A universal molecular method for identifying underground plant parts to species

As part of a large project to determine rooting depth and resource uptake on the Edwards Plateau of central Texas, we developed a DNA-based technique that allows the below-ground parts of all plants to be identified to the level of genus and usually to species. Identification is achieved by comparing DNA sequences of the internal transcribed spacer (ITS) region of the 18S-26S nuclear ribosomal DNA repeat, derived from below-ground plant material, with a reference ITS region database for plants at a site. The method works throughout plants because the plant ITS region can be PCR amplified using a set of universal primers. Congeneric species can usually be identified because the ITS region evolves relatively rapidly. In our study, all roots were easily identified to the level of genus; most congeneric species were identified solely by ITS sequence differences but some required a combination of ITS sequence data and above-ground surveys of species at a site. In addition to showing the feasibility and efficacy of our technique, we compare it with another DNA-based technique used to identify below-ground plant parts. Finally, we also describe a DNA extraction and purification technique that reliably provides high-quality DNA of sufficient quantity from roots so that PCR can be readily accomplished. Our technique should allow the below-ground parts of plants in any system to be identified and thereby open new possibilities for the study of below-ground plant communities.     

A phylogeny of Packera (Senecioneae; asteraceae) based on internal transcribed spacer region sequence data and a broad sampling of outgroups

A phylogeny of Packera is presented based on sequence data from the internal transcribed spacer region of nuclear ribosomal DNA of 26 species (28 populations) of Packera and 23 outgroup taxa, including representatives of all three subtribes of the Senecioneae. The results support a Mexican origin for Packera, with its closest relatives found among Old World taxa in the subtribe Senecioninae, such as Senecio jacobaea and Pericallis. Packera species from the west coast of the United States, previously included in the section Bolanderi of Greenman, are part of a basal assemblage including species of Greenman's Mexican section Sanguisorboidei. The rest of Packera separates into two sister groups, one containing species from the Arizona-New Mexico region and the other containing more geographically diverse taxa. Among the outgroups, New World Senecio species are monophyletic and two Tussilaginoid assemblages are strongly supported; the Tephroseroid group (Tephroseris and Sinosenecio) plus Petasites combine with the Luina complex to form a clade of north temperate taxa, and the four Mexican genera (Psacalium, Robinsonecio, Barkleyanthus, and Pittocaulon) form a monophyletic group.     

Sequencing of the internal transcribed spacer region ITS1 as a molecular tool detecting variation in the Stylosanthes guianensis species complex

 The internal transcribed spacer (ITS) regions 1 and 2 of the ribosomal DNA from Stylosanthes guianensis CIAT 1283 and cv ‘Schofield’ were amplified by polymerase chain reaction using conserved ITS primers from the 18S, 5.8S and 26S ribosomal genes flanking those regions. The entire region of 683 bp long was cloned, and seven clones were sequenced. Comparison of the ITS spacer regions with published DNA sequences of other plant species revealed limited homology only; this was in contrast to their comparison with the 5.8S rDNA sequences. The ITS1 region of 45 S. guianensis accessions was amplified by PCR and sequenced on both strands using the conserved primers ITS2-ITS5. These sequences, ranging from 201 to 204 bp, were aligned to each other to assess intra-specific polymorphism. Within the S. guianensis (Aubl.) Sw. species complex, 11 DNA sequence types could be distinguished based on an insertion/deletion (indel) event and 15 single base-pair substitutions. In 1 of the S. guianensis types, two kinds of ITS1 sequence were observed in each individual, reminiscent of an incomplete homogenization of the repeat structure in this type. Polymorphisms in the sequence of the ITS1 region were used to define molecular markers for S. guianensis on the basis of PCR-restriction fragment length polymorphism and selective PCR. Received: 24 June 1997 / Accepted: 31 October 1997     

Molecular criteria for determining new hybrid species--an application to the Sonneratia hybrids

The possible hybrid origin of new species can usually be corroborated by molecular means. Here, we suggest that the segregation patterns of the molecular markers be further analyzed. A true hybrid species should show the patterns under continuous breeding among its members, at least beyond the F2 generation. We applied the guidelines to the putative hybrid species of Sonneratia, a widespread mangrove genus, and concluded that all the observed hybrids in this genus are simple F1's. Thus, S. x gulngai and S. x hainanensis are not true hybrid species. The segregation patterns of molecular markers should be heeded in interpreting the existence of hybrid species.     

<Emphasis Type="Italic">Pterostylis nutans</Emphasis> (Orchidaceae) has a specific association with two <Emphasis Type="Italic">Ceratobasidium</Emphasis> root-associated fungi across its range in eastern Australia

In this study, we have identified the root-associated fungi of a common species of terrestrial orchid across its range in eastern Australia. We have amplified and cloned fungal ITS DNA extracted from roots of 15 Pterostylis nutans R. Br. plants from six separate geographic localities. Sequencing and GenBank comparison demonstrated two species of Ceratobasidium fungi as the main fungal partners of the orchid. Uncommon fungal associates included homobasidiomycete species such as a Gymnomyces sp. and a Tricholoma sp., Leptodontidium orchidicola, and an unidentified soil fungus. These results demonstrate that specificity for fungal partners occurs in P. nutans and reinforces the idea that conservation measures for endangered Australian orchids must include ex situ perpetuation of fungal symbionts as well as plant material.     

Chloroplast DNA-relationship in palaeoaustralLopidium concinnum (Hypopterygiaceae, Musci). An example of stenoevolution in mosses Studies in austral temperate rain forest bryophytes 2

Evolutionary relationship between disjunct populations of the palaeoaustral moss taxonLopidium concinnum (Hypopterygiaceae) from New Zealand and southern South America were studied using non-coding chloroplast DNA sequences. No or only slight changes could be observed within the sequences oftrnT_UGU —trnL_UAA 5exon intergenic spacer,trnL_UAA intron andtrnL_UAA 3exon —trnF_GAA intergenic spacer. This indicates nearly no genetic divergence between extant New Zealand and Chilean populations, i.e. no significant differing pathways of evolution within the 80–60 million years of disrupted areas with interrupted gene flow. Molecular data support the idea of an old Gondwanan relict species of stenoevolutionary character. Ecological data on short-range dispersal strengthen this assessment.     

Escaping introns in COI through cDNA barcoding of mushrooms: Pleurotus as a test case

DNA barcoding involves the use of one or more short, standardized DNA fragments for the rapid identification of species. A 648‐bp segment near the 5′ terminus of the mitochondrial cytochrome c oxidase subunit I (COI) gene has been adopted as the universal DNA barcode for members of the animal kingdom, but its utility in mushrooms is complicated by the frequent occurrence of large introns. As a consequence, ITS has been adopted as the standard DNA barcode marker for mushrooms despite several shortcomings. This study employed newly designed primers coupled with cDNA analysis to examine COI sequence diversity in six species of Pleurotus and compared these results with those for ITS. The ability of the COI gene to discriminate six species of Pleurotus, the commonly cultivated oyster mushroom, was examined by analysis of cDNA. The amplification success, sequence variation within and among species, and the ability to design effective primers was tested. We compared ITS sequences to their COI cDNA counterparts for all isolates. ITS discriminated between all six species, but some sequence results were uninterpretable, because of length variation among ITS copies. By comparison, a complete COI sequences were recovered from all but three individuals of Pleurotus giganteus where only the 5′ region was obtained. The COI sequences permitted the resolution of all species when partial data was excluded for P. giganteus. Our results suggest that COI can be a useful barcode marker for mushrooms when cDNA analysis is adopted, permitting identifications in cases where ITS cannot be recovered or where it offers higher resolution when fresh tissue is. The suitability of this approach remains to be confirmed for other mushrooms.     

Sequence variation in the cytochrome oxidase I, internal transcribed spacer 1, and Ts14 diagnostic antigen sequences of Taenia solium isolates from South and Central America, India, and Asia.

We examined the genetic variability in the pig–human tapeworm, Taenia solium, by sequencing the genes for cytochrome oxidase I, internal transcribed spacer 1, and a diagnostic antigen, Ts14, from individual cysts isolated from Peru, Colombia, Mexico, India, China, and the Philippines. For these genes, the rate of nucleotide variation was minimal. Isolates from these countries can be distinguished based on one to eight nucleotide differences in the 396 nucleotide cytochrome oxidase I (COI) sequence. However, all of the 15 isolates from within Peru had identical COI sequences. The Ts14 sequences from India and China were identical and differed from the Peru sequence by three nucleotides in 333. These data indicate that there is minimal genetic variability within the species T. solium. Minimal variability was also seen in the ITS1 sequence, but this variation was observed within the individual. Twenty-two cloned sequences from six isolates sorted into 13 unique sequences. The variability observed within the sequences from individual cysts was as great as the variability between the isolates.     

克隆植物构型及其对资源异质性的响应

密集型和游击型是两个极端的克隆植物构型,在它们之间还应存在中间过渡类型的连续统。作者给出了描述该连续统的一个指数ｖ＝ｌｎ ［Ｅ（ Ｒ２）］１２Ｅ（ｌ） ／ｌｎｎ （ １２ ≤ｖ≤１） 。并讨论了克隆植物的间隔物长度对生境斑块质量的响应     

核糖体DNA的内转录间隔区序列标记在真菌分类鉴定中的应用

传统的真菌分类主要根据真菌菌株的形态特征、生长特性与生理生化指标进行,而分子生物学技术的发展提升了真菌分类鉴定研究的手段。真菌核糖体DNA内转录间隔区(ITS)在进化上比编码区快,种内的不同菌株之间高度保守,但在种间变化极大,故可为真菌学的研究提供丰富的遗传信息。简要综述了ITS序列分析技术在真菌分类鉴定中的应用现状、相关问题及前景。