Transcriptome driven characterization of curly- and smooth-leafed endives reveals molecular differences in the sesquiterpenoid pathway

Endives (Cichorium endivia L.) are popular vegetables, diversified into curly/frisée- and smooth/broad-leafed (escaroles) cultivar types (cultigroups), and consumed as fresh and bagged salads. They are rich in sesquiterpene lactones (STL) that exert proven function on bitter taste and human health. The assembly of a reference transcriptome of 77,022 unigenes and RNA-sequencing experiments were carried out to characterize the differences between endives and escaroles at the gene structural and expression levels. A set of 3177 SNPs distinguished smooth from curly cultivars, and an SNP-supported phylogenetic tree separated the cultigroups into two distinct clades, consistently with the botanical varieties of origin (crispum and latifolium, respectively). A pool of 699 genes maintained differential expression pattern (core-DEGs) in pairwise comparisons between curly vs smooth cultivars grown in the same environment. Accurate annotation allowed the identification of 26 genes in the sesquiterpenoid biosynthesis pathway, which included several germacreneA synthase, germacreneA oxidase and costunolidesynthase members (GAS/GAO/COS module), required for the synthesis of costunolide, a key precursor of lactucopicrin- and lactucin-like sesquiterpene lactones. The core-DEGs contained a GAS gene (contig83192) that was positively correlated with STL levels and recurrently more expressed in curly than smooth endives, suggesting a cultigroup-specific behavior. The significant positive correlation of GAS/GAO/COS transcription and STL abundance (2.4-fold higher in frisée endives) suggested that sesquiterpenoid pathway control occurs at the transcriptional level. Based on correlation analyses, five transcription factors (MYB, MYB-related and WRKY) were inferred to act on contig83192/GAS and specific STL, suggesting the occurrence of two distinct routes in STL biosynthesis. Genetic analyses show molecular differences that could explain why curly endives taste bitterer than smooth ones. Donato Giannino of the Institute of Agricultural Biology and Biotechnology and colleagues in Italy analyzed the genetic differences between curly and smooth endives, a leafy vegetable used in salads. They found more than 3000 single sequence variations in genes distinguishing the two types from each other. Twenty six genes were involved in the biosynthesis of sesquiterpenoids, metabolites important for plant survival that also contribute to the bitter taste of endives and have antimalarial, sedative and analgesic effects when isolated for humans. Their levels were higher in curly than in smooth endives, potentially contributing to their more bitter taste. The information expands the genetic data available on endives for breeding programs.


Introduction
The Cichorium endivia (L.) species belong to the Asteraceae family and includes the botanical varieties crispum and latifolium (Lam.), which are respective sources (GRIN db, https://npgsweb.ars-grin.gov/ gringlobal/search.aspx) of two market cultivar types (cultigroups), the curly-and smooth-leafed endives. The former (synonyms: frisée, cut-type) bear green leaves with a narrow central vein, septate blade with incised margins (syn.: runcinated-bipinnatifid type), while the smooth types (syn.: escaroles) produce lighter green leaves with a large midrib, a broad and slightly lobed lamina and dentate margins. Agronomic and some physiological features of the two cultigroups have been wellcharacterized 1 . The consumption of endives has been increasing in fresh and minimally processed segments worldwide and greatly in Europe, where Italy, Spain, and France are major representatives of Cichorium spp. products (TrendEconomy, http://trendeconomy. com).
C. endivia life cycle is annual and flowering extends from May to August at the Mediterranean latitudes; the leaf rosette (head) develops an inflorescence stem bearing violet autogamous flowers. Self-compatibility prevails, the outcross rate is 1% 2 and leads to high inbreeding grade in natural populations that consist of a mixture of highly homozygous lines. Genetically, C. endivia (2n = 2x = 18) has a complex chromosomal organization 3 and its genome size 1 is alleged to share that of the close relative C. intybus in a range of 0.7-1.3 Gb 5,6 , it will be better defined after the genome sequence release. To date, the Cichorium spp. genetic consensus map 7 has included markers from a C. intybus × C. endivia cross 4 ; molecular marker assisted breeding of endive is expected to increase considering the recent development of genomic tools 8 . The strict autogamy compels the breeding strategies to mass or individual selections, pedigree breeding, and backcrossing 9 . Commercialized cultivars mostly consist of pure lines derived from repeated selfings of plants from local populations or of hybrids selected from parental line cross (F 1 hybrid production is poorly explored ). Breeding programs are mainly performed by private seed companies to develop varieties suitable for the fresh-salad or minimally processed-salad markets, able to span the whole year cultivation (outdoor or in greenhouse), namely cold resistant in spring and heat tolerant in summer. Major traits targeted include resistance to premature bolting, tip burn, root rot and mildew, preservation/ enhancement of nutritional quality, taste, and shelf life 1 .
Sesquiterpene lactones (STLs) are terpenoids with lactone rings, produced as secondary metabolites important for plant survival, typical of and used to classify Asteraceae species 10 , and known for exerting both positive and negative effects on human health 11 . Endive contains STLs, which can act on both nutraceutical and taste traits. The most abundant STLs of endive leaves are lactucin, 8deoxylactucin, lactucopicrin, and the respective 11 (S),13-dihydroderivatives 12 though novel STLs have been discovered 13 . Lactucopicrin has been used as antimalarial, sedative, and analgesic in humans 14,15 and recently as a protector against neurodegenerative diseases 16 . STLs also contribute to bitter taste, a crucial trait in terms of rejection or acceptance, which depends on consumers' use and culture 17 . Bitterness has been associated with lactucin (Lc-) and lactucopicrin (Lp-) classes in chicory 18 and lettuce 19 ; in endives, Lp has a dominant effect on bitterness perception, though complex equilibria between STL and phenolics also exert a significant impact 20 . STLs belong to the germacrene A type sesquiterpenoids. The sequential actions of germacrene A synthase and oxidase, and costunolide synthase lead to costunolide, which is the precursor of STLs 21 . These enzymes and genes (GAS, GAO, COS) have been specifically characterized in C. intybus [21][22][23][24] and not yet in C. endivia. Furthermore, the enzymes that use costunolide to synthesize both Lc-and Lp-like compounds have remained unknown in plants, so far.
The major aims of this work were to widen the knowledge on the differences between curly-and smoothleafed endives by analyzing allelic and gene transcriptional variation as well as to investigate on gene divergences in the STL pathway contextually with the notion that curly types have higher STL contents than escaroles 20 . A reference transcriptome was assembled and annotated using the "Domari" curly cultivar. RNA-sequencing of five cultivars produced both SNP markers, which could neatly separate the two cultigroups into two distinct phylogenetic clades, and pools of up-and down-regulated genes (core-DEGs), which maintained the differential pattern in curly vs smooth genotypes. One core-DEG GAS, belonging to the 26 genes of the STL pathway, was recurrently more expressed in curly than smooth endives and positively correlated with STL abundances. Co-expression/ correlations analyses based on biosynthesis genes/transcription factors expression and STL amounts supported that STL pathway control occurs at the transcriptional level; they also allowed inferring the roles of MYB, MYBrelated and WRKY transcription factors on GAS regulation and the depiction of likely networks that subtend Lp-and Lc-compound synthesis.
Fields were located in Tarquinia, Lazio, Italy (42°15′N 11°44′E, 31 m a.s.l.); soil characteristics and cultivation parameters were previously reported 25 . In this work, plants were shown in nursery (3 dm 2 /well) at the end of August 2012; 3-week-old seedlings were moved into open field (8.2 plants/m 2 ) and harvest occurred on the second half of November. The average temperature was of 18.4 ± 3.3°C (www.idrografico.roma.it/annali). Details on agrotechniques (basal dressing, fertirrigation, protection vs weeds, thrips, moths and powdery mildew) are available upon request.
Harvested heads (n = 9 per cultivar) were brought to laboratories and weighted ( Fig. 1 and Table S1); the external leaves were removed from the rosette and the following leaves were sampled (because assumed as representative of freshly consumed or fresh-cut products). More precisely, 10 leaves were excised from each plant (n = 3) of the same cultivar and pooled to form a replicate batch (RB) of 30 leaves; these had comparable weights among the cultivars, though significant differences for length and surface were scored ( Fig. 1 and Table S1). Three RB were rapidly generated (biological triplicates) and frozen in liquid nitrogen, gently crunched by hands and stored at −80°C. The content of each RB was either used for RNA isolation in transcriptional and allelic variation analyses or further lyophilized at −50°C for 72 h (lab freeze dryer with stoppering tray dryer, FreeZone®; Labconco Corp., Kansas City, MO, USA) and stored at −20°C for STL quantification.

RNA isolation, sequencing, and transcriptome assembly
For transcriptome reference assembly, ten "Domari" seedlings at the transplant (bearing 3-4 leaves) and ten plants at commercial maturation were selected. Apices, stems, leaves, or roots at the two developmental stages (n = 8) were used to isolate and purify total RNA (TRIzol, Invitrogen; RNAeasy kit, Qiagen). As for RNA sequencing, a mix of the eight samples (1 µg of total RNA each) was obtained; RNA yields and integrity (RIN > 7) were assessed (NanoDrop ND-1000, Thermo Scientific Inc; BioAnalyzer 2100; Agilent Technologies Inc.), cDNA libraries were synthesized (TruSeq RNA-seq kit, Illumina) and sequenced in 100 bp paired-end mode (Illumina HiSeq2000; IGA Technology Services, Udine, Italy). As for NGS transcriptional analyses and SNP mining, cDNA libraries were prepared from RNA of targeted leaves as described above and sequenced in 50 bp single-end. Three  Table S1 ("Myrna", "Confiance", "Flester") and two ("Domari" and "Imari") biological replicates were analyzed. RNA-seq datasets were stored in the National Centre for Biotechnology Information database (NCBI, www.ncbi.nlm. nih.gov) under the BioProject accession number PRJNA417356.
The transcriptome was assembled following the previously described one-step and two-step approaches 24 . Briefly, the output of one-step de novo assembly by Trinity v.2.2.0 26 was merged with the two-step assembly obtained from an EST-based backbone plus a de novo assemblies by Velvet v.1.2.10/Oasis v. 0.2.08 27,28 . Subsequently, the redundancies were removed by TGICL-CAP3 v. 2.1 29 and the transcript/isoform clustering was achieved by the CD-HIT package v. 4.6.6 30 with an identity threshold of 97%, and the longest transcripts were counted as representative for each cluster. BLASTX (cutoff E-value ≤10 −5 ) carried out annotation through these databases: Nr (NCBI non-redundant database; last update: 6 March 2017), RefSeq (NCBI Reference Sequence Database; release 79), TAIR10 (The Arabidopsis Information Resource, ver. 10), SwissProt and TrEMBL sections of the UniProt Knowledgebase (release-2017_05), KOG (euKaryotic Ortholog Groups) 31 . Full-length transcript analysis was carried out using the "analyze_-blastPlus_topHit_coverage.pl" script from the Trinity package. Blast2GO 4.1 32 was used to retrieve Gene Ontology (GO) and KEGG 33 annotations from the best hits from BLASTX analysis. GO functional classification was achieved by WEGO 34 . KEGG pathway annotation was improved by mining KAAS (KEGG Automatic Annotation Server) 35 . Protein domain/families annotation was achieved by InterProScan 5.1-44.0 36 . Transcription factors (TFs) were predicted using the PlantTFDB v.4 37 .
Multi-level quality evaluation of "Domari" transcriptome was achieved in three steps: (1) assessment of the number of reads that mapped back to the final assembly as properpaired matches by the "bowtie_PE_separate_then_join.pl script" from Trinity package; (2) evaluation of assemblies against a plant database containing near-universal singlecopy orthologue genes (BUSCO ver. 3) 38 ; (3) estimation of the number of full-length transcripts against Nr database by the Perl script "analyze_blastPlus_topHit_coverage.pl" of Trinity.
GAS, GAO, and COS from C. endivia (Ce) and C. intybus (Ci) were submitted to GeneBank and the numbers from MG383453 to MG383471 were assigned. Protein phylogenetic analysis was carried out using the above-mentioned sequences together with the following ones: CiGASsh, AAM21659. The reaction conditions were: enzyme activation at 95°C for 3 min; 45 amplification cycles of 5 s denaturation at 95°C and 30 s annealing/extension at 60°C; final melting step at 95°C, cooling to 60°C and heating at 95°C. Fluorescence data were collected every 0.1°C from 60 to 95°C. The melting curve were normalized between 100% and 0% fluorescent intensity by adjusting the pre-and post-melt normalization regions, respectively. Difference plots were generated by subtracting the normalized melting profiles against that of the "Domari" reference. The genotypes were discriminated visually from both normalized and difference melting curves.

Digital gene expression (DGE) analyses and quantitative PCR (qPCR)
The single-end reads were mapped on the reference assembly by Bowtie2 (v. 2.2.9) 42 and SAMtool pipeline, and read count for each transcript was scored in each replicate. The DGE levels were calculated and expressed as RPKM (Reads per kilobase per million mapped reads) values. Total RNA of leaf RBs was isolated (RNeasy Plant Mini Kit, Qiagen), DNase treated (RQ1, Promega), and 1 µg was reverse-transcribed at 55°C by SuperscriptIII (Life Technologies). One microliter of a 1:10 cDNA dilution was amplified by Eco Real-Time PCR System (Illumina) using 1× Quantimix easy master mix (Biotools) and 0.3 µM of each primer (Table S2) in a 10 µl final volume. PCR reaction conditions: 95°C for 10 min for polymerase activation, 45 cycles at 95°C for 10 s, 60°C for 30 s. The experiments included three biological and instrumental replicates. Gene expressions were normalized against the ACT reference gene 24 ; mean normalized expressions and log2 fold change (log 2 FC) were calculated by using the Q-Gene program 43 and by the 2 −ΔΔCt method, respectively.

Statistical analyses
ANOVA and Duncan Multiple Range Test were performed by Statistical Analysis System program (SAS software, Version 9.1, Cary, NC, USA). The principal component analysis (PCA) was based on mean centered and standardized data (unit variance scaled); results were pictured as bi-plots of scores (treatments) and loadings (variables) plots by using XLStat Pro (Addinsoft, Paris, France). As for DEG analysis, the Bioconductor edgeR package was used 44 . After sample normalization (based on trimmed mean of M values, TMM), unigenes with at least 1 read per million in at least three samples were selected; thresholds of gene expression difference significance were set on the co-occurrence of absolute value of log 2 FC ≥1 and a false discovery rate (FDR) value ≤0.05. Finally, genemetabolite correlation analyses were carried out by the R3.4.0 45 .

Transcriptome features
A cDNA library was synthesized from equal quantities of RNA isolated from apical tips, stems, leaves, and roots of C. endivia plants sampled at both transplant and harvest stages ( Table 1). The Illumina Hiseq2000 sequencing system generated approximately 246 million of raw reads (2 × 100 bp), which were processed to remove adaptors, ambiguous bases, and low-quality sequences, and 97.2% of them were retained for further processing (Table 1). Subsequently, the high-quality reads were assembled using two procedures as previously described 24 . The "onestep" procedure consisted of a de novo assembling by Trinity, which led to 255,105 sequences with an N50 and mean contig length of 1586 and 1048 bp, respectively   Table 2). The "two-step" pipeline included a templatebased assembly followed by a de novo assembly. Briefly, the endive high-quality reads were first mapped on 30,170 EST of a public database (The Compositae Genome Project, http://compgenomics.ucdavis.edu) that produced 27,179 read supported sequences. These were subjected to iterative contig extension process (SeqMan Pro, DNAStar) that expanded the mean length from 753 to 1044 bp. The unmapped reads were retrieved by Bowtie2 and assembled de novo into 51,038 contigs by Velvet/Oasis. Finally, the outputs from one-and two-step pipelines were merged into a final "Domari" reference transcriptome of 84,882 transcripts (N50 = 1591 bp; average contig length = 1214 bp), including all isoforms, and clustered into 77,022 unigenes ( Table 2). As for annotation and function classification, the unigenes with at least one BLASTX hit were 57,579 (74.8 %) and showed average length of 1429.2 bp; non-annotated unigenes were 19,443 (25.2%) and of short size (Fig. 2a). BLASTX searches (E-value ≤10 −5 ) against public protein databases showed that 73.0% unigenes had significant matches in the Nr database, the 74.0% in the TrEMBL, and the 70.8%, 67.1%, and 49.2% in the RefSeq, Tair, and SwissProt databases, respectively (Table S3). As for the functional categorization, the 52.7%, 20.0%, and 15.3% of unigenes were respectively annotated into GO, KEGG, and KOG (Table S3). Regarding GO clustering (Fig. 2b), the dominant subcategories were: "metabolic process" and "cellular process" in Biological Process (BP), "cell" and "cell part" in Cellular Component (CC), and "binding" and "catalytic activity" in Molecular Function (MF). Specifically, 27,935, 22,992, and 33,389, respectively, fell into BP, CC, and MF and 2371 had at least 10 GO terms (Fig.  S2). As for KEGG categorization, 15,430 annotated genes were assigned to 131 pathways belonging to 20 metabolic groups (Fig. 2c); "carbohydrate metabolism" and Final output from the merge of one-step and two-step assemblies b Contigs were clustered by CD-HIT; the longest transcripts were selected as representative for each isoform cluster (i.e. unigenes) "translation" represented the most abundant classes in "Metabolism" (M) and "Genetic information processing" (G), respectively. As for KOG (Fig. 2d) endivia unigene length distribution and annotation. a Unigenes distribution based on size (base pairs, bp) and BLASTX annotation. The black and gray arrows report the average length of annotated and non-annotated unigenes. b GO classification. The GO terms were classified into biological process (blue bars), cellular component (green bars), and molecular function (red bars). c KEGG classification; the histogram represents the unigene distribution into five major KEGG metabolic categories. M, metabolism; G, genetic information processing; E, environmental information processing; C, cellular processes; O, organismal systems. d Unigene functional classification into EuKaryotic Orthologous Groups (KOG). A, RNA processing and modification; B, chromatin structure and dynamics; C, energy production and conversion; D, cell cycle control, cell division, chromosome partitioning; E, amino acid transport and metabolism; F, nucleotide transport and metabolism; G, carbohydrate transport and metabolism; H, coenzyme transport and metabolism; I, lipid transport and metabolism; J, translation, ribosomal structure and biogenesis; K, transcription; L, replication, recombination and repair; M, cell wall/membrane/envelope biogenesis; N, cell motility; O, posttranslational modification, protein turnover, chaperones; P, inorganic ion transport and metabolism; Q, secondary metabolites biosynthesis, transport and catabolism; R, general function prediction only; S, function unknown; T, signal transduction mechanisms; U, intracellular trafficking, secretion, and vesicular transport; V, defense mechanisms; Y, nuclear structure; Z, cytoskeleton In addition, 33,277 unigenes were annotated into InterPro database scoring 54,881 domains, 17,749 families, 3314 repeats, and 6444 functional sites (Table S3 and Fig. S3a); Protein kinase-like domain and Cytochrome P450 were the most represented in domains and families, respectively (Fig. S3b, c). Finally, 5475 sequences showed hits in plant transcription factor database (PlantTFdb , Table S3) and could be assigned to 57 families (Fig. S4a). The most represented were bHLH (9%), ERF (7%), C2H2 (6%), MYB-related, NAC and WRKY (5%), MYB, bZIP, and C3H (4%), and G2-like (3%).
Regarding the transcriptome quality (Table 2), the final assembly included ca. 81% of properly paired reads (out of ca. 94% of the reads that mapped back to the assembly), and completeness was ca. 90% according to BUSCO evaluation. In addition, 24,152 unigenes (43% , Table S4) were either full-length or nearly full-length transcripts, which had at least 70% of the alignment coverage to respective hits in the Nr protein dataset (Table S5). Overall, these data supported a satisfactory assembly, which included over 71% of single copy-and ca. 6% of duplicated genes (Table 2).

Leaf-group differentiation based on sequence polymorphisms and gene expression
Referring to the "Domari" transcriptome, 15,940 unigenes contained 19,951 putative SSRs and 3,155 unigenes had more than one microsatellite (Table S6). Neglecting the mononucleotides, the di-and tri-nucleotide repeats were the most abundant (respectively 51.4% and 45.2 % out of 9284 SSR) and the AG/CT and ATC/ATG were the most frequent motifs of these repeats (Table 3). After mapping the reads of cultivar against those of "Domari" transcriptome, total SNP numbers of "Imari", "Myrna", "Confiance" and "Flester" were 5929, 5254, 10,647, and 10,607, respectively (Fig. 3a). The homozygous SNPs were ca. 90% in each cultivar (compare black vs gray boxes) and "Domari" contained 540 hetero-SNPs. The SNP average frequency was of ca. 1/9000 bp for both "Confiance" and "Flester", and 1/18,000 bp and 1/16,000 bp for "Myrna", and "Imari", respectively. The SNP number per unigene was greater in smooth than curly genotypes ("Confiance", "Flester" vs "Imari", "Myrna"); the former contained a mean of 0.14, which doubled that of the latter. Multiple pairwise comparisons allowed the identification of private SNPs (i.e. those that occur specifically in one population and not in all the others). Figure 3b reports a Venn diagram showing the number of cultivar-exclusive SNPs resulting from the different combinations. Overall, the number of private SNPs was highest in "Confiance" (4015), followed by "Flester" (3563), "Imari" (1622), and "Myrna" (1197); 3177 SNPs (core-SNPs) distinguished smooth vs curly cultivars. The core-SNPs spread over 1086 unigenes, 735 and 284 were respectively annotated into GO and KEGG (244 occurred in both), and these unigenes included 123 TFs. Enrichment analyses revealed the GO terms and KEGG pathways that were overrepresented in the core-SNP gene set (Table S7). Moreover, concatenated SNPs were used to depict a genetic relationship tree (Fig. 3c), which placed endives and escaroles into two well separated clades. Finally, lab-scale SNP validation was achieved by an HRM technique performed on 16 randomly selected events. Of these, all used primer couples produced amplicons and the 97% confirmed the predicted polymorphism ( Fig. S5 and Table  S8).

STP pathway: gene characterization and relationships with STL contents
Overall, the STP pathway included 26 unigenes encoding proteins ascribed to 11 distinct enzymes ( Table 5, Fig. 4b). DGE analysis of edible leaves revealed that two unigenes were below the transcription threshold (RPKM 0-0.1), one was lowly expressed (RPKM 0.1-1), 12 showed moderate expression (RPKM 1-8), and 11 were highly expressed (RPKM > 8). The reliability of DGE analysis was confirmed by qPCR based on eight STP unigenes (Fig. 4c). The GAS and QHS1 unigenes (Ce_contig83192 and 82792) showed respectively higher and lower expressions in curly than smooth genotypes. In order to enrich the gene pool of STP pathway, endive unigenes were blasted against the lettuce genome (phytozome.jgi.doe.gov) applying highly selective filters (identity ≥ 70%; full length ≥ 80%) and two two additional GAO (Ce_contig47698 and 11533) and two COS (Ce_contig69070 and 34331) orthologues were identified (Table S9). Phylogenetic trees (Fig. 5) of GAS, GAO, and COS proteins were constructed by using sequences from Cichorium spp. and Lactuca sativa of the Cichorieae tribe and excluding partial sequences (Ce_contig81731/GAS and Ce_contig34331/COS, Table S9). The analysis showed that new protein encoded by the Ce_contig16955 belonged to the type I GAS (Fig. 5a), which has lettuce LsGAS3 and chicory CiGASlo as reference proteins due to their assessed enzymatic function 23,46 . The Ce_con-tig52991 belonged to type II GAS, which have LsGAS1 and 2 and CiGASsh 23,47 as references. The Ce_con-tig83192 and 41447, which shared 89.7% sequence identity (Fig. S6), were in a Cichorium spp. cluster sited near that of lettuce GAS enzymes with uncharacterized function. As for GAO (Fig. 5b), the Ce_contig46043 fell in the Cichorium spp. sub-group of CiGAO near that of lettuce LsGAO1, both with ascertained functions 48,49 . The Ce_contigs 11533 and 47698 formed a group per se. As for COS (Fig. 5c), the Ce_contig84591 was within a sub-group of Cichorium spp. having CiCOS and LsCOS as landmarks 48,50 , whereas the Ce_contig69070 formed a separate group. As for polymorphic events, cultivar-specific SNPs (Table S10) were scored in five    (Table 6). Globally, the total STL content (STLTOT) was significantly higher in curly-than smooth-endives (2239 ± 531 vs 930.8 ± 181.7 mg/kg dry matter) consistently with both total amounts of lactucin-like and lactucopicrin-like compounds (LcTOT, 1453.5 ± 548.5 vs 584.1 ± 73.8; LpTOT, 786.4 ± 186.8 vs 346.7 ± 113.1) and the mean abundance of each STL molecule. The STLTOT, LpTOT, and LcTOT also differed significantly among all the cultivars though overlapping values occurred in some specific STL compounds (e.g.: dLc contents of "Imari" vs "Flester", Lp contents of "Domari" vs "Confiance"). The conversion of STL amounts into bitterness-deduced values indicated that curly endives had higher scores than escaroles (Table S11).
After scoring differences in STL biosynthesis gene transcriptions and contents between the curly and smooth cultivars, we carried out a search for TFs that could be involved in pathway regulation. Several families of TF were identified in the core-DEGs, and the MYB-related and CO-like ones were the most numerous (Fig. S4b). Subsequently, overall exploration of data was approached by PCA focusing on correlations among GAS/GAO/COS biosynthesis (BS) and TF gene expressions and STL contents from all cultivars. The criteria to select TF genes from the core-DEGs included transcript completeness (≥80%), protein identity (≥70%), and inferred involvement in STP pathway based on putative functional analogies with well-characterized orthologues. These thresholds led to identify five TFs (Table S12). The biplot picture (Fig. 6a) showed that the PC1 explained 54.4% of the variation; the contents of all STLs, the expression of all BS, and three TF genes (Ce_contigs: 72724/MYB-related, 74591/MYB, and 86458/WRKY) of the curly cultivars ("Domari", "Imari", and "Myrna") fell in the PC1 positive values. Oppositely, the smooth cultivars ("Flester", "Confiance") were on the PC1 negative side together with two MYB-related TF (Ce_contig32243 and 32240). The PC2 (see figure on previous page) Fig. 4 Differentially expressed gene analysis and STP biosynthesis pathway. a The Venn diagrams omit the numbers of differentially expressed genes in multiple comparisons (overlapping areas) and just report the number of up-(left) and down-regulated (right) genes specific to each comparison indicated at the vertexes. The number of genes that maintained the same differential transcription pattern in curly vs smooth cultivars (core-DEGs) is bolded. b Scheme of sesquiterpenoid and triterpenoid biosynthesis pathway in endive. Rectangles report the enzyme codes; and those assigned to endive contigs are in green (see Table 5). GAS/GAO/COS gene module acts in the costunolide synthesis branch. c Expression profiles of eight STP unigenes achieved by RNA-seq and qPCR (gray and black histograms, respectively). The unigene names in bold belong to the core-DEGs group. The last panel reports RNA-seq/qRT-PCR correlation analysis (bottom right) expressed as log2 fold change of curly vs smooth genotypes (six independent comparisons per gene); significant positive correlation occurred between the expression fold changes measured by the two methods (R 2 = 0.89; P < 0.001)    (Fig. 6b) and hereafter we refer to those that have r ≥ |0.7| and P ≤ 0.01 as thresholds. The 83192/GAS and 86458/WRKY transcriptions were positively correlated, and each of the two had positive correlation with total lactucopicrin-like contents. The two MYB-related TF (Ce_contig32240 and 32243) had negative correlation with LpTOT (Fig. 6a, bottom left  quadrant). Furthermore, the expressions of the six BS were positively correlated with DHdLc and dLc molecule contents (Fig. 6a, bottom right quadrants). No significant correlations occurred between TF and these BS genes. The analyses allowed the depiction of a putative gene/ metabolite network into distinct branches (Fig. 7): one encompassed all TF, the BS genes 83192/GAS and 11533/ GAO, and the Lp, Lc, and DHLc molecules; the other embraced the remaining GAS, GAO, and COS genes and the dLc, DHdLc, and DHdLp compounds.

Discussion
In the current scenario where the number of Asteraceae spp. sequenced genomes has increased for high profit crops 51-53 , the endive transcriptome mining of this work has been a sustainable strategy aimed to gene finding, expression analysis, and marker production. The "Domari" transcriptome assembly was achieved through a pipeline that combined the template-based methods accuracy and the ability of de novo assemblers to detect novel transcripts. The strategy was confirmed to be convenient 24 and led to a final transcriptome with better Type II Type I a b c Fig. 5 Phylogenetic analysis of STL biosynthesis proteins in the Cichorieae tribe. a-c Phylogenetic trees of germacrene A synthase (GAS), oxidase (GAO), and costunolide synthase (COS). They were constructed by neighbor-joining method, using deduced proteins of Cichorium endivia (Ce) contigs (in bold) and those retrieved from public databases of Lactuca sativa (Lsat or Ls) and Cichorium intybus (Ci) species. Bootstraps values were based on 1000 replicates. The line lengths indicate the relative distances between nodes. Underlined proteins have biochemically assessed functions (references in the text) parameters than those produced by the separate use of the one-and two-step approaches, achieving higher N50 and mean contig length, reduced duplication and fragmentation events, and high levels of completeness and reads representation. The transcriptome of C. endivia var. crispum widens the number of those available in the species 8 , though it differs for higher contig number, longer unigenes, and wider range of tissues from which RNA was isolated. Finally, it expands gene mining because it represents different vegetative tissues of young and ready-to-market plants and adds information on a recurrent parent cultivar.
The "Domari" transcriptome and cultivar re-sequencing provided a pool of SNPs that effectively fingerprinted the frisée and escarole cultigroups, respectively associated to the botanical var. crispum and latifolium 54 . Previously, the cultigroup classification was not fully supported by AFLPmarker analysis 55 , while the SNP-based phylogenetic analyses of this work neatly separated curly from smooth endives, supporting the cultigroup/taxa association, and provided tools for cultivar traceability. Moreover, this new SNP pool may turn useful to better characterize genetic differences between C. endive and C. intybus species, which share introgression and complex relatedness 56,57 . Contextually, C. endive cultivars showed lower SNP average frequency than that measured in the C. intybus (1/9000-1/18,000 vs 1/1068 bp) transcriptome 24 , which may reflect the prevalent cross-fertilization in the latter species 58 . The homozygous SNPs were ca. 90% in all endive cultivars, likely due to breeding process based on repeated self-fertilizations. Finally, the production of SNPs specific for parental cultivars provides useful tools to create endive specific genetic maps made of expressed genes .
A set of 699 of unigenes (core-DEGs) maintained a leafgroup specific transcription pattern within over five thousand differentially expressed genes in all endive vs escarole comparisons. The core-DEGs fell in nine overrepresented pathways (Table 4), including those of circadian rhythm and STP biosynthesis, and the MYBrelated and CO-like transcripts appeared as the most numerous TFs within the core-DEGs (Fig. S4). The MYBrelated and CO-like TFs are involved in circadian clock and photoperiod networks that control flowering time 59 . The common down-regulation of these genes in curly vs smooth endives may reflect leaf-group specific responses to growth cycle conditions and regulation of bolting time, a major breeding trait of Cichorieae leafy crops 58 . As for the STP biosynthesis pathway, the QHS1 gene, putatively encoding an enzyme that catalyzes the synthesis of βcaryophyllene, maintained the differential expression pattern in curly-vs smooth cultivars and was up regulated in the former. β-caryophyllene is one of the most widespread sesquiterpene floral volatiles that acts in defense  mechanisms 60 and the QHS1 expression pattern stimulates the speculation that different contents of β-caryophyllene, naturally found in Cichorium spp. 61 , may distinguish the leaf cultigroups and subtend different responses to biotic stress.
The costunolide biosynthesis branch of endive transcriptome consisted of 5 GAS, 3 GAO, and 3 COS transcripts that had significant sequence variability to suggest their origins from distinct genes. Consistently, in lettuce, each of these genes belong to families scattered in the genome and is able to encode isoforms by inferred alternative splicing (data retrievable from phytozome.jgi. doe.gov). The phylogenetic tree based on chicory, endive, and lettuce sequences highlighted the clustering of C.
(see figure on previous page) Fig. 6 Principal component analysis and correlation plot in endive STL pathway. a PCA biplot of STL contents and biosynthesis/transcription factor gene expressions in curly-and smooth-leafed endives. GAS germacrene A synthase, GAO germacrene A oxidase, COS costunolide synthase. Lp lactucopicrin, Lc lactucin, DHLc 11(S),13-dihydrolactucin, dLc 8-deoxylactucin, DHdLc 11(S),13-dihydro-8-deoxylactucin, DHLp 11(s),13dihydrolactucopicrin. b The Pearson's coefficient (r) and correlation significance (asterisks) are disposed in a symmetric matrix made with the same variables as in PCA. The heat map places variables in hierarchical clustering; negative and positive correlations assign (r) values in red and blue squares, respectively; bold values refer to significant correlations. *, **, *** = significant at P ≤ 0.05, 0.01, and 0.001, respectively; n.s. non-significant Fig. 7 Putative network subtending synthesis of Lp-and Lc-like compounds. a Heat map visualization of the relative gene expression levels (log2 fold change) in curly vs smooth endives. The column "Ex" reports the up-or down-regulation expression pattern that was maintained in each curly vs smooth pairwise comparison (log 2 FC and FDR values are listed in Table S13). b Blue and orange edge represent positive and negative correlations, respectively. Solid or dashed traits refers to correlation strength according to the r coefficient ranges (bottom right). Transcription factors were sited upstream the GAS-GAO-COS biosynthesis gene module leading to sesquiterpene lactones. TF transcription factors, BS biosynthesis genes, STL sesquiterpene lactones; explanatory notes for STL abbreviations are in the legend of Fig. 6 intybus and C. endive deduced proteins, confirming the species vicinity 54 . Some BS proteins (e.g. 52991 and 16955/GAS) fell in groups including members with ascertained function (Fig. 5) and they are likely to conserve it. However, the roles of other BS enzymes (e.g. GAS contigs: 83192 and 41447) need investigation, considering that amino acid stretch diversity (examples for GAS are in Fig. S6) suggests the occurrence of variation of catalytic functions and/or substrate specificity.
The PCA outcomes separated curly-from smooth types, the former were in association with STL contents and the expression of BS, MYB, and WRKY TFs, while the latter just grouped with two MYB-related factors (Fig. 6a). A set of BS genes (3 GAS, 1 GAO and 2 COS) showed positive correlation among themselves and vs the contents of DHdLc and dLc (Fig. 6b). Interestingly, DHLp was more significantly related to DHdLc/dLc than Lp/DHLc molecules (Fig. 6a) and showed significant positive correlation with just two COS and one GAO genes (Fig. 6b). Positive correlations between BS gene expression and STL contents were observed in chicory and artichoke 24,62 . Consistently, the higher expression of 41,447 and 52,991/GAS genes (Fig. 4c, qPCR panels) may explain the higher contents of dLc and DHdLc in the curly "Domari" and "Myrna" vs the smooth "Confiance" and "Flester", while the comparable messenger levels of "Imari" vs the smooth cultivars may subtend the low content differences in this STL class ( Table 6). The 83192/GAS / expression recurred as more abundant in curly than smooth cultivars (Fig. 4c,  Fig. 7), supporting a conserved role to determine higher amounts of lactucopicrin. A significant positive correlation of this GAS gene was found with a downstream GAO gene (11533/GAO), whereas no associated COS genes were identified. This may be due to a limited level of transcriptome functional annotation, caused by the fragmentary knowledge on the STL pathway that prevented the widening of COS gene pool. The identification of novel GAS and GAO putative enzymes involved in lactucopicrin biosynthesis may turn a relevant information, considering that they appear to be phylogenetically near a lettuce GAS cluster with uncharacterized function. Indeed, a three-way significant positive correlation is supportive for the control of 83192/GAS by the 86458/ WRKY and 72724/MYB TFs in Lp synthesis. Moreover, promoter sequence analysis of lettuce GAS gene 90% identical to endive 83192/GAS (Table S9) scored numerous target motifs for WRKY and MYB-like factors (Table S14). Inherently, the endive deduced proteins of 32240 and 32243/MYB_related genes were 65% identical to Arabidopsis LHY-CCA1-LIKE1 TFs (Table S13) that are co-expressed and highly correlated to several isoprenoid genes in photosynthetic tissues 63 . Moreover, a few WRKY factors can control sesquiterpene biosynthesis 64 ; the endive 86458/WRKY shares identity with the Arabidopsis WRKY70, an upstream effector of MYC2 factor 65 that regulates transcription of two terpene synthases 66 . Consequently, the presence of putative WRKY and MYB binding sites in the promoter of the L. sativa homologous of 83192/GAS gene may imply that the latter is also a direct target in endive. The strong positive correlation of both 86458/WRKY and 72724/MYB vs Lc and DHLc contents, which significantly grouped with Lp (Fig. 6), suggests that the branch leading to Lp, Lc, and DHLc might be under a common regulatory network. The negative correlation of 32240 and 32243/MYB_related gene expression vs those of 86458/WRKY and 72724/ MYB and the amounts of Lp, Lc, and DHLc reinforces the latter hypothesis. The transcription of all identified TF did not show significant correlation with dLc/DHdLc/DHLp levels, leading to hypothesize the existence of two separated routes in the synthesis of Lp/Lc/DHLc and DLc/ DHdLc/DHLp. Moreover, different GAS/GAO genes correlated with the two STL group types, which further supports the likeliness of a branching point at the germacrene synthase level before costunolide formation.
Looking at the STLTOT contents, ranges were higher than those found in a survey (128-264 and 235-2045 mg/ kg d.m. in curly and smooth types respectively) based on 32 accessions 12 , but, consistently, the average content of curly genotypes was maintained over 2.4-fold higher than escaroles. The relative levels of LcTOT and LpTOT subclasses (out of total STL) were 64.9% and 35.1% in curly endives similarly to those of escaroles (62.7% and 37.2%). These values differed from other results 20 reporting that LcTOT and LpTOT ratios were 77% and 22% in curly vs 82% and 16 % in smooth endives. Several factors may be evoked to explain these discrepancies including diversity of cultivars, leaf types and their positon in the rosette, cultivation and environmental conditions, which influence STL leaf content and composition of endive-related species such as chicory and lettuce 67,68 . Although sensory analyses were beyond the scope of this work, predicted bitterness based on STL perception thresholds 18 distinguished curly from smooth types, consistently with taste based bitterness indices of endive 20 . Moreover, Lc, DHLc, and Lp were the best discriminants of curly vs escarole types (respectively 3.7-, 2.1-, and 2.3-fold higher in the former). The Lp content sensibly altered predicted bitterness due to its much lower index than Lc and DHLc (0.5 vs 1.6-1.7 p.p.m.), and was consistent with the Lp dominant effect measured in sensory tests on endive 19 .
In conclusion, the assembled transcriptome was effective to assess differences between curly-and smoothleafed cultivars at both the allelic and gene expression levels, and useful to characterize the STL biosynthesis pathway in endive. Specifically, a set of GAS/GAO/COS genes with coordinated/correlated expression to the contents of DLc/DHdLc/DHLp was identified and the specific relationship among 86458/WRKY, 72724/MYB, and 83192/GAS was inferred for the Lp/Lc/DHLc branch. These findings open perspectives for further investigating these two key branches, considering that genes downstream the BS module have been unknown so far, as well as those of catabolism and transport.