Image_5_A SLAF-based high-density genetic map construction and genetic architecture of thermotolerant traits in maize (Zea mays L.).tif

The leaf scorching trait at flowering is a crucial thermosensitive phenotype in maize under high temperature stress (HS), yet the genetic basis of this trait remains poorly understood. In this study, we genotyped a 254 RIL-F2:8 population, derived from the leaf scorch-free parental inbred line Abe2 and the leaf scorching maternal inbred line B73, using the specific-locus amplified fragment sequencing (SLAF-seq) method. A total of 10,112 polymorphic SLAF markers were developed, and a high-density genetic map with a total length of 1,475.88 cM was constructed. The average sequencing depth of the parents was 55.23X, and that of the progeny was 12.53X. Then, we identified a total of 16 QTLs associated with thermotolerant traits at flowering, of which four QTLs of leaf scorching damage (LS) were distributed on chromosomes 1 (qLS1), 2 (qLS2.1, qLS2.2) and 3 (qLS3), which could explain 19.73% of phenotypic variation. Combining one qLS1 locus with QTL-seq results led to the identification of 6 candidate genes. Expression experiments and sequence variation indicated that Zm00001d033328, encoding N-acetyl-gamma-glutamyl-phosphate reductase, was the most likely candidate gene controlling thermotolerant traits at flowering. In summary, the high-density genetic map and genetic basis of thermotolerant traits lay a critical foundation for mapping other complex traits and identifying the genes associated with thermotolerant traits in maize.</p

Image_3_A SLAF-based high-density genetic map construction and genetic architecture of thermotolerant traits in maize (Zea mays L.).tif

The leaf scorching trait at flowering is a crucial thermosensitive phenotype in maize under high temperature stress (HS), yet the genetic basis of this trait remains poorly understood. In this study, we genotyped a 254 RIL-F2:8 population, derived from the leaf scorch-free parental inbred line Abe2 and the leaf scorching maternal inbred line B73, using the specific-locus amplified fragment sequencing (SLAF-seq) method. A total of 10,112 polymorphic SLAF markers were developed, and a high-density genetic map with a total length of 1,475.88 cM was constructed. The average sequencing depth of the parents was 55.23X, and that of the progeny was 12.53X. Then, we identified a total of 16 QTLs associated with thermotolerant traits at flowering, of which four QTLs of leaf scorching damage (LS) were distributed on chromosomes 1 (qLS1), 2 (qLS2.1, qLS2.2) and 3 (qLS3), which could explain 19.73% of phenotypic variation. Combining one qLS1 locus with QTL-seq results led to the identification of 6 candidate genes. Expression experiments and sequence variation indicated that Zm00001d033328, encoding N-acetyl-gamma-glutamyl-phosphate reductase, was the most likely candidate gene controlling thermotolerant traits at flowering. In summary, the high-density genetic map and genetic basis of thermotolerant traits lay a critical foundation for mapping other complex traits and identifying the genes associated with thermotolerant traits in maize.</p

Image_8_A SLAF-based high-density genetic map construction and genetic architecture of thermotolerant traits in maize (Zea mays L.).tif

The leaf scorching trait at flowering is a crucial thermosensitive phenotype in maize under high temperature stress (HS), yet the genetic basis of this trait remains poorly understood. In this study, we genotyped a 254 RIL-F2:8 population, derived from the leaf scorch-free parental inbred line Abe2 and the leaf scorching maternal inbred line B73, using the specific-locus amplified fragment sequencing (SLAF-seq) method. A total of 10,112 polymorphic SLAF markers were developed, and a high-density genetic map with a total length of 1,475.88 cM was constructed. The average sequencing depth of the parents was 55.23X, and that of the progeny was 12.53X. Then, we identified a total of 16 QTLs associated with thermotolerant traits at flowering, of which four QTLs of leaf scorching damage (LS) were distributed on chromosomes 1 (qLS1), 2 (qLS2.1, qLS2.2) and 3 (qLS3), which could explain 19.73% of phenotypic variation. Combining one qLS1 locus with QTL-seq results led to the identification of 6 candidate genes. Expression experiments and sequence variation indicated that Zm00001d033328, encoding N-acetyl-gamma-glutamyl-phosphate reductase, was the most likely candidate gene controlling thermotolerant traits at flowering. In summary, the high-density genetic map and genetic basis of thermotolerant traits lay a critical foundation for mapping other complex traits and identifying the genes associated with thermotolerant traits in maize.</p

Image_6_A SLAF-based high-density genetic map construction and genetic architecture of thermotolerant traits in maize (Zea mays L.).tif

The leaf scorching trait at flowering is a crucial thermosensitive phenotype in maize under high temperature stress (HS), yet the genetic basis of this trait remains poorly understood. In this study, we genotyped a 254 RIL-F2:8 population, derived from the leaf scorch-free parental inbred line Abe2 and the leaf scorching maternal inbred line B73, using the specific-locus amplified fragment sequencing (SLAF-seq) method. A total of 10,112 polymorphic SLAF markers were developed, and a high-density genetic map with a total length of 1,475.88 cM was constructed. The average sequencing depth of the parents was 55.23X, and that of the progeny was 12.53X. Then, we identified a total of 16 QTLs associated with thermotolerant traits at flowering, of which four QTLs of leaf scorching damage (LS) were distributed on chromosomes 1 (qLS1), 2 (qLS2.1, qLS2.2) and 3 (qLS3), which could explain 19.73% of phenotypic variation. Combining one qLS1 locus with QTL-seq results led to the identification of 6 candidate genes. Expression experiments and sequence variation indicated that Zm00001d033328, encoding N-acetyl-gamma-glutamyl-phosphate reductase, was the most likely candidate gene controlling thermotolerant traits at flowering. In summary, the high-density genetic map and genetic basis of thermotolerant traits lay a critical foundation for mapping other complex traits and identifying the genes associated with thermotolerant traits in maize.</p

Image_4_A SLAF-based high-density genetic map construction and genetic architecture of thermotolerant traits in maize (Zea mays L.).tif

The leaf scorching trait at flowering is a crucial thermosensitive phenotype in maize under high temperature stress (HS), yet the genetic basis of this trait remains poorly understood. In this study, we genotyped a 254 RIL-F2:8 population, derived from the leaf scorch-free parental inbred line Abe2 and the leaf scorching maternal inbred line B73, using the specific-locus amplified fragment sequencing (SLAF-seq) method. A total of 10,112 polymorphic SLAF markers were developed, and a high-density genetic map with a total length of 1,475.88 cM was constructed. The average sequencing depth of the parents was 55.23X, and that of the progeny was 12.53X. Then, we identified a total of 16 QTLs associated with thermotolerant traits at flowering, of which four QTLs of leaf scorching damage (LS) were distributed on chromosomes 1 (qLS1), 2 (qLS2.1, qLS2.2) and 3 (qLS3), which could explain 19.73% of phenotypic variation. Combining one qLS1 locus with QTL-seq results led to the identification of 6 candidate genes. Expression experiments and sequence variation indicated that Zm00001d033328, encoding N-acetyl-gamma-glutamyl-phosphate reductase, was the most likely candidate gene controlling thermotolerant traits at flowering. In summary, the high-density genetic map and genetic basis of thermotolerant traits lay a critical foundation for mapping other complex traits and identifying the genes associated with thermotolerant traits in maize.</p

Image_2_A SLAF-based high-density genetic map construction and genetic architecture of thermotolerant traits in maize (Zea mays L.).tif

The leaf scorching trait at flowering is a crucial thermosensitive phenotype in maize under high temperature stress (HS), yet the genetic basis of this trait remains poorly understood. In this study, we genotyped a 254 RIL-F2:8 population, derived from the leaf scorch-free parental inbred line Abe2 and the leaf scorching maternal inbred line B73, using the specific-locus amplified fragment sequencing (SLAF-seq) method. A total of 10,112 polymorphic SLAF markers were developed, and a high-density genetic map with a total length of 1,475.88 cM was constructed. The average sequencing depth of the parents was 55.23X, and that of the progeny was 12.53X. Then, we identified a total of 16 QTLs associated with thermotolerant traits at flowering, of which four QTLs of leaf scorching damage (LS) were distributed on chromosomes 1 (qLS1), 2 (qLS2.1, qLS2.2) and 3 (qLS3), which could explain 19.73% of phenotypic variation. Combining one qLS1 locus with QTL-seq results led to the identification of 6 candidate genes. Expression experiments and sequence variation indicated that Zm00001d033328, encoding N-acetyl-gamma-glutamyl-phosphate reductase, was the most likely candidate gene controlling thermotolerant traits at flowering. In summary, the high-density genetic map and genetic basis of thermotolerant traits lay a critical foundation for mapping other complex traits and identifying the genes associated with thermotolerant traits in maize.</p

DataSheet_1_A SLAF-based high-density genetic map construction and genetic architecture of thermotolerant traits in maize (Zea mays L.).zip

The leaf scorching trait at flowering is a crucial thermosensitive phenotype in maize under high temperature stress (HS), yet the genetic basis of this trait remains poorly understood. In this study, we genotyped a 254 RIL-F2:8 population, derived from the leaf scorch-free parental inbred line Abe2 and the leaf scorching maternal inbred line B73, using the specific-locus amplified fragment sequencing (SLAF-seq) method. A total of 10,112 polymorphic SLAF markers were developed, and a high-density genetic map with a total length of 1,475.88 cM was constructed. The average sequencing depth of the parents was 55.23X, and that of the progeny was 12.53X. Then, we identified a total of 16 QTLs associated with thermotolerant traits at flowering, of which four QTLs of leaf scorching damage (LS) were distributed on chromosomes 1 (qLS1), 2 (qLS2.1, qLS2.2) and 3 (qLS3), which could explain 19.73% of phenotypic variation. Combining one qLS1 locus with QTL-seq results led to the identification of 6 candidate genes. Expression experiments and sequence variation indicated that Zm00001d033328, encoding N-acetyl-gamma-glutamyl-phosphate reductase, was the most likely candidate gene controlling thermotolerant traits at flowering. In summary, the high-density genetic map and genetic basis of thermotolerant traits lay a critical foundation for mapping other complex traits and identifying the genes associated with thermotolerant traits in maize.</p

Image_7_A SLAF-based high-density genetic map construction and genetic architecture of thermotolerant traits in maize (Zea mays L.).tif

The leaf scorching trait at flowering is a crucial thermosensitive phenotype in maize under high temperature stress (HS), yet the genetic basis of this trait remains poorly understood. In this study, we genotyped a 254 RIL-F2:8 population, derived from the leaf scorch-free parental inbred line Abe2 and the leaf scorching maternal inbred line B73, using the specific-locus amplified fragment sequencing (SLAF-seq) method. A total of 10,112 polymorphic SLAF markers were developed, and a high-density genetic map with a total length of 1,475.88 cM was constructed. The average sequencing depth of the parents was 55.23X, and that of the progeny was 12.53X. Then, we identified a total of 16 QTLs associated with thermotolerant traits at flowering, of which four QTLs of leaf scorching damage (LS) were distributed on chromosomes 1 (qLS1), 2 (qLS2.1, qLS2.2) and 3 (qLS3), which could explain 19.73% of phenotypic variation. Combining one qLS1 locus with QTL-seq results led to the identification of 6 candidate genes. Expression experiments and sequence variation indicated that Zm00001d033328, encoding N-acetyl-gamma-glutamyl-phosphate reductase, was the most likely candidate gene controlling thermotolerant traits at flowering. In summary, the high-density genetic map and genetic basis of thermotolerant traits lay a critical foundation for mapping other complex traits and identifying the genes associated with thermotolerant traits in maize.</p

Image_1_A SLAF-based high-density genetic map construction and genetic architecture of thermotolerant traits in maize (Zea mays L.).tif

The leaf scorching trait at flowering is a crucial thermosensitive phenotype in maize under high temperature stress (HS), yet the genetic basis of this trait remains poorly understood. In this study, we genotyped a 254 RIL-F2:8 population, derived from the leaf scorch-free parental inbred line Abe2 and the leaf scorching maternal inbred line B73, using the specific-locus amplified fragment sequencing (SLAF-seq) method. A total of 10,112 polymorphic SLAF markers were developed, and a high-density genetic map with a total length of 1,475.88 cM was constructed. The average sequencing depth of the parents was 55.23X, and that of the progeny was 12.53X. Then, we identified a total of 16 QTLs associated with thermotolerant traits at flowering, of which four QTLs of leaf scorching damage (LS) were distributed on chromosomes 1 (qLS1), 2 (qLS2.1, qLS2.2) and 3 (qLS3), which could explain 19.73% of phenotypic variation. Combining one qLS1 locus with QTL-seq results led to the identification of 6 candidate genes. Expression experiments and sequence variation indicated that Zm00001d033328, encoding N-acetyl-gamma-glutamyl-phosphate reductase, was the most likely candidate gene controlling thermotolerant traits at flowering. In summary, the high-density genetic map and genetic basis of thermotolerant traits lay a critical foundation for mapping other complex traits and identifying the genes associated with thermotolerant traits in maize.</p

Citric Acid-Assisted Two-Step Enrichment with TiO₂ Enhances the Separation of Multi- and Monophosphorylated Peptides and Increases Phosphoprotein Profiling

Phosphopeptide enrichment is essential for large-scale phosphoprotein profiling. Titanium dioxide (TiO₂) is widely used in phosphopeptide enrichment, but it is limited in the isolation of multiphosphorylated peptides due to their strong binding. In this study, we found that citric acid greatly affects the binding of mono- and multiphosphopeptides with TiO₂, which can be used for stepwise phosphopeptide separation coupled with mass spectrum (MS) identification. We first loaded approximately 1 mg of peptide mixture of HeLa cell digests onto TiO₂ beads in highly concentrated citric acid (1 M). Then the flow-through fraction was diluted to ensure low concentration of citric acid (50 mM) and followed by loading onto another aliquot of TiO₂ beads. The two eluted fractions were subjected to nanoLC–MS/MS analysis. We identified 1,500 phosphorylated peptides, of which 69% were multiphosphorylated after the first enrichment. After the second enrichment, 2,167 phosphopeptides, of which 92% were monophosphorylated, were identified. In total, we successfully identified 3,136 unique phosphopeptides containing 3,973 phosphosites utilizing this strategy. Finally, more than 37% of the total phosphopeptides and 2.6-fold more of the multiphosphorylated peptides were identified as compared to the frequently used DHB/TiO₂ enrichment strategy. Combining SCX with CATSET, we identified 14,783 phosphopeptides and 15,713 phosphosites, of which 3,678 were unrecorded in PhosphoSitePlus database. This two-step separation procedure for sequentially enriching multi- and monophosphorylated peptides by using citric acid is advantageous in multiphosphorylated peptide separation, as well as for more comprehensive phosphoprotein profiling