Eighty-two European chestnut populations, from Greece, Italy, France, Spain and the UK were studied using 73 ISSR and 16 isoenzyme loci in a total exceeding 2100 trees and 25000 genotypic designations. A multivariate analysis was used in order to detect locus interactions in space, not directly observable with averaging techniques such as genetic distances, after data vector transformation. Discussion elaborates on the major gene pools of chestnut in Europe and on the differences and similarities observed between different populations, regions, and countries. Within-stand population dynamics were investigated via a gene flow analysis of 15 populations from six sites (two from Spain, two from Italy and two from Greece) using 12 SSR and 26 isoenzyme loci, through paternity analysis and spatial analysis of genetic diversity patterns. Within each stand, 26-43 trees were genotyped and 7-9 selected mother trees provided the half-sib progenies (16-22 seeds per tree) analysed (about 58000 genotypic designations). Cryptic and outcoming gene flow appeared reasonably high for a predominantly outcrossing species (about 64%), ensuring a number of incoming migrants per generation large enough to counterbalance loss of diversity by inbreeding and drift, while selfing was very low (< 41%). Mating probability dropped below 0.01 for distances larger than 2 km, and increased tip to about 10% for distances closer than 300 meters, indicating that in the presence of some clustering of like-genotypes, a certain amount of inbreeding is expected. These findings have important implications in gene conservation, stand restoration, and fruit orchard establishment and management, as well as tree improvement.

Hellenic orchard populations originating from distant geographic locations is reported, while genetic diversity of orchard populations is compared to that of wild ones in corresponding locations. Winter buds were sampled and nine loci (16 alleles) were revealed out of seven enzyme systems investigated. Results indicated that orchards are essentially low-number multiclonal plantations with respect to their grafted part. The percent unique genotypes in both orchards were estimated to be 21% of the total number of individuals, compared to 70-86% found in natural populations. A total of 650 pairwise comparisons has led to the identification of eleven unique clones present in orchard populations. Despite the significant distance among the orchards a common elite clone was identified. A clonal identification key was developed. The average probability that two trees may have the same genotypic multilocus array by chance alone is still high, indicating the need for future DNA fingerprinting. Evidence suggests the existence of a notable reduction of genetic diversity in orchard populations compared to wild ones in Greece. However, Hellenic orchards seem to possess higher amounts of genetic variability compared to western European ones. The implementation of these results in applied breeding research is discussed.

Chestnut (Castanea sativa Mill.) fruits were sampled from two distant geographic regions harboring different population types (old-growth natural, coppice and orchard). Fruits were measured for the quantitative traits: fruit length, length to the widest point, thickness, width, hylum length, hylum width, weight, while the ratios width to length, thickness to length and thickness to width were calculated. Combined analysis revealed significant differences among regions and populations within region for most of the fruit traits. Repeatability varied among fruit traits. High repeatability values were estimated for fruit length, width and weight. Coefficient of genetic variance for fruit traits (morphological and weight) ranged among 10.6% and 38.4% across regions. Correlation among traits was found to be high among fruit weight and width, fruit length and width, fruit weight and thickness, hylum length and weight, as well as hylum length and width. The quantitative genetic variation recorded for fruit traits indicates the potential for breeding and the necessity for conservation of the species' genetic resources.

Data regarding four leaf size parameters (leaf length, leaf width, petiole length, distance from leaf base to the leaf widest point), and four shape parameters (leaf length/leaf width, leaf length/petiole length, leaf length/distance from leaf base to the leaf widest point, distance from leaf base to the leaf widest point/petiole length), were recorded in six chestnut populations (three managed coppice, two old-growth natural and one fruit orchard population) originating from three different geographic localities. Ten leaves per tree and 27 trees per population were sampled. Univariate and multivariate analysis were carried out on a by population basis. Analysis of variance revealed significant population differences, which were extended in most parameters as found via t-tests. Multivariate methods involved both ordination and classification approaches (principal component analysis, multivariate analysis of variance and multiple discriminant analysis). Leaf size parameters emerged as most important variables in the corresponding eigenvectors. Most of the variation(> 85%) was resolved in low multidimensional space; nevertheless separation of populations in the first three components was poor. Redistribution results to original classes using the linear discriminant function presented a high misclassification of return (34%). It is concluded that leaf parameters may be suitable variables in order to detect levels of phenotypic variability among populations, nevertheless do not possess adequate discriminative capabilities to distinguish populations and population types.

The long range distribution of Castanea sativa indicates the probable existence of some adaptive variation among populations from extreme conditions. However their introduction in some European areas as well as the evidence of founder effects and the influence of grafted varieties on wild populations, pose the question about the existence of adaptations to local environments and of enough variability within populations. The variation of several traits related to adaptation was studied in a multi-site provenance-progeny test, for six European chestnut populations originating from contrasting environments of Spain, Greece and Italy. The experiment was established at six sites of contrasting European chestnut climate. At each site every population was represented by 26 open-pollinated progenies. The experimental design was a randomised complete block design with 20 blocks and one-tree plot. Assessment of height growth, flushing and bud set was carried out during the first three years after planting. The populations originating from Greece initiated growth earlier, followed by the populations from South Italy and South Spain, while the populations from Nor h Spain and North Italy initiated growth later. Cessation of the height growth occurred during summer and prior to reduction of day length. The North Italian population, growing in conditions of Central European climate, stopped growth later than the other five populations from Mediterranean conditions. Height growth of the northern populations was higher than growth of the southern ones.

During the past decade the use of molecular markers in plant genetics and breeding has flourished. Molecular markers such as RAPDs, RFLPs, AFLPs, DAFs, SCARs, SSRs and ISSRs have been introduced in tissue culture research and can potentially be used in various facets of pertinent studies. This review focuses on the employment of molecular markers in micropropagated plants for the: assessment of genetic fidelity, uniformity, stability and true-to-typeness among donor plants and tissue culture regenerants; determination of the precise origin of plants derived from microspore, anther culture, or protoplast fusion; investigation of the levels and mechanisms of somaclonal variation; verification of mutant phenotypes; identification of QTLs controlling vegetative propagation and somatic embryogenesis, etc. The pertinent literature is reviewed and the relative merits and shortcomings of the various molecular markers applied are presented with an emphasis on the nature of tissue culture-induced variation, marker choice and strategies for loci to be sampled and genomes to be studied towards an adequate evaluation of related research questions. The application of genomics in the identification of micropropagated plant's is also introduced.