Omics Studies And Fine Mapping Of The Major Effect QTL ARN6.1 For Adventitious Root Formation Under Waterlogging Stress In Cucumber

Cucumber(Cucumis sativus L.,2n=2x=14)is a climbing annual herbaceous plant in the Cucumis family,Cucurbitaceae.It is one of the most important vegetable crops for both open-field and greenhouse cultivation in China.As a consequence of global warming and frequent rainfall events,waterlogging has become one of the most critical limits for crop growth and production.Cucumber is generally considered as waterlogging sensitive,and is easily affected by heavy rain and subsequent periods of soil flooding,due to the shallow root system and strict oxygen requirement.Thus,ascertain the genetic architecture,breeding of new varieties with better adaptation to waterlogging stress is urgently required,and is one of the main objectives of plant breeders.Breeding for waterlogging tolerance is difficult as it is a complex quantitative trait,and the evaluation of tolerance can be easily influenced by environmental conditions.In our screening of germplasm collection,we identified a cucumber landrace,’Zaoer-N’ that exhibited high level of waterlogging resistance.Under waterlogging stress,Zaoer-N(South China type)was able to produce larger number of adventitious roots(ARs)on its hypocotyl,but almost no ARs were formed in the waterlogging-sensitive control line Pepino(American type).AR formation at the soil surface is considered a key step in the adaptation to waterlogging because ARs can obtain oxygen directly from the air.To understand the molecular mechanisms underlying AR emergence,we compared the response of two contrasting cucumber genotypes,’Zaoer-N’ and ’Pepino’ that differing in their ability of AR formation after 48 h of waterlogging through comparative RNA-seq based transcriptomic profiling and iTRAQ-based quantitative proteomics approach.We also investigated the inheritance of AR numbers(ARN)in six generations and performed a three-season QTL study for ARN in F2:3 families derived from a cross between Zaoer-N and Pepino.The main results are listed as follows:1,More than 27,000 transcripts were examined in cucumber hypocotyls,of which 1,494 and 1,766 genes in Zaoer-N and Pepino,respectively,were differentially expressed(FDR≤0.05,Fold changes≥2)two days after waterlogging.Comparative analysis revealed that genes functioning in carbohydrate mobilization,nitrate assimilation,hormone production and signaling pathways,transcription factors,cell division etc.might contribute to the waterlogging-triggered AR primordia initiation.2,Through iTRAQ-based quantitative proteomics approach,we identified 5,508 proteins and 146 were differentially regulated proteins(DRPs,FDR<0.05,Fold changes≥2),of which 47 and 56 DRPs were specific to tolerant and sensitive line,respectively.43 DRPs were share between Zaoer-N and Pepino,which manily involved in oxidoreductase activity,response to stress and heme binding.The 47 DRPs that specially regulated in Zaoer-N mainly fountioned in response to oxidative stress,response to chemical stimulus,peroxidase activity and antioxidant activity.On the contrary,protein functions related to carbohydrate metabolic process,hydrolase activity and cofactor binding were specially regulated in Pepino.The Pearson’s correlation coefficients(PCC values)of the DRPs and mRNA pairs were 0.26 in Zaoer-N,while PCC values were 0.18 for groups in Pepino,respectively.As expected,significant correlations existed in both of the two groups at the 0.05 level(p≤0.05,2-tailed).3,The mixed major gene plus polygene inheritance model was used to analyze waterlogging tolerance in the six generations of cucumber.The results showed that the genetic model D-4,mixed one negative dominance major-gene and additive-dominance polygenes,is the best-fitting genetic model for waterlogging triggered ARN phenotype.The additive effects of the major gene and polygene and the dominant effects of the polygene were positive rather than negative values,indicating that the inheritance of ARN is mainly controlled by the additive effects of the major gene and additive-dominant effect of the polygene.4,A genetic linkage map spanning 550.8 cM and consisting of 149 simple sequence repeat(SSR)markers segregating into seven linkage groups was constructed.Three QTLs(ARN3.1,ARN5.1 and ARN6.1)distributed on chromosomes 3,5 and 6 were identified by composite interval mapping(CIM).The major-effect QTL,ARN6.1,located between SSR12898 and SSR04751,was the only locus detected in three seasons,with least likelihood(LOD)scores of 8.8,10.4 and 9.5 and account for 17.6%,24%and 19.8%of the phenotypic variance,respectively.5,Two DNA pools,high ARN pool and low ARN pool were constructed,respectively,by mixing equal amount of DNA from 50 high ARN(ARN>40)and low ARN(<5)F2 plants from the 2014 Fall waterlogging experiment with 949 F2 individuals.Genomic DNAs of the two parental lines(Zaoer-N and Pepino)and the two bulks were used for Specific-Locus Amplified Fragment(SLAF)library construction and sequencing following our established protocol.After calculation of △(SNP-index)and △Euclidean distance value of the two pools,we narrowed the ARN6.1 to a 301-kb interval which is located within the 0.79 Mb major-effect QTL region identified by CIM.To narrow down the position of ARN6.1,2,274 F2 plants were genotyped with two KASP markers,KASP1 and KASP13,which were derived from two SNPs,SLAF 192310 and SLAF 192096 flanking the ARN6.1,respectively,from which 33 recombinant plants were obtained.These recombinant plants were further genotyped with 11 additional KASP markers(KASP2 to KASP 12).Based on the genotypic data of the 13 markers in the 33 recombinant plants,10 haplotypes could be recognized.In consideration of the ARN of these recombinant plants,it could be concluded that ARN6.1 must reside in the 61.5-kb interval defined by KASP10(25,477,136)and KASP11(25,538,672).Annotation of this 61.5-kb genomic region using FGENESH program identified 12 predicted genes.Transcripts of all 12 genes in the 61.5-kb candidate gene region were present in the transcriptomes;however,only Csa6G504460 was significantly upregulated(by 1.5-fold)in waterlogged Zaoer-N than in the non-waterlogged control,whereas no significant difference of its expression was found in Pepino.To validate the result,we examined the expression dynamics of Csa6G504460 with qPCR in Zaoer-N and Pepino using hypocotyl samples collected at 0,12,24,36,48,60 and 72 h after waterlogging.In Pepino,no significant difference was found in expression of this gene across multiple time pints in both waterlogged stressed and control plants.In contrast,the relative expression of Csa6G504460 was significantly higher in waterlogged hypocotyl of Zaoer-N than that in the control at 24,36 and 48 h after treatment;the peak expression occurred at 36 h after waterlogging.Thus,gene expression data suggested that Csa6G504460 is a possible candidate for the ARN6.1 locus.6,Csa6G504460 was predicted to encode an AAA-ATPase(ATPase associated with various cellular activities)domain-containing protein of～59 kDa(pI:5.61)with 511 amino acid residues with the AAA domain at positions 214-376 and a coiled-coil domain from residues 432 to 480 at the C-terminal region.Alignment of the cDNA sequences of this gene between the two lines identified three SNPs in the exon regions;however,two of them were silent mutations(CGA1791/Arg to CGT1791/Arg,CGT1797/Arg to CGA1797/Arg),and the third was a non-synonymous mutation(GA1519T/Asp in Zaoer-N to GG1519 T/Gly in Pepino).The non-synonymous SNP allele between Zaoer-N and Pepino was located in the coiled-coil domain.We investigated allelic variations among 30 re-sequenced cucumber inbred lines including 10 with high ARNs(>28.3)and 20 low ones(<4.8)under waterlogging stress.Among all SNPs detected,only one showed non-synonymous mutation between Zaoer-N and Pepino with the two alleles being consistent with ARN,which suggested this SNP may be the causal nucleotide variation.We investigated subcellular localization of the Csa6G504460 protein.A construct,p35S-Csa6G504460-GFP expressing the Csa6G504460-GFP fusion protein was developed.From the transient expression assay in Nicotiana benthamiana,Csa6G504460-GFP fluorescence was observed in the cytoplasm.Interestingly,Csa6G504460-GFP fusion proteins were also observed in endoplasmic reticulum(ER)-like structures in the tobacco mesophyll cells.Prokaryotic expression of ARN6.1 candidate gene and ATPase activity assay showed that Csa6G504460Asp protein exhibited a strong and dose-dependent ATPase activity,but the mutated protein Csa6G504460Gly nearly completely lost its ATPase activity.Further studies confirmed that Csa6G504460 regulates the ARN phenotype via its ATPase activity.Ectopic expression of 35S::Csa6G504460Asp in Arabidopsis resulted in better rooting ability and lateral root development in the transgenic plants.These findings confirmed the correlation of Csa6G504460 expression with root formation.