Identification of candidate gene associated with maize northern leaf blight resistance in a multi-parent population.

KEY MESSAGE: QTL mapping combined with genome-wide association studies, revealed a potential candidate gene for  resistance to northern leaf blight in the tropical CATETO-related maize line YML226, providing a basis for marker-assisted selection of maize varieties Northern leaf blight (NLB) is a foliar disease that can cause severe yield losses in maize. Identifying and utilizing NLB-resistant genes is the most effective way to prevent and control this disease. In this study, five important inbred lines of maize were used as parental lines to construct a multi-parent population for the identification of NLB-resistant loci. QTL mapping and GWAS analysis revealed that QTL qtl_YML226_1, which had the largest phenotypic variance explanation (PVE) of 9.28%, and SNP 5-49,193,921 were co-located in the CATETO-related line YML226. This locus was associated with the candidate gene Zm00001d014471, which encodes a pentatricopeptide repeat (PPR) protein. In the coding region of Zm00001d014471, YML226 had more specific SNPs than the other parental lines. qRT-PCR showed that the relative expressions of Zm00001d014471 in inoculated and uninoculated leaves of YML226 were significantly higher, indicating that the expression of the candidate gene was correlated with NLB resistance. The analysis showed that the higher expression level in YML226 might be caused by SNP mutations. This study identified NLB resistance candidate loci and genes in the tropical maize inbred line YML226 derived from the CATETO germplasm, thereby providing a theoretical basis for using modern marker-assisted breeding techniques to select genetic resources resistant to NLB.

Genome-Wide Association Studies on the Kernel Row Number in a Multi-Parent Maize Population.

Kernel row number (KRN) is a crucial trait in maize that directly influences yield; hence, understanding the mechanisms underlying KRN is vital for the development of high-yielding inbred lines and hybrids. We crossed four excellent panicle inbred lines (CML312, CML444, YML46, and YML32) with Ye107, and after eight generations of selfing, a multi-parent population was developed comprising four subpopulations, each consisting of 200 lines. KRN was accessed in five environments in Yunnan province over three years (2019, 2021, and 2022). The objectives of this study were to (1) identify quantitative trait loci and single nucleotide polymorphisms associated with KRN through linkage and genome-wide association analyses using high-quality genotypic data, (2) identify candidate genes regulating KRN by identifying co-localized QTLs and SNPs, and (3) explore the pathways involved in KRN formation and identify key candidate genes through Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. Our study successfully identified 277 significant Quantitative trait locus (QTLs) and 53 significant Single Nucleotide Polymorphism (SNPs) related to KRN. Based on gene expression, GO, and KEGG analyses, SNP-177304649, SNP-150393177, SNP-135283055, SNP-138554600, and SNP-120370778, which were highly likely to be associated with KRN, were identified. Seven novel candidate genes at this locus (Zm00001d022420, Zm00001d022421, Zm00001d016202, Zm00001d050984, Zm00001d050985, Zm00001d016000, and Zm00014a012929) are associated with KRN. Among these, Zm00014a012929 was identified using the reference genome Mo17. The remaining six genes were identified using the reference genome B73. To our knowledge, this is the first report on the association of these genes with KRN in maize. These findings provide a theoretical foundation and valuable insights into the genetic mechanisms underlying maize KRN and the development of high-yielding hybrids through heterosis.

Genome-Wide Association Study and Prediction of Tassel Weight of Tropical Maize Germplasm in Multi-Parent Population.

Tassel weight (TW) is a crucial agronomic trait that significantly affects pollen supply and grain yield development in maize breeding. To improve maize yield and develop new varieties, a comprehensive understanding of the genetic mechanisms underlying tassel weight is essential. In this study, tropical maize inbred lines, namely CML312, CML373, CML444, and YML46, were selected as female parents and crossed with the elite maize inbred line Ye107, which served as the common male parent, to develop a multi-parent population comprising four F8 recombinant inbred line (RIL) subpopulations. Using 6616 high-quality single nucleotide polymorphism (SNP) markers, we conducted genome-wide association analysis (GWAS) and genomic selection (GS) on 642 F8 RILs in four subpopulations across three different environments. Through GWAS, we identified 16 SNPs that were significantly associated with TW, encompassing two stable loci expressed across multiple environments. Furthermore, within the candidate regions of these SNPs, we discovered four novel candidate genes related to TW, namely Zm00001d044362, Zm00001d011048, Zm00001d011049, and Zm00001d031173 distributed on chromosomes 1, 3, and 8, which have not been previously reported. These genes are involved in processes such as signal transduction, growth and development, protein splicing, and pollen development, all of which play crucial roles in inflorescence meristem development, directly affecting TW. The co-localized SNP, S8_137379725, on chromosome 8 was situated within a 16.569 kb long terminal repeat retrotransposon (LTR-RT), located 22.819 kb upstream and 26.428 kb downstream of the candidate genes (Zm00001d011048 and Zm00001d011049). When comparing three distinct GS models, the BayesB model demonstrated the highest accuracy in predicting TW. This study establishes the theoretical foundation for future research into the genetic mechanisms underlying maize TW and the efficient breeding of high-yielding varieties with desired tassel weight through GS.

Studies on Candidate Genes Related to Flowering Time in a Multiparent Population of Maize Derived from Tropical and Temperate Germplasm.

A comprehensive study on maize flowering traits, focusing on the regulation of flowering time and the elucidation of molecular mechanisms underlying the genes controlling flowering, holds the potential to significantly enhance our understanding of the associated regulatory gene network. In this study, three tropical maize inbreds, CML384, CML171, and CML444, were used, along with a temperate maize variety, Shen137, as parental lines to cross with Ye107. The resulting F1s underwent seven consecutive generations of self-pollination through the single-seed descent (SSD) method to develop a multiparent population. To investigate the regulation of maize flowering time-related traits and to identify loci and candidate genes, a genome-wide association study (GWAS) was conducted. GWAS analysis identified 556 SNPs and 12 candidate genes that were significantly associated with flowering time-related traits. Additionally, an analysis of the effect of the estimated breeding values of the subpopulations on flowering time was conducted to further validate the findings of the present study. Collectively, this study offers valuable insights into novel candidate genes, contributing to an improved understanding of maize flowering time-related traits. This information holds practical significance for future maize breeding programs aimed at developing high-yielding hybrids.

Dissection of Common Rust Resistance in Tropical Maize Multiparent Population through GWAS and Linkage Studies.

Common rust (CR), caused by Puccina sorghi, is a major foliar disease in maize that leads to quality deterioration and yield losses. To dissect the genetic architecture of CR resistance in maize, this study utilized the susceptible temperate inbred line Ye107 as the male parent crossed with three resistant tropical maize inbred lines (CML312, D39, and Y32) to generate 627 F7 recombinant inbred lines (RILs), with the aim of identifying maize disease-resistant loci and candidate genes for common rust. Phenotypic data showed good segregation between resistance and susceptibility, with varying degrees of resistance observed across different subpopulations. Significant genotype effects and genotype × environment interactions were observed, with heritability ranging from 85.7% to 92.2%. Linkage and genome-wide association analyses across the three environments identified 20 QTLs and 62 significant SNPs. Among these, seven major QTLs explained 66% of the phenotypic variance. Comparison with six SNPs repeatedly identified across different environments revealed overlap between qRUST3-3 and Snp-203,116,453, and Snp-204,202,469. Haplotype analysis indicated two different haplotypes for CR resistance for both the SNPs. Based on LD decay plots, three co-located candidate genes, Zm00001d043536, Zm00001d043566, and Zm00001d043569, were identified within 20 kb upstream and downstream of these two SNPs. Zm00001d043536 regulates hormone regulation, Zm00001d043566 controls stomatal opening and closure, related to trichome, and Zm00001d043569 is associated with plant disease immune responses. Additionally, we performed candidate gene screening for five additional SNPs that were repeatedly detected across different environments, resulting in the identification of five candidate genes. These findings contribute to the development of genetic resources for common rust resistance in maize breeding programs.

Utilizing Two Populations Derived from Tropical Maize for Genome-Wide Association Analysis of Banded Leaf and Sheath Blight Resistance.

Banded leaf and sheath blight (BLSB) in maize is a soil-borne fungal disease caused by Rhizoctonia solani Kühn, resulting in significant yield losses. Investigating the genes responsible for regulating resistance to BLSB is crucial for yield enhancement. In this study, a multiparent maize population was developed, comprising two recombinant inbred line (RIL) populations totaling 442 F8RILs. The populations were generated by crossing two tropical inbred lines, CML444 and NK40-1, known for their BLSB resistance, as female parents, with the high-yielding but BLSB-susceptible inbred line Ye107 serving as the common male parent. Subsequently, we utilized 562,212 high-quality single nucleotide polymorphisms (SNPs) generated through genotyping-by-sequencing (GBS) for a comprehensive genome-wide association study (GWAS) aimed at identifying genes responsible for BLSB resistance. The objectives of this study were to (1) identify SNPs associated with BLSB resistance through genome-wide association analyses, (2) explore candidate genes regulating BLSB resistance in maize, and (3) investigate pathways involved in BLSB resistance and discover key candidate genes through Gene Ontology (GO) analysis. The GWAS analysis revealed nineteen SNPs significantly associated with BLSB that were consistently identified across four environments in the GWAS, with phenotypic variation explained (PVE) ranging from 2.48% to 11.71%. Screening a 40 kb region upstream and downstream of the significant SNPs revealed several potential candidate genes. By integrating information from maize GDB and the NCBI, we identified five novel candidate genes, namely, Zm00001d009723, Zm00001d009975, Zm00001d009566, Zm00001d009567, located on chromosome 8, and Zm00001d026376, on chromosome 10, related to BLSB resistance. These candidate genes exhibit association with various aspects, including maize cell membrane proteins and cell immune proteins, as well as connections to cell metabolism, transport, transcriptional regulation, and structural proteins. These proteins and biochemical processes play crucial roles in maize defense against BLSB. When Rhizoctonia solani invades maize plants, it induces the expression of genes encoding specific proteins and regulates corresponding metabolic pathways to thwart the invasion of this fungus. The present study significantly contributes to our understanding of the genetic basis of BLSB resistance in maize, offering valuable insights into novel candidate genes that could be instrumental in future breeding efforts to develop maize varieties with enhanced BLSB resistance.

Laser welding simulation of large-scale assembly module of stainless steel side-wall.

Due to the advantageous characteristics of laser welding technology, it is being increasingly used for constructing stainless steel rail vehicles. It can improve the appearance of a vehicle, enable designs with a relatively high degree of flatness, and ensure higher-quality connections between different parts of a vehicle. Moreover, it can improve the strength and stiffness of the components of the vehicle. In this study, a large-scale assembly module of a stainless steel side-wall was considered as the research object. The combined heat source model of a Gaussian heat source and a cylindrical volume heat source was used to obtain the heat source parameters of laser welding to match the experimental data. Based on the thermal cycle curve method (TCCM), the influence of the number of weld segments and mesh divisions of the local model on the efficiency and accuracy of laser welding simulations was investigated. Thereafter, the research results were applied to the welding simulation of the whole side-wall module. The shape of the molten pool obtained using the combined heat source was closer to that of the experiments (error < 10%), demonstrating the accuracy and effectiveness of the developed the heat source model for laser welding simulation. For local model laser welding using the TCCM, a coarse mesh was used, and the weld was divided into four segments, and highly accurate results were obtained. This calculation time was only 5.97% of that of a moving heat source in case of the thermo-elastic-plastic method (TEPM). Residual stress and welding deformation of the stainless steel side-wall module were calculated according to actual process parameters and the results of local model simulation. Residual stress was discontinuously distributed at the weld segments, and it only slightly influenced the overall stress distribution. The maximum residual stress (462.15 MPa) occurred at the weld of the large crossbeam. Welding eight small and two large crossbeams influenced the deformation change and the maximum deformation (1.26 mm) appeared in the middle position of the left side-wall. The findings of this study show that the TCCM has high calculation accuracy and is sufficiently economical for predicting laser welding of large structures.

Predicting heterosis via genetic distance and the number of SNPs in selected segments of chromosomes in maize.

A reliable method is needed for predicting heterosis to help maize (Zea mays L.) breeders develop new hybrids more efficiently. The objectives of this study were to 1) investigate if the numbers of selected PEUS SNPs (the SNP in the Promoters (1 kb upstream of the start codon), Exons, Untranslated region (UTR), and Stop codons) could be used for predicting MPH or BPH of GY; 2) if the number of PEUS SNPs is a better predictor of MPH and/or BPH of GY than genetic distance (GD). A line × tester experiment was conducted with 19 elite maize inbreds from three heterotic groups, which were crossed with five testers. The multi-location trial data on GY were recorded. Whole-genome resequencing of the 24 inbreds was carried out. After filtration, a total of 58,986,791 SNPs were called with high confidence. Selected SNPs in the promoters, exons, untranslated region (UTRs), and stop codons (PEUS SNPs) were counted, and the GD was calculated. The correlation between heterozygous PEUS SNPs/GD and mean MPH, BPH of GY revealed that 1) both the number of heterozygous PEUS SNP and the GD were highly correlated to both MPH_GY and BPH_GY at p<0.01 with correlation coefficients for the number of heterozygous PEUS SNP being higher than that for GD; 2) the mean number of heterozygous PEUS SNPs was also highly correlated with mean BPH_GY or mean MPH_GY (p<0.05) in the 95 crosses grouped by either male or female parents, implying that inbreds can be selected before making the actual crosses in the field. We concluded that the number of heterozygous PEUS SNPs would be a better predictor of MPH_GY and BPH_GY than GD. Hence, maize breeders could use heterozygous PEUS SNPs to select inbreds with high heterosis potential before actually making the crosses, thus improving the breeding efficiency.

Identification of Candidate QTLs and Genes for Ear Diameter by Multi-Parent Population in Maize.

Ear diameter (ED) is a critical component of grain yield (GY) in maize (Zea mays L.). Studying the genetic basis of ED in maize is of great significance in enhancing maize GY. Against this backdrop, this study was framed to (1) map the ED-related quantitative trait locus (QTL) and SNPs associated with ED; and (2) identify putative functional genes that may affect ED in maize. To accomplish this, an elite maize inbred line, Ye107, which belongs to the Reid heterotic group, was used as a common parent and crossed with seven elite inbred lines from three different heterotic groups (Suwan1, Reid, and nonReid) that exhibited abundant genetic variation in ED. This led to the construction of a multi-parent population consisting of 1215 F7 recombinant inbred lines (F7RILs). A genome-wide association study (GWAS) and linkage analysis were then conducted for the multi-parent population using 264,694 high-quality SNPs generated via the genotyping-by-sequencing method. Our study identified a total of 11 SNPs that were significantly associated with ED through the GWAS, and three QTLs were revealed by the linkage analysis for ED. The major QTL on chromosome 1 was co-identified in the region by the GWAS at SNP_143985532. SNP_143985532, located upstream of the Zm00001d030559 gene, encodes a callose synthase that is expressed in various tissues, with the highest expression level in the maize ear primordium. Haplotype analysis indicated that the haplotype B (allele AA) of Zm00001d030559 was positively correlated with ED. The candidate genes and SNPs identified in this study provide crucial insights for future studies on the genetic mechanism of maize ED formation, cloning of ED-related genes, and genetic improvement of ED. These results may help develop important genetic resources for enhancing maize yield through marker-assisted breeding.

Fine mapping of candidate quantitative trait loci for plant and ear height in a maize nested-association mapping population.

Plant height (PH) and ear height (EH) are two important traits in maize (Zea mays L.), as they are closely related to lodging resistance and planting density. Our objectives were to (1) investigate single-nucleotide polymorphisms (SNPs) that are associated with PH and EH for detecting quantitative trait loci (QTL) and new gene that determines PH and EH, (2) explore the value of the QTL in maize breeding, and (3) investigate whether the "triangle heterotic group" theory is applicable for lowering PH and EH to increase yield. Seven inbred female parents were crossed with a common founder male parent Ye 107 to create a nested association mapping (NAM) population. The analysis of phenotypic data on PH and EH revealed wide variation among the parents of the NAM population. Genome-wide association study (GWAS) and high-resolution linkage mapping were conducted using the NAM population, which generated 264,694 SNPs by genotyping-by-sequencing. A total of 105 SNPs and 22 QTL were identified by GWAS and found to be significantly associated with PH and EH. A high-confidence QTL for PH, Qtl-chr1-EP, was identified on chromosome 1 via GWAS and confirmed by linkage analysis in two recombinant inbred line (RIL) populations. Results revealed that the SNP variation in the promoter region of the candidate gene Zm00001d031938, located at Qtl-chr1-EP, which encoded UDP-N-acetylglucosamine-peptide N-acetyl-glucosaminyl-transferase, might decrease PH and EH. Furthermore, the triangle heterotic pattern adopted in maize breeding programs by our team is practicable in selecting high-yield crosses based on the low ratio of EH/PH (EP).

Anti-Diabetic Effect of Aster sphathulifolius in C57BL/KsJ-db/db Mice.

In this study, we investigated the anti-diabetic effect of Aster sphathulifolius (AS) extract in C57BL/KsJ-db/db mice. The db/db mice were orally administered with AS 50% ethanol extract at concentrations of 50, 100, and 200 mg/kg/day (db/db-AS50, db/db-AS100, and db/db-AS200, respectively) for 10 weeks. Food and water intake, fasting blood glucose concentrations, blood glycosylated hemoglobin levels, and plasma insulin levels were significantly lower in the db/db-AS200 group than in the vehicle-treated db/db group; whereas glucose tolerance was significantly improved in the db/db-AS200 group. Moreover, AS dose dependently increased both insulin receptor substrate 1 and glucose transporter type 4 expression in skeletal muscle, significantly increased glucokinase expression, and decreased glucose 6-phosphatase and phosphoenolpyruvate carboxykinase expressions in the liver. The expressions of transcription factors, such as sterol-regulatory element-binding protein, peroxisome proliferator-activated receptor γ, and adipocyte protein 2, were upregulated in adipose tissue. Furthermore, immunohistochemical analysis showed that AS upregulated insulin production by increasing pancreatic ß-cell mass. In summary, AS extract normalized hyperglycemia by multiple mechanisms: inhibition of glyconeogenesis, acceleration of glucose metabolism and lipid metabolism, and increase of glucose uptake. Using in vivo assays, this study has shown the potential of AS as a medicinal food and suggests the efficacy of AS for the use of prevention of diabetes.