Identification of Rice Transcription Factors Associated with Drought Tolerance Using the Ecotilling Method

Abstract

The drought tolerance (DT) of plants is a complex quantitative trait. Under natural and artificial selection, drought tolerance represents the crop survival ability and production capacity under drought conditions (Luo, 2010). To understand the regulation mechanism of varied drought tolerance among rice genotypes, 95 diverse rice landraces or varieties were evaluated within a field screen facility based on the ‘line–source soil moisture gradient’, and their resistance varied from extremely resistant to sensitive. The method of Ecotype Targeting Induced Local Lesions in Genomes (Ecotilling) was used to analyze the diversity in the promoters of 24 transcription factor families. The bands separated by electrophoresis using Ecotilling were converted into molecular markers. STRUCTURE analysis revealed a value of K = 2, namely, the population with two subgroups (i.e., indica and japonica), which coincided very well with the UPGMA clusters (NTSYS-pc software) using distance-based analysis and InDel markers. Then the association analysis between the promoter diversity of these transcription factors and the DT index/level of each variety was performed. The results showed that three genes were associated with the DT index and that five genes were associated with the DT level. The sequences of these associated genes are complex and variable, especially at approximately 1000 bp upstream of the transcription initiation sites. The study illuminated that association analysis aimed at Ecotilling diversity of natural groups could facilitate the isolation of rice genes related to complex quantitative traits.

Tilling and Eco-Tilling - A Reverse Genetic Approach for Crop Improvement

“…This technique is now applied to rice, maize, lotus, poplar , Brassica, zebrafish, Drosophila, Caenorhabditis and human , indicating its broad applicability. The implication of ECO-TILLING to identify potential novel functional alleles in the known and candidate genes/transcription factors (TFs) regulating qualitative and quantitative agronomic traits by association/genetic mapping is well-documented for expediting the genetic enhancement of crop plants (Mejlhede et al, 2006;Barkley and Wang, 2008;Ibiza et al, 2010;Negrao et al, 2011;Yu et al, 2012;Frerichmann et al, 2013). ECO-TILLING usually employs a mismatchspecific CEL-I nuclease to cleave the PCR amplified fragments at the site of heteroduplex formation involving nucleotide (SNP-allelic) polymorphism.…”

“…Consequently, these efforts led to the discovery and genotyping of novel potential alleles specifically derived from the trait-associated known and candidate genes in natural population of diverse crop plants (Perry et al, 2003;Caldwell et al, 2004;Comai et al, 2004;Henikoff et al, 2004;Yang et al, 2004;Suzuki et al, 2005). The added-advantage of agarose gel-based ECO-TILLING vis-à-vis the commonly utilized LICOR genotyper for large-scale mining and genotyping of allelic variants in accessions exhibiting low level polymorphism, is welldemonstrated in many crop plants (Raghavan et al, 2007;Negrao et al, 2011;Yu et al, 2012). This is merely because efficacy of an agarose gel-based ECO-TILLING approach in precise resolution of unlabeled CEL Icleaved heteroduplex PCR amplified fragments by a simpler, economical and timesaving agarose gel-based detection assay as compared to a standard ECO-TILLING method that requires labeled CEL I-cleaved heteroduplex PCR amplicons for resolution in a LICOR genotyper.…”

“…This is merely because efficacy of an agarose gel-based ECO-TILLING approach in precise resolution of unlabeled CEL Icleaved heteroduplex PCR amplified fragments by a simpler, economical and timesaving agarose gel-based detection assay as compared to a standard ECO-TILLING method that requires labeled CEL I-cleaved heteroduplex PCR amplicons for resolution in a LICOR genotyper. The broader utility and deployment of this agarose gel-based ECO-TILLING approach in manifold large-scale genotyping applications is well-documented by the research laboratories with minimal resources (Raghavan et al, 2007;Negrao et al, 2011;Yu et al, 2012). This includes understanding the natural allelic diversity, population genetic structure and domestication pattern among accessions, molecular mapping and genetic association analysis for identification of potential molecular tags like alleles and genes/QTLs (quantitative trait loci) governing vital agronomic traits and marker-assisted breeding for selecting desirable accessions for crop genetic improvement.…”

Biological Diversity and Public Health

“…Research has only recently characterized some of the molecular mechanisms that confer these abilities to plants. In rice, soy, corn and wheat, and other staple crops, researchers have identified candidate genes and hybridization strategies to endow them with resilience (15,44,47,65,88), and all crops in widespread use have been bred to be resilient to biotic and abiotic stressors.…”

Water Stress and Agriculture

“…The TFs are proteins that play a role in physiological and biological processes such as growth, development and responses to environmental stresses acting as key regulators involved in early stages of expression, gene regulation, signal transduction [92].…”