Zhonghuai Xiang

Silk production, a spectacular evolutionary innovation shared by approximately 143,000 arthropod species, is underpinned by genetic and cellular programs that remain incompletely resolved. Although a cellular atlas exists for the silkworm (Bombyx) silk gland (SG)—the premier model for this trait—the coordinated cellular dynamics and transcriptional logic that underlie its remarkable efficiency are unsolved. Here, we bridge this gap by an integrative meta-analysis that leverages single-cell deconvolution and high-dimensional weighted gene coexpression network analysis (hdWGCNA). Applied to existing atlases, public data, and de novo transcriptomes, this computational strategy enabled the identification of key cell subpopulations and the reconstruction of a high-resolution, global coexpression network orchestrating silk production. This network nominated the Hippo signaling pathway as a top regulatory hub. Functionally, conditional overexpression and knockout of its core effector, Yorkie (Yki), demonstrated that Yki is causal for SG development and silk protein synthesis. Further deconvolution of Yki-mutant transcriptomes confirmed the substantial impact of Hippo–Yki signaling on the cellular hierarchical framework of the SG. Our work not only deciphers a key regulatory axis controlling a classic biological trait but also provides an integrative computationally driven experimental framework, establishing a paradigm for obtaining mechanistic insights from complex omics landscapes.

The silkwormBombyx moriis an important economic insect for producing silk, the “queen of fabrics”. The currently available genomes limit the understanding of its genetic diversity and the discovery of valuable alleles for breeding. Here, we deeply re-sequence 1,078 silkworms and assemble long-read genomes for 545 representatives. We construct a high-resolution pan-genome dataset representing almost the entire genomic content in the silkworm. We find that the silkworm population harbors a high density of genomic variants and identify 7308 new genes, 4260 (22%) core genes, and 3,432,266 non-redundant structure variations (SVs). We reveal hundreds of genes and SVs that may contribute to the artificial selection (domestication and breeding) of silkworm. Further, we focus on four genes responsible, respectively, for two economic (silk yield and silk fineness) and two ecologically adaptive traits (egg diapause and aposematic coloration). Taken together, our population-scale genomic resources will promote functional genomics studies and breeding improvement for silkworm.

Multiple plant lineages have independently evolved sex chromosomes and variable karyotypes to maintain their sessile lifestyles through constant biological innovation. Morus notabilis, a dioecious mulberry species, has the fewest chromosomes among Morus spp., but the genetic basis of sex determination and karyotype evolution in this species has not been identified. In this study, three high-quality genome assemblies were generated for Morus spp. [including dioecious M. notabilis (male and female) and Morus yunnanensis (female)] with genome sizes of 301–329 Mb and were grouped into six pseudochromosomes. Using a combination of genomic approaches, we found that the putative ancestral karyotype of Morus species was close to 14 protochromosomes, and that several chromosome fusion events resulted in descending dysploidy (2n = 2x = 12). We also characterized a ∼ 6.2-Mb sex-determining region on chromosome 3. Four potential male-specific genes, a partially duplicated DNA helicase gene (named MSDH) and three Ty3_Gypsy long terminal repeat retrotransposons (named MSTG1/2/3), were identified in the Y-linked area and considered to be strong candidate genes for sex determination or differentiation. Population genomic analysis showed that Guangdong accessions in China were genetically similar to Japanese accessions of mulberry. In addition, genomic areas containing selective sweeps that distinguish domesticated mulberry from wild populations in terms of flowering and disease resistance were identified. Our study provides an important genetic resource for sex identification research and molecular breeding in mulberry.

Multiple plant lineages have independently evolved sex chromosomes and variable karyotypes to maintain their sessile lifestyles through constant biological innovation. Morus notabilis , a dioecious mulberry species, has the fewest chromosomes among Morus spp., but the genetic basis of sex determination and karyotype evolution in this species have not been identified. Three high-quality genome assemblies generated of Morus spp. (including those of dioecious M. notabilis and Morus yunnanensis ) were within the range 301-329 Mb in size which were grouped into six pseudochromosomes. Using a combination of genomic approaches, we showed that the putative ancestral karyotype of Morus was close to 14 protochromosomes, and that several chromosome fusion events resulted in descending dysploidy (2n = 2x = 12). We also characterized a ∼6.2-Mb sex-determining region on chromosome 3. The four potential male-specific genes, including a partially duplicated DNA helicase gene orthologue (named MSDH ) and three Ty3_Gypsy long terminal repeat retrotransposons (named MSTG ), were solely identified in the Y-linked area and considered to be strong candidate genes for sex determination or differentiation. Population genomic analysis showed that Guangdong accessions in China were genetically similar to Japanese accessions of mulberry. In addition, genomic areas containing selective sweeps that distinguish domesticated mulberry trees from wild populations in terms of flowering and disease resistance were identified. Our findings provide an important genetic resource for sex identification and molecular breeding in mulberry.

The endophytic microbiome is thought to play an important role in promoting plant growth and health. Using culture-independent and culture-dependent protocols, this study characterized the seasonal shifts in the endophytic fungal microbiota of four mulberry (Morus L.) cultivars having different levels of resistance to mulberry fruit sclerotiniosis. Core endophytes can be obtained by two approaches, and they were divided into two clusters by season. Spring samples harbored higher operational taxonomic units (OTUs) and α-diversity, while autumn samples had more sequences or isolates of the fungal class Dothideomycetes with the representative orders Capnodiales and Pleosporales. While comparing different mulberry cultivars, we found that the total number of OTUs in susceptible cultivars was higher than that of resistant cultivars, and Cladosporium sp. were observed in all. Notably, the causal agent of fruit sclerotiniosis (Scleromitrula shiraiana) was only detected in susceptible cultivars. Collectively, our work elucidated significant variations in the mulberry endophytic microbiome, mainly because of seasonal shifts, and the fact that the host cultivars and mulberry endophytic fungal community appeared to have a certain connection with the resistance level of mulberry fruit to sclerotiniosis. These results provided valuable information on the isolation and culturing of mulberry endophytes that could be applied to improve mulberry fruit production and health.

The endosomal-type Na⁺, K⁺/H⁺ antiporters (NHXs) play important roles in K⁺, vesicle pH homeostasis, and protein trafficking in plant. However, the structure governing ion transport mechanism and the key residues related to the structure–function of the endosomal-type NHXs remain unclear. Here, the structure-function relationship of the only endosomal-type NHX from mulberry, MnNHX6, was investigated by homology modeling, mutagenesis, and localization analyses in yeast. The ectopic expression of MnNHX6 in arabidopsis and Nhx1 mutant yeast can enhance their salt tolerance. MnNHX6’s three-dimensional structure, established by homology modeling, was supported by empirical, phylogenetic, and experimental data. Structure analysis showed that MnNHX6 contains unusual 13 transmembrane helices, but the structural core formed by TM5-TM12 assembly is conserved. Localization analysis showed that MnNHX6 has the same endosomal localization as yeast Nhx1/VPS44, and Arg402 is important for protein stability of MnNHX6. Mutagenesis analysis demonstrated MnNHX6 contains a conserved cation binding mechanism and a similar charge-compensated pattern as NHE1, but shares a different role in ion selectivity than the vacuolar-type NHXs. These results improve our understanding of the role played by the structure–function related key residues of the plant endosomal-type NHXs, and provide a basis for the ion transport mechanism study of endosomal-type NHXs.

Miniature inverted-repeat transposable elements (MITEs) are common in eukaryotic genomes, and are important for genomic evolution. In the present study, the identification of MITEs in the mulberry genome revealed 286,122 MITE-related sequences, including 90,789 full-length elements. The amplification of mulberry MITEs and the influence of MITEs on the evolution of the mulberry genome were analyzed. The timing of MITE amplifications varied considerably among the various MITE families. Fifty-one MITE families have undergone a single round of amplification, while the other families developed from multiple amplifications. Most mulberry MITEs were inserted near genes and some could regulate gene expression through small RNAs. An analysis of transgenic plants indicated that MITE insertions can upregulate the expression of a target gene. Moreover, MITEs are frequently associated with alternative splicing events (exonizations). The data presented herein provide insights into the generation of MITEs as well as their impact on gene regulation and evolution in mulberry.

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.

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.