Watermelon domestication was shaped by stepwise selection and regulation of the metabolome.

Although crop domestication has greatly aided human civilization, the sequential domestication and regulation of most quality traits remain poorly understood. Here, we report the stepwise selection and regulation of major fruit quality traits that occurred during watermelon evolution. The levels of fruit cucurbitacins and flavonoids were negatively selected during speciation, whereas sugar and carotenoid contents were positively selected during domestication. Interestingly, fruit malic acid and citric acid showed the opposite selection trends during the improvement. We identified a novel gene cluster (CGC1, cucurbitacin gene cluster on chromosome 1) containing both regulatory and structural genes involved in cucurbitacin biosynthesis, which revealed a cascade of transcriptional regulation operating mechanisms. In the CGC1, an allele caused a single nucleotide change in ClERF1 binding sites (GCC-box) in the promoter of ClBh1, which resulted in reduced expression of ClBh1 and inhibition of cucurbitacin synthesis in cultivated watermelon. Functional analysis revealed that a rare insertion of 244 amino acids, which arose in C. amarus and became fixed in sweet watermelon, in ClOSC (oxidosqualene cyclase) was critical for the negative selection of cucurbitacins during watermelon evolution. This research provides an important resource for metabolomics-assisted breeding in watermelon and for exploring metabolic pathway regulation mechanisms.

Mapping and functional verification of leaf yellowing genes in watermelon during whole growth period.

Increasing light energy utilization efficiency is an effective way to increase yield and improve quality of watermelon. Leaf is the main place for photosynthesis, and the color of leaf is directly related to the change of photosynthesis. In addition, leaf yellowing can be used as a marker trait to play an important role in watermelon hybrid breeding and improve seed breeding. It can not only be used to eliminate hybrids at seedling stage, but also be used to determine seed purity. In this study, transcriptome analysis was first carried out using the whole growth period leaf yellowing watermelon mutant w-yl and inbred line ZK, and identified 2,471 differentially expressed genes (DEGs) in the comparison group w-yl-vs-ZK. Among the top 20 terms of the gene ontology (GO) enrichment pathway, 17 terms were related to photosynthesis. KEGG pathway enrichment analysis showed that the most abundant pathway was photosynthesis-antenna proteins. The F2 population was constructed by conventional hybridization with the inbred line ZK. Genetic analysis showed that leaf yellowing of the mutant was controlled by a single recessive gene. The leaf yellowing gene of watermelon located between Ind14,179,011 and InD16,396,362 on chromosome 2 by using indel-specific PCR markers, with a region of 2.217 Mb. In the interval, it was found that five genes may have gene fragment deletion in w-yl, among which Cla97C02G036010, Cla97C02G036030, Cla97C02G036040, Cla97C02G036050 were the whole fragment loss, and Cla97C02G0360 was the C-terminal partial base loss. Gene function verification results showed that Cla97C02G036040, Cla97C02G036050 and Cla97C02G036060 may be the key factors leading to yellowing of w-yl leaves.

Transcriptomic and Metabolomic Analysis of the Effects of Exogenous Trehalose on Salt Tolerance in Watermelon (Citrullus lanatus).

Trehalose can effectively protect the biomolecular structure, maintain the balance of cell metabolism, and improve the tolerance to various abiotic stresses in plants. However, the molecular mechanism underlying the improvement in salt tolerance by exogenous trehalose in watermelon (Citrullus lanatus) seedlings is still unclear. To understand these molecular mechanisms, in this study, watermelon seedlings under salt stress were treated with various concentrations of exogenous trehalose. An amount of 20 mM exogenous trehalose significantly improved the physiological status; increased the activities of enzymes such as POD, SOD, and CAT; and increased the K+/Na+ ratio in watermelon seedlings under salt stress. RNA-seq and metabolomic analysis were performed to identify the specifically expressed genes and metabolites after trehalose treatment. Watermelon seedlings were divided into salt stress (CK2), control (CK1) and trehalose treatment (T) groups as per the treatment. Overall, 421 shared differentially expressed genes (DEGs) were identified in the two comparison groups, namely CK2-CK1 and T-CK2. Functional annotation and enrichment analysis revealed that the DEGs were mainly involved in MAPK signaling pathway for plant hormone signal transduction and phenylpropanoid biosynthesis. Furthermore, 129 shared differential expressed metabolites (DEMs) were identified in the two comparison groups using liquid chromatography-mass spectrometry, which were mainly involved in the metabolic pathway and phenylpropanoid biosynthesis. The combined transcriptomic and metabolomic analyses revealed that genes involved in phenylpropanoid biosynthesis, plant hormone signal transduction, and carbohydrate biosynthesis pathways, especially bHLH family transcription factors, played an important role in improving salt tolerance of watermelon seedlings after exogenous trehalose treatment.

Comparative Transcriptome Analysis Reveals Differential Gene Expression in Resistant and Susceptible Watermelon Varieties in Response to Meloidogyne incognita.

M. incognita is a major parasitic plant disease in watermelon production, causing serious economic losses. Although there are many studies on root-knot nematode, the resistance mechanism is still unclear. In this study, in order to fully understand the mechanism of watermelon resistance to root-knot nematode, the relatively strongly resistant 'Hongzi watermelon' variety and the susceptible 'M16' watermelon variety were used as materials, combined with RNA sequencing (RNA-seq), to analyze the expression abundance of resistant and susceptible varieties at 0, 2, 8 and 15 days post-infection (DPI) by M. incognita. The number of differentially expressed genes (DEGs) in the four comparison groups (A0_B0, A1_B1, A2_B2 and A3_B3) was 3645, 2306, 4449 and 2362, respectively, and there were 835 shared DEGs among them. GO annotation and KEGG pathway enrichment analysis showed that 835 DEGs were mainly involved in phenylpropane biosynthesis and carbon metabolism. Furthermore, lignin-biosynthesis-related genes (4CL (4-coumaric acid-CoA ligase), C3H (coumaric acid 3-hydroxylase), CSE (caffeoyl shikimate esterase), COMT (caffeic acid-O-methyltransferase), CCR (cinnamyl CoA reductase) and PRX (peroxidase)), defense-related proteins (UDP-glucoronosyl/UDP-glucosyl transferase, UGT84A13; salicylic acid binding protein, SABP2) and some transcription factors (TFs) were highlighted, which may be potential candidate genes for further analysis in the infection process of M. incognita. These results suggest that watermelon can achieve resistance to M. incognita by increasing the content of lignin and phenols in root or improving ROS level. These RNA-seq data provide new knowledge for future functional studies and will be helpful to further elucidate the molecular mechanism of resistance to M. incognita in watermelon.

Comparative Transcriptome Profiling Provides Insights into Plant Salt Tolerance in Watermelon (Citrullus lanatus).

Salt stress seriously reduced the yield and quality of watermelon and restricted the sustainable development of the watermelon industry. However, the molecular mechanism of watermelon in response to salt stress is still unclear. In this study, 150 mmol·L-1 NaCl was used to deal with the seedlings of salt-tolerant and salt-sensitive watermelon varieties. Physiological characteristics showed that salt stress significantly reduced the biomass of watermelon seedlings and the accumulation of K+ in roots and leaves and significantly increased the content of Na+, Cl-, and malondialdehyde (MDA). Compared with the salt-sensitive variety, the salt-tolerant variety had higher K+ accumulation, lower Cl-, Cl- accumulation, and MDA content in roots and leaves. Then, RNA-seq was performed on roots and leaves in normal culture and under 150 mmol·L-1 NaCl treatment. A total of 21,069 genes were identified by RNA-seq analysis, of which 1412 were genes encoding transcription factors (TFs). In the comparison groups of roots and leaves, 122 and 123 shared differentially expressed genes (DEGs) were obtained, respectively. Gene ontology (GO) annotation and KEGG enrichment results showed that there were many identical GO terms and KEGG pathways in roots and leaves, especially the pathways that related to sugar or energy (ATP or NADP+/NADPH). In addition, some DEGs related to salt tolerance were identified, such as plant hormone indole-3-acetic acid (IAA) and gibberellin (GA) signal transduction pathway-related genes, K+/Na+/Ca2+-related genes, lignin biosynthesis-related genes, etc. At the same time, we also identified some TFs related to salt tolerance, such as AP2-EREBP, bZIP, bHLH, MYB, NAC, OFP, TCP, and WRKY and found that these TFs had high correlation coefficients with salt tolerance-related genes, indicating that they might have a potential regulatory relationship. Interestingly, one TCP TF (Cla97C09G174040) co-exists both in roots and leaves, and it is speculated that it may be regulated by miR319 to improve the salt tolerance of watermelon.

Low Expression of Long Noncoding RNA SLC26A4 Antisense RNA 1 Is an Independent Prognostic Biomarker and Correlate of Immune Infiltrates in Breast Cancer.

BACKGROUND Aberrant expression of long noncoding RNA (lncRNA) SLC26A4 antisense RNA 1 (SLC26A4-AS1) plays an important role in some cancer types. However, the clinical significance of SLC26A4-AS1 in patients with breast cancer (BC) and the possible regulatory mechanisms of SLC26A4-AS1 are unclear. MATERIAL AND METHODS Statistical analysis was used to assess the correlation between SLC26A4-AS1 expression and patients' clinical characteristics. The Kaplan-Meier method and Cox regression analysis were used to assess the correlation between SLC26A4-AS1 expression and prognosis. Gene set enrichment analysis (GSEA) and immuno-infiltration analysis were used to investigate the possible regulatory mechanisms of SLC26A4-AS1. RESULTS Low SLC26A4-AS1 expression in BC was associated with age (P<0.001), estrogen-receptor status (P<0.001), PAM50 (P<0.001), and menopause status (P<0.001). Low SLC26A4-AS1 expression predicted a poorer overall survival (OS) (hazard ratio [HR]: 0.56; 95% confidence interval [CI]: 0.40-0.78; P=0.001) and disease-specific survival (DSS) (HR: 0.57; 95% CI: 0.37-0.88; P=0.011). Also, SLC26A4-AS1 expression (HR: 0.298; 95% CI: 0.154-0.579; P<0.001) was independently correlated with OS in patients with BC. SLC26A4-AS1 was related to CYP2E1 reactions, protein export, mitochondrial_ciii_assembly, formation of adenosine triphosphate by chemiosmotic coupling, budding and maturation of HIV virion, cristae formation, biocarta proteasome pathway, endosomal sorting complex required for transport, and histone modification. SLC26A4-AS1 expression was associated with some types of immune infiltrating cells. CONCLUSIONS SLC26A4-AS1 expression was significantly associated with poor survival and immune infiltration in patients with BC. It may be a promising prognostic biomarker for BC.

Genome-Wide Identification and Characterization of the Trehalose-6-phosphate Synthetase (TPS) Gene Family in Watermelon (Citrullus lanatus) and Their Transcriptional Responses to Salt Stress.

With the increase in watermelon cultivation area, there is an urgent need to explore enzymatic and genetic resources for the sustainable development of watermelon, especially under salt stress. Among the various compounds known, trehalose plays an important role in regulating abiotic stress tolerances in diverse organisms, including plants. Therefore, the present study comprehensively analyzed the trehalose-6-phosphate synthase (TPS) gene family in watermelon. The study analyzed the functional classification, evolutionary characteristics, and expression patterns of the watermelon TPS genes family. Seven ClTPSs were identified and classified into two distinct classes according to gene structure and phylogeny. Evolutionary analysis suggested the role of purifying selection in the evolution of the TPS family members. Further, cis-acting elements related to plant hormones and abiotic stress were identified in the promoter region of the TPS genes. The tissue-specific expression analysis showed that ClTPS genes were widely expressed in roots, stems, leaves, flowers, and fruits, while ClTPS3 was significantly induced under salt stress. The overexpression of ClTPS3 in Arabidopsis thaliana significantly improved salt tolerance. Finally, the STRING functional protein association networks suggested that the transcription factor ClMYB and ClbHLH regulate ClTPS3. Thus, the study indicates the critical role of ClTPS3 in watermelon response to salt stress.

A comprehensive genome variation map of melon identifies multiple domestication events and loci influencing agronomic traits.

Melon is an economically important fruit crop that has been cultivated for thousands of years; however, the genetic basis and history of its domestication still remain largely unknown. Here we report a comprehensive map of the genomic variation in melon derived from the resequencing of 1,175 accessions, which represent the global diversity of the species. Our results suggest that three independent domestication events occurred in melon, two in India and one in Africa. We detected two independent sets of domestication sweeps, resulting in diverse characteristics of the two subspecies melo and agrestis during melon breeding. Genome-wide association studies for 16 agronomic traits identified 208 loci significantly associated with fruit mass, quality and morphological characters. This study sheds light on the domestication history of melon and provides a valuable resource for genomics-assisted breeding of this important crop.