PCR-RFLP and AP-PCR of rbcL and ITS of rDNA Show That × Taxodiomeria peizhongii (Taxodium x Cryptomeria) Is not an Intergeneric Hybrid

×Taxodiomeria peizhongiiZ. J. Ye, J. J. Zhang et S. H. Pan was regarded as a new intergeneric hybrid between Taxodium mucronatum Tenore (as the female donor) and Cryptomeria fortunei Hooibrenk ex Otto et Dietr (as the male donor). To confirm the authenticity of the intergeneric hybrid, we analyzed the rbcL gene and the internal transcribed spacer (ITS) of 26S-18S ribosomal RNA gene of the three species using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and arbitrarily primed PCR (AP-PCR), and obtained the following results: i) Taxodiomeria peizhongii had the same RFLP maps of the rbcL gene and the ITS as Taxodium mucronatum, but was different from C. fortunei; ii) a 311-bp PCR amplification product was obtained in C. fortunei by AP-PCR of ITS, but was not found in Taxodiomeria peizhongii. Our results have demonstrated that C. fortunei did not provide any genome for Taxodiomeria peizhongii, implying that T. peizhongii is not an intergeneric hybrid between the two species.     

Research Note: Simple molecular discrimination of cultivated Porphyra species (Porphyra yezoensis and Porphyra tenera) and related wild species (Bangiales,Rhodophyta)

To discriminate between cultivated Porphyra species (Porphyra yezoensis and Porphyra tenera) and closely related wild Porphyra species, we developed a polymerase chain reaction‐restriction fragment length polymorphism (PCR‐RFLP) analysis of the rbcL gene using five restriction enzymes. Although our previous PCR‐RFLP analyses of internal transcribed spacer (ITS) rDNA and plastid RuBisCO spacer regions could not always discriminate wild P. yezoensis, wild P. tenera, and closely related wild species, the PCR‐RFLP profiles of the rbcL gene were useful in discriminating samples collected from natural habitats. Therefore, PCR‐RFLP analysis of the rbcL gene will help in the simple identification of a large number of samples, not only for the establishment of reliable cultures as breeding material, but also for the taxonomic investigations of species that are closely related to cultivated Porphyra.     

Genetic polymorphism in Caulerpa taxifolia (Ulvophyceae) chloroplast DNA revealed by a PCR‐based assay of the invasive Mediterranean strain

Abstract An invasive, cold‐tolerant strain of the tropical green alga Caulerpa taxifolia was introduced recently in the Mediterranean Sea and along the Californian coast. We screened 50 aquarium and open‐sea C. taxifolia specimens for the presence/absence of an intron located in the rbcL gene of chloroplast DNA. We also reanalysed a total of 229 sequences of the Internal Transcribed Spacer (ITS) of ribosomal DNA, combining previously published sequences from different studies with 68 new sequences to complement rbcL data. The introduced Mediterranean strain was found to be characterized by the absence of the rbcL intron and by the occurrence of a particular monomorphic ITS type. A PCR assay based on rbcL gene was developed to detect new introductions of the invasive strain of C. taxifolia. This rapid and inexpensive test could be useful to assist environment managers in the preservation of coastal marine ecosystems.     

GENETIC VARIATION AMONG STRAINS OF PSEUDOPFIESTERIA SHUMWAYAE AND PFIESTERIA PISCICIDA (DINOPHYCEAE)1

The putatively toxic dinoflagellates Pseudopfiesteria shumwayae (Glasgow et J. M. Burkh.) Litaker, Steid., P. L. Mason, Shields et P. A. Tester and Pfiesteria piscicida Steid. et J. M. Burkh. have been implicated in massive fish kills and of having negative impacts on human health along the mid‐Atlantic seaboard of the USA. Considerable debate still remains as to the mechanisms responsible for fish mortality (toxicity vs. micropredation) caused by these dinoflagellates. Genetic differences among these cultures have not been adequately investigated and may account for or correlate with phenotypic variability among strains within each species. Genetic variation among strains of Ps. shumwayae and P. piscicida was examined by PCR–RFLP analysis using cultures obtained from the Provasoli‐Guillard National Center for Culture of Marine Phytoplankton (CCMP), as well as those from our own and other colleagues’ collection efforts. Examination of restriction digest banding profiles for 22 strains of Ps. shumwayae revealed the presence of 10 polymorphic restriction endonuclease sites within the first and second internal transcribed spacers (ITS1 and ITS2) and the 5.8S gene of the rDNA complex, and the cytochrome oxidase subunit I (COI) gene. Three compound genotypes were represented within the 22 Ps. shumwayae strains. Conversely, PCR–RFLP examination of 14 strains of P. piscicida at the same ITS1, 5.8S, and ITS2 regions revealed only one variable restriction endonuclease site, located in the ITS1 region. In addition, a dinoflagellate culture listed as P. piscicida (CCMP 1928) and analyzed as part of this study was identified as closely related to Luciella masanensis P. L. Mason, H. J. Jeong, Litaker, Reece et Steid.     

Identification and Molecular Characterization of ‘Candidatus Phytoplasma mali’ Isolates in North‐western Italy

Apple proliferation (AP) is an important disease and is prevalent in several European countries. The causal agent of AP is ‘Candidatus Phytoplasma mali’ (‘Ca. Phytoplasma mali’). In this work, isolates of ‘Ca. Phytoplasma mali’ were detected and characterized through polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analyses of 16S rRNA gene and non‐ribosomal DNA fragment. The presence of three AP subtypes (AT‐1, AT‐2 and AP‐15) was identified in 31 symptomatic apple trees and two samples each constituted by a pool of five insects, collected in north‐western Italy, where AT‐1 is a dominant subtype. Subsequent nucleotide sequence analysis of the PCR‐amplified 1.8 kb (P1/P7) fragment, containing the 16S rDNA, the 16S–23S intergenic ribosomal region and the 5′‐end of the 23S rDNA, revealed the presence of at least two phytoplasmal genetic lineages within the AT‐1 subtype, designed AT‐1a and AT‐1b. Moreover, in silico single nucleotide polymorphism (SNP) analysis based on 16S rDNA sequence can differentiate AT‐1 subtype from AT‐2 and AP‐15 subtypes. Our data showed a high degree of genetic diversity among ‘Ca. Phytoplasma mali’ population in north‐western Italy and underlined the possible use of the 16S rDNA analysis for the identification and the geographical origin assignation of isolates of AP phytoplasma. Molecular markers on 16S rDNA, here identified, could be useful for studying the epidemiology of AP disease.     

Confirmation of cultivated Porphyra tenera (Bangiales, Rhodophyta) by polymerase chain reaction restriction fragment length polymorphism analyses of the plastid and nuclear DNA

Polymerase chain reaction restriction fragment length polymorphism (PCR‐RFLP) analysis of the plastid ribulose‐1,5‐bisphosphate carboxylase (RuBisCo) spacer region was developed for a more reliable and rapid species identification of cultivated Porphyra in combination with PCR‐RFLP analysis of the nuclear internal transcribed spacer (ITS) region. From the PCR‐RFLP analyses of the plastid and nuclear DNA, we examined seven strains of conchocelis that were used for cultivation as Porphyra tenera Kjellman but without strict species identification. The PCR‐RFLP analyses suggested that two strains, C‐32 and 90‐02, were cultivated P. tenera and that the other five strains, C‐24, C‐28, C‐29, C‐30 and M‐1, were Porphyra yezoensis f. narawaensis Miura. To identify species more accurately and to reveal additional genetic variation, the two strains C‐32 and 90‐02 were further studied by sequencing their RuBisCo spacer and ITS‐1 regions. Although RuBisCo spacer sequences of the two strains were identical to each other, each of their ITS‐1 sequences showed a single substitution. The sequence data again confirmed that the two strains (C‐32 and 90‐02) were cultivated P. tenera, and suggested that the two strains showed some genetic variation. We concluded that PCR‐RFLP analysis of the plastid and nuclear DNA is a powerful tool for reliable and rapid species identification of many strains of cultivated Porphyra in Japan and for the collection of genetically variable breeding material of Porphyra.     

Multiple gene analyses reveal extensive genetic diversity among ‘Candidatus Phytoplasma mali’ populations

This study focused on evaluating the genetic diversity among ‘Candidatus Phytoplasma mali’ (‘Ca. P. mali’) populations in orchards of north‐western Italy, where apple proliferation (AP) disease is widespread and induces severe economic losses. ‘Ca. P. mali’ was detected through restriction fragment length polymorphism (RFLP) analysis of PCR‐amplified 16S rDNA in 101 of 114 samples examined. Collective RFLP patterns, obtained by restriction analyses of four amplified genomic segments (16S/23S rDNA, PR‐1, PR‐2 and PR‐3 non‐ribosomal region, ribosomal protein genes rplV‐rpsC and secY gene), revealed the presence of 12 distinct genetic lineages among 60 selected representative ‘Ca. P. mali’ isolates, underscoring an unexpected high degree of genetic heterogeneity among AP phytoplasma populations in north‐western Italy. Prevalence of distinct genetic lineages in diverse geographic regions opens new interesting avenues for studying the epidemiology of AP disease. Furthermore, lineage‐specific molecular markers identified in this work could be useful for investigating the biological life cycle of ‘Ca. P. mali’.     

Comparative palynology and wood anatomy of <Emphasis Type="Italic">Taxodium distichum</Emphasis> (L.) Rich. and <Emphasis Type="Italic">Taxodium mucronatum</Emphasis> Ten.

A comparative study of Taxodium distichum (L.) Rich. and Taxodium mucronatum Ten. was carried out on the basis of pollen morphology and wood anatomy by light and scanning electron microscopy. We describe a detailed analysis of the anatomical characteristics of the wood, including the tracheids, ray parenchyma, axial parenchyma and number of cross-field pits. Palynological characters were also studied to reveal the shape, size and ultrastructure of the pollen grains. These studies give taxonomic support for the recognition of T. distichum and T. mucronatum as two different species.     

Survey of the efficacy of a short fragment of the rbcL gene as a supplemental DNA barcode for diatoms

DNA barcoding is a tool that uses a short, standard segment of DNA to identify organisms. In diatoms, a consensus on an appropriate DNA barcode has not been reached, but several markers show promise. These include the 5.8S gene plus a fragment of the internal transcribed spacer 2 (ITS‐2) of nuclear‐encoded ribosomal RNA, a 420‐bp segment of the 18S rRNA gene, and a 748‐bp fragment at the 3′‐end of the ribulose bisophosphate carboxylase large subunit (rbcL) gene. Here, we tested a 540‐bp fragment 417‐bp downstream of the start codon of the rbcL gene for its efficacy in distinguishing diatom species in a wide range of taxa. Overall, 381 sequences representing 66 genera and 245 species from the classes Mediophyceae and Bacillariophyceae were examined. Intra/interspecific thresholds were set at p = 0.01 differences per site (diff./site) for Mediophyceae and p = 0.02 diff./site for Bacillariophyceae and correctly segregated 96% and 93% of morphological congeners, respectively. When testing reproductively isolated or biological species, which are only available from Bacillariophyceae, 80% of species were discriminated. Therefore, we concluded that, alone, the rbcL region tested herein as potential a DNA barcode was not a sufficient discriminator of all diatoms. We suggest that this fragment could be used in a dual‐locus barcode with the more variable 5.8S+ITS‐2 to discriminate species without sufficient interspecific divergences in the tested rbcL region and to provide insight into species identity from a separately evolved genome.     

RESTRICTION FRAGMENT LENGTH POLYMORPHISM OF RIBOSOMAL DNA INTERNAL TRANSCRIBED SPACER AND 5.8S REGIONS IN JAPANESE ALEXANDRIUM SPECIES (DINOPHYCEAE)1

The 5.8S ribosomal RNA (rDNA) gene and flanking internal transcribed spacers (ITS1 and ITS2)from 9 isolates of Alexandrium catenella (Whedon and Kofoid) Taylor, 11 isolates of A. tamarense (Lebour) Taylor, and single isolates of A. affine (Inoue et Fukuyo) Balech, A. insuetum Balech, and A. pseudogonyaulax (Biecheler) Horiguchi ex Yuki et Fukuyo comb. nov. from various locations in Japan were amplified using the polymerase chain reaction (PCR) and subjected to restriction fragment-length polymorphism (RFLP) analysis. PCR products from all strains were approximately 610 bp, inclusive of a limited region of the 18S and 28S rRNA coding regions. RFLP analysis using four restriction enzymes revealed six distinct classes of rDNA (“ITS types”). Restriction patterns of A. catenella were uniform at the intra-specific level and clearly distinguishable from those of A. tamarense. The patterns associated with A. tamarense (“tamarense group”) were also uniform except for one strain, WKS-1. Some restriction fragments from WKS-1 were in common with those of A. catenella or A. tamarense, whereas some were distinct from all Alexandrium species tested. Alexandrium affine, A. insuetum, and A. pseudogonyaulax carry unique ITS types. The ITSs of the “tamarense group” exhibit sequence heterogeneity. In contrast, the ITSs of all other isolates (including WKS-1) appear homogeneous. RFLP analysis of the 5.8S rDNA and flanking ITSs regions from Alexandrium species reveals useful taxonomic and genetic markers at the species and/or population levels.     

A new PCR-RFLP-based method for an easier systematic affiliation of European water frogs

We describe a non‐invasive, PCR‐RFLP‐based method that allows reliable determination of the European water frog species Pelophylax lessonae and Pelophylax ridibundus and the hybrid form Pelophylax esculentus. Maximum‐likelihood analysis of ITS2 sequences revealed two robust monophyletic clades corresponding to water frogs of the P. lessonae and P. ridibundus groups. Three restriction enzymes (KpnI, HaeII, and SmaI) were used to digest three conserved ITS2 domains. Taxonomic identification was unambiguous; the three restriction enzymes gave the same results. A French reference sample was identified using allozyme electrophoresis. Our PCR‐RFLP method confirmed circa 83% of identification of the allozyme method. We conclude that the difference between identifications was caused by introgression.     

Comparative Analyses and Phylogenetic Relationships between <i>Cryptomeria fortunei</i> and Related Species Based on Complete Chloroplast Genomes

Cryptomeria fortunei (Chinese cedar) is a highly adaptable woody species and one of the main forest plantation trees in subtropical high-altitude areas in China. However, there are few studies on its chloroplast (cp) genome. In this study, the complete cp genome of C. fortunei was sequenced and evaluated via comparative analyses with those of related species (formerly the Taxodiaceae) in Cupressaceae. The C. fortunei cp genome was 131,580 bp in length, and the GC content of the whole genome was 35.38%. It lost one relevant large inverted repeat and contained 114 unique genes, including 82 protein-coding genes, 28 tRNAs and 4 rRNAs. The relative synonymous codon usage (RSCU) of codons ending with A/U was more than twice that of codons ending with G/C. Thirty long repeat structures (LRSs) and 213 simple sequence repeat (SSR) loci were detected in the C. fortunei cp genome. Comparative analyses of 10 cp genomes revealed that substantial rearrangements occurred in the gene organization. Additionally, 6 cp hotspot regions (trnS-GGA, ycf1, trnP-GGG, trnC-GCA, psbZ and accD) were identified, and 4 genes (petL, psbM, rpl22 and psaM) had likely underwent positive selection. Phylogenetic analysis showed that Cupressaceae, Taxaceae and Cephalotaxaceae clustered to form a clade and that C. fortunei was most closely related to C. japonica (Japanese cedar), C. japonica cv. Wogon Hort and Taxodium distichum (baldcypress). These results provide references for future studies of population genetics, phylogenetic status and molecular markers among Cupressaceae species and for the cultivation of improved varieties.     

Identification of insect cell lines and cell‐line cross‐contaminations by nuclear ribosomal ITS sequences

This report shows how the internal transcribed spacer (ITS) region of nuclear ribosomal DNA (rDNA) can be used to determine the species identity of insect cell lines and to distinguish between cell lines derived from closely related insect species. A PCR‐RFLP method with the endonucleases HincII and PstI produces restriction fragment profiles that could distinguish between insect cell lines at the species level. Another PCR‐based method used three species‐specific primer sets, Ly‐ITS1/Ly‐ITS2, ITS1‐1/Ld‐ITS1 and Sf9‐F2/ITS4, to identify the cell lines from Lymantria xylina, L. dispar and Spodoptera frugiperda, respectively. This method also detected cell‐line cross‐contaminations (CLCC) with contamination levels as low as 1% (10 cells in a population of 1000 cells) even when the contaminating cells were from a closely related species. Compared with conventional methods used for cell‐line identification and CLCC detection, the methods presented here are fast and sensitive and could easily be applied to other cell culture laboratories.     

GENETIC DIVERSITY AND INTROGRESSION IN TWO CULTIVATED SPECIES (PORPHYRA YEZOENSIS AND PORPHYRA TENERA) AND CLOSELY RELATED WILD SPECIES OF PORPHYRA (BANGIALES,RHODOPHYTA)1

We investigated the genetic variations of the samples that were tentatively identified as two cultivated Porphyra species (Porphyra yezoensis Ueda and Porphyra tenera Kjellm.) from various natural populations in Japan using molecular analyses of plastid and nuclear DNA. From PCR‐RFLP analyses using nuclear internal transcribed spacer (ITS) rDNA and plastid RUBISCO spacer regions and phylogenetic analyses using plastid rbcL and nuclear ITS‐1 rDNA sequences, our samples from natural populations of P. yezoensis and P. tenera showed remarkably higher genetic variations than found in strains that are currently used for cultivation. In addition, it is inferred that our samples contain four wild Porphyra species, and that three of the four species, containing Porphyra kinositae, are closely related to cultivated Porphyra species. Furthermore, our PCR‐RFLP and molecular phylogenetic analyses using both the nuclear and plastid DNA demonstrated the occurrence of plastid introgression from P. yezoensis to P. tenera and suggested the possibility of plastid introgression from cultivated P. yezoensis to wild P. yezoensis. These results imply the importance of collecting and establishing more strains of cultivated Porphyra species and related wild species from natural populations as genetic resources for further improvement of cultivated Porphyra strains.     

A COMBINED 18S rDNA AND rbcL PHYLOGENETIC ANALYSIS OF CHLOROMONAS AND CHLAMYDOMONAS (CHLOROPHYCEAE,VOLVOCALES) EMPHASIZING SNOW AND OTHER COLD‐TEMPERATURE HABITATS1

An extensive phylogenetic analysis of the biflagellate genera, Chlamydomonas Ehrenberg and Chloromonas Gobi emend. Wille, was undertaken using 18S rDNA and rbcL gene sequence analysis. Emphasis was placed on 21 cold‐tolerant taxa of which 10 are from snow. These taxa occurred in four distinct clades each in the 18S rDNA and rbcL phylogenies, and when taken together suggest at least five distinct origins in cold habitats. Most of these taxa occur in a single clade (A), and all snow species occurred in this clade. In the rbcL and combined rbcL–18S rDNA analyses, the snow taxa fell into three groups. Two groups occurred in subclade 1: Chlamydomonas augustae Skuja CU, Chlamydomonas augustae UTEX, and Chlamydomonas sp.‐A and Chloromonas clathrata Korshikov, Chloromonas rosae Ettl CU, and Chloromonas rosae v. psychrophila var. nov. The third snow group, subclade 2, included three species with unique cell divisions, Chloromonas brevispina (Fritsch) Hoham, Roemer et Mullet, Chloromonas pichinchae (Lagerheim) Wille, and Chloromonas sp.‐D, and the basal Chloromonas nivalis (Chodat) Hoham et Mullet with normal cell divisions. This suggests that the snow habitat has been colonized at least twice and possibly three times in the history of these biflagellates. In the 18S rDNA tree, one cold‐tolerant Chloromonas species fell outside clade A: Chloromonas subdivisa (Pascher et Jahoda) Gerloff et Ettl. In the rbcL tree, three cold‐tolerant Chloromonas species fell outside clade A: Chloromonas subdivisa, Chloromonas sp.‐ANT1, and Chloromonas sp.‐ANT3. These results support previous findings that pyrenoids have been gained and lost several times within this complex.     

Characterization of a Chromobacterium haemolyticum population from a natural tropical lake

Aim: To study genetic diversity of Chromobacterium haemolyticum isolates recovered from a natural tropical lake. Methods and Results: A set of 31 isolates were recovered from a bacterial freshwater community by conventional plating methods and subjected to genetic and phenotypic characterization. The 16S ribosomal RNA (rRNA) gene phylogeny revealed that the isolates were related most closely with C. haemolyticum. In addition to the molecular data, our isolates exhibited strong β‐haemolytic activity, were nonviolacein producers and utilized i‐inositol, d ‐mannitol and d ‐sorbitol in contrast with the other known chromobacteria. Evaluation of the genetic diversity in the 16S rRNA gene, tRNA intergenic spacers (tDNA) and 16S‐23S internal transcribed spacers (ITS) unveiled different levels of genetic heterogeneity in the population, which were also observed with repetitive extragenic palindromic (rep)‐PCR genomic fingerprinting using the BOX‐AR1 primer. tDNA‐ and ITS‐PCR analyses were partially congruent with the 16S rRNA gene phylogeny. The isolates exhibited high resistance to β‐lactamic antibiotics. Conclusion: The population genetic heterogeneity was revealed by 16S rRNA gene sequence, ITS and BOX‐PCR analysis. Significance and Impact of the Study: This study provides for the first time an insight into the genetic diversity of phylogenetically close isolates to C. haemolyticum species.     

Diversity of root-associated fungal endophytes in Rhododendron fortunei in subtropical forests of China

To investigate the diversity of root endophytes in Rhododendron fortunei, fungal strains were isolated from the hair roots of plants from four habitats in subtropical forests of China. In total, 220 slow-growing fungal isolates were isolated from the hair roots of R. fortunei. The isolates were initially grouped into 17 types based on the results of internal transcribed spacer-restriction fragment length polymorphism (ITS-RFLP) analysis. ITS sequences were obtained for representative isolates from each RFLP type and compared phylogenetically with known sequences of ericoid mycorrhizal endophytes and selected ascomycetes or basidiomycetes. Based on phylogenetic analysis of the ITS sequences in GenBank, 15 RFLP types were confirmed as ascomycetes, and two as basidiomycetes; nine of these were shown to be ericoid mycorrhizal endophytes in experimental cultures. The only common endophytes of R. fortunei were identified as Oidiodendron maius at four sites, although the isolation frequency (3–65%) differed sharply according to habitat. Phialocephala fortinii strains were isolated most abundantly from two habitats which related to the more acidic soil and pine mixed forests. A number of less common mycorrhizal RFLP types were isolated from R. fortunei at three, two, or one of the sites. Most of these appeared to have strong affinities for some unidentified root endophytes from Ericaceae hosts in Australian forests. We concluded that the endophyte population isolated from R. fortunei is composed mainly of ascomycete, as well as a few basidiomycete strains. In addition, one basidiomycete strain was confirmed as a putative ericoid mycorrhizal fungus.     

PSEUDOCODIUM MUCRONATUM,A NEW SPECIES FROM NEW CALEDONIA,AND AN ANALYSIS OF THE EVOLUTION OF CLIMATIC PREFERENCES IN THE GENUS (BRYOPSIDALES,CHLOROPHYTA)1

A new species, Pseudocodium mucronatum, is described from the Chesterfield platform off the west coast of New Caledonia. The species differs from its congeners in having mucronate utricules. A phylogenetic analysis of rbcL and tufA sequences showed that P. mucronatum is most closely associated with P. natalense De Clerck, Coppejans et Verbruggen and P. devriesii Weber Bosse, with which it shares compressed axes, depressed apices, and plastids in the utricles and the medullar siphons. We studied the evolution of climatic and ecological preferences in the genus using an interdisciplinary approach consisting of relaxed molecular clock analysis, extraction of macroecological data from satellite imagery in a geographic information system (GIS) framework, and ancestral character state estimation. It was shown that the genus originated in tropical waters during the Early Mesozoic. Whereas the P. floridanum‐okinawense lineage remained tropical, the lineage including P. natalense, P. devriesii, and P. mucronatum gradually invaded more temperate waters during Cenozoic times. Except for P. devriesii, which occurs in shallow and intertidal habitats, all Pseudocodium species grow in deep‐water habitats, and this ecological preference appears to be ancestral.     

Identification and characterisation of sugarcane intergeneric hybrids,Saccharum officinarum x Erianthus arundinaceus,with molecular markers and DNA in situ hybridisation

Molecular markers were used to characterise sugarcane intergeneric hybrids between S. officinarum and E. arundinaceus. Very simple diagnostic tools for hybrid identification among the progeny were derived from isozyme electrophoresis and a sequence-tagged PCR. Two enzyme systems (GOT and MDH B) and PCR amplification revealing spacer-size variation in the 5s-rDNA cluster were found most convenient. Specific characterisation of the two genomic components was possible using RFLP and in situ hybridisation. The strong molecular differentiation between S. officinarum and E. arundinaceus allows the identification of numerous Erianthus-specific RFLP bands in the hybrids. Genomic DNA in situ hybridisation allows for the differentiation of the chromosomes contributed by S. officinarum and E. arundinaceus in chromosome preparations of the hybrids. In situ hybridisation with the 18s-5.8s-25s rDNA probe highlights the basic chromosome numbers in the two parental species. The potential of these techniques to monitor the Erianthus genome during the introgression process is discussed.     

High mitochondrial differentiation levels between wild and domestic Bactrian camels: a basis for rapid detection of maternal hybridization

Hybridization between wild species and their domestic congeners often threatens the gene pool of the wild species. The last wild Bactrian camel (Camelus ferus) populations in Mongolia and China are examples of populations facing such a hybridization threat. To address this key issue in the conservation of wild camels, we analysed wild, hybrid and domestic Bactrian camels (Camelus bactrianus) originating from Mongolia, China and Austria. Through screening of an 804‐base‐pair mitochondrial fragment, we identified eight mitochondrial haplotypes and found high sequence divergence (1.9%) between C. ferus and C. bactrianus. On the basis of a mitochondrial DNA sequence fixed difference, we developed a diagnostic PCR restriction fragment length polymorphism (PCR‐RFLP) assay to differentiate between wild and domestic camel samples. We applied the assay to 81 individuals and confirmed the origin of all samples including five hybrids with known maternal ancestry. The PCR‐RFLP system was effective for both traditional (blood, skin) and non‐invasive samples (faeces, hair), as well as for museum specimens. Our results demonstrate high levels of mitochondrial differentiation between wild and domestic Bactrian camels and that maternal hybridization can be detected by a rapid and reliable PCR‐RFLP system.