Preservation of plant genetic resources in the biotechnology era

Thousands of years ago humans began domesticating crops as a food source. Among the wild germplasm available, they selected those that were best adapted for cultivation and utilization. Although wild ancestors have continued to persist in regions where domestication took place, there is a permanent risk of loss of the genetic variability of cultivated plants and their wild relatives in response to changing environmental conditions and cultural practices. Recognizing this danger, plant ex situ genebank collections were created since the beginning of the last century. World-wide, more than 6 million accessions have been accumulated including the German ex situ genebank in Gatersleben, one of the four largest global collections, housing 150,000 accessions belonging to 890 genera and 3032 species. This review summarizes the ex situ plant genetic resources conservation behavior with a special emphasis on German activities. Strategies for maintenance and management of germplasm collections are reviewed, considering modern biotechnologies (in vitro and cryo preservation). General aspects on genetic diversity and integrity are discussed.     

Simple sequence repeat (SSR) analysis for assessment of genetic variability in apricot germplasm

Thirty SSR primer combinations, developed from peach SSR-enriched genomic libraries and BAC libraries of peach [ Prunus persica (L.) Batsch.], were tested for cross amplification with 74 apricot ( Prunus armeniaca L.) germplasm accessions. Twelve primer pairs amplified 14 polymorphic SSR loci useful for discriminating most apricot cultivars, as well as for investigating patterns of variation in apricot germplasm. Levels of polymorphism were higher than the levels described using other codominant marker systems (i.e., isozymes, RFLP markers). Overall, 107 alleles were identified, and all but 11 accessions were unambiguously discriminated. Genetic differentiation of native germplasm into traditional ecogeographical groups was low, with a high level of genetic identity (> 0.75) between the groups. However, neighbor joining cluster analysis of marker distances between cultivars reflected the complex history of apricot domestication, producing groupings not evidently based on the geographical origin of the cultivars. Distant positioning of Chinese cultivars on UPGMA and neighbor joining dendrograms supports the authors' consideration of Chinese apricots as subspecies, Prunus armeniaca var. ansu Maxim., rather than a separate species.     

Tetrahydrocannabinol levels in hemp (cannabis sativa) germplasm resources

In most of the western world where industrial hemp, Cannabis sativa, is licensed for cultivation, the plants must not exceed a level of 0.3% tetrahydrocannabinol (THC), the principal intoxicating constituent of the species. Because there are no publicly available germplasm hemp collections in North America and only a very few, recent North American cultivars have been bred, the future breeding of cultivars suitable for North America is heavily dependent on European cultivars and European germplasm collections. Based mostly on material from Europe, this study surveyed THC levels of 167 accessions grown in southern Ontario, making this the largest survey to date of germplasm intended for breeding in North America. Forty-three percent of these had THC levels ≥0.3% and, therefore, are unsuitable for hemp development in North America. Discrepancies were found between THC levels reported for some germplasm holdings in Europe when they were grown in Canada and, accordingly, verification of THC levels developed in North America is necessary.     

Is It Still Necessary to Continue to Collect Crop Genetic Resources in the Mediterranean Area? A Case Study in Catalonia

Crop genetic resources have been extensively collected in Europe in the last century, creating large, publicly available ex situ collections. While this huge genetic diversity is often underutilized, in recent decades, several initiatives have emerged at the local level to collect germplasm cultivated on farm. Uncoordinated actors often carry out these collecting missions without considering previously collected data. To explore whether new collecting missions are likely to be worthwhile, we studied the crop genetic resources conservation network in Catalonia by analyzing the passport data and geographical distribution of germplasm stored in seed banks. Moreover, to determine whether this germplasm was representative of the diversity cultivated on farm, we performed new collecting missions in four randomly selected areas in the European Union’s Natura 2000 network and compared the results with the ex situ databases. Seed banks currently hold a large germplasm collection (2931 accessions), although most materials are conserved in private collections without regulated systems for seed regeneration and are not present as duplicates in the National Inventory. One important shortcoming of the ex situ network is that the germplasm conserved ex situ shows a low geographical coverage, representing only 35.3% of the municipalities in Catalonia. Our new missions allowed us to collect 234 accessions, mostly tomatoes (17.5%) and beans (16.2%). The ecological indicators’ richness (both at species (S) and variety (V) levels), total abundance (A), and the Shannon-Weaver diversity index calculated at species (H2, considering the different accessions of each variety as a single population) and variety levels (H3, considering the intra-varietal genetic diversity) were higher in the newly collected germplasm than in the ex situ collections, suggesting that seed banks do not accurately represent the genetic diversity still cultivated on farm. Moreover, some important landraces from each area were absent or underrepresented in the ex situ collections. Thus, it is necessary to continue to devote efforts to collecting germplasm; better organization between actors and targeting specific species/varieties can increase the efficiency of new collecting missions. As a conclusion, we propose different criteria to guide new missions and to improve the network’s conservation activities.     

How bryophytes came out of the cold: successful cryopreservation of threatened species

The use of ex situ techniques for the conservation of threatened plants has been increasing over the past decades. Cryopreservation is often used for the long-term storage of plant germplasm where conventional methods (i.e. seedbanking) are inappropriate. Simple encapsulation–dehydration protocols were developed for the cryopreservation of bryophytes at The Royal Botanic Gardens, Kew, as part of an ex situ project for the conservation of UK threatened species. The applicability of these methods was tested on 22 species with a broad range of ecological requirements and found to be highly successful across the board. Regeneration rates from frozen material were >68% for all species tested and half had regeneration rates of 100%. The high regeneration rate and broad applicability of the protocols across a range of species was attributed to a combination of the inherent totipotency of bryophytes and the in-built recovery periods in the pre-treatment protocol. In conclusion, bryophytes are well suited to cryopreservation and such techniques would be applicable for the long-term storage of similar conservation collections across the globe.     

Grafting versus seed propagated apricot populations: two main gene pools in Tunisia evidenced by SSR markers and model-based Bayesian clustering

Apricot was introduced into the Mediterranean Basin from China and Asian mountains through the Middle-East and the Central Europe. Traditionally present in Tunisia, we were interested in accessing the origin of apricot species in the country, and in particular in the number and the location of its introductions. A set of 82 representative apricot accessions including 49 grafted cultivars and 33 seed propagated ‘Bargougs’ were genotyped using 24 microsatellite loci revealing a total of 135 alleles. The model-based Bayesian clustering analysis using both Structure and InStruct programs as well as the multivariate method revealed five distinct genetic clusters. The genetic differentiation among clusters showed that cluster 1, with only four cultivars, was the most differentiated from the four remaining genetic clusters, which constituted the largest part of the studied germplasm. According to their geographic origin, the five identified groups (north, centre, south, Gafsa oasis and other oases groups) enclosed a similar variation within group, with a low level of differentiation. Overall results highlighted the distinction of two apricot gene pools in Tunisia related to the different mode of propagation of the cultivars: grafted and seed propagated apricot, which enclosed a narrow genetic basis. Our findings support the assumption that grafting and seed propagated apricots shared the same origin.     

Microsatellite variability in apricots (<Emphasis Type="Italic">Prunus armeniaca</Emphasis> L.) reflects their geographic origin and breeding history

A collection of 133 apricot cultivars and three related species originating from different geographical regions were studied with 10 polymorphic microsatellite markers developed in apricot. Altogether, 133 alleles were identified in the set of accessions, with an average of 13.30 alleles per locus. Out of them, 32 alleles occurred only once in the investigated samples, especially in apricots originating from different eco-geographic groups or in different species. The observed heterozygosity for individual loci ranged from 0.8636 to 0.3182, with an average of 0.6281. An unweighted pair group method with arithmetic mean dendrogram based on Nei's genetic distance grouped the accessions according to their eco-geographical origin and/or their pedigree information. Central Asian cultivars have a distinct position on the dendrogram, which supports the assumption that most cultivars have an Asian ancestor. Most East European cultivars analysed cluster together, and the data even revealed a few synonyms. Results show that American cultivars have not only European germ plasm in their pedigree, but they have also been enriched with germ plasm of Asian origin. The implications of these data for the use of simple sequence repeat (SSR) markers as a tool for fingerprinting cultivars in breeders' rights protection and apricot breeding are discussed. In this paper, we demonstrate for the first time the variability of apricot SSRs in a large collection of apricot cultivars and closely related species.     

Genetic Diversity and Population Structure of the Brachiaria brizantha Germplasm

Brachiaria brizantha (Hochst. ex A. Rich.) Stapf. (syn. Urochloa brizantha (Hochst. ex A. Rich.) R.D. Webster) is a species used primarily as forage in tropical America and Southeast Asia. B. brizantha has been extensively researched since the 1980s with the initiation of the Tropical Forages Breeding Program conducted by the Brazilian Agricultural Research Corporation (Empresa Brasileira de Pesquisa Agropecuária; EMBRAPA), holding one of the largest germplasm collections in the world. This work has identified 15 new microsatellite markers for this species, which have been used in addition to five previously reported markers, to estimate the genetic similarities among 172 accessions and six cultivars of this species. Similarity index values ranged from 0.40 to 1.00. Two duplications were found in the germplasm. A Bayesian analysis performed using the STRUCTURE 2.3.3 program revealed the presence of three clusters with different allelic pools. This analysis is valuable for the performance of crosses to explore heterosis; however, the mode of reproduction of the accessions and ploidy barriers must be observed for effective exploration. A grouping analysis using the neighbor-joining method was consistent with the STRUCTURE analysis, and a combination approach suggested that this germplasm collection does not exhibit considerable genetic variability despite the presence of three distinct allelic pools. The lack of correlation between the genetic and geographic distances is also discussed.     

Genetic fingerprinting of germplasm accessions as an aid for species conservation: a case study with Borderea chouardii (Dioscoreaceae), one of the most critically endangered Iberian plants

BACKGROUND AND AIMS: Molecular markers have changed previous expectations about germplasm collections of endangered plants, as new perspectives aim at holding a significant representation of all the genetic diversity in the studied species to accomplish further conservation initiatives successfully. Borderea chouardii is a critically endangered allotetraploid dioecious member of Dioscoreaceae, known from a single population in the Iberian pre-Pyrenees. This population was reported to be highly structured into two genetically distinct groups of individuals corresponding to their spatial separation along the vertical cliff where it grows. In 1999, the Spanish Government of Aragón launched the first conservation programme for the ex situ preservation of this species, and since then a seed collection has been conserved at the Germplasm Bank of the Universidad Politécnica de Madrid. However, as some seed samples had not been labelled clearly at the time of collection, their origin was uncertain. METHODS: Genetic variation in germplasm accessions of B. chouardii was investigated using microsatellite (simple sequence repeat; SSR) markers. KEY RESULTS: The 17 primer pairs used detected 62 SSR alleles in the 46 samples analysed from five different germplasm stocks. Eight alleles scored from the wild population were not detected in the germplasm samples analysed. The relatedness of the germplasm samples to the wild subpopulations through neighbour-joining clustering, principal coordinates analysis (PCO) and assignment tests revealed a biased higher representation of the genetic diversity of the lower cliff (43 samples) subpopulation than that of the upper cliff (three samples). CONCLUSIONS: The collection of additional samples from the upper cliff is recommended to achieve a better representation of the genetic diversity of this subpopulation. It is also recommended that these stocks should be managed separately according to their distinct microspatial origin in order to preserve the genetic substructuring of the wild population.     

Is there a future for the Cactaceae genera Copiapoa,Eriosyce and Eulychnia? A status report of a prickly situation

Many of Chile’s iconic cactus species are threatened by human activities and global change. In order to safeguard them from extinction, both in situ and ex situ conservation actions are urgently needed. In this paper, an overview is given of the in situ and ex situ conservation status of the mainly Chilean cactus genera Copiapoa, Eriosyce and Eulychnia, including a worldwide survey of living ex situ collections of the species of these genera. From our results, we can conclude that although the threats to Chile’s remarkable biodiversity are now recognized as an environmental problem, and efforts are underway to protect the threatened endemic flora, many of the most threatened species are currently not protected in situ. Although a higher percentage of living accessions of Copiapoa, Eriosyce and Eulychnia in ex situ collections are of known wild origin compared to results of previous studies on other plant groups, the number of available accessions is insufficient to adequately preserve the genetic diversity of the threatened species. Prospects to upscale both in situ and ex situ conservation of the studied genera are discussed.     

Could taxonomic misnaming threaten the ex situ conservation and the usage of plant genetic resources?

Ex situ conservation of plant germplasm, especially seed banking, is a favourable and widely used method for the conservation of plant genetic resources (PGR). The long-term conservation of these resources is fundamental for food security and plant breeding in order to stem the losses in agrobiodiversity and to meet the global challenges that agriculture is facing. The conservation and accessibility of PGR relies on their correct taxonomic labelling and on the building of a searchable database that links ex situ collections together. In the current study, we analysed the impact of taxonomic misnaming in the two major PGR databases (Genesys PGR, EURISCO), listing accessions conserved worldwide. The aim was to understand if taxonomic misnaming issues prevent PGR conservation. We chose as a case-study seed collections of accessions of the genus Citrullus (watermelon genepool), the taxonomy and nomenclature of which have been largely revised in recent times. We observed that taxonomic misnaming greatly limits PGR conservation with only 3% of the accessions listed in the databases correctly named; moreover, 28% were affected by taxonomic errors that prevent the establishment of the accessions’ taxonomic identity, with consequences on their conservation and exploitation. The existence of the problem was also confirmed by the experimental propagation of three misnamed accessions. We suggest herein a series of actions that, put in place, could solve the extant misnaming issues in the databases and prevent their reoccurrence, allowing the correct conservation and the usability by the stakeholders of all the accessions.     

Unused genetic resources: a case study of Polish common oat germplasm

Landraces and old, obsolete cultivars are a rich source of diversity and could become important and easy‐to‐use germplasm resources for breeding. They are characterised by yield stability, broad adaptation, tolerance to diseases and a greater competitiveness in the presence of weeds. The main objective of this study was to estimate and compare the genetic diversity among and within landraces, old cultivars and modern cultivars of common oat. Inter simple sequence repeats were used to study the genetic diversity of 12 modern Polish cultivars, 23 old Polish cultivars, 19 native landraces and 5 contemporary European cultivars. The results indicated a low amount of diversity among Polish modern cultivars, but an even lower diversity among old Polish cultivars, as well as large differences between these two gene pools. As expected, the landraces were the most diverse group and showed the highest internal variation. The landraces and old cultivars might serve as sources of useful alleles that have never been used in breeding. Additionally, it was possible to identify errors and inconsistencies in the passport data of gene‐bank accessions. These results can be applied to the maintenance and management of gene‐bank collections.     

Impact of Mapped SSR Markers on the Genetic Diversity of Apricot (<Emphasis Type="Italic">Prunus armeniaca</Emphasis> L.) in Tunisia

The impact of mapped microsatellites on the study of genetic diversity of Tunisian apricot accessions was assessed. The genetic variability of 47 traditional apricot cultivars originating from several areas in Tunisia was investigated with 32 polymorphic microsatellite loci selected for their location throughout the eight linkage groups of Prunus genome. The higher polymorphism and greater transportability of these markers among Prunus species were proved by the expected heterozygosity (He = 0.56) and Shannon’s index of diversity (I = 1.05), indicating that Tunisian apricot germplasm maintained a substantial level of genetic diversity. According to their geographical origin, the genetic differentiation among groups (north, center, and south; Fst = 0.04) was lower, while the gene flow among groups was consequent (Nm = 4.79), attesting a narrow genetic background of apricot in the country. Both unweighted pair-group method with arithmetic mean dendrogram, based on Nei’s genetic distances and factorial correspondence analysis, separated northern cultivars from central and southern cultivars, revealing the same molecular basis of apricot material in the Center and the South of Tunisia. These results revealed the efficiency of mapped markers for genetic variability measurements compared to randomly ones, however, no advantage was observed considering the genetic relationships among studied accessions.     

Molecular characterization and genetic diversity in an avocado collection of cultivars and local Spanish genotypes using SSRs

In this work, 75 avocado accessions maintained in an ex situ germplasm collection at the E.E. la Mayora in Málaga (Spain) were characterized with 16 microsatellites previously developed in this species. This avocado collection includes both local Spanish genotypes obtained through prospection and genotypes obtained by exchange with different countries. A total of 156 different amplification fragments were detected ranging from 4 to 16 per locus with an average of 9.75 alleles per locus. All the microsatellites were highly informative with an expected heterozygosity higher than 0.5 and a probability of identity below 0.36. The total probability of identity was 2.85x10(-14). Fifteen of the 16 loci studied showed a positive Wright's fixation index (F) indicating a deficit of heterozygotes with an average over all the SSRs of 0.18. A dendrogram was generated using UPGMA (unweighted pair group method with arithmetic averages) based on the Nei and Li similarity index. This dendrogram classified most of the genotypes analyzed into three major groups which mainly differed in racial origin although with low bootstrap support probably due to the presence of many interracial hybrids in the collection. All the genotypes studied could be unequivocally distinguished with the combination of SSRs used except some putative mutations of 'Hass' and an additional group of two cultivars. The results obtained indicate that the set of SSRs used is highly informative and are discussed in terms of their implications for avocado germplasm characterization and management.     

High genetic diversity of in situ and ex situ populations of Madagascan coffee species: further implications for the management of coffee genetic resources

The genetic diversity of endemic Madagascan Coffea species has not received any considerable attention, despite the high extinction threat facing most of the species on the island. In order to address this shortfall, we assessed and compared the genetic diversity and allelic richness of ex situ and in situ populations for four selected species, and 18 ex situ populations maintained at the Kianjavato Coffee Research Station. We also investigated the possibility of in situ and ex situ hybridization events for Madagascan coffee, both within and between species. For these purposes, we used 18 nuclear microsatellite markers, on a total of 37 Coffea populations (4 in situ and 33 ex situ) representing a total of 398 genotypes (96 in situ and 302 ex situ) for 23 species. Our study showed (1) strong differentiation between Madagascan Coffea species; (2) good differentiation between all the populations studied, including those from the same locality (even when the area of the locality was restricted); (3) good evidence for both in situ and ex situ hybridization, although in situ hybridization appeared to be limited; (4) much higher genetic diversity within in situ collections compared to ex situ collections; and (5) that, despite limitations, the Kianjavato Coffee Research Station collection is an extremely valuable resource of Madagascan coffee germplasm. Recommendations for in situ and ex situ germplasm conservation are discussed.     

Single seed Raman measurements allow taxonomical discrimination of Apiaceae accessions collected in gene banks

NIR-FT-Raman spectroscopy was applied for a nondestructive analysis of single seeds (fruit mericarps) of 36 accessions belonging to various species of the Apiaceae family. Main seed components such as fatty acids, polysaccharides, proteins, and lignin were identified based on the obtained Raman spectra. Variation at the species and genus level was related to differences observed between spectra. The application of cluster analysis discriminated among most of the species evaluated and grouped them according to their taxonomical classification. The spectroscopically analyzed seeds germinated and developed into normal seedlings to demonstrate the additional advantage that Raman spectroscopy is nondestructive and can be applied to living seed without harm. These results indicate that Raman spectroscopy is a valuable tool for the rational evaluation and management of genetic resources in ex situ seed collections by providing useful information for taxonomical validation of the accessions.     

Genetic diversity assessment and ex situ conservation strategy of the endangered Dendrobium officinale (Orchidaceae) using new trinucleotide microsatellite markers

Dendrobium officinale (Orchidaceae) is an endangered plant species with important medicinal value. To evaluate the effectiveness of ex situ collection of D. officinale genetic diversity, we developed 15 polymorphic trinucleotide microsatellite loci of D. officinale to examine the genetic diversity and structure of three D. officinale germplasm collections comprising 120 individuals from its germplasm collection base and their respective wild populations consisting of 62 individuals from three provinces in China. The three germplasm collections showed reductions in gene diversity and average number of alleles per locus, but an increase in average number of rare alleles (frequency?≤?0.05) per locus in comparison to their wild populations. However, the differences in gene diversity between the germplasm collections and wild populations were not statistically significant. The analysis using STRUCTURE revealed evident differences in genetic composition between each germplasm collection and its wild population, probably because the D. officinale individuals with distinct genotypes in each wild population were unevenly selected for establishing its germplasm collection. For conservation management plans, we propose that D. officinale individuals with rare alleles need to be conserved with top priority, and those individuals with the most common alleles also should be concerned. The 15 new microsatellite loci may be used as a powerful tool for further evaluation and conservation of the genetic diversity of D. officinale germplasm resources.