CsPM5.2, a phosphate transporter protein-like gene, promotes powdery mildew resistance in cucumber.

Powdery mildew (PM) is one of the most serious fungal diseases affecting cucumbers (Cucumis sativus L.). The mechanism of PM resistance in cucumber is intricate and remains fragmentary as it is controlled by several genes. In this study, we detected the major-effect Quantitative Trait Locus (QTL), PM5.2, involved in PM resistance by QTL mapping. Through fine mapping, the dominant PM resistance gene, CsPM5.2, was cloned and its function was confirmed by transgenic complementation and natural variation identification. In cultivar 9930, a dysfunctional CsPM5.2 mutant resulted from a single nucleotide polymorphism in the coding region and endowed susceptibility to PM. CsPM5.2 encodes a phosphate transporter-like protein PHO1; H3. The expression of CsPM5.2 is ubiquitous and induced by the PM pathogen. In cucumber, both CsPM5.2 and Cspm5.1 (Csmlo1) are required for PM resistance. Transcriptome analysis suggested that the salicylic acid (SA) pathway may play an important role in CsPM5.2-mediated PM resistance. Our findings help parse the mechanisms of PM resistance and provide strategies for breeding PM-resistant cucumber cultivars.

Mapping and identification of CsSF4, a gene encoding a UDP-N-acetyl glucosamine-peptide N-acetylglucosaminyltransferase required for fruit elongation in cucumber (Cucumis sativus L.).

KEY MESSAGE: The short fruit length phenotype in sf4 is caused by a SNP in Csa1G665390, which encodes an O-linked N-acetylglucosamine (GlcNAc) transferase in cucumber. Cucumber fruit is an excellent resource for studying fruit morphology due to its fast growth rate and naturally abundant morphological variations. The regulatory mechanisms underlying plant organ size and shape are important and fundamental biological questions. In this study, a short-fruit length mutant, sf4, was identified from an ethyl methanesulfonate (EMS) mutagenesis population derived from the North China-type cucumber inbred line WD1. Genetic analysis indicated that the short fruit length phenotype of sf4 was controlled by a recessive nuclear gene. The SF4 locus was located in a 116.7-kb genomic region between the SNP markers GCSNP75 and GCSNP82 on chromosome 1. Genomic and cDNA sequences analysis indicated that a single G to A transition at the last nucleotide of Csa1G665390 intron 21 in sf4 changed the splice site from GT-AG to GT-AA, resulting in a 42-bp deletion in exon 22. Csa1G665390 is presumed to be a candidate gene, CsSF4 that encodes an O-linked N-acetylglucosamine (GlcNAc) transferase (OGT). CsSF4 was highly expressed in the leaves and male flowers of wild-type cucumbers. Transcriptome analysis indicated that sf4 had alterations in expression of many genes involved in hormone response pathways, cell cycle regulation, DNA replication, and cell division, suggesting that cell proliferation-associated gene networks regulate fruit development in cucumber. Identification of CsSF4 will contribute to elucidating the function of OGT in cell proliferation and to understanding fruit elongation mechanisms in cucumber.

A Positive Feedback Loop Mediated by CsERF31 Initiates Female Cucumber Flower Development: ETHYLENE RESPONSE FACTOR31 mediates a positive feedback loop that initiates female cucumber flower development.

Sex determination is a crucially important developmental event that is pervasive throughout nature and enhances the adaptation of species. Among plants, cucumber (Cucumis sativus L.) can generate both unisexual and bisexual flowers, and the sex type is mainly controlled by several 1-aminocyclopropane-1-carboxylic acid (ACC) synthases. However, the regulatory mechanism of these synthases remains elusive. Here, we used gene expression analysis, protein-DNA interaction assays and transgenic plants to study the function of a gynoecium-specific gene, ETHYLENE RESPONSE FACTOR31 (CsERF31), in female flower differentiation. We found that in a predetermined female flower, ethylene signalling activates CsERF31 by CsEIN3, and then CsERF31 stimulates CsACS2, which triggers a positive feedback loop to ensure female rather than bisexual flower development. A similar interplay is functionally conserved in melon (Cucumis melo L.). Knockdown of CsERF31 by RNAi causes defective bisexual flowers to replace female flowers. Ectopic expression of CsERF31 suppresses stamen development and promotes pistil development in male flowers, demonstrating that CsERF31 functions as a sex switch. Taken together, our data confirm that CsERF31 represents the molecular link between female-male determination and female-bisexual determination, and provide mechanistic insight into how ethylene promotes female flowers, rather than bisexual flowers, in cucumber sex determination.

A positive feedback loop mediated by CsERF31 initiates female cucumber flower development.

Sex determination is a crucially important developmental event that is pervasive throughout nature and enhances the adaptation of species. Among plants, cucumber (Cucumis sativus L.) can generate both unisexual and bisexual flowers, and the sex type is mainly controlled by several 1-aminocyclopropane-1-carboxylic acid synthases (CsACSs). However, the regulatory mechanism of these synthases remains elusive. Here, we used gene expression analysis, protein-DNA interaction assays, and transgenic plants to study the function of a gynoecium-specific gene, ETHYLENE RESPONSE FACTOR31 (CsERF31), in female flower differentiation. We found that in a predetermined female flower, ethylene signaling activates CsERF31 by CsEIN3, and then CsERF31 stimulates CsACS2, which triggers a positive feedback loop to ensure female rather than bisexual flower development. A similar interplay is functionally conserved in melon (Cucumis melo L.). Knockdown of CsERF31 by RNAi causes defective bisexual flowers to replace female flowers. Ectopic expression of CsERF31 suppresses stamen development and promotes pistil development in male flowers, demonstrating that CsERF31 functions as a sex switch. Taken together, our data confirm that CsERF31 represents the molecular link between female-male determination and female-bisexual determination, and provide mechanistic insight into how ethylene promotes female flowers, rather than bisexual flowers, in cucumber sex determination.

A SNP of HD-ZIP I transcription factor leads to distortion of trichome morphology in cucumber (Cucumis sativus L.).

BACKGROUND: Trichomes are excellent model systems for the analysis of cell differentiation and play essential roles in plant protection. From cucumber inbred line 'WD1', we identified an EMS-induced trichome abnormally developing mutant, nps, which exhibited smaller, denser and no pyramid-shaped head trichomes. RESULTS: Using F2 and BC1 populations constructed from a cross between nps and '9930', the genetic analysis showed that the nps trait is controlled by a single recessive nuclear gene. We identified CsNps by map-based cloning with 576 individuals of the F2 population generated from the cross of nps and inbred line '9930'. The CsNps was located at a 13.4-kb genomic region on chromosome 3, which region contains three predicted genes. Sequence analysis showed that only one single nucleotide mutation (C → T) between 9930 and nps was found in the second exon of Csa3G748220, a plant-specific class I HD-Zip gene. The result of allelism test also indicated that nps is a novel allelic mutant of Mict (Micro-trichome). Thus, nps was renamed mict-L130F. By comparing the transcriptome of mict-L130F vs WD1 and 06-2 (mict) vs 06-1 (wildtype, near-isogenic line of 06-2), several potential target genes that may be related to trichome development were identified. CONCLUSIONS: Our results demonstrate that Mict-L130F is involved in the morphogenesis of trichomes. Map-based cloning of the Mict-L130F gene could promote the study of trichome development in cucumber.

TERMINAL FLOWER 1 and TERMINAL FLOWER 1d respond to temperature and photoperiod signals to inhibit determinate growth in cucumber.

Plants monitor environmental cues to balance their vegetative and productive growth by optimizing their inflorescence architecture. TERMINAL FLOWER 1 (TFL1) and its orthologs regulate the inflorescence structure in cucumber, yet the mechanisms underlying their responses to environmental factors and the formation of terminal flowers remain elusive. Here, we performed map-based cloning to identify the gene that controls a season-dependent determinate growth phenotype and found that it was caused by the complete deletion of CsTFL1 in the genome of cucumber line WI1983Hde. In the CsTFL1 deletion plants (CsTFL1del ), determinate growth could be partially rescued by high-temperature and long-day conditions. The expressions of CsTFL1 and its ortholog CsTFL1d could be upregulated by long-day and high-temperature signals. Knockdown of CsTFL1d resulted in determinate growth and the formation of terminal flowers in WT. These results indicate that the induction of CsTFL1d expression by long-day and high-temperature might partially rescue determinate growth of CsTFL1del . Furthermore, biochemical analyses showed that CsTFL1d interacts directly with CsNOT2a, which indicated that CsTFL1d and CsTFL1 function via similar regulatory mechanism. Our data suggest that CsTFL1 and CsTFL1d co-contribute to inhibit determinate growth by responding to temperature and photoperiod signals. It provides mechanistic insights into how environmental cues sculpt the inflorescence architecture of cucumber.

CsUFO is involved in the formation of flowers and tendrils in cucumber.

KEY MESSAGE: 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.

Mapping and identification of CsSh5.1, a gene encoding a xyloglucan galactosyltransferase required for hypocotyl elongation in cucumber (Cucumis sativus L.).

KEY MESSAGE: 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.

Genome-Wide Identification, Classification and Expression Analysis of the MYB Transcription Factor Family in Petunia.

A lot of researches have been focused on the evolution and function of MYB transcription factors (TFs). For revealing the formation of petunia flower color diversity, MYB gene family in petunia was identified and analyzed. In this study, a total of 155 MYB genes, including 40 1R-MYBs, 106 R2R3-MYBs, 7 R1R2R3-MYBs and 2 4R-MYBs, have been identified in the Petunia axillaris genome. Most R2R3 genes contain three exons and two introns, whereas the number of PaMYB introns varies from 0 to 12. The R2R3-MYB members could be divided into 28 subgroups. Analysis of gene structure and protein motifs revealed that members within the same subgroup presented similar exon/intron and motif organization, further supporting the results of phylogenetic analysis. Genes in subgroup 10, 11 and 21 were mainly expressed in petal, not in vegetative tissues. Genes in subgroup 9, 19, 25 and 27 expressed in all tissues, but the expression patterns of each gene were different. According to the promoter analysis, five R2R3-MYB and two MYB-related genes contained MBSI cis-element, which was involved in flavonoid biosynthetic regulation. PaMYB100/DPL has been reported to positively regulate to pigmentation. However, although PaMYB82, PaMYB68 and Pa1RMYB36 contained MBSI cis-element, their function in flavonoid biosynthesis has not been revealed. Consistent with existing knowledge, PaMYBs in subgroup 11 had similar function to AtMYBs in subgroup 6, genes in which played an important role in anthocyanin biosynthesis. In addition, PaMYB1 and PaMYB40 belonged to P9 (S7) and were potentially involved in regulation of flavonoid synthesis in petunia vegetative organs. This work provides a comprehensive understanding of the MYB gene family in petunia and lays a significant foundation for future studies on the function and evolution of MYB genes in petunia.

The HD-ZIP IV transcription factor Tril regulates fruit spine density through gene dosage effects in cucumber.

Trichomes and fruit spines are important traits that directly affect the appearance quality and commercial value of cucumber (Cucumis sativus). Tril (Trichome-less), encodes a HD-Zip IV transcription factor that plays a crucial role in the initiation of trichomes and fruit spines, but little is known about the details of the regulatory mechanisms involved. In this study, analysis of tissue expression patterns indicated that Tril is expressed and functions in the early stages of organ initiation and development. Expression of Tril under the control of its own promoter (the TrilPro::Tril-3*flag fragment) could partly rescue the mutant phenotypes of tril, csgl3 (cucumber glabrous 3, an allelic mutant of tril), and fs1 (few spines 1, a fragment substitution in the Tril promoter region), providing further evidence that Tril is responsible for the initiation of trichomes and fruit spines. In lines with dense spine, fs1-type lines, and transgenic lines of different backgrounds containing the TrilPro::Tril-3*flag foreign fragment, spine density increased in conjunction with increases in Tril expression, indicating that Tril has a gene dosage effect on fruit spine density in cucumber. Numerous Spines (NS) is a negative regulatory factor of fruit spine density. Characterization of the molecular and genetic interaction between Tril and NS/ns demonstrated that Tril functions upstream of NS with respect to spine initiation. Overall, our results reveal a novel regulatory mechanism governing the effect of Tril on fruit spine development, and provide a reference for future work on breeding for physical quality in cucumber.

A Mutation in CsYL2.1 Encoding a Plastid Isoform of Triose Phosphate Isomerase Leads to Yellow Leaf 2.1 (yl2.1) in Cucumber (Cucumis Sativus L.).

The leaf is an important photosynthetic organ and plays an essential role in the growth and development of plants. Leaf color mutants are ideal materials for studying chlorophyll metabolism, chloroplast development, and photosynthesis. In this study, we identified an EMS-induced mutant, yl2.1, which exhibited yellow cotyledons and true leaves that did not turn green with leaf growth. The yl2.1 locus was controlled by a recessive nuclear gene. The CsYL2.1 was mapped to a 166.7-kb genomic region on chromosome 2, which contains 24 predicted genes. Only one non-synonymous single nucleotide polymorphism (SNP) was found between yl2.1 and wt-WD1 that was located in Exon 7 of Csa2G263900, resulting in an amino acid substitution. CsYL2.1 encodes a plastid isoform of triose phosphate isomerase (pdTPI), which catalyzes the reversible conversion of dihydroxyacetone phosphate (DHAP) to glyceraldehyde-3-phosphate (GAP) in chloroplasts. CsYL2.1 was highly expressed in the cotyledons and leaves. The mesophyll cells of the yl2.1 leaves contained reduced chlorophyll and abnormal chloroplasts. Correspondingly, the photosynthetic efficiency of the yl2.1 leaves was impaired. Identification of CsYL2.1 is helpful in elucidating the function of ptTPI in the chlorophyll metabolism and chloroplast development and understanding the molecular mechanism of this leaf color variant in cucumber.

Mapping and identification of CsUp, a gene encoding an Auxilin-like protein, as a putative candidate gene for the upward-pedicel mutation (up) in cucumber.

BACKGROUND: Pedicel orientation can affect the female flower orientation and seed yield in cucumber. A spontaneous mutant possessing upward growth of pedicels was identified in the wild type inbred strain 9930 and named upward-pedicel (up). The morphological and genetic analyses of up were performed in this study. In order to clone the up gene, 933 F2 individuals and 524 BC1 individuals derived from C-8-6 (WT) and up were used for map-based cloning. RESULTS: up was mapped to a 35.2 kb physical interval on chromosome 1, which contains three predicted genes. Sequencing analysis revealed that a 5-bp deletion was found in the second exon of Csa1G535800, and it led to a frameshift mutation resulting in a premature stop codon. The candidate gene of CsUp (Csa1G535800) was further confirmed via genomic and cDNA sequencing in biparental and natural cucumber populations. Sequencing data showed that a 4-bp deletion was found in the sixth exon of Csa1G535800 in CGN19839, another inbred line, and there was also a mutation of an amino acid in Csa1G535800 that could contribute to the upward growth of pedicels in CGN19839. Moreover, it was found that Csa1G535800 exhibited strong expression in the pedicel of WT, suggesting its important role in development of pedicel orientation. Thus, Csa1G535800 was considered to be the candidate gene of CsUp. CONCLUSIONS: CsUp encodes an Auxilin-like protein and controls pedicel orientation in cucumber. The identification of CsUp may help us to understand the mechanism of pedicel orientation development and allow for investigation of novel functions of Auxilin-like proteins in cucumber.

STAYGREEN, STAY HEALTHY: a loss-of-susceptibility mutation in the STAYGREEN gene provides durable, broad-spectrum disease resistances for over 50 years of US cucumber production.

The Gy14 cucumber (Cucumis sativus) is resistant to oomyceteous downy mildew (DM), bacterial angular leaf spot (ALS) and fungal anthracnose (AR) pathogens, but the underlying molecular mechanisms are unknown. Quantitative trait locus (QTL) mapping for the disease resistances in Gy14 and further map-based cloning identified a candidate gene for the resistant loci, which was validated and functionally characterized by spatial-temporal gene expression profiling, allelic diversity and phylogenetic analysis, as well as local association studies. We showed that the triple-disease resistances in Gy14 were controlled by the cucumber STAYGREEN (CsSGR) gene. A single nucleotide polymorphism (SNP) in the coding region resulted in a nonsynonymous amino acid substitution in the CsSGR protein, and thus disease resistance. Genes in the chlorophyll degradation pathway showed differential expression between resistant and susceptible lines in response to pathogen inoculation. The causal SNP was significantly associated with disease resistances in natural and breeding populations. The resistance allele has undergone selection in cucumber breeding. The durable, broad-spectrum disease resistance is caused by a loss-of-susceptibility mutation of CsSGR. Probably, this is achieved through the inhibition of reactive oxygen species over-accumulation and phytotoxic catabolite over-buildup in the chlorophyll degradation pathway. The CsSGR-mediated host resistance represents a novel function of this highly conserved gene in plants.

Genome-Wide Mapping of Structural Variations Reveals a Copy Number Variant That Determines Reproductive Morphology in Cucumber.

Structural variations (SVs) represent a major source of genetic diversity. However, the functional impact and formation mechanisms of SVs in plant genomes remain largely unexplored. Here, we report a nucleotide-resolution SV map of cucumber (Cucumis sativas) that comprises 26,788 SVs based on deep resequencing of 115 diverse accessions. The largest proportion of cucumber SVs was formed through nonhomologous end-joining rearrangements, and the occurrence of SVs is closely associated with regions of high nucleotide diversity. These SVs affect the coding regions of 1676 genes, some of which are associated with cucumber domestication. Based on the map, we discovered a copy number variation (CNV) involving four genes that defines the Female (F) locus and gives rise to gynoecious cucumber plants, which bear only female flowers and set fruit at almost every node. The CNV arose from a recent 30.2-kb duplication at a meiotically unstable region, likely via microhomology-mediated break-induced replication. The SV set provides a snapshot of structural variations in plants and will serve as an important resource for exploring genes underlying key traits and for facilitating practical breeding in cucumber.

STAYGREEN (CsSGR) is a candidate for the anthracnose (Colletotrichum orbiculare) resistance locus cla in Gy14 cucumber.

KEY MESSAGE: 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.

GLABROUS (CmGL) encodes a HD-ZIP IV transcription factor playing roles in multicellular trichome initiation in melon.

KEY MESSAGE: Map-based cloning identified CmGL that encodes a HD-ZIP type IV transcription factor that controls multicellular trichome initiation in melon. Trichomes are small hairs covering the aerial parts of plants that originate from the epidermal cells, which can protect plants against the damage by insects and pathogens. The regulatory pathway of unicellular trichomes has been well studied in the model plant Arabidopsis. Little is known about the genetic control and regulation of trichome development in melon (Cucumis melo L.) which has multicellular trichomes. In this study, we identified a melon mutant, cmgl, which showed completely glabrous on all aerial organs. A bulked segregant analysis was conducted to identify polymorphic markers for linkage analysis in a population with 256 F2 plants, which allowed to locate the cmgl locus in melon chromosome VIII. Next-generation sequencing-aided marker discovery and fine mapping in a large population with 1536 F2 plants narrowed the candidate gene region to 12 kb that harbored only one candidate gene for cmgl, which encoded a class IV homeodomain-associated leucine zipper transcription factor. Four SNPs in the coding region of the CmGL gene were identified between the two parental lines; a single base substitution from C to A resulted in a premature termination codon and a truncated protein in the cmgl. The SNP was converted into a dCAPS marker, which showed co-segregation in the F2 population and 564 melon accessions. Result of this study will be helpful for better understanding of genetic control of trichome development in melon and marker-assisted selection in developing new cultivars.

Identification and mapping of ts (tender spines), a gene involved in soft spine development in Cucumis sativus.

KEY MESSAGE: 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.

Identification and mapping of Tril, a homeodomain-leucine zipper gene involved in multicellular trichome initiation in Cucumis sativus.

KEY MESSAGE: Using map-based cloning of Tril gene, we identified a homeodomain-leucine zipper gene involved in the initiation of multicellular trichomes (including the spines of fruit) in cucumber. ABSTRACT: Fruit spines are a special type of trichome that impacts the quality and appearance of cucumber (Cucumis sativus L.) fruit. Scanning electron microscopy revealed that the trichome-less (tril) mutant originating from European greenhouse cucumber has a completely glabrous phenotype on cotyledons, hypocotyls, young leaves, fruits, and fruit stalks. Genetic analysis revealed that tril was inherited as a recessive allele at a single locus. Using 1058 F2 individuals derived from a cross between cucumber tril mutant CGN19839 and the micro-trichome (mict) mutant 06-2, tril was mapped to chromosome 6, and narrowed down to a 37.4 kb genomic region which carries seven predicted genes. Genetic and molecular analyses revealed that gene Cucsa.045360 is a possible candidate gene for the differentiation of epidermal cells to trichomes. It is a member of the class IV homeodomain-leucine zipper (HD-Zip IV) family and encodes homeodomain and START domain, sharing 66.7% predicted amino acid sequence identity to PROTODERMAL FACTOR2 (PDF2) and 35.0% to GLABRA2 (GL2) of Arabidopsis. The homeobox domain had changed amino acid sequence because of an insertion in tril mutant. The results of genetic analysis and transcriptome profiling indicated that the Tril gene had an epistatic effect on the Mict gene in trichome development. Phenotypes of the tril mutant such as glabrous fruits and female flowers at every node could be used in developing new cultivars.

Transcriptome analysis in Cucumis sativus identifies genes involved in multicellular trichome development.

The regulatory gene network of unicellular trichome development in Arabidopsis thaliana has been studied intensively, but that of multicellular remains unclear. In the present study, we characterized cucumber trichomes as representative multicellular and unbranched structures, but in a spontaneous mutant, mict (micro-trichome), all trichomes showed a micro-size and stunted morphologies. We revealed the transcriptome profile using Illumina HiSeq 2000 sequencing technology, and determined that a total of 1391 genes exhibited differential expression. We further validated the accuracy of the transcriptome data by RT-qPCR and found that 43 genes encoding critical transcription factors were likely involved in multicellular trichome development. These 43 candidate genes were subdivided into seven groups: homeodomain, MYB-domain, WRKY-domain, bHLH-domain, ethylene-responsive, zinc finger and other transcription factor genes. Our findings also serve as a powerful tool to further study the relevant molecular networks, and provide a new perspective for investigating this complex and species-specific developmental process.

Tuberculate fruit gene Tu encodes a C2 H2 zinc finger protein that is required for the warty fruit phenotype in cucumber (Cucumis sativus L.).

Cucumber fruits that have tubercules and spines (trichomes) are known to possess a warty (Wty) phenotype. In this study, the tuberculate fruit gene Tu was identified by map-based cloning, and was found to encode a transcription factor (TF) with a single C2 H2 zinc finger domain. Tu was identified in all 38 Wty lines examined, and was completely absent from all 56 non-warty (nWty) lines. Cucumber plants transgenic for Tu (TCP) revealed that Tu was required for the Wty fruit phenotype. Subcellular localization showed that the fusion protein GFP-Tu was localized mainly to the nucleus. Based on analyses of semi-quantitative and quantitative reverse transcription polymerase chain reaction (RT-PCR), and mRNA in situ hybridization, we found that Tu was expressed specifically in fruit spine cells during development of fruit tubercules. Moreover, cytokinin (CTK) content measurements and cytological observations in Wty and nWty fruits revealed that the Wty fruit phenotype correlated with high endogenous CTK concentrations. As a result of further analyses on the transcriptomic profile of the nWty fruit epidermis and TCP fruit warts, expression of CTK-associated genes, and hormone content in nWty fruit epidermis, Wty fruit warts and epidermis, and TCP fruit warts and epidermis, we found that Tu probably promoted CTK biosynthesis in fruit warts. Here we show that Tu could not be expressed in the glabrous and tubercule-free mutant line gl that contained Tu, this result that futher confirmed the epistatic effect of the trichome (spine) gene Gl over Tu. Taken together, these data led us to propose a genetic pathway for the Wty fruit trait that could guide future mechanistic studies.