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An unusual flower and tendril (uft) mutant in cucumber was caused by a mutation in Csa1G056950 encoding an F-box protein. Flowers and tendrils are important agronomic and yield traits of cucumber (Cucumis sativus L.). In this study, we identified an unusual flower and tendril (uft) mutant from an ethyl methanesulfonate (EMS) mutagenesis population. Genetic analysis revealed that the phenotype of the uft mutant was regulated by a single recessive nuclear gene. Map-based cloning and MutMap+ results demonstrated that Csa1G056950 (CsUFO), encoding an F-box protein, was the causal gene for the uft mutant phenotype of cucumber. A single nucleotide polymorphism (SNP) mutation (C to T) in the second exon of CsUFO resulted in premature translation termination. The expression level of CsUFO was significantly decreased in apical buds of the uft mutant compared with the wild-type (WT) WD1. Transcriptome analysis indicated that many genes for organ development were down-regulated in uft plants, suggesting CsUFO-associated networks that regulate flower and tendril development. These findings provide a new insight into understanding the molecular mechanisms of flower organogenesis in cucumber.

CsSh5.1, which controls hypocotyl elongation under high temperature conditions in cucumber, was mapped to a 57.1 kb region on chromosome 5 containing a candidate gene encoding a xyloglucan galactosyltransferase. Hypocotyl growth is a vital process in seedling establishment. Hypocotyl elongation after germination relies more on longitudinal cell elongation than cell division. Cell elongation is largely determined by the extensibility of the cell wall. Here, we identified a spontaneous mutant in cucumber (Cucumis sativus L.), sh5.1, which exhibits a temperature-insensitive short hypocotyl phenotype. Genetic analysis showed that the phenotype of sh5.1 was controlled by a recessive nuclear gene. CsSh5.1 was mapped to a 57.1 kb interval on chromosome 5, containing eight predicted genes. Sequencing analysis revealed that the Csa5G171710 is the candidate gene of CsSh5.1, which was further confirmed via co-segregation analysis and genomic DNA sequencing in natural cucumber variations. The result indicated that hypocotyl elongation might be controlled by this gene. CsSh5.1 encodes a xyloglucan galactosyltransferase that specifically adds galactose to xyloglucan and forms galactosylated xyloglucans, which determine the strength and extensibility of the cell walls. CsSh5.1 expression in wild-type (WT) hypocotyl was significantly higher than that in sh5.1 hypocotyl under high temperature, suggesting its important role in hypocotyl cell elongation under high temperature. The identification of CsSh5.1 is helpful for elucidating the function of xyloglucan galactosyltransferase in cell wall expansion and understanding the mechanism of hypocotyl elongation in cucumber.

Map-based cloning identified a candidate gene for resistance to the anthracnose fungal pathogen Colletotrichum orbiculare in cucumber, which reveals a novel function for the highly conserved STAYGREEN family genes for host disease resistance in plants. Colletotrichum orbiculare is a hemibiotrophic fungal pathogen that causes anthracnose disease in cucumber and other cucurbit crops. No host resistance genes against the anthracnose pathogens have been cloned in crop plants. Here, we reported fine mapping and cloning of a resistance gene to the race 1 anthracnose pathogen in cucumber inbred lines Gy14 and WI 2757. Phenotypic and QTL analysis in multiple populations revealed that a single recessive gene, cla, was underlying anthracnose resistance in both lines, but WI2757 carried an additional minor-effect QTL. Fine mapping using 150 Gy14 × 9930 recombinant inbred lines and 1043 F2 individuals delimited the cla locus into a 32 kb region in cucumber Chromosome 5 with three predicted genes. Multiple lines of evidence suggested that the cucumber STAYGREEN (CsSGR) gene is a candidate for the anthracnose resistance locus. A single nucleotide mutation in the third exon of CsSGR resulted in the substitution of Glutamine in 9930 to Arginine in Gy14 in CsSGR protein which seems responsible for the differential anthracnose inoculation responses between Gy14 and 9930. Quantitative real-time PCR analysis indicated that CsSGR was significantly upregulated upon anthracnose pathogen inoculation in the susceptible 9930, while its expression was much lower in the resistant Gy14. Investigation of allelic diversities in natural cucumber populations revealed that the resistance allele in almost all improved cultivars or breeding lines of the U.S. origin was derived from PI 197087. This work reveals an unknown function for the highly conserved STAYGREEN (SGR) family genes for host disease resistance in plants.

Using map-based cloning of ts gene, we identified a new sort of gene involved in the initiation of multicellular tender spine in cucumber. The cucumber (Cucumis sativus L.) fruit contains spines on the surface, which is an extremely valuable quality trait affecting the selection of customers. In this study, we elaborated cucumber line NC072 with wild type (WT) hard fruit spines and its spontaneous mutant NC073, possessing tender and soft spines on fruits. The mutant trait was named as tender spines (ts), which is controlled by a single recessive nuclear gene. We identified the gene ts by map-based cloning with an F2 segregating population of 721 individuals generated from NC073 and WT line SA419-2. It was located between two markers Indel6239679 and Indel6349344, 109.7 kb physical distance on chromosome 1 containing fifteen putative genes. With sequencing and quantitative reverse transcription-polymerase chain reaction analysis, the Csa1G056960 gene was considered as the most possible candidate gene of ts. In the mutant, Csa1G056960 has a nucleotide change in the 5′ splicing site of the second intron, which causes different splicing to delete the second exon, resulting in a N-terminal deletion in the predicted amino acid sequence. The gene encodes a C-type lectin receptor-like tyrosine-protein kinase which would play an important role in the formation of cucumber fruit. This is firstly reported of a receptor kinase gene regulating the development of multicellular spines/trichomes in plants. The ts allele could accelerate the molecular breeding of cucumber soft spines.

Fruit spine size is one of the importantly external quality traits effected the economic value of cucumber fruit. Morphological–cytological observation of the fruit spine size phenotype indicated that large spine formation arises from an increasing of spiny pedestal cell number caused by cell division, and best periods to accurately score fruit spine size trait was 4th day before flowering to 7th day after flowering according the continuous observation. Genetic analysis showed that a single dominant gene determined the fruit spine size trait in cucumber. BC1 population (189 individuals) of two inbred lines (large spine PI197088 and small spine SA0422) was used for primary mapping of the SS/ss locus with 7 markers covering an interval of 37.1 cM. An F2 segregating population of 1032 individuals constructed from the same two parents (PI197088 and SA0422) was used to fine mapping of the SS/ss locus. Six new markers linked to the gene were successfully screened for construction of a fine linkage map, in which the SS/ss locus was located in the region flanked by marker SE1 (3 recombinants) and SSR43 (2 recombinants) with a 189 kb physical distance. Markers from this study will be valuable for candidate gene cloning and marker-assisted selection for cucumber breeding.

Mottled/uniform color at the flower end of immature fruit is a highly important external quality trait that affects the market value of cucumber. Genetic analysis of different F2 and backcross populations revealed that one single recessive gene, u (uniform immature fruit color), determines the uniform immature fruit color trait in cucumber. Based on earlier studies, the u locus is located on chromosome 5 (Chr. 5). By combining bulked segregant analysis using 60 published molecular markers on Chr. 5, we found that eight markers are polymorphic and are linked to the u locus. In addition, we developed five new relevant polymorphic simple sequence repeat (SSR) markers between markers SSR16203 and SSR15818. Subsequently, the F2 population (477 individuals) from the cross of S06 (uniform fruit color line) × S94 (mottled fruit color line) was used for fine mapping of the u gene. The u gene was mapped to a 313.2-kb region between markers SSR10 and SSR27, at a genetic distance of 0.8 and 0.5 cM, respectively. Moreover, validity analysis of the codominant markers SSR10 and SSR27 was performed using 50 lines with mottled/uniform fruit color, demonstrating that these two SSR markers can be used for marker-assisted selection of the mottled/uniform fruit color trait in cucumber breeding. The results of this study will facilitate the cloning of the u gene.

Dull/glossy fruit skin is a highly valuable external quality trait that affects the market value of cucumbers. In this study, genetic analysis showed that one single dominant gene, D (dull fruit skin), determines the dull fruit skin trait in cucumber. By combining bulked segregant analysis with 11 published polymorphic molecular markers on chromosome 5, the D/d gene was preliminarily mapped between markers SCZ69 and SSR16203, at genetic distances of 0.3 and 0.6 cM, respectively. Subsequently, a larger F2 (S06 × S94) population (842 individuals in total) was used for high-resolution mapping of the D/d gene. Finally, the D/d gene was fine-mapped between markers SSR37 and SSR112, at a physical distance of 244.9 kb (containing 31 candidate genes), using eight newly developed polymorphic simple sequence repeat (SSR) markers between SCZ69 and SSR16203. Based on semi-quantitative RT-PCR analysis, the possible candidate gene D was identified as Csa016880 or Csa016887. Meanwhile, validity analysis of the markers SSR37 and SSR112 was performed with 72 dull/glossy fruit lines, and showed that the two co-dominant SSR markers could be used for marker-assisted selection of the dull/glossy fruit trait in cucumber breeding. Moreover, this study will be helpful for cloning of the D gene in cucumber.

The high-density consensus map was constructed based on the GY14 × PI 183967 map from an inter-subspecific cross and the extended S94 × S06 map from an intra-subspecific cross. The consensus map was composed of 1,369 loci, including 1,152 SSR loci, 192 SRAP loci, 21 SCAR loci and one STS locus as well as three gene loci of fruit external quality traits in seven chromosomes, and spanned 700.5 cM, of which 682.7 cM (97.5%) were covered by SSR markers. The average genetic distance and physical interval between loci were 0.51 cM and ~268 kbp, respectively. Additionally, the physical position of the sequence-associated markers aligned along the assembled cucumber genome sequence established a relationship between genetic maps and cucumber genome sequence and to a great extent validated the order of markers in individual maps and consensus map. This consensus map with a high marker density and well-ordered markers is a saturated and reliable linkage map for genetic analysis of cucumber or the Cucurbitaceae family of plants.

To elucidate the genetic and molecular mechanisms of plant architecture, a Cucumber Lateral Suppressor gene (CLS) for the lateral branching development in cucumber branching line S06 was cloned and characterized. Full-length cDNA of CLS (EU503217) was isolated by performing rapid amplification of cDNA ends polymerase chain reaction. Phylogenetic analysis showed that the gene CLS is closely related to Lateral Suppressor that encodes transcriptional regulator proteins belonging to GRAS family of known transcription factor. Southern blot analysis revealed that CLS gene has one copy in cucumber genome. The expression patterns revealed that transcripts were detected in all organs, but not in internodes. In addition, the transcript level in male flowers is higher than the female flowers and fruit. Introduction of the Pro35s:CLS overexpression vector into Arabidopsis las plants led to a full complementation of the mutant phenotypes. Moreover, RNA in situ hybridization showed that the pattern of CLS transcript was accumulated in the leaf axils in different developmental stage, which was similar to LAS in Arabidopsis. These results together indicate that CLS may play an important role in cucumber axillary meristem initiation and thus the lateral branch formation.

The cucumber lines, S94 (Northern China open-field type, powdery mildew (PM) susceptible) and S06 (European greenhouse type, PM resistant), and their F(6:7) populations were used to investigate PM resistance under seedling spray inoculation in 2005/Autumn and 2006/Spring. QTL analysis was undertaken based on a constructed molecular linkage map of the corresponding F(6) population using composite interval mapping. A total of four QTLs (pm1.1, pm2.1, pm4.1 and pm6.1) for PM resistance were identified and located on LG 1, 2, 4 and 6, respectively, explaining 5.2%-21.0% of the phenotypic variation. Three consistent QTLs (pm1.1, pm2.1 and pm4.1) were detected under the two test conditions. The QTL pm6.1 was only identified in 2005/Autumn. The total phenotypic variation explained by the QTLs was 52.0% and 42.0% in 2005/Autumn and 2006/Spring, respectively. Anchor markers tightly linked to those loci (

Powdery mildew is one of the most important diseases of cucumber (Cucumis sativus L.). For many years, researchers have studied and attempted to isolate resistance genes for this disease for breeding powdery mildew-resistant cucumber. In this study, bulked segregant analysis and an initial quantitative trait loci (QTLs) analysis based on a local linkage map were performed using an F2 population. A major locus on the long arm of chromosome 5 was identified for powdery mildew resistance and named pm5.1. For fine mapping of the pm5.1 locus, BC3F1 and BC2F2 populations that segregated only at the target genomic region were developed by marker-assisted selection. The location of the pm5.1 locus was confirmed by QTL analysis using a subset of the BC3F1 population and was delimited in a 1.8-cM interval between flanking markers UW065011 and UW065190. Using the large BC3F1 (n = 480) and BC2F2 (n = 483) populations, pm5.1 was mapped to a ~170-kb region between markers UW065021 and UW065094. Based on the annotations for the genes in this region, a MLO-like gene was identified as the most likely candidate for the pm5.1. Sequence alignment analysis of the MLO-like genes from susceptible and resistant cucumber lines revealed two types of mutations in this candidate gene that may result in recessive resistance to powdery mildew in the resistant cucumber lines.

Using SRAP (sequence-related amplified polymorphism) markers a genetic linkage map of cucumber was constructed with a population consisting of 138 F2 individuals derived from a cross of the two cucumber lines, S06 and S52. In the survey of parental polymorphisms with 182 primer combinations, 64 polymorphism-revealing primer pairs were screened out, which generated totally 108 polymorphic bands with an average of 1.7 bands per primer pair and at most 6 bands from one primer pair. The constructed molecular linkage map included 92 loci, distributed in seven linkage groups and spanning 1164.2 cM in length with an average genetic distance of 12.6 cM between two neighboring loci. Based on this linkage map, the quantitative trait loci (QTL) for the lateral branch number (Ibn) and the lateral branch average length (Ibl) in cucumber were identified by QTLMapperl.6. A major QTL Ibn1 located between ME11 SA4B and ME5EM5 in LG2 could explain 10.63% of the total variation with its positively effecting allele from S06. A major QTL Ibl1 located between DC1OD3 and DC1EM14 in LG2 could account for 10.38% of the total variation with its positively effecting allele from S06.