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Project Article | Sichuan Agricultural University Maize Research Institute Utilizes MTP to Assist in the Creation of Disease-resistant, Stress-tolerant Dwarf Maize Germplasm
Release time:
2024-12-31
Introduction
Dwarfing traits represent desirable characteristics such as high yield, fertilizer tolerance, and lodging resistance. These traits are an important research focus in the breeding of high-yielding varieties of crops like wheat, rice, and maize.
In maize, dwarfing breeding has mainly focused on the Br2 gene. However, the application of a single gene limits genetic diversity. Therefore, new dwarfing resources need to be created and identified through methods such as natural mutation, artificial mutagenesis, and genetic engineering.
Wide hybridization is an effective strategy that can overcome hybridization barriers and introduce wild-type genes into crops, thereby creating new dwarfing germplasm resources.
Recently, Dr. Zhou Yang, a PhD student at the Maize Research Institute of Sichuan Agricultural University, and Professor Tang Qilin, corresponding author, published a research paper titled "Maize-Tripsacum-Teosinte allopolyploid (MTP), a novel dwarf mutant inducer tool in maize" in the International Journal of Molecular Sciences (PBJ, CAS Q1, IF = 10.1). This study utilized the maize-Tripsacum-teosinte allopolyploid MTP as a bridging material, crossing and backcrossing it with maize to create MTP-maize introgression lines with important agronomic traits such as disease and pest resistance and abiotic stress tolerance. The study identified a series of new semi-dwarf traits.
Research Content
Establishment and Analysis of a Dwarfing Germplasm Bank from MTP Wide Hybridization in Maize
Through wide hybridization and mutagenesis with maize lines using MTP, more than 10 new semi-dwarf materials with desirable traits were successfully created. Among them, five dwarf materials exhibited stable and excellent dwarfing phenotypes, with traits such as uniformly shortened plants and coordinated male and female flowering.
Further systematic identification of allelic variation, inheritance patterns, and hormone responses in the five semi-dwarf materials revealed that they were not allelic to the Br2 dwarfing gene. Additionally, the dwarfing genes in these five germplasm lines were not allelic to each other. These traits are mainly controlled by single-gene inheritance. The hormone responses were found to vary, including gibberellin sensitivity, brassinosteroid sensitivity, and gibberellin and auxin insensitivity. Among them, d024 was found to be sensitive to brassinosteroids (BR).
Figure 1. Schematic diagram of the creation of the dwarf maize germplasm bank through distant hybridization of MTP
Localization of the Dwarfing Gene d024 and Analysis of the Source of Variation
A systematic study was conducted on the early-discovered MTP-maize dwarfing line d024. Compared to the wild type (H024), d024 exhibited similar internode number to H024, but the internodes at the ear position and above were significantly shortened.
Cytological observations revealed that, compared to the wild type, the length of longitudinal cut cells in the earstem internodes of d024 was significantly reduced. This suggests that the reduction in cell length within the stem internodes contributes to the dwarfing phenotype of d024.
Through genetic analysis, d024 showed a recessive single-gene inheritance pattern. Initially, extreme high and low plants from the F2 population of d024 X H024 were pooled and subjected to BSA-seq (Bulked Segregant Analysis) for preliminary localization of d024. Based on the B73 reference genome (RefGen_v4), a unique genomic region on chromosome 3 (4.1 Mb) was identified as being associated with the dwarfing phenotype of d024.
Subsequently, the candidate region for the d024 dwarfing trait was further narrowed, and additional polymorphic markers were used for genotypic identification. Among 708 dwarf plants in the F2 population, molecular markers 558, indel-5589, and indel-5598 revealed 21, 20, and 27 recombinant events, respectively. This indicated that the candidate gene was located in a physical region approximately 7.6 kb between markers 558 and indel-5589. According to B73_RefGen_v4, only one candidate gene (Zm00001d039453) was located in this region.
Cloning of the candidate gene revealed 9 SNP/Indel variations. SNP-8 and indel-7 showed nucleotide changes, both of which led to amino acid changes.
Moreover, a 191 bp indel-4 deletion was found in the promoter region of the candidate gene, leading to the loss of the BRRE (brassinosteroid response element) and a reduction in promoter activity.
More importantly, genotyping of the indel-4 promoter was performed in 28 maize inbred lines, 5 Tripsacum accessions, and 1 Teosinte (MTP parent). None of the maize inbred lines or Tripsacum accessions carried the indel-4 deletion. Only Teosinte (the MTP parent) showed nucleotide sequence homology with d024’s indel-4.
Therefore, this study identifies the key candidate gene controlling the height of d024 as Zm00001d039453, ZmCYP90D1
.
Figure 2. Localization, cloning, and analysis of the mutation source of the dwarfing gene d024
Gene Knockout Validation of ZmCYP90D1 Function
CRISPR/Cas9 technology was used to generate Zm00001d039453 gene knockout mutants in the maize inbred line B73 background to evaluate whether mutations in this gene caused the dwarfing phenotype. The results showed that the generated knockout lines, KO#1 (zmcyp90d1–1) and KO#2 (zmcyp90d1–2), exhibited a semi-dwarf phenotype similar to that of the d024 mutant. Moreover, the expression level of ZmCYP90D1 in the stems of these knockout lines was significantly lower than in B73. This confirmed that the dwarfing phenotype in d024 plants is caused by the deletion of the ZmCYP90D1 gene.
Overexpression of ZmCYP90D1 in Arabidopsis lines AtOE#1 and AtOE#2 showed that the height and gene expression levels of ZmCYP90D1 in both AtOE#1 and AtOE#2 were higher than in the B37 control line.
To verify the function of the homologous gene of ZmCYP90D1 in Arabidopsis, the T-DNA insertion mutant SALK-036476 (AT3G13730) was screened. At the end of flowering, the height and gene expression levels of AT3G13730 in the SALK-036476 line were lower than those in the control group. This suggests that ZmCYP90D1 positively regulates plant height in crops
.
Figure 3. ZmCYP90D1 positively regulates plant height in crops
Interaction between ZmCYP90D1 and the Transcription Factor ZmBES1/BZR1-5
ZmBES1/BZR1-5 is an important transcription factor that can directly bind to the BRRE element in the promoter of target genes. Motif analysis of the indel-4 region in ZmCYP90D1 identified a conserved core BRRE sequence, suggesting that these elements are potential targets for ZmBES1/BZR1-5.
A Y1H (Yeast One-Hybrid) assay was conducted using the CYP90D1B73(indel-4) and CYP90D1Tripsacum(indel-4) promoters. The results showed that ZmBES1/BZR1-5 exhibited stronger transcriptional activity on the CYP90D1B73(indel-4) promoter compared to the CYP90D1Tripsacum(indel-4) promoter.
In tobacco transient expression analysis, the coding sequence of ZmBES1/BZR1-5 driven by the 35S promoter was used as an effector, while the CYP90D1 indel-4 promoters from B73 and Tripsacum were used as reporter genes. The results indicated that the LUC (luciferase) activity of the CYP90D1Tripsacum reporter gene was lower than that of the CYP90D1B73 reporter gene.
Electrophoretic mobility shift assays (EMSA) demonstrated that ZmBES1/BZR1-5 could bind to the BRRE motif in the indel-4 region. Therefore, the 191 bp deletion in indel-4 diminished its effect on ZmBES1/BZR1-5 transcription.
A Y2H (Yeast Two-Hybrid) assay was used to screen for interactions between ZmFDXs, ZmNAD(P)H, and ZmCYP90D1. Bimolecular fluorescence complementation (BiFC) and split-luciferase imaging were performed in Nicotiana benthamiana plants. The results showed that when ZmCYP90D1-nYFP and ZmFDXs-CYFP, or ZmCYP90D1-nYFP and ZmNAD(P)H-CYFP were co-infiltrated, yellow fluorescence protein (YFP) was recombined in the membrane system of epidermal cells, and luciferase activity was detected in tobacco leaf cells. These results indicate that ZmCYP90D1 interacts with ZmFDXs and ZmNAD(P)H in vivo.
Figure 4. The ZmBES1/BZR1-5-ZmCYP90D1-ZmFDXs/ZmNAD(P)H module regulates the BR biosynthesis pathway
ZmCYP90D1 Regulates Hormones and Their Stages
Treatment with three plant hormones (IAA, GA3, and epiBL) showed that a concentration of 100 μg/L epiBL promoted the elongation of the longitudinal stem internodes in d024. Hydroponic growth of seedlings revealed that as the concentration of epiBL increased, the plant height of d024 increased, whereas GA3 and IAA had no significant effect on stem elongation in d024. The levels of BRs (brassinosteroids) in d024, KO#1, and WT plants were measured, and no significant differences were found between the three.
Further analysis of BR precursors showed that the contents of castasterone (CS) and teasterone (TY) were lower in the dwarf phenotypes (d024 and KO#1) compared to the WT. The content of 28-hydroxy-brassinosteroid (28-high-BR), a functional form of BR, was also lower in d024 and KO#1. These results indicate that d024 is a BR-deficient dwarf mutant.
The expression levels of six BR-related genes in the internodes of B73 and KO#1 plants were analyzed. It was found that there were no significant changes in the early stages of BR biosynthesis, such as in the expression of ZmDWF1/na2. However, in later stages, significant expression of ZmDwf4 and ZmBrd1 was observed, accompanied by significant downregulation of genes related to the BR signaling pathway.
Thus, it is concluded that the regulation of ZmCYP90D1 primarily occurs in the later stages of BR biosynthesis
.
Figure 5. Downstream regulation of the BR biosynthesis pathway by ZmCYP90D1
Regulatory Mechanism of ZmCYP90D1
A working model has been proposed to illustrate how ZmCYP90D1 regulates maize plant height by controlling the formation of internodal meristems and the elongation of internodal cells.
The expression of the dwarfing gene ZmCYP90D1 is regulated by the upstream transcription factor ZmBES1/BZR1-5. This, in turn, influences the interaction between ZmCYP90D1 and ZmFDXs, ZmNAD(P)H, thereby regulating the changes in brassinosteroid (BR) content in the internodes. These changes control the elongation of internodal cells, ultimately affecting the plant height.
Figure 6. Working model of the effects of CYP90D1B73 and CYP90D1Tripsacum on maize plant height
Application of indel-4 in Field Performance
The indel-4 from tetraploid Tripsacum was successfully introduced into the Mo17 inbred line through repeated backcrossing and molecular marker-assisted selection, creating a new dwarf maize material, Mo17d024.
In field trials conducted in 2023 in Chongzhou and Wenjiang, China, the improved F1 (B73d024 X Mo17d024) exhibited shorter plant height and larger stem diameter under high planting density compared to the control hybrid. Compared to the original F1 (B73 X Mo17), the improved F1 showed comparable and stable yield under high-density planting, with a higher harvest index.
These results indicate that the application of indel-4 can enhance the yield potential and harvest index of maize under intensive planting conditions.
Figure 7. The dwarf allele from tetraploid Tripsacum ensures yield potential and a higher harvest index under high-density planting
In summary, the team developed a maize-Tripsacum—Tripsacum hexaploid allopolyploid (MTP) using a bridge material formed by the tetraploid perennial Tripsacum, Tripsacum, and maize. This MTP was then crossed and backcrossed with maize to create new germplasm with important agronomic traits such as disease resistance, insect resistance, abiotic stress tolerance, and dwarfing. This approach provides opportunities for developing maize varieties with stronger resistance to biotic and abiotic stresses and utilizing new traits such as dwarfism. This study, which uses MTP to create new dwarf maize resources, offers a novel pathway and new resources for dwarf maize breeding.
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