Huanle He

Construction of a cucumber genetic linkage map with SRAP markers and location of the genes for lateral branch traits.

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 (lbn) and the lateral branch average length (lbl) in cucumber were identified by QTLMapper1.6. A major QTL lbn1 located between ME11SA4B and ME5EM5 in LG2 could explain 10.63% of the total variation with its positively effecting allele from S06. A major QTL lbl1 located between DC1OD3 and DC1EM14 in LG2 could account for 10.38% of the total variation with its positively effecting allele from S06.

Tuberculate fruit gene Tu encodes a C2H2 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.

Micro‐trichome as a class I homeodomain‐leucine zipper gene regulates multicellular trichome development in Cucumis sativus

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.

A High-Density Genetic Linkage Map for Cucumber (Cucumis sativus L.): Based on Specific Length Amplified Fragment (SLAF) Sequencing and QTL Analysis of Fruit Traits in Cucumber

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.

Transcriptome profiling of trichome-less reveals genes associated with multicellular trichome development in Cucumis sativus

Trichomes on plants, similar to fine hairs on animal and human bodies, play important roles in plant survival and development. They also represent a useful model for the study of cell differentiation. Although the regulatory gene network of unicellular trichome development in Arabidopsis thaliana has been well studied, the genes that regulate multicellular trichome development remain unclear. We confirmed that Cucumis sativus (cucumber) trichomes are multicellular and unbranched, but identified a spontaneous mutant, trichome-less (tril), which presented a completely glabrous phenotype. We compared the transcriptome profilings of the tril mutant and wild type using the Illumina HiSeq 2000 sequencing technology. A total of 991 genes exhibited differential expression: 518 were up-regulated and 473 were down-regulated. We further identified 62 differentially expressed genes that encoded crucial transcription factors and were subdivided into seven categories: homeodomain, MADS, MYB, and WRKY domains, ethylene-responsive, zinc finger, and other transcription factor genes. We further analyzed the tissue-expression profiles of two candidate genes, GLABRA2-like and ATHB51-like, using qRT-PCR and found that these two genes were specifically expressed in the epidermis and trichomes, respectively. These results and the tril mutant provide useful tools to study the molecular networks associated with multicellular trichome development.

Loss-of-Function Mutations in CsMLO1 Confer Durable Powdery Mildew Resistance in Cucumber (Cucumis sativus L.).

Powdery mildew (PM) is a serious fungal disease of cucumber worldwide. The identification of resistance genes is very important for resistance breeding to ensure cucumber production. Here, natural loss-of-function mutations at an MLO homologous locus, CsMLO1, were found to confer durable PM resistance in cucumber. CsMLO1 encoded a cell membrane protein, was mainly expressed in leaves and cotyledons, and was up-regulated by PM at the early stage of host–pathogen interaction. Ectopic expression of CsMLO1 rescued the phenotype of the PM resistant Atmlo2 Atmlo12 double mutant to PM susceptible in Arabidopsis. Domesticated and wild resistant cucumbers originating from various geographical regions of the world were found to harbor three independent natural mutations that resulted in CsMLO1 loss of function. In addition, between the near-isogenic lines (NILs) of PM resistant and susceptible, S1003 and NIL(Pm5.1), quantitative RT-PCR revealed that there is no difference at expression levels of several genes in the pathways of ethylene, jasmonic acid or salicylic acid. Moreover, the two NILs were used for transcriptome profiling to explore the mechanism underlying the resistance. Several genes correlated with plant cell wall thickening are possibly involved in the PM resistance. This study revealed that loss of function of CsMLO1 conferred durable PM resistance, and that this loss of function is necessary but alone may not be sufficient for PM resistance in cucumber. These findings will facilitate the molecular breeding of PM resistant varieties to control this destructive disease in cucumber.

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.

The Cucumber Lateral Suppressor Gene (CLS) Is Functionally Associated with Axillary Meristem Initiation

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.

Ectopic Expression of CsCTR1, a Cucumber CTR-Like Gene, Attenuates Constitutive Ethylene Signaling in an Arabidopsis ctr1-1 Mutant and Expression Pattern Analysis of CsCTR1 in Cucumber (Cucumis sativus)

The gaseous plant hormone ethylene regulates many aspects of plant growth, development and responses to the environment. Constitutive triple response 1 (CTR1) is a central regulator involved in the ethylene signal transduction pathway. To obtain a better understanding of this particular pathway in cucumber, the cDNA-encoding CTR1 (designated CsCTR1) was isolated from cucumber. A sequence alignment and phylogenetic analyses revealed that CsCTR1 has a high degree of homology with other plant CTR1 proteins. The ectopic expression of CsCTR1 in the Arabidopsis ctr1-1 mutant attenuates constitutive ethylene signaling of this mutant, suggesting that CsCTR1 indeed performs its function as negative regulator of the ethylene signaling pathway. CsCTR1 is constitutively expressed in all of the examined cucumber organs, including roots, stems, leaves, shoot apices, mature male and female flowers, as well as young fruits. CsCTR1 expression gradually declined during male flower development and increased during female flower development. Additionally, our results indicate that CsCTR1 can be induced in the roots, leaves and shoot apices by external ethylene. In conclusion, this study provides a basis for further studies on the role of CTR1 in the biological processes of cucumber and on the molecular mechanism of the cucumber ethylene signaling pathway.

QTL molecular marker location of powdery mildew resistance in cucumber (Cucumis sativus L.)

The cucumber lines, S94 (Northern China open-field type, powdery mildew (PM) susceptible) and S06 (European greenhouse type, PM resistant), and their F6:7 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 F6 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 (<5 cM) could lay a basis for both molecular marker-assisted breeding and map-based gene cloning of the PM-resistance gene in cucumber.