Advances in sequencing and key character analysis of mango (Mangifera indica L.).

Mango (Mangifera indica L.) is an important fruit crop in tropical and subtropical countries associated with many agronomic and horticultural problems, such as susceptibility to pathogens, including powdery mildew and anthracnose, poor yield and quality, and short shelf life. Conventional breeding techniques exhibit significant limitations in improving mango quality due to the characteristics of long ripening, self-incompatibility, and high genetic heterozygosity. In recent years, much emphasis has been placed on identification of key genes controlling a certain trait through genomic association analysis and directly breeding new varieties through transgene or genotype selection of offspring. This paper reviews the latest research progress on the genome and transcriptome sequencing of mango fruit. The rapid development of genome sequencing and bioinformatics provides effective strategies for identifying, labeling, cloning, and manipulating many genes related to economically important traits. Preliminary verification of the functions of mango genes has been conducted, including genes related to flowering regulation, fruit development, and polyphenol biosynthesis. Importantly, modern biotechnology can refine existing mango varieties to meet the market demand with high economic benefits.

Independent flavonoid and anthocyanin biosynthesis in the flesh of a red-fleshed table grape revealed by metabolome and transcriptome co-analysis.

BACKGROUND: Red flesh is a desired fruit trait, but the regulation of red flesh formation in grape is not well understood. 'Mio Red' is a seedless table grape variety with light-red flesh and blue-purple skin. The skin color develops at veraison whereas the flesh color develops at a later stage of berry development. The flesh and skin flavonoid metabolomes and transcriptomes were analyzed. RESULTS: A total of 161 flavonoids were identified, including 16 anthocyanins. A total of 66 flavonoids were found at significantly different levels in the flesh and skin (fold change ≥ 2 or ≤ 0.5, variable importance in projection (VIP) ≥ 1). The main anthocyanins in the flesh were pelargonidin and peonidin, and in the skin were peonidin, delphinidin, and petunidin. Transcriptome comparison revealed 57 differentially expressed structural genes of the flavonoid-metabolism pathway (log2fold change ≥ 1, FDR < 0.05, FPKM ≥ 1). Two differentially expressed anthocyanin synthase (ANS) genes were annotated, ANS2 (Vitvi02g00435) with high expression in flesh and ANS1 (Vitvi11g00565) in skin, respectively. One dihydro flavonol 4-reductase (DFR, Vitvi18g00988) gene was differentially expressed although high in both skin and flesh. Screened and correlation analysis of 12 ERF, 9 MYB and 3 bHLH genes. The Y1H and dual luciferase assays showed that MYBA1 highly activates the ANS2 promoter in flesh and that ERFCBF6 was an inhibitory, EFR23 and bHLH93 may activate the DFR gene. These genes may be involved in the regulation of berry flesh color. CONCLUSIONS: Our study revealed that anthocyanin biosynthesis in grape flesh is independent of that in the skin. Differentially expressed ANS, MYB and ERF transcription factors provide new clues for the future breeding of table grapes that will provide the health benefits as red wine.

AP2/ERF genes associated with superfast fig (Ficus carica L.) fruit ripening.

Fig fruits have significant health value and are culturally important. Under suitable climatic conditions, fig fruits undergo a superfast ripening process, nearly doubling in size, weight, and sugar content over three days in parallel with a sharp decrease in firmness. In this study, 119 FcAP2/ERF genes were identified in the fig genome, namely 95 ERFs, 20 AP2s, three RAVs, and one soloist. Most of the ERF subfamily members (76) contained no introns, whereas the majority of the AP2 subfamily members had at least two introns each. Three previously published transcriptome datasets were mined to discover expression patterns, encompassing the fruit peel and flesh of the 'Purple Peel' cultivar at six developmental stages; the fruit receptacle and flesh of the 'Brown Turkey' cultivar after ethephon treatment; and the receptacle and flesh of parthenocarpic and pollinated fruits of the 'Brown Turkey' cultivar. Eighty-three FcAP2/ERFs (68 ERFs, 13 AP2s, one RAV, and one soloist) were expressed in the combined transcriptome dataset. Most FcAP2/ERFs were significantly downregulated (|log2(fold change) | ≥ 1 and p-adjust < 0.05) during both normal fruit development and ethephon-induced accelerated ripening, suggesting a repressive role of these genes in fruit ripening. Five significantly downregulated ERFs also had repression domains in the C-terminal. Seven FcAP2/ERFs were identified as differentially expressed during ripening in all three transcriptome datasets. These genes were strong candidates for future functional genetic studies to elucidate the major FcAP2/ERF regulators of the superfast fig fruit ripening process.

Genome-Wide Analysis of Anthocyanin Biosynthesis Regulatory WD40 Gene FcTTG1 and Related Family in Ficus carica L.

WD40 proteins serve as crucial regulators in a broad spectrum of plant developmental and physiological processes, including anthocyanin biosynthesis. However, in fig (Ficus carica L.), neither the WD40 family nor any member involved in anthocyanin biosynthesis has been elucidated. In the present study, 204 WD40 genes were identified from the fig genome and phylogenetically classified into 5 clusters and 12 subfamilies. Bioinformatics analysis prediction localized 109, 69, and 26 FcWD40 proteins to the cytoplasm, nucleus and other cellular compartments, respectively. RNA-seq data mining revealed 127 FcWD40s expressed at FPKM > 10 in fig fruit. Most of these genes demonstrated higher expression in the early stages of fruit development. FcWD40-97 was recruited according to three criteria: high expression in fig fruit, predicted nuclear localization, and closest clustering with TTG1s identified in other plants. FcWD40-97, encoding 339 amino acids including 5 WD-repeat motifs, showed 88.01 and 87.94% amino acid sequence similarity to apple and peach TTG1, respectively. The gene is located on fig chromosome 4, and is composed of 1 intron and 2 exons. Promoter analysis revealed multiple light-responsive elements, one salicylic acid-responsive element, three methyl jasmonate-responsive elements, and one MYB-binding site involved in flavonoid biosynthesis gene regulation. FcWD40-97 was in the FPKM > 100 expression level group in fig fruit, and higher expression was consistently found in the peel compared to the flesh at the same development stages. Expression level did not change significantly under light deprivation, whereas in leaves and roots, its expression was relatively low. Transient expression verified FcWD40-97's localization to the nucleus. Yeast two-hybrid (Y2H) and biomolecular fluorescence complementation (BiFC) assays revealed that FcWD40-97 interacts with FcMYB114, FcMYB123, and FcbHLH42 proteins in vitro and in vivo, showing that FcWD40-97 functions as a member of the MYB-bHLH-WD40 (MBW) complex in anthocyanin-biosynthesis regulation in fig. We therefore renamed FcWD40-97 as FcTTG1. Our results provide the first systematic analysis of the FcWD40 family and identification of FcTTG1 in fig pigmentation.

Identification and characterization of auxin response factor (ARF) family members involved in fig (Ficus carica L.) fruit development.

The auxin response factor (ARF) combines with AuxREs cis-acting elements in response to auxin to regulate plant development. To date, no comprehensive analysis of ARF genes expressed during fruit development has been conducted for common fig (Ficus carica L.). In this study, members of the FcARF gene family were screened, identified in the fig genome database and their features characterized using bioinformatics. Twenty FcARF genes were clustered into three classes, with almost similar highly conserved DBD (B3-like DNA binding domain), AUX/IAA (auxin/indole-3-acetic acid gene family) and MR domain structure among class members. Analysis of amino acid species in MR domain revealed 10 potential transcription activators and 10 transcription inhibitors, and 17 FcARF members were predicted to be located in the nucleus. DNA sequence analysis showed that the ARF gene family consisted of 4-25 exons, and the promoter region contained 16 cis-acting elements involved in stress response, hormone response and flavonoid biosynthesis. ARF genes were expressed in most tissues of fig, especially flower and peel. Transcriptomics analysis results showed that FcARF2, FcARF11 and FcARF12, belonging to class-Ia, were stably and highly expressed in the early development stage of flower and peel of 'Purple peel' fig. However, their expression levels decreased after maturity. Expression of class-Ic member FcARF3 conformed to the regularity of fig fruit development. These four potential transcription inhibitors may regulate fruit growth and development of 'Purple Peel' fig. This study provides comprehensive information on the fig ARF gene family, including gene structure, chromosome position, phylogenetic relationship and expression pattern. Our work provides a foundation for further research on auxin-mediated fig fruit development.

Integrated Metabolome and Transcriptome Analysis of Fruit Flavor and Carotenoids Biosynthesis Differences Between Mature-Green and Tree-Ripe of cv. "Golden Phoenix" Mangoes (Mangifera indica L.).

The commodity value of fruits is directly affected by fruit flavor and color. Secondary metabolites, such as amino acids, organic acids, esters, and ß-carotene, are important synthetic products, which are of great significance in the flavor formation of mango fruits. In this study, a total of 309 different metabolites, consisting of organic acids, amino acids, phenolic acids, and saccharides, and a further 84 types of volatile organic compounds (VOCs) were identified in differential levels in TR vs. MG mango fruit stages. The major volatile compounds found were ester [2(3H)-furanone, 5-ethyldihydro; N-(2,5-ditrifluoromethylbenzoyl)-D-alanine, pentyl ester; and Octanoic acid, ethyl ester], aldehyde (benzaldehyde, 3-ethyl, and nonanal), and phenol [2-(1,1-dimethylethyl)-6-(1-methylethyl) phenol]. The analysis of carotenoid contents identified 68 carotenoids and we report for the first-time significant contents of zeaxanthin palmitate and (E/Z)-phytoene in mango fruits. α-carotene was a further major contributor to carotene contents with lesser contributions from 5,6epoxy-lutein-caprate-palmitate, ß-carotene, lutein oleate, and ß-cryptoxanthin. What is more, lutein content was significantly decreased in TR vs. MG fruit. RT-qPCR analysis revealed that relative to the MG stage, the expression of carotenogenic genes GGPS, PSY, LCYB, and ZEP was downregulated in TR mango fruit, whereas the transcript levels of PSD, CHYB, and NCED were downregulated. Additionally, the transcription level of some transcription factors (MYB, bHLH, and NAC) was highly correlated with pigment content in the pulp and may be responsible for carotenoid accumulation. The results describe major differences in metabolic pathways during the transition from MG to the TR stage of fruit ripening that are likely to contribute alterations in fruit flavor and provide several associated genes to be further studied in mango fruit.

Genome-wide analysis of JAZ family genes expression patterns during fig (Ficus carica L.) fruit development and in response to hormone treatment.

BACKGROUND: Jasmonate-ZIM domain (JAZ) repressors negatively regulate signal transduction of jasmonates, which regulate plant development and immunity. However, no comprehensive analysis of the JAZ gene family members has been done in the common fig (Ficus carica L.) during fruit development and hormonal treatment. RESULTS: In this study, 10 non-redundant fig JAZ family genes (FcJAZs) distributed on 7 chromosomes were identified in the fig genome. Phylogenetic and structural analysis showed that FcJAZ genes can be grouped into 5 classes. All the classes contained relatively complete TIFY and Jas domains. Yeast two hybrid (Y2H) results showed that all FcJAZs proteins may interact with the identified transcription factor, FcMYC2. Tissue-specific expression analysis showed that FcJAZs were highly expressed in the female flowers and roots. Expression patterns of FcJAZs during the fruit development were analyzed by RNA-Seq and qRT-PCR. The findings showed that, most FcJAZs were significantly downregulated from stage 3 to 5 in the female flower, whereas downregulation of these genes was observed in the fruit peel from stage 4 to 5. Weighted-gene co-expression network analysis (WGCNA) showed the expression pattern of FcJAZs was correlated with hormone signal transduction and plant-pathogen interaction. Putative cis-elements analysis of FcJAZs and expression patterns of FcJAZs which respond to hormone treatments revealed that FcJAZs may regulate fig fruit development by modulating the effect of ethylene or gibberellin. CONCLUSIONS: This study provides a comprehensive analysis of the FcJAZ family members and provides information on FcJAZs contributions and their role in regulating the common fig fruit development.

AGAMOUS Gene as a New Sex-Identification Marker in Fig (Ficus carica L.) Is More Efficient Than RAN1.

Fig is an ancient gynodioecious fruit tree with females for commercial fruit production and hermaphrodites (males) sometimes used as pollen providers. An early sex-identification method would improve breeding efficiency. Three AGAMOUS (AG) genes were recruited from the Ficus carica genome using AG sequences from Ficus microcarpa and Ficus hispida. FcAG was 5230 bp in length, with 7 exons and 6 introns, and a 744-bp coding sequence. The gene was present in both female and male fig genomes, with a 15-bp deletion in the 7th exon. The other two AG genes (FcAG2-Gall_Stamen and FcAG3-Gall_Stamen) were male-specific, without the 15-bp deletion (759-bp coding sequence), and were only expressed in the gall and stamen of the male fig fruit. Using the deletion as the forward primer (AG-Marker), male plants were very efficiently identified by the presence of a 146-bp PCR product. The previously reported fig male and female polymorphism gene RESPONSIVE-TO-ANTAGONIST1 (RAN1) was also cloned and compared between male and female plants. Fifteen SNPs were found in the 3015-bp protein-coding sequence. Among them, 12 SNPs were identified as having sex-differentiating capacity by checking the sequences of 27 known male and 24 known female cultivars. A RAN1-Marker of 608 bp, including 6 SNPs, was designed, and a PCR and sequencing-based method was verified with 352 fig seedlings from two hybrid populations. Our results confirmed that the newly established AG-Marker is as accurate as the RAN1-Marker, and provide new clues to understanding Ficus sex determination.

Genome-Wide Characterization and Analysis of bHLH Transcription Factors Related to Anthocyanin Biosynthesis in Fig (Ficus carica L.).

The basic helix-loop-helix (bHLH) transcription factor family is the second largest transcription factor family in plants, and participates in various plant growth and development processes. A total of 118 bHLH genes were identified from fig (Ficus carica L.) by whole-genome database search. Phylogenetic analysis with Arabidopsis homologs divided them into 25 subfamilies. Most of the bHLHs in each subfamily shared a similar gene structure and conserved motifs. Seventy-two bHLHs were found expressed at fragments per kilobase per million mapped (FPKM) > 10 in the fig fruit; among them, 15 bHLHs from eight subfamilies had FPKM > 100 in at least one sample. bHLH subfamilies had different expression patterns in the female flower tissue and peel during fig fruit development. Comparing green and purple peel mutants, 13 bHLH genes had a significantly different (≥ 2-fold) expression. Light deprivation resulted in 68 significantly upregulated and 22 downregulated bHLH genes in the peel of the fruit. Sixteen bHLH genes in subfamily III were selected by three sets of transcriptomic data as candidate genes related to anthocyanin synthesis. Interaction network prediction and yeast two-hybrid screening verified the interaction between FcbHLH42 and anthocyanin synthesis-related genes. The transient expression of FcbHLH42 in tobacco led to an apparent anthocyanin accumulation. Our results confirm the first fig bHLH gene involved in fruit color development, laying the foundation for an in-depth functional study on other FcbHLH genes in fig fruit quality formation, and contributing to our understanding of the evolution of bHLH genes in other horticulturally important Ficus species.

Metabolome and transcriptome analysis of flavor components and flavonoid biosynthesis in fig female flower tissues (Ficus carica L.) after bagging.

BACKGROUND: Bagging can improve the appearance of fruits and increase the food safety and commodification, it also has effects on intrinsic quality of the fruits, which was commonly reported negative changes. Fig can be regarded as a new model fruit with its relatively small genome size and long fruit season. RESULTS: In this study, widely targeted metabolomics based on HPLC MS/MS and RNA-seq of the fruit tissue of the 'Zibao' fig before and after bagging were analyzed to reveal the metabolites changes of the edible part of figs and the underneath gene expression network changes. A total of 771 metabolites were identified in the metabolome analysis using fig female flower tissue. Of these, 88 metabolites (including one carbohydrate, eight organic acids, seven amino acids, and two vitamins) showed significant differences in fruit tissue before and after bagging. Changes in 16 structural genes, 13 MYB transcription factors, and endogenous hormone (ABA, IAA, and GA) metabolism and signal transduction-related genes in the biosynthesis pathway of flavonoids after bagging were analyzed by transcriptome analysis. KEGG enrichment analysis also determined significant differences in flavonoid biosynthesis pathways in female flower tissue before and after bagging. CONCLUSIONS: This work provided comprehensive information on the composition and abundance of metabolites in the female flower tissue of fig. The results showed that the differences in flavor components of the fruit before and after bagging could be explained by changes in the composition and abundance of carbohydrates, organic acids, amino acids, and phenolic compounds. This study provides new insights into the effects of bagging on changes in the intrinsic and appearance quality of fruits.

Papain-Like Cysteine Protease Gene Family in Fig (Ficus carica L.): Genome-Wide Analysis and Expression Patterns.

The papain-like cysteine proteases (PLCPs) are the most abundant family of cysteine proteases in plants, with essential roles in biotic/abiotic stress responses, growth and senescence. Papain, bromelain and ficin are widely used in food, medicine and other industries. In this study, 31 PLCP genes (FcPCLPs) were identified in the fig (Ficus carica L.) genome by HMM search and manual screening, and assigned to one of nine subfamilies based on gene structure and conserved motifs. SAG12 and RD21 were the largest subfamilies with 10 and 7 members, respectively. The FcPCLPs ranged from 1,128 to 5,075 bp in length, containing 1-10 introns, and the coding sequence ranged from 624 to 1,518 bp, encoding 207-505 amino acids. Subcellular localization analysis indicated that 24, 2, and 5 PLCP proteins were targeted to the lysosome/vacuole, cytoplasm and extracellular matrix, respectively. Promoter (2,000 bp upstream) analysis of FcPLCPs revealed a high number of plant hormone and low temperature response elements. RNA-seq revealed differential expression of 17 FcPLCPs in the inflorescence and receptacle, and RD21 subfamily members were the major PLCPs expressed in the fruit; 16 and 5 FcPLCPs responded significantly to ethylene and light, respectively. Proteome analyses revealed 18 and 5 PLCPs in the fruit cell soluble proteome and fruit latex, respectively. Ficins were the major PLCP in fig fruit, with decreased abundance in inflorescences, but increased abundance in receptacles of commercial-ripe fruit. FcRD21B/C and FcALP1 were aligned as the genes encoding the main ficin isoforms. Our study provides valuable multi-omics information on the FcPLCP family and lays the foundation for further functional studies.

Differential color development and response to light deprivation of fig (Ficus carica L.) syconia peel and female flower tissues: transcriptome elucidation.

BACKGROUND: Color directly affects fruit quality and consumer preference. In fig syconia, the female flower tissue is contained in a receptacle. Anthocyanin pigmentation of this tissue and the peel differs temporally and spatially. A transcriptome study was carried out to elucidate key genes and transcription factors regulating differences in fig coloring. RESULTS: Anthocyanins in the female flower tissue were identified mainly as pelargonidin-3-glucoside and cyanidin-3-rutinoside; in the peel, the major anthocyanins were cyanidin 3-O-glucoside and cyanidin-3-rutinoside. Anthocyanin content was significantly higher in the female flower tissue vs. peel before fig ripening, whereas at ripening, the anthocyanin content in the peel was 5.39 times higher than that in the female flower tissue. Light-deprivation treatment strongly inhibited peel, but not female flower tissue, anthocyanin pigmentation. RNA-Seq revealed 522 differentially expressed genes (recruited with criteria log2 ≥ 2 and P < 0.05) at fig ripening, with 50 upregulated and 472 downregulated genes in the female flower tissue. Light deprivation upregulated 1180 and downregulated 856 genes in the peel, and upregulated 909 and downregulated 817 genes in the female flower tissue. KEGG enrichment revealed significantly changed expression in the phenylpropanoid-biosynthesis and flavonoid-biosynthesis pathways in the peel, but not in the female flower tissue, with significant repression of FcCHS, FcCHI, FcF3H, FcF3'H, FcDFR and FcUFGT transcripts. Light deprivation led to differential expression of 71 and 80 transcription factor genes in the peel and female flower tissue, respectively. Yeast one-hybrid screen revealed that FcHY5 and FcMYB114 bind the promoter regions of FcCHS and FcDFR, respectively in the flavonoid-biosynthesis pathway. CONCLUSIONS: Phenylpropanoid- and flavonoid-biosynthesis pathways were differentially expressed spatially and temporally in the peel and female flower tissue of fig syconia; pathway expression in the peel was strongly regulated by light signal. Differentially expressed transcription factors were recruited as candidates to screen important expression regulators in the light-dependent and light-independent anthocyanin-synthesis pathway. Our study lays the groundwork for further elucidation of crucial players in fig pigmentation.

The potassium channel FaTPK1 plays a critical role in fruit quality formation in strawberry (Fragaria × ananassa).

Potassium (K+ ), an abundant cation in plant cells, is important in fruit development and plant resistance. However, how cellular K+ is directed by potassium channels in fruit development and quality formation of strawberry (Fragaria × ananassa) is not yet fully clear. Here, a two-pore K+ (TPK) channel gene in strawberry, FaTPK1, was cloned using reverse transcription-PCR. A green fluorescent protein subcellular localization analysis showed that FaTPK1 localized in the vacuole membrane. A transcription analysis indicated that the mRNA expression level of FaTPK1 increased rapidly and was maintained at a high level in ripened fruit, which was coupled with the fruit's red colour development, suggesting that FaTPK1 is related to fruit quality formation. The down- and up-regulation of the FaTPK1 mRNA expression levels using RNA interference and overexpression, respectively, inhibited and promoted fruit ripening, respectively, as demonstrated by consistent changes in firmness and the contents of soluble sugars, anthocyanin and abscisic acid, as well as the transcript levels of ripening-regulated genes PG1 (polygalacturonase), GAL6 (beta-galactosidase), XYL2 (D-xylulose reductase), SUT1 (sucrose transporter), CHS (chalcone synthase) and CHI (chalcone flavanone isomerase). Additionally, the regulatory changes influenced fruit resistance to Botrytis cinerea. An isothermal calorimetry analysis showed that the Escherichia coli-expressed FaTPK1 recombinant protein could bind K+ with a binding constant of 2.1 × 10-3  m-1 and a dissociation constant of 476 µm. Thus, the strawberry TPK1 is a ubiquitously expressed, tonoplast-localized two-pore potassium channel that plays important roles in fruit ripening and quality formation.