A review of factors that influence the production of quality seed for long-term conservation in genebanks

Seed quality is a critical aspect in agriculture as well as in the long-term conservation of plant genetic resources in genebanks. Since potential seed longevity depends on initial quality, genebank curators need to be aware of the best management practices that contribute to the production of high quality seed during routine germplasm regeneration/multiplication. Among the factors influencing initial seed quality, those related to crop management, including plant nutrient and water supply during crop growth, climatic conditions during seed development and maturation, as well as the harvest and drying practices are of considerable significance. Seeds of high quality can be obtained by planting in suitable areas/fields and at appropriate times, applying good crop management practices, adoption of proper harvesting and drying techniques, careful handling and processing to minimize mechanical injuries and unwanted seed mixing with other accessions, and ensuring minimum deterioration before reaching the designated storage. However, seed production and post-harvest handling highly depend on the biology and agronomy of the species. As germplasm collections contain a wide range of diversity for morphological and agronomic characters and that there might well be critical gaps in knowledge among genebank staff or about the species in question, genebanks may also need to embark on research to gain crop specific knowledge on optimal seed production procedures to improve seed quality.     

Preservation of seed viability during 25 years of storage under standard genebank conditions

Maintaining sufficient viability is critical to the sustainability of ex situ conserved seed collections. For this reason, accessions are regenerated when viability falls below a predefined threshold. Viability is monitored by determining the germination ability of accessions at predefined time intervals. Optimizing the frequency of these germination tests, in order to avoid waste of resources, is hampered by the scarce availability of data about seed longevity, particularly for material maintained under genebank conditions. Here we report on the analysis of nearly 40,000 germination test results collected for a wide range of crop species over a 25-years period by the centre for genetic resources, the Netherlands (CGN), where seeds of genebank accessions are dried to 3–7 % moisture content and stored for the long term under near-vacuum in aluminium foil bags at ?20 °C. The results indicate that seed viability is well maintained for the large majority of seed lots during the first 25 years after regeneration as only 3.3 % of the monitoring tests revealed below-threshold germination values. It is argued that the majority of these sub-standard seed lots are due to other causes than seed ageing, including dormancy problems and estimation error in germination testing. For material, maintained under the seed management procedures and storage conditions practiced by CGN, it is therefore recommended to delay the first germination monitoring tests to 25 years after regeneration.     

Who is sowing our seeds? A systematic review of the use of plant genetic resources in research

Collections of plant genetic resources managed by genebanks function to conserve the range of genetic diversity present in crop genepools. They can facilitate access to valuable allelic variation for both plant breeders and researchers who are able to request germplasm for use in crop improvement and both basic and applied scientific research. The direct impact of genebank collections is often unclear as downstream uses of germplasm samples may not be reported back to the genebank of origin. This study aims to systematically review scientific use of germplasm using the UK Vegetable Genebank (UKVGB) as a model. Between the years of 1980–2016, a total of 271 publications were identified as using UKVGB material. The frequency of publications and the international nature of use increased significantly over the time period studied. Accessions directly sourced from the UKVGB made up the majority of material used by researchers, but material from research-derived resources such as differential sets and core collections or diversity sets have also been used. Resistance to pests and diseases and genetic diversity were the main topics of study although germplasm was used to address a wide range of other research questions. Genebanks such as UKVGB provide an essential resource of allelic diversity in crop genepools which supports a diverse range of research projects. The utilisation of these plant genetic resources has increased over time, contributing to a substantial number of publications. Developments in sequencing technologies have no doubt played a part as larger numbers of accessions can be utilized in a single experiment, but the increase also no doubt reflects a greater interest in the use of allelic diversity to overcome challenges in crop improvement and research.     

AFLP analysis of genetic diversity in leafy kale (<Emphasis Type="Italic">Brassica oleracea</Emphasis> L. convar. <Emphasis Type="Italic">acephala</Emphasis> (DC.) Alef.) landraces,cultivars and wild populations in Europe

AFLP markers were used to characterize diversity and asses the genetic structure among 17 accessions of kale landraces, cultivars and wild populations from Europe. The range of average gene diversity in accessions was 0.11–0.27. Several landraces showed higher levels of diversity than the wild populations and one cultivar had the lowest diversity measures. The landraces that were most genetically diverse were from areas where kales are known to be extensively grown, suggesting in situ conservation in these areas as a supplement to storage of seeds in gene banks. An analysis of molecular variance (AMOVA) showed that 62% of the total variation was found within accessions. For most accessions, genetic distance was not related to geographic distance. Similarities among accessions were probably not caused by recent gene flow since they were widely separated geographically; more likely the relationship among them is due to seed dispersal through human interactions. Our results indicate that a kale population found in a natural habitat in Denmark was probably not truly wild but most likely an escape from a cultivated Danish kale that had subsequently become naturalized.     

Dispersal of durum wheat [<Emphasis Type="Italic">Triticum turgidum</Emphasis> L. ssp. <Emphasis Type="Italic">turgidum</Emphasis> convar. <Emphasis Type="Italic">durum</Emphasis> (Desf.) MacKey] landraces across the Mediterranean basin assessed by AFLPs and microsatellites

A comprehensive characterization of crop germplasm is critical to the optimal improvement of the quality and productivity of crops. Genetic relationships and variability were evaluated among 63 durum wheat landraces from the Mediterranean basin using amplified fragment length polymorphisms (AFLPs) and microsatellites markers. The genetic diversity indices found were comparable to those of other crop species, with average polymorphism information content (PIC) values of 0.24 and 0.70 for AFLP and microsatellites, respectively. The mean number of alleles observed for the microsatellites loci was 9.15. Non-metric multi-dimensional scaling clustered the accessions according to their geographical origin with the landraces from the South shore of the Mediterranean Sea closely related. The results support two dispersal patterns of durum wheat in the Mediterranean basin, one through its north side and a second one through its south side.     

Phenotypic Variation and Relationships in Landraces and Improved Varieties of Rye (Secale cereale L.) from Northern Europe

Phenotypic diversity for agronomic characteristics was determined in an experiment with 29 landraces and 14 improved varieties of rye from the Nordic area, Germany and Poland. The accessions were scored for 12 characters. Effects of the location, year, type, and country of origin for landraces were investigated with analysis of variance. Phenotypic variations for the traits were estimated using the Shannon–Weaver diversity index. The genetic variation was high, with an average of H ₀ = 0.566. The landraces from Norway, Sweden and Finland showed the highest variation, whereas the improved varieties had the lowest. The German material also had low variation but the status of this material is uncertain. The genetic diversity showed that 70% of the variation was found within the accessions. A cluster analysis was carried out to identify the relationship between the accessions. The material grouped into eight clusters, where clusters I to V included landraces from Sweden, Finland and Norway, except for cluster III, which included one improved variety from Denmark. Cluster VI comprised a single Swedish landrace from Gotland. Most of the improved varieties were in cluster VII and the last cluster contained accessions from Germany and Sweden.     

Morphological Diversity of Bambara Groundnut [Vigna subterranea (L.) Verdc.] Landraces in Tanzania

A field experiment was conducted in Tanzania for two seasons to assess the genetic diversity of bambara groundnut landraces based on morphological characters. One hundred accessions collected from a wide range of agricultural zones in the country were evaluated in a 10 × 10 triple lattice block design at Maruku station in Bukoba, Tanzania. For the qualitative characters evaluated, considerable morphological variations were observed for growth habit, pod shape and pod colour. Quantitative morphological characters such as peduncle length, number of leaves per plant, terminal leaflet width, terminal leaflet length, petiole length, plant spread, plant length, pod width, seed length, seed width, number of pods per plant, shelling percentage and days to 50% flowering showed significant variation among accessions for the two test seasons. Multivariate analysis for 15 quantitative morphological traits that showed significant variation indicated that the first four PCs with eigenvalues ≥1 accounted for 63.0 and 65.0% of the total variance among the accessions during the two test seasons, respectively. The most important loadings for PC1 and PC2 for the two seasons were terminal leaflet width, terminal leaflet length, petiole length, plant spread, plant height and pod length, pod width, seed length, seed width, numbers of pods per plant. Cluster analysis grouped bambara groundnut accessions into three major groups with respect to their geographic origins. Based on this present study, bambara groundnut landraces from Tanzania displayed a considerable diversity for morphological and agronomic traits useful for germplasm management and utilization into crop improvement.     

Genetic, morphological and cyanogen content evaluation of a new collection of Caribbean Lima bean (Phaseolus lunatus L.) landraces

Lima bean is a species cultivated broadly in the Americas and has been cultivated in the Caribbean for at least 500 years. In order to determine the genetic structure and diversity of Lima bean from the Caribbean, 50 landraces from Haiti, the Dominican Republic and Puerto Rico were collected and analyzed using 24 SSR markers. All landraces in this Caribbean collection were found to be of Middle-American descent. The genetic diversity (H_E) was highest in the landraces from Puerto Rico and lowest in Haitian landraces. The observed proportion of heterozygotes (H_O) was higher in the Haitian landraces and lowest in Puerto Rican landraces. Unweighted pair-group method with arithmetic averaging analysis showed that the landraces clustered into 3 clusters with all Haitian landraces grouping in one cluster. We also assessed the agro-morphological characteristics of the collection as well as the content of cyanogenic glucoside, linamarin, in leaves and dry seeds. Lima bean, which is a model crop for indirect plant defenses against herbivory, also possess linamarin as a source of direct plant defense. Upon tissue damage, linamarin is converted to toxic hydrogen cyanide. In our collection 44.6 % of the landraces had average seed HCN content ≤200 ppm which is the permitted level for Lima bean seed in the US. Our results also identified the landraces in this collection which have high linamarin levels in the leaves while having low levels in the seeds. Such landraces have the desirable combination of traits and will be the focus of our future plans for agronomic trait improvement though breeding.     

Molecular diversity of Omani wheat revealed by microsatellites: II. Hexaploid landraces

For millennia, wheat (Triticum spp.) has been grown in traditional aflaj-irrigation systems of remote mountain oases in Oman. However, little is known about the diversity of the ancient landraces used. Given recent reports about the occurrence of novel germplasm in such material, the objective of this study was to evaluate the genetic diversity of hexaploid wheat (Triticum aestivum L.) landraces in relation to their geographic origin using microsatellites. The collection covered most of the cultivation areas in northern Oman where wheat landraces are growing. Total genomic DNA was extracted from six pooled plants representing each accession. A total of 161 wheat accessions were assayed using 35 microsatellite loci in which a total of 305 polymorphic bands were recorded for the 35 microsatellites. The polymorphic information content (PIC) across the 35 microsatellite loci ranged from 0.02 to 0.89 with an average of 0.50. A heterozygosity percentage value of 9.09 was determined and the highest level recorded for accessions from the Batinah district. Rare alleles averaged 1.85 with the highest value being from the Dakhilia district. The results indicated a significant correlation between gene diversity and number of alleles across districts. The correlation coefficient between these two variables over the 35 loci was 0.657, whereby correlation coefficients of 0.718, 0.706, 0.657 and 0.651, respectively, were found for the Batinah, Dhahira, Dakhilia and Sharqia materials. Genetic distances indicated that all landraces were closely related. The cluster analysis discriminated most of the landraces accessions. However, it failed to achieve region-specific groupings of landraces. The present study demonstrated the presence of high diversity in Omani landraces and also indicated the effectiveness of microsatellites to describe it.     

The Spanish barley core collection

Spanish barleys constitute a germplasm group of particular interest for breeding purposes, as Spain has been proposed as a possible centre of origin of the crop. The Spanish National Germplasm Bank (Banco Nacional de Germoplasma, BNG), holds a collection of about 2000 barley accessions, mostly landraces collected in Spain prior to extensive introduction of modern varieties. The objective of this work is to create a core collection of barleys representative of old barley genotypes grown in Spain. The core collection will be constituted by three groups of germplasm: successful old varieties (15); entries in common with previously existing barley core collections (15); and 2-row (8) and 6-row (122) entries from the BNG, for a total of 160 entries. Entries were allocated by stratified sampling in agro-ecological uniform zones of barley cultivation in Spain. Classification of agro-ecological regions for barley was based on historical yield records for Spanish provinces. The number of entries for each region was determined in proportion to the logarithm of historical barley acreage. Final choice of accessions within provinces tried to maximize the diversity and avoid duplications by looking at passport data, and to agronomic evaluation data available for a group of about 900 accessions.     

Target region amplification polymorphism (TRAP) for assessing genetic diversity and marker-trait associations in chickpea (Cicer arietinum L.) germplasm

The effective utilization of crop diversity held in genebanks depends on our knowledge of useful traits and available markers associated with the target traits. Target region amplification polymorphism (TRAP) was used to evaluate the genetic diversity and underlying relationships among 263 accessions of chickpea landraces maintained by the USDA-ARS Western Regional Plant Introduction Station in Pullman, WA, USA. Two-hundred sixty-two TRAP markers were amplified by eight primer combinations. Altogether, 110 (42 %) markers were polymorphic, the other 152 (58 %) displayed no variation. These polymorphic markers revealed important differences among the accessions, with an estimated, mean pair-wise genetic distance of 25.82 %, ranging from 2.8 to 50.0 %. Genetic distance analysis divided the accessions into two major groups, with 113 and 150 accessions each, and substantial association between molecular diversity and geographic origin was evident. Bayesian analysis of population structure revealed two groups (K = 2) with evidence for six sub-groups. Additionally, the population structure of a subset of 110 lines was determined (K = 3) for testing marker-trait associations (MTAs). Phenotypic traits included the concentrations of protein and nine mineral elements in the seeds. Two MTAs were significant (p < 0.01) for concentrations of Ca and K, and three MTAs were significant for Cu and Ni concentrations. The results indicate that this population is useful for genome-wide association studies on other economic traits given the level of genetic diversity uncovered and the marker-trait associations in seed minerals discovered.     

Using GIS to check co-ordinates of genebank accessions

The geographic co-ordinates of the locations where germplasm accessions have been collected are usually documented in genebank databases. However, the co-ordinate data are often incomplete and may contain errors. This paper describes procedures to check for errors, to determine the cause of these errors and to assign new co-ordinates, using Geographical Information Systems (GIS). These procedures can assist in improving the quality of genebank databases, and with that, increase the capability for analysis and use of crop genetic diversity.     

Genetic diversity in local cultivars of garden pea (Pisum sativum L.) conserved ‘on farm’ and in historical collections

During a national Swedish collection mission of vegetable varieties conserved ‘on farm’ more than 70 pea accessions were obtained, many of which had been grown locally for more than 100 years. In spite of a likely origin in the multitude of obsolete commercial pea varieties available on the Swedish seed market in the nineteenth century, the rediscovered local cultivars have lost their original names and cultivar identity while being maintained ‘on farm’. To analyze genetic diversity in the repatriated material, 20 accessions were genotyped with twelve SSR markers and compared with 15 obsolete cultivars kept in genebanks and 13 cultivars preserved as non-viable seeds collected in 1877–1918. Most of the local cultivars were genetically distinct from each other, and in only a few cases could a possible origin in a tested obsolete cultivar be suggested. These results reflect the wide diversity of pea cultivars present in Sweden during the nineteenth century. Both between and within accession genetic diversity was larger among the historical samples of obsolete cultivars compared to local cultivars and cultivars preserved in genebanks, indicating genetic erosion over time both in genebanks and during conservation ‘on farm’. The constraints on identifying and verifying historical cultivars using genetic markers are discussed.     

Genetic diversity among Polish landraces of common oat (Avena sativa L.)

All over the world about 220,000 accessions of the genus Avena is preserved in gene banks. Polish oats collection, in the National Centre for Plant Genetic Resources, consists of about 2,500 accessions. More than 80 % of them belong to the species Avena sativa. Only 136 accessions have the landrace or traditional cultivar status and 91 of them were collected in Poland. The main objective of this study was to estimate genetic diversity of a set of 67 Polish landraces using inter simple sequence repeat (ISSR) markers. We also tried to determine whether genetic diversity depended on the region and altitude of collection site and if there was a relationship between DNA polymorphism and some morphological traits. The accessions were collected between years 1973 and 1999 from areas of Northern, Southern and Eastern Poland. The obtained results indicated a relatively low diversity (0.09–0.37) of the common oat landraces. Analysis of molecular variation (AMOVA) showed that there were no significant differences within designated geographic regions. It indicated that at the same time the altitude of collection site had significant effect on the genetic differentiation of the accessions. AMOVA also showed the presence of variance between groups formed on the basis of the colour of lemma. White lemma accessions were also characterized by highest genetic distinctiveness. Considering the evident impact of local climatic conditions on the genetic distinctiveness of Polish landraces utility of them in the Polish breeding programs should be considered.     

Genetic structure of Lima bean (<Emphasis Type="Italic">Phaseolus lunatus</Emphasis> L.) landraces grown in the Mayan area

Lima bean (Phaseolus lunatus L.) is an important crop in the Mayan culture. The Mayan area, considered as a main center of Mesoamerican diversity, has been divided into two subareas: the Mayan lowlands and the Mayan highlands. The Yucatan Peninsula is part of the Mayan lowlands and holds the highest number of Lima bean landraces of Mexico, but Lima beans are in high risk of genetic erosion due to intensification of the traditional Mayan agriculture. However, information on genetic diversity of Lima beans of the Mayan highlands is lacking. By using 46 landraces collected in the Mayan area (23 from each subarea) and 73 ISSR loci (inter-simple sequence repeats), we analyzed the structure, diversity and genetic relationships of Lima beans of this part of Mesoamerica. High levels of diversity (H _BAY = 0.45) and genetic structure (F _ST = 0.66) were found for the whole Mayan area. Genetic diversity in the Mayan lowlands was apparently higher than the Mayan highlands (H _BAY = 0.44 and 0.36, respectively); but differences were not statistically significant. Genetic structure between the subareas was high (AMOVA = 30% of total variation), most landraces grouping according to their geographic origin. This study shows the importance of the Mayan culture in the diversification and conservation of Lima beans. The results provide important information that should be considered when implementing strategies to collect Lima bean landraces and planning in situ and ex situ programs to conserve these landraces in the Mayan region.     

Temporal variation of diversity in Italian durum wheat germplasm

The aim of this work is to analyse the temporal change of genetic diversity in Italian durum wheat germplasm. The germplasm deployed in this study (158 accessions), belonging to 5 different historical classes, was characterised for its microsatellite and gliadin markers. The level of genetic diversity (He), based on gliadin and SSR markers results – on average – greater in indigenous landraces present in Italy before 1915, with the exception of pure line material which had been selected from landraces (showing highest level of heterozigosity for gliadin markers). Genotypes obtained from crosses or mutagenesis (referring to the 1950–1960 period) along with those resulting from crosses between CIMMYT lines and old materials (1970s and beyond) were also genetically more diverse. Forty-nine percent of indigenous landraces were genetically heterogeneous. Nine out of 53 landrace accessions were able to capture 4 different SSR private alleles. It is speculated that the reduction of allele richness is an indicator of the genetic erosion of the pre-breeding germplasm and it is pointed out that the implementation of appropriate methods of genetic conservation of this germplasm is a priority for breeding and food safety.