Cloning and Characterizing Candidate Genes Underlying Major QTLs for Wheat Stem Pith Thickness and Stem Diameter

Qier Liu

Wheat varieties with a strong stem phenotype are desirable for enhancing resistance to lodging, drought, and insect. The strong stem trait is determined by multiple factors including stem diameter (SD), stem pith-thickness (PT), and the lignin content in the stem wall. Based on forward genetic strategies, a single gene-controlled major QTL for PT and a closely linked QTL for SD were identified on Chromosome 3BL. In this study, bulked segregant RNA-seq (BSR-seq) analysis was applied to screening differentially expressed SNP genes and identify candidate genes. Twenty-four highprobability SNPs in eight genes were identified by comparing the mRNA sequences between the two bulks. A putative vacuolar processing enzyme gene, TaVPE3, which functions as an executor of plant cell death, was screened out as a candidate gene of PT. Two allelic variants, TaVPE3-3Ba and TaVPE3-3Bb, were identified with a 9-bp insertion/deletion (InDel) and 36 SNPs in the coding region. A co-dominant genespecific marker based on the SNP was developed. A total of 171 Australian historical cultivars were used to validate the associations between allelic patterns and PT. The PT of cultivars with TaVPE3-3Bb was significantly (P<0.01) higher than that of those with TaVPE3-3Ba, indicating that the marker can be effectively used in wheat breeding. In addition to TaVPE3 for PT, a O-methyltransferase gene, TaOMT2, with a 222-bp InDel in the 3′-untranslated region was screened out as a candidate gene of SD. To investigate the gene function of TaOMT2 and TaVPE3, CRISPR/Cas 9 gene-editing technology was applied to create knockout (KO) mutants. Transgenic assays suggested that TaOMT2 affects stem diameter by regulating vascular development, as the number and size of vascular bundles in the stems were significantly increased in the TaOMT2-overexpressing (OE) plant but decreased in the TaOMT2-bd knockout (KO) mutant plants. Apart from that, overexpression of TaOMT2 enhances the stem mechanical strength and lignin content in the transgenic wheat lines. Meanwhile, a heterozygous TaVPE3-d mutant was generated and analyzed for its programmed cell death (PCD). Confocal microscopy showed delayed PCD in pith cells in the mutant. However, due to the redundancy of gene function in wheat, the single homozygous mutants in 3D homeologs did not completely inhibit the PCD process. Therefore, no significant difference in stem PT was found, and the overall growth performance of the mutant was similar to WT. The findings not only demonstrated that TaOMT2 is the causal gene of the 3BL QTL for stem diameter in wheat but also provided a selection strategy for designing strong stem wheat cultivars through breeding.

Cloning and Characterizing Candidate Genes Underlying Major QTLs for Wheat Stem Pith Thickness and Stem Diameter

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