Trichosanthes anguina L.), which belongs to the Cucurbitaceae family, is a popular ornamental and food crop species with medicinal value and is grown in many parts of the world. Although progress has been made in its genetic improvement, the organization, composition, and evolution of the snake gourd genome remain largely unknown. Here, we report a high-quality genome assembly for snake gourd, comprising 202 contigs, with a total size of 919.8 Mb and an N50 size of 20.1 Mb. These findings indicate that snake gourd has one of the largest genomes of Cucurbitaceae species sequenced to date. The snake gourd genome assembly harbors 22,874 protein-coding genes and 80.0% of the genome consists of repetitive sequences. Phylogenetic analysis reveals that snake gourd is closely related to sponge gourd but diverged from their common ancestor ~33–47 million years ago. The genome sequence reported here serves as a valuable resource for snake gourd genetic research and comparative genomic studies in Cucurbitaceae and other plant species. In addition, fruit transcriptome analysis reveals the candidate genes related to quality traits during snake gourd fruit development and provides a basis for future research on snake gourd fruit development and ripening at the transcript level.Snake gourd ( Snake gourd  is a diploid annual woody climber (liana) of the genus Trichosanthes in the family Cucurbitaceae2. This species is commonly called viper gourd, snake tomato, or long tomato, and has several synonyms . Snake gourd originated in India or the Indo-Malayan region in tropical Asia4 and is widely distributed in Asian countries7. Its green, tender stems, leaves, and fruits are consumed as edible vegetables6, which have high nutritional value, because they are rich in vitamins, essential minerals, dietary fiber, and other nutrients, and are a wholesome, healthy addition to diets8. The fruits of snake gourd are frequently consumed when immature. As the fruits mature, the rind and flesh turn red, and the red flesh is used as a tomato substitute2. Snake gourd fruits can grow to 1.5 m in length, are serpentine in shape (hence the name snake gourd), and are used for ornamental purposes when they are mature. Their functional constituents include flavonoids, β-carotenoids, lycopene, and phenolic acids, which have beneficial pharmacological and therapeutic effects for humans11. For these reasons, snake gourd is a potentially economically important crop species with food, medicinal and ornamental value, and is worthy of further study and scientific research.As the second largest vegetable family, the Cucurbitaceae family comprises one of the most genetically diverse groups of plants. The members of this family are prevalent in tropical regions17, melon20, zucchini21, bottle gourd22, watermelon25, pumpkin27, wax gourd28, sponge gourd29, and bitter gourd30. However, no reference genome of snake gourd is available. Recent studies have shown that there is likely a common tetraploid ancestor of Cucurbitaceae species, and it has been inferred that cucurbits diverged from their common ancestor with grape 107–121 million years ago (Mya)31. Watermelon is believed to have differentiated from within the Cucurbitaceae family ~20.4 Mya and the divergence between melon and cucumber occurred 9.0–10.2 Mya33. However, the evolutionary history of the snake gourd genome remains largely unexplored.In the past decade, owing to the rapid advances in sequencing technology and bioinformatic algorithms, reference genomes of a number of cucurbit species have been assembled, including cucumberIn this study, we assembled the genome of snake gourd using Nanopore long reads combined with Hi-C chromatin interaction maps. Protein-coding genes and noncoding RNAs (ncRNAs) were predicted from the genome assembly, after which the predicted genes were functionally annotated. Phylogenetic and comparative genomic analyses indicated that snake gourd is closely related to sponge gourd. Transcriptome analysis revealed candidate genes that are involved in fruit texture, pigment accumulation, plant hormones, and resistance, and the expression profiles during fruit development and ripening, which may contribute to the unique characteristics of snake gourd fruits. The genome sequence presented in this study provides insights into both the structural characteristics of the snake gourd genome and the evolutionary relationship of snake gourd and related species and serves as a valuable resource for genomic research and comparative genomics within the Cucurbitaceae.For this study, snake gourd plants were grown in a greenhouse, as shown in Fig. A nanopore library was constructed and sequenced, generating a total of 135.9 Gb of raw data. After cleaning, the data were reduced to 111.8 Gb, representing ~108.5× the total data composing snake gourd genome, with an N50 read length of 32.7 kb. De novo assembly of the Nanopore reads resulted in an initial assembly of 919.8 Mb consisting of 167 contigs with an N50 length of 21.9 Mb Table , which s34, which had a mapping rate of 99.4%. Further evaluation using BUSCO (https://busco.ezlab.org/) with a database of 2,326 conserved core plant genes indicated that 95.7% of the core genes were found to be complete in the snake gourd genome assembly, including 93.5% that were single copies and 2.2% that were duplicated copies. In addition, 1.1% were found to be fragmented and only 3.2% were missing. Taken together, these results supported the integrity and high quality of the assembled snake gourd genome.To evaluate the quality of the assembly, we mapped the cleaned Illumina short reads to the assembled genome using BWABased on the principles of structure prediction and de novo prediction, we constructed a repeat sequence database of the snake gourd genome, which was used to screen the assembly for repeat sequences. A total of 736.1 Mb (80.0%) of repetitive sequences were identified in the assembled genome. Among these repetitive sequences, long terminal repeat (LTR) retrotransposons were predominant, constituting 66.7% of the snake gourd genome assembly, with 45.6% belonging to the copia type and 19.8% belonging to the gypsy type  strategy—were used to predict protein-coding genes in the snake gourd genome and the predictions from these three methods were integrated using EVMT. anguina L., Luffa cylindrica L., Cucumis sativus L., Cucumis melo, Citrullus lanatus, Lagenaria siceraria, Cucurbita moschata, Cucurbita pepo, and Momordica charantia), two rosid species , one monocot (Oryza sativa), and one in the basal lineage of angiosperms (Amborella trichopoda), clustered into 34,827 gene families. A total of 17,057 gene families were identified in snake gourd, 125 of which (comprising 451 genes) were specific to the snake gourd genome  events occurred, but is substantially higher than that in C. moschata and C. pepo, whose genomes underwent a recent WGD event  encoding flavonol synthase/flavanone 3-hydroxylase in the flavonoid biosynthetic pathway, two autophagy-related genes associated with the regulation of the autophagy pathway, and two genes encoding alcohol dehydrogenases (ADHs) (EVM0001187.1 and EVM0017122.1), which are involved in fatty acid degradation, tyrosine metabolism, α-linolenic acid metabolism, and glycolysis/gluconeogenesis. Furthermore, a number of other genes were identified that are known to participate in fruit ripening, such as those encoding cell wall-associated hydrolase, phenylalanine ammonia-lyase (PAL) 1, malonate–CoA ligase, and aquaporin PIP2-2. Together, these specific genes and gene families may contribute to the unique fruit features of snake gourd  and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed for gene families specific to snake gourd. GO analysis showed that these families were enriched in genes involved in metabolic processes, cellular processes, and biological regulation or encoded products that have binding, nucleic acid binding, DNA binding, and catalytic activity function  relative to those of the last common ancestor, whereas 268 gene families showed contraction -γ-cadinene synthase, myrcene synthase, 1-aminocyclopropane-1-carboxylate oxidase (ACO), auxin-induced protein 22D, 23 kDa jasmonate-induced protein, anthocyanidin 3-O-glucosyltransferase, and flavonoid hydroxylase. These genes are involved in cell wall biosynthesis and degradation, flavor and aromatic compound generation, phytohormone synthesis and signal transduction, and flavonoid biosynthesis. Other gene families involved in plant defense and response to pathogens were also found to be expanded in snake gourd, such as those whose members encode UDP-glucosyltransferase, salicylate carboxymethyltransferase, tobacco mosaic virus (TMV) resistance protein N, and other disease resistance proteins.Based on 970 single-copy genes in snake gourd and in 12 other plant species, a phylogenetic tree was constructed. The phylogenetic tree showed that, of all the Cucurbitaceae species examined, snake gourd is most closely related to sponge gourd Fig. . Snake gion Fig. . KEGG anion Fig. , and inc36. However, our in-depth genomic analysis indicated that no recent WGD events have occurred in the snake gourd genome, which is consistent with the findings of a recent report37. The Ks distribution among these species suggested that snake gourd diverged from sponge gourd ~42 Mya and from watermelon ~47 Mya  in the fruits at different stages. The results showed that there were 362 common DEGs in the development and ripening stages (20 d vs. 40 d and 40 d vs. 60 d) Fig. . Among tperoxidase 31 (increase of 3.4-fold), peroxidase 55-like (increase of 4.0-fold), peroxidase 5 (increase of 6.4-fold), peroxidase 64-like (increase of 8.8-fold), peroxidase 2-like (increase of 11.6-fold), peroxidase 21-like (increase of 10.5-fold), and probable mannitol dehydrogenase (increase of 2.1-fold), markedly changed in snake gourd fruit  upregulated and 124 (26%) downregulated genes, were differentially expressed in the 20 d vs. 40 d comparison. KEGG pathway analysis of the upregulated genes showed that they were mainly enriched in the pathways involved in phenylpropanoid biosynthesis, amino sugar and nucleotide sugar metabolism, protein processing in the endoplasmic reticulum and starch and sucrose metabolism Fig. . The expPAL, cysteine protein inhibitor 1-like, MLP-like protein 329, poly transporter 5-like, and PAL 3. Their expression profiles were found to be downregulated after 60 d compared with 40 d. The results of the KEGG pathway analysis of the upregulated genes suggested that the most enriched pathways were involved in fatty acid biosynthesis, carbon metabolism, pyruvate metabolism, and carotenoid biosynthesis  (increase of 1.7-fold), 15-cis-phytoene desaturase (increase of 4.6-fold), 15-cis-zeta-carotene isomerase (increase of 5.1-fold), zeta-carotene desaturase (ZDS) (increase of 19.0-fold), prolycopene isomerase (CRTISO) (increase of 25-fold), and β-carotene 3-hydroxylase 1 (increase of 40.0-fold), and 5 were downregulated, including lycopene epsilon cyclase, lycopene β-cyclase, probable 9-cis-epoxycarotenoid dioxygenase NCED5, and abscisic acid 8-hydroxylase 4-like. Moreover, 11 genes related to the accumulation of flavonoids and anthocyanidins in the fruits were also found, including anthocyanidin 3-O-glucosyltransferase 2-like, flavonoid 3-monooxygenase-like, flavonoid 3,5-methyltransferase-like, flavonol synthase/flavanone 3-hydroxylase-like, and isoflavone reductase-like protein, which exhibited biphasic expression patterns. PAL is involved in the phenylalanine metabolism pathway and functions prior to lignin synthesis, and the expression of many peroxidases related to enzymes associated with lignification, the cell wall, auxin catabolism, defense against pathogens, and scavenging of reactive oxygen species is downregulated. In addition, the expression levels of numerous genes related to plant hormones synthesis and signal transduction were significantly altered: the levels of ACO homolog 1/3-like, ethylene receptor 2-like, ethylene response sensor 1, the serine/threonine-protein kinase CTR1, ethylene insensitive 3-like 3 protein, auxin response factor 18 (ARF), auxin-responsive protein SAUR71-like, gibberellin-regulated protein 1-like (GRP), and indole-3-acetic acid-amido synthetase GH3.17 were upregulated, and the expression levels of others, such as ACO1/3, ethylene-responsive transcription factor 1B-like (ERF), indole-3-acetic acid-amido synthetase GH3.10/GH3.6-like, auxin-induced protein 15A/22D/22B-like, ARF 4/9/18, auxin-responsive protein IAA/SAUR50-like, auxin transporter-like protein 1/2, the jasmonic acid-amido synthetase JAR1, abscisic acid receptor PYR1-like, GRP 9/4-like, and the AP2-like ethylene-responsive transcription factor ANT, were downregulated. Furthermore, the mRNAs for some enzymes related to fruit cell wall structure also changed significantly, with PE, PE 3-like, pectate lyase 5/8/12/18 (PL), pectin acetylesterase 8-like, PG-like, PG At1g48100-like, PG QRT3-like, GAL 3/10/5-like, BGLU 18/44-like, glucan endo-1,3-β-glucosidase, expansin (EXP), EG 6/10/11/17/24-like, and cellulose synthase-like protein G3 downregulated while others such as β-d-xylosidase1-like, GAL 13-like, GAL 16, GAL-like, cellulose synthase-like protein E1/H1, and EG 9-like were downregulated. These changes in mRNAs of cell wall enzymes may be related to fruit texture changes, as the fruits were softer after 60 d compared with 40 d.We identified 4801 genes that were differentially expressed by comparing the transcriptome at 60 d with that at 40 d. A total of 1957 (41%) genes were upregulated and 2844 (59%) genes were downregulated. In specific gene families of snake gourd, five genes were found to be differentially expressed: sis Fig. . Among tArabidopsis, pumpkin, and sponge gourd, respectively; moreover, there were no proteins that clustered within the GH-9 subfamily in these three plant species  had much higher expression in SG3, whereas the expression levels of abscisic acid 8-hydroxylase 4-like (EVM0017015 and EVM0004622) and 9-cis-epoxycarotenoid dioxygenase NCED5 (EVM0015273) decreased during fruit maturation  related to cell wall structure was selected to construct a phylogenetic tree. The GH families were divided into four subfamilies: GH-1, GH-9, GH-28, and GH-35. The members of the GH-28 and GH-35 subfamilies encode the majority of GH proteins. The genes belonging to the GH-28 and GH-35 subfamilies in snake gourd showed the closest phylogenetic relationships with those of pumpkin and sponge gourd Fig. . In the ies Fig. . Based oies Fig. . In the ies Fig. . In the ion Fig. .Fig. 5Ph2. However, research on this species is limited3, and related molecular-level studies are scarce. We aimed to sequence, assemble, annotate, and comparatively analyze the snake gourd genome to provide a basis for future investigations and improvement of this species. This is the first report of a high-quality genome for snake gourd, which has one of the largest genomes of Cucurbitaceae species sequenced to date.Snake gourd is an excellent vegetable species that is popular for both its unique flavor and its nutritional, health, medicinal and ornamental value and has high utilization potential30 -based assessment revealed that the snake gourd assembly is more complete than or comparable to other reported cucurbit reference genomes  but larger than those of other Cucurbitaceae species, such as watermelon, melon, pumpkin, bottle gourd, cucumber, zucchini, bitter gourd 200–400 Mb), and sponge gourd (669 Mb)00 Mb, an40, root tip cell growth and differentiation41, and chloroplast degradation in senescing leaves42. The autophagy pathway has also been reported to be involved in plant innate immunity43 and responses to a variety of abiotic stresses44, and in assisting plants in surviving nutrient-limitation stress45. ADH enzymes play multiple roles in anaerobic fermentation, aerobic fermentation, and the production of scents that discourage predation, attract pollinators, and facilitate seed dispersal46. In petunia, both the ADH2 and ADH3 genes act in the lipoxygenase pathway to produce floral scents47 and flavor volatiles in ripening fruits. A previous study reported a strawberry fruit-specific aquaporin, FaPIP1;1, which showed an expression profile associated with fruit ripening48. Further study indicated that the FaPIP aquaporins showed an expression pattern associated with fruit firmness48. In addition, several specific genes were found to be differentially expressed between 40 d and 60 d, which may contribute to the unique snake gourd fruit features.In specific gene families of snake gourd, a number of genes have been found to potentially participate in fruit ripening, such as several genes encoding autophagy-related proteins, ADHs, and the aquaporin PIP2-2. Autophagy is involved in plant processes such as floret ripening (in wheat)49. We identified transcripts for highly expressed cell wall-modifying genes similar to those expressed in expanding cucumber51, tomato52, melon53, and watermelon54 fruits, such as EXPs, endo-1,2-B-glucanase, BGLUs, PLs, and pectin methylesterases. Major increases in transcripts for mRNAs encoding enzymes related to cell wall metabolism were detected, including GAL 10 (increase of 14.3-fold), PE-like (increase of 21.6-fold), EG 17-like (increase of 21.2-fold), expansin-A10-like (increase of 27.1-fold), UDP-glucuronate 4-epimerase 1-like (increase of 29.8-fold), and PE 53 (increase of 30.4-fold), which may be involved in the production of longer and harder fruits. We also detected the upregulated expression of auxin-related genes. These genes may be involved in fruit elongation, as many studies have shown that phytohormones, especially auxin and gibberellins, are related to fruit growth and changes in fruit shape55. These results are consistent with those of previous studies that found that three homologs of auxin-related genes were differentially expressed in developing watermelon fruits54. In addition, we found an increase in resistance (R) gene transcripts, such as those of TMV resistance protein N, leucine-rich repeat extensin-like protein 3, and LRR receptor-like serine/threonine-protein kinase FEI 1. Studies have shown that younger and smaller fruits are more susceptible to infection than older and larger fruits56, and changes in the expression of resistance genes may explain the stronger resistance of larger fruits compared with smaller ones. These genes mainly regulate the resistance of plants to pathogens and insects, and in cucumber, eight DEGs have been identified that may be associated with aphid resistance57.Between 20 d and 40 d, the snake gourd fruits became longer and harder, and we detected increased transcript levels of genes related to cell division, cell expansion, fruit growth, and protection of the fruit surface58, and the key genes involved in the regulation of flesh texture were differentially expressed in cultivated and wild watermelon59. In addition, the genes involved in the upstream and downstream steps of the carotenoid biosynthesis or degradation pathway were upregulated and downregulated, respectively. These changes may contribute to the increased carotenoid accumulation, making the fruit turn orange after ripening. This is supported by research on carotenoid accumulation and related gene expression during squash fruit development60.Transcripts for several enzymes mentioned above related to cell wall metabolism were found to be downregulated during ripening from 40 to 60 d, which may be the cause of fruit softening at 60 d. The expression levels of these enzymes are different among various fruits and at different fruit developmental stages. Studies on watermelon have shown that there are differences in the expression of genes related to cell wall components in two kinds of flesh with different texturesYoung fresh leaf samples were collected from snake gourd plants grown in the greenhouse of the Shouguang vegetable expo garden. Genomic DNA was extracted from young leaves and used to construct two Illumina DNA libraries whose fragment size was ~350 bp, according to the standard protocols provided by the Illumina company. The libraries were sequenced on an Illumina HiSeq X platform in paired-end mode and with read length of 150 bp. The sequence data were evaluated  and filtered to obtain high-quality clean reads, which were subsequently used for estimation of the genome size, GC content, heterozygosity level, and postassembly error correction and evaluation. The raw sequencing reads were processed for quality control and for the removal of adapter and low-quality sequences using a custom Perl script. After trimming, reads with lengths less than 100 bp or a Q30 value < 85% were discarded.61 and the corrected reads were assembled into contigs using SMARTdenovo (https://github.com/ruanjue/smartdenovo). The assembled contigs were further polished using the Racon62 and Pilon63 programs in conjunction with cleaned Illumina reads.A Nanopore library was constructed from high-molecular-weight DNA using a Ligation Sequencing Kit (SQK-LSK109) and sequenced on a PromethION R9 flow cell with a PromethION Flow Cell Priming Kit (EXP-FLP001.PRO.6)  according to the manufacturer’s instructions. The Oxford Nanopore reads were self-corrected using Canu34 (version 0.7.10-r789). Only uniquely aligned paired-end reads with a mapping quality greater than 20 were used for further analysis. Invalid read pairs including dangling-end and self-cycle, religation and dumped products were filtered by HiC-Pro64 v2.8.1. Approximately 75% of the uniquely mapped read pairs were valid interaction pairs and were used for correction of the assembled contigs, which were then clustered, ordered, and oriented onto chromosomes by LACHESIS65 with the parameters “CLUSTER_MIN_RE_SITES, 52; CLUSTER_MAX_LINK_DENSITY, 2; ORDER_MIN_N_RES_IN_TRUN, 51; ORDER_MIN_N_RES_IN_SHREDS, 50.”We also constructed Hi-C fragment libraries (insert size of 300–700 bp) and sequenced them using the Illumina platform. The raw Hi-C reads were processed to trim adapter sequences and to remove low-quality reads. The cleaned Hi-C reads were first truncated at the putative Hi-C junctions, and then the resulting trimmed reads were aligned to the genome assembly with BWA66 and RepeatScout67. Repeat sequences in the database were classified using PASTEClassifier68 and then merged together with the repeats in RepBase69 to construct the final repetitive sequence database. Repetitive sequences of the snake gourd genome were predicted using RepeatMasker70 based on the constructed repetitive sequence database.A repetitive sequence database of the snake gourd genome was constructed using LTR_FINDER71, Augustus72 v2.4, GlimmerHMM73 v3.0.4, GeneID74 v1.4, and SNAP75 (version 2006-07-28) for ab initio predictions, and GeMoMa76 v1.3.1 was used for homology-based predictions using protein sequences from A. thaliana, C. lanatus, C. sativus, and L. cylindrica. For transcript-based predictions, a mixture of four tissues  of snake gourd was used to construct an Illumina RNA-Seq library, which was subsequently subjected to PE (2 × 150 bp) sequencing on an Illumina HiSeq X Ten platform . After discarding the reads with low-quality bases, adapter sequences, and duplicated sequences, the retained high-quality clean reads were mapped to the snake gourd genome assembly using HISAT277 v2.0.4 and, based on the mapping results, the reads were assembled into transcripts using StringTie78 v1.2.3, TransDecoder v2.0 (http://transdecoder.github.io) and GeneMarkS-T79 v5.1; the assembled transcripts were subsequently used for gene prediction. RNA-Seq reads were also de novo-assembled using Trinity80 and PASA81 v2.0.2 was used for gene prediction from these de novo-assembled transcripts. Finally, EVM35 v1.1.1 was used to integrate the prediction results obtained by the above three methods. In addition, we also predicted different ncRNAs. MicroRNA and rRNA sequences were predicted based on the comparison of the snake gourd genome to the content of the Rfam82 database via BLASTn and tRNA sequences were predicted using tRNAscan-SE83. Pseudogene prediction was also performed. Using the predicted protein sequence, through a BLAST84 comparison, we identified possible homologous gene sequences in the genome and then used GeneWise85 to identify premature termination codons and frameshift mutations in the gene sequences to identify pseudogenes.Protein-coding genes were predicted from the snake gourd genome using three different strategies: ab initio prediction, homology-based prediction, and transcript-based prediction. We used GENSCAN86, KOG87, GO88, KEGG89, and TrEMBL90 databases using BLAST91 v2.2.31, with an E-value cutoff of 1 × 10−5, and functional annotations of the snake gourd genes were derived from the homologous sequences in these databases.The protein sequences of the snake gourd genes were compared to the content of the NCBI nr92 v2.3.7. The obtained orthologous groups (gene families) were annotated using the PANTHER v15 database93. GO and KEGG enrichment analyses of gene families unique to snake gourd were ultimately performed using ClusterProfile94 v3.14.0.The protein sequences from snake gourd and 12 other representative plant species were clustered into orthologous groups using OrthoFinder95 v1.6.11, with A. trichopoda as the root and the number of bootstraps set to 1000. We then used MCMCTee software in the PAML package96 v4.9i to calculate the divergence time. The number of iterations of the Markov chain included a burn-in number of 700,000, a sampfreq of 30, and an nSample of 6,000,000.By the use of 970 single-copy protein sequences, an evolutionary tree was constructed using the maximum likelihood method implemented in IQ-TREE97 v4.2, we estimated the number of gene family members of each branch’s ancestor via a birth mortality model, which was used to predict the contraction and expansion of gene families of the snake gourd relative to its ancestors. We determined whether the expansion or contraction was significant using a P-value cutoff of 0.05. The expanded and contracted gene families identified in snake gourd were annotated with PANTHER, and GO and KEGG enrichment analyses on these families were performed with ClusterProfile.Using the results of the evolutionary tree with differentiation time and gene family clustering by CAFE98 v0.9.29.130 was used to compare the protein sequences of two species and determine similar gene pairs . Based on the diamond results, collinear blocks between the genomes of the two species were identified using MCScanX99.DiamondKs) and fourfold degenerate (4DTv) sites of paralogous genes, which were calculated using WGD software100 in conjunction with a Perl script (https://github.com/JinfengChen/Scripts), respectively.WGD events were determined based on the distribution of the synonymous substitution rate  < 0.01 were used. As a screening standard, the fold change (0.01) represents the FDR between two samples by the use of a corrected P-value denoting significant differences. The gene functions were annotated on the basis of the content from six databases: the Nr, Pfam, KOG/COG, Swiss-Prot, KEGG, and GO databases.Fresh snake gourd fruit samples at 20, 40, and 60 d were collected for transcriptome analysis. cDNA libraries were obtained by PCR enrichment. After the library was checked for quality by quantitative PCR, the Illumina platform was used for sequencing. The data were cleaned by removing low-quality sequences and those containing adapter reads and used for sequence alignment with the specified reference genome. The transcriptome was assembled using StringTiehttp://hmmer.org/download.html) employing the hidden Markov model (HMM) corresponding to the Pfam GH family. From the proteins obtained using the raw HMM, a high-quality protein set  was aligned and used to construct a specific GH family HMM using hmmbuild from the HMMER v3 suite. With this new specific HMM, all proteins with an E-value lower than 0.01 were selected. The GH genes were further filtered based on manual curation and functional annotations (supplied by Beijing Biomarker).Predicted proteins from the genome were scanned using HMMER v3 (SupplementarySupplementary Table 3Supplementary Table 5


Epeorus herklotsi (Ephemeroptera: Heptageniidae) from Longquan, Zhejiang province, China was a circular molecule of 15,499 bp in length, containing 13 protein-coding genes, two ribosomal RNAs, 23 transfer RNAs, and a control region. The A + T content of the overall base composition of H-strand is 65.7%. All of the 13 protein-coding genes were begun with ATN as start codon except ND5 using GTG. ATP6, ATP8, COI, COIII, ND1, ND2, ND4L, and ND6 genes were terminated with TAA as stop codon, Cyt b ended with TAG and the other three protein-coding genes end with an incomplete stop codon (TA or T). In BI phylogenetic trees, the monophyly of the families Caenidae, Heptageniidae, Isonychiidae and Viemamellidae, and the genus Epeorus were strongly supported. Epeorus is a sister clade to Parafronurus, then cluster with Paegniodes.The mitochondrial genome of  Epeorus herklotsi is widely distributed throughout the rivers of China. For the convenience of identifying this species, we sequenced the mitochondrial genome of E. herklotsi. In Ephemeroptera, gene rearrangements were found in six sequences of twenty-three known mayfly mitochondrial genomes with IMQM tRNA cluster found in Heptageniidae  were used for normal and long PCRs.The samples of ATP6, ATP8, COI, COIII, ND1, ND2, ND4L, ND6 genes were terminated with TAA as stop codon, Cyt b ended with TAG and the other protein-coding genes ended with an incomplete stop codon (TA or T). An IMQM tRNA cluster at the upstream of ND2 gene was found, which was consistent with P. youi , C (21.3%), A (32.7%), G (13%), and the A + T content (65.7%). All of the 13 protein-coding genes were begun with ATN as start codon except ND5 using GTG. S. chinensis  and S. immanis (FJ606783) were cluster with Ameletus sp. 1 (KM244682) and Ephemera orientalis, respectively. Within the Heptageniidae clade, Epeorus was a sister clade to Parafronurus, then (Epeorus + Parafronurus) cluster with Paegniodes. Two species of Epeorus herklotsi were cluster together.Bayesian inference (BI) tree was constructed using the 13 PCGs from 21 species using E. herklotsi has been assigned deposited to GenBank with the following accession number MH752075.The complete mitochondrial genome of


Cell cycle progression is a tightly regulated process by which DNA replicates and cell reproduces. The major driving force underlying cell cycle progression is the sequential activation of cyclin-dependent kinases (CDKs), which is achieved in part by the ubiquitin-mediated proteolysis of their cyclin partners and kinase inhibitors (CKIs). In eukaryotic cells, two families of E3 ubiquitin ligases, anaphase-promoting complex/cyclosome and Skp1-Cul1-F-box protein complex, are responsible for ubiquitination and proteasomal degradation of many of these CDK regulators, ensuring cell cycle progresses in a timely and precisely regulated manner. In the past couple of decades, accumulating evidence have demonstrated that the dysregulated cell cycle transition caused by inefficient proteolytic control leads to uncontrolled cell proliferation and finally results in tumorigenesis. Based upon this notion, targeting the E3 ubiquitin ligases involved in cell cycle regulation is expected to provide novel therapeutic strategies for cancer treatment. Thus, a better understanding of the diversity and complexity of ubiquitin signaling in cell cycle regulation will shed new light on the precise control of the cell cycle progression and guide anticancer drug development.  The cell cycle is a tightly orchestrated cellular process that governs the timely DNA replication and cell division events.Sequential activation of cyclin-dependent kinases (CDKs) drives cell cycle progression in a timely and precisely regulated manner.The activity of CDKs is modulated by cyclin partners and CDK inhibitors (CKIs), which are tightly controlled by the ubiquitin–proteasome system.Two important types of E3 ligases, the anaphase-promoting complex or cyclosome (APC/C) and Skp1-Cul1-F-box (SCF) complexes, are dedicated to cell cycle control.Targeting E3 ubiquitin ligases provides effective therapeutic strategies for cancer treatment.Unlike proteolytic signals, relatively little is known regarding the roles and mechanisms of non-proteolytic signals, such as the ones mediated by K6, K27, and K29 polyubiquitin chain, underlying the cell cycle control.The specificity and diversity of deubiquitinating enzymes (DUBs) in regulating mitosis need to be further investigated.How is the balance of ubiquitination–deubiquitination achieved to ensure accurate cell cycle progression remains elusive.Unlike CDC20, the regulation of the enzymatic activity of CDH1 is not well defined yet.The detailed mechanisms underlying spindle checkpoint imposed various E3 ligase activities of CDC20 toward different substrates need to be further investigated.How those ubiquitination signaling events at the spindle checkpoint are integrated and orchestrated in a space–time-dependent manner remains not fully understood.The cell cycle is a series of tightly orchestrated molecular events that coordinately regulate DNA replication and chromosome segregation, eventually resulting in cell division and genetic material transmission. In eukaryotic cells, the cell cycle consists of four distinct phases, G1 phase (gap 1), S phase (DNA synthesis), G2 phase (gap 2), and M phase (mitotic) that proceed in a unidirectional manner  complex and control progression through mitosis, while Skp1-Cul1-F-box (SCF) ubiquitin ligase complex catalyzed K48-linked polyubiquitination and subsequent proteasomal degradation of substrates to modulate cell cycle progression –23. To gProgression through the cell cycle is determined by phosphorylation of CDK substrates , 28. To CDC20 then mediates the proteasomal degradation of Cyclin B1 and Securin, facilitating chromosome segregation and anaphase onset [CDH1 E3 ligase [CDC20 to APC/CCDH1 during anaphase. Activation of CDH1 then mediates a large number of mitotic and G1 regulators for ubiquitination and proteasomal degradation, such as Cyclin B1, PLK1, CDC20, FOXM1, and SKP2, facilitating irreversible mitotic exit and G1 maintenance [The APC/C is a multi-subunit cullin-RING E3 ubiquitin ligase that functions in mitotic phase and G1 phase, regulating cell cycle progression through M phase and entry into S phase , 32. These onset , 35, 36.se onset . Simulta3 ligase , 39. Togntenance . As cellntenance . CollectThe SCF complex contains three core subunits Cullin, SKP1, and RBX1, as well as a variable F-box protein. In comparison to APC/C, the number of substrates of SCF complex is enormous due to the variety of F-box proteins. Although almost 70 F-box proteins have been reported in mammals , only foCDH1 [CDH1-mediated proteasomal degradation, conferring activation of the SCFSKP2 E3 ligase complex [In the early G1 phase, the E3 ligase activity of SKP2 is suppressed due to the active presence of APC/CCDH1 , 46. How complex , 47. Mea complex , 49. Sub complex . Consequ complex , 52. Giv complex , 53, it  complex .When cells entry into G2 phase, Cyclin F ubiquitinates and restricts the activity of E2Fs, the main and most critical transcriptional engines of the cell cycle , 56; medCDC20 is turned on in prometaphase, Cyclin A is ubiquitinated and degraded by the proteasome [CDC20 for proteasomal degradation, is initiated during the metaphase, and occurs significantly later than the destruction of Cyclin A [CDC20 is blocked by MAD2 in prometaphase when chromosomes are not fully attached to the mitotic spindles [During the early stage of mitosis, Cyclin A associates and activates CDK1, driving the initiation of chromosome condensation –63. Onceoteasome , 65. Of Cyclin A , 67. InvCyclin A , 69. Thespindles , 71. Morspindles . Collect The protein abundance of CDH1 diminished during late G1 phase Fig. , which iβTrCP-dependent manner [CDC20 and delays cell division until accurate chromosome segregation can be guaranteed [CDC20 mediates Cyclin B1 and Securin for ubiquitination and proteasomal degradation, allowing for chromosome segregation and metaphase-to-anaphase transition [It is well established that three checkpoints operate in eukaryotic cells to ensure ordered and accurate cell cycle progression Fig. . In addit manner , 83. Supt manner . Moreovet manner . Of notet manner . In addit manner , while dt manner . The M caranteed . By contansition , 35, 36.Recognition of the substrates by corresponding E3 ligases is achieved by short destruction-mediating sequence elements, which is named degron . The besCDC20 results in efficient tumor regression [CDH1-deficient cells proliferate inefficiently and CDH1 heterozygous animals show increased susceptibility to spontaneous tumors, largely conferring CDH1 a tumor suppressor role [CDH1 is considered to promote proteasomal degradation of CIP/KIP family of CKIs and thus facilitate tumorigenesis.Accumulating evidence have shown that CDC20 is frequently overexpressed in a wide range of cancers, indicating that it might function as an oncoprotein , 100. Frgression , while tgression , advocatgression . In contgression , 100. CDsor role . In addiRegarding the role of SCF E3 complex in cancer development, emerging evidence suggest that it acts in a F-box protein- and context-dependent manner –109. SpeIn the present review, we mainly summarized the proteolytic signals involved in the cell cycle control. Moreover, non-proteolytic ubiquitination of the cell cycle regulators also plays crucial roles in controlling cell cycle progression. For example, the endoplasmic reticulum lipid associated protein 2 was found to interact and facilitate K63-linked ubiquitination and stabilization of Cyclin B1, facilitating mitosis exit . Di-ubiqCDC20, ensuring the fidelity of chromosome segregation [With respect to the well-established APC/C E3 ligase complex, activation of CDC20 is required for anaphase onset, while CDH1 plays a central role in mediating mitosis exit and G1 maintenance. The ordered activation of CDC20 and CDH1 is essential for accurate mitosis progression. Mitotic phosphorylation of APC/C relieves its auto-inhibition and facilitates CDC20 engagement , 34, whiregation . Althougregation , 74, regregation . Anotherregation . HoweverOverall, the cell cycle progression is tightly regulated to ensure the genomic integrity and identity in daughter cells, and ubiquitin signaling involves almost each step of the cell cycle. Dysregulation of the ubiquitination modification led to uncontrolled cell cycle progression and eventually resulted in tumorigenesis . Based u


Saccharomyces cerevisiae, a popular platform organism in industrial biotechnology, carry the genetic information required for synthesis of biotin, thiamine, pyridoxine, para-aminobenzoic acid, pantothenic acid, nicotinic acid, and inositol. However, omission of these B vitamins typically leads to suboptimal growth. This study demonstrates that, for each individual B vitamin, it is possible to achieve fast vitamin-independent growth by adaptive laboratory evolution (ALE). Identification of mutations responsible for these fast-growing phenotypes by whole-genome sequencing and reverse engineering showed that, for each compound, a small number of mutations sufficed to achieve fast growth in its absence. These results form an important first step toward development of S. cerevisiae strains that exhibit fast growth on inexpensive, fully supplemented mineral media that only require complementation with a carbon source, thereby reducing costs, complexity, and contamination risks in industrial yeast fermentation processes.Many strains of  Saccharomyces cerevisiae commonly use synthetic media (SM) that contain a set of water-soluble growth factors that, based on their roles in human nutrition, are referred to as B vitamins. Previous work demonstrated that in S. cerevisiae CEN.PK113-7D, requirements for biotin were eliminated by laboratory evolution. In the present study, this laboratory strain was shown to exhibit suboptimal specific growth rates when either inositol, nicotinic acid, pyridoxine, pantothenic acid, para-aminobenzoic acid (pABA), or thiamine was omitted from SM. Subsequently, this strain was evolved in parallel serial-transfer experiments for fast aerobic growth on glucose in the absence of individual B vitamins. In all evolution lines, specific growth rates reached at least 90% of the growth rate observed in SM supplemented with a complete B vitamin mixture. Fast growth was already observed after a few transfers on SM without myo-inositol, nicotinic acid, or pABA. Reaching similar results in SM lacking thiamine, pyridoxine, or pantothenate required more than 300 generations of selective growth. The genomes of evolved single-colony isolates were resequenced, and for each B vitamin, a subset of non-synonymous mutations associated with fast vitamin-independent growth was selected. These mutations were introduced in a non-evolved reference strain using CRISPR/Cas9-based genome editing. For each B vitamin, the introduction of a small number of mutations sufficed to achieve a substantially increased specific growth rate in non-supplemented SM that represented at least 87% of the specific growth rate observed in fully supplemented complete SM.Quantitative physiological studies on IMPORTANCE Many strains of Saccharomyces cerevisiae, a popular platform organism in industrial biotechnology, carry the genetic information required for synthesis of biotin, thiamine, pyridoxine, para-aminobenzoic acid, pantothenic acid, nicotinic acid, and inositol. However, omission of these B vitamins typically leads to suboptimal growth. This study demonstrates that, for each individual B vitamin, it is possible to achieve fast vitamin-independent growth by adaptive laboratory evolution (ALE). Identification of mutations responsible for these fast-growing phenotypes by whole-genome sequencing and reverse engineering showed that, for each compound, a small number of mutations sufficed to achieve fast growth in its absence. These results form an important first step toward development of S. cerevisiae strains that exhibit fast growth on inexpensive, fully supplemented mineral media that only require complementation with a carbon source, thereby reducing costs, complexity, and contamination risks in industrial yeast fermentation processes. Saccharomyces yeasts (7), nicotinic acid (B3), inositol (B8), pantothenic acid (B5), para-aminobenzoic acid (pABA) (formerly known as B10), pyridoxine (B6), and thiamine (B1). Based on their water solubility and roles in the human diet, these compounds are all referred to as B vitamins, but their chemical structures and cellular functions are very different , and (iii) inositol, which is a precursor for phosphatidylinositol and glycosylphosphatidylinositol anchor proteins  are essential for fundamental and applied research. In contrast to complex media, which contain non-defined components such as yeast extract and/or peptone, defined media enable generation of highly reproducible data, independent variation of the concentrations of individual nutrients, and, in applied settings, design of balanced media for high-biomass-density cultivation and application of defined nutrient limitation regimes , 2. CDMYs yeasts . In addiifferent . Taking ic acid B, para-amSaccharomyces species are bradytroph for some B vitamins; growth does not strictly depend on addition of all of these compounds, but their omission from CDMY typically results in reduced specific growth rates , but fast biotin-independent growth was obtained through prolonged adaptive laboratory evolution (ALE) in a biotin-free CDMY. Reverse engineering of mutations acquired by evolved strains showed that, along with mutations in the plasma membrane transporter genes TPO1 and PDR12, a massive amplification of BIO1 was crucial for fast biotin-independent growth of evolved strains  thaS. cerevisiae for inositol, nicotinic acid, pantothenic acid, pABA, pyridoxine or thiamine could be achieved by ALE and to identify mutations that support fast growth in the absence of each of these vitamins. To this end, the laboratory strain S. cerevisiae CEN.PK113-7D was subjected to parallel aerobic ALE experiments that encompassed serial transfer in different synthetic media, which each lacked a single B vitamin. Independently evolved strains from each medium type were then characterized by whole-genome resequencing and the relevance of selected identified mutations was assessed by their reverse engineering in the parental non-evolved strain.The goals of the present study were to investigate whether single-vitamin prototrophy of (This article was submitted to an online preprint archive .)S. cerevisiae strains belonging to the CEN.PK lineage, which was developed in an interdisciplinary project supported by the German Volkswagen Stiftung between 1993 and 1994  .–22.−1, wete SMD –, few trathiamine . At leashiamine) . The spehiamine) .To identify mutations contributing to vitamin independence, the genomes of the sets of three independently evolved isolates for each SMDΔ version were sequenced with Illumina short-read technology. After aligning reads to the reference CEN.PK113-7D genome sequence , mapped THI4, was observed in strain IMS0749  on chromosome VII, which harbors  IMS0749 , which hturnover . Segmentturnover . Since tturnover , 25, theSNV analysis was systematically performed, and data from the three sequenced isolates were compared. To eliminate false positives caused by mapping artifacts, reads of the CEN.PK113-7D strains were mapped back on its own reference assembly. Identified SNVs found were systematically subtracted. SNV analysis was restricted to non-synonymous mutations in coding sequences .RPG1 and PMR1, while a second strain (IMS0725) showed SNVs in MTO1 and VTH2. A mutation in YFR054W was identified in a single strain (IMS0730) evolved for inositol-independent growth. The absence of mutations in several strains subjected to serial transfer in SMDΔ lacking nicotinic acid or inositol is consistent with the fast growth of the parental strain CEN.PK113-7D in these media  and one in ARO7 (IMS0728), while SNVs in NUP57 and HTS2 were found in strains IMS0728 and IMS0729, respectively , 27pABA ynthesis .In line with the much longer ALE experiments (approximately 332 generations), strains evolved in SMDΔ lacking thiamine, pantothenate, or pyridoxine showed larger numbers of SNVs, with a maximum number of 30 SNVs in the isolate IMS0738 evolved in SMDΔ lacking pyridoxine .CNB1 and ERR3. A third isolate, strain IMS0748, contained two SNVs in PMR1 and FRE2. CNB1, PMR1, and FRE2 all encode proteins that have been implicated in divalent cation homeostasis . TUP1 codes for a general transcriptional corepressor  and IMX1722 (fre2Δ) still grew significantly slower than the evolved isolates (CNB1L82F), IMX1986 (PMR1S104Y), and IMX1987 (FRE2T110S). In addition, THI4 was overexpressed (strain IMX2290) to simulate the copy number increase observed in IMS0749. Strains IMX1987 (FRE2T110S) and IMX2290 (THI4↑) grew as fast as the evolved isolates on SMDΔ lacking thiamine (0.35 to 0.36 h−1)  was also tested. None of these combinations yielded a higher specific growth rate than observed in the evolved strains and in the reverse-engineered FRE2T110S and THI4↑ strains.The specific growth rate of lete SMD . Neverthlete SMD . The rolisolates . Subsequ.36 h−1) . CombinapABA, strain CEN.PK113-7D grew 50% slower than in complete SMD. However, it took only a few transfers to achieve fast pABA-independent growth. The independently evolved isolates IMX2057 and IMX1989 harbored mutations affecting genes that encode chorismate-utilizing enzymes, the precursor of pABA   and IMX1989 (ARO7L205S) were not statistically different from those of the corresponding evolved isolates (In SMDΔ lacking ctively) . As thesynthesis , 27. Intisolates .ISW2, GAL11, TUP1, SPE2, and FMS1 were analyzed by reverse engineering. Single deletions of SPE2, FMS1, and GAL11 resulted in an inability to grow on SMDΔ lacking pantothenate. This result was anticipated for the spe2Δ and fms1Δ mutants, in view of the roles of these genes in pantothenate biosynthesis. However, GAL11 was not previously implicated in pantothenate biosynthesis. The gal11Δ strain was conditional, as the mutant did grow on complex yeast extract-peptone-dextrose (YPD) and SMD media. Of the remaining two deletion mutants, the tup1Δ strain IMX1817 showed a 68% higher specific growth rate on SMDΔ than strain CEN.PK113-7D, while deletion of ISW2 did not result in faster growth on this medium  did not result in faster pyridoxine-independent growth , IMX2136 (BAS1D101N), and IMX2137 (BAS1S41P). All three BAS1 mutant strains grew faster on SMDΔ lacking pyridoxine than strain CEN.PK113-7D, reaching specific growth rates on this medium that were not significantly different from the average of those of evolved strains IMS736, IMS737, and IMS738  are strictly required for growth. Remarkably, the impact of individually eliminating these six vitamins from glucose-containing CDMY differently affected specific growth rates in aerobic glucose-grown cultures, with growth rate reductions varying from 0% to 57%. It should, however, be noted that requirements for these growth factors, which for aerobic yeast cultivation cannot be formally defined as vitamins, and their absolute and relative requirements may well be condition and strain dependent. For example, it is well documented that synthesis of nicotinic acid and pantothenic acid by S. cerevisiae is strictly oxygen dependent , (ii) genes encoding transcriptional regulator proteins (TUP1 and GAL11 for pantothenate and BAS1 for pyridoxine), and (iii) non-transcriptional-regulator proteins whose functions were not previously associated with vitamin biosynthesis .In total, the role of 12 genes that were found to be mutated in the ALE experiments were selected for further analysis by reverse engineering of the evolved alleles and/or deletion mutations in the parental non-evolved genetic background . These gSPE2 and FMS1 were not found to contribute to faster growth in the absence of the relevant vitamin. The second group yielded interesting information on the regulation of vitamin biosynthesis in S. cerevisiae. In particular, the key role of mutations in BAS1 in enabling fast pyridoxine-independent growth and the role of GAL11 and TUP1 mutations in fast pantothenate-independent growth dependency provided interesting insights and leads for further research.Of the first group of mutations defined above, only those in S. cerevisiae transcriptional activator Bas1 is involved in the regulation of purine and histidine  encodes a subunit of the mediator complex required for initiation by RNA polymerase II and consequently plays a critical role in transcription of a large number of RNA polymerase II-dependent genes   for recruitment of the RNA polymerase II machinery by an as yet unidentified transcription factor involved in regulation of pantothenate biosynthesis. Further research is required to resolve and understand the role of the wild-type and evolved alleles of GAL11 in regulation of pantothenate metabolism.ALE experiments in pantothenate-free medium yielded different mutations in epressed –49. GAL1nt genes , 51. Dese medium . The ina., Gcn4) . Of two ISW2, which encodes an ATP-dependent DNA translocase involved in chromatin remodeling  and a PMR1S104Y strain (IMX1986)  , top indS. cerevisiae, synthesis of the thiazole moiety of thiamine biosynthesis involves sulfide transfer from an active-site cysteine (Cys205) residue of the thiazole synthase Thi4. This sulfur transfer reaction is iron dependent and generates inactive enzyme by formation of a dehydroalanine. Fe(II) plays an essential role in this sulfide transfer, which remains poorly understood -biotin, 1.0 g/liter d-calcium pantothenate, 1.0 g/liter nicotinic acid, 25 g/liter myo-inositol, 1.0 g/liter thiamine hydrochloride, 1.0 g/liter pyridoxol hydrochloride, 0.20 g/liter 4-aminobenzoic acid]. Vitamin dropout media were prepared using vitamin solutions lacking either thiamine, pyridoxine, pantothenic acid, inositol, nicotinic acid, or para-aminobenzoic acid, yielding SMΔthiamine, SMΔpyridoxine, SMΔpantothenic acid, SMΔinositol, SMΔnicotinic acid, and SMΔpABA, respectively. A concentrated glucose solution was autoclaved at 110°C for 20 min and then added to the SM and YP media at a final concentration of 20 g/liter, yielding SMD and YPD, respectively. Five hundred-milliliter shake flasks containing 100 ml medium and 100-ml shake flasks containing 20 ml medium were incubated at 30°C and at 200 rpm in an Innova incubator . Solid media were prepared by adding 1.5% Bacto agar and, when indicated, 200 mg/liter G418 or 200 mg/liter hygromycin. Escherichia coli strains were grown in LB  supplemented with 100 mg/liter ampicillin or kanamycin. S. cerevisiae and E. coli cultures were stored at −80°C after the addition of 30% (vol/vol) glycerol.The E. coli using a Sigma GenElute Plasmid kit (Sigma-Aldrich). Plasmids used in this study are shown in E. coli XL1-Blue was used for chemical transformation  and desalted or PAGE-purified oligonucleotide primers  was performed according to the manufacturers’ instructions. DreamTaq polymerase (Thermo Scientific) was used for diagnostic PCR. Primers used in this study are shown in ormation . PlasmidS. cerevisiae CEN.PK113-7D for fast growth in SMD medium lacking a single vitamin was performed by sequential transfer in aerobic shake-flask batch cultures. A frozen aliquot of strain CEN.PK113-7D was inoculated in a preculture shake flask containing SMD medium supplemented with all vitamins. Cells were then spun down, washed twice with sterile water, and used to inoculate a second shake flask containing SMD lacking one of the vitamins. The culture was then grown until stationary phase and transferred in a third shake flask containing the same fresh medium. At each transfer, 0.2 ml culture broth was transferred to 20 ml fresh medium, corresponding to approximately 6.7 generations in each growth cycle. The evolution experiment was performed in SMΔthiamine, SMΔpyridoxine, SMΔpantothenic acid, SMΔinositol, SMΔnicotinic acid, and SMΔpABA media. Each evolution experiment was performed in triplicates. After a defined number of transfers, intermediate strains were stocked and characterized for the growth rate. The experiment was stopped once the target specific growth rate of 0.35 h−1 was reached. From each evolved population, three single colonies were then isolated and stored. The specific growth rate of these single cell lines was measured to verify that they were representative of the evolved population. The best performing isolate from each evolution line was selected for whole-genome sequencing.Laboratory evolution of 660) of 0.1 or 0.2 was used as a starting point. The flasks were then incubated, and growth was monitored using a 7200 Jenway spectrometer . Specific growth rates were calculated from at least four time points in the exponential growth phase of each culture.For specific growth rate measurements of strains (evolved populations as well as single cell lines), an aliquot was used to inoculate a shake flask containing 100 ml of fresh medium. For specific growth rate measurements of the engineered strains, a frozen aliquot was thawed and used to inoculate a 20-ml starter culture that was then used to inoculate the 100-ml flask. An initial optical density at 660 nm  according to the manufacturer’s protocol. Illumina-based paired-end sequencing with 150-bp reads was performed on 300-bp insert libraries  with a minimum resulting coverage of 50×. Data mapping was performed against the CEN.PK113-7D genome , 71.in vitro Gibson assembly as previously described  according to the manufacturer’s instructions. Transformants were selected on LB plates supplemented with 100 mg/liter ampicillin.Plasmids carrying two copies of the same genomic RNA (gRNA) were cloned by escribed . In brieescribed . The twoCNB1, PMR1, FRE2, ABZ1, GAL11, SynPAM, TUP1, ISW2, BAS1, hphNT1, YPRcTau3, FMS1, and SPE2, respectively. The fragment targeting GAL11 was cloned in a pROS12 backbone yielding plasmid pUDR441. The fragments targeting CNB1, PMR1, FRE2, ABZ1, SynPAM, TUP1, ISW2, BAS1, hphNT1, YPRcTau3, FMS1, and SPE2 were cloned in a pROS13 backbone yielding plasmids pUDR388, pUDR389, pUDR390, pUDR438, pUDR471, pUDR472, pUDR473, pUDR566, pUDR592, pUDR514, pUDR652, and pUDR651, respectively.Primers 13520, 13521, 13522, 13686, 13518, 14229, 13517, 13519, 15037, 15728, 12985, 16598, and 16601 were used to amplify the 2-μm fragments targeting THI4 was cloned by golden gate assembly using the yeast toolkit (YTK) parts  parts . The THIStrains carrying the target mutations were all constructed starting from IMX585 expressing the Cas9 protein . For alllocus that were previously combined at a 1:1 molar ratio, boiled for 5 min, and annealed by cooling down the solution at room temperature on the bench.In the first step, IMX585 was targeted at the gene of interest by transforming the strain with the relative pUDR plasmid. The double-strand break was then repaired by cotransforming the strain with two cDNA oligonucleotides carrying the SynPAM sequence flanked by 60-bp homology sequences to the targeted Five hundred nanograms of annealed primer pairs 13612/13613, 13541/13542, 13539/13540, 14988/14989, 15075/15076, 13533/13534, 13531/13532, 13535/13536, and 13527/13528 were cotransformed with 500 ng pUDR388, pUDR389, pUDR390, pUDR438, pUDR412, pUDR441, pUDR472, pUDR473, and pUDR652, respectively, yielding IMX1721, IMX1722, IMX1723, IMX1988, IMX1820, IMX1819, IMX1817, IMX1818, and IMX2292, respectively. IMX1819 and IMX1820 transformants were selected on YPD plates with 200 mg/liter hygromycin, while IMX1721, IMX1722, IMX1723, IMX1988, IMX1817, IMX1818, and IMX2292 transformants were selected on YPD plates with 200 mg/liter G418.BAS1 knockout strain could not be obtained with the marker-free SynPAM strategy. Therefore, the hphNT1 marker cassette was amplified by using primers 15584/15585 to add 60-bp homology flanks and pROS12 as a template. The PCR fragment was then gel purified, and 500 ng was cotransformed with 500 ng pUDR592 to yield IMX2128. Transformants were selected on YPD plates with 200 mg/liter G418 and 200 mg/liter hygromycin.The CNB1L82F, PMR1S104Y, FRE2T110S, ARO7L205S, ABZ1R593H, GAL11I541N, GAL11Q383stop, TUP1V374A, TUP1Q99stop, ISW2S181stop, and FMS1Q33K, respectively, using IMS0747, IMS0748, IMS0748, IMS0728, IMS0727, IMS0734, IMS0735, IMS0734, IMS0735, IMS0733, and IMS0735 genomic DNA as the templates, respectively. Transformants were selected on YPD plates with 200 mg/liter G418, yielding IMX1985, IMX1986, IMX1987, IMX1989, IMX2057, IMX1991, IMX1992, IMX2002, IMX2003, IMX1990, and IMX2292, respectively. The BAS1Q152R, BAS1D101N, and BAS1S41P mutant alleles were amplified from IMS736, IMS737, and IMS738 genomic DNA, respectively, using the primer pair 13687/13688. After gel purification, 500 ng of each PCR product was cotransformed in IMX2128, together with the hphNT1-targeting plasmid pUDR650, yielding IMX2135, IMX2136, and IMX2137, respectively. The strain IMX2289 carrying the SPE2A278T mutant allele was constructed by transforming IMX585 with the SPE2-targeting plasmid pUDR651 together with the annealed primer pair 16602/16603 containing the desired single base change plus a synonymous mutation causing the removal of the PAM sequence. After transformation, strains IMX2135, IMX2136, IMX2137, and IMX2289 were plated on YPD plates with 200 mg/liter G418 for selection.In the second step, the SynPAM target sequence in each knockout strain was targeted for the insertion of the mutant allele. The mutant gene flanked by approximately 400-bp upstream and downstream sequences was amplified using the evolved strain genomic DNA as the template. The PCR product was then gel purified, and 500 ng was cotransformed with 500 ng of pUDR471. Primer pairs 13523/13524, 11292/11293, 13525/13526, 12052/12053, 11725/11726, 13498/13499, 13498/13499, 15077/15078, 15077/15078, 13496/13497, and 13527/13528 were used to amplify the mutant alleles of GAL11, TUP1, and FMS1 were deleted in IMX2002, IMX2003, and IMX2127, respectively, by cotransforming the relative gRNA plasmid and the relative double-stranded DNA (dsDNA) oligonucleotide pair as performed for the single-knockout strains, yielding the intermediate strains IMX2066, IMX2110, and IMX2294, respectively. Then, the SynPAM sequence was targeted in IMX2066, IMX2110, and IMX2294 as previously described for the single mutant strains, yielding IMX2087, IMX2127, and IMX2307, respectively. IMX2043 carrying the PMR1S104Y-FRE2T110S double mutation was constructed by cotransforming IMX1987 with pUDR390 and the linear fragment containing the FRE2T110S mutant allele that was previously amplified as described above. The SPE2A278T mutant allele was combined with the GAL11I541NTUP1V374A mutant alleles present in IMX2127 by cotransforming the strain with the SPE2-targeting plasmid pUDR651 together with the annealed primer pair 16602/16603, yielding IMX2308. The THI4 overexpression cassette was amplified by using pUDI180 as a template and primers 12174/12175. Five hundred nanograms of gel-purified PCR product was cotransformed together with the YPRcTau3-targeting plasmid pUDR514 in IMX585 and IMX1985 yielding IMX2290 and IMX2291, respectively.Mutant alleles found in the same evolved strains were combined in a single strain by repeating the strategy described above but this time using a mutant strain as a starting point instead of IMX585. In this way, BAS1 could not be verified by Sanger sequencing; therefore, whole-genome resequencing of IMX2135, IMX2136, and IMX2137 was performed as explained above for the evolved single-colony isolates.To verify the correct gene editing, single colonies were picked from each transformation plate and genomic DNA was extracted as previously described . The tarAfter genotyping of the transformants, correct isolates were grown in 20 ml YPD in a 50-ml vented Greiner tube at 30°C overnight by inoculating a single colony. The next day, 1 μl was transferred to a new tube containing the same amount of medium and the sample was grown overnight. The day after, each liquid culture was restreaked to single colony by plating on YPD agar plates. Plates were incubated at 30°C overnight, and the next day, single colonies were patched on both YPD and YPD plus the relative antibiotic (either G418 or hygromycin) to assess which clones had lost the gRNA plasmid. One clone for each strain that had lost the plasmid was then grown in YPD, and 30% (vol/vol) glycerol was added prior to stocking samples at −80°C.BAS1 deletion Saccharomyces cerevisiae strains were deposited at NCBI (https://www.ncbi.nlm.nih.gov/) under BioProject accession number PRJNA603441. All measurement data used to prepare the figures of the manuscript are available at the data.4TU.nl repository (https://doi.org/10.4121/uuid:53c9992f-d004-4d26-a3cd-789c524fe35c).The sequencing data of the evolved and of the


Genes related to the response to drought stress in leaf and root tissue ofdrought-susceptible (DS) and tolerant (DT) genotypes were characterized byRNA-Seq. In total, 54,750 transcripts, representative of 28,590 genes, wereidentified; of these, 1,648 were of high-fidelity (merge of 12 libraries) anddescribed for the first time in the Andean germplasm. From the 1,239differentially expressed genes (DEGs), 458 were identified in DT, with apredominance of genes in categories of oxidative stress, response to stimulusand kinase activity. Most genes related to oxidation-reduction terms in rootswere early triggered in DT (T75) compared to DS (T150) suggestive of a mechanismof tolerance by reducing the damage from ROS. Among the KEGG enriched by DEGsup-regulated in DT leaves, two related to the formation of Sulfur-containingcompounds, which are known for their involvement in tolerance to abioticstresses, were common to all treatments. Through qPCR, 88.64% of the DEGs werevalidated. A total of 151,283 variants were identified and functional effectsestimated for 85,780. The raw data files were submitted to the NCBI database. Atranscriptome map revealed new genes and isoforms under drought. These resultssupports a better understanding of the drought tolerance mechanisms inbeans. Drought has become increasingly intense and worrisome because the worldwide demandfor agricultural products is expected to increase over the coming decade . During Phaseolus vulgaris L.) is a new world crop  and Pérola (DS) using RNA sequencing (RNA-Seq) and multiplebioinformatics resources to explore the potential candidate drought-responsive genesin leaves and roots. Based on the Andean genome, new genes and isoforms wereidentified in this study and provide a drought transcriptome map for common bean,and more than 150,000 variants  were identified asdifferentially expressed in response to drought conditions. From the KyotoEncyclopedia of Genes and Genomes (KEGG), 70 metabolic pathways were identified fornewly identified genes, and 97 metabolic pathways for DEGs. These results provide abroad overview of transcriptional regulation in response to drought, enriching thepublic database of genes potentially involved in abiotic stress.Two well-known genotypes of the common bean, BAT 477 and Pérola, characterizedaccording to their contrasting drought tolerances , 75 min (T75), or 150min (T150). The experiment was conducted in the form of randomcomplete blocks with five replications and in a double factorial arrangement,with two sampled genotypes and water regime as factors.Both varieties were maintained in a controlled environment (hydroponics system)with a specific nutrient solution, according to the recommendations of 0;control), 25 min (T25), 50 min (T50), 75 min(T75), 100 min (T100), 125 min (T125) and150 min (T150). In all treatments, the photosynthetic rate (μmolCO2 m-2s-1), stomatal conductance (mmolH2O m-2s-1), leaf temperature (°C) and leaftranspiration rate (mmol H2O m-2 s-1) wereevaluated using an LI-6400 Portable Photosynthesis System model . Measurements were taken on an expanded leaf subjectedto progressive water deficit treatments for the periods of 0 min, 25 min, 50min, 75 min, 100 min, 125 min, or 150 min. The osmotic (Ψs) andhydric (Ψw) potentials in megapascals (MPa), were taken according tothe methodology described by The physiological evaluations consisted of seven treatments: 0min  for both lineages (BAT 477 and Pérola) were sampled from15-day-old seedlings and were stored at -80 °C, totaling twelve differentRNA-Seq libraries. From the five biological replicates conducted for eachtreatment, two were individually collected, processed and bulked with equimolaramounts of RNA. Total RNA was extracted using a commercial PureLink®RNA Mini Kit (Thermo Fisher) following the manufacturer’s instructions. RNAquantity and purity were estimated through spectrophotometry using a NanoVuePlus Spectrophotometer , andthe integrity was verified using microfluidics technology and an Agilent 2100Bioanalyzer . The RNA-Seq libraries were prepared using theIllumina TruSeq RNA kit  followingthe manufacturer’s instructions and were paired-end sequenced in a technicalreplicate system using two Illumina platforms (GAII and HiSeq 2000).Fresh leaves and roots from three treatments . The transcriptome wasassembled using reads that mapped to each of those twelve RNA-Seq librariesusing the Cufflinks v2.2.1  using the reference genome. After that, theDEGs were identified between-sample comparison  using thhttps://transdecoder.github.io/). In addition, the loci with nohit with the Andean genome were annotated against theMesoamerican transcriptome and LncRNAs (http://denovo.cnag.cat/genomes/bean/) usingBLASTn v.2.2.30+. The BLAST2GO tool v4.1. The GO terms withsignificant changes in abundance were identified using Fisher’s exact test, withFDR correction (p≤0.05). The identification of genes involvedin specific metabolic pathways was performed using the KEGG database  through the BLASTx v.2.2.30+algorithm (BQSR) and indel realignment (IR).The mapping step was performed using STAR v.2.4.1 (http://broadinstitute.github.io/picard/). Quality controlfilters were used, such as a Fisher strand (FS)>30, a quality depth(QD)<2, and a minimum of 35bp of physical distance between two consecutiveSNPs according to GATK recommendations  v.3.4-46 (® Gene Expression assay (Thermo Fisher Scientific) wereperformed using the Applied Biosystems® 7500 Real-Time PCR (ThermoFisher Scientific), followed by the determination of the Cq(quantification cycle) values using 7500 Software v.2.3 (Thermo FisherScientific). The Cq values were subjected to the 2-ΔΔCT method (https://cran.r-project.org/) using the packages agricolaev.1.2-4 and multcomp v.1.4-6. A set of three reference genes was used, and the stability assessed with the geNorm Plusapplication  integrated into the qbasePLUS/qPCR dataanalysis software package .The dataset(s) supporting the conclusions of this article are available in theSRA/NCBI database repository, accession number: TableS1). The photosynthetic rate (μmolCO2 m-2 s-1), stomatal conductance (molH2O m-2s-1) and leaf transpiration rate(mmol H2O m-2s-1) decreased, while the leaftemperature (°C) increased, and the internal CO2 concentration (μmolCO2 mol-1) varied across the dehydration process. Ingeneral, the DT presented lower values of the photosynthetic rate(p<0.05 at T0 and T50) andstomatal conductance (p<0.05 at T50) than did theDS. For leaf transpiration, DT presented a reduced value at T0(p<0.05). Similar values of leaf temperature andCO2 concentration were obtained for both genotypes.The physiological parameters measured on plants grown hydroponically exposed to ashort-term water deficit indicated that the dehydration treatments triggeredchanges in the plant physiological behaviour at both genotypes(TableS2 and deposited in the Sequence ReadArchive (SRA)/NCBI database repository through accession numberSRP077562 (http://www.ncbi.nlm.nih.gov/sra/SRP077562). The coverage of thebean genome per library was ~4x, while that of the total RNA-Seq was 94.96x, andthat of the coding DNA sequence (CDS) was 476.39x. The total mapped reads perlibrary was 87.52% (SD ± 3.72%). A P. vulgaris drought-specifictranscriptome was assembled into 28,590 gene loci with 54,750 transcripts, ofwhich 1,648 loci (5.76%) and 23,112 isoforms were newly described based on theAndean genome.All raw sequence data generated a total of ~580,000,000 reads, totalling 5.8 Gigabase pairs (Gbp) of RNA-Seq data; ~329 and ~250 millions of reads were obtained,and 73.83% and 85.57% were retained (Q30) for further assembly in HiSeq andGAII, respectively. The raw data are summarized inTable S3), of which 1,301 revealed asignificantly relative abundance compared with thepreviously annotated P. vulgaris genes, and 1,300 terms wereover-represented (Table S4) in biological process (69.02%),molecular function (18.52%) and cellular component (12.45%). Through KEGGanalysis 70 metabolic pathways were identified containing 77 associated enzymes(TableS5).From the 1,648 loci newly predicted, 790 showed hits with previous sequencesevaluated using both BLASTx and PSI-BLAST, 858 showed no homology with the NR.From the 790 loci with BLAST hit, 1,886 GO terms (GO IDs) were identified, and466 GO terms were directly annotated (Table S6), and 403 had no hit .From the 858 loci with no hit, 58 were categorized as non-coding RNA and wereclassified into seven biotypes: a) gene, snRNA, snoRNA, CD-box (56.8%); b) gene,miRNA (25.3%); c) gene, snRNA, snoRNA, HACA-box (11.6%); d) intron (3.2%); e)gene (1.1%); f) gene, antisense (1.1%); and g) gene, tRNA (1.1%). From theremaining 800 loci, 261 presented high similarity with transcripts encodingproteins and 136 were lncRNAs, both identified through homology to theMesoamerican genome . For the leaves and roots, thenumber of expressed genes (CPM >1) was similar, although the set of genes andtissue distribution varied considerably. In total, 21,696 genes were identifiedfor the DT, with 17,425 (80.31%) common to both tissues (leaf and root), 2,423(11.17%) exclusive in the roots, and 1,848 (8.52%) exclusive in the leaves. ForDS, a set of 21,699 genes was identified under control conditions, of which17,484 (80.58%) were expressed in both tissues, 2,550 (11.75%) were exclusive inthe roots, and 1,665 (7.67%) were exclusive in the leaves. Between thecontrasting genotypes, considering the leaf tissues, 816 genes were expressedonly in DT, and 692 in DS; for the roots, 489 only in DT and 675 in DS.For the whole set of genes , the comparative transcriptome revealeddifferences in gene expression patterns between DT and DS, even under controlconditions (TableS7. Compared with the control conditions(T0), T75 showed an increase in the overall number ofexpressed genes in the DS (326) and DT (42) genotypes. Interestingly, withingenotypes, while drought sensitivity-related genes increased in both tissues(189), drought tolerance-related genes increased in the roots (332) butdecreased in the leaves. Between genotypes, an increase in the total number ofgenes expressed specifically in the DS genotype  was observed. By contrast, in T150, there was anincrease in the number of expressed genes in the DT genotype (301), mainly inthe roots (63 genes), compared with the DS genotype (30 genes), whose genes werepreferentially expressed in the leaves. Overall, between the genotypes, a highernumber of genes were expressed in the leaves for the DS (900) than for the DT(653) genotype; however, in the roots, an opposite trend was noted: more geneswere observed for the DT genotype (615) than for the DS (536) genotype. In general, a greater number of KEGGpathways linked to DEGs were observed in the comparison of T0vs T150 within genotypes for the up- anddown-regulated genes in both tissues and genotypes(TableS5).The gene expression level, based on fold change (FC), is presented in75, 192 DEGs for the DT genotype and123 for the DS were observed, of which 177 (91.71%) and 110 (89.43%) wereup-regulated, respectively. In T150, root tissue, 681 DEGs werereported in the DT (569 up- and 112 down-regulated), of which 184 (27.02%) werealso identified in T75. However, in the DS, 403 DEGs were identified(338 up- and 65 down-regulated), of which 103 (25.56%) were common to bothtreatments. For the leaf tissue in T150, a set of 251 DEGs wereidentified in the DT (82.07% up-regulated) and 208 in the DS genotype (71.64%up-regulated). For the leaf tissue, only a small portion of DEGs (5.58% for theDT genotype and 11.53% for the DS genotype) identified in T150 weredifferentially expressed in relation to T75. Among the DEGs in theroots, 73 in T75 were common to both genotypes (69 up-regulated andfour down-regulated); however, in T150, this total was 245 (227 up-and 18 down-regulated). Of the 353 DGEs in T150 leaves, 106 were thesame in both genotypes (92 up- and 14 down-regulated). Between the genotypes, 17DEGs were identified in the roots in T75, and only four wereidentified in T150; however, in the leaves in T75 andT150, 97 and 57 DEGs were identified, respectively.For DGE, in each pairwise comparison , the valFrom the DEGs, only one lncRNA was verified in the leaf tissue (DS genotype),while in the root tissue, six lncRNAs were identified that responded to droughtstress with high reliability (fold change ≥ 4 and FDR < 0.01), four in the DTgenotype, of which three were up-regulated, and two in the DS genotype.TableS3). Regarding functional annotation, inT75 roots, 27 enriched terms were associated with up-regulateddrought tolerance-associated genes, while for the drought sensitivity-associatedgenes, only one term related to oxidoreductase activity was reported. Noenriched terms were identified for down-regulated DEGs in the roots inT75 or in the leaves in T75 and T150. Forthe DT genotype in the long-term dehydration treatment (T150), 51over-represented GO terms for up-regulated genes were identified in the roots.For the DS genotype, enriched GPO terms reported were similar to thosepreviously found related to the T75 and T150 treatments inthe DT (Table S4).The GO categorization for all 1,239 DEGs was conducted with emphasis in the mostrepresentative distributions filtered by node score within GO categories(Table S5). In DT root tissues, 75 pathwaysfor up-regulated genes and 47 for down-regulated genes were identified betweentreatments; however, in DS root tissues, these values were 49 and 24,respectively. In leaf tissue, 51 and 18 pathways for up- and down-regulatedgenes were reported for the DT genotype; however, for the DS genotype, thesevalues were 55 and 14, respectively. In both comparisons for root tissue, thenumber of pathways exclusively represented in up-regulated DEGs in the DTgenotype was 17 for T0vs T75 and 28 for T0vs T150; however, in the DS genotype, one and twoKEGG pathways were identified in the same comparisons. In leaf tissues, four andeight pathways were exclusively identified for T0 vs T150in the DT and DS genotypes, respectively, and another 12 were verified in DEGsfrom the T75vs T150 comparison in the DS genotype.Through KEGG pathway, 97 metabolic processes were identified containing 145associated enzymes (TableS5).In the KEGG analysis of leaf tissues, nine metabolic pathways were identified inthe DT genotype. Of these, four were exclusive to this genotype , of which two were common to allthree treatments (cysteine and methionine metabolism and sulphur metabolism). Inthe DS genotype, 15 pathways were identified, five of which were common to allthree treatments and 10 of which were exclusive to this genotype. In the roots,10 pathways were identified in the DS genotype and none in the DT genotype. A total of 151,283 variants wasidentified, including 135,167 SNPs and 16,116 indels, of which 120,726 SNPs and13,631 indels occurred in genes. The variants were placed on all P.vulgaris chromosomes, with one variant for every 3,413 bases . The gene expression profile obtained byqPCR was 88.64%, concordant with the results obtained in RNA-Seq analysis . Of the 15 DEGs identified byRNA-Seq, most  correlated wellwith the qPCR results, presenting high correspondence (100%) and a strongcoefficient of correlation (≥60%), which are important technical validationparameters in RNA-Seq. Among the genes with high correspondence between RNA-Seqand qPCR methodologies, those that were up-regulated in the DT genotype undergreater drought stress were glutathione S-transferase and peroxidase . GST presented increased expression inthe leaf tissue of the DT genotype in T0, T75 andT150, compared to DS. The POX enzyme was up-regulated inT0 and T75 in the DT genotype, followed by weakup-regulation in subsequent T150 treatments in the leaves, while POXin the DS genotype showed the opposite pattern of expression, with increasedup-regulation in T150. In the roots, POX expression graduallyincreased with treatment time, with significant levels of expression inT150 in the DT genotype.The reference genes used present high stability of expression among all samples,with the combined value below the cutoff of 1.5 . These enzymes play a fundamentalrole in various regulatory mechanisms of the cell through signal transductionpathways during biotic and abiotic stress  within the Andean and Mesoamerican gene pools   and one involved in ABAbiosynthesis (XLOC_011282) were identified. ABA is a well-characterizedphytohormone that triggers several pathways for plant stress responses, mainlyby the activation of a transcriptional regulatory network involved in drought. TFs werloci). Chaperones play an important role in proteinhomeostasis and, although constitutively expressed, are extremely active in thesignalling of stresses such as drought, temperature extremes and salinity  have been identified as regulators of manyfundamental biological processes, such as tissue development, as well as theresponse to osmotic, saline and drought stresses . This differential expression pattern over timedemonstrated the effectiveness of the implemented stress-induced protocol. Inthe root tissue there was an increased number of DEGs up-regulated in the DTgenotype  compared with that inthe DS genotype , with a predominance ofenriched categories related to osmotic and redox processes, signal transductionmechanisms, TFs, and the development of cellular metabolism. Regardingphysiological aspects, in dry conditions, the DT genotype develops root tissuefor access to deeper layers of the soil, as an important adaptive advantage inthe mechanism of bean tolerance (In T2O2), hydroxyl radical,and singlet oxygen , dioxygenases (gibberellin andleucoanthocyanidins), oxidases (1-aminocyclopropane-1-carboxylate),NADP-dependent malic enzymes and cytochrome P450 (CYP) proteins; in the leavesthe main DEGs were cationic peroxidase, oxidases , lipoxygenases (seed linoleate 9s-lipoxygenase),lysine-ketoglutarate reductase saccharopine dehydrogenase, zinc-binding alcoholdehydrogenase family protein and protein hothead-like. These DEGs identifiedbetween tissues indicated that the DT genotype triggers distinct mechanisms ofdrought tolerance, inducing expressive antioxidant responses.75, termsrelated to metabolic processes of polysaccharides, aminoglycans, chitin, andglucosamine were still highlighted, which are related to plant adaptationprocess under stress, such as restructuring of the cell wall at lower watercontent and secondary wall formation (75 and T150) in the DT and DS genotypes. Theenzymatic hydrolysis of chitin involves an initial cleavage by chitinases intochitin oligosaccharides and further cleavage to N-acetylglucosamine (GlcNAc) andmonosaccharides by chitobiases , but in late stress inDS roots (T150), suggesting that the susceptibility also seemed to berelated to the late response to stress. On the other hand, the tolerance couldbe attributed to the ability to activate genes promoting an immediate responseat the beginning of stress, increasing the chances of adaptation and minimizingand/or fighting the effects of the lack of water. These findings are supportedby previous results on the physiological characterization of BAT 477 and Pérolagenotypes evaluated under water deficit  and sulphur metabolism, both of which are essential for plantgrowth . Thefin0, T75) and roots (T150) of the DTgenotype when compared to DS, is closely associated with the response to abioticstresses, such as drought tolerance  of the RNA-seqexperiments using qPCR, with an overall moderate correlation (61%) between bothmethods. As the experiment was carried out in biological replicates with thesame genotypes and design as used for the RNA-Seq study, this similarity wasexpected. Although qPCR-based methods have been considered the gold standard formeasuring gene expression, these methods are negatively affected by severalfactors and hydrolase activity (GO:0004553), was previously described in soybean asLOC100801147, and acts in the breakdown of hemicellulose, suggesting thedisassembly of cell wall components during drought stress. The high levels ofexpression in the roots of the DT genotype in all treatments probably suggestthat cell expansion necessary to maintain root growth under drought to absorbwater in deeper soil layers and thereby maintain the water status of the plant.In leaves, although gene expression in the DT genotype was greater than that inthe DS genotype, down-regulation was observed (greater than 50%), and theexpression level was three times smaller than that in the root. Loss of leafarea is the most important morphological adaptation to drought in common bean(The results of RNA-Seq and qPCR for the enzyme involved in cell wall degradation,GH10 xylanase, were not perfectly correlated but were in the same direction andshowed a similar trend, being up-regulated in the roots of the DT genotype inTmon bean. Under dmon bean.Through genome re-sequencing, numerous SNPs have been identified , whoseaIn this study, we constructed a bean transcriptome under drought conditions and noveltranscripts and novel isoforms were identified in the Andean common bean genome,contribution to improve the currently available database to the community of beans.As more genes become available, the chance to perform more refined mining willincrease, which would obtain better knowledge of the control of specific routesrelated to the response to drought stress, which could be used for breedingdrought-resistant plants. In addition, a broad genome SNP coverage improves theability to predict target genic regions and/or genomic candidate variants, allowingmore refined studies. The results of this study contribute to knowledge of the genemechanisms, as well as their functional variants, related to two contrasting commonDT bean genotypes.


A structurally diverse chemical pool is produced after the successive regioselective and stereoselective oligomerization of resveratrol. This review describes the current status and knowledge of the structure of resveratrol oligomers (ROs) in Dipterocarpaceaeous plants (DPs). Beginning with the recently validated formation of ROs in DPs, each downstream conversion is described from the perspective of the resveratrol coupling mode. Particular emphasis is placed upon the regioselectivity of monomer- and dimer-derived radical–radical coupling processes, which are responsible for producing dimers, trimers, and tetramers with various cyclic frame skeletons, as well as related processes that result in highly condensed scaffolds, such as hexamers and octamers. Trimers in oxidized, dearomatized, and rearranged forms are also summarized, as well as the biogenic relationship between the compounds. Furthermore, emphasis is placed on the O- and C-glucosides of ROs, as well as on the hetero-coupled ROs. In addition, several stereoisomers that originate from asymmetric carbons and the stereochemistry with respect to the conformation due to the chiral axis are described. Besides, NMR spectroscopic properties such as coalescence and anisotropy are briefly described. Approaches to determine absolute configuration are also summarized.Oligostilbenoids are a group of natural products derived from the oxidative coupling of C The D in 1966 ; in the  in 1966 . In esse in 1966 . Since wVatica  , 631A–1DRO Figs. . In addi5 is one of the most abundant dimeric resveratrols, which has been isolated from the majority of RO-containing plant families, such as Dipterocarpaceae, Vitaceae, Cyperaceae, Fabaceae, Paeoniaceae, and Gnetaceae, among which DPs are known to produce (−)-form. Indeed, this issue is vital in considering an absolute configuration of biogenetically downstream chemicals. Because the structural diversity of RO can be attributed to skeletal variations and the presence of stereoisomerism, analyzing two- and three-dimensional structures is an interesting challenge, academically speaking.Generally, asymmetric carbons exist in proportion to the oligomerization degree, e.g., in many cases, the dimers and tetramers of resveratrol have four and eight chiral atoms, respectively. Among the 200 ROs isolated from the DPs, dimers, trimers, and tetramers are common. This trend can be observed in other families, including Vitaceae –4; howevIn this section, representative ROs are discussed from the viewpoint of oligomerization degree and skeletal diversity with biosynthetic aspects. The listed compounds are prioritized to systematically depict a plausible biogenic relationship between compounds with topological differences. Indeed, our findings, which are based on experiments in the last 2 decades in combination with the existing literature, support the proposed biosynthetic aspects. In this section, each RO is delineated as a planar structure Figs. , 6, 7, 81B–1C-QM (6), further undergoes particular cyclization(s) and tautomerization, thereby generating several different frame skeletons. As a result of the oxa-conjugate addition, dimers bearing heterocyclic dihydrobenzofuran ) are generated. Alternatively, 5-exo-trig cyclization through C7a–C8b bond formation results in another QM (1B–1C-QM′ (7)), which undergoes further tautomerization and 6-exo-trig cyclization through the C10a–C7b bond, which, in turn, results in resveratrol dimers with non-heterocyclic ring systems, such as indane  . 2-. 2-61) (62) . Wh. Wh39 iss B (97)  and H (997) 32, for er of 16) . The NMR 32 Fig. . The enengs Fig. . For exa42 and 37 with the 3--6-hydroxy-2-(4-hydroxyphenyl)-2,3-dihydrobenzofuran-4-yl group  connected to the dibenzobicyclo[5.3.1]octadiene core, where H8c and H14c are situated in syn orientation. The hindered rotation and conformational stability in such molecules can also be enhanced by attractive forces, such as CH–π and OH–π interactions [When the partial structures connected through the C–C bond increase in size, the rotational barriers also increase, which results in a stable conformer. Examples of this can be seen in ractions , 95.55 exhibited broad signals in the entire region due to unstable conformation at ambient temperatures [1 in various conditions (temperatures and solvents) as well as the completely overlapping methine signals. Successful isolation of glucosides of 55 (vaticasides E (114) and F [12–C7e bond, which, in turn, results in the successful determination of 55  and F ) finally55 Figs. , 21.Fig.39 [99 [5 [39 [65 and 66 [63) and 64) [85) [42) [68 and 69 [5 ((+)-101 [38) [d-β-glucopyranosyl group (70–72)) [31) [58 and 59 [Some ROs with absolute configurations determined by different approaches have been summarized in the existing literature : X-ray c39  and 99 [39 [99 ; the com9 [99 [5 , (+)-39 9 [5 [39 , 65 and 5 and 66 , 53, 63) and 64) , and mod64) [85) ; the com85) [42) ; the app8 and 69 , 53 as w8 and 69 ); the re((+)-101 ); the ac101 [38) ; assignm(70–72)) , 59, 60;8 and 59 ). Curren8 and 59  is promiO-atom(s), dearomatization, rearrangement, tautomerization, and a hetero-coupling with other BBs), as well as deducing further stereochemical diversity in the chemical pool. Much work remains in clarifying the differences in physicochemical properties among the diverse stereoisomers that arise from enantiomerism, diastereomerism, and atropisomerism; these will be the subject of future work, as will be defining further RO structural diversity.Even though a considerable amount of knowledge is available with respect to the structural diversity of ROs in DPs , compounds with other link modes have not yet been comprehensively studied. This includes defining versatile structural motifs stemming from minor couplings and structural modifications (i.e., in terms of introduction of