC4 were not required for H. pylori-induced IL-1ß production in neutrophils. Instead, bacterial motility is essential for the production of IL-1ß in response to H. pylori. In conclusion, our study shows that host TLR2 and NLRP3 inflammasome and bacterial T4SS and motility are essential factors for IL-1ß production by neutrophils in response to H. pylori.

Study of Quantitative Trait Loci (QTLs) Associated with Allelopathic Trait in Rice.

In rice there are few genetic studies reported for allelopathy traits, which signify the ability of plants to inhibit or stimulate growth of other plants in the environment, by exuding chemicals. QTL analysis for allelopathic traits were conducted with 98 F8 RILs developed from the cross between the high allelopathic parents of 'Sathi' and non-allelopathic parents of 'Nong-an'. The performance of allelopathic traits were evaluated with inhibition rate on root length, shoot length, total length, root weight, shoot weight, and total weight of lettuce as a receiver plant. With 785 polymorphic DNA markers, we constructed a linkage map showing a total of 2489.75 cM genetic length and 3.17 cM of average genetic distance between each adjacent marker. QTL analysis detected on QTL regions on chromosome 8 responsible for the inhibition of shoot length and inhibition of total length. The qISL-8 explained 20.38% of the phenotypic variation for the inhibition on the shoot length. The qITL-8 explained 14.93% of the phenotypic variation for the inhibition on total length. The physical distance of the detected QTL region was 194 Kbp where 31 genes are located.

Increasing Coverage of Proteome Identification of the Fruiting Body of Agaricus bisporus by Shotgun Proteomics.

To increase coverage of protein identification of an Agaricus bisporus fruiting body, we analyzed the crude protein fraction of the fruiting body by using a shotgun proteomics approach where 7 MudPIT (Multi-Protein identification Technology) runs were conducted and the MS/MS spectra from the 7 MudPIT runs were merged. Overall, 3093 non-redundant proteins were identified to support the expression of those genes annotated in the genome database of Agaricus bisporus. The physicochemical properties of the identified proteins, i.e., wide pI value range and molecular mass range, were indicative of unbiased protein identification. The relative quantification of the identified proteins revealed that K5XI50 (Aldedh domain-containing protein) and K5XEW1 (Ubiquitin-like domain-containing protein) were highly abundant in the fruiting body. Based on the information in the Uniprot (Universal Protein Resource) database for A. bisporus, only approximately 53% of the 3093 identified proteins have been functionally described and approximately 47% of the proteins remain uncharacterized. Gene Ontology analysis revealed that the majority of proteins were annotated with a biological process, and proteins associated with coiled-coil (12.8%) and nucleotide binding (8.21%) categories were dominant. The Kyoto Encyclopedia of Genes and Genome analysis revealed that proteins involved in biosynthesis of secondary metabolites and tyrosine metabolism were enriched in a fruiting body of Agaricus bisporus, suggesting that the proteins are associated with antioxidant metabolites.