SilkMeta: a comprehensive platform for sharing and exploiting pan-genomic and multi-omic silkworm data.

The silkworm Bombyx mori is a domesticated insect that serves as an animal model for research and agriculture. The silkworm super-pan-genome dataset, which we published last year, is a unique resource for the study of global genomic diversity and phenotype-genotype association. Here we present SilkMeta (http://silkmeta.org.cn), a comprehensive database covering the available silkworm pan-genome and multi-omics data. The database contains 1082 short-read genomes, 546 long-read assembled genomes, 1168 transcriptomes, 294 phenotype characterizations (phenome), tens of millions of variations (variome), 7253 long non-coding RNAs (lncRNAs), 18 717 full length transcripts and a set of population statistics. We have compiled publications on functional genomics research and genetic stock deciphering (mutant map). A range of bioinformatics tools is also provided for data visualization and retrieval. The large batch of omics data and tools were integrated in twelve functional modules that provide useful strategies and data for comparative and functional genomics research. The interactive bioinformatics platform SilkMeta will benefit not only the silkworm but also the insect biology communities.

Dehydrogenase MnGutB1 catalyzes 1-deoxynojirimycin biosynthesis in mulberry.

As the prevalence of diabetes continues to increase, the number of individuals living with diabetes complications will reach an unprecedented magnitude. Continuous use of some synthetic agents to reduce blood glucose levels causes severe side effects, and thus, the demand for nontoxic, affordable drugs persists. Naturally occurring compounds, such as iminosugars derived from the mulberry (Morus spp.), have been shown to reduce blood glucose levels. In mulberry, 1-deoxynojirimycin (DNJ) is the predominant iminosugar. However, the mechanism underlying DNJ biosynthesis is not completely understood. Here, we showed that DNJ in mulberry is derived from sugar and catalyzed through 2-amino-2-deoxy-D-mannitol (ADM) dehydrogenase MnGutB1. Combining both targeted and nontargeted metabolite profiling methods, DNJ and its precursors ADM and nojirimycin (NJ) were quantified in mulberry samples from different tissues. Purified His-tagged MnGutB1 oxidized the hexose derivative ADM to form the 6-oxo compound DNJ. The mutant MnGutB1 D283N lost this remarkable capability. Furthermore, in contrast to virus-induced gene silencing of MnGutB1 in mulberry leaves that disrupted the biosynthesis of DNJ, overexpression of MnGutB1 in hairy roots and light-induced upregulation of MnGutB1 enhanced DNJ accumulation. Our results demonstrated that hexose derivative ADM, rather than lysine derivatives, is the precursor in DNJ biosynthesis, and it is catalyzed by MnGutB1 to form the 6-oxo compound. These results represent a breakthrough in producing DNJ and its analogs for medical use by metabolic engineering or synthetic biology.

Mno-miR164a and MnNAC100 regulate the resistance of mulberry to Botrytis cinerea.

Although microRNAs (miRNAs) regulate the defense response of a variety of plant species against a variety of pathogenic fungi, the involvement of miRNAs in mulberry's defense against Botrytis cinerea has not yet been documented. In this study, we identified responsive B. cinerea miRNA mno-miR164a in mulberry trees. After infection with B. cinerea, the expression of mno-miR164a was reduced, which was fully correlated with the upregulation of its target gene, MnNAC100, responsible for encoding a transcription factor. By using transient infiltration/VIGS mulberry that overexpressed mno-miR164a or knocked-down MnNAC100, our study revealed a substantial enhancement in mulberry's resistance to B. cinerea when mno-miR164a was overexpressed or MnNAC100 expression was suppressed. This enhancement was accompanied by increased catalase (CAT) activity and reduced malondialdehyde (MDA) content. In addition, mno-miR164a-mediated inhibition of MnNAC100 enhanced the expression of a cluster of defense-related genes in transgenic plants upon exposure to B. cinerea. Meanwhile, MnNAC100 acts as a transcriptional repressor, directly suppressing the expression of MnPDF1.2. Our study indicated that the mno-miR164a-MnNAC100 regulatory module manipulates the defense response of mulberry to B. cinerea infection. This discovery has great potential in breeding of resistant varieties and disease control.

Chromosome constitution and genetic relationships of Morus spp. revealed by genomic in situ hybridization.

BACKGROUND: Mulberry (Morus spp.) is an economically important woody plant, which has been used for sericulture (silk farming) for thousands of years. The genetic background of mulberry is complex due to polyploidy and frequent hybridization events. RESULTS: Comparative genomic in situ hybridization (cGISH) and self-GISH were performed to illustrate the chromosome constitution and genetic relationships of 40 mulberry accessions belonging to 12 species and three varietas in the Morus genus and containing eight different ploidy levels. We identified six homozygous cGISH signal patterns and one heterozygous cGISH signal pattern using four genomic DNA probes. Using cGISH and self-GISH data, we defined five mulberry sections (Notabilis, Nigra, Wittiorum, and Cathayana, all contained only one species; and Alba, which contained seven closely related species and three varietas, was further divided into two subsections) and proposed the genetic relationships among them. Differential cGISH signal patterns detected in section Alba allowed us to refine the genetic relationships among the closely related members of this section. CONCLUSIONS: We propose that GISH is an efficient tool to investigate the chromosome constitution and genetic relationships in mulberry. The results obtained here can be used to guide outbreeding of heterozygous perennial crops like mulberry.

The silkworm gustatory receptor BmGr63 is dedicated to the detection of isoquercetin in mulberry.

Gustatory systems in phytophagous insects are used to perceive feeding stimulants and deterrents, and are involved in insect decisions to feed on particular plants. During the process, gustatory receptors (Grs) can recognize diverse phytochemicals and provide a molecular basis for taste perception. The silkworm, as a representative Lepidoptera species, has developed a strong feeding preference for mulberry leaves. The mulberry-derived flavonoid glycoside, isoquercetin, is required to induce feeding behaviours. However, the corresponding Grs for isoquercetin and underlying molecular mechanisms remain unclear. In this study, we used molecular methods, voltage clamp recordings and feeding assays to identify silkworm BmGr63, which was tuned to isoquercetin. The use of qRT-PCR confirmed that BmGr63 was highly expressed in the mouthpart of fourth and fifth instar larvae. Functional analysis showed that oocytes expressing BmGr63 from the 'bitter' clade responded to mulberry extracts. Among 20 test chemicals, BmGr63 specifically recognized isoquercetin. The preference for isoquercetin was not observed in BmGr63 knock-down groups. The tuning between BmGr63 and isoquercetin has been demonstrated, which is meaningful to explain the silkworm-mulberry feeding mechanism from molecular levels and thus provides evidence for further feeding relationship studies between phytophagous insects and host plants.

The Mulberry SPL Gene Family and the Response of MnSPL7 to Silkworm Herbivory through Activating the Transcription of MnTT2L2 in the Catechin Biosynthesis Pathway.

SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) genes, as unique plant transcription factors, play important roles in plant developmental regulation and stress response adaptation. Although mulberry is a commercially valuable tree species, there have been few systematic studies on SPL genes. In this work, we identified 15 full-length SPL genes in the mulberry genome, which were distributed on 4 Morus notabilis chromosomes. Phylogenetic analysis clustered the SPL genes from five plants (Malus × domestica Borkh, Populus trichocarpa, M. notabilis, Arabidopsis thaliana, and Oryza sativa) into five groups. Two zinc fingers (Zn1 and Zn2) were found in the conserved SBP domain in all of the MnSPLs. Comparative analyses of gene structures and conserved motifs revealed the conservation of MnSPLs within a group, whereas there were significant structure differences among groups. Gene quantitative analysis showed that the expression of MnSPLs had tissue specificity, and MnSPLs had much higher expression levels in older mulberry leaves. Furthermore, transcriptome data showed that the expression levels of MnSPL7 and MnSPL14 were significantly increased under silkworm herbivory. Molecular experiments revealed that MnSPL7 responded to herbivory treatment through promoting the transcription of MnTT2L2 and further upregulating the expression levels of catechin synthesis genes (F3'H, DFR, and LAR).

Hierarchical Action of Mulberry miR156 in the Vegetative Phase Transition.

The vegetative phase transition is a prerequisite for flowering in angiosperm plants. Mulberry miR156 has been confirmed to be a crucial factor in the vegetative phase transition in Arabidopsis thaliana. The over-expression of miR156 in transgenic Populus × canadensis dramatically prolongs the juvenile phase. Here, we find that the expression of mno-miR156 decreases with age in all tissues in mulberry, which led us to study the hierarchical action of miR156 in mulberry. Utilizing degradome sequencing and dual-luciferase reporter assays, nine MnSPLs were shown to be directly regulated by miR156. The results of yeast one-hybrid and dual-luciferase reporter assays also revealed that six MnSPLs could recognize the promoter sequences of mno-miR172 and activate its expression. Our results demonstrate that mno-miR156 performs its role by repressing MnSPL/mno-miR172 pathway expression in mulberry. This work uncovered a miR156/SPLs/miR172 regulation pathway in the development of mulberry and fills a gap in our knowledge about the molecular mechanism of vegetative phase transition in perennial woody plants.

Origin and functional differentiation of (E)-ß-ocimene synthases reflect the expansion of monoterpenes in angiosperms.

The acquisition of new metabolic activities is a major force driving evolution. We explored, from the perspectives of gene family expansion and the evolutionary adaptability of proteins, how new functions have arisen in which terpene synthases diverged. Monoterpenoids are diverse natural compounds that can be divided into cyclic and acyclic skeleton forms according to their chemical structure. We demonstrate, through phylogenetic reconstructions and genome synteny analyses, that the (E)-ß-ocimene synthases, which are acyclic monoterpene synthases (mTPSs), appear to have arisen several times in independent lineages during plant evolution. Bioinformatics analyses and classical mutation experiments identified four sites (I388, F420, S446, and F485) playing important roles in the neofunctionalization of mTPSs. Incubation of neryl diphosphate with Salvia officinalis 1,8-cineole synthase (SCS) and mutated proteins show that these four sites obstruct the isomerization of geranyl diphosphate. Quantum mechanical/molecular mechanical molecular dynamics simulations of models of SCS, SCSY420F/I446S, and SCSN338I/Y420F/I446S/L485F with (3R)-linalyl diphosphate suggest that mutations changed the configuration of the intermediate to obtain new activities. These results provide new perspectives on the evolution of mTPSs, explain the convergent evolution of (E)-ß-ocimene synthases at the molecular level, and identify key residues to control the specificity of engineered mTPSs.

An R1R2R3 MYB Transcription Factor, MnMYB3R1, Regulates the Polyphenol Oxidase Gene in Mulberry (Morus notabilis).

The aim of this study was to determine how the mulberry (Morus notabilis) polyphenol oxidase 1 gene (MnPPO1) is regulated during plant stress responses by exploring the interaction between its promoter region and regulatory transcription factors. First, we analyzed the cis-acting elements in the MnPPO1 promoter. Then, we used the MnPPO1 promoter region [(1268 bp, including an MYB3R-binding cis-element (MSA)] as a probe to capture proteins in DNA pull-down assays. These analyses revealed that the MYB3R1 transcription factor in M. notabilis (encoded by MnMYB3R1) binds to the MnPPO1 promoter region. We further explored the interaction between the MnPPO1 promoter and MYB3R1 with the dual luciferase reporter, yeast one-hybrid, and chromatin immunoprecipitation assays. These analyses verified that MnMYB3R1 binds to the MSA in the MnPPO1 promoter region. The overexpression of MnMYB3R1 in tobacco upregulated the expression of the tobacco PPO gene. This observation as well as the quantitative real-time PCR results implied that MnMYB3R1 and PPO are involved in the abscisic acid-responsive stress response pathway.

Preparation of Polysaccharides from Ramulus mori, and Their Antioxidant, Anti-Inflammatory and Antibacterial Activities.

The extraction of Ramulus mori polysaccharides (RMPs) was optimized using response surface methodology (RSM). The optimal process conditions, which gave the highest yield of RMPs (6.25%) were 80 °C, 50 min, and a solid⁻liquid ratio of 1:40 (g/mL), with the extraction performed twice. The RMPs contained seven monosaccharides, namely, mannose, rhamnose; glucuronic acid, glucose, xylose, galactose, and arabinose, in a 1.36:2.68:0.46:328.17:1.53:21.80:6.16 molar ratio. The glass transition and melting temperatures of RMPs were 83 and 473 °C, respectively. RMPs were α-polysaccharides and had surfaces that resembled a porous sponge, as observed by scanning electron microscopy. RMPs inhibited the proliferation of Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa and showed antioxidant activity (assessed by three different methods), although it was generally weaker than that of vitamin C. RMPs showed anti-inflammatory activity in a concentration-dependent manner. This study provides a basis for exploring the potential uses of RMPs.

Horizontal transfers of LTR retrotransposons in seven species of Rosales.

Horizontal transposable element transfer (HTT) events have occurred among a large number of species and play important roles in the composition and evolution of eukaryotic genomes. HTTs are also regarded as effective forces in promoting genomic variation and biological innovation. In the present study, HTT events were identified and analyzed in seven sequenced species of Rosales using bioinformatics methods by comparing sequence conservation and Ka/Ks value of reverse transcriptase (RT) with 20 conserved genes, estimating the dating of HTTs, and analyzing the phylogenetic relationships. Seven HTT events involving long terminal repeat (LTR) retrotransposons, two HTTs between Morus notabilis and Ziziphus jujuba, and five between Malus domestica and Pyrus bretschneideri were identified. Further analysis revealed that these LTR retrotransposons had functional structures, and the copy insertion times were lower than the dating of HTTs, particularly in Mn.Zj.1 and Md.Pb.3. Altogether, the results demonstrate that LTR retrotransposons still have potential transposition activity in host genomes. These results indicate that HTT events are another strategy for exchanging genetic material among species and are important for the evolution of genomes.

LysM1 in MmLYK2 is a motif required for the interaction of MmLYP1 and MmLYK2 in the chitin signaling.

KEY MESSAGE: Two LysM-containing proteins, namely, MmLYP1 and MmLYK2, were identified in mulberry. These proteins might be involved in chitin signaling. The LysM1 of MmLYK2 is critical for their interactions. Chitin is a major component of fungal cell walls and acts as an elicitor in plant innate immunity. Lysin motif (LysM)-containing proteins are essential for chitin recognition. However, related studies have been rarely reported in woody plants. In this study, in mulberry, the expression of a LysM-containing protein, MmLYP1, was significantly up-regulated after treatment with chitin and pathogenic fungi. In addition, MmLYP1 has an affinity for insoluble chitin polymers. Thus, MmLYP1 might function in chitin signaling. Since MmLYP1 lacks an intracellular domain, additional protein kinases are required for this signaling. An LysM-containing kinase, MmLYK2, was then identified. Expression of the MmLYK2 did not change significantly after chitin treatment, and the affinity of MmLYK2 for insoluble chitin was not high. The structure of MmLYP1 is similar to that of the chitin elicitor-binding proteins in rice and Arabidopsis. However, MmLYK2 has two LysM motifs, while the chitin elicitor receptor kinase 1 proteins in rice and Arabidopsis have one and three LysM motifs, respectively. The LysM1 of MmLYK2 interacted with all four LysM motifs in MmLYP1 and MmLYK2 in yeast. The chimera lacking the LysM1 of MmLYK2 did not interact with MmLYP1 and MmLYK2 in yeast and Nicotiana benthamiana cells. The LysM1 in MmLYK2 is the key motif in the interaction between MmLYP1 and MmLYK2, which may be involved in chitin signaling.

Analysis of codon usage patterns in Morus notabilis based on genome and transcriptome data.

Codons play important roles in regulating gene expression levels and mRNA half-lives. However, codon usage and related studies in multicellular organisms still lag far behind those in unicellular organisms. In this study, we describe for the first time genome-wide patterns of codon bias in Morus notabilis (mulberry tree), and analyze genome-wide codon usage in 12 other species within the order Rosales. The codon usage of M. notabilis was affected by nucleotide composition, mutation pressure, nature selection, and gene expression level. Translational selection optimal codons were identified and highly expressed genes of M. notabilis tended to use the optimal codons. Genes with higher expression levels have shorter coding region and lower amino acid complexity. Housekeeping genes showed stronger translational selection, which, notably, was not caused by the large differences between the expression level of housekeeping genes and other genes.

Laccase Gene Sh-lac Is Involved in the Growth and Melanin Biosynthesis of Scleromitrula shiraiana.

Scleromitrula shiraiana causes the popcorn disease in mulberry trees resulting in severe economic losses. Previous studies have shown that melanin may play a vital role in establishing the pathogenicity of fungi. In the present study, we identified the melanin produced in S. shiraiana belongs to DHN melanin by gas chromatography-mass spectrometry, and cloned the laccase Sh-lac, a potential DHN melanin biosynthesis gene from S. shiraiana. We obtained two stable Sh-lac silenced transformants using RNAi, ilac-4 and 8 to elucidate the DHN melanin biosynthetic pathway in S. shiraiana. The melanin production of ilac-4 and ilac-8 was significantly reduced, and their vegetative growth was also suppressed. Results such as these led to a proposal that Sh-lac played a key role in DHN melanin formation in S. shiraiana and may function differentially with other melanin biosynthetic genes. The inhibition of melanin was accompanied by the decrease of oxalic acid and the adhesion of hyphae was impaired. Our results indicated that laccase was an important enzyme in the synthesis of melanin and might play a critical role in the pathogenicity of S. shiraiana.

Antinociceptive and Antibacterial Properties of Anthocyanins and Flavonols from Fruits of Black and Non-Black Mulberries.

Anthocyanins and flavones are important pigments responsible for the coloration of fruits. Mulberry fruit is rich in anthocyanins and flavonols, which have multiple uses in traditional Chinese medicine. The antinociceptive and antibacterial activities of total flavonoids (TF) from black mulberry (MnTF, TF of Morus nigra) and non-black mulberry (MmTF, TF of Morus mongolica; and MazTF, TF of Morus alba 'Zhenzhubai') fruits were studied. MnTF was rich in anthocyanins (11.3 mg/g) and flavonols (0.7 mg/g) identified by ultra-performance liquid chromatography-tunable ultraviolet/mass single-quadrupole detection (UPLC-TUV/QDa). Comparatively, MmTF and MazTF had low flavonol contents and MazTF had no anthocyanins. MnTF showed significantly higher antinociceptive and antibacterial activities toward Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus than MmTF and MazTF. MnTF inhibited the expression of interleukin 6 (IL-6), inducible nitric oxide synthase (iNOS), phospho-p65 (p-p65) and phospho-IκBα (p-IκBα), and increased interleukin 10 (IL-10). Additionally, mice tests showed that cyanidin-3-O-glucoside (C3G), rutin (Ru) and isoquercetin (IQ) were the main active ingredients in the antinociceptive process. Stronger antinociceptive effect of MnTF was correlated with its high content of anthocyanins and flavonols and its inhibitory effects on proinflammatory cytokines, iNOS and nuclear factor-κB (NF-κB) pathway-related proteins.

Evolutionary and functional analysis of mulberry type III polyketide synthases.

BACKGROUND: Type III polyketide synthases are important for the biosynthesis of flavonoids and various plant polyphenols. Mulberry plants have abundant polyphenols, but very little is known about the mulberry type III polyketide synthase genes. An analysis of these genes may provide new targets for genetic improvement to increase relevant secondary metabolites and enhance the plant tolerance to biotic and abiotic stresses. RESULTS: Eighteen genes encoding type III polyketide synthases were identified, including six chalcone synthases (CHS), ten stilbene synthases (STS), and two polyketide synthases (PKS). Functional characterization of four genes representing most of the MnCHS and MnSTS genes by coexpression with 4-Coumaroyl-CoA ligase in Escherichia coli indicated that their products were able to catalyze p-coumaroyl-CoA and malonyl-CoA to generate naringenin and resveratrol, respectively. Microsynteny analysis within mulberry indicated that segmental and tandem duplication events contributed to the expansion of the MnCHS family, while tandem duplications were mainly responsible for the generation of the MnSTS genes. Combining the evolution and expression analysis results of the mulberry type III PKS genes indicated that MnCHS and MnSTS genes evolved mainly under purifying selection to maintain their original functions, but transcriptional subfunctionalization occurred during long-term species evolution. Moreover, mulberry leaves can rapidly accumulated oxyresveratrol after UV-C irradiation, suggesting that resveratrol was converted to oxyresveratrol. CONCLUSIONS: Characterizing the functions and evolution of mulberry type III PKS genes is crucial for advancing our understanding of these genes and providing the basis for further studies on the biosynthesis of relevant secondary metabolites in mulberry plants.

[Introduction trial of medicine mulberry (Morus nigra) in Chongqing].

Medicine mulberry (Morus nigra) mainly distributed in southern areas of Xinjiang Uighur Autonomous Region and introduced by grafting, is a unique Morus species, whose plant number is little. As a traditional herbal medicine, medicine mulberry with high levels of secondary metabolites has important values of scientific research and utilization. In order to solve the introduction problems for medicine mulberry, we have established its rapid propagation system through tissue culture since 2011. The shoots of medicine mulberry through tissue culture were transplanted into the field to carry out an introduction experiment. Here, we firstly reported that the growth status and pest and disease occurrence of medicine mulberry in the field of Chongqing and found that the medicine mulberry through tissue culture had well-developed root system, it showed better growth than medicine mulberry by grafting technique, and Pseudodendrothrips moil was a major pest of medicine mulberry. The introduction technique for medicine mulberry established successfully in this study could lay the foundation for large-scale cultivation and high efficiency utilization of medicine mulberry.

Biotic stress-induced expression of mulberry cystatins and identification of cystatin exhibiting stability to silkworm gut proteinases.

MAIN CONCLUSION: Biotic stresses induce the expression of mulberry cystatins. MaCPI-4 protein is stable in silkworm digestive fluid and accumulates in gut food debris and frass. Plant cystatins are considered to be involved in defense responses to insect herbivores though little is known about how cystatins from the natural host respond to a specialist herbivory and the following postingestive interaction is also poorly understood. Here, we studied the biotic stress-mediated inductions of cystatins from mulberry tree, and examined the stability of mulberry cystatin proteins in the gut of silkworm, Bombyx mori, a specialist insect feeding on mulberry leaf. First, we cloned and characterized six cystatin genes from a mulberry cultivar, Morus atropurpurea Roxb., named as MaCPI-1 to MaCPI-6. The recombinant MaCPI-1, MaCPI-3 and MaCPI-4 proteins, which showed inhibitory effects against papain in vitro, were produced. Silkworm herbivory as well as methyl jasmonate (MeJA) treatment induced the expression of five mulberry cystatin genes, and the highest inductions were observed from MaCPI-1 and MaCPI-6. Mechanical wounding led to the inductions of four cystatin genes. The differential induction occurred in MaCPI-2. The induced protein changes were detected from three mulberry cystatins comprising MaCPI-1, MaCPI-3 and MaCPI-4. In vivo and in vitro assays showed that MaCPI-1 and MaCPI-3 proteins were susceptible to silkworm digestive fluid and MaCPI-4 had an antidigestive stability, and was detected in silkworm gut and frass. Collectively, our data indicated that biotic stresses resulted in the transcriptional inductions and protein changes of mulberry cystatins (MaCPIs), and identified MaCPI-4 with stability in the gut of its specialist herbivore.

Identification of the mulberry genes involved in ethylene biosynthesis and signaling pathways and the expression of MaERF-B2-1 and MaERF-B2-2 in the response to flooding stress.

The phytohormone ethylene is essential to plant growth and development. It plays crucial roles in responses to biotic and abiotic stress. The mulberry tree is an important crop plant in countries in which people rear silkworms for silk production. The availability of the mulberry genome has made it possible to identify mulberry genes involved in ethylene biosynthesis and signal pathways. A total of 145 mulberry genes were identified by both homology-based and hidden Markov model (HMM) search, including 29 genes associated with ethylene biosynthesis and 116 genes in the AP2/ERF family. Studies on gene structure have provided a genetic basis for understanding the functions of these genes. The differences in gene expression were also observed in different tissues. The expression of two mulberry genes in the AP2/ERF family, MaERF-B2-1 and MaERF-B2-2, was found to be associated with the response to flooding stress.

Cloning and expression analyses of the anthocyanin biosynthetic genes in mulberry plants.

Anthocyanins are natural food colorants produced by plants that play important roles in their growth and development. Mulberry fruits are rich in anthocyanins, which are the most important active components of mulberry and have many potentially beneficial effects on human health. The study of anthocyanin biosynthesis will bring benefits for quality improvement and industrial exploration of mulberry fruits. In the present study, nine putative genes involved in anthocyanin biosynthesis in mulberry plants were identified and cloned. Sequence analysis revealed that the mulberry anthocyanin biosynthetic genes were conserved and had counterparts in other plants. Spatial transcriptional analysis showed detectable expression of eight of these genes in different tissues. The results of expression and UPLC analyses in two mulberry cultivars with differently colored fruit indicated that anthocyanin concentrations correlated with the expression levels of genes associated with anthocyanin biosynthesis including CHS1, CHI, F3H1, F3'H1, and ANS during the fruit ripening process. The present studies provide insight into anthocyanin biosynthesis in mulberry plants and may facilitate genetic engineering for improvement of the anthocyanin content in mulberry fruit.