Genebank Collections Research Articles

Molecular characterization of Greek pepper (Capsicum annuum L) landraces with neutral (ISSR) and gene-based (SCoT and EST-SSR) molecular markers

Thirty landraces were selected from a Gene Bank collection comprising of pepper (Capsicum annuum L.) landraces from across Greece. The genetic variability among the 30 landraces and one commercial greek cultivar, intended for industrial use, was assessed by Inter-Simple Sequence Repeat (ISSR), Start Codon Targeted (SCoT) and Expressed Sequence Tag (EST) - Simple Sequence Repeat (SSR) molecular markers. All genotypes were clearly distinguished in the dendrograms produced, with the ISSR and SCoT dendrograms being the most consistent. Three landraces were grouped together in all dendrogams revealing a possible phylogenetic relationship. Furthermore, EST-SSR markers were combined with High Resolution Melting (HRM) analysis by which the more adequate and confident separation of the landraces was accomplished due to the unique HRM profiles produced. Genotyping the rich Greek pepper germplasm by molecular markers will aid the breeding process towards well adopted Greek pepper cultivars of higher quality and productivity.

Selecting High Amylose Rice Germplasm Combined with NIR Spectroscopy at the RDA Genebank Conserved

Rice (Oryza sativa L.) germplasms with an amylose content above 30% were identified by screening 9481 accessions from the Rural Development Administration (RDA) gene bank. The total set of accessions came from 65 countries, including Korea, China, Japan, the Philippines, India, Taiwan, the United States, and Russia. High-throughput near infrared reflectance (NIR) spectroscopy equipped with a fiberoptic probe (700~2500 nm) was used to estimate the amylose content. The amylose contents ranged from 5 to 40% based on NIR spectroscopy; divided into 5% increments, the amylose content of 2820 accessions was found to be between 15 and 20%. To select rice accessions high in amylose, 239 accessions with an amylose content of 30% or greater were selected based on NIR spectroscopy data and cultivated in the field for final selection. Among the 239 accessions selected and cultivated, 151 were deemed agronomically satisfactory. Among the 151 accessions, based on laboratory analysis, 14 had an amylose content higher than 30% and 33 had an amylose content between 28 and 30%. The amylose contents of the reference cultivars Hopum-byeo and Sobi-byeo were 20.7% and 19.9%, respectively. Finally, successful selection of accessions with high amylose content from the large RDA gene bank collection was achieved based on a combination of NIR spectroscopy and laboratory data.

High Resolution Melting (HRM) analysis in eggplant (Solanum melongena L.): A tool for microsatellite genotyping and molecular characterization of a Greek Genebank collection

The conservation and characterization of eggplant (Solanum melongena L.) genetic resources in germplasm banks has been the basis of their use in breeding projects, which has resulted in the development of new cultivars. High Resolution Melting (HRM) analysis, combined with eight microsatellite markers, has been integrated in order to facilitate the molecular identification and characterization of the eggplant germplasm, collected from the National Genebank Collection of Greece. The eight microsatellite loci used were highly informative and generated sixty three HRM profiles, which were sufficient to discriminate all eggplant landraces and cultivars studied, highlighting its potential use for cultivar genotyping. The thirty six eggplant genotypes were classified into four clusters. Hence, this assay provided a fast, cost-effective and closed-tube microsatellite genotyping method, well suited for molecular characterization of eggplant cultivars.

Aegilops conservation and collection evaluation in the Czech Republic

AbstractAegilops distribution was revised on localities in the territory of the Czech Republic and the former Czechoslovakia, based on herbarium data, literature and floristic databases. The only surviving species Ae. cylindrica forms the northern limit of distribution in the Danube basin, other sites are secondary. In situ conservation was proposed for two Czech sites where Aegilops have been naturalized. An ex situ gene bank collection has been maintained in the Gene Bank Prague since 1985. The collection includes 21 species and 1099 accessions. The whole collection was evaluated for breeding‐valuable characters: resistance to leaf diseases, cereal aphids, viral diseases, and qualitative parameters. Recently, the collection was retested for the present races of leaf and stem rust including new introductions from Kazakhstan. Six different leaf rust races and five different stem rust races collected from the Czech Republic were applied in the older tests, and three races from each rust species in the recent tests. The highest number of accessions resistant both to wheat leaf rust and wheat stem rust and powdery mildew was found in Ae. speltoides (90%). Different reactions of individual accessions of the tested species to the three different pathotypes of each rust species indicates that several diverse genes for specific resistance are present in the accessions of the tested species.

Oil content, fatty acid composition and tocopherol concentration in the Spanish almond genebank collection

Total oil content and concentrations of the major fatty acids and of the tocopherol homologues were determined in the kernel oil of 44 local almond cultivars originated in the different Spanish growing regions. The ranges of variability for the different parameters was similar to those already reported for almond, such as 50.58–64.95% of dry matter (DM) for oil content, 64.97–79.59% of oleic acid in the total oil, and 313.0–616.1mg/kg oil for α-tocopherol. Despite these ranges of variability, extremely high values were obtained in some genotypes, such as very high content of oleic acid in ‘Muel’ and of α-tocopherol in ‘Araguayo-2’. These ranges of variability indicate that this collection reflects a high level of diversity and the importance of maintaining and characterising such a diverse germplasm collection. The knowledge of this diversity may be extremely useful in identifying interesting parents to be included in a breeding programme as a response to new selection objectives, such as the improvement of the chemical quality of the almond kernel.

Separating the wheat from the chaff – a strategy to utilize plant genetic resources from ex situ genebanks

The need for higher yielding and better-adapted crop plants for feeding the world's rapidly growing population has raised the question of how to systematically utilize large genebank collections with their wide range of largely untouched genetic diversity. Phenotypic data that has been recorded for decades during various rounds of seed multiplication provides a rich source of information. Their usefulness has remained limited though, due to various biases induced by conservation management over time or changing environmental conditions. Here, we present a powerful procedure that permits an unbiased trait-based selection of plant samples based on such phenotypic data. Applying this technique to the wheat collection of one of the largest genebanks worldwide, we identified groups of plant samples displaying contrasting phenotypes for selected traits. As a proof of concept for our discovery pipeline, we resequenced the entire major but conserved flowering time locus Ppd-D1 in just a few such selected wheat samples – and nearly doubled the number of hitherto known alleles.

The 3,000 rice genomes project: new opportunities and challenges for future rice research.

Rice is the world’s most important staple grown by millions of small-holder farmers. Sustaining rice production relies on the intelligent use of rice diversity. The 3,000 Rice Genomes Project is a giga-dataset of publically available genome sequences (averaging 14× depth of coverage) derived from 3,000 accessions of rice with global representation of genetic and functional diversity. The seed of these accessions is available from the International Rice Genebank Collection. Together, they are an unprecedented resource for advancing rice science and breeding technology. Our immediate challenge now is to comprehensively and systematically mine this dataset to link genotypic variation to functional variation with the ultimate goal of creating new and sustainable rice varieties that can support a future world population that will approach 9.6 billion by 2050.

Next-generation genebanking: plant genetic resources management and utilization in the sequencing era

Advances in sequencing technologies have made it possible to analyse large amounts of germplasm against low production costs, which has opened the door to screen genebank collections more efficiently for DNA sequence variation. The present study explores how these developments may affect the operations of genebanks and, consequently, how genebank agendas may change. It is argued that the new developments are more likely to have an impact on the user-oriented activities than the housekeeping operations of genebanks. To better facilitate the user community, genebanks may have to strengthen their core business, in particular, by improving quality management procedures and by providing access to a wider diversity of a crop's gene pool. In addition, genebanks may have to provide novel services, such as the introduction of specific user-oriented collection types, including research populations and genetically purified lines, and the development of novel information services, including plant genetic resources portals that should guide users to the information and materials of specific interest. To improve their user-oriented services, genebanks need to increase their communication and collaboration with the user community and to develop strategic alliances with this sector.

Phenotypic evaluation and characterization of a collection of Malus species

A collection of 337 Malus accessions representing 46 species of the Fruit Genebank in Dresden-Pillnitz, Germany, were evaluated for 64 morphological and phenological traits to proof the classification of the accession and to analyze genetic relationships. Morphological characteristics were determined for the whole tree, shoots, leaves, flowers and fruits. Data were subjected to principal component analysis (PCA), and a dendrogram was constructed to demonstrate morphological relationship. According to PCA the most important variables integrated in the first principal component were mainly fruit descriptors and three traits characterizing pubescence (1 year old shoot, leaf blade, and pedicel). Cluster analysis clearly separated the accessions in Malus series. The results showed the polyphyletic origin of the section Sorbomalus. The North American apple species, M. fusca, was closely related to the species contributing to the East Asian center of the genus origin. The relict species of the European continent (M. trilobata) was grouped in near distance to the North American species belonging to section Chloromeles. The species of Sieversinae and Baccatae dominated in one main cluster indicating their close relatedness. Most of M. ×domestica accessions were grouped with M. sieversii and M. orientalis; this supports the Sievers apple as an ancestor of domesticated apple. The morphological evaluations in this study indicated a high level of phenotypic diversity in the genebank collection. These phenotypical data will increase the feasibility of prioritizing accessions based on the uniqueness of desirable morphological traits.

Retrotransposon-based molecular markers for analysis of genetic diversity within the Genus Linum.

SSAP method was used to study the genetic diversity of 22 Linum species from sections Linum, Adenolinum, Dasylinum, Stellerolinum, and 46 flax cultivars. All the studied flax varieties were distinguished using SSAP for retrotransposons FL9 and FL11. Thus, the validity of SSAP method was demonstrated for flax marking, identification of accessions in genebank collections, and control during propagation of flax varieties. Polymorphism of Fl1a, Fl1b, and Cassandra insertions were very low in flax varieties, but these retrotransposons were successfully used for the investigation of Linum species. Species clusterization based on SSAP markers was in concordance with their taxonomic division into sections Dasylinum, Stellerolinum, Adenolinum, and Linum. All species of sect. Adenolinum clustered apart from species of sect. Linum. The data confirmed the accuracy of the separation in these sections. Members of section Linum are not as closely related as members of other sections, so taxonomic revision of this section is desirable. L. usitatissimum accessions genetically distant from modern flax cultivars were revealed in our work. These accessions are of utmost interest for flax breeding and introduction of new useful traits into flax cultivars. The chromosome localization of Cassandra retrotransposon in Linum species was determined.

Screening and identification of potentialTrichodermasp. against soil borne pathogens of vanilla (Vanilla planifolia)

This study was conducted to evaluate the biocontrol potential of indigenous Trichoderma sp. against fungal pathogens of Vanilla. The pathogens causing wilt and rot diseases of vanilla Fusarium oxysporum, Rhizoctonia solani and Sclerotium rolfsii were isolated from naturally infected vanilla plants. Among the ten isolates, two species Trichoderma harzianum T2 and Trichoderma virens T5 which showed 80 to 90% inhibitions against the selected fungal pathogens were selected. The Trichoderma isolates were identified using Internal transcribed spacer (ITS) region amplification and sequencing. The sequences generated in this study were deposited in the NCBI gene bank and culture collection centre and got the accession number JN 863298 for the isolate Trichoderma virens and JN 000305 for the isolate Trichoderma harzianum.The compatibility of the bioagents with selected fungicides were also investigated under in vitro conditions.Bordeaux mixture was found to be inhibitory to Trichoderma at all given concentrations, whereas the bioagents found to be compatible with potassium phosphonate. Trichoderma harzianum inoculation gave 90-95 % disease suppression under green house experiment and was superior than Trichoderma virens and the standard (MTCC 801). Application of biocontrol agents for crop protection is very significant as it has several advantages such as possibility of multiple pathogen suppression, low cost and promotion of soil fertility

GERMPLASM OF VEGETABLE CROPS (Department of vegetable crops of VIR H 90th anniversary) All

Analysis of investigation and application of genetic resources of vegetable and melon crops since Vavilov's time up to nowadays has been done. The main trends of investigations of the gene bank collection in recent times are described. The results of breeding and seed production are shown. The initial breeding materials are recommended.

It background of the medium-term storage of Martonvásár Cereal Genebank resources in phytotron cold rooms

Genebanks are storage facilities designed to maintain the plant genetic resources of crop varieties (and their wild relatives) and to ensure that they are made available and distributed for use by plant breeders, researchers and farmers. The Martonvásár Cereal Genebank (MV-CGB) collection evolved from the working collections of local breeders and consists predominantly of local and regional materials. Established in 1992 by the Agricultural Research Institute of the Hungarian Academy of Sciences (Bedő, 2009), MVCGB with its over 10,000 accessions of the major species (Triticum, Aegilops, Agropyron, Elymus, Thinopyrum, Pseudoroegneria, Secale, Hordeum, Avena, Zea mays), became one of the approx. 80 cereal germplasm collections that exist globally. In Martonvásár breeding is underway on a number of cereal species, and large numbers of genotypes are tested each year in the field and under laboratory conditions. The increasing size of the research programmes assisted by a modern genebank background involve an enormous increase in the quantity of data that must be handled during research activities such as traditional breeding, pre-breeding and organic breeding. A computerized system is of primary importance to synchronize breeding and genebank activities, to monitor the quality and quantity of seed accessions in cold storage, to assist the registration of samples, and to facilitate characterization, regeneration and germplasm distribution.

FROST HARDINESS OF PEACH AND APRICOT FLOWER BUDS

The determination of the frost resistance and winter hardiness of peach and apricot varieties is an important task in Hungary, as production can only be competitive if the location and variety are wisely chosen. Various overwintering organs of trees have very different levels of frost resistance, and this resistance also varies greatly over time. During dormancy flower buds are the most frost sensitive organs of peach and apricot trees. From the practical point of view frost resistance and winter hardiness of the flower buds deserve detailed study. The development rate of the overwintering organs of peach and apricot varieties and changes in their frost resistance during the dormancy period have been regularly investigated in recent years in the gene bank collection of Corvinus University of Budapest. The present paper provides an analysis of frost hardiness of flower buds based on the results obtained in three consecutive winters with very diverse weather conditions. The hardening processes of overwintering organs are fundamentally determined by environmental factors, particularly by temperature. Thus, even at the same location, the development rate of the flower buds and the development of frost resistance may differ from year to year. The determination of the maximum frost resistance genetically attainable and the description of changes in frost resistance are only possible in a series of experiments over several years.

An assessment of the genetic integrity of ex situ germplasm collections of three endangered species of Coffea from Madagascar: implications for the management of field germplasm collections

Madagascar has 59 species of Coffea, of which 42 are listed as Critically Endangered, Endangered, or Vulnerable by criteria of the Red List Category system of the World Conservation Union. In an attempt to assess the conservation value of ex situ collections of Malagasy coffee species, a study was undertaken using the field genebank collections maintained at the Kianjavato Coffee Research Station. Three species were selected for this purpose, C. kianjavatensis, C. montis-sacri, and C. vatovavyensis, and for comparative purposes extant, in situ populations of the same species were studied. Parentage analyses of ex situ propagated offspring of C. kianjavatensis and C. montis-sacri were performed to assess if crossing with other Coffea species maintained in the field genebank is compromising the genetic integrity of the collection. For these three species, higher genetic diversity was observed in the ex situ populations compared to the in situ populations, highlighting the importance of preserving the plants currently in ex situ collections. Parentage analyses of seed-propagated offspring of C. kianjavatensis and C. montis-sacri revealed that cross contamination with pollen from other Coffea species in the ex situ field genebank is occurring. These results have significant implications for the conservation management of wild Coffea species and for the management of ex situ genebanks.

Genetic Selection and Conservation of Genetic Diversity*

For 100s of years, livestock producers have employed various types of selection to alter livestock populations. Current selection strategies are little different, except our technologies for selection have become more powerful. Genetic resources at the breed level have been in and out of favour over time. These resources are the raw materials used to manipulate populations, and therefore, they are critical to the past and future success of the livestock sector. With increasing ability to rapidly change genetic composition of livestock populations, the conservation of these genetic resources becomes more critical. Globally, awareness of the need to steward genetic resources has increased. A growing number of countries have embarked on large scale conservation efforts by using in situ, ex situ (gene banking), or both approaches. Gene banking efforts have substantially increased and data suggest that gene banks are successfully capturing genetic diversity for research or industry use. It is also noteworthy that both industry and the research community are utilizing gene bank holdings. As pressures grow to meet consumer demands and potential changes in production systems, the linkage between selection goals and genetic conservation will increase as a mechanism to facilitate continued livestock sector development.

Molecular analysis of temporal genetic structuring in pea (Pisum sativum L.) cultivars bred in the Czech Republic and in former Czechoslovakia since the mid-20th century

Changes in genetic diversity of peas bred in the Czech Republic and in former Czechoslovakia since the mid-20<sup>th</sup> century were analysed using 38 molecular marker loci, including retrotransposons and microsatellites, differentiating a total of 84 alleles. Both marker types were comparably effective in revealing the genetic diversity, with a high correlation (r = 0.81), although the pairwise genetic distances of each marker type differed. In total, 175 accessions, selected from the Czech pea gene bank collection and representing the pea cultivars collected or bred in the country, were divided into three groups according to their date of sampling or variety registration. The first group contained 70 old cultivars and landraces collected prior to 1961. The second group contained 46 cultivars released from 1961 to 1980. The third group contained 59 cultivars released between 1981 and 2004. In spite of the decline in several diversity measures, differences in allele frequencies and even allele loss in three microsatellite loci were recorded over the 70-year period, while these differences between the groups were not statistically significant. In addition, genetic heterogeneity was detected in 29 accessions (15%). This indicates that although no genetic erosion could be observed since then, it is important to monitor the genetic diversity, furthermore it highlights the vital role of germplasm collections for the crop diversity conservation. 

COMPARATIVE STUDY ON THE BEHAVIOR OF OLIVE CULTIVARS FROM DIFFERENT ORIGINS CULTIVATED IN THE SAME ENVIRONMENT, WITH REGARD TO SOME IMPORTANT CHARACTERISTICS OF THE OLIVE OIL PRODUCED

ISHS VI International Symposium on Olive Growing COMPARATIVE STUDY ON THE BEHAVIOR OF OLIVE CULTIVARS FROM DIFFERENT ORIGINS CULTIVATED IN THE SAME ENVIRONMENT, WITH REGARD TO SOME IMPORTANT CHARACTERISTICS OF THE OLIVE OIL PRODUCED

A Bayesian analysis of gene flow from crops to their wild relatives: cultivated (Lactuca sativa L.) and prickly lettuce (L.serriola L.) and the recent expansion of L.serriola in Europe.

Interspecific gene flow can lead to the formation of hybrid populations that have a competitive advantage over the parental populations, even for hybrids from a cross between crops and wild relatives. Wild prickly lettuce (Lactuca serriola) has recently expanded in Europe and hybridization with the related crop species (cultivated lettuce, L.sativa) has been hypothesized as one of the mechanisms behind this expansion. In a basically selfing species, such as lettuce, assessing hybridization in natural populations may not be straightforward. Therefore, we analysed a uniquely large data set of plants genotyped with SSR (simple sequence repeat) markers with two programs for Bayesian population genetic analysis, STRUCTURE and NewHybrids. The data set comprised 7738 plants, including a complete genebank collection, which provided a wide coverage of cultivated germplasm and a fair coverage of wild accessions, and a set of wild populations recently sampled across Europe. STRUCTURE analysis inferred the occurrence of hybrids at a level of 7% across Europe. NewHybrids indicated these hybrids to be advanced selfed generations of a hybridization event or of one backcross after such an event, which is according to expectations for a basically selfing species. These advanced selfed generations could not be detected effectively with crop-specific alleles. In the northern part of Europe, where the expansion of L.serriola took place, the fewest putative hybrids were found. Therefore, we conclude that other mechanisms than crop/wild gene flow, such as an increase in disturbed habitats and/or climate warming, are more likely explanations for this expansion.

Applications of next‐generation sequencing in plant biology

The last several years have seen revolutionary advances in DNA sequencing technologies with the advent of next-generation sequencing (NGS) techniques. NGS methods now allow millions of bases to be sequenced in one round, at a fraction of the cost relative to traditional Sanger sequencing. As costs and capabilities of these technologies continue to improve, we are only beginning to see the possibilities of NGS platforms, which are developing in parallel with online availability of a wide range of biological data sets and scientific publications and allowing us to address a variety of questions not possible before. As techniques and data sets continue to improve and grow, we are rapidly moving to the point where every organism, not just select "model organisms", is open to the power of NGS. This volume presents a brief synopsis of NGS technologies and the development of exemplary applications of such methods in the fields of molecular marker development, hybridization and introgression, transcriptome investigations, phylogenetic and ecological studies, polyploid genetics, and applications for large genebank collections.