Efficient genetic transformation and CRISPR/Cas9-mediated genome editing of watermelon assisted by genes encoding developmental regulators / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Cucurbitaceae is an important family of flowering plants containing multiple species of important food plants, such as melons, cucumbers, squashes, and pumpkins. However, a highly efficient genetic transformation system has not been established for most of these species (Nanasato and Tabei, 2020). Watermelon (Citrullus lanatus), an economically important and globally cultivated fruit crop, is a model species for fruit quality research due to its rich diversity of fruit size, shape, flavor, aroma, texture, peel and flesh color, and nutritional composition (Guo et al., 2019). Through pan-genome sequencing, many candidate loci associated with fruit quality traits have been identified (Guo et al., 2019). However, few of these loci have been validated. The major barrier is the low transformation efficiency of the species, with only few successful cases of genetic transformation reported so far (Tian et al., 2017; Feng et al., 2021; Wang JF et al., 2021; Wang YP et al., 2021). For example, Tian et al. (2017) obtained only 16 transgenic lines from about 960 cotyledon fragments, yielding a transformation efficiency of 1.67%. Therefore, efficient genetic transformation could not only facilitate the functional genomic studies in watermelon as well as other horticultural species, but also speed up the transgenic and genome-editing breeding.

Rootstock mediates transcriptional regulation of citrulline metabolism in grafted watermelon / Mediação do porta-enxerto na regulação transcricional do metabolismo da citrulina na melancia enxertada

Abstract Citrulline is a non-essential amino acid, involved in key biological functions in plants and humans. Rootstocks have a major impact on citrulline accumulation in grafted watermelon. Information regarding rootstock induced changes in citrulline metabolism is elusive. To understand the regulatory mechanism, parallel changes in the expression profiles of citrulline metabolic genes and citrulline content of watermelon were monitored during the development of self-rooted watermelon and watermelon grafted onto pumpkin, wild and bottle gourd rootstocks. Results demonstrated that rootstocks regulated the expression profiles in different ways to influence the citrulline content. GAT, NAGPR, ASS3 ASS2 and Asl2 showed the negative correlation with citrulline content in pumpkin grafted watermelon. Pumpkin rootstock promoted the citrulline content by high down-regulation and synergistic effect of ASS2, ASS3, ASL1 and ASl2 genes. In wild grafted watermelon, citrulline was accumulated as a result of down regulation of GAT, NAGS and ASL2 genes, which showed an inverse correlation with citrulline. In gourd grafted watermelon, changes in citrulline content were observed to be linked with lower expressions of GAT, NAGK, ASS2, ASS3, ASL1 and ARG which were negatively correlated with citrulline content. Our study will provide the basis to understand the molecular mechanism of citrulline accumulation in various rootstocks.

Resumo A citrulina é um aminoácido não essencial, envolvida em importantes funções biológicas de plantas e seres humanos. Os porta-enxertos têm um grande impacto no acúmulo de citrulina na melancia enxertada. Informações sobre alterações induzidas por porta-enxertos no metabolismo da citrulina ainda não foram descritas. Para entender o mecanismo regulatório, foram monitoradas mudanças paralelas nos perfis de expressão dos genes metabólicos de citrulina e no teor de citrulina da melancia durante o desenvolvimento da melancia e da melancia enxertada em porta-enxertos de abóbora, silvestre e cabaça. Os resultados demonstraram que o porta-enxerto regulou os perfis de expressão de diferentes maneiras para influenciar no conteúdo de citrulina. GAT, NAGPR, ASS3, ASS2 e ASL2 apresentaram correlação negativa com o teor de citrulina em melancia enxertada de abóbora. O porta-enxerto de abóbora promoveu o conteúdo de citrulina por meio de baixa regulação e efeito sinérgico de duas famílias de genes ASS e ASL. Na melancia enxertada, a acumulação de citrulina resultou na regulação negativa de GAT, NAGS e ASL2, que mostraram uma correlação inversa com a citrulina. Na melancia enxertada, observou-se que as alterações no conteúdo de citrulina foram associadas a menores expressões de GAT, NAGK, ASS2, ASS3, ASL1 e ARG, que foram negativamente correlacionadas com o conteúdo de citrulina. Esses resultados fornecem a base para identificar o mecanismo molecular do acúmulo de citrulina em vários porta-enxertos.

Screening and analysis on the differentially expression genes between diploid and autotetraploid watermelon by using of digital gene expression profile / Triagem e análise da expressão diferencial dos genes entre melancia diplóide e autotetraplóide através do perfil de expressão gênica digital

Abstract Synthetic polyploids are key breeding materials for watermelon. Compared with diploid watermelon, the tetraploid watermelon often exhibit wide phenotypic differences and differential gene expression. Digital gene expression (DGE) profile technique was performed in this study to present gene expression patterns in an autotetraploid and its progenitor diploid watermelon, and deferentially expressed genes (DEGs) related to the abiotic and biotic stress were also addressed. Altogether, 4,985 DEGs were obtained in the autotetraploid against its progenitor diploid, and 66.02% DEGs is up-regulated. GO analysis shows that these DEGs mainly distributed in 'metabolic process', 'cell' and 'catalytic activity'. KEGG analysis revealed that these DEGs mainly cover 'metabolic pathways', 'secondary metabolites' and 'ribosome'. Moreover, 134 tolerance related DEGs were identified which cover osmotic adjustment substance, protective enzymes/protein, signaling proteins and pathogenesis-related proteins. This study present the differential expression of stress related genes and global gene expression patterns at background level in autotetraploid watermelons. These new evidences could supplement the molecular theoretical basis for the better resistance after the genome doubling in the gourd family.

Resumo Poliploides sintéticos são materias fundamentais para melhoramento genético da melancia. Comparativamente ao seu homólogo diploide, a melancia tetraploide apresenta amplas diferenças genotípica e fenotípica e diferença de expressão gênica. A expressão gênica digital ou DGE (digital gene expression) foi utilizada neste estudo para representar o perfil de expressão gênica da melancia autotetraploide e seu progenitor diploide e a expressão diferencial de genes relacionados ao estresse biótico e abiótico. Os resultados mostraram que 4.985 DEGs foram observados no organismo autotetraploide, sendo que, deste total, 66.02%foram supra-regulados. A análise de ontologia gênica (GO) mostrou que estes DEGs estão relacionados principalmente com processos metabólicas, célula e atividade catalítica, abrangendo de acordo com a análise de genes e genoma (KEGG) rotas metabólicas, metabolismo secundário e ribossomos. Além disso, 134 genes de defesa foram identificados, abrangendo substâncias de ajuste osmótico, enzimas/proteínas de proteção, proteínas sinalizadoras e proteínas relacionadas à patogênese. Este estudo mostrou a expressão diferencial de genes relacionados ao estresse e o perfil global de expressão gênica de melancia autotetraploide, estes resultados podem complementar, a nível molecular, o entendimento do fator resistência após a duplicação do genoma em cucurbitáceas.

Construction of cDNA expression library of watermelon for isolation of ClWRKY1 transcription factors gene involved in resistance to Fusarium wilt.

Full-length cDNAs are very important for genome annotation and functional analysis of genes. The number of full-length cDNAs from watermelon remains limited. Here we report first the construction of a full-length enriched cDNA library from Fusarium wilt stressed watermelon (Citrullus lanatus Thunb.) cultivar PI296341 root tissues using the SMART method. The titer of primary cDNA library and amplified library was 2.21 × 106 and 2.0 × 1010 pfu/ml, respectively and the rate of recombinant was above 85%. The size of insert fragment ranged from 0.3 to 2.0 kb. In this study, we first cloned a gene named ClWRKY1, which was 1981 bp long and encoded a protein consisting of 394 amino acids. It contained two characteristic WRKY domains and two zinc finger motifs. Quantitative real-time PCR showed that ClWRKY1 expression levels reached maximum level at 12 h after inoculation with Fusarium oxysporum f. sp. niveum. The full-length cDNA library of watermelon root tissues is not only essential for the cloning of genes which are known, but also an initial key for the screening and cloning of new genes that might be involved in resistance to Fusarium wilt.