Transcriptome Analysis For Resistance To Orange Wheat Blossom Midge In Wheat

Orange wheat blossom midge(Sitodiplosis mosellana Géhin)is one of the economically important pest insects which adversely affected wheat yield and quality worldwide.Breeding and applying of migde-resistant wheat is documented one of the most effective way to reduce kernel losses.However,faced much more challenging for the delicate invasion and complex resistance,leading to the traditional midge-resistant breeding methods can not develop midge-resistant wheat cultivars effectively and quickly,thus making midge-resistant breeding based on molecular marker assisted selection(MAS)the choice of priority.Previously,a major QTL was mapped to chromosome 4AL,and showed stably resistance to OWBM among years,in our group,and the phenotypes of RIL population parents 6218 and jimai 24 used for linkage analysis were stable.The objective of present study,based on the previous mapping results,was to discover midge-resistant candidate regions and deferentially expressed genes(DEGs)via BSA&RNA-Seq(BSR-Seq)and a series of bioinformatics tools,using recombinant inbred lines(RILs)and near-isogenic line(NILs)with their parent 6218(highly susceptible to midge)and Jimai24(highly resistant to midge),and to develop midge-resistant markers according to polymorphic loci in gene sequences.The above study results are great significance to the molecular breeding for wheat resistance to midge.The main research results are as follows:1.Based on transcriptomic analysis for 22 resistant/susceptible wheat samples,seven differentially expressed genes(DEGs)with SNPs were revealed via differential expression analysis,of which two and five were located on chromosome 3A and 4A,respectively.Nine DEGs were revealed by BSR-Seq in candidate regions of 4AL,of which four DEGs were consistent with the results of differential expression analysis.Moreover,the candidate region on 4A was corresponding with the previously-mapped major QTL region.Three homologous genes annotated as OMT were revealed by homologous analysis,of which two and one were located on 1D and 7D.Two insect-resistant genes,SPI-like gene and Malectin-like gene,were discovered based on gene functional annotation.2.Based on transcriptomic analysis for three pairs of NILs,22 DEGs were revealed via differential expression analysis and according to candidate regions by BSR-Seq analysis,of which three,two and seventeen were located on chromosome 2D,3A and 4A,respectively.Among these DEGs,fourteen DEGs exist polymorphic SNPs between resistant and susceptible NILs,of which TraesCS4A01G436100 and TraesCS4A01G437800 were consistent with the results of transcriptomic analysis for 22 resistant/susceptible wheat samples.3.qRT-PCR assay was subjected to eleven resistance-related genes designed with chromosome specific primers.qRT-PCR assay showed that the expression level of three genes in resistant and susceptible samples were not consistent with the corresponding resistance level;the expression level of four genes were only significantly different between resistant and susceptible wheat parents;the expression level of other four genes were significantly different between the selected resistant and susceptible RILs and their parents,that is,the expression level of these four genes in resistant and susceptible samples were consistent with the corresponding resistance level,so these four genes were determined to midge resistance-related genes.4.Sequence analysis was performed for eight genes which amplified with targeting size as expected in the parents.The sequencing results showed that the sequences of seven genes in two parents were consistent with the reference sequence,and SNPs did exist with non-synonymous mutations within them,of which two genes had insertion-deletion(Indel).After that,eight chromosome specific SNPs and two Indels could be used to develop newly molecular markers for the midge resistance.5.Based on the sequence variation(SNPs and Indels)in differential genes obtained in present study,two EST,one tetra-primer ARMS PCR and eight KASP markers were developed.The results showed that the detection rates of these markers in RIL lines were about 90%,of which the detection rates of markers E1-2,T3-1-1,K3-1-1,K3-7-1,K3-7-3,K3-16-1,K10-10-6 and K10-10-13 x in resistant wheat cultivars were 30.77% to 62.5%,and that in susceptible wheat cultivars were 76.92% to 92.31%.Among these markers,K3-1-1,K3-7-1 and K3-7-3 had the highest detection rates(92.31%)in susceptible cultivars,so they could be used for the screening of midge-resistant wheat germplasm.Based on the genotyping of eleven markers,it was found that thirteen midge-resistant wheat cultivars had more resistant loci,and hadn't susceptible loci,which could be used as midge-resistant gene donors for midge-resistant MAS breeding.Further analysis showed that,out of the thirteen midge-resistant wheat cultivars with all resistance marker alleles,most were old wheat cultivars,which were rarely used in present breeding programs,as highlighted the importance of wheat germplasm enhancement on midge-resistance.In conclusion,this study revealed 17 OWBM-resistant genes through the transcriptomic analysis of all wheat samples,four of which were confirmed to be related with OWBM resistance by qRT-PCR analysis and gene sequence analysis.According to the specificity SNP and Indel of the gene sequence,eleven markers were successfully developed,of which eight markers could effectively detect OWBM resistance for wheat varieties.