DNA extraction from processed wood: A case study for the identification of an endangered timber species (Gonystylus bancanus)

We applied human forensic techniques to the extraction of whole genomic DNA from processed wood samples to explore the possibility of identifying an endangered tropical timber species by using DNA sequencing technology. High-yield and high-quality DNA samples were obtained from 2 commercial wood and 3 herbarium samples. Large PCR fragments ranging from 500–800 bp were successfully amplified from 2 chloroplast and 1 mitochondrial regions in all 5 samples, indicating limited degradation of the cytoplasmic genomes. DNA extraction from stem wood taken from herbarium specimens appeared superior to that from stem wood with bark intact or from leaf samples. DNA sequences from thetrn regions allowed for easy identification of the focal species based on GenBank Blast search. Little sequence variation was observed in the 3 regions, with the mitochondrialcox3 region completely conserved. Extraction of high-quality and large intact DNA fragments makes dry wood materials amenable to various DNA marker-based applications, including fingerprinting and historical approaches. By sampling stemwood, the wealth of historical information housed in international herbaria can be explored with minimal damage to taxonomically important features.     

叶绿体基因组研究进展

作为植物细胞器的重要组成部分和光合作用的器官,叶绿体在生物进化的漫长历史中发挥了重要作用.伴随着生物技术的深入发展,人们发现叶绿体基因组结构和序列的信息在揭示物种起源、进化演变及其不同物种之间的亲缘关系等方面具有重要价值.与此同时,比核转化具有明显优势的叶绿体转化技术在遗传改良、生物制剂的生产等方面显示出巨大潜力,而叶绿体基因组结构和序列分析则是叶绿体转化的基石.基于叶绿体的这些重要作用,收集整理了有关的资料,从几个方面归纳了本领域最近的研究进展,希望能使读者对迅速发展的叶绿体基因组研究有更全面的了解,以及对叶绿体基因组在物种的进化、遗传、系统发育关系等方面的作用有更深刻的认识,同时也希望对叶绿体转化技术的研究和广泛应用产生积极作用.     

The Mitochondrial Genome of Soybean Reveals Complex Genome Structures and Gene Evolution at Intercellular and Phylogenetic Levels

Determining mitochondrial genomes is important for elucidating vital activities of seed plants. Mitochondrial genomes are specific to each plant species because of their variable size, complex structures and patterns of gene losses and gains during evolution. This complexity has made research on the soybean mitochondrial genome difficult compared with its nuclear and chloroplast genomes. The present study helps to solve a 30-year mystery regarding the most complex mitochondrial genome structure, showing that pairwise rearrangements among the many large repeats may produce an enriched molecular pool of 760 circles in seed plants. The soybean mitochondrial genome harbors 58 genes of known function in addition to 52 predicted open reading frames of unknown function. The genome contains sequences of multiple identifiable origins, including 6.8 kb and 7.1 kb DNA fragments that have been transferred from the nuclear and chloroplast genomes, respectively, and some horizontal DNA transfers. The soybean mitochondrial genome has lost 16 genes, including nine protein-coding genes and seven tRNA genes; however, it has acquired five chloroplast-derived genes during evolution. Four tRNA genes, common among the three genomes, are derived from the chloroplast. Sizeable DNA transfers to the nucleus, with pericentromeric regions as hotspots, are observed, including DNA transfers of 125.0 kb and 151.6 kb identified unambiguously from the soybean mitochondrial and chloroplast genomes, respectively. The soybean nuclear genome has acquired five genes from its mitochondrial genome. These results provide biological insights into the mitochondrial genome of seed plants, and are especially helpful for deciphering vital activities in soybean.     

Pervasive migration of organellar DNA to the nucleus in plants

A surprisingly large number of plant nuclear DNA sequences inferred to be remnants of chloroplast and mitochondrial DNA migration events were detected through computer-assisted database searches. Nineteen independent organellar DNA insertions, with a median size of 117 by (range of 38 to >785 bp), occur in the proximity of 15 nuclear genes. One fragment appears to have been passed through a RNA intermediate, based on the presence of an edited version of the mitochondrial gene in the nucleus. Tandemly arranged fragments from disparate regions of organellar genomes and from different organellar genomes indicate that the fragments joined together from an intracellular pool of RNA and/or DNA before they integrated into the nuclear genome. Comparisons of integrated sequences to genes lacking the insertions, as well as the occurrence of coligated fragments, support a model of random integration by end joining. All transferred sequences were found in noncoding regions, but the positioning of organellar-derived DNA in introns, as well as regions 5 and 3 to nuclear genes, suggests that the random integration of organellar DNA has the potential to influence gene expression patterns. A semiquantitative estimate was performed on the amount of organellar DNA being transferred and assimilated into the nucleus. Based on this database survey, we estimate that 3–7% of the plant nuclear genomic sequence files contain organellar-derived DNA. The timing and the magnitude of genetic flux to the nuclear genome suggest that random integration is a substantial and ongoing process for creating sequence variation.Correspondence to: J.L. Blanchard     

Oldies (but goldies!): extracting DNA from cryopreserved allozyme homogenates

In this work, we describe a method that allows the extraction of plant genomic DNA from frozen enzyme homogenates. To test if reliable DNA was recovered from allozyme extracts, we compared these results with that obtained from fresh frozen and dried material. Sequencing and microsatellite fragments were analyzed for samples from 5 plant families in order to check for chloroplast and nuclear DNA quality. High-molecular weight DNA was recovered from frozen homogenates, and it was found that the quality was independent of storage time, which varied from 1 to 9 years. High-quality PCR products along with sequences and microsatellite fragments were obtained for nearly all analyzed samples. Frozen protein homogenates from previous population studies could thus be used for further phylogeographical and phylogenetic analyses using DNA molecular tools. This simple method provides an opportunity to take advantage of stored cryopreserved samples.     

Cucumber: a model angiosperm for mitochondrial transformation?

Plants possess three major genomes, carried in the chloroplast, mitochondrion, and nucleus. The chloroplast genomes of higher plants tend to be of similar sizes and structure. In contrast both the nuclear and mitochondrial genomes show great size differences, even among closely related species. The largest plant mitochondrial genomes exist in the genus Cucumis at 1500 to 2300 kilobases, over 100 times the sizes of the yeast or human mitochondrial genomes. Biochemical and molecular analyses have established that the huge Cucumis mitochondrial genomes are due to extensive duplication of short repetitive DNA motifs. The organellar genomes of almost all organisms are maternally transmitted and few methods exist to manipulate these important genomes. Although chloroplast transformation has been achieved, no routine method exists to transform the mitochondrial genome of higher plants. A mitochondrial-transformation system for a higher plant would allow geneticists to use reverse genetics to study mitochondrial gene expression and to establish the efficacy of engineered mitochondrial genes for the genetic improvement of the mitochondrial genome. Cucumber possesses three unique attributes that make it a potential model system for mitochondrial transformation of a higher plant. Firstly, its mitochondria show paternal transmission. Secondly, microspores possess relatively few, huge mitochondria. Finally, there exists in cucumber unique mitochondrial mutations conditioning strongly mosaic (msc) phenotypes. The msc phenotypes appear after regeneration of plants from cell culture and sort with specific rearranged and deleted regions in the mitochondrial genome. These mitochondrial deletions may be a useful genetic tool to develop selectable markers for mitochondrial transformation of higher plants.     

Extracting DNA from submerged pine wood.

A DNA extraction protocol for submerged pine logs was developed with the following properties: (i) high molecular weight DNA, (ii) PCR amplification of chloroplast and nuclear sequences, and (iii) high sequence homology to voucher pine specimens. The DNA extraction protocol was modified from a cetyltrimehtylammonium bromide (CTAB) protocol by adding stringent electrophoretic purification, proteinase K, RNAse, polyvinyl pyrrolidone (PVP), and Gene Releaser. Chloroplast rbcL (ribulose-1,5-bisphosphate carboxylase) could be amplified. Nuclear ribosomal sequences had >95% homology to Pinus taeda and Pinus palustris. Microsatellite polymorphism for PtTX2082 matched 2 of 14 known P. taeda alleles. Our results show DNA analysis for submerged conifer wood is feasible.     

Extraction, amplification and characterization of wood DNA from dipterocarpaceae

A successful DNA extraction from wood yielding appropriate DNA quality for PCR amplification allows molecular genetic investigations of wood tissue. Genotypes, the origin of sampled material, and species can be identified based on an investigation of wood if suitable information on genetic variation patterns within and among species is available. Potential applications are in forensics and in the control of the timber and wood trade. We extracted DNA from wood of Dipterocarpaceae, a family that dominates rainforests and comprises many important timber species in Southeast Asia. Several different DNA isolation techniques were compared and optimized for wood samples from natural populations and from wood processing enterprises. The quality of the DNA was tested by spectrophotometry, PCR amplification, and PCR inhibitor tests. An average DNA yield of 2.2 μg was obtained per 50–100 mg of dried wood sample. Chloroplast DNA (cpDNA) regions of different length were amenable to PCR amplification from the extracted DNA. Modification of DNA isolation techniques by the addition of polyvinylpyrrolidone (PVP) addition up to 3.1% into lysis buffer reduced PCR inhibition effectively. In order to evaluate the extraction method, we analyzed leaves and wood from the same tree by PCR amplification, genotyping and sequencing of chloroplast microsatellites.     

Problems and paradigms: Parochial,visionary and factual thinking on the origins of life

The existence and properties of the chloroplast genome were established by a combination of genetic methods which identified chloroplast mutations and placed them into a linear sequence or map; and by chemical methods, CsCl density gradient ultracentrifugation and base analysis, which identified non-nuclear DNA extracted from isolated chloroplasts. These studies, carried out in the 1950s and 1960s, primarily with Chlamydomonas, as well as parallel studies of mitochondrial DNA with yeast and Neurospora, laid the framework for distinguishing organelle and nuclear genomes. On this basis, the coding and regulatory functions of three genomes – nuclear, chloroplast, and mitochondrial – are being addressed in modern plant molecular biology.     

Comprehensive molecular characterization of tissue-culture-derived Hordeum marinum plants

Summary Scuttelar calli of Hordeum marinum readily and efficiently regenerate functional plants. In order to assess genetic variability among the regenerants we employed multiple analytic tools, which included molecular and biochemical assays. Total DNA extract from regenerated plants was digested with at least two restriction enzymes and hybridized to four nuclear and six mitochondrial coding sequences, in addition to one nuclear and three mitochondrial noncoding probes. SDS-PAGE analyses of hordein extracted from seeds of regenerated plants and activity assays of -amylase were also performed. The nuclear and mitochondrial genomes of 50 regenerated plants demonstrated relative stability when assessed with coding sequences and by biochemical analyses. However, the mitochondrial noncoding probes revealed one qualitative somaclonal variant characterized by a loss of a hybridizing fragment. Moreover, changes in the methylation patterns of the rRNA genes and the nontranscribed spacer were revealed in another regenerated plant. The albino plant regenerated was characterized by a loss of three chloroplast DNA BamHI fragments.     

Checking the geographical origin of oak wood: molecular and statistical tools

New methods for better identification of timber geographical origin would constitute an important technical element in the forest industry, for phytosanitary certification procedures or in the chain of custody developed for the certification of timber from sustainably managed forests. In the case of the European white oaks, a detailed reference map of chloroplast (cp) DNA variation across the range exists, and we propose here to use the strong geographical structure, characterized by a differentiation of western vs. eastern populations, for the purpose of oak wood traceability. We first developed cpDNA markers permitting the characterization of haplotype on degraded DNA obtained from wood samples. The techniques were subsequently validated by confirming the full correspondence between genotypes obtained from living tissues (buds) and from wood collected from the same individual oak. Finally, a statistical procedure was used to test if the haplotype composition of a lot of wood samples is consistent with its presumed geographical origin. Clearly, the technique cannot permit the unambiguous identification of wood products of unknown origin but can be used to check the conformity of genetic composition of wood samples with the region of alleged origin. This could lead to major applications not only in the forest industry but also in archaeology or in palaeobotany.     

Transfer of genetic material between the chloroplast and nucleus: how is it related to stress in plants?

Background

The presence of chloroplast-related DNA sequences in the nuclear genome is generally regarded as a relic of the process by which genes have been transferred from the chloroplast to the nucleus. The remaining chloroplast encoded genes are not identical across the plant kingdom indicating an ongoing transfer of genes from the organelle to the nucleus.

Scope

This review focuses on the active processes by which the nuclear genome might be acquiring or removing DNA sequences from the chloroplast genome. Present knowledge of the contribution to the nuclear genome of DNA originating from the chloroplast will be reviewed. In particular, the possible effects of stressful environments on the transfer of genetic material between the chloroplast and nucleus will be considered. The significance of this research and suggestions for the future research directions to identify drivers, such as stress, of the nuclear incorporation of plastid sequences are discussed.

Conclusions

The transfer to the nuclear genome of most of the protein-encoding functions for chloroplast-located proteins facilitates the control of gene expression. The continual transfer of fragments, including complete functional genes, from the chloroplast to the nucleus has been observed. However, the mechanisms by which the loss of functions and physical DNA elimination from the chloroplast genome following the transfer of those functions to the nucleus remains obscure. The frequency of polymorphism across chloroplast-related DNA fragments within a species will indicate the rate at which these DNA fragments are incorporated and removed from the chromosomes.Key words: Stress, DNA transfer, organelles and nucleus, genome integration     

藻类植物叶绿体基因组研究进展

藻类植物的cpDNA结构复杂,普遍缺失反向重复序列IR,且存在IR的藻类植物种类的cpDNA也有IR变短退化迹象.藻类植物的cpDNA包含的基因一般比高等植物要多,编码能力更强.藻类植物cpDNA全序列的测定方法主要是Fosmid文库构建,配合使用Long-PCR技术.该文对国内外有关藻类植物叶绿体基因组结构、叶绿体编码基因、叶绿体基因组在藻类系统发育中的应用以及藻类植物叶绿体基因组的提取和序列测定方法等进行综述,为藻类植物的系统发育和叶绿体起源以及功能基因组学的研究提供理论依据.     

Successful amplification of rice chloroplast microsatellites from century-old grass samples from the park grass experiment

We report the successful amplification of microsatellite markers for the chloroplast genome from century-old samples of 2 grasses growing in the Park Grass Experiment (PGE):Anthoxanthum ordoratum andFestuca rubra. This opens the possibility of establishing a long-term genetic time series for the PGE, which began in 1856 and is believed to be the oldest ecological experiment in existence. Although the plant samples used were not originally prepared or stored with molecular analysis in mind, the hexadecyltrimethylammonium bromide (CTAB) method of DNA extraction was successfully used. Obtained DNA was degraded but could be amplified by means of PCR. It produced bands around the expected size for chloroplast microsatellite primers derived from rice. When sequenced, bands showed good homology with sequences from rice chloroplast genomes listed in GenBank (accession No X15901).     

Cre-lox recombination: Cre-ative tools for plant biotechnology

Targeted insertion and the precise deletion of DNA from transgenic plant chromosomes increase the potential of plant biotechnology for commercial applications and basic research. The Cre–lox recombination system is one of the best characterized and most widely used systems for these purposes. Cre–lox has many applications, but it is primarily used for the controlled excision of DNA fragments, in particular selectable marker genes, from the nuclear and chloroplast genomes, and for the targeted insertion of DNA into specific sites in the nuclear genome. Recent developments, including regulated expression of cre and the creative use of wild-type and modified lox sites, have improved the potential of these applications. After almost 15 years of research and development in plants, the Cre–lox system continues to provide an efficient and precise tool for plant biotechnologists.     

Chloroplast DNA insertions into the nuclear genome of rice: the genes,sites and ages of insertion involved

Rice (Oryza sativa) is one of three predominant grain crops, and its nuclear and organelle genomes have been sequenced. Following genome analysis revealed many exchanges of DNA sequences between the nuclear and organelle genomes. In this study, a total of 45 chloroplast DNA insertions more than 2 kb in length were detected in rice nuclear genome. A homologous recombination mechanism is expected for those chloroplast insertions with high similarity between their flanking sequences. Only five chloroplast insertions with high sequence similarity between two flanking sequences from an insertion were found in the 45 insertions, suggesting that rice might follow the non-homologous end-joining (NHEJ) repair of double-stranded breaks mechanism, which is suggested to be common to all eukaryotes. Our studies indicate that the most chloroplast insertions occurred at a nuclear region characterized by a sharp change of repetitive sequence density. One potential explanation is that regions such as this might be susceptible target sites or “hotspots” of DNA damage. Our results also suggest that the insertion of retrotransposon elements or non-chloroplast DNA into chloroplast DNA insertions may contribute significantly to their fragmentation process. Moreover, based on chloroplast insertions in nuclear genomes of two subspecies (indica and japonica) of cultivated rice, our results strongly suggest that they diverged during 0.06–0.22 million years ago. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Amplification of oak DNA from ancient and modern wood

A polymorphic noncoding region of chloroplast DNA (cpDNA) was successfully amplified by the polymerase chain reaction (PCR) from various oak wood samples, including recent and more ancient (about 600-years-old) samples from different oak species. Adaptation of DNA isolation and amplification protocols was necessary to obtain this result. Polymorphisms useful to distinguish species or geographical origin of these samples could be scored through sequencing. These polymorphisms include one substitution and two microsatellite-type polymorphisms, due to a variable number of A/T repeats. Identical results were obtained independently in two separate laboratories.