Genetic mapping and identification of Rht8-B1 that regulates plant height in wheat.

BACKGROUND: Plant height (PH) and spike compactness (SC) are important agronomic traits that affect yield improvement in wheat crops. The identification of the loci or genes responsible for these traits is thus of great importance for marker-assisted selection in wheat breeding. RESULTS: In this study, we used a recombinant inbred line (RIL) population with 139 lines derived from a cross between the mutant Rht8-2 and the local wheat variety NongDa5181 (ND5181) to construct a high-density genetic linkage map by applying the Wheat 40 K Panel. We identified seven stable QTLs for PH (three) and SC (four) in two environments using the RIL population, and found that Rht8-B1 is the causal gene of qPH2B.1 by further genetic mapping, gene cloning and gene editing analyses. Our results also showed that two natural variants from GC to TT in the coding region of Rht8-B1 resulted in an amino acid change from G (ND5181) to V (Rht8-2) at the 175th position, reducing PH by 3.6%~6.2% in the RIL population. Moreover, gene editing analysis suggested that the height of T2 generation in Rht8-B1 edited plants was reduced by 5.6%, and that the impact of Rht8-B1 on PH was significantly lower than Rht8-D1. Additionally, analysis of the distribution of Rht8-B1 in various wheat resources suggested that the Rht8-B1b allele has not been widely utilized in modern wheat breeding. CONCLUSIONS: The combination of Rht8-B1b with other favorable Rht genes might be an alternative approach for developing lodging-resistant crops. Our study provides important information for marker-assisted selection in wheat breeding.

Fine mapping and genetic analysis identified a C2H2-type zinc finger as a candidate gene for heading date regulation in wheat.

KEY MESSAGE: A minor-effect QTL, Qhd.2AS, that affects heading date in wheat was mapped to a genomic interval of 1.70-Mb on 2AS, and gene analysis indicated that the C2H2-type zinc finger protein gene TraesCS2A02G181200 is the best candidate for Qhd.2AS. Heading date (HD) is a complex quantitative trait that determines the regional adaptability of cereal crops, and identifying the underlying genetic elements with minor effects on HD is important for improving wheat production in diverse environments. In this study, a minor QTL for HD that we named Qhd.2AS was detected on the short arm of chromosome 2A by Bulked Segregant Analysis and validated in a recombinant inbred population. Using a segregating population of 4894 individuals, Qhd.2AS was further delimited to an interval of 0.41 cM, corresponding to a genomic region spanning 1.70 Mb (from 138.87 to 140.57 Mb) that contains 16 high-confidence genes based on IWGSC RefSeq v1.0. Analyses of sequence variations and gene transcription indicated that TraesCS2A02G181200, which encodes a C2H2-type zinc finger protein, is the best candidate gene for Qhd.2AS that influences HD. Screening a TILLING mutant library identified two mutants with premature stop codons in TraesCS2A02G181200, both of which exhibited a delay in HD of 2-4 days. Additionally, variations in its putative regulatory sites were widely present in natural accession, and we also identified the allele which was positively selected during wheat breeding. Epistatic analyses indicated that Qhd.2AS-mediated HD variation is independent of VRN-B1 and environmental factors. Phenotypic investigation of homozygous recombinant inbred lines (RILs) and F2:3 families showed that Qhd.2AS has no negative effect on yield-related traits. These results provide important cues for refining HD and therefore improving yield in wheat breeding programs and will deepen our understanding of the genetic regulation of HD in cereal plants.

Gene Mapping and Identification of a Missense Mutation in One Copy of VRN-A1 Affects Heading Date Variation in Wheat.

Heading date (HD) is an important trait for wide adaptability and yield stability in wheat. The Vernalization 1 (VRN1) gene is a key regulatory factor controlling HD in wheat. The identification of allelic variations in VRN1 is crucial for wheat improvement as climate change becomes more of a threat to agriculture. In this study, we identified an EMS-induced late-heading wheat mutant je0155 and crossed it with wide-type (WT) Jing411 to construct an F2 population of 344 individuals. Through Bulk Segregant Analysis (BSA) of early and late-heading plants, we identified a Quantitative Trait Locus (QTL) for HD on chromosome 5A. Further genetic linkage analysis limited the QTL to a physical region of 0.8 Mb. Cloning and sequencing revealed three copies of VRN-A1 in the WT and mutant lines; one copy contained a missense mutation of C changed to T in exon 4 and another copy contained a mutation in intron 5. Genotype and phenotype analysis of the segregation population validated that the mutations in VRN-A1 contributed to the late HD phenotype in the mutant. Expression analysis of C- or T-type alleles in exon 4 of the WT and mutant lines indicated that this mutation led to lower expression of VRN-A1, which resulted in the late-heading of je0155. This study provides valuable information for the genetic regulation of HD and many important resources for HD refinement in wheat breeding programs.

A Bioinformatics Investigation into the Pharmacological Mechanisms of Sodium-Glucose Co-transporter 2 Inhibitors in Diabetes Mellitus and Heart Failure Based on Network Pharmacology.

PURPOSE: Diabetes mellitus (DM) is a major risk factor for the development of heart failure (HF). Sodium-glucose co-transporter 2 (SGLT2) inhibitors have demonstrated consistent benefits in the reduction of hospitalization for HF in patients with DM. However, the pharmacological mechanism is not clear. To investigate the mechanisms of SGLT2 inhibitors in DM with HF, we performed target prediction and network analysis by a network pharmacology method. METHODS: We selected targets of SGLT2 inhibitors and DM status with HF from databases and studies. The "Drug-Target" and "Drug-Target-Disease" networks were constructed using Cytoscape. Then the protein-protein interaction (PPI) was analyzed using the STRING database. Gene Ontology (GO) biological functions and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were performed to investigate using the Bioconductor tool for analysis. RESULTS: There were 125 effective targets between SGLT2 inhibitors and DM status with HF. Through further screening, 33 core targets were obtained, including SRC, MAPK1, NARS, MAPK3 and EGFR. It was predicted that the Rap1 signaling pathway, MAPK signaling pathway, EGFR tyrosine kinase inhibitor resistance, AGE-RAGE signaling pathway in diabetic complications and other signaling pathways were involved in the treatment of DM with HF by SGLT2 inhibitors. CONCLUSION: Our study elucidated the possible mechanisms of SGLT2 inhibitors from a systemic and holistic perspective based on pharmacological networks. The key targets and pathways will provide new insights for further research on the pharmacological mechanism of SGLT2 inhibitors in the treatment of DM with HF.

Identification of the vernalization gene VRN-B1 responsible for heading date variation by QTL mapping using a RIL population in wheat.

BACKGROUND: Heading time is one of the most important agronomic traits in wheat, as it largely affects both adaptation to different agro-ecological conditions and yield potential. Identification of genes underlying the regulation of wheat heading and the development of diagnostic markers could facilitate our understanding of genetic control of this process. RESULTS: In this study, we developed 400 recombinant inbred lines (RILs) by crossing a γ-ray-induced early heading mutant (eh1) with the late heading cultivar, Lunxuan987. Bulked Segregant Analysis (BSA) of both RNA and DNA pools consisting of various RILs detected a quantitative trait loci (QTL) for heading date located on chromosomes 5B, and further genetic linkage analysis limited the QTL to a 3.31 cM region. We then identified a large deletion in the first intron of the vernalization gene VRN-B1 in eh1, and showed it was associated with the heading phenotype in the RIL population. However, it is not the mutation loci that resulted in early heading phonotype in the mutant compared to that of wildtype. RNA-seq analysis suggested that Vrn-B3 and several newly discovered genes, including beta-amylase 1 (BMY1) and anther-specific protein (RTS), were highly expressed in both the mutant and early heading pool with the dominant Vrn-B1 genotype compared to that of Lunxuan987 and late heading pool. Enrichment analysis of differentially expressed genes (DEGs) identified several key pathways previously reported to be associated with flowering, including fatty acid elongation, starch and sucrose metabolism, and flavonoid biosynthesis. CONCLUSION: The development of new markers for Vrn-B1 in this study supplies an alternative solution for marker-assisted breeding to optimize heading time in wheat and the DEGs analysis provides basic information for VRN-B1 regulation study.

Genome-wide characterization of two homeobox families identifies key genes associated with grain-related traits in wheat.

Homeodomain proteins encoded by BEL1- and KNAT1-type genes are ubiquitously distributed across plant species and play important roles in growth and development, whereby a comprehensive investigation of their molecular interactions and potential functions in wheat is of great significance. In this study, we systematically investigated the phylogenetic relationships, gene structures, conserved domains, and cis-acting elements of 34 TaBEL and 34 TaKNAT genes in the wheat genome. Our analysis revealed these genes evolved under different selective pressures and showed variable transcript levels in different wheat tissues. Subcellular localization analysis further indicated the proteins encoded by these genes were either exclusively located in the nucleus or both in the nucleus and the cytoplasm. Additionally, a comprehensive protein-protein interaction network was constructed with representative genes in which each TaBEL or TaKNAT proteins interact with at least two partners. The evaluation of wheat mutants identified key genes, including TaBEL-5B, TaBEL-4A.4, and TaKNAT6, which are involved in grain-related traits. Finally, haplotype analysis suggests TaKNAT-6B is associated with grain-related traits and is preferentially selected among a large set of wheat accessions. Our study provides important information on BEL1- and KNAT1-type gene families in wheat, and lays the foundation for functional research in the future.

Induction of Semi-Dwarf Trait to a Three Pistil Tall Mutant Through Breeding With Increased Grain Numbers in Wheat.

Multi-ovary wheat (three pistil) is a unique germplasm for the seed production of hybrid wheat. The purpose of the present study was to transfer the multi-ovary trait to semi-dwarf plants to increase the production of grains in wheat crops. Therefore, tall, semi-dwarf, and dwarf plants were crossed with plants with the three-pistil trait. A three-pistil tall plant was used as the female parent, while tall (Synthetic hexaploid), semi-dwarf, and dwarf plants were used as male parents. F1 and F2 progenies with parents were planted in 2015-16 using RCBD. The outcome of the crosses showed that multi-ovary tall plants gave significant difference for all five traits (days to maturity, plant height, number of seeds per spike, grain weight per spike, and grain yield per unit area) in both generations. The greatest number of grains per spike and grain yield per unit area were obtained from the cross of three-pistil tall and dwarf parent (P1/P6) in the F1 and F2 generations. The cross also resulted in a significant reduction in height (96 cm). Further heterosis studies conducted with crosses between three-pistil tall and dwarf parent (P1/P6) showed the greatest heterosis and heterobeltiosis for the number of grains per spike (60.0 and 26.19%, respectively) and grain yield per m2 (27.68 and 2.85%, respectively). In the case of grain weight per spike, the heterosis value was also positive and significant (17.7). Meanwhile, for other traits, their values were negative for heterosis and heterobeltiosis. High numbers of grains and grain weight were found to be associated with positive heterobeltiosis and in turn the grain yield per m2, but plant height and maturity had negative affirmation with heterobeltiosis. Heterosis studies also indicated the dominant gene action for the three-pistil trait. Thus, the study clearly signified that grain yield can be increased by using the multi-ovary genotype with the semi-dwarf height. This new germplasm will be helpful for breeders to increase the production of wheat crops in the southern climate of Pakistan.

Exploring the Pleiotropic Genes and Therapeutic Targets Associated with Heart Failure and Chronic Kidney Disease by Integrating metaCCA and SGLT2 Inhibitors' Target Prediction.

BACKGROUND: Previous studies have shown that heart failure (HF) and chronic kidney disease (CKD) have common genetic mechanisms, overlapping pathophysiological pathways, and therapeutic drug-sodium-glucose cotransporter 2 (SGLT2) inhibitors. METHODS: The genetic pleiotropy metaCCA method was applied on summary statistics data from two independent meta-analyses of GWAS comprising more than 1 million people to identify shared variants and pleiotropic effects between HF and CKD. Targets of SGLT2 inhibitors were predicted by SwissTargetPrediction and DrugBank databases. To refine all genes, we performed using versatile gene-based association study 2 (VEGAS2) and transcriptome-wide association studies (TWAS) for HF and CKD, respectively. Gene enrichment and KEGG pathway analyses were used to explore the potential functional significance of the identified genes and targets. RESULTS: After metaCCA analysis, 4,624 SNPs and 1,745 genes were identified to be potentially pleiotropic in the univariate and multivariate SNP-multivariate phenotype analyses, respectively. 21 common genes were detected in both metaCCA and SGLT2 inhibitors' target prediction. In addition, 169 putative pleiotropic genes were identified, which met the significance threshold both in metaCCA analysis and in the VEGAS2 or TWAS analysis for at least one disease. CONCLUSION: We identified novel variants associated with HF and CKD using effectively incorporating information from different GWAS datasets. Our analysis may provide new insights into HF and CKD therapeutic approaches based on the pleiotropic genes, common targets, and mechanisms by integrating the metaCCA method, TWAS and VEGAS2 analyses, and target prediction of SGLT2 inhibitors.

Genome-Wide and Exome-Capturing Sequencing of a Gamma-Ray-Induced Mutant Reveals Biased Variations in Common Wheat.

Induced mutagenesis is a powerful approach for the creation of novel germplasm and the improvement of agronomic traits. The evaluation of mutagenic effects and functional variations in crops is needed for breeding mutant strains. To investigate the mutagenic effects of gamma-ray irradiation in wheat, this study characterized genomic variations of wheat early heading mutant (eh1) as compared to wild-type (WT) Zhongyuan 9 (ZY9). Whole-genome resequencing of eh1 and ZY9 produced 737.7 Gb sequencing data and identified a total of 23,537,117 homozygous single nucleotide polymorphism (SNP) and 1,608,468 Indel. Analysis of SNP distribution across the chromosome suggests that mutation hotspots existed in certain chromosomal regions. Among the three subgenomes, the variation frequency in subgenome D was significantly lower than in subgenomes A and B. A total of 27.8 Gb data were obtained by exome-capturing sequencing, while 217,948 SNP and 13,554 Indel were identified. Variation annotation in the gene-coding sequences demonstrated that 5.0% of the SNP and 5.3% of the Indel were functionally important. Characterization of exomic variations in 12 additional gamma-ray-induced mutant lines further provided additional insights into the mutagenic effects of this approach. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genome (KEGG) analysis suggested that genes with functional variations were enriched in several metabolic pathways, including plant-pathogen interactions and ADP binding. Kompetitive allele-specific PCR (KASP) genotyping with selected SNP within functional genes indicated that 85.7% of the SNPs were polymorphic between the eh1 and wild type. This study provides a basic understanding of the mechanism behind gamma-ray irradiation in hexaploid wheat.

Integrative Analysis of Transcriptome-Wide Association Study and mRNA Expression Profiles Identified Candidate Genes and Pathways Associated With Acute Myocardial Infarction.

BACKGROUND: Acute myocardial infarction (AMI), characterized by an event of myocardial necrosis, is a common cardiac emergency worldwide. However, the genetic mechanisms of AMI remain largely elusive. METHODS: A genome-wide association study dataset of AMI was obtained from the CARDIoGRAMplusC4D project. A transcriptome-wide association study (TWAS) was conducted using the FUSION tool with gene expression references of the left ventricle and whole blood. Significant genes detected by TWAS were subjected to Gene Ontology (GO) enrichment analysis. Then the TWAS results of AMI were integrated with mRNA expression profiling to identify common genes and biological processes. Finally, the identified common genes were validated by RT-qPCR analysis. RESULTS: TWAS identified 1,050 genes for the left ventricle and 1,079 genes for whole blood. Upon comparison with the mRNA expression profile, 4 common genes were detected, including HP (PTWAS = 1.22 × 10-3, PGEO = 4.98 × 10-2); CAMP (PTWAS = 2.48 × 10-2, PGEO = 2.36 × 10-5); TNFAIP6 (PTWAS = 1.90 × 10-2, PGEO = 3.46 × 10-2); and ARG1 (PTWAS = 8.35 × 10-3, PGEO = 4.93 × 10-2). Functional enrichment analysis of the genes identified by TWAS detected multiple AMI-associated biological processes, including autophagy of mitochondrion (GO: 0000422) and mitochondrion disassembly (GO: 0061726). CONCLUSION: This integrative study of TWAS and mRNA expression profiling identified multiple candidate genes and biological processes for AMI. Our results may provide a fundamental clue for understanding the genetic mechanisms of AMI.

Novel biomarkers for post-contrast acute kidney injury identified from long non-coding RNA expression profiles.

Background: Post-contrast acute kidney injury (PC-AKI) is a severe complication of cardiac catheterization. Emerging evidence indicated that long non-coding RNAs (lncRNAs) could serve as biomarkers for various diseases. However, the lncRNA expression profile and potential biomarkers in PC-AKI remain unclear. This study aimed to investigate novel lncRNA biomarkers for the early detection of PC-AKI. Methods: lncRNA profile in the kidney tissues of PC-AKI rats was evaluated through RNA sequencing. Potential lncRNA biomarkers were identified through human-rat homology analysis, kidney and blood filtering in rats and verified in 112 clinical samples. The expression patterns of the candidate lncRNAs were detected in HK-2 cells and rat models to evaluate their potential for early detection. Results: In total, 357 lncRNAs were found to be differentially expressed in PC-AKI. We identified lnc-HILPDA and lnc-PRND were conservative and remarkably upregulated in both kidneys and blood from rats and the blood of PC-AKI patients; these lncRNAs can precisely distinguish PC-AKI patients (area under the curve (AUC) values of 0.885 and 0.875, respectively). The combination of these two lncRNAs exhibited improved accuracy for predicting PC-AKI, with 100% sensitivity and 83.93% specificity. Time-course experiments showed that the significant difference was first noted in the blood of PC-AKI rats at 12 h for lnc-HILPDA and 24 h for lnc-PRND. Conclusion: Our study revealed that lnc-HILPDA and lnc-PRND may serve as the novel biomarkers for early detection and profoundly affect the clinical stratification and strategy guidance of PC-AKI.

Transcriptome sequencing reveals hotspot mutation regions and dwarfing mechanisms in wheat mutants induced by γ-ray irradiation and EMS.

Induced mutation is an important approach for creating novel plant germplasms. The introduction of dwarf or semi-dwarf genes into wheat has led to great advancements in yield improvement. In this study, four elite dwarf wheat mutants, named dm1-dm4, induced from γ-ray irradiation or ethyl methanesulfonate (EMS) mutagenesis, were used to identify transcriptome variations and dwarfing mechanisms. The results showed that the hotspot regions of mutations distributed on the chromosomes were consistent among the four mutant lines and these regions were mainly located around the 50, 360 and 400 Mb positions of chromosome 1A and the distal regions of chromosomes 2A and 2BL. Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses suggested that 'protein processing in endoplasmic reticulum' was the most common significantly enriched pathway based on the differentially expressed genes (DEGs) between wildtype (WT) and the mutants. Notably, 18 out of 20 genes involved in this process encode heat shock proteins (HSPs). The results implied that HSPs might participate in wheat dwarfism response and function in the dwarfism process through protein folding and/or degradation. Moreover, seven genes in dm4 involved in modulating auxin levels were down-regulated and dm4 was more sensitive to auxin treatment compared with WT, indicating the important roles of auxin in regulation of dwarf phenotype in dm4. This study not only identified transcriptome sequence variation induced by physical and chemical mutagenesis but also revealed potential dwarfing mechanisms in the wheat mutant lines.

A combined association mapping and t-test analysis of SNP loci and candidate genes involving in resistance to low nitrogen traits by a wheat mutant population.

Crop productivity is highly dependent on the application of N fertilizers, but ever-increasing N application is causing serious environmental impacts. To facilitate the development of new wheat cultivars that can thrive in low N growth conditions, key loci and genes associated with wheat responses to low N must be identified. In this GWAS and t-test study of 190 M6 mutant wheat lines (Jing 411-derived) based on genotype data from the wheat 660k SNP array, we identified a total of 221 significant SNPs associated four seedling phenotypic traits that have been implicated in resistance to low N: relative root length, relative shoot length, relative root weight, and relative shoot weight. Notably, we detected large numbers of significantly associated SNP in what appear to be genomic 'hotspots' for resistance to low N on chromosomes 2A and 6B, strongly suggesting that these regions are functionally related to the resistance phenotypes that we observed in some of the mutant lines. Moreover, the candidate genes, including genes encoding high-affinity nitrate transporter 2.1, gibberellin responsive protein, were identified for resistance to low N. This study raises plausible mechanistic hypotheses that can be evaluated in future applied or basic efforts by breeders or plant biologists seeking to develop new high-NUE wheat cultivars.