A. Karp

Reproducibility testing of RAPD, AFLP and SSR markers in plants by a network of European laboratories

A number of PCR-based techniques can be used to detect polymorphisms in plants. For their wide-scale usage in germplasm characterisation and breeding it is important that these marker technologies can be exchanged between laboratories, which in turn requires that they can be standardised to yield reproducible results, so that direct collation and comparison of the data are possible. This article describes a network experiment involving several European laboratories, in which the reproducibility of three popular molecular marker techniques was examined: random-amplified fragment length polymorphism (RAPD), amplified fragment length polymorphism (AFLP) and sequence-tagged microsatellites (SSR). For each technique, an optimal system was chosen, which had been standardised and routinely used by one laboratory. This system (genetic screening package) was distributed to different participating laboratories in the network and the results obtained compared with those of the original sender. Different experiences were gained in this exchange experiment with the different techniques. RAPDs proved difficult to reproduce. For AFLPs, a single-band difference was observed in one track, whilst SSR alleles were amplified by all laboratories, but small differences in their sizing were obtained.

Bioenergy from plants and the sustainable yield challenge

Bioenergy from plants, particularly from perennial grasses and trees, could make a substantial contribution to alleviation of global problems in climate change and energy security if high yields can be sustained. Here, yield traits in a range of key bioenergy crops are reviewed, from which several targets for future improvement can be identified. Some are already the focus of genetically modified (GM) and non-GM approaches. However, the efficient growth strategies of perennial bioenergy crops rely on newly assimilated and recycled carbon and remobilized nitrogen in a continually shifting balance between sources and sinks. This balance is affected by biotic (e.g. pest, disease) and abiotic (e.g. drought) stresses. Future research should focus on three main challenges: changing (photo)thermal time sensitivity to lengthen the growing season without risking frost damage or limiting remobilization of nutritional elements following senescence; increasing aboveground biomass without depleting belowground reserves required for next years growth and thus without increasing the requirement for nutrient applications; and increasing aboveground biomass without increasing water use.

Somaclonal variation as a tool for crop improvement

Somaclonal variation is a tool that can be used by plant breeders. The review examines where this tool can be applied most effectively and the factors that limit or improve its chances of success. The main factors that influence the variation generated from tissue culture are (1) the degree of departure from organised growth, (2) the genotype, (3) growth regulators and (4) tissue source. Despite an increasing understanding of how these factors work it is still not possible to predict the outcome of a somaclonal breeding programme. New varieties have been produced by somaclonal variation, but in a large number of cases improved variants have not been selected because (1) the variation was all negative, (2) positive changes were also altered in negative ways, (3) the changes were not novel, or (4) the changes were not stable after selfing or crossing. Somaclonal variation is cheaper than other methods of genetic manipulation. At the present time, it is also more universally applicable and does not require ‘containment’ procedures. It has been most successful in crops with limited genetic systems and/or narrow genetic bases, where it can provide a rapid source of variability for crop improvement.

Chromosome variation in wheat plants regenerated from cultured immature embryos

SummaryA cytological study has been made of plants regenerated from cultured immature embryos of four wheat cultivars (Triticum aestivum, 2n = 6x = 42). In total, 29% of the 192 plants examined were aneuploid with a range in chromosome numbers of 38–45. Evidence of chromosome structural changes was also found. This variation occurred in regenerants of all four cultivars, but there were large differences in the proportions of aneuploids arising from individual cultures which meant that no significant differences could be demonstrated between cultivars. Chromosome abnormalities were present in plants regenerated both from embryogenic cultures and from cultures in which the origin of shoots could not be distinctly defined.

Chromosome variation in protoplast-derived potato plants.

SummaryChromosomes have been studied in protoplast-derived potato plants of the tetraploid cultivars Maris Bard and Fortyfold. A high degree of aneuploidy was found amongst the regenerants of both cultivars but the nature of the chromosome variation differed. The Maris Bard regenerants were characterised by high chromosome numbers, a wide range of aneuploidy (46–92) and a low percentage of plants with the normal chromosome number (2n = 48), whereas a much higher proportion of the Fortyfold regenerants had 48 chromosomes and the variants were within a more limited aneuploid range. In both cultivars chromosome variation was found between calluses, within calluses and even within shoot cultures. The origin of the chromosome variation and the differences found between the two cultivars are discussed.

Genetic modification of potato development using Ri T-DNA

SummaryForty-two potato plants were regenerated from a hairy-root line obtained after infection of a shoot of Solanum tuberosum cv ‘Desiree’ with Agrobacterium rhizogenes strain LBA 9402 (pRil855). Transformed plants were uniform and had a distinct phenotype and development compared with untransformed controls. Their growth was vigorous, especially early in their development, their roots were abundant and showed reduced geotropism, their leaves were slightly crinkled and glossy and they produced longer tubers with more frequent, prominent eyes. Cytological examination showed that ten of the forty-two transformed plants had either 47 or 49 chromosomes instead of the normal 48. In two of these aneuploids structural changes were observed.

Genetic improvement of willow for bioenergy and biofuels.

Willows (Salix spp.) are a very diverse group of catkin-bearing trees and shrubs that are widely distributed across temperate regions of the globe. Some species respond well to being grown in short rotation coppice (SRC) cycles, which are much shorter than conventional forestry. Coppicing reinvigorates growth and the biomass rapidly accumulated can be used as a source of renewable carbon for bioenergy and biofuels. As SRC willows re-distribute nutrients during the perennial cycle they require only minimal nitrogen fertilizer for growth. This results in fuel chains with potentially high greenhouse gas reductions. To exploit their potential for renewable energy, willows need to be kept free of pests and diseases and yields need to be improved without significantly increasing the requirements for fertilizers and water. The biomass composition needs to be optimized for different end-uses. Yields also need to be sustainable on land less productive for food crops to reduce conflicts over land use. Advances in understanding the physiology and growth of willow, and in the identification of genes underlying key traits, are now at the stage where they can start to be used in breeding programs to help achieve these goals.

Variation in oil palm (Elaeis guineensis Jacq.) tissue culture-derived regenerants revealed by AFLPs with methylation-sensitive enzymes.

Abstract Tissue culture-derived plants of oil palm (Elaeis guineensis Jacq.) can develop abnormal flowers in which stamen primordia are converted into carpel-like tissues (mantled fruit). This abnormality can be heritable; individual palms may show variation in mantling and reversion to the normal phenotype over time has been observed. Four sets of ortets (mother plant used as tissue source) and ramets (regenerated plants) were compared using standard amplified fragment length polymorphism (AFLP) analysis and AFLPs using methylation-sensitive enzymes. No polymorphisms were found when standard AFLPs were produced with ten different primer combinations. In contrast, when methylation-sensitive AFLPs were used, polymorphisms were detectable. Polymorphisms appeared as new bands in the ramets, suggesting that a reduction in methylation had occurred during tissue culture. The highest number of polymorphic bands (0.3%) was obtained when HpaII was used as the restriction endonuclease, indicating that the loss of methylation had occurred most frequently at the internal C within the HpaII recognition sequence 5’-CCGG-3’. Conversion of nine of the polymorphic bands into probes for Southern analysis confirmed that these were not due to partial digestion of the AFLP templates and showed that the majority were single-copy sequences. The exceptions were fragments showing homology to 25S ribosomal RNA genes and the chalcone synthase gene family. Examination of the Southern blots suggested that most of the single-copy sequences were partially de-methylated, and one example was found in which de-methylation affected only one allele. No polymorphism was consistently different between normal and abnormal clones in all the sets. This suggests that, whilst this method is an effective way of detecting variation in tissue culture-derived plants, different approaches will be required to identify the causal basis of the mantled fruit abnormality.

A study of genetic diversity in Populus nigra subsp. betulifolia in the Upper Severn area of the UK using AFLP markers

A survey of amplified fragment length polymorphism (AFLP) was conducted to examine genetic diversity in 146 individuals of British, native black poplar (Populus nigra subsp. betulifolia) and three individuals considered by collectors to be non‐betulifolia poplars. Using two primer pairs, a total of 147 bands were detected of which 82 (56%) were polymorphic in at least one individual. Cluster analysis and principal coordinates analysis of the calculated similarity matrix revealed a low level of genetic diversity, although a loose clustering into five groups could be identified, one of which contained the non‐betulifolia individuals. Examination of the spatial distribution of the other four groups (all betulifolia) revealed a general correlation between geographic proximity and genetic similarity. On the basis of the polymorphism observed, it was possible to identify a small number of individual plants which exhibit maximum diversity and might therefore be suitable for inclusion in a replanting programme designed to maintain at least the existing low level of polymorphism observed within British black poplar.

An analysis of genetic diversity in coconut (Cocos nucifera) populations from across the geographic range using sequence-tagged microsatellites (SSRs) and AFLPs.

Abstract Genetic diversity in 31 individuals from 14 coconut populations across the entire geographic range (2–3 individuals per population) was assessed using sequence-tagged microsatellites (or simple sequence repeats, SSRs) and amplified fragment length polymorphism (AFLP). From the 39 SSR primer sets tested, only two gave patterns that could not be scored and used in the data analysis. The remainder included five SSRs that gave double-locus profiles in which one locus could still be scored separately. The 37 SSRs revealed between 2 and 16 alleles per locus and a total of 339 alleles in the 14 populations. Gene diversity (D = 1-Σpi2) ranged from 0.47 to 0.90. Two of the four Dwarf populations were homozygous at all 37 loci, which is consistent with their autogamous (self-fertilising) reproduction. One Dwarf population was heterozygous at one locus but the other (Niu Leka Dwarf), which is known to be cross-pollinating, showed high levels of heterozygosity. Generally, diversity was higher in populations from the South Pacific and South East Asia. Three SSR loci (CNZ46, CN2A5, CN11E6) gave distinct genotypes for all but two populations. The East African populations had higher heterozygosities than those from West Africa, and the populations from Tonga and Fiji generally had distinct alleles from those of the South Pacific. AFLP analysis with 12 primer combinations gave a total of 1106 bands, of which 303 were polymorphic (27%). Similarity matrices were constructed from the two data sets using the proportion of shared alleles for SSRs and a Jaccard coefficient for AFLPs. In each case cluster and principal co-ordinates analyses were performed, with the resultant dendrograms and plots revealing similar relationships among the populations for both approaches. There was generally a good separation of populations, and phenetic relationships were in agreement with those previously shown by RFLPs. The use of SSRs and AFLPs in genetic-diversity analysis for the establishment of germplasm collections is discussed.