Metabolomic and transcriptomic analysis of flavonoids biosynthesis mechanisms in mulberry fruit (Hongguo 2) under exogenous hormone treatments.

The mulberry fruit is prized for its superior nutrition value and abundant color due to its high flavone content. To enhance comprehension of flavone biogenesis induced by external hormones, we sprayed exogenous ethylene (ETH), indoleacetic acid (IAA) and spermine (SPM) on mulberry fruit (Hongguo 2) during its color-changed period. The levels of anthocyanin, titratable acid, soluble sugar and endogenous hormones were determined after hormone treatment, integrated transcriptome and metabolome analysis were performed for mechanism exploration. Our results indicated that exogenous ETH, SPM, and IAA play important roles in mulberry ripening, including acid reduction, sugar increase and flavonoid synthesis.

Phylogenomics of mulberries (Morus, Moraceae) inferred from plastomes and single copy nuclear genes.

Mulberries (genus Morus), belonging to the order Rosales, family Moraceae, are important woody plants due to their economic values in sericulture, as well as for nutritional benefits and medicinal values. However, the taxonomy and phylogeny of Morus, especially for the Asian species, remains challenging due to its wide geographical distribution, morphological plasticity, and interspecific hybridization. To better understand the evolutionary history of Morus, we combined plastomes and a large-scale nuclear gene analyses to investigate their phylogenetic relationships. We assembled the plastomes and screened 211 single-copy nuclear genes from 13 Morus species and related taxa. The plastomes of Morus species were relatively conserved in terms of genome size, gene content, synteny, IR boundary and codon usage. Using nuclear data, our results elucidated identical topologies based on coalescent and concatenation methods. The genus Morus was supported as monophyletic, with M. notabilis as the first diverging lineage and the two North American Morus species, M. microphylla and M. rubra, as sister to the other Asian species. In the Asian Morus species, interspecific relationships were completely resolved. However, cyto-nuclear discordances and gene tree-species tree conflicts were detected in the phylogenies of Morus, with multiple evidences supporting hybridization/introgression as the main cause of discordances between nuclear and plastid phylogenies, while gene tree-species tree conflicts were mainly caused by ILS.

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.

Unraveling the Contribution of MulSOS2 in Conferring Salinity Tolerance in Mulberry (Morus atropurpurea Roxb).

Salinity is one of the most serious threats to sustainable agriculture. The Salt Overly Sensitive (SOS) signaling pathway plays an important role in salinity tolerance in plants, and the SOS2 gene plays a critical role in this pathway. Mulberry not only has important economic value but also is an important ecological tree species; however, the roles of the SOS2 gene associated with salt stress have not been reported in mulberry. To gain insight into the response of mulberry to salt stress, SOS2 (designated MulSOS2) was cloned from mulberry (Morus atropurpurea Roxb), and sequence analysis of the amino acids of MulSOS2 showed that it shares some conserved domains with its homologs from other plant species. Our data showed that the MulSOS2 gene was expressed at different levels in different tissues of mulberry, and its expression was induced substantially not only by NaCl but also by ABA. In addition, MulSOS2 was exogenously expressed in Arabidopsis, and the results showed that under salt stress, transgenic MulSOS2 plants accumulated more proline and less malondialdehyde than the wild-type plants and exhibited increased tolerance to salt stress. Moreover, the MulSOS2 gene was transiently overexpressed in mulberry leaves and stably overexpressed in the hairy roots, and similar results were obtained for resistance to salt stress in transgenic mulberry plants. Taken together, the results of this study are helpful to further explore the function of the MulSOS2 gene, which provides a valuable gene for the genetic breeding of salt tolerance in mulberry.

Transcriptomic and metabolomic analyses reveal the importance of ethylene networks in mulberry fruit ripening.

Mulberry (Morus alba L.) is a climacteric and highly perishable fruit. Ethylene has been considered to be an important trigger of fruit ripening process. However, the role of ethylene in the mulberry fruit ripening process remains unclear. In this study, we performed a comprehensive analysis of metabolomic and transcriptomic data of mulberry fruit and the physiological changes accompanying the fruit ripening process. Our study revealed that changes in the accumulation of specific metabolites at different stages of fruit development and ripening were closely correlated to transcriptional changes as well as underlying physiological changes and the development of taste biomolecules. The ripening of mulberry fruits was highly associated with the production of endogenous ethylene, and further application of exogenous ethylene assisted the ripening process. Transcriptomic analysis revealed that differential expression of diverse ripening-related genes was involved in sugar metabolism, anthocyanin biosynthesis, and cell wall modification pathways. Network analysis of transcriptomics and metabolomics data revealed that many transcription factors and ripening-related genes were involved, among which ethylene-responsive transcription factor 3 (MaERF3) plays a crucial role in the ripening process. The role of MaERF3 in ripening was experimentally proven in a transient overexpression assay in apples. Our study indicates that ethylene plays a vital role in modulating mulberry fruit ripening. The results provide a basis for guiding the genetic manipulation of mulberry fruits towards sustainable agricultural practices and improve post-harvest management, potentially enhancing the quality and shelf life of mulberry fruits for sustainable agriculture and forestry.

The evolutionary origin of naturally occurring intermolecular Diels-Alderases from Morus alba.

Biosynthetic enzymes evolutionarily gain novel functions, thereby expanding the structural diversity of natural products to the benefit of host organisms. Diels-Alderases (DAs), functionally unique enzymes catalysing [4 + 2] cycloaddition reactions, have received considerable research interest. However, their evolutionary mechanisms remain obscure. Here, we investigate the evolutionary origins of the intermolecular DAs in the biosynthesis of Moraceae plant-derived Diels-Alder-type secondary metabolites. Our findings suggest that these DAs have evolved from an ancestor functioning as a flavin adenine dinucleotide (FAD)-dependent oxidocyclase (OC), which catalyses the oxidative cyclisation reactions of isoprenoid-substituted phenolic compounds. Through crystal structure determination, computational calculations, and site-directed mutagenesis experiments, we identified several critical substitutions, including S348L, A357L, D389E and H418R that alter the substrate-binding mode and enable the OCs to gain intermolecular DA activity during evolution. This work provides mechanistic insights into the evolutionary rationale of DAs and paves the way for mining and engineering new DAs from other protein families.

Integrated Analysis of lncRNAs and mRNAs Reveals Complex Gene Network Mediated by lncRNAs and Regulatory Function of MuLRR-RLK-AS in Response to Phytoplasma Infection in Mulberry.

Phytoplasma disease is one of the most serious infectious diseases that affects the growth and development of mulberry. Long non-coding RNAs (lncRNAs) play an important role in plants' defense systems; however, the contribution of lncRNAs in the response to phytoplasma infection in mulberry is still largely unknown. Herein, strand-specific RNA sequencing was performed to profile the mRNAs and lncRNAs involved in the response to phytoplasma infection in mulberry, and a total of 4169 genes were found to be differentially expressed (DE) between healthy and phytoplasma-infected leaves. Moreover, 1794 lncRNAs were identified, of which 742 lncRNAs were DE between healthy and infected leaves. Target prediction showed that there were 68 and 44 DE lncRNAs which may function as cis and trans-regulators, targeting 54 and 44 DE genes, respectively. These DE target genes are associated with biological processes such as metabolism, signaling, development, transcriptional regulation, etc. In addition, it was found that the expression of the antisense lncRNA (MuLRR-RLK-AS) of the leucine-rich repeat receptor-like protein kinase gene (MuLRR-RLK) was decreased in the phytoplasma-infected leaves. Interestingly, it was found that overexpression of MuLRR-RLK-AS can inhibit the expression of MuLRR-RLK. Moreover, it was found that the expression levels of PTI-related and MAPK genes in the transgenic MuLRR-RLK Arabidopsis plants were significantly higher than those in the wild-type plants when inoculated with pathogens, and the transgenic plants were conferred with strong disease resistance. Our results demonstrate that MuLRR-RLK-AS, as a trans-regulatory factor, can inhibit the expression of the MuLRR-RLK gene and is a negative regulatory factor for mulberry resistance. The information provided is particularly useful for understanding the functions and mechanisms of lncRNAs in the response to phytoplasma infection in mulberry.

Mining and functional characterization of NADPH-cytochrome P450 reductases of the DNJ biosynthetic pathway in mulberry leaves.

BACKGROUND: 1-Deoxynojirimycin (DNJ), the main active ingredient in mulberry leaves, with wide applications in the medicine and food industries due to its significant functions in lowering blood sugar, and lipids, and combating viral infections. Cytochrome P450 is a key enzyme for DNJ biosynthesis, its activity depends on the electron supply of NADPH-cytochrome P450 reductases (CPRs). However, the gene for MaCPRs in mulberry leaves remains unknown. RESULTS: In this study, we successfully cloned and functionally characterized two key genes, MaCPR1 and MaCPR2, based on the transcriptional profile of mulberry leaves. The MaCPR1 gene comprised 2064 bp, with its open reading frame (ORF) encoding 687 amino acids. The MaCPR2 gene comprised 2148 bp, and its ORF encoding 715 amino acids. The phylogenetic tree indicates that MaCPR1 and MaCPR2 belong to Class I and Class II, respectively. In vitro, we found that the recombinant enzymes MaCPR2 protein could reduce cytochrome c and ferricyanide using NADPH as an electron donor, while MaCPR1 did not. In yeast, heterologous co-expression indicates that MaCPR2 delivers electrons to MaC3'H hydroxylase, a key enzyme catalyzing the production of chlorogenic acid from 3-O-p-coumaroylquinic acid. CONCLUSIONS: These findings highlight the orchestration of hydroxylation process mediated by MaCPR2 during the biosynthesis of secondary metabolite biosynthesis in mulberry leaves. These results provided a foundational understanding for fully elucidating the DNJ biosynthetic pathway within mulberry leaves.

Grafting seedling rootstock strengthens tolerance to drought stress in polyploid mulberry (Morus alba L.).

The economically adaptable mulberry (Morus alba L.) has a long history of grafting in China, yet the physiological mechanisms and advantages in drought tolerance remain unexplored. In our study, we investigated the responses of self-rooted 2X (diploid), 3X (triploid), and 4X (tetraploid) plants, as well as polyploid plants grafted onto diploid seedling rootstocks (2X/2X, 3X/2X, and 4X/2X) under drought stress. We found that self-rooted diploid plants exhibited the most severe phenotypic damage, lowest water retention, photosynthetic capacity, and the least effective osmotic stress adjustment compared to tetraploid and triploid plants. However, grafted diploid and triploid plants showed effective mitigation of drought-induced damage, with higher relative water content and improved soil water retention. Grafted plants also improved the photosystem response to drought stress through elevated photosynthetic potential, closed stomatal aperture, and faster recovery of chlorophyll biosynthesis in the leaves. Additionally, grafted plants altered osmotic protective compound levels, including starch, soluble sugar, and proline content, thereby enhancing drought resistance. Absolute quantification PCR indicated that the expression levels of proline synthesis-related genes in grafted plants were not influenced after drought stress, whereas they were significantly increased in self-rooted plants. Consequently, our findings support that self-rooted triploid and tetraploid mulberries exhibited superior drought resistance compared to diploid plants. Moreover, grafting onto seedling rootstocks enhanced tolerance against drought stress in diploid and triploid mulberry, but not in tetraploid. Our study provides valuable insights for a comprehensive analysis of physiological effects in response to drought stress between stem-roots and seedling rootstocks.

Integrated transcriptomic and metabolomic analyses elucidate the mechanism of flavonoid biosynthesis in the regulation of mulberry seed germination under salt stress.

Seed propagation is the main method of mulberry expansion in China, an important economic forest species. However, seed germination is the most sensitive stage to various abiotic stresses, especially salinity stress. To reveal the molecular regulatory mechanism of mulberry seed germination under salt stress, flavonoid metabolomics and transcriptomics analyses were performed on mulberry seeds germinated under 50 and 100 mmol/L NaCl stress. Analysis of the flavonoid metabolome revealed that a total of 145 differential flavonoid metabolites (DFMs) were classified into 9 groups, 40 flavonols, 32 flavones, 16 chalcones and 14 flavanones. Among them, 61.4% (89) of the DFMs accumulated continuously with increasing salt concentration, reaching the highest level at a 100 mmol/L salt concentration; these DFMs included quercetin-3-O-glucoside (isoquercitrin), kaempferol (3,5,7,4'-tetrahydroxyflavone), quercetin-7-O-glucoside, taxifolin (dihydroquercetin) and apigenin (4',5,7-trihydroxyflavone), indicating that these flavonoids may be key metabolites involved in the response to salt stress. Transcriptional analysis identified a total of 3055 differentially expressed genes (DEGs), most of which were enriched in flavonoid biosynthesis (ko00941), phenylpropanoid biosynthesis (ko00940) and biosynthesis of secondary metabolites (ko01110). Combined analysis of flavonoid metabolomic and transcriptomic data indicated that phenylalanine ammonia-lyase (PAL), 4-coumarate-CoA ligase (4CL), chalcone synthase (CHS), flavonol synthase (FLS), bifunctional dihydroflavonol 4-reductase/flavanone 4-reductase (DFR) and anthocyanidin reductase (ANR) were the key genes involved in flavonoid accumulation during mulberry seed germination under 50 and 100 mmol/L NaCl stress. In addition, three transcription factors, MYB, bHLH and NAC, were involved in the regulation of flavonoid accumulation under salt stress. The results of quantitative real-time PCR (qRT‒PCR) validation showed that the expression levels of 11 DEGs, including 7 genes involved in flavonoid biosynthesis, under different salt concentrations were consistent with the transcriptomic data, and parallel reaction monitoring (PRM) results showed that the expression levels of 6 key enzymes (proteins) involved in flavonoid synthesis were consistent with the accumulation of flavonoids. This study provides a new perspective for investigating the regulatory role of flavonoid biosynthesis in the regulation of mulberry seed germination under salt stress at different concentrations.

Genome-wide transcriptome profiling of mulberry (Morus alba) response to boron deficiency and toxicity reveal candidate genes associated with boron tolerance in leaves.

Mulberry (Morus alba) is an essential plant with countless economic benefits; however, its growth and metabolic processes are hampered by boron (B) stresses. Very little research has been performed to elucidate boron tolerance and detoxification mechanisms in this species. The M. alba cultivar, Yu-711, was exposed to five different concentrations of boric acid (H3BO3), including deficient (T1; 0 mM) moderate B deficiency (T2; 0.02 mM), sufficient (CK; 0.1 mM) and toxic (T3 and T4; 0.5 and 1 mM) levels for 18 days of growth in pots experiment. Transcriptome analysis of B deficiency and toxicity treatments was performed on mulberry leaves. The transcriptome data reveal that a total of 6114 genes were differentially expressed (DEGs), of which 3830 were up-regulated and 2284 were down-regulated. A comparative analysis between treatment groups CK-vs-T1 (deficiency) and CK-vs-T4 (toxicity) indicates that 590 and 1383 genes were down-regulated in both deficiency and B toxicity, respectively. The results show that 206 genes were differentially expressed in all treatments. B deficiency and toxicity significantly altered the expression of the key aquaporins (PIP2-1, PIP2-7, PIP2-4 and NIP3-1) and high-affinity boron transporter genes (BOR1 and BOR7). In addition, boron stress also altered the expression of antioxidants and photosynthesis-related genes. B stresses were found to alter several transcription factors including ERF1B, which is associated with the regulation of boron uptake and the synthesis and signaling of phytohormones. Unravelling the mechanisms of B tolerance and detoxification is important and would give us further insight into how B stresses affect mulberry plants.

Mulberry-derived miR168a downregulates BmMthl1 to promote physical development and fecundity in silkworms.

Plant-derived miRNAs and their interactions with host organisms are considered important factors in regulating host physiological processes. In this study, we investigated the interaction between the silkworm, an oligophagous insect, and its primary food source, mulberry, to determine whether mulberry-derived miRNAs can penetrate silkworm cells and regulate their functions. Our results demonstrated that miR168a from mulberry leaves enters the silkworm hemolymph and binds to the silkworm Argonaute1 BmAGO1, which is transported via vesicles secreted by silkworm cells to exert its regulatory functions. In vivo and in vitro functional studies revealed that miR168a targets the mRNA of silkworm G protein-coupled receptor, BmMthl1, thereby inhibiting its expression and activating the JNK-FoxO pathway. This activation reduces oxidative stress responses, prolongs the lifespan of silkworms, and improves their reproductive capacity. These findings highlight the challenges of replacing mulberry leaves with alternative protein sources and provide a foundation for developing silkworm germplasms suitable for factory rearing.

Molecular and metabolic insights into purplish leaf coloration through the investigation of two mulberry (Morus alba) genotypes.

BACKGROUND: Leaf coloration in plants, attributed to anthocyanin compounds, plays a crucial role in various physiological functions, and also for pharmaceutical and horticultural uses. However, the molecular mechanisms governing leaf coloration and the physiological significance of anthocyanins in leaves remain poorly understood. RESULTS: In this study, we investigated leaf color variation in two closely related mulberry genotypes, one with purplish-red young leaves (EP) and another with normal leaf color (EW). We integrated transcriptomic and metabolomic approaches to gain insights into the metabolic and genetic basis of purplish-red leaf development in mulberry. Our results revealed that flavonoid biosynthesis, particularly the accumulation of delphinidin-3-O-glucoside, is a key determinant of leaf color. Additionally, the up-regulation of CHS genes and transcription factors, including MYB family members, likely contributes to the increased flavonoid content in purplish-red leaves. CONCLUSION: These findings enhance our understanding of the molecular mechanisms responsible for the purplish coloration observed in mulberry leaves and also offer supporting evidence for the hypothesis that anthocyanins serve a protective function in plant tissues until the processes of light absorption and carbon fixation reach maturity, thereby ensuring a balanced equilibrium between energy capture and utilization.

Functional characterization of MaEXPA11 and its roles in response to biotic and abiotic stresses in mulberry.

Mulberry is a traditional economic tree with various values in sericulture, ecology, food industry and medicine. Expansins (EXPs) are known as cell wall expansion related proteins and have been characterized to involve in plant development and responses to diverse stresses. In present study, twenty EXP and expansin-like (EXL) genes were identified in mulberry. RNA-seq results indicated that three EXP and EXL genes showed up-regulated expression level under sclerotiniose pathogen infection in three independent RNA-seq datasets. The most significant upregulated EXPA11 was selected as key EXP involving in response to sclerotiniose pathogen infection in mulberry. Furthermore, a comprehensive functional analysis was performed to reveal subcellular location, tissue expression profile of MaEXPA11 in mulberry. Down-regulation of MaEXPA11 using virus induced gene silence (VIGS) was performed to explore the function of MaEXPA11 in Morus alba. Results showed that MaEXPA11 can positively regulate mulberry resistance to Ciboria shiraiana infection and negatively regulate mulberry resistance to cold or drought stress.

Two genes encoded by mulberry crinkle leaf virus (MCLV): The V4 gene enhances viral replication, and the V5 gene is needed for MCLV infection in Nicotiana benthamiana.

Mulberry crinkle leaf virus (MCLV) is a member of the genus Mulcrilevirus, family Geminiviridae. The expression and functions of the V4 and V5 genes encoded by the MCLV genome remain unknown. Here, we confirmed the expression of V4 and V5 by analyzing the V4 and V5 mRNAs and the promoter activity of individual ORFs upstream sequences. The functions of V4 and V5 were investigated by constructing Agrobacterium-mediated infectious clones of wild-type MCLV variant П (MCLV vII), MCLVwt and MCLV vП mutants, such as MCLVmV4 (start codon of V4 ORF mutated), MCLVdV4 (5'-end partial deletion of V4 ORF sequence) and MCLVmV5 (V5 ORF start codon mutated). Although MCLVwt, MCLVmV4, and MCLVdV4 could infect natural host mulberry and experimental tomato plants systematically, the replication of the MCLVmV4 and MCLVdV4 genomes was obviously reduced compared to MCLVwt in both mulberry and tomato plants. MCLV vП expressing V5 could infect Nicotiana benthamiana plants systematically, but MCLVmV5 could not, implying that V5 is needed for MCLV vП to infect N. benthamiana plants. Taken together, V4 is involved in replication of the MCLV genome in host plants, and V5 potentially might extend the host range. Our findings lay a foundation for in-depth insight into the functions of MCLV-encoded proteins and provide a novel perspective for the subsequent study of MCLV-host plant interactions.

Post-harvest ultraviolet irradiation induces changes in physical-chemical properties and levels of polycyclic aromatic hydrocarbons and gene expression in mulberry fruit.

BACKGROUND: Earlier studies reported that post-harvest ultraviolet (UV) irradiation could increase the health-promoting compounds in fruit but the effects of UV irradiation on the reduction of the polycyclic aromatic hydrocarbon (PAH) content in mulberries remain less known. Black mulberry fruit were exposed to two UV illumination dosages (3.5 and 7 kJ m-2 ) and were stored for 4, 8, and 12 days. RESULTS: Mulberries treated in this way displayed higher antioxidant enzyme activity and phenolic compound content in comparison with a control condition. The transcription factors (TFs) MdoMYB121, MdoMYB155, MdbZIP2, and MdbZIP48 were strongly expressed in two UV illumination dosages (about 45-95% higher than the control). The fluorine (Flu) and naphthalene (Nap) content in treated fruit decreased by 21-85% in comparison with the control condition. CONCLUSION: The findings of this study indicate that UV irradiation can be considered as a promising technique to remove some PAHs in black mulberries, to increase their health-promoting potential, and indirectly to improve their aesthetic quality due to the resulting desirable color parameters. © 2023 Society of Chemical Industry.

Biosynthetic pathways and related genes regulation of bioactive ingredients in mulberry leaves.

Mulberry leaves are served not only as fodder for silkworms but also as potential functional food, exhibiting nutritional and medical benefits due to the complex and diverse constituents, including alkaloids, flavonoids, phenolic acids, and benzofurans, which possess a wide range of biological activities, such as anti-diabete, anti-oxidant, anti-inflammatory, and so on. Nevertheless, compared with the well-studied phytochemistry and pharmacology of mulberry leaves, the current understanding of the biosynthesis mechanisms and regulatory mechanisms of active ingredients in mulberry leaves remain unclear. Natural resources of these active ingredients are limited owing to their low contents in mulberry leaves tissues and the long growth cycle of mulberry. Biosynthesis is emerging as an alternative means for accumulation of the desired high-value compounds, which can broaden channels for their large-scale green productions. Therefore, this review summarizes the recent research advance on the correlative key genes, enzyme biocatalytic reactions and biosynthetic pathways of valuable natural ingredients (i.e. alkaloids, flavonoids, phenolic acids, and benzofurans) in mulberry leaves, thereby offering important insights for their further biomanufacturing.

Mulberry MnGolS2 Mediates Resistance to Botrytis cinerea on Transgenic Plants.

Galactitol synthetase (GolS) as a key enzyme in the raffinose family oligosaccharides (RFOs) biosynthesis pathway, which is closely related to stress. At present, there are few studies on GolS in biological stress. The expression of MnGolS2 gene in mulberry was increased under Botrytis cinerea infection. The MnGolS2 gene was cloned and ectopically expressed in Arabidopsis. The content of MDA in leaves of transgenic plants was decreased and the content of CAT was increased after inoculation with B. cinerea. In this study, the role of MnGolS2 in biotic stress was demonstrated for the first time. In addition, it was found that MnGolS2 may increase the resistance of B. cinerea by interacting with other resistance genes. This study offers a crucial foundation for further research into the role of the GolS2 gene.

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.

Morpho-cultural, pathological and molecular variability in Phloeospora maculans causing leaf spot of mulberry (Morus species) in India.

BACKGROUND: Leaf spot disease (LSD) of mulberry caused by Phloeospora maculans is a major threat to the silk industry of Jammu and Kashmir, India, therefore, it was necessary to study the population structure of the pathogen for successful management of the disease. METHODS AND RESULTS: To understand the diversity in the Phloeospora maculans, a combination of conventional (morphological, cultural and pathological) and molecular (ISSR markers) approaches were employed to discern the variability in 27 isolates collected from Srinagar, Bandipora, and Baramulla districts of Jammu and Kashmir, India. The studies revealed a high level of variability in the pathogen. Based on the morpho-cultural and pathological studies, the pathogen isolates were grouped into different categories based on colony growth, texture, margin and colour besides changes in colour of medium, incubation period, leaf area infected, etc.A high level of polymorphism was observed in different isolates of P. maculans using ISSR markers, which indicated that these markers are suitable for studying the genetic diversity in this pathogen. All the isolates (27) of P. maculans were clustered into two groups or populations as indicated by mean delta K value. Analysis of molecular variance revealed the low genetic variation among the populations (1.08%) and a high level of genetic variation within the populations (98.91%). Fst value was found to be 0.01 indicating smaller amount of genetic differentiation between the populations against calculated P-value of 0.29. CONCLUSION: A high level of diversity based on morphological, cultural, pathological and molecular levels was observed in Phloeospora maculans collected from various districts of Kashmir valley, which indicates that the study of population structure is necessary for successful management of the disease.