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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.

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.

The spines of cucumber fruit not only have important commercial value but are also a classical tissue to study cell division and differentiation modes of multicellular trichomes. It has been reported that CsTs (C-type Lectin receptor-like kinase) can influence the development of fruit spines. In this study, we took a pair of cucumber materials defined as hard (Ts, wild type) and tender spines (ts, mutant) and defined the developmental process of fruit spines as consisting of four stages (stage I to stage IV) by continuously observing by microscope and SEM. Comparisons of transcriptome profiles at different development stages of wild-type spines showed that 803 and 722 genes were upregulated in the stalk (stage II and stage III) and base (stage IV) development stages of fruit spines, respectively. The function analysis of DEGs showed that genes related to auxin polar transport and HD-ZIP transcription factor are significantly upregulated during the development of the stalk. bHLH transcription factors and cytoskeleton-related genes were significantly upregulated during the development of the base. In addition, stage III is the key point for the difference between wild-type and mutant spines. We detected 628 DEGs between wild type and mutant at stage III. These DEGs are mainly involved in the calcium signaling of the cytoskeleton and auxin polar transport. Coincidentally, we found that CsVTI11, a factor involved in auxin signal transmission, can interact with CsTs in vivo, but this interaction does not occur between CsVTI11 and Csts, further suggesting that CsTs may regulate the development of fruit spines by influencing cell polarity. These results provide useful tools to study the molecular networks associated with cucumber fruit spine development and elucidate the biological pathways that C-type Lectin receptor-like kinase plays in regulating the development of fruit spines.

Trichomes that cover the epidermis of aerial plant organs play multiple roles in plant protection. Compared with a unicellular trichome in model plants, the development mechanism of the multicellular trichome is largely unclear. Notably, variations in trichome development are often accompanied by defects in the biosynthesis of cuticle and secondary metabolites; however, major questions about the interactions between developmental differences in trichomes and defects in metabolic pathways remain unanswered. Here, we characterized the glabrous mutantmict/csgl1/cstbhvia combined metabolomic and transcriptomic analyses to extend our limited knowledge regarding multicellular trichome development and metabolism in cucumber.Mictwas found to be explicitly expressed within trichome cells. Transcriptomic analysis indicated that genes involved in flavonoid and cuticle metabolism are significantly downregulated inmictmutants. Further metabolomic analysis confirmed that flavonoids, lipids, and cuticle compositions are dramatically altered inmictmutants. Additional studies revealed thatMictregulates flavonoid, lipid, and cuticle biosynthesis by likely directly binding to downstream functional genes, such asCsTT4,CsFLS1,CsCER26, andCsMYB36. These findings suggest that specific metabolic pathways (e.g., flavonoids and cuticle components) are co-regulated byMictand provide insights into transcriptional regulation mechanisms of multicellular trichome development and its specific metabolism in cucumber.

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.

Unisexual flowers provide a useful system for studying plant sex determination. In cucumber (Cucumis sativus L.), three major Mendelian loci control unisexual flower development, Female (F), androecious [a; 1-aminocyclopropane-1-carboxylate {ACC} synthase 11, acs11], and Monoecious (M; ACS2), referred to here as the Female, Androecious, Monoecious (FAM) model, in combination with two genes, gynoecious (g, the WIP family C2H2 zinc finger transcription factor gene WIP1) and the ethylene biosynthetic gene ACC oxidase 2 (ACO2). The F locus, conferring gynoecy and the potential for increasing fruit yield, is defined by a 30.2-kb tandem duplication containing three genes. However, the gene that determines the Female phenotype, and its mechanism, remains unknown. Here, we created a set of mutants and revealed that ACS1G is responsible for gynoecy conferred by the F locus. The duplication resulted in ACS1G acquiring a new promoter and expression pattern; in plants carrying the F locus duplication, ACS1G is expressed early in floral bud development, where it functions with ACO2 to generate an ethylene burst. The resulting ethylene represses WIP1 and activates ACS2 to initiate gynoecy. This early ACS1G expression bypasses the need for ACS11 to produce ethylene, thereby establishing a dominant pathway for female floral development. Based on these findings, we propose a model for how these ethylene biosynthesis genes cooperate to control unisexual flower development in cucumber.

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.

Background The trichomes of cucumber on the fruit are also called spines, it not only has important commercial value but also is a classical tissue to study cell division and differentiation mode of multicellular trichomes. Although there have been many researches about the development of unicellular trichomes in model plants, the molecular mechanism of multicellular trichomes formation remains elusive. In this study, we took a pair of cucumber materials defined as hard (Ts, wild-type) and tender spines (ts, mutant) in previous study, the whole developmental process of fruit spines was continuously observed by microscope and SEM, in an attempt to define the development stage of fruit spines, transcriptome profiles at different stages was made to explore the molecular mechanism in the process of spines development. Results With significant phenotypic differences, the developmental process of fruit spines was clearly defined as four stages. Comparion of transcriptome profilings showed a total of 2,788 differential expression genes (DEGs) between the wild-type and mutant at different developmental stages of fruit spines, these genes exhibited different expression patterns at different spines developmental stage. Some DEGs related to cell cycle and meristem had also been identified in transcriptome data. By correlating the expression patterns of several transcription factors that have been reported to be involved in the development of spines, we identified some genes that may be involved in the formation of cucumber fruit spines. The cluster analysis of transcription factors revealed that there were 26 transcription factor families that may be involved in the development of fruit spines, among them, the ERF-ERF, bHLH and WAKY family transcription factor gene families were the top three gene families. Conclusions We defined the four stages of fruit spines development and identified a number of genes that may be involved in the development of multicellular trichomes based on comparative transcription. The results provided a step stone for further analysis of the molecular mechanism in the developmental process of multicellular trichomes.

The woodland strawberry, Fragaria vesca, is a model plant for the diverse Rosaceae family, which contains many valuable fruit and ornamental crops. Light is an important external environment factor which influences all aspects of plant growth and development, including fruit ripening. Transcription regulation of light will provide insights into effect of light for fruit ripening. We treat strawberry fruit with dark or light, and by the RNA-Seq method, compare transcriptional level between dark-treated and light-treated strawberry fruits. Additionally, we detect anthocyanin and sugar accumulation in two-treated conditions of fruits. Moreover, we detect the protein change of FvMYB10, a key regulator of anthocyanin biosynthesis, under dark and light. Light can promote anthocyanin and soluble sugar accumulation in mature fruit, and also increases the expression of aroma-related genes and stabilizes FvMYB10 protein. Our findings reveal that light is essential for anthocyanin and sugar accumulation and regulates FvMYB10 at transcriptional level and post-translation level.

Tnt1 is an active retrotransposon originally identified in tobacco (Nicotiana tabacum L.) (Grandbastien et al., 1989), but its transposition activity could be activated through tissue culture in other plant species. The insertions are stable and inheritable in the progeny, which has made it a valuable and versatile tool for developing insertional mutagenesis libraries in several plant species. Here, we explored its utility for mutagenesis in cucumber (Cucumis sativus L.). T3 Tnt1 transgenic cucumber plants were subjected to tissue culture to regenerate self-pollinated progeny. With PCR and analyses and Southern hybridization, we found regenerated plants maintained the original Tnt1 insertion and created new insertions suggesting characteristic re-transposition activity of Tnt1 during this process. Using genome walking, some flanking sequences of Tnt1 insertions were recovered in regenerated plants. The results demonstrated that Tnt1 could be stably inherited and re-transposable during tissue culture in cucumber and that it is feasible to use for developing an insertional mutagenesis library for cucumber. Keywords: cucumber, Tnt1, retrotransposon, tissue culture, mutagenesis library

The development of trichomes (spines) on cucumber fruits is an important agronomic trait. It has been reported that two MYB family members, CsMYB6 (Csa3G824850) and CsTRY (Csa5G139610) act as negative regulators of trichome or fruit spine initiation. To further study the functions of these two genes, we overexpressed them in tobacco, and found that the flowers and seed coats of transformants overexpressing CsTRY displayed an unexpected defect in pigmentation that was not observed in plants overexpressing CsMYB6. Moreover, the expression of key genes in the flavonoid synthesis pathway was repressed in CsTRY overexpressing plants, which resulted in the decrease of several important flavonoid secondary metabolites. In addition, CsTRY could interact with the AN1 homologous gene CsAN1 (Csa7G044190) in cucumber, which further confirmed that CsTRY not only regulates the development of fruit spines, but also functions in the synthesis of flavonoids, acting as the repressor of anthocyanin synthesis.

In cucumber (Cucumis sativus L.), the differentiation and development of female flowers are important processes that directly affect the fruit yield and quality. Sex differentiation is mainly controlled by three ethylene synthase genes, F (CsACS1G), M (CsACS2), and A (CsACS11). Thus, ethylene plays a key role in the sex differentiation in cucumber. The "one-hormone hypothesis" posits that F and M regulate the ethylene levels and initiate female flower development in cucumber. Nonetheless, the precise molecular mechanism of this process remains elusive. To investigate the mechanism by which F and M regulate the sex phenotype, three cucumber near-isogenic lines, namely H34 (FFmmAA, hermaphroditic), G12 (FFMMAA, gynoecious), and M12 (ffMMAA, monoecious), with different F and M loci were generated. The transcriptomic analysis of the apical shoots revealed that the expression of the B-class floral homeotic genes, CsPI (Csa4G358770) and CsAP3 (Csa3G865440), was immensely suppressed in G12 (100% female flowers) but highly expressed in M12 (∼90% male flowers). In contrast, CAG2 (Csa1G467100), which is an AG-like C-class floral homeotic gene, was specifically highly expressed in G12. Thus, the initiation of female flowers is likely to be caused by the downregulation of B-class and upregulation of C-class genes by ethylene production in the floral primordium. Additionally, CsERF31, which was highly expressed in G12, showed temporal and spatial expression patterns similar to those of M and responded to the ethylene-related chemical treatments. The biochemical experiments further demonstrated that CsERF31 could directly bind the promoter of M and promote its expression. Thus, CsERF31 responded to the ethylene signal derived from F and mediated the positive feedback regulation of ethylene by activating M expression, which offers an extended "one-hormone hypothesis" of sex differentiation in cucumber.

Plant trichomes serve as a highly suitable model for investigating cell differentiation at the single-cell level. The regulatory genes involved in unicellular trichome development in Arabidopsis thaliana have been intensively studied, but genes regulating multicellular trichome development in plants remain unclear. Here, we characterized Cucumis sativus (cucumber) trichomes as representative multicellular and unbranched structures, and identified Micro-trichome (Mict), using map-based cloning in an F2 segregating population of 7,936 individuals generated from a spontaneous mict mutant. In mict plants, trichomes in both leaves and fruits, are small, poorly developed, and denser than in the wild type. Sequence analysis revealed that a 2,649-bp genomic deletion, spanning the first and second exons, occurred in a plant-specific class I homeodomain-leucine zipper gene. Tissue-specific expression analysis indicated that Mict is strongly expressed in the trichome cells. Transcriptome profiling identified potential targets of Mict including putative homologs of genes known in other systems to regulate trichome development, meristem determinacy, and hormone responsiveness. Phylogenic analysis charted the relationships among putative homologs in angiosperms. Our paper represents initial steps toward understanding the development of multicellular trichomes.

Dihydroflavonol 4-reductase (DFR) genes from Rosa chinensis (Asn type) and Calibrachoa hybrida (Asp type), driven by a CaMV 35S promoter, were integrated into the petunia (Petunia hybrida) cultivar 9702. Exogenous DFR gene expression characteristics were similar to flower-color changes, and effects on anthocyanin concentration were observed in both types of DFR gene transformants. Expression analysis showed that exogenous DFR genes were expressed in all of the tissues, but the expression levels were significantly different. However, both of them exhibited a high expression level in petals that were starting to open. The introgression of DFR genes may significantly change DFR enzyme activity. Anthocyanin ultra-performance liquid chromatography results showed that anthocyanin concentrations changed according to DFR enzyme activity. Therefore, the change in flower color was probably the result of a DFR enzyme change. Pelargonidin 3-O-glucoside was found in two different transgenic petunias, indicating that both CaDFR and RoDFR could catalyze dihydrokaempferol. Our results also suggest that transgenic petunias with DFR gene of Asp type could biosynthesize pelargonidin 3-O-glucoside.

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.

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.

Dihydroflavonol-4-reductase (DFR) catalyzes the reduction of dihydroflavonols in the anthocyanin biosynthetic pathway. In this study, we obtained the full-length cDNA of DFR from Calibrachoa hybrida , CaDFR , through RACE-based technique. The cDNA of CaDFR is 1, 351 bp with an open reading frame of 1, 152 bp, which encodes a putative protein of 383 amino acids. The CaDFR protein is predicted with 42.74 KD in molecular weight and a p I value of 5.83. Sequence alignment showed that CaDFR had close relationship with DFRs from other species of the Solanaceae family, and it belonged to the Asp-type. We constructed 35S:: CaDFR overexpression vector and transformed it into three cultivars of Petunia hybrida using the Agrobacterium-mediated method. Multiple transformants were selected in the presence of 50 mg/l kanamycin antibiotic and further verified by polymerase chain reaction and Southern blotting analyses. We observed that the bud and flower color of transgenic petunia altered comparing with that of control plants. The DFR activities varied in most color-changed transgenic petunias. These results confirm the identity and function of CaDFR and further suggest that integration of the CaDFR can be used to change flower color of transgenic plants.

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.

High-density genetic linkage map plays an important role in genome assembly and quantitative trait loci (QTL) fine mapping. Since the coming of next-generation sequencing, makes the structure of high-density linkage maps much more convenient and practical, which simplifies SNP discovery and high-throughput genotyping. In this research, a high-density linkage map of cucumber was structured using specific length amplified fragment sequencing, using 153 F2 populations of S1000 × S1002. The high-density genetic map composed 3,057 SLAFs, including 4,475 SNP markers on seven chromosomes, and spanned 1061.19 cM. The average genetic distance is 0.35 cM. Based on this high-density genome map, QTL analysis was performed on two cucumber fruit traits, fruit length and fruit diameter. There are 15 QTLs for the two fruit traits were detected.

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.

A maize genetic linkage map derived from 115 simple sequence repeat (SSR) markers was constructed from an F 2 population. The F 2 was generated from a cross between a stay-green inbred line (Q319) and a normal inbred line (Mol7). The map resolved 10 linkage groups and spanned 1431.0 cM in length with an average genetic distance of 12.44 cM between two neighbouring loci. A total of 14 quantitative trait loci (QTL) were detected for stay-green traits at different postflowering time intervals and identified by composite interval mapping. The respective QTL contribution to phenotypic variance ranged from 5.40% to 11.49%, with trait synergistic action from Q319. Moreover, maize stay-green traits were closely correlated to grain yield. Additional QTL analyses indicated that multiple intervals of stay-green QTL overlapped with yield QTL.

Plants are exposed to many potentially pathogenic microbes in the environment, but each species is only susceptible to a limited number of pathogens. The broad resistance is referred to as nonhost resistance. To date, little is known about the underlying mechanism of nonhost resistance and the signaling transduction process. Here we describe a simple method for isolating Arabidopsis nonhost resistance mutants against a nonadapted bacterial pathogen. A RAP2.6 promoter-driven LUC reporter system was developed to replace the tedious bacterial growth assay during the primary screening. The RAP2.6-LUC reporter gene is normally induced by the virulent bacterium Pseudomonas syringae pv tomato but not the nonadapted bacterium P. syringae pv phaseolicola. By using this method we isolated 4 mutants displaying strong reporter activity in response to P. syringae pv phaseolicola, which were characterized in some details. ebs1, ebs2, ebs3, and ebs4 (enhanced bacterial susceptibility) were compromised in resistance against P. syringae pv phaseolicola and/or P. syringae pv tomato. In addition, ebs4 showed enhanced hypersensitive response to the incompatible bacterium P. syringae pv tomato (avrB). These results demonstrated that the method is suited for large scale screening for nonhost resistance mutants.

A 173-point genetic linkage map of cucumber (Cucumis sativus L.), consisting of 116 SRAPs, 33 RAPDs, 11 SSRs, 9 SCARs, 3 ISSRs, and 1 STS, was constructed using 130 F 2 progeny derived from a narrow cross between line S94 (Northern China open-field type) and line S06 (greenhouse European type). The seven linkage groups spanned 1016 cM with a mean marker interval of 5.9 cM. Using the F 2 population and its F 3 derived families, a total of 38 QTLs were detected on five linkage groups with an LOD threshold of 3.0 for nine fruit-related traits: fruit weight, length, and diameter, fruit flesh thickness, seed-cavity diameter, fruit-stalk length, fruit pedicel length, length/diameter and length/stalk ratio. Of the identified QTLs, /?/4.3 for fruit-stalk length explained the largest portion of phenotypic variation (r 2 =∼30%). Several QTLs were detected, in the same linkage region in different generations and different seasons. Additionally, several QTLs for various fruit traits were mapped to the same or neighbouring marker intervals, suggesting they are possible character associations for controlling cucumber fruit development.

A bacterial artificial chromosome (BAC) library consisting of 19,200 clones with an average insert size of 105 kb has been constructed from a cucumber ( Cucumis sativus L.) inbred line S94, derived from a cultivar in North China. The entire library was equivalent to approximately 5 haploid cucumber genomes. To facilitate chromosome engineering and anchor the cucumber genetic linkage map to its chromosomes, 15 sequence-characterized amplified regions (SCAR) and seven simple sequence repeats (SSR) markers from each linkage group of cucumber were used to screen an ordered array of pooled BAC DNA with polymerase chain reaction (PCR). Fifteen markers gave at least two positive clones. As a result, 22 BAC clones representing 7 linkage groups of cucumber were identified, which further validated the genome coverage and utility of the library. This BAC library and linkage group specific clones provide essential resources for future research of the cucumber genome.

Rice is a typical silicon-accumulating plant and the beneficial effect of silicon on rice has long been recognized. In a previous study using 244 recombinant inbred lines (RILs) of an indica rice cross, Zhenshan 97B/Milyang 46 grown in 2003, four QTLs were detected for hull silicon content. QTL qHUS-6 had the largest effect among these, and the same interval also had significant effects on yield traits in the same population. The primary objective of this study was to validate the QTL effect in this region on HUS and yield traits. The same RIL population and another RIL population of lower heterogeneity were grown in 2004. QTL qHUS-6 was found to have significant additive effects on hull silicon content with a consistent direction in the two populations. From a residual heterozygous line selected from RILs of the same cross, 15 F2:3 lines that differed only in a 2.15-Mb segment extending from RM587 to RM6119 on the short arm of chromosome 6 were derived. In these lines, qHUS-6 displayed a major effect, so did QTLs for yield traits previously detected in the same region. Two more QTLs for HUS detected in 2003, qHUS-1-1 and qHUS-1-2, also had consistent effects in the Zhenshan 97B/Milyang 46 RIL population in 2004. Thus this study verified three candidate regions for fine mapping HUS QTLs and determining the genetic relationship between silicon content and yield traits in rice.

By using a set of recombinant inbred line (RIL) population involving in 195 lines derived from a cross of Zhenshan 97B (lowland variety) and IRAT109 (upland variety), the correlation analysis between coleoptile length (CL) and drought resistance index (DRI) and their QTL identification were conducted. There existed a significantly positive relationship between CL and DRI with the correlation coefficient of 0.2206** under water stress conditions. Under normal and water stress conditions, a total of eleven and four QTLs for CL and DRI, respectively, were detected on chromosomes 1, 2, 4, 5, 6, 7, 9, 11 and 12 by using a linkage map including 213 SSR markers, which explained 4.84% to 22.65% of phenotypic variance. Chromosomes 1 and 9 possessing the QTLs for DRI harbored simultaneously QTLs for CL, and qCL9 shared the same chromosome location with qDRI9 (RM160-RM215). Comparing the QTLs related to drought resistance in other studies, QTLs for CL and DRI were located in the same or adjacent marker interval as those related to root traits, such as number, dry weight, depth, and length of root. Moreover, sixteen and three pairs of epistatic loci for CL and DRI were found, which accounted for 56.17% and 11.93% of the total variation in CL and DRI, respectively.

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. 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.

Polarity formation is central to leaf morphogenesis, and several key genes that function in adaxial-abaxial polarity establishment have been identified and characterized extensively. We previously reported that Arabidopsis thaliana ASYMMERTIC LEAVES1 (AS1) and AS2 are important in promoting leaf adaxial fates. We obtained an as2 enhancer mutant, asymmetric leaves enhancer3 (ae3), which demonstrated pleiotropic plant phenotypes, including a defective adaxial identity in some leaves. The ae3 as2 double mutant displayed severely abaxialized leaves, which were accompanied by elevated levels of leaf abaxial promoting genes FILAMENTOUS FLOWER, YABBY3, KANADI1 (KAN1), and KAN2 and a reduced level of the adaxial promoting gene REVOLUTA. We identified AE3, which encodes a putative 26S proteasome subunit RPN8a. Furthermore, double mutant combinations of as2 with other 26S subunit mutations, including rpt2a, rpt4a, rpt5a, rpn1a, rpn9a, pad1, and pbe1, all displayed comparable phenotypes with those of ae3 as2, albeit with varying phenotypic severity. Since these mutated genes encode subunits that are located in different parts of the 26S proteasome, it is possible that the proteolytic function of the 26S holoenzyme is involved in leaf polarity formation. Together, our findings reveal that posttranslational regulation is essential in proper leaf patterning.

Quantitative trait loci (QTLs) analysis for Si content in rice (Oryza sativa L.) was performed to study the inheritance of Si content in rice. A recombinant inbred line (RIL) population of 244 lines derived from the indica rice cross 'Zhenshan 97B' (ZS97B)/'Milyang 46' (MY46) were grown in 2003 with two replications. Silicon content in the hull (HUS), flag leaf (FLS), and stem (STS) were measured. On the basis of a linkage map consisting of 207 DNA marker loci, a total of 10 QTLs showing significant additive effects and 14 digenic interactions having significant additive-by-additive (AA) epistatic effects were detected. The number of QTLs and AA interactions for individual traits were four and four for HUS, four and four for FLS, and two and six for STS, respectively. General contributions to the phenotype variance due to additive effects and AA effects were 29.3 and 18.6% for HUS, 14.8 and 13.6% for FLS, and 8.6 and 28.6% for STS, respectively. This indicates that gene actions at both the one- and two-locus levels play an important role to genetically control Si content in rice. In addition, it was shown that the detection of QTLs at the one-locus level, as well as magnitude and direction of the additive effect, might be influenced greatly by digenic interactions involving loci linked to the given QTL. By comparing these results with other reports, genetic association between QTLs for Si content and QTLs for yield component traits and lodging-tolerance related traits were evident.

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.

Warty fruit is one of the highly valuable external quality traits related to the market values of cucumber. Genetic analysis has shown that a single dominant gene, Tu (Tuberculate fruit), determines the warty fruit trait in the cucumber plant. An F(2) population (247 individuals) from the cross of S06 x S52 was used for the mapping of the Tu/tu locus. By combining bulked segregant analysis with the sequence-related amplified polymorphism (SRAP) and simple sequence repeat (SSR) markers, 15 markers (9 SRAPs and 6 SSRs) linked to the Tu/tu locus were identified. Of nine SRAP markers, three closely linked to the Tu/tu locus were successfully converted into sequence characterized amplified region (SCAR) markers. The Tu/tu locus was mapped between the co-dominant SSR marker SSR16203 and the SCAR marker C_SC933, at a genetic distance of 1.4 and 5.9 cM, respectively. Then the linked SSR markers in the study were used as anchor loci to locate the Tu/tu locus on cucumber chromosome 5. Moreover, the validity analysis of the C_SC69 and C_SC24 markers was performed with 62 cucumber lines of diverse origins, showing that the two SCAR markers can be used for marker-assisted selection (MAS) of the warty fruit trait in cucumber breeding. The information provided in this study will facilitate the map-based cloning of the Tu/tu gene.