The Pattern of Genetic Variability in Apomictic Clones of Taraxacum officinale Indicates the Alternation of Asexual and Sexual Histories of Apomicts

Dandelions (genus Taraxacum) comprise a group of sexual diploids and apomictic polyploids with a complicated reticular evolution. Apomixis (clonal reproduction through seeds) in this genus is considered to be obligate, and therefore represent a good model for studying the role of asexual reproduction in microevolutionary processes of apomictic genera. In our study, a total of 187 apomictic individuals composing a set of nine microspecies (sampled across wide geographic area in Europe) were genotyped for six microsatellite loci and for 162 amplified fragment length polymorphism (AFLP) markers. Our results indicated that significant genetic similarity existed within accessions with low numbers of genotypes. Genotypic variability was high among accessions but low within accessions. Clustering methods discriminated individuals into nine groups corresponding to their phenotypes. Furthermore, two groups of apomictic genotypes were observed, which suggests that they had different asexual histories. A matrix compatibility test suggests that most of the variability within accession groups was mutational in origin. However, the presence of recombination was also detected. The accumulation of mutations in asexual clones leads to the establishment of a network of clone mates. However, this study suggests that the clones primarily originated from the hybridisation between sexual and apomicts.     

Plastic reproductive strategies in a clonal marine invertebrate

Plastic reproductive allocation may allow individuals to maximize their fitness when conditions vary. Mate availability is one condition that may determine the fitness of an individual's allocation strategy. Using a variety of methods, I detected evidence of plastic allocation to asexual (clonal) reproduction in response to mate availability in the brittle star Ophiactis savignyi. There were more mature individuals in populations in which both sexes were present, and clones from these populations had fewer clone-mates than clones from single-sex populations. Animals placed with mates in a field experiment divided less frequently than animals without a mate. These findings demonstrate that animals reduce their allocation to asexual reproduction when mates are present and when a loss of fecundity associated with cloning would decrease offspring production. This plasticity is probably adaptive because it maximizes sexual-reproductive potential when fertilization is more likely, but maximizes survival of the clone when mates are absent and gametes are unlikely to be converted to offspring.     

ITS Polymorphisms Shed Light on Hybrid Evolution in Apomictic Plants: A Case Study on the Ranunculus auricomus Complex

The reconstruction of reticulate evolutionary histories in plants is still a major methodological challenge. Sequences of the ITS nrDNA are a popular marker to analyze hybrid relationships, but variation of this multicopy spacer region is affected by concerted evolution, high intraindividual polymorphism, and shifts in mode of reproduction. The relevance of changes in secondary structure is still under dispute. We aim to shed light on the extent of polymorphism within and between sexual species and their putative natural as well as synthetic hybrid derivatives in the Ranunculus auricomus complex to test morphology-based hypotheses of hybrid origin and parentage of taxa. We employed direct sequencing of ITS nrDNA from 68 individuals representing three sexuals, their synthetic hybrids and one sympatric natural apomict, as well as cloning of ITS copies in four representative individuals, RNA secondary structure analysis, and landmark geometric morphometric analysis on leaves. Phylogenetic network analyses indicate additivity of parental ITS variants in both synthetic and natural hybrids. The triploid synthetic hybrids are genetically much closer to their maternal progenitors, probably due to ploidy dosage effects, although exhibiting a paternal-like leaf morphology. The natural hybrids are genetically and morphologically closer to the putative paternal progenitor species. Secondary structures of ITS1-5.8S-ITS2 were rather conserved in all taxa. The observed similarities in ITS polymorphisms suggest that the natural apomict R. variabilis is an ancient hybrid of the diploid sexual species R. notabilis and the sexual species R. cassubicifolius. The additivity pattern shared by R. variabilis and the synthetic hybrids supports an evolutionary and biogeographical scenario that R. variabilis originated from ancient hybridization. Concerted evolution of ITS copies in R. variabilis is incomplete, probably due to a shift to asexual reproduction. Under the condition of comprehensive inter- and intraspecific sampling, ITS polymorphisms are powerful for elucidating reticulate evolutionary histories.     

Mitochondrial and ribosomal internal transcribed spacer (ITS2) diversity of the African malaria vector Anopheles funestus

The pattern of sequence variation in the mitochondrial DNA cytochrome b gene (cyt-b) and ribosomal DNA internal transcribed spacer 2 (ITS2) was examined in Anopheles funestus from Senegal and Burkina Faso in West Africa and Kenya in East Africa. From both West African countries, samples included individuals hypothesized to represent reproductively isolated taxa based upon different karyotypes and behaviours. Analysis of the cyt-b data revealed high haplotypic diversity (86%) and an average pairwise difference per site of 0.42%. Sequence variation was not partitioned by geographical origin or karyotype class. The most common haplotype was sampled across Africa (approximately 6000 km). Analysis of the ITS2 data revealed one of the longest spacers yet found in anophelines (approximately 704 bp). In common with other anopheline ITS2 sequences, this one had microsatellites and frequent runs of individual nucleotides. Also in common with data from other anopheline ITS2 studies, the An. funestus sequences were almost monomorphic, with only two rare polymorphisms detected. The results from both markers are congruent and do not support the hypothesis of reproductively isolated chromosomal taxa within An. funestus. Whether the lack of support by mitochondrial DNA (mtDNA) and ribosomal DNA (rDNA) sequences is a result of the recent origin of the presumptive taxa, or of the absence of barriers to gene flow, remains to be elucidated, using more rapidly evolving markers such as microsatellites.     

Formation of a hybrid triploid agamosperm on a sexual diploid plant: evidence from progeny tests in Taraxacum platycarpum Dahlst.

Agamospermous dandelions of hybrid origin between a native sexual diploid species (Taraxacum platycarpum Dahlst. or T. japonicum Koidz.) and an alien agamospermous triploid [T. officinale Weber and T. laevigatum (Willd.) DC.] are now widely distributed throughout mainland Japan. These hybrid dandelions are known to be genetically variable. We hypothesized that this variability is maintained by repeated ongoing hybridization, based on the fact that triploid dandelions not only produce seeds agamospermously, but also produce some functional pollen grains that are able to sire seeds of sexual dandelions. To test this hypothesis, we examined whether heads of Japanese diploid dandelions produce new hybrid seeds after fertilization by pollen from triploid agamosperms under field conditions. One of the 430 tested plants grown from sexual dandelion seeds had morphological and molecular characteristics, which are consistent with a hybrid origin. The plant formed a hybrid surrounded by many individuals having recurved involucral bracts, in which frequency of T. officinale was very low (3.5 %). Cytological data and bagging experiments demonstrate triploidy and asexual seed production of the hybrid. Taken together, these results supported that the new hybrid is probably derived from a backcross of a hybrid to the native sexual species. Our findings provide evidence for the evolution of a new agamosperm through interspecific hybridization as a contemporary population process.     

No evidence for accumulation of deleterious mutations and fitness degradation in clonal fish hybrids: Abandoning sex without regrets

Despite its inherent costs, sexual reproduction is ubiquitous in nature, and the mechanisms to protect it from a competitive displacement by asexuality remain unclear. Popular mutation‐based explanations, like the Muller's ratchet and the Kondrashov's hatchet, assume that purifying selection may not halt the accumulation of deleterious mutations in the nonrecombining genomes, ultimately leading to their degeneration. However, empirical evidence is scarce and it remains particularly unclear whether mutational degradation proceeds fast enough to ensure the decay of clonal organisms and to prevent them from outcompeting their sexual counterparts. To test this hypothesis, we jointly analysed the exome sequences and the fitness‐related phenotypic traits of the sexually reproducing fish species and their clonal hybrids, whose evolutionary ages ranged from F1 generations to 300 ky. As expected, mutations tended to accumulate in the clonal genomes in a time‐dependent manner. However, contrary to the predictions, we found no trend towards increased nonsynonymity of mutations acquired by clones, nor higher radicality of their amino acid substitutions. Moreover, there was no evidence for fitness degeneration in the old clones compared with that in the younger ones. In summary, although an efficacy of purifying selection may still be reduced in the asexual genomes, our data indicate that its efficiency is not drastically decreased. Even the oldest investigated clone was found to be too young to suffer fitness consequences from a mutation accumulation. This suggests that mechanisms other than mutation accumulation may be needed to explain the competitive advantage of sex in the short term.     

Circumscription and phylogeny of Saxifraga sect. Ciliatae: Evidence from nrDNA ITS sequences

The largest section of the genus Saxifraga (Saxifragaceae), sect. Ciliatae, consists of 175 morphologically diverse species. This section is mainly distributed in the Qinghai-Tibetan Plateau and adjacent regions of southwest China and more than 80% of the total number of species are endemic to this region. It remains unknown whether this section is monophyletic and up to now no study has been conducted on the infra-sectional phylogeny. In this study, ITS sequences of the nuclear ribosomal DNA were firstly determined for 33 species mainly from this section and related sections. We further downloaded the corresponding sequences of the same DNA region for the other 22 species of Saxifraga and Mitella from GenBank. All sequences were together used to construct the phy-logenetic trees. The main implications of the phylogenetic analyses include: (1) sect. Ciliatae, as traditionally defined, constitutes as a monophyletic clade and its sister group is a well supported clade that includes species from 8 sections such as sect. Porphyrion, sect. Saxifraga and sect. Mesogyne; (2) three morphological subsections, i.e., subsect. Gemmiparae, subsect. Hirculoideae and subsect. Rosulares were tentatively recovered despite the relatively low statistic bootstrap support for the last one; however, subsect. Flagellares and subsect. Hemi-sphaericae were not recognized as separate entities, and nested within subsect. Gemmiparae; (3) subsect. Hircu-loideae and subsect. Rosulares clustered together as sister subclades while subsect. Gemmiparae diverged early. In addition, our results suggest that the paired variation of ITS sequences in sect. Ciliatae is relatively low between the sampled species in spite of their diverse morphology. It is suggested that such a scenario may mirror rapid speciation in this section that probably trigged by the uplifts of the Qinghai-Tibetan Plateau and the extensive selection pressure under the alpine environments.     

Linkage studies of polymorphic, repeated DNA sequences in centromeric regions of human chromosomes.

The DNA at human centromeric regions was characterized by using a repetitive sequence, 308, which localizes in situ exclusively to centromeres of all chromosomes. We previously noted that this sequence is enriched on chromosome 6 and has chromosome-specific organization on 6, 3, 7, 14, X, and Y. In addition to this basic organization, sequences homologous to 308 are polymorphic among normal individuals. The variants are transmitted in a Mendelian manner within a family. To determine the chromosome origin of the variants, we studied their linkage to markers of various chromosomes. Linkage analysis of one pedigree segregating two polymorphisms shows that the 2.6-kilobase (kb) BamHI and 2.6-kb TaqI fragments are linked to each other and to the HLA loci on chromosome 6. Data from another family shows that 2.8-kb TaqI, 4.0-kb TaqI, and 1.3-kb BamHI polymorphic fragments are linked and are probably near the Fy locus on chromosome 1. By dot blot analysis, we determined that the relative amount of these sequences in the genome is not measurably different between unrelated individuals. Thus, the polymorphisms represent changes in homologous 308 sequences on specific chromosomes and can be used as chromosome-specific markers. Linkage studies using polymorphisms of repeated sequences will be most useful within a kindred, especially from an inbred population, because polymorphic repeats of the same restriction size may be heterogeneous in origin.     

Genetic diversity and mycelial compatibility groups of the plant-pathogenic fungus Sclerotinia sclerotiorum in Brazil

The genetic variability of 40 Sclerotinia sclerotiorum isolates from various fields widely distributed throughout Brazil and different host crops was analyzed using RAPD markers and mycelial compatibility groupings (MCGs). The isolates were characterized using 16 random primers of the OPERON series, which produced 121 DNA fragments. UPGMA cluster analysis using Jaccard's genetic distance and MCGs allowed separation of the isolates into three clusters, with similarity indices of 68.2, 61.8, and 61.8%, and five MCGs. The haplotypes obtained with RAPD markers provided very characteristic groupings of S. sclerotiorum isolates according to MCG, but did not show any relationship with geographic origin or host type. Furthermore, analysis of molecular variance demonstrated that 99.1% of the observed variation was a result of genetic differences between individuals; the host culture did not have a significant effect. This is the first report of high level variability of S. sclerotiorum in Brazil based on the study of isolates of wide geographical origin, supported by RAPD markers and MCGs. These results endorse the prevalence of sexual reproduction in tropical and subtropical regions in contrast to clonal reproduction in temperate regions.     

Population structure,seasonal genotypic differentiation,and clonal diversity of weedy dandelions in three Boston area populations (Taraxacum sp.)

Weedy dandelions have a worldwide distribution and thrive in urban environments despite a lack of sexual reproduction throughout most of its range. North American dandelions, introduced from Eurasia, are believed to be primarily, if not exclusively, apomictic triploids. In some European populations, apomicts co‐occur with diploid sexual individuals and hybridizations can create genetically unique apomicts, which may subsequently disperse and establish new populations globally. Using six nuclear microsatellite markers and a cpDNA intergenic spacer, we investigate the impact of this unusual natural history on population structure and diversity in three urban Boston area dandelion populations. Our results show high levels of genetic diversity within populations, spatial population structure, and seasonal genotypic differentiation in flowering times. We find evidence that sexual reproduction and recombination, presumably in Europe, and extensive gene flow drive these patterns of diversity and create the appearance of panmixia despite the lack of evidence for local sexual reproduction.     

Nuclear vs. plastid data: complex Pleistocene history of a circumpolar key species

To fully understand the contemporary genetic structure of plants, both nuclear and plastid markers are needed. Three chloroplast DNA (cpDNA) lineages, which probably diverged before the major Pleistocene glaciations, have been identified in the circumpolar/circumboreal Vaccinium uliginosum. Here we investigate its nuclear DNA variation using nuclear ribosomal internal transcribed spacer (ITS) sequences, DNA ploidy level measurements and amplified fragment length polymorphisms (AFLPs). We also extend the cpDNA dataset. Two ITS lineages, corresponding to diploids and tetraploids, respectively, were identified. However, both main sequence types apparently occurred in most individual plants but showed ploidy-biased homogenization and possibly reflect paralogy predating the origin of V. uliginosum. The ploidy levels were largely consistent with the cpDNA lineages, suggesting that the initial cpDNA divergence followed early polyploidizations. Five main AFLP groups were identified, consistent with recent glacial refugia in Beringia, western Siberia, the southern European mountains and areas south/east of the Scandinavian and Laurentide ice sheets. Except from the southern European mountains, there has been extensive expansion from all refugia, resulting in several contact zones. Surprisingly, the presumably older ploidy and cpDNA patterns were partly inconsistent with the main AFLP groups and more consistent with AFLP subgroups. A likely major driver causing the inconsistencies is recent nuclear gene flow via unreduced pollen from diploids to tetraploids. This may prevent cytoplasmic introgression and result in overlayed patterns formed by processes dominating at different time scales. The data also suggest more recent polyploidizations, as well as several chloroplast capture events, further complicating this scenario. This study highlights the importance of combining different marker systems to unravel intraspecific histories.     

Clonal diversity and structure within a population of the pondweed Potamogeton pectinatus foraged by Bewick's swans

Clonal diversity within plant populations is affected by factors that influence genet (clone) survival and seed recruitment, such as resource availability, disturbance, seed dispersal mechanism, propagule predation and the age of the population. Here we studied a population of Potamogeton pectinatus, a pseudo-annual aquatic macrophyte. Within populations reproduction appears to be mainly asexually through subterranean propagules (tubers), while recruitment via seeds is believed to be relatively unimportant. RAPD markers were used to analyse clonal diversity and genetic variation within the population. Ninety-seven genets were identified among 128 samples taken from eight plots. The proportion of distinguishable genets (0.76) and Simpson's diversity index (0.99) exhibited high levels of clonal diversity compared to other clonal plants. According to an analysis of molecular variance (amova) most genetic variation occurred between individuals within plots (93-97%) rather than between plots (8-3%). These results imply that sexual reproduction plays an unexpectedly important role within the population. Nevertheless, autocorrelation statistics revealed a spatial genetic structure resulting from clonal growth. In contrast to genetic variation, clonal diversity was affected by several ecological factors. Water depth and silt content had direct negative effects on clonal diversity. Tuber predation by Bewick's swans had an unexpected indirect negative effect on clonal diversity through reducing the tuber-bank biomass in spring, which on its turn was positively correlated to clonal diversity. The disturbance by swans, therefore, did not enhance seed recruitment and thus clonal diversity; on the contrary, heavily foraged areas are probably more prone to stochastic loss of genets leading to reduced clonal diversity.     

Genetic differentiation and delimitation of Pugionium dolabratum and Pugionium cornutum (Brassicaceae)

Species delimitation has been a major research focus in evolutionary biology. However, the genetic delimitation of recently diverged species varies depending on the markers examined. In this study, we aimed to examine genetic differentiation and delimitations between only two species of Pugionium Gaertn (Brassicaceae)—Pugionium cornutum (L.) Gaertn and Pugionium dolabratum Maxim—that occur in the desert habitats of central Asia and have parapatric distributions. We genotyped 169 individuals from 25 populations, using two chloroplast (cp) DNA fragments (trnV-trnM and trnS-trnfM), seven simple repeated sequence (SSR) loci and the nuclear ribosomal internal transcribed spacer (ITS). Four cp haplotypes were identified, three of which commonly occur in the two species, suggesting incomplete species-specific lineage sorting. Between-species cpDNA differentiation (F _CT) was low, even lower than among populations of the same species. However, we found higher than average SSR F _CT values, and both Bayesian clustering of SSR variables and maximum-likelihood genetic analyses divided all sampled individuals into two groups, agreeing well with morphological separation, although gene flow between species was obvious according to the SSR loci data. However, two types of ITS sequences were highly consistent with the morphological delimitation of the two species in all sampled individuals. These results together suggest that these two species shared numerous ancestral cpDNA polymorphisms and point to the importance of nuclear DNA (ITS or genetic accumulation at multiple loci) in delimiting recently diverged species.     

Molecular diversity of the syndinean genus Euduboscquella based on single-cell PCR analysis

The genus Euduboscquella is one of a few described genera within the syndinean dinoflagellates, an enigmatic lineage with abundant diversity in marine environmental clone libraries based on small subunit (SSU) rRNA. The region composed of the SSU through to the partial large subunit (LSU) rRNA was determined from 40 individual tintinnid ciliate loricae infected with Euduboscquella sampled from eight surface water sites in the Northern Hemisphere, producing seven distinct SSU sequences. The corresponding host SSU rRNA region was also amplified from eight host species. The SSU tree of Euduboscquella and syndinean group I sequences from environmental clones had seven well-supported clades and one poorly supported clade across data sets from 57 to 692 total sequences. The genus Euduboscquella consistently formed a supported monophyletic clade within a single subclade of group I sequences. For most parasites with identical SSU sequences, the more variable internal transcribed spacer (ITS) to LSU rRNA regions were polymorphic at 3 to 10 sites. However, in E. cachoni there was variation between ITS to LSU copies at up to 20 sites within an individual, while in a parasite of Tintinnopsis spp., variation between different individuals ranged up to 19 polymorphic sites. However, applying the compensatory base change model to the ITS2 sequences suggested no compensatory changes within or between individuals with the same SSU sequence, while one to four compensatory changes between individuals with similar but not identical SSU sequences were found. Comparisons between host and parasite phylogenies do not suggest a simple pattern of host or parasite specificity.     

珠芽蓼种群克隆多样性及克隆结构的初步研究

珠芽蓼(Polygonum viviparum)是青藏高原东缘广泛分布的克隆植物,具有有性和无性(根状茎和珠芽)两种生殖方式。该研究采用RAPD技术对分布于不同海拔的珠芽蓼7个自然种群进行了克隆结构和克隆多样性(是单克隆种群还是多克隆种群)以及克隆多样性与海拔因子之间的相关性研究,为了解高山克隆植物对环境的适应性策略及揭示克隆植物的繁殖和分布特点提供科学依据。研究结果表明:1)采用13条RAPD引物对珠芽蓼7个种群共140个样本进行扩增分析,共扩增到117个位点,其中多态性位点84个,多态位点百分率PPL达到71.79%,检测到43个基因型,且全部为局限型基因型;2)与Ellstrand和Roose(1987)总结的克隆植物的克隆多样性平均值相比(PD=0.17,D=0.62),珠芽蓼种群克隆多样性水平稍高,Simpson指数平均为0.639,基因型比率PD平均为0.307;3)克隆结构分析表明,珠芽蓼种群内克隆之间的镶嵌明显,这可能与珠芽蓼过渡型的克隆构型有关。研究中珠芽蓼种群的构型有游击型、密集型以及这两者之间的过渡类型;4)采用SPSS软件对珠芽蓼种群的克隆多样性与海拔高度进行相关性分析,结果显示它们之间并无明显的相关性。     

A new Pyrenean hybrid Cirsium (Asteraceae) as revealed by morphological and molecular analyses

A new nothosubspecies Cirsium  ×  vivantii is described after a molecular and morphological characterization of eight individuals collected in two separate valleys of the French central Pyrenees. Both hypervariable Amplified Fragment Length Polymorphism (AFLP) markers and nuclear rDNA (ITS) and plastid ( trn L-F, TRNT-L) DNA sequences were analysed. The profiles of these hybrid samples were compared to those of 43 individuals belonging to their presumed parental taxa C. carniolicum ssp. rufescens and C. palustre . A total of 133 AFLP bands were scored from three primer-pair combinations. All 130 AFLP bands that amplified in the hybrid samples were present in either C. carniolicum ssp. rufescens , C. palustre or both taxa, supporting the participation of these plant genomes in the resulting hybrids. Several Additive Polymorphic Sites (APS) detected in the ITS sequences of the hybrid samples also confirmed their derived origins from ribotypes of the two parental taxa. The lack of exclusive AFLP markers and the nonconcerted evolution of rDNA polymorphisms towards either of the parental ribotypes indicated their recent origin. Plastid trn T-L sequences were used to identify C. palustre as the maternal parent of six of these hybrid individuals; either parent could have acted as the plastid genome donor for the other two individuals. The morphological study revealed that all hybrid individuals were morphologically intermediate between their parents showing largely lobed and less spiny basal leaves as in C. carniolicum ssp. rufescens and decurrent leaf bases and pinkish corollas as in C. palustre .  © 2007 The Linnean Society of London, Botanical Journal of the Linnean Society , 2007, 154 , 421–434.     

Recombination between Clonal Lineages of the Asexual Fungus Verticillium dahliae Detected by Genotyping by Sequencing

Most asexual species of fungi have either lost sexuality recently, or they experience recombination by cryptic sexual reproduction. Verticillium dahliae is a plant-pathogenic, ascomycete fungus with no known sexual stage, even though related genera have well-described sexual reproduction. V. dahliae reproduces mitotically and its population structure is highly clonal. However, previously described discrepancies in phylogenetic relationships among clonal lineages may be explained more parsimoniously by recombination than mutation; therefore, we looked for evidence of recombination within and between clonal lineages. Genotyping by sequencing was performed on 141 V. dahliae isolates from diverse geographic and host origins, resulting in 26,748 single-nucleotide polymorphisms (SNPs). We found a strongly clonal population structure with the same lineages as described previously by vegetative compatibility groups (VCGs) and molecular markers. We detected 443 recombination events, evenly distributed throughout the genome. Most recombination events detected were between clonal lineages, with relatively few recombinant haplotypes detected within lineages. The only three isolates with mating type MAT1-1 had recombinant SNP haplotypes; all other isolates had mating type MAT1-2. We found homologs of eight meiosis-specific genes in the V. dahliae genome, all with conserved or partially conserved protein domains. The extent of recombination and molecular signs of sex in (mating-type and meiosis-specific genes) suggest that V. dahliae clonal lineages arose by recombination, even though the current population structure is markedly clonal. Moreover, the detection of new lineages may be evidence that sexual reproduction has occurred recently and may potentially occur under some circumstances. We speculate that the current clonal population structure, despite the sexual origin of lineages, has arisen, in part, as a consequence of agriculture and selection for adaptation to agricultural cropping systems.     

Dynamic grapevine clones��an AFLP-marker study of the Vitis vinifera cultivar Riesling comprising 86 clones

Grapevine cultivars are planted in worldwide viticulture and are asexually propagated. Horticultural clones are asexually derived from a single individual, and clonal variation can only occur through mutations. Molecular markers are an important tool for the differentiation and identification of clones and mutations. For breeding purposes and clonal selection, knowledge upon the variability of a given clone is essential. The aim of this study was to assess amplified fragment length polymorphism (AFLP) markers for classifying mutations in 86 Riesling clones of Vitis vinifera and to enhance our understanding on the dynamic of grapevine clones analysed by AFLP fingerprints. AFLP markers detected 135 polymorphic bands of a total amount of 305 bands. AFLP markers detected two different types of mutations: single-event mutations, only detected once in one clone, displaying the variation of the grape genome and specific loci mutations where the mutation could be found frequently in the set of clones and therefore stand for the stability of grapevine genome. A general grouping of clones according to age, sub-clonal lineage or origin could not be determined by the set of AFLP markers employed.     

Fingerprinting trifoliate orange germ plasm accessions with isozymes, RFLPs, and inter-simple sequence repeat markers

 Trifoliate orange [Poncirus trifoliata (L.) Raf.] is frequently used as a parent in citrus rootstock breeding, but the origin and amount of genetic diversity in germ plasm collections are poorly understood. Most accessions are self-compatible, but produce a mixture of sexual and apomictic seedlings. Variation among 48 vegetatively propagated trifoliate orange accessions was assessed at seven isozyme loci, together with the restriction fragment length polymorphisms (RFLPs) detected by 38 probe-enzyme combinations and the inter-simple sequence repeat (ISSR) markers generated by 11 primers. Isozymes and RFLPs detected few polymorphisms among accessions, although genetic analysis has shown that the common phenotype is heterozygous for four isozyme and at least four RFLP loci. ISSR amplification generated multiple banding profiles with an average of 58 fragments/primer/accession. These fragments were repeatable across DNA samples extracted from different trees of the same accession or extracted at different times, and across separate PCR runs. Seventeen unique marker phenotypes were identified. The 48 trifoliate orange accessions were classified into four major groups based on polymorphic ISSR markers. All large-flowered accessions are in group 4, while small-flowered accessions are in group 3. Many ISSR markers segregated in progeny derived by open-pollination (probably mostly selfing) of a common accession, indicating that these ISSR markers are also heterozygous. Accessions having identical genotypes for a large number of heterozygous markers are unlikely to have diverged by recombination. Thus the limited divergence we detected among most accessions most likely originated by mutation. ‘Monoembryonic’ and ‘Simmons’ differed from other accessions only in the loss of specific markers, indicating that they originated as zygotic seedlings of individuals similar to the common genotype. Three accessions recently introduced from China have relatively different fingerprints with 3–14 unique ISSR markers, and probably represent a much more divergent germ plasm that may be a valuable breeding resource. Received: 8 August 1996 / Accepted: 21 March 1997     

Persistent nuclear ribosomal DNA sequence polymorphism in the Amelanchier agamic complex (Rosaceae)

Individual plants of several Amelanchier taxa contain many polymorphic nucleotide sites in the internal transcribed spacers (ITS) of nuclear ribosomal DNA (nrDNA). This polymorphism is unusual because it is not recent in origin and thus has resisted homogenization by concerted evolution. Amelanchier ITS sequence polymorphism is hypothesized to be the result of gene flow between two major North American clades resolved by phylogenetic analysis of ITS sequences. Western North American species plus A. humilis and A. sanguinea of eastern North America form one clade (A), and the remaining eastern North American Amelanchier make up clade B. Five eastern North American taxa are polymorphic at many of the nucleotide sites where clades A and B have diverged and are thought to be of hybrid origin, with A. humilis or A. sanguinea as one parent and various members of clade B as the other parent. Morphological evidence suggests that A. humilis is one of the parents of one of the polymorphic taxa, a microspecies that we refer to informally as A. "erecta." Sequences of 21 cloned copies of the ITS1- 5.8S gene-ITS2 region from one A. "erecta" individual are identical to A. humilis sequence or to the clade B consensus sequence, or they are apparent recombinants of A. humilis and clade B ITS repeats. Amelanchier "erecta" and another polymorphic taxon are suspected to be relatively old because both grow several hundred kilometers beyond the range of one of their parents. ITS sequence polymorphisms have apparently persisted in these two taxa perhaps because of polyploidy and/or agamospermy (asexual seed production), which are prevalent in the genus.