Chromosome-level genome assembly and population genomics reveals crucial selection for subgynoecy development in chieh-qua.

Chieh-qua is an important cucurbit crop and very popular in South China and Southeast Asia. Despite its significance, its genetic basis and domestication history are unclear. In this study, we have successfully generated a chromosome-level reference genome assembly for the chieh-qua 'A36' using a hybrid assembly strategy that combines PacBio long reads and Illumina short reads. The assembled genome of chieh-qua is approximately 953.3 Mb in size and is organized into 12 chromosomes, with contig N50 of 6.9 Mb and scaffold N50 of 68.2 Mb. Notably, the chieh-qua genome is comparable in size to the wax gourd genome. Through gene prediction analysis, we have identified a total of 24 593 protein-coding genes in the A36 genome. Additionally, approximately 56.6% (539.3 Mb) of the chieh-qua genome consists of repetitive sequences. Comparative genome analysis revealed that chieh-qua and wax gourd are closely related, indicating a close evolutionary relationship between the two species. Population genomic analysis, employing 129 chieh-qua accessions and 146 wax gourd accessions, demonstrated that chieh-qua exhibits greater genetic diversity compared to wax gourd. We also employed the GWAS method to identify related QTLs associated with subgynoecy, an interested and important trait in chieh-qua. The MYB59 (BhiCQ0880026447) exhibited relatively high expression levels in the shoot apex of four subgynoecious varieties compared with monoecious varieties. Overall, this research provides insights into the domestication history of chieh-qua and offers valuable genomic resources for further molecular research.

A nonsynonymous mutation in BhLS, encoding an acyl-CoA N-acyltransferase leads to fruit and seed size variation in wax gourd (Benincasa hispida).

Wax gourd (Benincasa hispida (Thunb.) Cogn., 2n = 2x = 24) is an economically important vegetable crop cultivated widely in many tropical and subtropical regions, including China, India, and Japan. Both fruit and seeds are prized agronomic attributes in wax gourd breeding and production. However, the genetic mechanisms underlying these traits remain largely unexplored. In this study, we observed a strong correlation between fruit size and seed size variation in our mapping population, indicating genetic control by a single gene, BhLS, with large size being dominant over small. Through bulk segregant analysis sequencing and fine mapping with a large F2 population, we precisely located the BhLS gene within a 47.098-kb physical interval on Chromosome 10. Within this interval, only one gene, Bhi10M000649, was identified, showing homology to Arabidopsis HOOKLESS1. A nonsynonymous mutation (G to C) in the second exon of Bhi10M000649 was found to be significantly associated with both fruit and seed size variation in wax gourd. These findings collectively highlight the pleiotropic effect of the BhLS gene in regulating fruit and seed size in wax gourd. Our results offer molecular insights into the variation of fruit and seed size in wax gourd and establish a fundamental framework for breeding wax gourd cultivars with desired traits.

Fine mapping and candidate gene analysis of gynoecy trait in chieh-qua (Benincasa hispida Cogn. var. chieh-qua How).

Gynoecy demonstrates an earlier production of hybrids and a higher yield and improves the efficiency of hybrid seed production. Therefore, the utilization of gynoecy is beneficial for the genetic breeding of chieh-qua. However, little knowledge of gynoecious-related genes in chieh-qua has been reported until now. Here, we used an F2 population from the cross between the gynoecious line 'A36' and the monoecious line 'SX' for genetic mapping and revealed that chieh-qua gynoecy was regulated by a single recessive gene. We fine-mapped it into a 530-kb region flanked by the markers Indel-3 and KASP145 on Chr.8, which harbors eight candidate genes. One of the candidate genes, Bhi08G000345, encoding networked protein 4 (CqNET4), contained a non-synonymous SNP resulting in the amino acid substitution of isoleucine (ATA; I) to methionine (ATG; M). CqNET4 was prominently expressed in the female flower, and only three genes related to ethylene synthesis were significantly expressed between 'A36' and 'SX.' The results presented here provide support for the CqNET4 as the most likely candidate gene for chieh-qua gynoecy, which differed from the reported gynoecious genes.

A chromosome-level reference genome of the wax gourd (Benincasa hispida).

The wax gourd (Benincasa hispida), the only species in the genus Benincasa, is an important crop native to Asia that has been widely planted for multi-purpose uses. The first wax gourd draft genome was published three years ago, but it was incomplete and highly-fragmented due to data and technical limitations. Herein, we report a new chromosome-level genome assembly and annotation of B. hispida. We generated 974.87 Mb of unitigs with N50 size of 2.43 Mb via a hybrid assembly strategy by using PacBio long reads and Illumina short reads. We then joined them into scaffolds with Hi-C data, resulting 1862 scaffolds with a total length of 975.62 Mb, and 94.92% of the length (926.05 Mb) is contained in the 12 largest scaffolds corresponding to the 12 chromosomes of B. hispida. We predicted 37,092 protein-coding genes, and 85.05% of them were functionally annotated. This chromosome-level reference genome provides significant improvement to the earlier version of draft genome and would be valuable resource for research and molecular breeding of the wax gourd.

Genome-Wide Identification of the SAUR Gene Family in Wax Gourd (Benincasa hispida) and Functional Characterization of BhSAUR60 during Fruit Development.

The wax gourd (Benincasa hispida) is an important vegetable crop whose fruits contain nutrients and metabolites. Small auxin upregulated RNA (SAUR) genes constitute the largest early auxin-responsive gene family and regulate various biological processes in plants, although this gene family has not been studied in the wax gourd. Here, we performed genome-wide identification of the SAUR gene family in wax gourds and analyzed their syntenic and phylogenetic relationships, gene structures, conserved motifs, cis-acting elements, and expression patterns. A total of 68 SAUR (BhSAUR) genes were identified, which were distributed on nine chromosomes with 41 genes in two clusters. More than half of the BhSAUR genes were derived from tandem duplication events. The BhSAUR proteins were classified into seven subfamilies. BhSAUR gene promoters contained cis-acting elements involved in plant hormone and environmental signal responses. Further expression profiles showed that BhSAUR genes displayed different expression patterns. BhSAUR60 was highly expressed in fruits, and overexpression led to longer fruits in Arabidopsis. In addition, the plants with overexpression displayed longer floral organs and wavy stems. In conclusion, our results provide a systematic analysis of the wax gourd SAUR gene family and facilitate the functional study of BhSAUR60 during wax gourd fruit development.

Genome-wide association study and genetic mapping of BhWAX conferring mature fruit cuticular wax in wax gourd.

BACKGROUND: Wax gourd [Benincasa hispida (Thunb) Cogn. (2n = 2x = 24)] is an economically important vegetable crop of genus Benincasa in the Cucurbitaceae family. Fruit is the main consumption organ of wax gourd. The mature fruit cuticular wax (MFCW) is an important trait in breeding programs, which is also of evolutionary significance in wax gourd. However, the genetic architecture of this valuable trait remains unrevealed. RESULTS: In this study, genetic analysis revealed that the inheritance of MFCW was controlled by a single gene, with MFCW dominant over non-MFCW, and the gene was primarily named as BhWAX. Genome-wide association study (GWAS) highlighted a 1.1 Mb interval on chromosome 9 associated with MFCW in wax gourd germplasm resources. Traditional fine genetic mapping delimited BhWAX to a 0.5 Mb region containing 12 genes. Based on the gene annotation, expression analysis and co-segregation analysis, Bhi09G001428 that encodes a membrane bound O-acyltransferase (MBOAT) was proposed as the candidate gene for BhWAX. Moreover, it was demonstrated that the efficiency of a cleaved amplified polymorphic sequences (CAPS) marker in the determination of MFCW in wax gourd reached 80%. CONCLUSIONS: In closing, the study identified the candidate gene controlling MFCW and provided an efficient molecular marker for the trait in wax gourd for the first time, which will not only be beneficial for functional validation of the gene and marker-assisted breeding of wax gourd, but also lay a foundation for analysis of its evolutionary meaning among cucurbits.

A time-course transcriptome analysis of wax gourd fruit development reveals predominant genes regulating taste and nutrition.

Wax gourd, which belongs to Cucurbitaceae, is an excellent plant resource with the concomitant function of both medicine and foodstuff. Its unique taste and rich nutrition are deeply accepted by consumers. However, the main flavor and nutrients are still unclear, which restricts the quality breeding process of wax gourd. Here, we discovered that monosaccharides, malic acid and citrulline affect the flavor and nutrition of wax gourd and clarified the dynamic accumulation process of these metabolites. To gain insights into the underlying predominant genes regulating accumulation of these metabolites, we performed a time-course transcriptome analysis using RNA-sequencing analysis and compared the expression of screened genes among twenty-four germplasms with different metabolites levels. In addition, the expression abundance among the homologous genes were also analyzed. Finally, a total of 8 genes related to sugar [AGA2 (Bhi03G001926), SUS (Bhi12G001032)], malic acid [MDH (Bhi12G001426, Bhi01G000427), PEPC (Bhi12G000721, Bhi09G002867), ME (Bhi01G002616)] and citrulline [ASS (Bhi02G000401)], respectively were determined. In summary, understanding the core genes influencing taste or nutrition will provide a theoretical basis for fruit quality improvement in wax gourd.

Genetic mapping and genome-wide association study identify BhYAB4 as the candidate gene regulating seed shape in wax gourd (Benincasa hispida).

Wax gourd is an important vegetable crop of the Cucurbitaceae family. According to the shape and structure of the seed coat, the seeds of the wax gourd can be divided into bilateral and unilateral. Bilateral seeds usually germinate quickly and have a high germination rate than unilateral seeds. Thereby, wax gourd varieties with bilateral seeds are more welcomed by seed companies and growers. However, the genetic basis and molecular mechanism regulating seed shape remain unclear in the wax gourd. In this study, the genetic analysis demonstrated that the seed shape of wax gourd was controlled by a single gene, with bilateral dominant to unilateral. Combined with genetic mapping and genome-wide association study, Bhi04G000544 (BhYAB4), encoding a YABBY transcription factor, was identified as the candidate gene for seed shape determination in the wax gourd. A G/A single nucleotide polymorphism variation of BhYAB4 was detected among different germplasm resources, with BhYAB4G specifically enriched in bilateral seeds and BhYAB4A in unilateral seeds. The G to A mutation caused intron retention and premature stop codon of BhYAB4. Expression analysis showed that both BhYAB4G and BhYAB4A were highly expressed in seeds, while the nuclear localization of BhYAB4A protein was disturbed compared with that of BhYAB4G protein. Finally, a derived cleaved amplified polymorphic sequence marker that could efficiently distinguish between bilateral and unilateral seeds was developed, thereby facilitating the molecular marker-assisted breeding of wax gourd cultivars.

A transcriptome analysis of Benincasa hispida revealed the pathways and genes involved in response to Phytophthora melonis infection.

Wilt disease caused by Phytophthora melonis infection is one of the most serious threats to Benincasa hispida production. However, the mechanism of the response of B. hispida to a P. melonis infection remains largely unknown. In the present study, two B. hispida cultivars with different degrees of resistance to P. melonis were identified: B488 (a moderately resistant cultivar) and B214 (a moderately susceptible cultivar). RNA-seq was performed on P. melonis-infected B488 and B214 12 hours post infection (hpi). Compared with the control, 680 and 988 DEGs were respectively detected in B488 and B214. A KEGG pathway analysis combined with a cluster analysis revealed that phenylpropanoid biosynthesis, plant-pathogen interaction, the MAPK signaling pathway-plant, and plant hormone signal transduction were the most relevant pathways during the response of both B488 and B214 to P. melonis infection, as well as the differentially expressed genes in the two cultivars. In addition, a cluster analysis of transcription factor genes in DEGs identified four genes upregulated in B488 but not in B214 at 6 hpi and 12 hpi, which was confirmed by qRT-PCR. These were candidate genes for elucidating the mechanism of the B. hispida response to P. melonis infection and laying the foundation for the improvement of B. hispida.

Magnesium Deficiency Reduced the Yield and Seed Germination in Wax Gourd by Affecting the Carbohydrate Translocation.

Magnesium (Mg) is a particular mineral nutrient greatly affecting the size and activity of sink organs. Wax gourd crop with its fruits having fresh weight up to 20-50 kg per single fruit serves as an excellent experimental plant species for better understanding the role of varied Mg nutrition in sink strength and yield formation. This study aimed to investigate the effects of Mg deficiency on fruit yield and seed vigor in wax gourd grown under field conditions. Plants were grown under field conditions until maturity with increasing soil Mg applications. At the beginning of fruit formation, leaves were used to analyze concentrations of sucrose, starch and Mg as well as phloem export of sucrose. At maturity, fruit yield was determined and the seeds collected were used in germination studies and starch analysis. Low Mg supply resulted in a significant impairment in fruit fresh yield, which was closely associated with higher accumulation of starch and sucrose in source leaves and lower amount of sucrose in phloem exudate. Seeds obtained from Mg deficiency plants exhibited lower amount of starch and substantial reduction in both germination capacity and seedling establishment when compared to the seeds from the Mg adequate plants. Our study revealed that magnesium deficiency significantly diminished fruit yield of field-grown wax gourd, most probably by limiting the carbohydrate transport from source organs to developing fruit. Ensuring sufficient Mg supply to plant species with high sink size such as wax gourd, during the reproductive growth stage, is a critical factor for achieving higher fruit yield formation and also better vigor of next-generation seeds.

Understanding the heat resistance of cucumber through leaf transcriptomics.

Heat stress is a major environmental factor limiting plant productivity and quality in agriculture. Cucumber, one of the most important vegetables among cucurbitaceae, prefers to grow in a warm environment. Until now the molecular knowledge of heat stress in cucumber remained unclear. In this study, we performed transcriptome analysis using two diverse genetic cucumber cultivars, L-9 and A-16 grown under normal and heat stress. L-9 displayed heat-tolerance phenotype with higher superoxide dismutase enzyme (SOD) enzyme activity and lower malondialdehyde (MDA) content than A-16 under heat stress. RNA-sequencing revealed that a total of 963 and 2778 genes are differentially expressed between L-9 and A-16 under normal and heat stress respectively. In addition, we found that differentially expressed genes (DEGs) associated with plant hormones signally pathway, transcription factors, and secondary metabolites showed significantly change in expression level after heat stress, which were confirmed by quantitative real-time PCR assay. Our results not only explored several crucial genes involved in cucumber heat resistance, but also provide a new insight into studying heat stress.

Metabolic and transcriptomic analysis of two Cucurbita moschata germplasms throughout fruit development.

BACKGROUND: Pumpkins (Cucurbita moschata; Cucurbitaceae) are valued for their fruits and seeds and are rich in nutrients. Carotenoids and sugar contents, as main feature of pumpkin pulp, are used to determine the fruit quality. RESULTS: Two pumpkin germplasms, CMO-X and CMO-E, were analyzed regarding the essential quality traits such as dry weight, soluble solids, organic acids, carotenoids and sugar contents. For the comparison of fruit development in these two germplasms, fruit transcriptome was analyzed at 5 different developmental stages from 0 d to 40 d in a time course manner. Putative pathways for carotenoids biosynthesis and sucrose metabolism were developed in C. moschata fruit and homologs were identified for each key gene involved in the pathways. Gene expression data was found consistent with the accumulation of metabolites across developmental stages and also between two germplasms. PSY, PDS, ZEP, CRTISO and SUS, SPS, HK, FK were found highly correlated with the accumulation of carotenoids and sucrose metabolites, respectively, at different growth stages of C. moschata as shown by whole transcriptomic analysis. The results of qRT-PCR analysis further confirmed the association of these genes. CONCLUSION: Developmental regulation of the genes associated with the metabolite accumulation can be considered as an important factor for the determination of C. moschata fruit quality. This research will facilitate the investigation of metabolic profiles in other cultivars.

The dynamic transcriptome of pepper (Capsicum annuum) whole roots reveals an important role for the phenylpropanoid biosynthesis pathway in root resistance to Phytophthora capsici.

Phytophthora root rot, caused by the soilborne oomycete pathogen Phytophthora capsici (Leon.), is a devastating disease causing significant losses in pepper production worldwide. To uncover the mechanism of root-mediated resistance to P. capsici we elucidated the dynamic transcriptome of whole pepper roots of the resistant accession CM334 and the susceptible accession NMCA10399 after P. capsici infection at 0, 12 and 36 hpi using RNA-Seq method. We detected that the roots of the resistant CM334 and the susceptible NMCA10399 had different transcriptional responses to P. capsici, suggesting the former activated a response to P. capsici earlier than the latter. KEGG enrichment analysis showed the pathways involved in the synthesis of secondary metabolites were those in which the most DEGs were enriched. Focusing on the gene regulation of phenylpropanoid biosynthesis-related genes, we found genes related to the key enzyme phenylalanine ammonia-lyase (PAL) were activated earlier with greater changes in the resistant accession than in the susceptible one. Moreover, genes related to cinnamoyl-CoA reductase (CCR1) were also upregulated in resistant roots but downregulated with great folder changes in susceptible roots. Briefly, we inferred that the phenylpropanoid biosynthesis pathway, especially cinnamaldehyde and lignin derived from its branches, played significant roles in pepper root resistance to P. capsici. These results provide new insight into root-mediated resistance to P. capsici in pepper.

The wax gourd genomes offer insights into the genetic diversity and ancestral cucurbit karyotype.

The botanical family Cucurbitaceae includes a variety of fruit crops with global or local economic importance. How their genomes evolve and the genetic basis of diversity remain largely unexplored. In this study, we sequence the genome of the wax gourd (Benincasa hispida), which bears giant fruit up to 80 cm in length and weighing over 20 kg. Comparative analyses of six cucurbit genomes reveal that the wax gourd genome represents the most ancestral karyotype, with the predicted ancestral genome having 15 proto-chromosomes. We also resequence 146 lines of diverse germplasm and build a variation map consisting of 16 million variations. Combining population genetics and linkage mapping, we identify a number of regions/genes potentially selected during domestication and improvement, some of which likely contribute to the large fruit size in wax gourds. Our analyses of these data help to understand genome evolution and function in cucurbits.

A high-density genetic map developed by specific-locus amplified fragment (SLAF) sequencing and identification of a locus controlling anthocyanin pigmentation in stalk of Zicaitai (Brassica rapa L. ssp. chinensis var. purpurea).

BACKGROUND: Caixin and Zicaitai (Brassica rapa) belong to Southern and Central China respectively. Zicaitai contains high amount of anthocyanin in leaf and stalk resulting to the purple color. Stalk is the major edible part and stalk color is an economically important trait for the two vegetables. The aim of this study is to construct a high density genetic map using the specific length amplified fragment sequencing (SLAF-seq) technique to explore genetic basis for anthocyanin pigmentation traits via quantitative trait loci (QTL) mapping. RESULTS: We constructed a high generation linkage map with a mapping panel of F2 populations derived from 150 individuals of parental lines "Xianghongtai 01" and "Yinong 50D" with purple and green stalk respectively. The map was constructed containing 4253 loci, representing 10,940 single nucleotide polymorphism (SNP) markers spanning 1030.04 centiMorgans (cM) over 10 linkage groups (LGs), with an average distance between markers of 0.27 cM. Quantitative trait loci (QTL) analysis revealed that a major locus on chromosome 7 and 4 minor QTLs explaining 2.69-61.21% of phenotypic variation (PVE) were strongly responsible for variation in stalk color trait. Bioinformatics analysis of the major locus identified 62 protein-coding genes. Among the major locus, there were no biosynthetic genes related to anthocyanin. However, there were several transcription factors like helix-loop-helix (bHLH) bHLH, MYB in the locus. Seven predicted candidate genes were selected for the transcription level analysis. Only bHLH49 transcription factor, was significantly higher expressed in both stalks and young leaves of Xianghongtai01 than Yinong50D. An insertion and deletion (InDel) marker developed from deletion/insertion in the promoter region of bHLH49 showed significant correlation with the stalk color trait in the F2 population. CONCLUSION: Using the constructed high-qualified linkage map, this study successfully identified QTLs for stalk color trait. The identified valuable markers and candidate genes for anthocyanin accumulation in stalk will provide useful information for molecular regulation of anthocyanin biosynthesis. Overall our findings will lay a foundation for functional gene cloning, marker-assisted selection (MAS) and molecular breeding of important economic traits in B. rapa.

Cloning and characterization of WRKY gene homologs in Chieh-qua (Benincasa hispida Cogn. var. Chieh-qua How) and their expression in response to fusaric acid treatment.

The WRKY transcription factors play an important role in plant resistance for biotic and abiotic stresses. In the present study, we cloned 10 WRKY gene homologs (CqWRKY) in Chieh-qua (Benincasa hispida Cogn. var. Chieh-qua) using the rapid-amplification of cDNA ends (RACE) or homology-based cloning methods. We characterized the structure of these CqWRKY genes. Phylogenetic analysis of these sequences with cucumber homologs suggested possible structural conservation of these genes among cucurbit crops. We examined the expression levels of these genes in response to fusaric acid (FA) treatment between resistant and susceptible Chieh-qua lines with quantitative real-time PCR. All genes could be upregulated upon FA treatment, but four CqWRKY genes exhibited differential expression between resistant and susceptible lines before and after FA application. CqWRKY31 seemed to be a positive regulator while CqWRKY1, CqWRKY23 and CqWRKY53 were negative regulators of fusaric resistance. This is the first report of characterization of WRKY family genes in Chieh-qua. The results may also be useful in breeding Chieh-qua for Fusarium wilt resistance.

High-density genetic map construction and gene mapping of pericarp color in wax gourd using specific-locus amplified fragment (SLAF) sequencing.

BACKGROUND: High-density map is a valuable tool for genetic and genomic analysis. Although wax gourd is a widely distributed vegetable of Cucurbitaceae and has important medicinal and health value, no genetic map has been constructed because of the lack of efficient markers. Specific-locus amplified fragment sequencing (SLAF-seq) is a newly developed high-throughput strategy for large-scale single nucleotide polymorphism (SNP) discovery and genotyping. RESULTS: In our present study, we constructed a high-density genetic map by using SLAF-seq and identified a locus controlling pericarp color in wax gourd. An F2 population of 140 individuals and their two parents were subjected to SLAF-seq. A total of 143.38 M pair-end reads were generated. The average sequencing depth was 26.51 in the maternal line (B214), 27.01 in the parental line (B227), and 5.11 in each F2 individual. When filtering low-depth SLAF tags, a total of 142,653 high-quality SLAFs were detected, and 22,151 of them were polymorphic, with a polymorphism rate of 15.42 %. And finally, 4,607 of the polymorphic markers were selected for genetic map construction, and 12 linkage groups (LGs) were generated. The map spanned 2,172.86 cM with an average distance between adjacent markers for 0.49 cM. The inheritance of pericarp color was also studied, which showed that the pericarp color was controlled by one single gene. And based on the newly constructed high-density map, a single locus locating on chromosome 5 was identified for controlling the pericarp color of wax gourd. CONCLUSIONS: This is the first report of high-density genetic map construction and gene mapping in wax gourd, which will be served as an invaluable tool for gene mapping, marker assisted breeding, map-based gene cloning, comparative mapping and draft genome assembling of wax gourd.

Characterization and chromosomal organization of Ty1-copia retrotransposons in wax gourd.

Wax gourd (2n=2x=24) is an important vegetable species in Cucurbitaceae. Because it can be stored for a very long period of time, it plays an important role in ensuring the annual supply and regulating off-season supply of the vegetables. However, the availability of genetic information about wax gourd is limited. This study aimed to identify the useful genetic information for wax gourd. The conserved domains of reverse transcriptase (RT) genes of Ty1-copia retrotransposons were isolated from the genome of wax gourd using degenerate oligonucleotide primers. A total of twenty eight RT sequences were obtained, which showed high heterogeneity with the similarity ranging from 47.5% to 94.3%. Sixteen (57.1%) of them were found to be defective, being disrupted by stop codons and/or frameshift mutations. These 28 sequences were divided into five subfamilies. The comparative phylogenetic analysis with other Cucurbitaceae species from GenBank database showed that most retrotransposons derived from the same genus tended to cluster together, although there were a few exceptions. These results indicate that both vertical transmission and horizontal transmission are the sources of Ty1-copia retrotransposons in wax gourd. Fluorescent in situ hybridization (FISH) with Ty1-copia retrotransposon sequences as probes revealed that this kind of retrotransposons had a dispersed genomic organization, physically distributed among all the chromosomes of wax gourd, with clusters in the heterochromatin regions. This is the first report of Ty1-copia retrotransposons in wax gourd, which would be helpful for our understanding about the organization and evolutions of wax gourd genome and also provide valuable information for our utilization of wax gourd retrotransposons.

De novo assembly and characterization of the transcriptome, and development of SSR markers in wax gourd (Benicasa hispida).

BACKGROUND: Wax gourd is a widely used vegetable of Cucuribtaceae, and also has important medicinal and health values. However, the genomic resources of wax gourd were scarcity, and only a few nucleotide sequences could be obtained in public databases. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we examined transcriptome in wax gourd. More than 44 million of high quality reads were generated from five different tissues of wax gourd using Illumina paired-end sequencing technology. Approximately 4 Gbp data were generated, and de novo assembled into 65,059 unigenes, with an N50 of 1,132 bp. Based on sequence similarity search with known protein database, 36,070 (55.4%) showed significant similarity to known proteins in Nr database, and 24,969 (38.4%) had BLAST hits in Swiss-Prot database. Among the annotated unigenes, 14,994 of wax gourd unigenes were assigned to GO term annotation, and 23,977 were found to have COG classifications. In addition, a total of 18,713 unigenes were assigned to 281 KEGG pathways. Furthermore, 6,242 microsatellites (simple sequence repeats) were detected as potential molecular markers in wax gourd. Two hundred primer pairs for SSRs were designed for validation of the amplification and polymorphism. The result showed that 170 of the 200 primer pairs were successfully amplified and 49 (28.8%) of them exhibited polymorphisms. CONCLUSION/SIGNIFICANCE: Our study enriches the genomic resources of wax gourd and provides powerful information for future studies. The availability of this ample amount of information about the transcriptome and SSRs in wax gourd could serve as valuable basis for studies on the physiology, biochemistry, molecular genetics and molecular breeding of this important vegetable crop.

[Field resistance of Phytophthora melonis to metalaxyl in South China].

OBJECTIVE: Phytophthora melonis is the casual agent of wax gourd and cucumber Phytophthora blight which becomes a constraint for sustainable production of the related crops. Metalaxyl is one of the principal fungicides for controlling the disease now. The objectives of the present study were: (1) to investigate the baseline sensitivity and field resistance of P. melonis to metalaxyl in South China; (2) to test the occurrence of metalaxyl-resistant mutants from metalaxyl-sensitive wild type strains exposed to the fungicide; and (3) to monitor the development of metalaxyl resistance in P. melonis population. METHODS: Over 400 samples of wax gourd and cucumber Phytophthora blight were collected from Guangxi Zhuang Autonomous Region and Guangdong province during 2007-2010, and 193 strains of P. melonis were isolated and purified. The sensitivity of the isolated strains to metalaxyl was tested using mycelial growth rate method in vitro and floating-leaf-disk method in vivo, respectively. The metalaxyl-sensitive strains were induced on PDA plates containing 10 microg/mL metalaxyl. RESULTS: The sensitive, moderately resistant and resistant strains were recorded as 29.0% , 18.1% and 52.8%, respectively, among 193 tested strains. The frequency and level of resistance of P. melonis from Guangdong were higher than that from Guangxi. The strains from cucumber was generally more resistant to metalaxyl than those from wax gourd. The metalaxyl-resistant strains were frequently detected as predominant populations in most of the sampling sites and the highest resistance index (4226.9) was confirmed. Metalaxyl-resistant (M1r) mutants could be isolated from approximately 60% of the sensitive wild-type strains. The resistance level of the M mutants was 189-407 times higher than that of their sensitive parental strains. The EC50 values of 9 sensitive strains from a sampling site without a record of phenylamide fungicide application ranged from 0.0429 to 0.5461 microg/mL. Their mean EC50 value (0.3200 +/- 0.1617 microg/mL) was considered as the baseline sensitivity of P. melonis to metalaxyl in South China. CONCLUSION: Metalaxyl-resistant strains universally occur in South China, especially in the vegetable-growing areas with a longer history of metalaxyl application. The establishment of the baseline sensitivity of P. melonis to metalaxyl will provide a science-based guide for evaluating and further monitoring resistance of the pathogen to the fungicide.