Mulberry is a fundamental component of the global sericulture industry, and its positive impact on our health and the environment cannot be overstated. However, the mulberry reference genomes reported previously remained unassembled or unplaced sequences. Here, we report the assembly and analysis of the telomere-to-telomere gap-free reference genome of the mulberry species, Morus notabilis, which has emerged as an important reference in mulberry gene function research and genetic improvement. The mulberry gap-free reference genome produced here provides an unprecedented opportunity for us to study the structure and function of centromeres. Our results revealed that all mulberry centromeric regions share conserved centromeric satellite repeats with different copies. Strikingly, we found that M. notabilis is a species with polycentric chromosomes and the only reported polycentric chromosome species up to now. We propose a compelling model that explains the formation mechanism of new centromeres and addresses the unsolved scientific question of the chromosome fusion-fission cycle in mulberry species. Our study sheds light on the functional genomics, chromosome evolution, and genetic improvement of mulberry species.

Abiotic stress, especially drought stress, poses a significant threat to terrestrial plant growth, development, and productivity. Although mulberry has great genetic diversity and extensive stress-tolerant traits in agroforestry systems, only a few reports offer preliminary insight into the biochemical responses of mulberry leaves under drought conditions. In this study, we performed a comparative metabolomic and transcriptomic analysis on the “drooping mulberry” (Morus alba var. pendula Dippel) under PEG-6000-simulated drought stress. Our research revealed that drought stress significantly enhanced flavonoid accumulation and upregulated the expression of phenylpropanoid biosynthetic genes. Furthermore, the activities of superoxide dismutase (SOD), catalase (CAT) and malondialdehyde (MDA) content were elevated. In vitro enzyme assays and fermentation tests indicated the involvement of flavonol synthase/flavanone 3-hydroxylase (XM_010098126.2) and anthocyanidin 3-O-glucosyltransferase 5 (XM_010101521.2) in the biosynthesis of flavonol aglycones and glycosides, respectively. The recombinant MaF3GT5 protein was found to recognize kaempferol, quercetin, and UDP-glucose as substrates but not 3-/7-O-glucosylated flavonols and UDP-rhamnose. MaF3GT5 is capable of forming 3-O- and 7-O-monoglucoside, but not di-O-glucosides, from kaempferol. This implies its role as a flavonol 3, 7-O-glucosyltransferase. The findings from this study provided insights into the biosynthesis of flavonoids and could have substantial implications for the future diversified utilization of mulberry.

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.

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).

Abiotic stress, especially drought stress poses a significant threat to the terrestrial plant growth, development, and productivity. Although mulberry have great genetic diversity and extensive stress tolerant traits in agroforestry systems, only a few reports offer preliminary insight into biochemical responses of mulberry leaves under drought conditions. In this study, we performed a comparative metabolomic and transcriptomic analysis on the “drooping mulberry” (Morus alba var. Pendula Dippel) under PEG-6000-simulated drought stress. Our research revealed that drought stress significantly enhanced flavonoid accumulation and up-regulated the expression of phenylpropanoid biosynthetic genes. Furthermore, the activities of superoxide dismutase (SOD), catalase (CAT) and malondialdehyde (MDA) content were elevated. In vitro enzyme assays and fermentation tests indicated the involvement of flavonol synthase/flavanone 3-hydroxylase (XM_010098126.2) and anthocyanidin 3-O-glucosyltransferase 5 (XM_010101521.2) in the biosynthesis of flavonol aglycones and glycosides, respectively. The recombinant MaF3GT5 protein was found to recognize kaempferol, quercetin, and UDP-glucose as substrates, but not 3-/7-O-glucosylated flavonols and UDP-rhamnose. MaF3GT5 is capable of forming 3-O- and 7-O-monoglucoside, but not di-O-glucosides, form kaempferol. This implies its role as a flavonol 3, 7-O-glucosyltransferase. The findings from this study provided insights into the biosynthesis of flavonoids and could have substantial implications for the future diversified utilization of mulberry.

Mulberry is a critical economic tree with a high diversity of germplasm resources. However, the lack of primary data on genome size, chromosome number, and ploidy for this species limits the exploitation of mulberry resources. In this study, the genome size of 323 mulberry germplasm resources were examined using flow cytometry and chromosome numbers analyzed. The genome sizes ranged from 0.36 to 3.08 Gb, and seven different ploidies of mulberry germplasm resources were identified, with chromosome numbers ranging from 14 to 308. Correlation analysis indicated that genome size (1C) and chromosome number positively correlated. Here, the genome size, chromosome number, and ploidy database MGCPdb (https://mgcpdb.biodb.org) were constructed for mulberry plants, which contains 323 core mulberry germplasm resources and provides raw data of flow cytometry analysis, genome size, and chromosome count. This database is significant and valuable for mulberry genome evolution, polyploidy breeding, and genetic diversity research.