Daniela Seelenfreund

Comparative genomics of Ceriporiopsis subvermispora and Phanerochaete chrysosporium provide insight into selective ligninolysis

Efficient lignin depolymerization is unique to the wood decay basidiomycetes, collectively referred to as white rot fungi. Phanerochaete chrysosporium simultaneously degrades lignin and cellulose, whereas the closely related species, Ceriporiopsis subvermispora, also depolymerizes lignin but may do so with relatively little cellulose degradation. To investigate the basis for selective ligninolysis, we conducted comparative genome analysis of C. subvermispora and P. chrysosporium. Genes encoding manganese peroxidase numbered 13 and five in C. subvermispora and P. chrysosporium, respectively. In addition, the C. subvermispora genome contains at least seven genes predicted to encode laccases, whereas the P. chrysosporium genome contains none. We also observed expansion of the number of C. subvermispora desaturase-encoding genes putatively involved in lipid metabolism. Microarray-based transcriptome analysis showed substantial up-regulation of several desaturase and MnP genes in wood-containing medium. MS identified MnP proteins in C. subvermispora culture filtrates, but none in P. chrysosporium cultures. These results support the importance of MnP and a lignin degradation mechanism whereby cleavage of the dominant nonphenolic structures is mediated by lipid peroxidation products. Two C. subvermispora genes were predicted to encode peroxidases structurally similar to P. chrysosporium lignin peroxidase and, following heterologous expression in Escherichia coli, the enzymes were shown to oxidize high redox potential substrates, but not Mn2+. Apart from oxidative lignin degradation, we also examined cellulolytic and hemicellulolytic systems in both fungi. In summary, the C. subvermispora genetic inventory and expression patterns exhibit increased oxidoreductase potential and diminished cellulolytic capability relative to P. chrysosporium.

DNA Isolation and AFLP Fingerprinting of Nectarine and Peach Varieties (Prunus persica)

Traditional identification of peach and nectarine varieties relies on the assessment of agronomic traits of the adult plant. This leads to a significant delay of time, constraints to breeders in the surveillance of germplasm and a risk for fruit growers and exporters. We describe a method for rapid assessment of peach and nectarine varieties based on AFLP fingerprinting and extraction of high quality DNA. The best primer pairs were selected from 64 primer combinations that reliably distinguished 8 peach and 6 nectarine varieties. A graphical representation of the detected polymorphisms was shown to simplify the analysis.

A holistic picture of Austronesian migrations revealed by phylogeography of Pacific paper mulberry

Significance Paper mulberry, a common East Asian tree used for paper making, is propagated across the Pacific for making barkcloth, a practical and symbolic component of Austronesian material culture. Using chloroplast DNA sequences, we demonstrate a tight genealogical link between its populations in South China and North Taiwan, and South Taiwan and Remote Oceania by way of Sulawesi and New Guinea, presenting the first study, to our knowledge, of a commensal plant species transported to Polynesia whose phylogeographic structure concurs with expectations of the “out of Taiwan” hypothesis of Austronesian expansion. As a commensal plant likely transported across the full range of Austronesian expansion from South China to East Polynesia, paper mulberry may also be the most widely transported fiber crop in human prehistory. The peopling of Remote Oceanic islands by Austronesian speakers is a fascinating and yet contentious part of human prehistory. Linguistic, archaeological, and genetic studies have shown the complex nature of the process in which different components that helped to shape Lapita culture in Near Oceania each have their own unique history. Important evidence points to Taiwan as an Austronesian ancestral homeland with a more distant origin in South China, whereas alternative models favor South China to North Vietnam or a Southeast Asian origin. We test these propositions by studying phylogeography of paper mulberry, a common East Asian tree species introduced and clonally propagated since prehistoric times across the Pacific for making barkcloth, a practical and symbolic component of Austronesian cultures. Using the hypervariable chloroplast ndhF-rpl32 sequences of 604 samples collected from East Asia, Southeast Asia, and Oceanic islands (including 19 historical herbarium specimens from Near and Remote Oceania), 48 haplotypes are detected and haplotype cp-17 is predominant in both Near and Remote Oceania. Because cp-17 has an unambiguous Taiwanese origin and cp-17–carrying Oceanic paper mulberries are clonally propagated, our data concur with expectations of Taiwan as the Austronesian homeland, providing circumstantial support for the “out of Taiwan” hypothesis. Our data also provide insights into the dispersal of paper mulberry from South China “into North Taiwan,” the “out of South China–Indochina” expansion to New Guinea, and the geographic origins of post-European introductions of paper mulberry into Oceania.

Paper mulberry (Broussonetia papyrifera) as a commensal model for human mobility in Oceania: anthropological, botanical and genetic considerations.

Paper mulberry (Broussonetia papyrifera (L.) Vent.) was one of the most widely distributed crop species in prehistoric Oceania, occurring from continental East Asia to the Polynesian islands. Its broad distribution is largely due to human-mediated dispersal during colonization of the islands of Near and Remote Oceania. We explore the potential for analyses of genetic variation in paper mulberry and the value of such data for the development of a new commensal model species for reconstructing patterns of human mobility in Oceania. We introduce and discuss paper mulberry as another commensal species and outline key features for its contribution to the understanding of human migration and post-colonization interaction. Here, we describe some of the extant B. papyrifera populations in Remote Oceania and Taiwan that were sampled for initial studies. We argue that the unique characteristics of this species and its importance in ancient Pacific island societies may provide the opportunity to collect valuable genetic data with which we can address several key questions in Pacific prehistory.

DNA Extraction and Amplification from Contemporary Polynesian Bark-Cloth

Background Paper mulberry has been used for thousands of years in Asia and Oceania for making paper and bark-cloth, respectively. Museums around the world hold valuable collections of Polynesian bark-cloth. Genetic analysis of the plant fibers from which the textiles were made may answer a number of questions of interest related to provenance, authenticity or species used in the manufacture of these textiles. Recovery of nucleic acids from paper mulberry bark-cloth has not been reported before. Methodology We describe a simple method for the extraction of PCR-amplifiable DNA from small samples of contemporary Polynesian bark-cloth (tapa) using two types of nuclear markers. We report the amplification of about 300 bp sequences of the ITS1 region and of a microsatellite marker. Conclusions Sufficient DNA was retrieved from all bark-cloth samples to permit successful PCR amplification. This method shows a means of obtaining useful genetic information from modern bark-cloth samples and opens perspectives for the analyses of small fragments derived from ethnographic materials.

Molecular analysis of Broussonetia papyrifera (L.) Vent. (Magnoliophyta: Urticales) from the Pacific, based on ribosomal sequences of nuclear DNA

Abstract Broussonetia papyrifera (L.) Vent. (Magnoliophyta: Urticales), or paper mulberry, is a species of Asian origin dispersed by humans throughout the Pacific. Our aim is to evaluate the genetic variability of this plant in order to determine its potential as a commensal species for studying the mobility and/or migratory movements of the people that carried it. For this study, we analysed the non-coding transcribed spacer sequences (ITS) of ribosomal nuclear DNA found in samples of B. papyrifera collected in Remote Oceania and Taiwan. Our results show three genotypes: the Pacific samples form a distinct and homogenous subgroup, while the Taiwanese accessions present two genotypes. We discuss the relevance of these results in the context of the dispersal of B. papyrifera in the Pacific and its association with Austronesian migration history.

Ancient and modern introduction of Broussonetia papyrifera ([L.] Vent.; Moraceae) into the Pacific: genetic, geographical and historical evidence

Broussonetia papyrifera (L.) Vent. (Moraceae), or paper mulberry, is a species of cultural importance in South East Asia, East Asia and the Pacific. Originally from mainland South East Asia or East Asia, this plant was introduced into the Pacific range by prehistoric Austronesian voyagers. We used non-coding internal transcribed spacer (ITS) sequences of nuclear ribosomal DNA and inter-simple sequence repeat (ISSR) on 79 samples of B. papyrifera from different islands of Remote Oceania, and South East Asia and East Asia. Our results show an absence of genetic diversity in the introduced range of Remote Oceania, with the sole exception of Hawaii. By contrast, Asian samples show genetic diversity. The data obtained suggest a prehistoric human-mediated introduction of this species from East Asia to Remote Oceania and a second, possibly historic, human-mediated introduction to Hawaii.

Analysis of manganese-regulated gene expression in the ligninolytic basidiomycete Ceriporiopsis subvermispora.

In this work, we explore the use of the unbiased cDNA–AFLP strategy to identify genes involved in Mn2+ homeostasis in Ceriporiopsis subvermispora. In this ligninolytic white-rot fungus, whose genome has not yet been sequenced, three Mn peroxidase genes responding to Mn2+ have been characterized. Using cDNA–AFLP to identify transcript-derived fragments (TDFs), a total of 37 differentially expressed cDNA fragments were identified by comparing band intensities among cDNA–AFLP patterns obtained from mycelia from cultures supplemented with different concentrations of Mn2+. Of 21 differentially expressed TDFs, nine were classified as upregulated, five as downregulated and seven as unregulated. Of these, six upregulated and two downregulated TDFs were selected for further characterization. The expected TDFs for the known Mn peroxidases were not isolated, but several genes encoding proteins related to protein sorting, storage and excretion of excess Mn2+ were identified. Transcripts induced under Mn2+ supplementation exhibited homologies to the elongation factor eEF3, a HDEL sequence binding protein and the ARD1 subunit of the N-acetyltransferase complex, among others. Overall, the results obtained in this study suggest a complex picture of Mn2+ homeostasis and provide the possibility to search for common regulatory elements in the promoters of the novel putatively identified genes.

Analysis of the intronic single nucleotide polymorphism rs#466452 of the nephrin gene in patients with diabetic nephropathy

We present the analysis of an intronic polymorphism of the nephrin gene and its relationship to the development of diabetic nephropathy in a study of diabetes type 1 and type 2 patients. The frequency of the single nucleotide polymorphism rs#466452 in the nephrin gene was determined in 231 patients and control subjects. The C/T status of the polymorphism was assessed using restriction enzyme digestions and the nephrin transcript from a kidney biopsy was examined. Association between the polymorphism and clinical parameters was evaluated using multivariate correspondence analysis. A bioinformatics analysis of the single nucleotide polymorphism rs#466452 suggested the appearance of a splicing enhancer sequence in intron 24 of the nephrin gene and a modification of proteins that bind to this sequence. However, no change in the splicing of a nephrin transcript from a renal biopsy was found. No association was found between the polymorphism and diabetes or degree of renal damage in diabetes type 1 or 2 patients. The single nucleotide polymorphism rs#466452 of the nephrin gene seems to be neutral in relation to diabetes and the development of diabetic nephropathy, and does not affect the splicing of a nephrin transcript, in spite of a splicing enhancer site.

A pilot study on genetic variation in purine-rich elements in the nephrin gene promoter in type 2 diabetic patients

Diabetic nephropathy (DN) is one of the major complications of type 2 diabetes and is associated with coronary disease. Nephrin, a protein mainly expressed in glomeruli, is decreased in DN and other kidney diseases. Since insulin levels are misregulated in type 2 diabetes, a possible connection between DN and its decreased nephrin expression could be the presence of regulatory elements responsive to insulin in the nephrin gene (NPHS1) promoter region. In this work, using bioinformatic tools, we identified a purine-rich GAGA element in the nephrin gene promoter and conducted a genomic study in search of the presence of polymorphisms in this element and its possible association with DN in type 2 diabetic patients. We amplified and sequenced a 514 bp promoter region of 100 individuals and found no genetic variants in the purine-rich GAGA-box of the nephrin gene promoter between groups of patients with diabetes type 2 with and without renal and coronary complications, control patients without diabetes and healthy controls.