Poly(A) tailing of ancient DNA: a method for reproducible microsatellite genotyping

Microsatellites could be of great potential use in the analysis of ancient remains, but so far such analyses have failed to be reproducible mainly because of the high degree of ancient DNA (aDNA) degradation. During PCR, annealing of the primers to the complementary sequences of microsatellites occurs together with cross-annealing of partially degraded repeated sequences. This could create chimeric alleles that do not correspond to the authentic ones. Here we report a simple method for processing aDNA fragments prior to PCR that greatly reduces the production of chimeric alleles. This approach eliminates aDNA molecules broken within the repeats as targets for Taq polymerase by adding poly(A) tails at the 3(') ends of the DNA fragments, which disrupts the homology in the region and thus prevents annealing out of register. We have analyzed one dinucleotide- (D6S337) and two trinucleotide-containing loci (IT15 and SCA1) using poly(A)-tailed and the same untreated aDNA as template. aDNAs were isolated from 28 human remains, 600 and 7000 years of age. In repeated experiments with untreated aDNAs we obtained three to five times more alleles compared to poly(A)-tailed aDNAs. According to our results, modification of aDNA by poly(A) tailing is an efficient pretreatment for accurate genotyping.     

Panicum spikelets from the Early Holocene Takarkori rockshelter (SW Libya): Archaeo-molecular and -botanical investigations

This paper deals with the extraction, amplification and sequencing of ancient DNA (aDNA) from spikelets of wild cereals dated at ca. 9000 cal yr BP, representing the most ancient plants with preserved genetic material from the Sahara desert. The sub-fossil records were collected from the archaeological excavation carried out at Takarkori, an archaeological site located in south-western Libya. Morphological and genetic analyses were made on 100 well preserved dried spikelets. Ten DNA extraction protocols were performed to evaluate nucleic acid recovery in terms of DNA yield, purity and amplification success of the chloroplast barcode region matK. The extraction protocol that returned the most suitable DNA to be amplified is the Kistler and Shapiro (2011: J Archaeol Sci 38: 3549-3554) modified protocol. In our study, the results from matK amplification suggested that four specimens are the most appropriate number of spikelets for these analyses. DNA was then used for PCR amplifications of four chloroplast barcode genes: rbcL, matK, trnH-psbA and trnL. A phylogenetic analysis shows the strict relation between the archaeological specimens and modern Panicoideae, supporting the morphological identification. The results indicate that spikelets have a close relation to Panicum laetum Kunth, a wild cereal still collected in tropical Africa.     

More on contamination: the use of asymmetric molecular behavior to identify authentic ancient human DNA

Authentication of ancient human DNA results is an exceedingly difficult challenge due to the presence of modern contaminant DNA sequences. Nevertheless, the field of ancient human genetics generates huge scientific and public interest, and thus researchers are rarely discouraged by problems concerning the authenticity of such data. Although several methods have been developed to the purpose of authenticating ancient DNA (aDNA) results, while they are useful in faunal research, most of the methods have proven complicated to apply to ancient human DNA. Here, we investigate in detail the reliability of one of the proposed criteria, that of appropriate molecular behavior. Using real-time polymerase chain reaction (PCR) and pyrosequencing, we have quantified the relative levels of authentic aDNA and contaminant human DNA sequences recovered from archaeological dog and cattle remains. In doing so, we also produce data that describes the efficiency of bleach incubation of bone powder and its relative detrimental effects on contaminant and authentic ancient DNA. We note that bleach treatment is significantly more detrimental to contaminant than to authentic aDNA in the bleached bone powder. Furthermore, we find that there is a substantial increase in the relative proportions of authentic DNA to contaminant DNA as the PCR target fragment size is decreased. We therefore conclude that the degradation pattern in aDNA provides a quantifiable difference between authentic aDNA and modern contamination. This asymmetrical behavior of authentic and contaminant DNA can be used to identify authentic haplotypes in human aDNA studies.     

De Novo Assembly and Species-Specific Marker Development as a Useful Tool for the Identification of Scutellaria L. Species

Scutellaria L. (family Lamiaceae) includes approximately 470 species found in most parts of the world and is commonly known as skullcaps. Scutellaria L. is a medicinal herb used as a folk remedy in Korea and East Asia, but it is difficult to identify and classify various subspecies by morphological methods. Since Scutellaria L. has not been studied genetically, to expand the knowledge of species in the genus Scutellaria L., de novo whole-genome assembly was performed in Scutellaria indica var. tsusimensis (H. Hara) Ohwi using the Illumina sequencing platform. We aimed to develop a molecular method that could be used to classify S. indica var. tsusimensis (H. Hara) Ohwi, S. indica L. and three other Scutellaria L. species. The assembly results for S. indica var. tsusimensis (H. Hara) Ohwi revealed a genome size of 318,741,328 bp and a scaffold N50 of 78,430. The assembly contained 92.08% of the conserved BUSCO core gene set and was estimated to cover 94.65% of the genome. The obtained genes were compared with previously registered Scutellaria nucleotide sequences and similar regions using the NCBI BLAST service, and a total of 279 similar nucleotide sequences were detected. By selecting the 279 similar nucleotide sequences and nine chloroplast DNA barcode genes, primers were prepared so that the size of the PCR product was 100 to 1000 bp. As a result, a species-specific primer set capable of distinguishing five species of Scutellaria L. was developed.     

Genista sessilifolia DC. and Genista tinctoria L. inhibit UV light and nitric oxide-induced DNA damage and human melanoma cell growth

Many Genista species (Leguminosae), containing isoflavones as biologically active substances, show interesting biological properties such as hypoglycemic, antiinflammatory, antiulcer, spasmolytic, antioxidant, estrogenic and cytotoxic activity against different human cancer cell lines. In this work, we describe the chemical composition of the methanolic extracts from aerial parts of Genista sessilifolia DC. and Genista tinctoria L., and their biological activity testing the effect on pBR322 DNA cleavage induced by hydroxyl radicals (OH), generated from UV-photolysis of hydrogen peroxide (H₂O₂) and by nitric oxide (NO). In addition, we investigated the growth inhibitory activity of these natural products against human melanoma cell line (M14). The extracts of G. sessilifolia and G. tinctoria, for their isoflavone components, showed a protective effect on UV light and nitric oxide-mediated plasmid DNA damage, and inhibited the growth of melanoma cells. The data of the present study also suggest that these natural products could trigger apoptotic death in M14 cells. In fact, a high DNA fragmentation (COMET assay) and a significant increase of caspase-3 activity, not correlated to LDH release, a marker of membrane breakdown, occurred in melanoma cells exposed to these extracts. The significant production of reactive oxygen species (ROS) evidenced in these experimental conditions could contribute to trigger the apoptosis cascades.     

一种实用可靠的古代 DNA 获取方法

古代DNA序列信息能够为物种演化研究提供最直接的分子证据,但获取古代DNA的技术仍存在诸多瓶颈,尤其是扩增中存在受损伤DNA模板的干扰、获取成本高和实验周期长等问题．改进了异丙醇沉淀提取法,并采用了尿嘧啶糖苷酶(UNG)去除受损伤DNA模板后进行扩增的方法,最终可以高效地获取真实的古代DNA序列．实验利用距今4 300～3 900年前的猪牙样本,将改进的古 DNA 获取方法与常规方法进行比较研究,结果表明,改进的异丙醇沉淀法提取结合UNG处理后进行PCR扩增的方法,可以在保证古代DNA获取成功率并提高获得的DNA序列可靠性的前提下,将经费投入和实验周期都各减少至常规方法的50%以下．这可以为开展大规模古代样本检测提供一种切实可行的 DNA 获取方法．     

Application of DNA barcodes in Hedyotis L. (Spermacoceae,Rubiaceae)

The potential application of DNA barcodes of plastid (matK, trnH–psbA, petD, and rbcL) and nuclear (internal transcribed spacer (ITS) of rDNA) DNA regions was investigated for 25 Hedyotis taxa. The ITS showed the best species discrimination by resolving 23 of the species as exclusive lineages with no shared alleles between any of the 24 distinct species (H. assimilis and H. mellii are not supported as distinct species based on our molecular and morphological data). Conversely, rbcL performed the worst and only resolved 10 of the species as exclusive lineages, and 10 species with shared alleles. Using ITS has the advantage of high PCR amplification success and it provides good intra- and interspecific variation distribution patterns. The most powerful plastid markers were petD and trnH–psbA, but we could amplify and sequence trnH–psbA for only 83% of the accessions sampled. Combination of ITS and petD performed extremely well, with all 24 of the distinct species resolved as exclusive lineages and no shared alleles between any of the distinct species. We therefore recommend ITS, or a combination of ITS and petD, as the standard DNA barcode in Hedyotis, but acknowledge that there are no shared alleles between distinct species for matK and rbcL combined.     

Technical note: improved ancient DNA purification for PCR using ion-exchange columns

A novel method of ancient DNA (aDNA) purification was developed using ion-exchange columns to improve PCR-amplifiable DNA extraction from ancient bone samples. Thirteen PCR-resistant ancient bone samples aged 500-3,300 years were tested to extract aDNA using a recently reported, silica-based aDNA extraction method and an ion-exchange column method for the further purification. The PCR success rates of the aDNA extracts were evaluated for the amplification ability of the fragments of mitochondrial DNA, a high-copy DNA, and amelogenin, a low-copy DNA. The results demonstrate that the further purification of silica-based aDNA extracts using ion-exchange columns considerably improved PCR amplification. We suggest that the ion-exchange column-based method will be useful for the improvement of PCR-amplifiable aDNA extraction, particularly from the poorly preserved, PCR-resistant, ancient samples.     

Palaeogenetic analysis of (pre)historic artifacts and its significance for anthropology

The possibility of isolating ancient DNA (aDNA) from all kinds of (pre)historic anthropogenetic artifacts opens new perspectives. This study applies palaeogenetic techniques to three anthropological issues: 1. Palaeodiet. DNA sequences from organic residues in vessels identify Precolumbian Aztec diet. 2. (Pre)historic husbandry and economic structures. aDNA data can reveal the species and the genetic evolutionary stage of animals and plants and show the manner and the extent of their growth, cultivation, or domestication. 3. Production techniques, use, and functionality. Identification of the plant or animal source of an archaeological find can reveal the use or the function of the find. Examples from a Celtic "sausage-end" and an Aztec "eye salve" are given.     

Paleo‐metagenomics of North American fossil packrat middens: Past biodiversity revealed by ancient DNA

Fossil rodent middens are powerful tools in paleoecology. In arid parts of western North America, packrat (Neotoma spp.) middens preserve plant and animal remains for tens of thousands of years. Midden contents are so well preserved that fragments of endogenous ancient DNA (aDNA) can be extracted and analyzed across millennia. Here, we explore the use of shotgun metagenomics to study the aDNA obtained from packrat middens up to 32,000 C¹⁴ years old. Eleven Illumina HiSeq 2500 libraries were successfully sequenced, and between 0.11% and 6.7% of reads were classified using Centrifuge against the NCBI “nt” database. Eukaryotic taxa identified belonged primarily to vascular plants with smaller proportions mapping to ascomycete fungi, arthropods, chordates, and nematodes. Plant taxonomic diversity in the middens is shown to change through time and tracks changes in assemblages determined by morphological examination of the plant remains. Amplicon sequencing of ITS2 and rbcL provided minimal data for some middens, but failed at amplifying the highly fragmented DNA present in others. With repeated sampling and deep sequencing, analysis of packrat midden aDNA from well‐preserved midden material can provide highly detailed characterizations of past communities of plants, animals, bacteria, and fungi present as trace DNA fossils. The prospects for gaining more paleoecological insights from aDNA for rodent middens will continue to improve with optimization of laboratory methods, decreasing sequencing costs, and increasing computational power.     

Ancient Mitochondrial DNA Analyses of Ascaris Eggs Discovered in Coprolites from Joseon Tomb

Analysis of ancient DNA (aDNA) extracted from Ascaris is very important for understanding the phylogenetic lineage of the parasite species. When aDNAs obtained from a Joseon tomb (SN2-19-1) coprolite in which Ascaris eggs were identified were amplified with primers for cytochrome b (cyt b) and 18S small subunit ribosomal RNA (18S rRNA) gene, the outcome exhibited Ascaris specific amplicon bands. By cloning, sequencing, and analysis of the amplified DNA, we obtained information valuable for comprehending genetic lineage of Ascaris prevalent among pre-modern Joseon peoples.     

Optimization of DNA Recovery and Amplification from Non-Carbonized Archaeobotanical Remains

Ancient DNA (aDNA) recovered from archaeobotanical remains can provide key insights into many prominent archaeological research questions, including processes of domestication, past subsistence strategies, and human interactions with the environment. However, it is often difficult to isolate aDNA from ancient plant materials, and furthermore, such DNA extracts frequently contain inhibitory substances that preclude successful PCR amplification. In the age of high-throughput sequencing, this problem is even more significant because each additional endogenous aDNA molecule improves analytical resolution. Therefore, in this paper, we compare a variety of DNA extraction techniques on primarily desiccated archaeobotanical remains and identify which method consistently yields the greatest amount of purified DNA. In addition, we test five DNA polymerases to determine how well they replicate DNA extracted from non-charred ancient plant remains. Based upon the criteria of resistance to enzymatic inhibition, behavior in quantitative real-time PCR, replication fidelity, and compatibility with aDNA damage, we conclude these polymerases have nuanced properties, requiring researchers to make educated decisions as to which one to use for a given task. The experimental findings should prove useful to the aDNA and archaeological communities by guiding future research methodologies and ensuring precious archaeobotanical remains are studied in optimal ways, and may thereby yield important new perspectives on the interactions between humans and past plant communities.     

中国蛛缘蝽科物种DNA条形码研究(英文)

【目的】本研究旨在探讨DNA条形码对中国蛛缘蝽科(半翅目：缘蝽总科)物种界定的适用性。【方法】对中国蛛缘蝽科13属23种207个样本的线粒体COI基因DNA条形码序列进行扩增,并扩增稻缘蝽属Leptocorisa 3个物种的31条内转录间隔区1(ITS-1)序列作为辅助标记。使用MEGA 11软件计算种间和种内遗传距离(Kimura 2-parameter, K2P);采用邻接法(neighbor-joining, NJ)进行物种聚类分析;利用中介邻接网络算法构建单倍型网络图。【结果】基于线粒体COI DNA条形码序列得出测试的中国蛛缘蝽科所有23个种的种内平均K2P距离在2%以下,种间K2P距离在0.98%~23.98%之间(平均17.50%)。多数物种彼此能够被较好地分开,且支持率较高。其中,中稻缘蝽Leptocorisa chinensis和大稻缘蝽L. oratoria共享部分COI单倍型,造成COI条形码无法区分二者,可通过ITS-1序列在单倍型网络分析中将二者区分。【结论】本研究得出的中国蛛缘蝽科中绝大部分物种的DNA条形码数据分析结果与基于形态特征的分类单元一致。然而,对于其中亲缘关系极近的物种,单靠线粒体数据尤其是COI条形码序列无法进行准确界定,需引入其他DNA序列或其他类型数据进行区分。     

The chloroplast DNA locus psbZ-trnfM as a potential barcode marker in Phoenix L. (Arecaceae)

The genus Phoenix (Arecaceae) comprises 14 species distributed from Cape Verde Islands to SE Asia. It includes the economically important species Phoenix dactylifera. The paucity of differential morphological and anatomical useful characters, and interspecific hybridization, make identification of Phoenix species difficult. In this context, the development of reliable DNA markers for species and hybrid identification would be of great utility. Previous studies identified a 12 bp polymorphic chloroplast minisatellite in the trnG (GCC)-trnfM (CAU) spacer, and showed its potential for species identification in Phoenix. In this work, in order to develop an efficient DNA barcode marker for Phoenix, a longer cpDNA region (700 bp) comprising the mentioned minisatellite, and located between the psbZ and trnfM (CAU) genes, was sequenced. One hundred and thirty-six individuals, representing all Phoenix species except P. andamanensis,were analysed. The minisatellite showed 2-7 repetitions of the 12 bp motif, with 1-3 out of seven haplotypes per species. Phoenix reclinata and P. canariensis had species-specific haplotypes. Additional polymorphisms were found in the flanking regions of the minisatellite, including substitutions, indels and homopolymers. All this information allowed us to identify unambiguously eight out of the 13 species, and overall 80% of the individuals sampled. Phoenix rupicola and P. theophrasti had the same haplotype, and so had P. atlantica, P. dactylifera, and P. sylvestris (the “date palm complex” sensu Pintaud et al. 2013). For these species, additional molecular markers will be required for their unambiguous identification. The psbZ-trnfM (CAU) region therefore could be considered as a good basis for the establishment of a DNA barcoding system in Phoenix, and is potentially useful for the identification of the female parent in Phoenix hybrids.     

Reconstructing long‐term human impacts on plant communities: an ecological approach based on lake sediment DNA

Paleoenvironmental studies are essential to understand biodiversity changes over long timescales and to assess the relative importance of anthropogenic and environmental factors. Sedimentary ancient DNA (sedaDNA) is an emerging tool in the field of paleoecology and has proven to be a complementary approach to the use of pollen and macroremains for investigating past community changes. SedaDNA‐based reconstructions of ancient environments often rely on indicator taxa or expert knowledge, but quantitative ecological analyses might provide more objective information. Here, we analysed sedaDNA to investigate plant community trajectories in the catchment of a high‐elevation lake in the Alps over the last 6400 years. We combined data on past and present plant species assemblages along with sedimentological and geochemical records to assess the relative impact of human activities through pastoralism, and abiotic factors (temperature and soil evolution). Over the last 6400 years, we identified significant variation in plant communities, mostly related to soil evolution and pastoral activities. An abrupt vegetational change corresponding to the establishment of an agropastoral landscape was detected during the Late Holocene, approximately 4500 years ago, with the replacement of mountain forests and tall‐herb communities by heathlands and grazed lands. Our results highlight the importance of anthropogenic activities in mountain areas for the long‐term evolution of local plant assemblages. SedaDNA data, associated with other paleoenvironmental proxies and present plant assemblages, appear to be a relevant tool for reconstruction of plant cover history. Their integration, in conjunction with classical tools, offers interesting perspectives for a better understanding of long‐term ecosystem dynamics under the influence of human‐induced and environmental drivers.     

Application de la technique de PCR en temps réel à l’étude de l’ADN ancien

Real-time Polymerase Chain Reaction (PCR) for the study of ancient DNA. The properties of ancient DNA (aDNA) make difficult the retrieval of DNA sequence. The advantage of Real-Time PCR was exploited, for the first time, in the study of aDNA. We determined the optimal condition to amplify, in one round of PCR, aDNA, which should be directly sequenced. Beside the verification of aDNA authenticity, we compared two cleaning bone methods: scalpel and ethanol. The ethanol specimens showed the best DNA yield. The aDNA was extracted and amplified (mitochondrial hypervariable region I) from five skeletons exhumed from the archaeological site of Notre-Dame-du-Bourg (France), dated from 3rd to 17th century. To cite this article: R. Kefi et al., C. R. Palevol 2 (2003) 125–132.     

德国鸢尾对Cd胁迫的生理生态响应及积累特性

通过盆栽研究了Cd胁迫下德国鸢尾的生长状况、生态效应、生理特性及吸收和富集Cd的能力.结果表明:德国鸢尾对小于5 mg/kg的Cd有较强的耐性,适用于城区土壤修复;Cd浓度大于5 mg/kg时抑制德国鸢尾生长,降低了其生态效应.随着Cd浓度的增大,德国鸢尾根系活力、叶绿素含量和含水量逐渐降低,游离脯氨酸和可溶性糖含量先升高后降低,细胞膜透性逐渐升高.Cd在德国鸢尾体内分布为根系＞地上部分,随着Cd浓度的增大,德国鸢尾根系和地上部分Cd积累浓度逐渐升高、富集系数和转运系数逐渐降低;Cd浓度为20 mg/kg时德国鸢尾对Cd的积累量最大,为2.122 mg/plant.     

Cytochrome c oxidase I primers for corbiculate bees: DNA barcode and mini‐barcode

Bees (Apidae), of which there are more than 19 900 species, are extremely important for ecosystem services and economic purposes, so taxon identity is a major concern. The goal of this study was to optimize the DNA barcode technique based on the Cytochrome c oxidase (COI) mitochondrial gene region. This approach has previously been shown to be useful in resolving taxonomic inconsistencies and for species identification when morphological data are poor. Specifically, we designed and tested new primers and standardized PCR conditions to amplify the barcode region for bees, focusing on the corbiculate Apids. In addition, primers were designed to amplify small COI amplicons and tested with pinned specimens. Short barcode sequences were easily obtained for some Bombus century‐old museum specimens and shown to be useful as mini‐barcodes. The new primers and PCR conditions established in this study proved to be successful for the amplification of the barcode region for all species tested, regardless of the conditions of tissue preservation. We saw no evidence of Wolbachia or numts amplification by these primers, and so we suggest that these new primers are of broad value for corbiculate bee identification through DNA barcode.     

Mitochondrial DNA evidence for the spread of Pacific rats through Oceania

In the 10 years since we published our first full analysis of mitochondrial DNA (mtDNA) variation in Rattus exulans as a means for tracking human migration in Polynesia, we have extended the commensal approach through time and space with the use of ancient DNA (aDNA) and by analysing samples from across the Pacific. Not only can mtDNA phylogenies provide information regarding population origins and paths of migration, they have also provided information regarding degrees of contact and interaction between islands. An important extension of the R. exulans project is the creation and on-going development of a genetic database for the identification of Rattus species based on mtDNA sequences. The phylogenetic analysis of sequences from 18 species and 1 subspecies of Rattus thus far have raised some questions regarding species identification and species distributions in the Pacific.