Fine Mapping Of QBlsr5a, A QTL For Resistance To Bacterial Leaf Streak And Expression Analysis Of Candidate Genes In Rice

Bacterial leaf streak(BLS),caused by pathogen Xanthomonas campestris pv.Oryzicola,is a major rice disease subject to quarantine regulations in China.BLS can cause 10%-20%loss of grain yield in general and may even result in up to 40%yield loss when serious.Now the disease is widely distributed in the rice production area of southern China.Compared with other two major rice diseases,bacterial blight and blast,rice resistance to BLS is a typical quantitative trait.No major resistance genes against BLS have been found in rice. Consequently,identifying the quantitative resistance loci(QRL)conferring the resistance to BLS in rice is very important for the breeding of resistant rice varieties and for the understanding of the mechanism of quantitative resistance.So far,13 QTLs conferring the resistance to BLS have been mapped in rice.Among them, 11 QTLs were mapped by Tang et al.using a recombinant inbred line population derived from a cross between a highly-resistant variety Acc8558 and a highly-susceptive variety H359 and the QTL qBlsr5a(the resistant allele was from the resistant parent Acc8558)on the short arm of chromosome 5 showed the largest contribution to phenotypic variation.Subsequent studies confirmed the existence of qBlsr5a.These results suggested that qBlsr5a would be useful for rice breeding.In this study,qBlsr5a was further investigated based on the results of previous studies.With Acc8558 as the donor and H359 as the recipient,an near-isogenic line H359-BLSR5a was created by backcross breeding and marker-assisted selection,in which only the QTL qBlsr5a was introgressed from the donor parent.A large F₂ population was constructed by hybridizing the near-isogenic line with H359.By selecting individuals with extreme phenotypes and examining their progeny(F_2:3)lines,individuals with the resistant homozygous genotype at the target QTL in the F₂ population were identified.By genotyping these individuals with SSR markers and performing linkage analysis,qBlsr5a was mapped to an interval between SSR markers RM153 and RM159,covering a range of 2.4 cM or 290 kb.To identify BLS-resistant genes in rice,high-throughput detection of genes responding to the infection of BLS pathogen in Acc8558 and H359 was performed using Affymetrix microrarray.A total of 956 differentially expressed genes(DEGs)were identified in the two varieties,of which 12 genes were consistently up-regulated and 54 consistently down-regulated in the two varieties,while 20 genes showed opposite expression change directions in the two varieties.Gene ontologies,pathways and promoters of the DEGs were analyzed.Comparative analysis showed that 30 DEGs matched the positions of 13 QTLs conferring resistance to BLS reported before.But only one DEG(LOC_Os05g01330)encoding an unknown protein was found in the interval between SSR markers RM153 and RM159.By searching Gramene and rice TIGR databases,we found that 51 putative genes were annotated in the 290 kb region where qBlsr5a was located.Thirty-three(64.7%)of them encode proteins with known functions and the remaining eighteen genes encode either unknown or hypothetical proteins.According to the annotation information,nineteen genes (including the DEG identified by the microarray analysis in this region)were selected as candidate genes of qBlsr5a for expression analysis.Among them,three genes are predicted to encode putative polygalacturonase inhibitor precursor protein(PGIP);one encodes putative ankyrin-1 protein with ANK repeats structure;one encodes transcription initiation factorâ…¡A, gamma subunit(TFâ…¡A_γ),one encodes ZIK1 protein(zinc finger protein interacting with K protein 1)and two encode putative nucleic acid binding protein and putative chromosome condensation factor,respectively,both of which contain putative zinc finger structure.Using H359 and H359-Blsr5a as materials,the expression of the nineteen candidate genes were analyzed with semi-quantitative RT-PCR.Results indicated that:1)four genes did not express in the two rice accessions;2)seven genes showed expression in the two rice accessions but did not respond to the infection of BLS pathogen;3)eight genes exhibited differential expression due to the inoculation of BLS pathogen,of which seven genes were upregulated and one was downregulated.However,only two of these genes showed difference between H359 and H359-BLSR5a.The most interesting gene is LOC_Os05g01444,which was induced to express by inoculation in H359-BLSR5a but not in H359.This gene is 1,206 bp long without intron, encoding a putative inhibitor protein PGIP,which is known to play important roles in disease resistance in plants.Therefore,LOC_Os05g01444 should be an important candidate gene of qBlsr5a.