| Cucurbitaceae crops are seriously damaged by the southern root-knot nematode(Meloidogyne incognita).However,as important vegetable,especially cucumber and melon,no resistant materials have been found.This is a big bottleneck for resistance breeding in cucumber and melon.Horned melon(Cucumis metuliferus)is an important cucurbit wild resistance resource.However,due to the reproductive isolation between horned melon and other cucurbit crops,its resistance has not been applied in production.Therefore,in this study,the whole genome of C.metuliferus(CM27)was sequenced;CG-MS was used to identify the volatile substances in the root of CM3 and cucumber(XTMC),The chemotaxis and nematicidal activities of three volatile compounds were tested;one F2 population constructed by crossing CM3(resistant to southern root-knot nematode)and CM27(Susceptible to southern root-knot nematode)was usedfor BSA-Seq.The main findings of this study are as follows:(1)Using PacBio SMART platform and Hi-C to obtain a high-quality chromosome-level of C.metuliferus(CM27)genome,and to perfrom genome annotation.The genome assembly of CM27 obtained 432 contigs,with a total size of 329 Mb.The contig N50 size was 2.9 Mb.After Hi-C assembly and manual adjustment,the CM27 genome was further anchored on 12 chromosomes.Through genome annotation,29,214 protein-coding genes were predicted in the CM27 genome,with a total length of 100.68 Mb.The complete BUSCO score of 93.54% was identified,suggesting that the high-quality genome of CM27 genome was obtained.(2)Whole-genome comparison between wild material C.metuliferus(CM27)and cultivated species(cucumber and melon)found that C.metuliferus genome contains more NBS-LRR genes.Based on the comparative genomic results,we focused on analyzing the evolutionary relationship of NBS-LRR genes among cucurbit crops,and identified 102 NBS-LRR-like coding genes in C.metuliferus(CM27).A total of 66NBS-LRR genes were detected in the melon(IVF77)genome and 65 NBS-LRR genes in the cucumber genome.The number of NBS-LRR genes between C.metuliferus(CM27)and IVF77/cucumber was significantly different(P < 0.01).Comparative genomics analysis showed that large indels were the main reason for the difference in the number of NBS-LRR genes between C.metuliferus and melon.Indels may also contribute to the difference in the number of NBS-LRR genes between C.metuliferus and cucumber.In addition,pseudogeneization may also be another reason for the loss of the NBS-LRR gene in cultivars(cucumber and melon).(3)Based on the phenotype of CM3 and cucumber(XTMC,as a control),we used CG-MS technology and explained why nematodes don’t like to attack horned melon roots.The results of the behavioral selection test of nematodes on the root tips and root systems of the two materials showed that a small number of nematodes gathered around the root tips of CM3.The two tests indicated root volatiles were one of the reasons for the resistance of C.metuliferus.CG-MS was used to analyze the volatile substances in the root system of CM3 and XTMC.After comparison,it was confirmed that there are18 unique volatile compounds in CM3.Three of these were diluted to four concentrations and assayed for chemotaxis and nematicidal activity.The results showed that all tested concentrations of(methoxymethyl)-benzene strongly repelled nematodes,while cresol and(Z)-2-penten-1-ol attracted nematodes at certain concentrations.While(methoxymethyl)-benzene could not significantly kill the nematodes at all the tested concentrations,the other two tested compounds showed a significant increase in nematicidal activity with increasing concentrations.In order to further confirm whether the natural volatile in CM3 root can help cucumbers resist the infestation of root knot nematodes or not.We used a sealed double-pot cultivation device.Compared with the two control groups,the number of galls in the experimental group was significantly reduced.This shows that the natural volatile substances of horned melon have the potential to help the cucumber to resist nematodes.(4)F2 population was constructed by using CM27(as susceptible parent)and CM3(C.metuliferus,as resistant parent).From the results of genetic analysis of the F2 population,the resistance of CM3 was controlled by a single dominant gene.Variation detection among the four sequencing pools found that a total of 213,982 SNPs were obtained between the parents,including 5,248 SNPs with non-synonymous mutations;a total of 20,384 SNPs were obtained between the resistance pool and the susceptibility pool,and 714 SNPs caused non-synonymous mutations.There were 49,296 Small InDels between the parents,and 21,220 Small InDels were detected between the resistance pool and the susceptibility pool.Combining the SNP and InDel associated regions,5 candidate regions related to the trait were obtained after taking the intersection,with 0.54 Mb length.98 genes were annotated in the associated region,including 25 non-synonymous mutations and 0 frameshift mutation.Combined with the BSA mapping results and the transcriptome results of CM3,76 of the 98 genes in the candidate regions were expressed in CM3 roots.The expression trend results showed that 16 genes were up-regulated at one or two consecutive time points after inoculation with nematodes.The gene annotation results show that the candidate genes EVM0021559 and EVM0024197 are SNAP25 homologous protein SNAP30-like proteins,which are homologous to the cloned soybean anti-cyst nematode Rhg1 siteα-SNAP gene;another candidate gene EVM0006692 encodes LRR receptors.serine/threonine-like protein kinase,is an immunoreceptor kinase gene.To sum up,in order to analyze the resistance mechanism of C.metuliferus to M.incognita,a high-quality reference genome(CM27),GC-MS analysis and BSA-Seq and were used to analyze in various aspects.In this study,a high-quality chromosome-level C.metuliferus(CM27)genome was successfully obtained,and the unique root volatile substances of CM3 were identified.Finally,the resistance of CM3 was successfully located in the region of 0.54 Mb.These results lay the foundation for further analysis of the genetic mechanism of resistance to M.incognita in C.metuliferus. |
