Comparative Genetic Mapping In Genus Cucumis And Developing NBS Resistance Markers In Cucumber And Pepper

Cucumber and melon are two important vegetable species in the genus Cucumis (familyCucurbitaceae). Comparative genetic mapping not only help us understand the evolution andphylogenetic relationships of plants in genus Cucumis, but also help us develop many geneticmarkers, predict and map-based clone gene. Most plant resistance genes are belonged toNBS-LRR. Identifying and developing NBS molecular markers play an important role inmolecular assisted selection.1In total,2,487SSR markers were used to screen for polymorphisms between the twoparental lines, Q3-2-2and Top Mark of the F2mapping population. Of the2,442cucumberSSRs,196were polymorphic between Top Mark and Q3-2-2. In total,154markers werefinally placed on the F2genetic map (145from cucumber,2from watermelon and7fromother species). Using this F2mapping population, Cuevas mapped169co-dominant markers.Genotypic data from the154additional cucumber or watermelon markers developed hereinwere combined with the previously mapped169markers for linkage analysis. All323codominant markers were placed onto this melon genetic map in13LG. The F2-based mapconsisted of13LG spanning1,012cM, with a mean marker interval of3.3cM.22448SSR markers were used to screen for polymorphisms between the two parentallines, Top Mark and WI846-1of the RIL mapping population. The SSRs detectedpolymorphisms between Top Mark and WI846-1was127. Of the127polymorphic markersidentified herein (i.e.,120from cucumber,6from melon or Arabidopsis, and1fromwatermelon),89were placed on the RIL map. Using the same melon RIL population, Cuevasmapped256marker loci. The two sets of marker data were pooled for linkage analysis using80RILs. The resulting linkage map possessed335marker loci including203codominant(SSR, CAPS, and SNP) and132dominant markers (103RAPDs and29AFLPs) in22LGwith cumulative genetic distances of879.1cM.3The F2and RIL maps developed herein were merged to produce a consensus map usingthe JoinMap3.0program. The132dominant AFLP and RAPD markers resident on the RILmap were excluded from map integration because they were deemed ineffective for definingcucumber-melon synteny. The consensus melon genetic map consisted of401co-dominantmarker loci (199from cucumber,202from melon)positioned in12LGs spanning1,029.0cMwith an average marker interval of2.6cM.4. The cucumber Gy14and9930draft genome scaffold locations of the markers onmelon genetic map were predicted by in silico PCR or BLAST searches of the cucumber draft genome sequences. The locations of these cucumber scaffolds in the seven chromosomes ofcucumber were deduced from published cucumber genetic maps. Thus, we could analysis thesyntenic relationship between melon and cucumber. It was determined that cucumberChromosome7was syntenic to melon Chromosome I. Cucumber Chromosomes2and6eachcontained genomic regions that were syntenic with melon chromosomes III+V+XI andIII+VIII+XI, respectively. Likewise, cucumber Chromosomes1,3,4, and5each was syntenicwith genomic regions of two melon chromosomes previously designated as II+XII, IV+VI,VII+VIII, and IX+X, respectively. However, the marker orders in several syntenic blocks onthese consensus linkage maps were not co-linear suggesting that more complicated structuralchanges beyond simple chromosome fusion events have occurred during the evolution ofcucumber.5. To verify the syntenic relationships from comparative mapping conducted herein, asimilar strategy was used with molecular markers from a melon linkage map developed byDeleu et al. The genomic or EST sequences from which these markers were developed wereused in BLAST analysis against the cucumber draft genome assemblies. Of the414markers,only3(0.7%) did not yield either in silico PCR products or BLAST hits in the cucumber draftgenomes examined. Melon-cucumber syntenic relationships revealed from the present studywere confirmed by this independent study.6. We found the number of NBS-encoding R genes from the cucumber inbred lineGy14was70using genome-wide software, most of which were cluster located on thechromosome. Phylogenetic analysis of the58cucumber NBS-encoding R genes that hadcomplete NBS domain showed that R genes were divided into two subfamilies, CC-NBS andTIR-NBS.The CC-NBS and TIR-NBS subfamilies in cucumber consist of34and24members, respectively. According to the genome sequence of cucumber inbred lines Gy14and9930, We developed SSR, Indel and SNPs markers within RGAs or just flanking them.Among all developed markers,48of them were finally mapped on the cucumber genetic map.67RGAs were mapped on the Gy14×9930map by comparing the physical position betweenRGAs markers and RGAs. Most of the RGAs were mapped on the genetic map within1cM.7. According to the NBS domain of potato phytophthora resistance gene Rpi-blb1, wedesigned specific primers and transferred it to a CAPs marker.12pepper inbred line weretested with this primers, all the inbred lines had a about590bp PCR production. BcgI wasused to digest the PCR fragments amplified from the primer pair among12pepper genotypes,of which resistance genotypes had330bp and230bp two bands, suspect gynotypes only hada590bp band. The results of disease resistance identification using root-irrigating methodshowed that the molecular marker related to Phytophthora capsici resistance was consistentto disease resistance identification and was suitable for molecular assisted selection inPhytophthora capsici resistance breeding.