Characterization Of Ta LecRK-Ⅳ.1 Gene In Wheat And Development Of Molecular Markers Derived From Wheat Genes Induced By Sharp Eyespot

Wheat sharp eyespot caused mainly by the soil-borne fungus Rhizoctonia cerealis,is a stem based disease.It has become an important yield limitation for the production of wheat in some regions of the globe,especially in China.As a limiting factor for wheat cultivation,control of sharp eyespot is a major concern for sustainable agriculture.Among several approaches to curb this disease,developing resistant wheat varieties is the most environmentally safe means to reduce yield losses caused by sharp eyespot.After the identification of the resistant genotypes,genetic studies for resistance and resistance-related genes can be undertaken to dissect the nature of genes playing important roles in the resistance reaction against sharp eyespot.RNA sequencing(RNA-Seq)and Microarray technologies allow the comparison of differential expression of genes under stress conditions in order to distinguish those involved in stress responses.Using these techniques(RNA-seq and microarray),genes responsive to Rhizoctonia cerealis infection can be detected by differential expression after the pathogen inoculation.The identified genes can be subjected to further study for cloning and functional analysis.Reversely,the function of a candidate gene can then be analyzed by virus-induced gene silencing(VIGS)to dissect whether a candidate gene is functionally implicated in the regulation of a particular trait.This strategy is often deployed to investigate the possible involvement of candidate gene(s)related to stress resistance or tolerance if they are remarkably expressed under stress conditions.Once the resistance or important resistance-related genes/transcripts have been identified and cloned,they provide the basis for the application of molecular markers,which efficiently assist phenotypic selection in all phases of resistance breeding.A gene sequence or expressed sequence tag can serve for molecular markers development with practical use in breeding research for germplasm characterization and marker-assisted selection for screening large breeding population.With the progress in wheat genomics,knowledge on gene sequences allows the development of molecular markers associated with important traits.In this study,we have identified an L-type lectin receptor kinase gene,designated TaLecRKⅣ.1,which was transcriptionally up-regulated in response to Rhizoctonia cerealis infection in the partially-resistant wheat line CI12633.Time course expression analysis by the mean of quantitative polymerase chain reaction(qPCR)showed that the gene was induced in CI12633 in comparison to the expression levels in Wenmai 6,a susceptible wheat cultivar to sharp eyespot.The sequence of the gene was cloned and sequence analysis revealed that the gene encodes an L-type lectin-like receptor kinase protein consisting of 676 amino acid residues.Knock-down of TaLec RK-Ⅳ.1 by VIGS led to dwarf plant phenotype in wheat line CI12633,suggesting that TaLecRK-Ⅳ.1 is a negative regulator of dwarfism in plant.Analysis of the expression of TaLecRK-Ⅳ.1 in a dwarf wheat line carrying Rht-D1b(formerly Rht2),showed that TaLecK-Ⅳ.1 was less expressed in this dwarf variety.In addition,TaLecKⅣ.1 expression was induced in plants treated with gibberellic acid(GA)and auxin,two phyohormones involved in growth and development as compared to TaLecK-Ⅳ.1 transcript levels in plants sprayed with abscisic acid and salicylic acid.These data suggest that TaLecKⅣ.1 might be involved in plant developmental pathway.On the other hand,evaluation of the resistance response of TaLecK-Ⅳ.1-silenced plants to sharp eyespot did not reveal an increased susceptibility of these plants to sharp eyespot,implying that the gene might not play a major role in the resistance response to this disease(sharp eyespot).A population of recombinant inbred lines(RILs)of 114 individuals generated by crossing sharp eyespot resistant wheat line,Shanhongmai with the susceptible wheat cultivar Wenmai 6 along with the two parents was evaluated by field trials to assess their resistance reaction to sharp eyespot.Phenotypic assessment revealed that none of the RIL plants was highly resistant to sharp eyespot.Individuals of this population were continuously distributed over the range of the disease index(DI),suggesting that sharp eyespot resistance was quantitatively inherited in this population.Eight molecular markers were developed based on the cDNA sequences of the putative sharp eyespot resistance-related genes identified by microarray analysis.These markers were used to genotype the RIL population.Of these,three molecular markers(RGAleft,RGA-right and F3F-TC371509R)were found to be linked to sharp eyespot resistance allele(s)based on the segregation ratio of the individuals of the recombinant inbred lines.However,the analysis for the quantitative trait locus(QTL)did not detect any significant QTL associated with the resistance in this population as the LOD values of all QTLs identified in the study were lower than that of the threshold value above which a QTL is declared as significant.This might be attributed to a variation during the test of the population to the disease infection.Alternatively,sharp eyespot resistance in Shanhongmai × Wenmai 6 –derived population might be controlled by minor QTL or the markers developed in this study were not associated with the genes/alleles with major contribution to sharp eyespot resistance.In further study,improvement of the disease assessment and employment of a larger population size and other molecular markers development strategies will be helpful in developing effective molecular markers that can be used in resistance breeding and to clarify the nature of sharp eyespot resistance trait in the employed population for the study.