DataSheet_1_Leaf rolling in bread wheat (Triticum aestivum L.) is controlled by the upregulation of a pair of closely linked/duplicate zinc finger homeodomain class transcription factors during moisture stress conditions.docx

Zinc finger-homeodomain (ZF-HDs) class IV transcriptional factors (TFs) is a plant-specific transcription factor and play a key role in stress responses, plant growth, development, and hormonal signaling. In this study, two new leaf rolling TFs genes, namely TaZHD1 and TaZHD10, were identified in wheat using comparative genomic analysis of the target region that carried a major QTL for leaf rolling identified through multi-environment phenotyping and high throughput genotyping of a RIL population. Structural and functional annotation of the candidate ZHD genes with its closest rice orthologs reflects the species-specific evolution and, undoubtedly, validates the notions of remote-distance homology concept. Meanwhile, the morphological analysis resulted in contrasting difference for leaf rolling in extreme RILs between parental lines HD2012 and NI5439 at booting and heading stages. Transcriptome-wide expression profiling revealed that TaZHD10 transcripts showed significantly higher expression levels than TaZHD1 in all leaf tissues upon drought stress. The relative expression of these genes was further validated by qRT-PCR analysis, which also showed consistent results across the studied genotypes at the booting and anthesis stage. The contrasting modulation of these genes under drought conditions and the available evidenced for its epigenetic behavior that might involve the regulation of metabolic and gene regulatory networks. Prediction of miRNAs resulted in five Tae-miRs that could be associated with RNAi mediated control of TaZHD1 and TaZHD10 putatively involved in the metabolic pathway controlling rolled leaf phenotype. Gene interaction network analysis indicated that TaZHD1 and TaZHD10 showed pleiotropic effects and might also involve other functions in wheat in addition to leaf rolling. Overall, the results increase our understanding of TaZHD genes and provide valuable information as robust candidate genes for future functional genomics research aiming for the breeding of wheat varieties tolerant to leaf rolling.</p

Representative gel picture of PCR amplification of promoter region of <i>TaGW2-6A</i> gene in 15 Indian wheat genotypes.

<p>Representative gel picture of PCR amplification of promoter region of <i>TaGW2-6A</i> gene in 15 Indian wheat genotypes.</p

Frequency distribution curve for nine agronomic traits including 1000 grain weight in 207 Indian wheat genotypes used in association mapping study.

<p>Frequency distribution curve for nine agronomic traits including 1000 grain weight in 207 Indian wheat genotypes used in association mapping study.</p

A hypothetical pathway of CGCG mediated regulation of <i>TaGW2-6A</i>.

<p>A hypothetical pathway of CGCG mediated regulation of <i>TaGW2-6A</i>.</p

Descriptive statistics for 1000 grain weight and other agronomic traits.

<p>Descriptive statistics for 1000 grain weight and other agronomic traits.</p

Bar diagrams showing (A) relative expression level of <i>TaGW2-6A</i> in immature seeds at 15dpf.

<p>Actin gene was used as the endogenous control and variety HI 1500 used as reference; (B) TGW of varieties having SNP-494_A on the left panel and those with SNP-494_G in the right panel.</p

Validation of CAPS in genotypes with causal SNP_G and SNP_A on 2% agarose gel.

<p>G and A represent undigested PCR products of genotypes with SNP_G and SNP_A; G1–G4 and A1–A4 are genotypes with SNP_G and SNP_A after digestion with <i>FauI</i>, M = marker.</p

Summary of four SNPs (identified by sequence alignment) with their position, variation, frequency and count.

<p>Summary of four SNPs (identified by sequence alignment) with their position, variation, frequency and count.</p

Summary of marker-trait association using single SNP for 1000 grain weight.

<p>Significantly associated SNP is marked with *.</p

Plot showing significant regression of (A) <i>TaGW2-6A</i> expression level and thousand grain weight (TGW) in genotypes with two different alleles (A and G) of SNP-494 identified in promoter region, and (B) TGW with relative expression level in same genotypes.

<p>* and ** indicate significance at 0.05 and 0.01 levels, respectively.</p