Genome-wide identification and characterization of SRLK gene family reveal their roles in self-incompatibility of Erigeron breviscapus.

Self-incompatibility (SI) is a reproductive protection mechanism that plants acquired during evolution to prevent self-recession. As the female determinant of SI specificity, SRK has been shown to be the only recognized gene on the stigma and plays important roles in SI response. Asteraceae is the largest family of dicotyledonous plants, many of which exhibit self-incompatibility. However, systematic studies on SRK gene family in Asteraceae are still limited due to lack of high-quality genomic data. In this study, we performed the first systematic genome-wide identification of S-locus receptor like kinases (SRLKs) in the self-incompatible Asteraceae species, Erigeron breviscapus, which is also a widely used perennial medicinal plant endemic to China.52 SRLK genes were identified in the E. breviscapus genome. Structural analysis revealed that the EbSRLK proteins in E. breviscapus are conserved. SRLK proteins from E. breviscapus and other SI plants are clustered into 7 clades, and the majority of the EbSRLK proteins are distributed in Clade I. Chromosomal and duplication analyses indicate that 65% of the EbSRLK genes belong to tandem repeats and could be divided into six tandem gene clusters. Gene expression patterns obtained in E. breviscapus multiple-tissue RNA-Seq data revealed differential temporal and spatial features of EbSRLK genes. Among these, two EbSRLK genes having high expression levels in tongue flowers were cloned. Subcellular localization assay demonstrated that both of their fused proteins are localized on the plasma membrane. All these results indicated that EbSRLK genes possibly involved in SI response in E. breviscapus. This comprehensive genome-wide study of the SRLK gene family in E. breviscapus provides valuable information for understanding the mechanism of SSI in Asteraceae.

CUCUME: An RNA methylation database integrating systemic mRNAs signals, GWAS and QTL genetic regulation and epigenetics in different tissues of Cucurbitaceae.

As an internal modification of transcripts, RNA methylation determines RNA fate by changing RNA-protein binding affinity. In plants, RNA methylation is ubiquitous and is involved in all aspects of RNA post-transcriptional regulation. For instance, long-distance mobile RNAs, strongly influenced by their methylation status, play important roles in plant growth, development and environmental adaptation. Cucumber/pumpkin heterografts are widely used to improve stress tolerance of cucumber and to study mobile RNA signals due to their strong developed vasculature system. Here, we developed the Cucume (Cucurbit RNA methylation, http://cucume.cn/) database for these two important vegetables, cucumber (Cucumis sativus L.) and pumpkin (Cucurbita moschata) with high productivity worldwide. We identified mRNAs harboring 5-methylcytosine (m5C) and N6-methyladenosine (m6A) sites in pumpkin and cucumber at the whole genome level via Methylated RNA Immunoprecipitation sequencing (MeRIP-seq) of different tissues and the vascular exudates. In addition to RNA methylation sites, the Cucume database includes graft-transmissible systemic mRNAs identified in previous studies using cucumber/pumpkin heterografts. The further integration of cucumber genome-wide association analysis (GWAS) and quantitative trait loci (QTL) allows the study of RNA methylation-related genetic and epigenetic regulation in cucurbits. Therefore, the here developed Cucume database will promote understanding the role of cucurbit RNA methylation in RNA mobility and QTL, ultimately benefitting future breeding of agronomic crop germplasms.

Rootstock-scion exchanging mRNAs participate in the pathways of amino acids and fatty acid metabolism in cucumber under early chilling stress.

Cucumber (Cucumis sativus L.) often experiences chilling stress that limits their growth and productivity. Grafting is widely used to improve abiotic stress resistance by alternating a vigorous root system, suggesting there exists systemic signals communication between distant organs. mRNAs are reported to be evolving in fortification strategies by long-distance signaling when plants suffering from chilling stress. However, the potential function of mobile mRNAs alleviating chilling stress in grafted cucumber is still unknown. Here, the physiological changes, mobile mRNAs profiling, transcriptomic and metabolomic changes in above- and underground tissues of all graft combinations of cucumber and pumpkin responding to chilling stress were established and analyzed comprehensively. The co-relationship between the cluster of chilling-induced pumpkin mobile mRNAs with Differentially Expressed Genes (DEGs) and Differentially Intensive Metabolites (DIMs) revealed that four key chilling-induced pumpkin mobile mRNAs were highly related to glycine, serine and threonine synthesis and fatty acid ß-oxidative degradation metabolism in cucumber tissues of heterografts. The verification of mobile mRNAs, potential transport of metabolites and exogenous application of key metabolites of glycerophospholipid metabolism pathway in cucumber seedlings confirmed that the role of mobile mRNAs in regulating chilling responses in grafted cucumber. Our results build a link between the long-distance mRNAs of chilling-tolerant pumpkin and the fatty acid ß-oxidative degradation metabolism of chilling-sensitive cucumber. It helps to uncover the mechanism of signaling interaction between scion and stock responding to chilling tolerant in grafted cucumber.

Transcriptomic and Physiological Analysis Reveal That α-Linolenic Acid Biosynthesis Responds to Early Chilling Tolerance in Pumpkin Rootstock Varieties.

Climate changes especially chilling stress affects cucurbit crops during winter seasonal production. Grafting to pumpkin rootstocks is widely used to improve the vigor of cucurbits, especially cucumber (Cucumis sativus L.) plants, in the face of chilling stress. In our study, multi-disciplinary aspect approaches were used to investigate growth changes of pumpkin under chilling stress. Firstly, the morphological and physiological characteristics of 14 pumpkin (Cucurbita moschata) varieties following different periods of chilling stress was analyzed by using physiological means. Mathematical results of principal component analysis (PCA) with chlorophyll-a, chlorophyll-b, carotenoid contents, chilling injury index and relative electrolyte permeability indicated that relative electrolyte permeability as the primary judgment index was best associated with the comparison of chilling tolerance in pumpkin rootstock varieties. Then, transcriptomic and DCMU (Diuron) application and chlorophyll fluorescence examination analysis of pumpkin leaves revealed that 390 Cucurbita moschata differentially expressed genes (CmoDEGs) that affect photosynthesis were upregulated in leaves. 127 CmoDEGs both in leaves and roots were enriched for genes involved in unsaturated fatty acid metabolism, suggesting that plasma membrane lipids are involved in chilling perception. The results of increased composition of unsaturated fatty acid in leaves and qRT-PCR analysis of relative mRNA abundance confirmed that α-linolenic acid biosynthesis was responding to pumpkin chilling tolerance. The integration of physiological, mathematical bioinformatical and biological analysis results contributes to our understanding of the molecular mechanisms underlying chilling tolerance and its improvement in cucumber grafted on pumpkin rootstocks. It provided an important theoretical basis and reference for further understanding on the impact of climate change on plant physiological changes.

Genetic Mapping and Identification of the Candidate Gene for White Seed Coat in Cucurbita maxima.

Seed coat color is an important agronomic trait of edible seed pumpkin in Cucurbita maxima. In this study, the development pattern of seed coat was detected in yellow and white seed coat accessions Wuminglv and Agol. Genetic analysis suggested that a single recessive gene white seed coat (wsc) is involved in seed coat color regulation in Cucurbita maxima. An F2 segregating population including 2798 plants was used for fine mapping and a candidate region containing nine genes was identified. Analysis of 54 inbred accessions revealed four main Insertion/Deletion sites in the promoter of CmaCh15G005270 encoding an MYB transcription factor were co-segregated with the phenotype of seed coat color. RNA-seq analysis and qRT-PCR revealed that some genes involved in phenylpropanoid/flavonoid metabolism pathway displayed remarkable distinction in Wuminglv and Agol during the seed coat development. The flanking InDel marker S1548 was developed to predict the seed coat color in the MAS breeding with an accuracy of 100%. The results may provide valuable information for further studies in seed coat color formation and structure development in Cucurbitaceae crops and help the molecular breeding of Cucurbita maxima.

Identification of Long-Distance Transmissible mRNA between Scion and Rootstock in Cucurbit Seedling Heterografts.

: Grafting has been widely used to improve plant growth and tolerance in crop production, as well as for clarifying systemic mRNA signaling from donor to recipient tissues in organ-to-organ communication. In this study, we investigated graft partner interaction mechanisms of Cucumis sativus (Csa) and Cucurbita moschata (Cmo) using a large-scale endogenous mRNA transport. The results indicated that most mobile transcripts followed an allocation pathway from source to sink. Gene ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that mRNA mobility functions are universally common and individually specific. Identification of mRNA mobility between distant tissues in heterografts with RT-PCR (reverse transcription PCR), RT-qPCR (reverse transcriptional quantitative real time PCR), and clone sequencing were used to estimate 78.75% of selected mobile transcripts. Integration of bioinformatic analysis and RT-qPCR identification allowed us to hypothesize a scion-to-rootstock-to-scion feedback signal loop of Csa move-down and Cmo move-up mRNAs, where Csa scion move-down mRNAs were involved in carbon fixation and biosynthesis of amino acid pathways, and Cmo root received Csa move-down mRNA and then delivered the corresponding Cmo upward mRNA to scion to improve photosynthesis of cucumber scion. This formed a feedback signal loop of scion-to-rootstock-to scion to explain why pumpkin rootstock enhanced cucumber production in the industry, which was utilized for organ communication and mediates photosynthesis processes in heterograft cucurbit crops.

Genome-Wide Characterization of GRAS Family and Their Potential Roles in Cold Tolerance of Cucumber (Cucumis sativus L.).

Cucumber (Cucumis sativus L.) is one of the most important cucurbit vegetables but is often subjected to stress during cultivation. GRAS (gibberellic acid insensitive, repressor of GAI, and scarecrow) genes encode a family of transcriptional factors that regulate plant growth and development. In the model plant Arabidopsis thaliana, GRAS family genes function in formation of axillary meristem and root radial structure, phytohormone (gibberellin) signal transduction, light signal transduction and abiotic/biological stress. In this study, a gene family was comprehensively analyzed from the aspects of evolutionary tree, gene structure, chromosome location, evolutionary and expression pattern by means of bioinformatics; 37 GRAS gene family members have been screened from cucumber. We reconstructed an evolutionary tree based on multiple sequence alignment of the typical GRAS domain and conserved motif sequences with those of other species (A. thaliana and Solanum lycopersicum). Cucumber GRAS family was divided into 10 groups according to the classification of Arabidopsis and tomato genes. We conclude that tandem and segmental duplication have played important roles in the expansion and evolution of the cucumber GRAS (CsaGRAS) family. Expression patterns of CsaGRAS genes in different tissues and under cold treatment, combined with gene ontology annotation and interaction network analysis, revealed potentially different functions for CsaGRAS genes in response to cold tolerance, with members of the SHR, SCR and DELLA subfamilies likely playing important roles. In conclusion, this study provides valuable information and candidate genes for improving cucumber tolerance to cold stress.

A High-Density EST-SSR-Based Genetic Map and QTL Analysis of Dwarf Trait in Cucurbita pepo L.

As one of the earliest domesticated species, Cucurbita pepo (including squash and pumpkin) is rich in phenotypic polymorphism and has huge economic value. In this research, using 1660 expressed sequence tags-simple sequence repeats (EST-SSRs) and 632 genomic simple sequence repeats (gSSRs), we constructed the highest-density EST-SSR-based genetic map in Cucurbita genus, which spanned 2199.1 cM in total and harbored 623 loci distributed in 20 linkage groups. Using this map as a bridge, the two previous gSSR maps were integrated by common gSSRs and the corresponding relationships around chromosomes in three sets of genomes were also collated. Meanwhile, one large segmental inversion that existed between our map and the C. pepo genome was detected. Furthermore, three Quantitative Trait Loci (QTLs) of the dwarf trait (gibberellin-sensitive dwarf type) in C. pepo were located, and the candidate region that covered the major QTL spanned 1.39 Mb, which harbored a predicted gibberellin 2-ß-oxidase gene. Considering the rich phenotypic polymorphism, the important economic value in the Cucurbita genus species and several advantages of the SSR marker were identified; thus, this high-density EST-SSR-based genetic map will be useful in Pumpkin and Squash breeding work in the future.