Genome-wide identification of HMT gene family explores BpHMT2 enhancing selenium accumulation and tolerance in Broussonetia papyrifera.

Broussonetia papyrifera, a valuable feed resource, is known for its fast growth, wide adaptability, high protein content and strong selenium enrichment capacity. Selenomethionine (SeMet), the main selenium form in selenium fortification B. papyrifera, is safe for animals and this enhances its nutritional value as a feed resource. However, the molecular mechanisms underlying SeMet synthesis remain unclear. This study identified three homocysteine S-methyltransferase genes from the B. papyrifera genome. The phylogenetic tree demonstrated that BpHMTs were divided into two classes, and BpHMT2 in the Class 2-D subfamily evolved earlier and possesses more fundamental functions. On the basis of the correlation between gene expression levels and selenium content, BpHMT2 was identified as a key candidate gene associated with selenium tolerance. Subcellular localization experiments confirmed the targeting of BpHMT2 in nucleus, cell membrane and chloroplasts. Moreover, three BpHMT2 overexpression Arabidopsis thaliana lines were confirmed to enhance plant selenium tolerance and SeMet accumulation. Overall, our finding provides insights into the molecular mechanisms of selenium metabolism in B. papyrifera, highlighting the potential role of BpHMT2 in SeMet synthesis. This research contributes to our understanding of selenium-enriched feed resources, with increased SeMet content contributing to the improved nutritional value of B. papyrifera as a feed resource.

Induction of tetraploids in Paper Mulberry (Broussonetia papyrifera (L.) L'Hér. ex Vent.) by colchicine.

BACKGROUND: Broussonetia papyrifera (L.) L'Hér. ex Vent. has the characteristics of strong stress resistance, high crude protein content, and pruning tolerance. It is an ecological, economic, and medicinal plant. Polyploid plants usually perform better than their corresponding diploid plants in terms of nutrients, active substances, and stress resistance. RESULTS: In this study, the leaves, calli, and seeds of diploid B. papyrifera were used for tetraploid induction by colchicine. The induction effect of colchicine on B. papyrifera was summarized through the early morphology, chromosome count and flow cytometry. It was concluded that the best induction effect (18.6%) was obtained when the leaves of B. papyrifera were treated in liquid MS (Murashige and Skoog) medium containing 450 mg·L-1 colchicine for 3 d. The comparative analysis of the growth characteristics of diploid and tetraploid B. papyrifera showed that tetraploid B. papyrifera has larger ground diameter, larger stomata, thicker palisade tissue and thicker sponge tissue than diploid B. papyrifera. In addition, the measurement of photosynthetic features also showed that tetraploids had higher chlorophyll content and higher photosynthetic rates. CONCLUSION: This study showed that tetraploid B. papyrifera could be obtained by treating leaves, callus and seeds with liquid and solid colchicine, but the induction efficiency was different. Moreover, there were differences in stomata, leaf cell structure and photosynthetic features between tetraploid B. papyrifera and its corresponding diploid. The induced tetraploid B. papyrifera can provide a technical basis and breeding material for the creation of B. papyrifera germplasm resources in the future.

Screening and Validation of Appropriate Reference Genes for Real-Time Quantitative PCR under PEG, NaCl and ZnSO4 Treatments in Broussonetia papyrifera.

Real-time quantitative PCR (RT-qPCR) has a high sensitivity and strong specificity, and is widely used in the analysis of gene expression. Selecting appropriate internal reference genes is the key to accurately analyzing the expression changes of target genes by RT-qPCR. To find out the most suitable internal reference genes for studying the gene expression in Broussonetia papyrifera under abiotic stresses (including drought, salt, and ZnSO4 treatments), seven different tissues of B. papyrifera, as well as the roots, stems, and leaves of B. papyrifera under the abiotic stresses were used as test materials, and 15 candidate internal reference genes were screened based on the transcriptome data via RT-qPCR. Then, the expression stability of the candidate genes was comprehensively evaluated through the software geNorm (v3.5), NormFinder (v0.953), BestKeeper (v1.0), and RefFinder. The best internal reference genes and their combinations were screened out according to the analysis results. rRNA and Actin were the best reference genes under drought stress. Under salt stress, DOUB, HSP, NADH, and rRNA were the most stable reference genes. Under heavy metal stress, HSP and NADH were the most suitable reference genes. EIF3 and Actin were the most suitable internal reference genes in the different tissues of B. papyrifera. In addition, HSP, rRNA, NADH, and UBC were the most suitable internal reference genes for the abiotic stresses and the different tissues of B. papyrifera. The expression patterns of DREB and POD were analyzed by using the selected stable and unstable reference genes. This further verified the reliability of the screened internal reference genes. This study lays the foundation for the functional analysis and regulatory mechanism research of genes in B. papyrifera.

The complete chloroplast genome sequences of three Broussonetia species and comparative analysis within the Moraceae.

Background: Species of Broussonetia (family Moraceae) are commonly used to make textiles and high-grade paper. The distribution of Broussonetia papyrifera L. is considered to be related to the spread and location of humans. The complete chloroplast (cp) genomes of B. papyrifera, Broussonetia kazinoki Sieb., and Broussonetia kaempferi Sieb. were analyzed to better understand the status and evolutionary biology of the genus Broussonetia. Methods: The cp genomes were assembled and characterized using SOAPdenovo2 and DOGMA. Phylogenetic and molecular dating analysis were performed using the concatenated nucleotide sequences of 35 species in the Moraceae family and were based on 66 protein-coding genes (PCGs). An analysis of the sequence divergence (pi) of each PCG among the 35 cp genomes was conducted using DnaSP v6. Codon usage indices were calculated using the CodonW program. Results: All three cp genomes had the typical land plant quadripartite structure, ranging in size from 160,239 bp to 160,841 bp. The ribosomal protein L22 gene (RPL22) was either incomplete or missing in all three Broussonetia species. Phylogenetic analysis revealed two clades. Clade 1 included Morus and Artocarpus, whereas clade 2 included the other seven genera. Malaisia scandens Lour. was clustered within the genus Broussonetia. The differentiation of Broussonetia was estimated to have taken place 26 million years ago. The PCGs' pi values ranged from 0.0005 to 0.0419, indicating small differences within the Moraceae family. The distribution of most of the genes in the effective number of codons plot (ENc-plot) fell on or near the trend line; the slopes of the trend line of neutrality plots were within the range of 0.0363-0.171. These results will facilitate the identification, taxonomy, and utilization of the Broussonetia species and further the evolutionary studies of the Moraceae family.

Broussonetia × hanjiana (Moraceae) microsatellite markers for species and ploidy determination developed using next-generation sequencing.

BACKGROUND: Broussonetia × hanjiana has been considered a hybrid owing to its morphology, which is intermediate between that of B. papyrifera (L.) L'Her. ex Vent. and B. kazinoki Siebold. A recent study demonstrated the hybrid origin of B. × hanjiana in Korea using molecular markers. In this study, we developed microsatellite markers for B. × hanjiana using next-generation sequencing and cross-species transferability analysis. METHODS AND RESULTS: A total of 432 primers were designed from 205,819 contigs. Among them, 24 microsatellite markers showing polymorphisms were used to evaluate the population genetic characteristics. The observed heterozygosity (HO) and expected heterozygosity (HE) were 0.835 and 0.628, respectively. The cross-species transferability of these markers was evaluated in two closely related species of Broussonetia; all 24 markers showed cross-species amplification. Using flow cytometry, diploid and triploid individuals were identified in B. × hanjiana. In particular, the BR137 marker showed evidence of two parent species (B. papyripera and B. kazinoki), with a hybrid pattern observed in B. × hanjiana, demonstrating its utility for species identification and ploidy assessment. CONCLUSIONS: The new B. × hanjiana microsatellite markers can be useful in genetic studies of closely related B. papyripera, B. kazinoki, and B. × hanjiana.

Comprehensive Profiling of Paper Mulberry (Broussonetia papyrifera) Crotonylome Reveals the Significance of Lysine Crotonylation in Young Leaves.

Lysine crotonylation is a newly discovered and reversible posttranslational modification involved in various biological processes, especially metabolism regulation. A total of 5159 lysine crotonylation sites in 2272 protein groups were identified. Twenty-seven motifs were found to be the preferred amino acid sequences for crotonylation sites. Functional annotation analyses revealed that most crotonylated proteins play important roles in metabolic processes and photosynthesis. Bioinformatics analysis suggested that lysine crotonylation preferentially targets a variety of important biological processes, including ribosome, glyoxylate and dicarboxylate metabolism, carbon fixation in photosynthetic organisms, proteasome and the TCA cycle, indicating lysine crotonylation is involved in the common mechanism of metabolic regulation. A protein interaction network analysis revealed that diverse interactions are modulated by protein crotonylation. These results suggest that lysine crotonylation is involved in a variety of biological processes. HSP70 is a crucial protein involved in protecting plant cells and tissues from thermal or abiotic stress responses, and HSP70 protein was found to be crotonylated in paper mulberry. This systematic analysis provides the first comprehensive analysis of lysine crotonylation in paper mulberry and provides important resources for further study on the regulatory mechanism and function of the lysine crotonylated proteome.

Genome-Wide Analysis of BpYABs and Function Identification Involving in the Leaf and Silique Development in Transgenic Arabidopsis.

YABs play an important role in the leaf development of the paper mulberry (Broussonetia papyrifera) and of the heterophylly. Thus, we investigated the function of BpYABs. Gene cloning, phylogenetic analysis, motif identification, subcellular localization, transactivation activity assay, qRT-PCR, in situ hybridization, and ectopic expression were used in our study. Six BpYABs were isolated, and four of them had transcriptional activity. BpYAB1, BpYAB3, BpYAB4, and BpYAB5 were localized to the nucleus. BpYAB1 was only expressed in the flower, while BpYAB6 was not expressed in any detected tissues; the four remaining BpYABs were expressed in the bud, leaf and flower, and their expression level decreased with leaf development. Further in situ hybridization showed that BpYAB3 and BpYAB5 were expressed in the vascular tissues and lamina, but neither showed the adaxial-abaxial polarity distribution pattern in the mature leaf lamina. Ectopic expression of BpYAB2, BpYAB3, BpYAB4 and BpYAB5 induced increased expression of AtWOX1 and caused the leaf of Arabidopsis to become smaller and curl downwards. Ectopic expression also led to shorter siliques and smaller seeds, but not for BpYAB5. These results suggest that BpYABs have functional divergency and redundancy in regulating leaf and silique development.

Plastome phylogenomics of Allaeanthus, Broussonetia and Malaisia (Dorstenieae, Moraceae) and the origin of B. × kazinoki.

Species of Broussonetia have been essential in the development of papermaking technology. In Japan and Korea, a hybrid between B. monoica and B. papyrifera (= B. × kazinoki) known as kozo and daknamu is still the major source of raw materials for making traditional paper washi and hanji, respectively. Despite their cultural and practical significance, however, the origin and taxonomy of kozo and daknamu remain controversial. Additionally, the long-held generic concept of Broussonetia s.l., which included Sect. Allaeanthus and Sect. Broussonetia, was challenged as phylogenetic analyses showed Malaisia is sister to the latter section. To re-examine the taxonomic proposition that recognizes Allaeanthus, Broussonetia, and Malaisia (i.e., Broussonetia alliance), plastome and nuclear ribosomal DNA (nrDNA) sequences of six species of the alliance were assembled. Characterized by the canonical quadripartite structure, genome alignments and contents of the six plastomes (160,121-162,594 bp) are highly conserved, except for the pseudogenization and/or loss of the rpl22 gene. Relationships of the Broussonetia alliance are identical between plastome and nrDNA trees, supporting the maintenance of Malaisia and the resurrection of Allaeanthus. The phylogenomic relationships also indicate that the monoecy in B. monoica is a derived state, possibly resulting from hybridization between the dioecious B. kaempferi (♀) and B. papyrifera (♂). Based on the hypervariable ndhF-rpl32 intergenic spacer selected by sliding window analysis, phylogeographic analysis indicates that B. monoica is the sole maternal parent of B. × kazinoki and that daknamu carries multiple haplotypes, while only one haplotype was detected in kozo. Because hybridizations between B. monoica and B. papyrifera are unidirectional and have occurred rarely in nature, our data suggest that daknamu might have originated via deliberate hybrid breeding selected for making hanji in Korea. On the contrary, kozo appears to have a single origin and the possibility of a Korean origin cannot be ruled out.

Genome-Wide Analysis of the UGT Gene Family and Identification of Flavonoids in Broussonetia papyrifera.

Broussonetia papyrifera is a multifunctional deciduous tree that is both a food and a source of traditional Chinese medicine for both humans and animals. Further analysis of the UGT gene family is of great significance to the utilization of B. papyrifera. The substrates of plant UGT genes include highly diverse and complex chemicals, such as flavonoids and terpenes. In order to deepen our understanding of this family, a comprehensive analysis was performed. Phylogenetic analysis showed that 155 BpUGTs were divided into 15 subgroups. A conserved motif analysis showed that BpUGT proteins in the same subgroups possessed similar motif structures. Tandem duplication was the primary driving force for the expansion of the BpUGT gene family. The global promoter analysis indicated that they were associated with complex hormone regulatory networks and the stress response, as well as the synthesis of secondary metabolites. The expression pattern analysis showed that the expression level of BpUGTs in leaves and roots was higher than that in fruits and stems. Next, we determined the composition and content of flavonoids, the main products of the BpUGT reaction. A total of 19 compounds were isolated and analyzed by UPLC-ESI-MS/MS in 3 species of Broussonetia including B. kazinoki, B. papyrifera, and B. kazinoki × B. papyrifera, and the number of compounds was different in these 3 species. The total flavonoid content and antioxidant capacities of the three species were analyzed respectively. All assays exhibited the same trend: the hybrid paper mulberry showed a higher total flavonoid content, a higher total phenol content and higher antioxidant activity than the other two species. Overall, our study provides valuable information for understanding the function of BpUGTs in the biosynthesis of flavonoids.

Identification of the PP2C gene family in paper mulberry (Broussonetia papyrifera) and its roles in the regulation mechanism of the response to cold stress.

OBJECTIVES: To study the possible roles of type-2C protein phosphatases (PP2Cs) which have been confirmed to play roles in the response to diverse abiotic stresses in paper mulberry, we launched a series of genomic and functional studies of BpPP2Cs. RESULTS: Sixty-three PP2C proteins in paper mulberry (Broussonetia papyrifera) were classified into 13 clades. Four BpPP2Cs with kinase domains were verified to be highly conserved in organisms ranging from algae to dicots. Seven pairs of BpPP2C genes were found to be expanding, and 18 BpPP2C genes had orthologues in Arabidopsis. BpPP2Cs showed broad expression in different tissues; the expression levels of 18 BpPP2Cs were changed and the phosphorylation levels of seven BpPP2C proteins increased at low temperature. Cold-response elements were found in the promoter region of 31 BpPP2Cs. Finally, Bp01g0320 was found to act as a hub protein and Bp01g0512 and Bp09g1278 played key roles in the ABA-signaling pathway and MAPK cascades, respectively. CONCLUSION: These results suggest that the PP2C gene family of paper mulberry is evolutionarily conserved and participates the regulation of the response to cold stress, which will play a vital role in further research on phosphatases in paper mulberry.

Cooperation between Broussonetia papyrifera and Its Symbiotic Fungal Community To Improve Local Adaptation of the Host.

The genetic basis of plant local adaptation has been extensively studied, yet the interplay between local adaptation, plant genetic divergence, and the microbial community remains unclear. Our study used the restriction-site associated DNA sequencing (RAD-seq) approach to explore genetic divergence in Broussonetia papyrifera and used internal transcribed spacers (ITS) to characterize fungal community. RAD-seq results show that B. papyrifera individuals could be divided into three genotypes; this genotyping result was consistent with the classification of climate type at the sample site. Most of the 101 highly differentiated genes were related to stress resistance and the microbiome. Moreover, ß-diversity results indicated that genetic divergence had a significant effect on fungal community across all compartments (P < 0.01). At genus and operational taxonomic unit (OTU) level, Mortierella, Hannaella oryzae, OTU81578 (Mortierella), and OTU1665209 (H. oryzae) were found to be the major OTUs that contribute to differences in fungal community. The properties of cooccurrence networks vary greatly among three genotypes. The results of redundancy analysis (RDA) indicated that B. papyrifera-associated fungal community was significantly related to its local adaptability. Our findings suggest that genetic divergence of B. papyrifera is closely related to local adaptation, with significant effects on the associated fungal community, which in turn would enhance host local adaptability. This improves present understanding about the coevolution of microbial communities and the host plant.IMPORTANCE The coevolution of plants with the associated fungal community and its effect on plant adaptability are not clear, especially for native trees. This study focuses on the genetic basis of local adaptation in plants and the effect of genetic divergence of Broussonetia papyrifera on the associated fungal community. We identified genes related to the microbiome that are important for local adaptation of the host. Our results show that genetic divergence in B. papyrifera significantly affects the fungal community, which has a close connection with local adaptation. This helps us to understand the relationship between local adaptation, genetic divergence, and associated fungal communities. This study highlights the effect of plant genetic divergence on associated fungal community for native trees and establishes a close connection between this effect and local adaptability in the host. In addition, these observations lay a foundation for the research of coevolution of plants and their symbiotic microbiome through genome-wide association study (GWAS).

A tale of textiles: Genetic characterization of historical paper mulberry barkcloth from Oceania.

Humans introduced paper mulberry (Broussonetia papyrifera) from Taiwan into the Pacific over 5000 years ago as a fiber source to make barkcloth textiles that were, and still are, important cultural artifacts throughout the Pacific. We have used B. papyrifera, a species closely associated to humans, as a proxy to understand the human settlement of the Pacific Islands. We report the first genetic analysis of paper mulberry textiles from historical and archaeological contexts (200 to 50 years before present) and compare our results with genetic data obtained from contemporary and herbarium paper mulberry samples. Following stringent ancient DNA protocols, we extracted DNA from 13 barkcloth textiles. We confirmed that the fiber source is paper mulberry in nine of the 13 textiles studied using the nuclear ITS-1 marker and by statistical estimates. We detected high genetic diversity in historical Pacific paper mulberry barkcloth with a set of ten microsatellites, showing new alleles and specific genetic patterns. These genetic signatures allow tracing connections to plants from the Asian homeland, Near and Remote Oceania, establishing links not observed previously (using the same genetic tools) in extant plants or herbaria samples. These results show that historic barkcloth textiles are cultural materials amenable to genetic analysis to reveal human history and that these artifacts may harbor evidence of greater genetic diversity in Pacific B. papyrifera in the past.

Human mediated translocation of Pacific paper mulberry [Broussonetia papyrifera (L.) L'Hér. ex Vent. (Moraceae)]: Genetic evidence of dispersal routes in Remote Oceania.

Paper mulberry, Broussonetia papyrifera (L.) L'Hér. ex Vent. (Moraceae), a dioecious species, was transported by humans from Taiwan to the islands of Remote Oceania. Its introduction and cultivation in Remote Oceania was intentional due to its cultural importance as a fiber source for barkcloth textiles. The aim of this study was to explore the genetic diversity and structure of paper mulberry populations within Remote Oceania in order to infer dispersal patterns that may reflect past human interaction among island groups. We present the integrated analysis of 380 samples (313 contemporary and 67 herbarium specimens) collected in Near and Remote Oceania. Genetic characterization was based on a set of ten microsatellites developed for B. papyrifera and complemented with the analysis of the ribosomal internal transcribed spacer ITS-1 sequence, a sex marker and the chloroplast ndhF-rpl32 intergenic spacer. Microsatellite data identify a total of 64 genotypes, despite this being a clonally propagated crop, and show three major dispersal hubs within Remote Oceania, centered on the islands of Fiji, Tonga, and Pitcairn. Of 64 genotypes identified, 55 correspond to genotypes associated to female-sexed plants that probably descend from plants introduced by the prehistoric Austronesian-speaking voyagers. The ratio of accessions to genotypes between herbarium and contemporary samples, suggests recent loss of genetic diversity. In addition to the chloroplast haplotypes described previously, we detected two new haplotypes within Remote Oceania both originating in Taiwan. This is the first study of a commensal species to show genetic structuring within Remote Oceania. In spite of the genetic bottleneck, the presence of only one sex, a timespan of less than 5000 years, and asexual propagation of this crop in Remote Oceania, we detect genetic diversity and regional structuring. These observations suggest specific migration routes between island groups within Remote Oceania.

A Chromosome-Scale Genome Assembly of Paper Mulberry (Broussonetia papyrifera) Provides New Insights into Its Forage and Papermaking Usage.

Paper mulberry (Broussonetia papyrifera) is a well-known woody tree historically used for Cai Lun papermaking, one of the four great inventions of ancient China. More recently, Paper mulberry has also been used as forage to address the shortage of feedstuff because of its digestible crude fiber and high protein contents. In this study, we obtained a chromosome-scale genome assembly for Paper mulberry using integrated approaches, including Illumina and PacBio sequencing platform as well as Hi-C, optical, and genetic maps. The assembled Paper mulberry genome consists of 386.83 Mb, which is close to the estimated size, and 99.25% (383.93 Mb) of the assembly was assigned to 13 pseudochromosomes. Comparative genomic analysis revealed the expansion and contraction in the flavonoid and lignin biosynthetic gene families, respectively, accounting for the enhanced flavonoid and decreased lignin biosynthesis in Paper mulberry. Moreover, the increased ratio of syringyl-lignin to guaiacyl-lignin in Paper mulberry underscores its suitability for use in medicine, forage, papermaking, and barkcloth making. We also identified the root-associated microbiota of Paper mulberry and found that Pseudomonas and Rhizobia were enriched in its roots and may provide the source of nitrogen for its stems and leaves via symbiotic nitrogen fixation. Collectively, these results suggest that Paper mulberry might have undergone adaptive evolution and recruited nitrogen-fixing microbes to promote growth by enhancing flavonoid production and altering lignin monomer composition. Our study provides significant insights into genetic basis of the usefulness of Paper mulberry in papermaking and barkcloth making, and as forage. These insights will facilitate further domestication and selection as well as industrial utilization of Paper mulberry worldwide.

New insight into the molecular basis of cadmium stress responses of wild paper mulberry plant by transcriptome analysis.

BACKGROUND: Heavy metal contamination is becoming a limitation to the utilization of soil and the distribution of vegetation. In particular, cadmium (Cd) pollution has had a serious impact on the food chain. Broussonetia papyrifera is a widely distributed pioneer tree species of heavy metal contaminated areas with important economic value. However, little is known about the genomic background of the Cd-tolerance mechanism in B. papyrifera. RESULTS: The CdCl2 responsive physiology was evaluated and proved to be involved in antioxidase activity and active oxygen species (ROS) accumulation. The leaf and root transcriptomes derived from B. papyrifera grown under normal and CdCl2 stress conditions were systematically investigated using the Illumina HiSeq method. A total of 180,678,660 bp (27.1 GB) clean reads were assembled into 589,487 high-quality unigenes, of which 256,025 (43.43% of the total) and 250,251 (42.45% of the total) were aligned in Gene Ontology (GO) and Protein family (Pfam), respectively. A total of 24,414 differentially expressed genes (DEGs) were GO-annotated into 53, 23, 55, and 60 terms from the transcriptomes of root and leaf tissues under Cd stress and control conditions. A total of 117,547 Kyoto Encyclopedia of Genes and Genomes (KEGG) Orthology (KO)-annotated DEGs were enriched in at least 47 KEGG pathway terms among the four comparisons. Many genes encoding important transcription factors (e.g., auxin/indole-3-acetic acid (AUX/IAA), basic helix-loop-helix (bHLH), DNA-binding one zinc finger (Dof), and MYB) and proteins involved in plant-pathogen interactions, phenylpropanoid biosynthesis, plant hormone signal transduction, oxidative phosphorylation, carbon fixation, peroxisomes, flavonoid biosynthesis, and glutathione metabolism, among others, were substantially upregulated under CdCl2 stress. CONCLUSIONS: These genes represent important candidates for studying Cd-response mechanisms and molecular biology of B. papyrifera and related species. Our findings provide a genomic sequence resource for functional genetic assignments in B. papyrifera, which will help elucidate the molecular mechanisms of its Cd-stress responses and facilitate the bioremediation of heavy metal contaminated areas via breeding of new stress-tolerant cultivars.

Not stealing from the treasure chest (or just a bit): Analyses on plant derived writing supports and non-invasive DNA sampling.

Written communication plays a crucial role in the history of modern civilizations as manuscripts do not only exist contemporarily, but are passed on to subsequent generations. Besides a document's content, information is stored in the materials used for its production. Analyses of the composition allow, for example, identifying the biological origins of materials, dating, and help to understand degradation patterns. A combination of microscopic and DNA approaches was applied in order to analyze various plant derived writing sheets. Given their diversity and abundance in museum collections, plant based writing supports are yet an underexplored target for DNA studies. DNA retrieval of paper is low compared to raw paper plant material, which is likely due to the loss of organic components during paper production. Optimizing DNA extraction for each respective material drastically increased DNA recovery. Finally, we present a non-invasive DNA sampling method that utilizes nylon membranes, commonly used for bacterial DNA sampling and that is applicable to delicate material. Although bacterial infestation was visible on one sample, as indicated by scanning electron microscopy, endogenous DNA was retrieved. The results presented here are promising as they extend the scope of sources for DNA analyses by demonstrating that DNA molecules can be retrieved from a variety of plant derived writing supports. In future, such analyses can help to explore the biological diversity not only of plants and of additives utilized for producing writing supports, but also of the plenty products made from paper.

Phylogeography of herbarium specimens of asexually propagated paper mulberry [Broussonetia papyrifera (L.) L'Hér. ex Vent. (Moraceae)] reveals genetic diversity across the Pacific.

Background and Aims: Paper mulberry or Broussonetia papyrifera (L.) L'Hér. ex Vent. (Moraceae) is a dioecious species native to continental South-east Asia and East Asia, including Taiwan, that was introduced to the Pacific by pre-historic voyagers and transported intentionally and propagated asexually across the full range of Austronesian expansion from Taiwan to East Polynesia. The aim of this study was to gain insight into the dispersal of paper mulberry into Oceania through the genetic analysis of herbaria samples which represent a more complete coverage of the historical geographical range of the species in the Pacific before later introductions and local extinctions occurred. Methods: DNA from 47 herbarium specimens of B. papyrifera collected from 1882 to 2006 from different islands of the Pacific was obtained under ancient DNA protocols. Genetic characterization was based on the ribosomal internal transcribed spacer ITS-1 sequence, a sex marker, the chloroplast ndhF-rpl32 intergenic spacer and a set of ten microsatellites developed for B. papyrifera. Key Results: Microsatellites allowed detection of 15 genotypes in Near and Remote Oceanian samples, in spite of the vegetative propagation of B. papyrifera in the Pacific. These genotypes are structured in two groups separating West and East Polynesia, and place Pitcairn in a pivotal position. We also detected the presence of male plants that carry the Polynesian chloroplast DNA (cpDNA) haplotype, in contrast to findings in contemporary B. papyrifera populations where only female plants bear the Polynesian cpDNA haplotype. Conclusions: For the first time, genetic diversity was detected among paper mulberry accessions from Remote Oceania. A clear separation between West and East Polynesia was found that may be indicative of pulses during its dispersal history. The pattern linking the genotypes within Remote Oceania reflects the importance of central Polynesia as a dispersal hub, in agreement with archaeological evidence.

Sex Distribution of Paper Mulberry (Broussonetia papyrifera) in the Pacific.

BACKGROUND: Paper mulberry (Broussonetia papyrifera (L.) L'Hér. ex Vent) is a dioecious tree native to East Asia and mainland Southeast-Asia, introduced prehistorically to Polynesia as a source of bark fiber by Austronesian-speaking voyagers. In Oceania, trees are coppiced and harvested for production of bark-cloth, so flowering is generally unknown. A survey of botanical records of paper mulberry revealed a distributional disjunction: the tree is apparently absent in Borneo and the Philippines. A subsequent study of chloroplast haplotypes linked paper mulberry of Remote Oceania directly to a population in southern Taiwan, distinct from known populations in mainland Southeast-Asia. METHODOLOGY: We describe the optimization and use of a DNA marker designed to identify sex in paper mulberry. We used this marker to determine the sex distribution in selected localities across Asia, Near and Remote Oceania. We also characterized all samples using the ribosomal internal transcribed spacer sequence (ITS) in order to relate results to a previous survey of ITS diversity. RESULTS: In Near and Remote Oceania, contemporary paper mulberry plants are all female with the exception of Hawaii, where plants of both sexes are found. In its natural range in Asia, male and female plants are found, as expected. Male plants in Hawaii display an East Asian ITS genotype, consistent with modern introduction, while females in Remote Oceania share a distinctive variant. CONCLUSIONS: Most paper mulberry plants now present in the Pacific appear to be descended from female clones introduced prehistorically. In Hawaii, the presence of male and female plants is thought to reflect a dual origin, one a prehistoric female introduction and the other a modern male introduction by Japanese/Chinese immigrants. If only female clones were dispersed from a source-region in Taiwan, this may explain the absence of botanical records and breeding populations in the Philippines and Borneo, and Remote Oceania.

Genetic diversity of Broussonetia papyrifera populations in southwest China.

Broussonetia papyrifera is an important native tree species with high economic value in southwest China. Its resources are drastically reduced because of over-harvesting and habitat fragmentation. In this study, 17 natural populations of B. papyrifera were analyzed using inter-simple sequence repeat (ISSR) markers to assess the genetic diversity and population structure. In total, 100 bands were obtained from 16 ISSR primers. The B. papyrifera populations showed relatively high genetic diversity at the species level [percentage of polymorphic bands (PPB): 96%; Nei's genetic diversity (HE): 0.3074; Shannon's information index (I): 0.4617], while the genetic diversity at the population level was relatively low (PPB: 53.2%; HE: 0.1826; I: 0.2735). Relatively high level of genetic differentiation among populations (41%) was disclosed by analysis of molecular variance, which agrees with the Nei's genetic diversity statistics (40.59%) and Shannon's information measure (40.76%). Gene flow among populations (NM) was only 0.7318. A significant correlation was observed between genetic and geographic distance among the studied populations (r=0.2948). We conjectured that the genetic diversity of B. papyrifera resulted from human disturbance, habitat fragmentation, small effective population size, and geographic barrier. Given the high genetic differentiation among populations, some utilization and conservation strategies were proposed. This study provides a reference for the sustainable use of the species in southwest China.

De Novo assembly of expressed transcripts and global transcriptomic analysis from seedlings of the paper mulberry (Broussonetia kazinoki x Broussonetia papyifera).

The paper mulberry is one of the multifunctional tree species in agroforestry systems and is also commonly utilized in traditional medicine in China and other Asian countries. However, little is known about its molecular genetics, which hinders research on and exploitation of this valuable resource. To discern the correlation between gene expression and the essential properties of the paper mulberry, we performed a transcriptomics analysis, assembling a total of 37,725 unigenes from 54,638,676 reads generated by RNA-seq. Among these, 22,692 unigenes showed greater than 60% similarity with genes from other species. The lengths of 13,566 annotated unigenes were longer than 1,000 bp. Functional clustering analysis with COG (Cluster of Orthologous Groups) revealed that 17,184 unigenes are primarily involved in transcription, translation, signal transduction, carbohydrate metabolism, secondary metabolism, and energy metabolism. GO (Gene Ontology) annotation suggests enrichment of genes encoding antioxidant activity, transporter activity, biosynthesis, metabolism and stress response, with a total of 30,659 unigenes falling in these categories. KEGG (Kyoto Encyclopedia of Genes and Genomes) metabolic pathway analysis showed that 7,199 unigenes are associated with 119 metabolic pathways. In addition to the basic metabolism, these genes are enriched for plant pathogen interaction, flavonoid metabolism and other secondary metabolic processes. Furthermore, differences in the transcriptomes of leaf, stem and root tissues were analyzed and 7,233 specifically expressed unigenes were identified. This global expression analysis provided novel insights about the molecular mechanisms of the biosynthesis of flavonoid, lignin and cellulose, as well as on the response to biotic and abiotic stresses including the remediation of contaminated soil by the paper mulberry.