Plant genetic resources (PGR) include cultivars, landraces, wild species closely related to cultivated varieties, breeder's elite lines and mutants. The loss of genetic diversity caused by the practice of agriculture and the availability of genetic information has resulted in a great effort dedicated to the collection of PGR. Prior to the advent of molecular profiling, accessions in germplasm collections were examined based on morphology. The development of molecular techniques now allows a more accurate analysis of large collections. Next-generation sequencing (NGS) with de novo assembly and resequencing has already provided a substantial amount of information, which warrants the coordination of existing databases and their integration into genebanks. Thus, the integration and coordination of genomic data into genebanks is very important and requires an international effort. From the determination of phenotypic traits to the application of NGS to whole genomes, every aspect of genomics will have a great impact not only on PGR conservation, but also on plant breeding programmes.

We now understand that many different types of DNA structural polymorphisms contribute to functional diversity of plant genomes, including single nucleotide polymorphisms, insertions of retrotransposons and DNA transposons, including Helitrons carrying pseudogenes, and other types of insertion–deletion polymorphisms, many of which may contribute to the phenotype by affecting gene expression through a variety of mechanisms including those involving non-coding RNAs. These polymorphisms can now be probed with tools such as array comparative genomic hybridization and, most comprehensively, genomic sequencing. Rapid developments in next generation sequencing will soon make genomic sequencing of germplasm collections a reality. This will help eliminate an important difficulty in the estimation of genetic relationships between accessions caused by ascertainment bias. Also, it has now become obvious that epigenetic differences, such as cytosine methylation, also contribute to the heritable phenotype, although detailed understanding of their transgenerational stability in crop species is lacking. The degree of linkage disequilibrium of epialleles with DNA sequence polymorphisms has important implications to the analysis of genetic diversity. Epigenetic marks in complete linkage disequilibrium (LD) with DNA polymorphisms do not add additional diversity information. However, epialleles in partial or low LD with DNA sequence alleles constitute another layer of genetic information that should not be neglected in germplasm analysis, especially if they exhibit transgenerational stability.

The recombinant inbred line mapping population Opata85 × Synthetic W7984 was used to map Jeli long terminal repeat retrotransposon insertion sites in the hexaploid wheat genome. Sequence-specific amplified polymorphism technique was applied to reveal Jeli insertions. Jeli was found to provide multiple genetic markers for common wheat. Our marker system revealed A-genome Jeli insertions, and therefore can be used for targeted analysis of the A genome.

WRKY transcription factors are one of the largest families of transcriptional regulators and form an integral part of signalling webs which modulate many plant processes, such as abiotic stress tolerance. In the present paper, an innovative method has been applied to identify novel WRKY-1 alleles involved in the responses to salt and drought stresses in Triticum durum. This technique involves scanning for sequencing variations in cDNA-derived PCR amplicons, using high-resolution melting (HRM) followed by direct Sanger sequencing of only those amplicons which were predicted to carry nucleotide changes. HRM represents a novel advance in detection of single-nucleotide polymorphisms (SNPs) by measuring temperature-induced strand separation of short PCR amplicons. The use of this approach is still limited in the field of plant biology. Here, HRM analysis has been applied to the discovery and genotyping of durum wheat SNPs. Specific primers have been designed, starting at multi-alignment of WRKY-1-conserved portions. The PCR amplicons, containing single SNPs, produce distinctive HRM profiles, and by sequencing the PCR products identified, SNPs have been characterized and validated. The results showed that all the revealed SNPs are located on salt-tolerant varieties, confirming their value in breeding activities.

In this study, the targetting-induced local lesions in genomes approach was used to identify mutants for genes related to starch metabolism in barley. Starch is the major reserve of plants and serves as primary carbohydrate component in human and livestock diets and has also numerous industrial applications. Mutants for biosynthetic or regulatory genes of starch metabolism often produce starch granules with abnormal morphological and molecular features that could be of interest for technological applications. We report the identification of 29 mutations in five starch-related barley genes (Bmy1, GBSSI, LDA1, SSI and SSII) through the molecular screening of TILLMore, a sodium azide-mutagenized population. Almost all the mutations detected were CG–TA transitions and several (c. 60%) implied a change in amino-acid sequence and therefore possible phenotypic effects. Four mutants showed non-sense or splice-junction alterations, which could drastically affect the protein function.

We have established mutant collections of the model species Medicago truncatula according to current protocols. In particular, we used a transposon (Tnt1) tagging method and an ethyl methanesulfonate (EMS) mutagenesis approach (TILLING). The collections were subjected to both forward and reverse genetics screenings, and several mutants were isolated that affect plant traits (e.g. shoot, root developments, flower morphology, etc.) and also biosynthetic pathways of secondary compounds (saponins and tannins). Genes responsible for some of the mutations were cloned and further characterized.

Alternative oxidase (AOX) has been proposed as a promising functional marker candidate for multiple plant stress behaviour. The present paper describes natural polymorphism in AOX2b of Daucus carota L. (DcAOX2b). Exon-primed intron crossing-PCR (EPIC-PCR) revealed length variation (intron length polymorphisms, ILPs) in intron 1. Six fragment patterns were identified in 40 genotypes. However, no more than two fragments were found per genotype, suggesting the presence of two alleles. The ILPs were able to discriminate between single plant genotypes in cv. Rotin and to distinguish individual wild carrot plants. The repetitive pattern of intron 1 length variation allows the grouping of genotypes for functional analysis in future studies. Sequence analysis in intron 1 of polymorphic but also of obviously identical PCR-fragments revealed underlying high levels of sequence polymorphisms between alleles and genotypes. Variation was due to repetitive insertion/deletion (InDel) events and single-nucleotide polymorphisms (SNPs). The results suggest that high AOX2b gene diversity in D. carota may be a source of functional markers for agronomic traits related to environmental stress responses.

Triticale possesses favourable agronomic attributes originating from both its wheat and rye progenitors, including high grain and biomass yields. Triticale, primarily used as animal feed in North America, is an excellent candidate for production of industrial bio-products. Little is known about the coordination of gene expression of rye and wheat genomes in this intergeneric hybrid, but significant DNA losses from the parental genomes have been reported. To clarify the regulation of gene expression in triticale, we carried out 454 sequencing of cDNAs obtained from root, leaf, stem and floral tissues in different lines of triticale and rye exhibiting different phenotypes and assembled reads into contigs. Related to the data assembly were the absence of reference genomes and the paucity of rye sequences in GenBank or other public databases. Consequently, we have sequenced cDNA libraries from roots, seedlings, leaves, floral tissues and immature seeds to facilitate the identification of triticale sequences originating from rye. To further characterize the wheat-derived cDNAs, we also developed a database close to 25,000 non-redundant full-length wheat coding sequence genes, based on existing databases and contigs that were verified against protein sequences from the grass genomes of Brachypodium distachyon, rice, sorghum and maize.

In Arabidopsis, a variety of mapping populations have been used for the detection of quantitative trait loci (QTLs) responsible for natural variation. In this study, we present an overview of the advantages and disadvantages of the different types of populations used. To do this, we compare the results of both experimental and natural populations for the commonly analysed trait flowering time. It is expected that genome wide association (GWA) mapping will be an increasingly important tool for QTL mapping because of the high allelic richness and mapping resolution in natural populations. In Arabidopsis, GWA mapping becomes ever more facilitated by the increasing availability of re-sequenced genomes of many accessions. However, specifically designed mapping populations such as recombinant inbred lines and near isogenic lines will remain important. The high QTL detection power of such experimental populations can identify spurious GWA associations, and their unique genomic structure is superior for investigating the role of low-frequency alleles. Future QTL studies will therefore benefit from a combined approach of GWA and classical linkage analysis.

Apple cultivars are an integral part of the Latvian State Program for Preservation and Sustainable Use of Genetic Resources for Food and Agriculture. Latvian apple (n = 109) cultivars of local origin, nominated as National Plant Genetic Resources, were analyzed with eight simple sequence repeat (SSR) markers recommended by the European Cooperative Programme for Plant Genetic Resources (ECPGR) Malus/Pyrus working group and a marker for scab resistance gene (Vf). All SSR loci exhibited a high level of polymorphism – 12 to 39 alleles, 18.75 in average, with high observed heterozygosity (Ho) ranging from 0.64 to 0.89 and a mean of 0.78. The gene diversity (polymorphism information content value) varied from 0.79 to 0.90, with an average of 0.86. All cultivars could be distinguished with the tested set of SSR loci, with a high frequency of rare (38%) or unique (26%) alleles. For the Vf gene, all three possible genotypes (VfVf, Vfvf and vfvf) were detected for 1, 6 and 102 varieties, respectively. The analyzed germplasm showed high genetic diversity, particularly landraces, while the modern cultivars clustered mainly into two groups. Cluster analysis did not reveal a clear pattern with well-defined groups, but confirmed some relationships based on known or putative pedigrees, as well as suggesting the possible parentage of some cultivars.

Sustainable utilization of plant genetic resources for food and agriculture has been increasingly discussed at both national and international forums. Besides exploitation, conservation of plant genetic resources has become an integral part of these discussions. Conservation aims at maintaining the diversity of living organisms, their habitat and the interrelationship between organisms and their environment. For achieving such goals, appropriate conservation strategies have to be adopted. Determining the genetic makeup of a particular plant species is of critical importance when planning a suitable conservation strategy. In this study, we sequenced the chloroplast trnK intron, matK and rbcL gene aimed at understanding the rarity of Nymphaea tetragona, a critically rare and endangered plant of India found at only one location. We extended our investigation to other Nymphaea species such as N. nouchali, N. pubescens and N. rubra that are commonly available throughout India. Interestingly, matK gene of N. tetragona revealed high number of non-synonymous substitutions. Molecular evolutionary analysis indicated that three of these sites may be under mild selective pressures. Such adaptive changes at the DNA and protein sequence level of matK gene may have been associated with the colonization of N. tetragona, suggesting that it could have migrated from China.

Here, we present a brief overview of the main studies conducted on the common bean (Phaseolus vulgaris L.) in Europe and other countries outside its centres of origin. We focus on the proportions of the Andean and Mesoamerican gene pools, and on the inter-gene pool hybridization events. In Europe, for chloroplast microsatellites, 67% of European germplasm is of Andean origin. Within Europe, interesting trends have been seen; indeed, the majority of the Andean type is found in the three macro-areas of the Iberian Peninsula, Italy and central-northern Europe, while, in eastern and south-eastern Europe, the proportion of the Mesoamerican type increased. On a local scale, the contribution of the Mesoamerican type is always low. On other continents, various situations are seen using different markers: in China and Brazil, the Mesoamerican gene pool prevails, while in an African sample, overall, both gene pools are equally represented, with differences in individual countries. The frequency of European bean genotypes deriving from at least one hybridization event was 44% with an uneven distribution. Interestingly, hybrids tend to have intermediate seed size in comparison with ‘pure’ Andean or Mesoamerican types. On other continents, very few hybrids are found, probably because of the different marker systems used.

The N.I. Vavilov Institute of Plant Industry (VIR) holds and maintains collections of various crop plants among the largest and oldest worldwide. Among them, small berry trees have gained attention because of their potential for human health. Small berries, usually containing various valuable compounds such as vitamins or antioxidants in significant quantities, could be used for easily improving the human diet. Subsets of VIR collections of raspberry (Rubus idaeus L.) and blue honeysuckle (Lonicera caerulea L.) were investigated for genetic diversity. Ninety-five raspberry accessions were genotyped with eight nuclear simple-sequence repeat (microsatellite) markers. Results indicated a fair level of genetic diversity, but also a structure of three main groups in the collection. Blue honeysuckle accessions were genotyped with five intersimple-sequence repeat markers, yielding more than 1100 polymorphic fragments across the 194 accessions. Statistical analysis of these data showed that the subspecies level was key in explaining blue honeysuckle diversity. This study shows that the collections constitute important resources that could be used for either direct consumption goals or breeding of new cultivars. Results may also be used to establish recommendations for efficient conservation of these genetic resources.

Woad (Isatis tinctoria L.) was the most important source of natural blue indigo, a pigment used mainly for dyestuff until the beginning of the 20th century, when the increased use of synthetic dyes by the industry lead to a decrease in the interest of natural dyes and to the abandonment of dye crops. The aim of this study was to characterize, according to morphological and agronomical traits, 11 woad accessions from different countries and to quantify the indigo precursor content by high-performance liquid chromatograph-diode array detection (HPLC-DAD). Qualitative traits revealed a low variability and a great variation was observed in quantitative traits. Principal component analysis (PCA) diagrams divided the accessions into four groups, primarily according to their geographic origin. The first three components of the PCA accounted for 76.8% of the total variation. Reciprocal interactions between indigo precursors and fresh leaf weight were compared through genetic diversity, with significant differences in isatan B (0.5–5.1 g/kg), indoxyl (0.3–2.0 g/kg) and residual indigo (0.3–0.5 g/kg). This information, together with genetic data, can be used to assist local farmers to re-introduce Isatis species in the European agricultural system, not only indicating the higher indigo yielding genotypes, but also the most suitable harvest time.

Woad (Isatis tinctoria L.) was introduced in Europe in ancient times to produce indigo, a natural blue pigment used mainly for dyestuff. This species was cultivated in Portugal until the beginning of the 20th century, especially in the inner North and South. A set of nine inter-simple sequence repeat (ISSR) markers generated 177 reproducible fragments, of which 171 were polymorphic. The mean number of fragments/accession was 111, ranging between 100 (Portugal-Coimbra) and 124 (Poland). The total polymorphism observed was 0.3272, the average polymorphism was 0.1784 and the gene differentiation between accessions was 0.4546. Polymorphism ranged between 53.8% (Austria) and 73.1% (Belgium). The genetic relationship among woad accessions was obtained with unweighted pair group method with arithmetic mean dendrogram based on a molecular marker, clearly clustering the woad accessions according to their geographic origin. The genetic diversity observed in this collection shows that there is a considerable potential for its improvement and that ISSR could be used to evaluate intra- and inter-accession similarities in I. tinctoria species.

The genetic relationships among 13 melon inodorus populations that were collected in southern Italy were assessed using 100 inter-simple-sequence repeat (ISSR) primers and 15 morphological traits. The dihaploid line Nad-1 and the cultivar Charentais-T, both of which belong to the botanical variety cantalupensis, were used as reference accessions in the molecular analysis. A total of 358 polymorphic bands were obtained from 39 of the 100 ISSR primers used, and 15 phenotypic traits were scored and used for genetic-similarity calculations and cluster analysis. The resulting dendrograms based on the ISSR and phenotypic data allowed almost all of the melon genotypes to be distinguished on the basis of the skin colour of the fruits. Mantel's test revealed a good correlation between the morphological and molecular data in their ability to detect genetic relationships among melon ecotypes (r = 0.50, P = 0.99). The data obtained confirm the effectiveness of this approach, and open new perspectives to reveal possible molecular associations with the phenotypic traits analysed.

Sugar and acidity levels are the main criteria of general fruit quality and for citrus juices pulp, in particular. The constituents of the acidity (organic acids) and the sweetness (glucose, fructose and sucrose) and the genes involved in their regulation have seldom been used to explore Citrus genetic diversity. We evaluated the juice composition of primary metabolic components for 87 varieties belonging to the eight major Citrus species grown under the same environmental and cultivation conditions by HPLC. We investigated the sequence polymorphism of nine candidate genes encoding for key enzymes of sugars and organic acids metabolic pathways by single strand conformation polymorphism (SSCP). Whatever the biochemical or molecular analyses, the observed structure of Citrus diversity was organized around three groups corresponding to the ancestral species (mandarin, pummelo and citron). As expected, the secondary species were closely related to their putative ancestors except for Citrus aurantium. Biochemical diversity was strongly correlated to molecular SSCP diversity at the genus level but not at the intraspecific level. Compared with other molecular marker types, higher diversity has been observed with SSCP technology, which makes it suitable for future quantitative trait loci mapping approach on gene polymorphism in citrus pulp acidity and sweetness regulation.

Variations in seed storage protein patterns were investigated for six accessions of jointed goatgrass (Aegilops cylindrica) populations collected from Slovakia within the framework of the bilateral Co-operation in Science and Technology between the Slovak Republic and Hungary. The study covered populations collected from the southwestern (localities: Sered and Dunajská Streda), southern (localities: Chlaba and Kamenica nad Hronom) and southeastern (localities: Cierna nad Tisou and Dobra) parts of Slovakia. Analysis of profiles of seed storage proteins – glutenins and gliadins – was carried out using acid polyacrylamide gel electrophoresis and sodium dodecyl sulphate polyacrylamide gel electrophoresis. All accessions have a uniform three-band high molecular weight glutenin pattern with CxCyDy subunit composition. The highest variations in gliadin bands among the populations were observed from Cierna nad Tisou. There were small differences among the populations from Chlaba and Dobra. The lowest variations were in populations from Sered, Dunajska Streda and Kamenica nad Hronom. The present investigation showed that these jointed goatgrass populations are valuable genetic resources for wheat crop improvement programmes.

Sri Lanka has a valuable repository of germplasm collection due to the availability of a large number of different traditional and improved rice varieties. Molecular techniques can increase the effectiveness of traditional technologies in assessing genetic diversity. Amplified fragment length polymorphism (AFLP) was used to evaluate the genetic diversity among rice varieties available in the germplasm collection of Plant Genetic Resources Centre, Sri Lanka. AFLP analysis of rice varieties using ten different primer combinations yielded a total of 772 polymorphic bands (98.4%). Genetic similarities were estimated using Jaccard's (J) similarity coefficient. Unweighted pair group method with arithmetic mean (UPGMA)-based dendrogram was constructed. Genetic similarities varied from 0.073 to 0.565. Cluster analysis by genetic similarity divided the accessions into four main groups. The Cophenetic correlation with r = 0.781 indicated high confidence of AFLP data to group the varieties in UPGMA clusters. Principal component analysis further confirmed the patterns obtained by the cluster analysis. The results revealed very high genetic diversity at molecular level among the Sri Lankan rice varieties used in this study.