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Mesocarp color is an important agronomic trait of Cucurbita maxima and is determined mainly by the contents and compositions of the carotenoids. The two inbred lines with significant differences in fruit flesh color were used in the study, the orange ‘312-1’ and white ‘98-2’. Changes in seven carotenoid contents and compositions in the flesh of fruit produced by inbred lines ‘312-1’ and ‘98-2’ were analyzed during fruit development. The expression of eight key carotenoid biosynthesis genes in the fruit flesh were investigated during five fruit development stage in two inbred lines. As the flesh color intensified, the orange flesh color of ‘312-1’ was determined mainly by the increased contents of lutein, β-carotene and zeaxanthin and the lack of carotenoid accumulation led to the formation of white flesh in ‘98-2’ fruit. The expression of the LCY-e and CHYb genes was significantly stronger in ‘312-1’ than in ‘98-2’, and their expression was strongly correlated with lutein and β-carotene contents during fruit development. This study provides a deep understanding of the molecular mechanism of carotenoid biosynthesis in fruit flesh and provides a basis for additional studies on the highly refined improvement of squash quality.

Powdery mildew resistance in zucchini is controlled by one major dominant locus, CpPM10.1. CpPM10.1 was fine mapped. The expression of candidate gene Cp4.1LG10g02780 in resistant individuals was significantly upregulated after inoculation with the powdery mildew. Powdery mildew (PM) is one of the most destructive fungal diseases, reducing the productivity of Cucurbita crops globally. PM influences the photosynthesis, growth and development of infected zucchini and seriously reduces fruit yield and quality. In the present study, the zucchini inbred line ‘X10′ had highly stable PM resistance, and the inbred line 'Jin234′ was highly susceptible to PM in the seedling stage and adult stages. Genetic analysis revealed that PM resistance in ‘X10′ is controlled by one major dominant locus. Based on the strategy of QTL-seq combined with linkage analysis and developed molecular markers, the major locus was found to be located in a 382.9-kb candidate region on chromosome 10; therefore, the major locus was named CpPM10.1. Using 1,400 F2 individuals derived from a cross between ‘X10′ and ‘JIN234′ and F2:3 offspring of the recombinants, the CpPM10.1 locus was defined in a region of approximately 20.9 kb that contained 5 coding genes. Among them, Cp4.1LG10g02780 contained a conserved domain (RPW8), which controls resistance to a broad range of PM pathogens. Cp4.1LG10g02780 also had nonsynonymous SNPs between the resistant ‘X10′ and susceptible ‘Jin234.′ Furthermore, the expression of Cp4.1LG10g02780 was strongly positively involved in PM resistance in the key period of inoculation. Further allelic diversity analysis in zucchini germplasm resources indicated that PM resistance was associated with two SNPs in the Cp4.1LG10g02780 RPW8 domain. This study not only provides highly stable PM resistance gene resources for cucurbit crops but also lays the foundation for the functional analysis of PM resistance and resistance breeding in zucchini.

Dwarfism is an important agronomic trait in pumpkin that can increase yield. In this study, the dwarf Cucurbita pepo L. line X10 exhibited significantly longitudinally shorter cell length in the stem than did the normal-vine line JIN234. The dwarf stature of X10 was recovered with exogenous gibberellin (GA3) application, suggesting that X10 might be sensitive to GA biosynthesis. Genetic analysis revealed that this dwarf trait is controlled by a single completely dominant locus: CpDw (Cucurbita pepo L. Dwarf). Using 1,300 F2 individuals derived from a cross between X10 and JIN234, we mapped the CpDw locus to a region of approximately 24.6 kb on chromosome 10 that contain 5 annotated genes. The high expression level of Cp4.1LG10g05910.1 and high GA2ox enzyme activity in X10 revealed that the GA 2-oxidase gene Cp4.1LG10g05910.1 is a candidate gene for CpDw. Alignment of the Cp4.1LG10g05910.1 gene revealed two nonsynonymous single nucleotide polymorphism (SNP) mutations in the two exons, as well as several SNPs and InDels in the important functional elements of promoter between parental lines. Further allelic diversity analysis of the Cucurbita spp. germplasm resources indicated that Cp4.1LG10g05910.1 may be involved in vine growth during the early developmental stage in C. pepo but not in C. maxima or C. moschata. This study provides an important theoretical basis for the genetic regulation of vine length and crop breeding in pumpkin.

This study was aimed at revealing the dynamic regulation of mRNAs, long noncoding RNAs (lncRNAs), and microRNAs (miRNAs) in hepatocellular carcinoma (HCC) and to identify HCC biomarkers capable of predicting prognosis. Differentially expressed mRNAs (DEmRNAs), lncRNAs, and miRNAs were acquired by comparing expression profiles of HCC with normal samples, using an expression data set from The Cancer Genome Atlas. Altered biological functions and pathways in HCC were analyzed by subjecting DEmRNAs to Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analysis. Gene modules significantly associated with disease status were identified by weighted gene coexpression network analysis. An lncRNA-mRNA and an miRNA-mRNA coexpression network were constructed for genes in disease-related modules, followed by the identification of prognostic biomarkers using Kaplan-Meier survival analysis. Differential expression and association with the prognosis of 4 miRNAs were verified in independent data sets. A total of 1220 differentially expressed genes were identified between HCC and normal samples. Differentially expressed mRNAs were significantly enriched in functions and pathways related to “plasma membrane structure,” “sensory perception,” “metabolism,” and “cell proliferation.” Two disease-associated gene modules were identified. Among genes in lncRNA-mRNA and miRNA-mRNA coexpression networks, 9 DEmRNAs and 7 DEmiRNAs were identified to be potential prognostic biomarkers. MIMAT0000102, MIMAT0003882, and MIMAT0004677 were successfully validated in independent data sets. Our results may advance our understanding of molecular mechanisms underlying HCC. The biomarkers may contribute to diagnosis in future clinical practice.

Cucurbits (Cucurbitaceae family) include many economically important fruit vegetable crops such as watermelon, pumpkin/squash, cucumber, and melon. Seed size (SS) is an important trait in cucurbits breeding, which is controlled by quantitative trait loci (QTL). Recent advances have deciphered several signaling pathways underlying seed size variation in model plants such as Arabidopsis and rice, but little is known on the genetic basis of SS variation in cucurbits. Here we conducted literature review on seed size QTL identified in watermelon, pumpkin/squash, cucumber and melon, and inferred 14, 9 and 13 consensus SS QTL based on their physical positions in respective draft genomes. Among them, four from watermelon (ClSS2.2, ClSS6.1, ClSS6.2, and ClSS8.2), two from cucumber (CsSS4.1 and CsSS5.1), and one from melon (CmSS11.1) were major-effect, stable QTL for seed size and weight. Whole genome sequence alignment revealed that these major-effect QTL were located in syntenic regions across different genomes suggesting possible structural and functional conservation of some important genes for seed size control in cucurbit crops. Annotation of genes in the four watermelon consensus SS QTL regions identified genes that are known to play important roles in seed size control including members of the zinc finger protein and the E3 ubiquitin-protein ligase families. The present work highlights the utility of comparative analysis in understanding the genetic basis of seed size variation, which may help future mapping and cloning of seed size QTL in cucurbits.

QTL analysis revealed 11 QTL underlying flowering time and fruit size variation in the semi-wild Xishuangbanna cucumber, of which, FT6.2 and FS5.2 played the most important roles in determining photoperiod-dependent flowering time and round-fruit shape, respectively. Flowering time and fruit size are two important traits in domestication and diversifying selection in cucumber, but their genetic basis is not well understood. Here we reported QTL mapping results on flowering time and fruit size with F2 and F2:3 segregating populations derived from the cross between WI7200, a small fruited, early flowering primitive cultivated cucumber and WI7167, a round-fruited, later flowering semi-wild Xishuangbanna (XIS) cucumber. A linkage map with 267 microsatellite marker loci was developed with 138 F2 plants. Phenotypic data of male and female flowering time, fruit length and diameter and three other traits (mature fruit weight and number, and seedling hypocotyl length) were collected in multiple environments. Three flowering time QTL, FT1.1, FT5.1 and FT6.2 were identified, in which FT6.2 played the most important role in conferring less photoperiod sensitive early flowering during domestication whereas FT1.1 seemed more influential in regulating flowering time within the cultivated cucumber. Eight consensus fruit size QTL distributed in 7 chromosomes were detected, each of which contributed to both longitudinal and radial growth in cucumber fruit development. Among them, FS5.2 on chromosome 5 exhibited the largest effect on the determination of round fruit shape that was characteristic of the WI7167 XIS cucumber. Possible roles of these flowering time and fruit size QTL in domestication of cucumber and crop evolution of the semi-wild XIS cucumber, as well as the genetic basis of round fruit shape in cucumber are discussed.

A high-density genetic map can provide a basis for quantitative trait locus (QTL) mapping and marker-assisted selection (MAS) based on single-nucleotide polymorphisms (SNPs). Specific length amplified fragment sequencing (SLAF-seq) has been successfully applied to construct high-density maps of many species but not Cucurbita maxima. In this study, 100 F2 individuals from two morphologically diverse parents, namely “2013-12” and “9-6”, were used to successfully construct a high-density genetic map of C. maxima using the SLAF-seq method. A total of 8,406 SLAF markers were used to construct a 3,376.87 cM genetic map for 20 linkage groups (LGs) with an average genetic distance of 0.47 cM. With these markers, total ten QTLs for seed-related traits were identified with composite interval mapping (CIM) method. The QTLs affecting seed width (SW), seed length (SL) and hundred-seed weight (HSW) explained a maximum 38.6%, 28.9% and 17.2% of the phenotypic variation and were detected in LG6, LG6 and LG17, respectively. To validate the locations determined by SLAF-seq, additional 150 F2 individuals were used for QTL mapping of SW and SL using cleaved amplified polymorphic sequence (CAPS) markers. The major QTL SL6-1 and SW6-1 locations identified by SLAF-seq overlapped the region identified in the F2 population analysis by the CAPS markers. Based on nonsynonymous SNPs and gene descriptions of the major SW- and SL-associated regions identified by SLAF-seq, we found that the VQ motif, E3 ubiquitin-protein ligase and F-box and Leucine Rich Repeat domains containing protein may play roles in SL and SW in C. maxima. This study reports the construction of the first high-density linkage map of C. maxima by using SNPs developed using SLAF-seq technology. The information obtained from the genetic map is a valuable tool for association mapping of important agronomic traits, map-based gene cloning and MAS-based breeding in C. maxima.

In this study, two pumpkin lines were used as experimental materials to study the accumulation of soluble sugars and the expression of related genes during the development of pumpkin fruits. There were differences in the soluble sugars contents and regulatory mechanisms between the two pumpkin lines. The glucose content of ‘312-1’ decreased rapidly in the early stage of fruit development, which may be due to the high expression of Sucrose invertase (INV) and Hexokinase (HK) at this stage. There were significant differences in sucrose contents between mature fruits of ‘312-1’ and ‘98-2’, and the high expression of Sucrose synthase (SUS) and Sucrose phosphate synthase (SPS) led to sucrose accumulation in these pumpkins. The relative expression of SPS in ‘312-1’ was higher than that in ‘98-2’, which led to more sucrose accumulation in the former. In contrast, the Fructokinase (FK), which is responsible for fructose metabolism, was differentially expressed at individual stages of fruit development. These results indicate that INV, SUS, SPS, HK and FK may play significant roles by contributing coordinately to sucrose biosynthesis during pumpkin fruit development. This study provides a deeper understanding of the molecular mechanism of sucrose biosynthesis and a basis for further studies on the improvement of fruit eating quality in pumpkin.

Emerging evidence suggests that microRNAs (miRNAs) play important roles in regulating HCC development and progression; however, the mechanisms by which their specific functions and mechanisms remained to be further explored. miR-129 has been reported in gastric cancers, lung cancer and colon cancer. In this study, we disclosed a new tumor suppresser function of miR-129 in HCC. We also found the downregulation of miR-129 occurred in nearly 3/4 of the tumors examined (56/76) compared with adjacent nontumorous tissues, which was more importantly, correlated to the advanced stage and vascular invasion. We then demonstrated that miR-129 overexpression attenuated HCC cells proliferation and invasion, inducing apoptosis in vitro. Moreover, we used miR-129 antagonist and found that anti-miR-129 promoted HCC cells malignant phenotypes. Mechanistically, our further investigations revealed that miR-129 suppressed cell proliferation and invasion by targeting the 3’-untranslated region of PAK5, as well as miR-129 silencing up-regulated PAK5 expression. Moreover, miR-129 expression was inversely correlated with PAK5 expression in 76 cases of HCC samples. RNA interference of PAK5 attenuated anti-miR-129 mediated cell proliferation and invasion in HCC cells. Taken together, these results demonstrated that miR-129 suppressed tumorigenesis and progression by directly targeting PAK5, defining miR-129 as a potential treatment target for HCC. - Highlights:more » • Decreased of miR-129 is found in HCC and associated with advanced stage and metastasis. • miR-129 suppresses proliferation and invasion of HCC cells. • miR-129 directly targets the 3′ UTR of PAK5 and diminishes PAK5 expression. • PAK5 is involved in miR-129 mediated suppression functions.« less

Seeded pumpkins are important economic crops; the seeds contain various unsaturated fatty acids, such as oleic acid and linoleic acid, which are crucial for human and animal nutrition. The fatty acid desaturase-2 (FAD2) gene encodes delta-12 desaturase, which converts oleic acid to linoleic acid. However, little is known about sequence variations in FAD2 in seeded pumpkins. Twenty-seven FAD2 clones from 27 accessions of Cucurbita moschata, Cucurbita maxima, Cucurbita pepo, and Cucurbita ficifolia were obtained (totally 1152 bp; a single gene without introns). More than 90% nucleotide identities were detected among the 27 FAD2 clones. Nucleotide substitution, rather than nucleotide insertion and deletion, led to sequence polymorphism in the 27 FAD2 clones. Furthermore, the 27 FAD2 selected clones all encoded the FAD2 enzyme (delta-12 desaturase) with amino acid sequence identities from 91.7 to 100% for 384 amino acids. The same main-function domain between 47 and 329 amino acids was identified. The four species clustered separately based on differences in the sequences that were identified using the unweighted pair group method with arithmetic mean. Geographic origin and species were found to be closely related to sequence variation in FAD2.

Cucurbita maxima is one of the most widely cultivated vegetables in China and exhibits distinct morphological characteristics. In this study, genetic linkage analysis with 57 simple-sequence repeats, 21 amplified fragment length polymorphisms, 3 random-amplified polymorphic DNA, and one morphological marker revealed 20 genetic linkage groups of C. maxima covering a genetic distance of 991.5 cM with an average of 12.1 cM between adjacent markers. Genetic linkage analysis identified the simple-sequence repeat marker 'PU078072' 5.9 cM away from the locus 'Rc', which controls rind color. The genetic map in the present study will be useful for better mapping, tagging, and cloning of quantitative trait loci/gene(s) affecting economically important traits and for breeding new varieties of C. maxima through marker-assisted selection.

Sex expression is a complex process, and in-depth knowledge of its mechanism in pumpkin is important. In this study, young shoot apices at the one-true-leaf stage and 10-leaf stage in Cucurbita maxima trimonoecious line &lsquo, 2013&ndash, 12&rsquo, and subandroecious line &lsquo, 9&ndash, 6&rsquo, were collected as materials, and transcriptome sequencing was performed using an Illumina HiSeqTM 2000 System. 496 up-regulated genes and 375 down-regulated genes were identified between shoot apices containing mostly male flower buds and only female flower buds. Based on gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, the differentially expressed genes were mainly enriched in the ethylene and auxin synthesis and signal transduction pathways. In addition, shoot apices at the 4-leaf stage were treated with the ethylene-releasing agent 2-chloroethylphosphonic acid (Ethrel), aminoethoxyvinyl glycine (AVG), AgNO3 and indoleacetic acid (IAA). The number of female flowers up to node 20 on the main stem of &lsquo, increased significantly after Ethrel and IAA treatment and decreased significantly after AVG and AgNO3 treatment. The female flowers in &lsquo, showed slight changes after treatment with the exogenous chemicals. The expression of key genes in ethylene synthesis and signal transduction (CmaACS7, CmaACO1, CmaETR1 and CmaEIN3) was determined using quantitative RT-PCR, and the expression of these four genes was positively correlated with the number of female flowers in &lsquo, The variations in gene expression, especially that of CmaACS7, after chemical treatment were small in &lsquo, From stage 1 (S1) to stage 7 (S7) of flower development, the expression of CmaACS7 in the stamen was much lower than that in the ovary, stigma and style. These transcriptome data and chemical treatment results indicated that IAA might affect pumpkin sex expression by inducing CmaACS7 expression and indirectly affecting ethylene production, and the ethylene synthesis and signal transduction pathways play crucial roles in pumpkin flower sex expression. A possible reason for the differences in sex expression between pumpkin lines &lsquo, and &lsquo, was proposed based on the key gene expression. Overall, these transcriptome data and chemical treatment results suggest important roles for ethylene in pumpkin sex expression.

Seed pumpkin is an important economic crop grown in Heilongjiang Province, China that comprises distinct cultivars of Cucurbita maxima and C. pepo. However, little is known about the genetic diversity of seed pumpkin. Morphological and molecular characterizations were used to investigate the genetic diversity and genetic relationships within a germplasm collection that included 38 Chinese and 28 Russian seed cultivars of C. maxima, and 10 Chinese seed genotypes of C. pepo. The most variable morphological characters were all usually associated with the fruit. Thirty-five SSR markers yielded a total of 85 amplified DNA fragments, of which 85.89% were polymorphic, indicating a high degree of genetic diversity. The information provided by the SSR markers was agreed with the morphological diversity. Principal components analysis (PCA), principal coordinates analysis (PCO), and UPGMA clustering clearly distinguished the accessions based on species and geographic regions for both morphological and molecular characterizations. This indicated that genetic divergence has occurred between germplasm from China and that from Russia. On the other hand, geographical location also appeared to have affected genetic diversity due to adaptation of the plants to the different environments. This finding suggested that molecular information on genetic relationships in combination with morphological characteristics would serve to (1) improve the classification system of seed pumpkin cultivars in cultivation for germplasm conservation, (2) enhance the economic value of seed pumpkin.

HrpNEa is a harpin protein produced by the bacterial plant pathogen Erwinia amylovora. When applied to aerial parts of plants, the protein induces systemic acquired resistance in a variety of plant species. Here, we report that treating Chinese cabbage roots with HrpNEa induces resistance of the plant to Pectobacterium carotovora subsp. carotovora, the pathogen that invades roots and causes bacterial soft rot in cruciferous plants. Treating Chinese cabbage roots with HrpNEa significantly decreased severities of soft rot symptoms on the plant. The root treatment decreased the number of P. carotovora subsp. carotovora cells attached to root surfaces and inhibited the ability of P. carotovora subsp. carotovora to produce quorum-sensing signals, which regulate pathogenicity in a bacterial population-dependent manner. The inhibitory effects of HrpNEa on the root attachment and quorum-sensing signals accompanied the induced expression of several defense response genes. These results suggest that HrpNEa induces Chinese cabbage resistance to bacterial soft rot by inhibiting the bacterial attachment to root surfaces.

This research using Jinhuiyihao (Cucurbita maxima Duch) cotyledon studied many influencing factors by agrobacterium-mediated method. They are pre-culture time, infection time and concentration of acetosyringone (AS), co-infection time and the concentration of carbenicillin (Carb), cefotaxime (Cef) and kanamycine (Kan). Then a suitable genetic transformation system was established. The results indicated that no pre-culturation, infection for 30 min, acetosyringone (AS) with a concentration of 100mg/L and co-infection for 5 days would get high transformation efficiency. The optimal bactericide is Cef, and the medium with Cefotaxime 500 mg/L, kanamycin 100 mg/L. Eventually, culturing the resistance shoots in MS, the result of Southern blot and PCR showed that the kanamycin-resistant foreign gene was integrated into the genome of Cucurbita.

Harpins of phytopathogenic bacteria stimulate defense and plant growth in many types of plants, conferring disease resistance and enhanced yield. In a previous study, we characterized nine fragments of the harpin protein HpaGXooc from Xanthomonas oryzae pv. oryzicola for plant defense elicitation and plant growth stimulation activity relative to the intact protein. In plants grown under controlled conditions, the fragment HpaG10-42 was more active in both regards than HpaGXooc. Here, we demonstrate that the activity of HpaG10-42 in rice under field conditions significantly exceeds that of HpaGXooc, stimulating resistance to three important diseases and increasing grain yield. We carried out tests in 672 experimental plots with nine cultivars of rice planted at three locations. Application protocols were optimized by testing variations in application rate, frequency, and timing with respect to rice growth stage. Of the concentrations (24, 24, 12, and 6 μg/ml), and number and timing of applications (at one to four different stages of growth) tested, HpaG10-42 at 6 μg/ml applied to plants once at nursery seedling stage and three times in the field was most effective. Bacterial blight, rice blast, and sheath blight were reduced 61.6 and 56.4, 93.6 and 76.0, and 93.2 and 55.0% in indica and japonica cultivars, respectively, relative to controls. Grain yields were 22 to 27% greater. These results are similar to results obtained with typical local management practices, including use of chemicals, to decrease disease severities and increase yield in rice. Our results demonstrate that the HpaG10-42 protein fragment can be used effectively to control diseases and increase yield of this staple food crop.

HrpN, a protein produced by the plant pathogenic bacterium Erwinia amylovora, has been shown to stimulate plant growth and resistance to pathogens and insects. Here we report that HrpN activates abscisic acid (ABA) signalling to induce drought tolerance (DT) in Arabidopsis thaliana L. plants grown with water stress. Spraying wild-type plants with HrpN-promoted stomatal closure decreased leaf transpiration rate, increased moisture and proline levels in leaves, and alleviated extents of damage to cell membranes and plant drought symptoms caused by water deficiency. In plants treated with HrpN, ABA levels increased; expression of several ABA-signalling regulatory genes and the important effector gene rd29B was induced or enhanced. Induced expression of rd29B, promotion of stomatal closure, and reduction in drought severity were observed in the abi1-1 mutant, which has a defect in the phosphatase ABI1, after HrpN was applied. In contrast, HrpN failed to induce these responses in the abi2-1 mutant, which is impaired in the phosphatase ABI2. Inhibiting wild-type plants to synthesize ABA eliminated the role of HrpN in promoting stomatal closure and reducing drought severity. Moreover, resistance to Pseudomonas syringae developed in abi2-1 as in wild-type plants following treatment with HrpN. Thus, an ABI2-dependent ABA signalling pathway is responsible for the induction of DT but does not affect pathogen defence under the circumstances of this study.

Harpin proteins from gram-negative plant-pathogenic bacteria can stimulate hypersensitive cell death (HCD) and pathogen defense as well as enhance growth in plants. Two of these diverse activities clearly are beneficial and may depend on particular functional regions of the proteins. Identification of beneficial and deleterious regions might facilitate the beneficial use of harpin-related proteins on crops without causing negative effects like cell death. Here, we report the identification and testing of nine functional fragments of HpaGXooc, a 137-amino-acid harpin protein from Xanthomonas oryzae pv. oryzicola, the pathogen that causes bacterial leaf streak of rice. Polymerase chain reaction-based mutagenesis generated nine proteinaceous fragments of HpaGXooc; these caused different responses following their application to Nicotiana tabacum (tobacco) and Oryza sativa (rice). Fragment HpaG62-137, which spans the indicated amino acid residues of the HpaG, induced more intense HCD; in contrast, HpaG10-42 did not cause evident cell death in tobacco. However, both fragments stimulated stronger defense responses and enhanced more growth in rice than the full-length parent protein, HpaGXooc. Of the nine fragments, the parent protein and one deletion mutant of HpaGXooc tested, HpaG10-42, stimulated higher levels of rice growth and resulted in greater levels of resistance to X. oryzae pv. oryzae and Magnaporthe grisea. These pathogens cause bacterial leaf blight and rice blast, respectively, the two most important diseases of rice world-wide. HpaG10-42 was more active than HpaGXooc in inducing expression of several genes that regulate rice defense and growth processes and activating certain signaling pathways, which may explain the greater beneficial effects observed from treatment with that fragment. Overall, our results suggest that HpaG10-42 holds promise for practical agricultural use to induce disease resistance and enhance growth of rice.