Fine Mapping And Cloning Of The Sm Gene Conferring Resistance To Gray Leaf Spot In Tomato

Tomato gray leaf spot,caused by Stemphylium,is a foliar disease in warm regions worldwide and made severe yield losses in tomato（Solanum lycopersicum L.）.Breeding resistant cultivars in marker-assisted selection（MAS）is the most essential method to control the disease.Resistance against gray leaf spot has been identified in the wild species S.pimpinellifolium and conferred by a single incompletely dominant gene,Sm,which was located on chromosome 11 between RFLP markers TG110 and T10 at distances of 4.1 cM and 6.8 cM from the markers,respectively.In this study,the genome-wide association study and a map-based cloning approach were used for fine mapping the Sm gene.The results in this study will facilitate marker assisted selection in tomato breeding for gray leaf spot resistance and be beneficial to elucidate mechanisms underlying the resistance.The main contents are as follows:1.Two hundred and eighty-nine diverse accessions were analyzed using a genome-wide association study which a genome resequencing data set was available.A strong association signal,SNP:SL2.50ch11₉317565,P=1.12×10-20,was identified for tomato gray leaf spot resistance.2.An F2 segregating population derived from the susceptible line Heinz 1706 and the resistant line 9706 was used for fine mapping of tomato gray leaf spot resistance gene Sm.The disease evaluation of 441 F2 individuals showed the segregation between resistant（R）and susceptible（S）plants was in agreement with a 3:1 segregating ratio（x23:1=1.03 P=0.31）,which suggesting the presence of a single dominant resistance gene.Combination with the genotypes of F2 recombinants individuals and phenotypes of F2:3 families,Sm gene was mapped between makers InDe1343 and InDel-FT-32 on chromosome 11.3.Based on Heinz 1706 genome sequence,the physical distance between the two flanking markers,InDe1343 and InDel-FT-32,was approximately 185 kb,which contained 5 genes.However,none of them encoded a typical disease resistance protein.Additional,these annotated genes expression levels were not significantly different in the two parents across all of the time points before and after inoculation.The 185-kb genomic DNA region in the Heinz 1706 draft genome was manually annotated by the FGENESH program and thirty-three genes were predicted.Primers were designed based on predicted genes sequences and twenty-five PCR products were assembled into the tobacco rattle virus-based virus-induced gene silencing（VIGS）vector.Agroinfiltration were used to deliver VIGS vectors into the Sm-resistant line 9706 with sprout vacuum-infiltration（SVI）method.Ultimately,the ORF9-silenced plants showed susceptible after inoculation S.solani.This result indicated ORF9 was involved in Sm mediated resistance and it seems to be good candidate of the Sm locus.4.The bacterial artificial chromosome（BAC）library constructed with the Ty2 donor 12g-60 that resistant to S.solani was screened,from which a single clone B80B15 covering the ORF9 region was identified.The full length of this clone was sequenced and predicted.The candidate gene consists of 14 exons of 5256 nucleotides,encoding a predicted polypeptide of 1751 amino acids.5.In addition,a co-dominant marker Sm-InDel was developed that produced a 122 bp or a 140 bp fragment for resistant or susceptible alleles,respectively.It can detect gray leaf spot resistance in 289 germplasm materials with 96.5%accuracy.It is expected to be used in MAS for gray leaf spot resistance...