Mike J. Wilkinson

A new system of comparing PCR primers applied to ISSR fingerprinting of potato cultivars

Abstractu2002Commercial scale fingerprinting of potato cultivars is made difficult by the need for speed, reliability and the ability to distinguish between large numbers of genotypes. There are also problems in extrapolating the results of small experimental studies to predict the performance of techniques or primers for larger applications. The potential of ISSR-PCR for fingerprinting purposes was evaluated using four primers on 34 potato cultivars. The complex band profiles generated were reproducible between repeat PCRs, DNA extractions, electrophoreses and gel scorings. Two primers were each able to distinguish all cultivars. The combined use of any two of the four primers also allowed complete diagnosis. It is concluded that ISSR-PCR provides a quick, reliable and highly informative system for DNA fingerprinting that is amenable for routine applications. Two possible correlates of the ability of primers to distinguish between genotypes were then examined. Marker Index failed to correlate significantly with genotype diagnosis, but a strong and seemingly linear relationship was observed between Resolving Power of a primer and its ability to distinguish genotypes (r2=0.98). Resolving Power of one or a pair of primers was found to provide a moderately accurate estimate of the number of genotypes identified. Possible implications for future studies on DNA fingerprinting are discussed.

Land plants and DNA barcodes: short-term and long-term goals

Land plants have had the reputation of being problematic for DNA barcoding for two general reasons: (i) the standard DNA regions used in algae, animals and fungi have exceedingly low levels of variability and (ii) the typically used land plant plastid phylogenetic markers (e.g. rbcL, trnL-F, etc.) appear to have too little variation. However, no one has assessed how well current phylogenetic resources might work in the context of identification (versus phylogeny reconstruction). In this paper, we make such an assessment, particularly with two of the markers commonly sequenced in land plant phylogenetic studies, plastid rbcL and internal transcribed spacers of the large subunits of nuclear ribosomal DNA (ITS), and find that both of these DNA regions perform well even though the data currently available in GenBank/EBI were not produced to be used as barcodes and BLAST searches are not an ideal tool for this purpose. These results bode well for the use of even more variable regions of plastid DNA (such as, for example, psbA-trnH) as barcodes, once they have been widely sequenced. In the short term, efforts to bring land plant barcoding up to the standards being used now in other organisms should make swift progress. There are two categories of DNA barcode users, scientists in fields other than taxonomy and taxonomists. For the former, the use of mitochondrial and plastid DNA, the two most easily assessed genomes, is at least in the short term a useful tool that permits them to get on with their studies, which depend on knowing roughly which species or species groups they are dealing with, but these same DNA regions have important drawbacks for use in taxonomic studies (i.e. studies designed to elucidate species limits). For these purposes, DNA markers from uniparentally (usually maternally) inherited genomes can only provide half of the story required to improve taxonomic standards being used in DNA barcoding. In the long term, we will need to develop more sophisticated barcoding tools, which would be multiple, low-copy nuclear markers with sufficient genetic variability and PCR-reliability; these would permit the detection of hybrids and permit researchers to identify the ‘genetic gaps’ that are useful in assessing species limits.

Developing an appropriate strategy to assess genetic variability in plant germplasm collections

Abstractu2002The value of molecular biology for monitoring the genetic status of germplasm collections is subject to practical limitations. The large number and variability of accessions held usually dictates the approach that can be employed. A quick, simple but reliable molecular protocol must be combined with an appropriate strategy for handling large sample sizes. In this study, ISSR-PCR was used to reveal genetic variability within and between accessions held in a collection of lupin germplasm. Pooling of DNA from individuals within accessions was found to be the most appropriate strategy for assessing large quantities of plant material. Band profiles generated from pools containing five individuals were fully representative of all constituent individuals used in the mix. Pools comprising 10 or 20 individuals, however, sometimes failed to contain minor bands that had been present only in the profile of one individual. Variation was observed between pools containing five different genotypes from the same accession. Routine large-scale screens are required to assess the genetic diversity and homogeneity of the lupin germplasm collection held in Reading. It is concluded that 2–3 pools of five genotypes may be sufficient to represent the genetic variability within and between accessions in the lupin and similar collections.

Low probability of chloroplast movement from oilseed rape (Brassicanapus) into wild Brassica rapa

Pollen-mediated movement of transgenes from transplastomic oilseed rape (Brassica napus) into wild relatives will be avoided if chloroplasts are maternally transmitted. We assess the probability of chloroplast exchange between conventional oilseed rape and wild Brassica rapa to model the future behavior of transplastomic cultivars. Primers specific to cpDNA were used to demonstrate maternal inheritance of chloroplasts in 47 natural hybrids between cultivated B. napus and wild B. rapa. We conclude that there will be no or negligible pollen-mediated chloroplast dispersal from oilseed rape. Transgene introgression could still occur in mixed populations, however, if B. napus acted as the recurrent female parent. Rate of transfer would then depend on the abundance of mixed populations, their persistence as mixtures, and hybridization frequency within stands. A low incidence of sympatry (0.6–0.7%) between wild B. rapa and cultivated B. napus along the river Thames, UK, in 1997 and 1998, suggests mixed stands will form only rarely. Eighteen feral populations of B. napus also showed a strong tendency toward rapid decline in plant number, seed return, and ultimately, extinction within 3 years. Conversely, hybrid production is significant in mixed stands, and the absence of control practices means that oilseed rape will have slightly greater persistence. We infer that some introgression from transplastomic B. napus into B. rapa is inevitable in mixed populations even though such populations will occur infrequently and will tend to lose B. napus plants relatively quickly. Chloroplast exchange will be extremely rare and scattered.

A genetic linkage map of microsatellite, gene-specific and morphological markers in diploid Fragaria

Diploid Fragaria provide a potential model for genomic studies in the Rosaceae. To develop a genetic linkage map of diploid Fragaria, we scored 78 markers (68 microsatellites, one sequence-characterised amplified region, six gene-specific markers and three morphological traits) in an interspecific F2 population of 94 plants generated from a cross of F.vesca f. semperflorensxa0×xa0F. nubicola. Co-segregation analysis arranged 76 markers into seven discrete linkage groups covering 448xa0cM, with linkage group sizes ranging from 100.3xa0cM to 22.9xa0cM. Marker coverage was generally good; however some clustering of markers was observed on six of the seven linkage groups. Segregation distortion was observed at a high proportion of loci (54%), which could reflect the interspecific nature of the progeny and, in some cases, the self-incompatibility of F. nubicola. Such distortion may also account for some of the marker clustering observed in the map. One of the morphological markers, pale-green leaf (pg) has not previously been mapped in Fragaria and was located to the mid-point of linkage groupxa0VI. The transferable nature of the markers used in this study means that the map will be ideal for use as a framework for additional marker incorporation aimed at enhancing and resolving map coverage of the diploid Fragaria genome. The map also provides a sound basis for linkage map transfer to the cultivated octoploid strawberry.

Risk assessment of GM plants: avoiding gridlock?

Cultivation of genetically modified crops is presently based largely on four crops containing few transgenes and grown in four countries. This will soon change and pose new challenges for risk assessment. A more structured approach that is as generic as possible is advocated to study consequences of gene flow. Hazards should be precisely defined and prioritized, with emphasis on quantifying elements of exposure. This requires coordinated effort between large, multidisciplinary research teams.

Extensive pyrosequencing reveals frequent intra-genomic variations of internal transcribed spacer regions of nuclear ribosomal DNA.

Background Internal transcribed spacer of nuclear ribosomal DNA (nrDNA) is already one of the most popular phylogenetic and DNA barcoding markers. However, the existence of its multiple copies has complicated such usage and a detailed characterization of intra-genomic variations is critical to address such concerns. Methodology/Principal Findings In this study, we used sequence-tagged pyrosequencing and genome-wide analyses to characterize intra-genomic variations of internal transcribed spacer 2 (ITS2) regions from 178 plant species. We discovered that mutation of ITS2 is frequent, with a mean of 35 variants per species. And on average, three of the most abundant variants make up 91% of all ITS2 copies. Moreover, we found different congeneric species share identical variants in 13 genera. Interestingly, different species across different genera also share identical variants. In particular, one minor variant of ITS2 in Eleutherococcus giraldii was found identical to the ITS2 major variant of Panax ginseng, both from Araliaceae family. In addition, DNA barcoding gap analysis showed that the intra-genomic distances were markedly smaller than those of the intra-specific or inter-specific variants. When each of 5543 variants were examined for its species discrimination efficiency, a 97% success rate was obtained at the species level. Conclusions Identification of identical ITS2 variants across intra-generic or inter-generic species revealed complex species evolutionary history, possibly, horizontal gene transfer and ancestral hybridization. Although intra-genomic multiple variants are frequently found within each genome, the usage of the major variants alone is sufficient for phylogeny construction and species determination in most cases. Furthermore, the inclusion of minor variants further improves the resolution of species identification.

The development of ISSR-derived SCAR markers around the SEASONAL FLOWERING LOCUS ( SFL ) in Fragaria vesca

Fragaria vesca is a short-lived perennial with a seasonal-flowering habit. Seasonality of flowering is widespread in the Rosaceae and is also found in the majority of temperate polycarpic perennials. Genetic analysis has shown that seasonal flowering is controlled by a single gene in F. vesca, the SEASONAL FLOWERING LOCUS (SFL). Here, we report progress towards the marker-assisted selection and positional cloning of SFL, in which three ISSR markers linked to SFL were converted to locus-specific sequence-characterized amplified region (SCAR1–SCAR3) markers to allow large-scale screening of mapping progenies. We believe this is the first study describing the development of SCAR markers from ISSR profiles. The work also provides useful insight into the nature of polymorphisms generated by the ISSR marker system. Our results indicate that the ISSR polymorphisms originally detected were probably caused by point mutations in the positions targeted by primer anchors (causing differential PCR failure), by indels within the amplicon (leading to variation in amplicon size) and by internal sequence differences (leading to variation in DNA folding and so in band mobility). The cause of the original ISSR polymorphism was important in the selection of appropriate strategies for SCAR-marker development. The SCAR markers produced were mapped using a F. vesca f. vesca × F. vesca f. semperflorens testcross population. Marker SCAR2 was inseparable from the SFL, whereas SCAR1 mapped 3.0xa0cM to the north of the gene and SCAR3 1.7xa0cM to its south.

Transgene risk is low

The prospect of a commercial release in Europe of genetically modified oilseed rape (Brassica napus L.) has intensified concern over the chances of transgene movement into wild relatives. We have found that potential transgene recruitment is likely to be slow and uncertain.

Polyploid speciation inHedera (Araliaceae): Phylogenetic and biogeographic insights based on chromosome counts and ITS sequences

Variation in chromosome number and internal transcribed sequences (ITS) of nrDNA is used to infer phylogenetic relationships of a wide range ofHedera species. Polyploidy was found to be frequent inHedera, with diploid, tetraploid, hexaploid and octoploid populations being detected. Nucleotide additivity occurs in the ITS sequences of one tetraploid (H. hibernica) and two hexaploid species (H. maderensis, H. pastuchovii), suggesting that all three species originated by allopolyploidisation. ITS sequence polymorphism and nucleotide characters may indicate the presence of an ancient genome persistent only in some allopolyploid species. Phylogenetic analyses of ITS sequence data reveal two lineages ofHedera: one containing all sequences belonging to extant diploids plus the tetraploidH. algeriensis, and a second that includes this ancient ITS type and others exclusive to several polyploid species. The origin of the polyploids is evaluated on the basis of morphology, chromosome counts, ITS sequence polymorphism, and phylogenetic analyses. Reconstruction of reticulate evolution inHedera agrees with two allopolyploid areas on both sides of the Mediterranean basin. Morphological, molecular and cytological evidence also suggests an active dispersal ofHedera populations that may account for three independent introductions in Macaronesia.