Ex situ conservation of underutilised fruit tree species: establishment of a core collection for Ficus carica L. using microsatellite markers (SSRs)

Ex situ germ plasm collections of woody crops are necessary to ensure the optimal use of plant genetic resources. The fig tree (Ficus carica L.) germ plasm bank, consisting of 229 accessions, is located in Centro de Investigación ‘La Orden’. Despite great progress in conservation, ex situ collections face size and organization problems. Core collections obtained from structured samples of bigger collections are a useful tool to improve germ plasm management. In this work, we used simple sequence repeat (SSR) markers to establish a core collection in this underutilised Mediterranean fruit tree species. Four approaches have been carried out (random sampling, maximization, simulated annealing and stepwise clustering) to determine the best method to develop a core collection in this woody plant. The genetic diversity obtained with each subset was compared with that of the complete collection. It was found that the most efficient way to achieve the maximum diversity was the maximization strategy, which, with 30 accessions, recovers all the SSR alleles and does not show significant differences in allele frequency distribution in any of the loci or in the variability parameters (H _O, H _E) between the whole and core collections. Thus, this core collection, a representative of most fig diversity conserved in the germ plasm bank, could be used as a basis for plant material exchange among researchers and breeders.     

High‐throughput genotyping for species identification and diversity assessment in germplasm collections

Germplasm collections provide an extremely valuable resource for breeders and researchers. However, misclassification of accessions by species often hinders the effective use of these collections. We propose that use of high‐throughput genotyping tools can provide a fast, efficient and cost‐effective way of confirming species in germplasm collections, as well as providing valuable genetic diversity data. We genotyped 180 Brassicaceae samples sourced from the Australian Grains Genebank across the recently released Illumina Infinium Brassica 60K SNP array. Of these, 76 were provided on the basis of suspected misclassification and another 104 were sourced independently from the germplasm collection. Presence of the A‐ and C‐genomes combined with principle components analysis clearly separated Brassica rapa, B. oleracea, B. napus, B. carinata and B. juncea samples into distinct species groups. Several lines were further validated using chromosome counts. Overall, 18% of samples (32/180) were misclassified on the basis of species. Within these 180 samples, 23/76 (30%) supplied on the basis of suspected misclassification were misclassified, and 9/105 (9%) of the samples randomly sourced from the Australian Grains Genebank were misclassified. Surprisingly, several individuals were also found to be the product of interspecific hybridization events. The SNP (single nucleotide polymorphism) array proved effective at confirming species, and provided useful information related to genetic diversity. As similar genomic resources become available for different crops, high‐throughput molecular genotyping will offer an efficient and cost‐effective method to screen germplasm collections worldwide, facilitating more effective use of these valuable resources by breeders and researchers.     

Lessons from leeches: a call for DNA barcoding in the lab

Many evolution of development labs study organisms that must be periodically collected from the wild. Whenever this is the case, there is the risk that different field collections will recover genetically different strains or cryptic species. Ignoring this potential for genetic variation may introduce an uncontrolled source of experimental variability, leading to confusion or misinterpretation of the results. Leeches in the genus Helobdella have been a workhorse of annelid developmental biology for 30 years. Nearly all early Helobdella research was based on a single isolate, but in recent years isolates from multiple field collections and multiple sites across the country have been used. To assess the genetic distinctness of different isolates, we obtained specimens from most Helobdella laboratory cultures currently or recently in use and from some of their source field sites. From these samples, we sequenced part of the mitochondrial gene cytochrome oxidase I (COI). Sequence divergences and phylogenetic analyses reveal that, collectively, the Helobdella development community has worked on five distinct species from two major clades. Morphologically similar isolates that were thought to represent the same species (H. robusta) actually represent three species, two of which coexist at the same locality. Another isolate represents part of a species complex (the "H. triserialis" complex), and yet another is an invasive species (H. europaea). We caution researchers similarly working on multiple wild-collected isolates to preserve voucher specimens and to obtain from these a molecular "barcode," such as a COI gene sequence, to reveal genetic variation in animals used for research.     

Züchtungsforschung auf lange Sicht

Summary Plant breeding research on agricultural crops in the Netherlands is described. A generally high level of breeding can be expected, when there is close cooperation between free research in a university department, directed research in an institute (based on a program planned to produce practical results), and private plant breeders. The increasing activities and efficiency of private breeders call for more basic research and more help on a higher level. Therefore long term programs of the Foundation for Agricultural Plant Breeding (S.V.P.) are necessary to point out to all interested groups in which direction research may be expected to go in the coming period. For long term programs private breeders know in which parts of their work the S.V.P. is supposed to help them.The board and heads of the institute will have formed a better overall picture of the situation so that priority of the different problems can be estimated and flexibility in research work can be maintained. It is necessary that the research institute and private breeders continually be in contact to avoid misunderstanding, duplication and unwanted gaps. To realize this the research workers from the institute should regularly visit private breeders and vice versa.Furthermore working groups in each of the crops with which S.V.P. is working should meet at least once a year to exchange ideas and discuss the actual situation. The S.V.P. itself should not breed varieties but should leave this entirely to private breeders. In the Netherlands this system has proved to be very workable and satisfactory.The main tasks of this research work are: (1) Research in collections. — (2) Detection of important characters and their genetical analyses. — (3) Introduction of new characters. — (4) Building up parental lines for the benefit of private breeders. — (5) Research on breeding methods. — (6) Research on testing methods. — (7) Studies on the influence of environmental conditions.Consequently the type of specialized research workers has to be changed from crop experts to problem solvers. The plant material resulting from their research serves as a building stone in the program of the private breeders.The increasing number of specialized problems makes it impossible to do all the necessary fundamental research at a plant breeding institute. Cooperation with other institutes, specialized in the discipline involved, is very important. To maintain a high level of research activities it is suggested that strong cooperation between institutes in different countries should be aimed at.

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Herrn Prof. Dr.R. v. Sengbusch zum 70. Geburtstag gewidmet.     

Developing a common bean core collection suitable for association mapping studies

Because of the continuous introduction of germplasm from abroad, some collections have a high number of accessions, making it difficult to explore the genetic variability present in a germplasm bank for conservation and breeding purposes. Therefore, the aim of this study was to quantify and analyze the structure of genetic variability among 500 common bean accessions to construct a core collection. A total of 58 SSRs were used for this purpose. The polymorphism information content (PIC) in the 180 common bean accessions selected to compose the core collection ranged from 0.17 to 0.86, and the discriminatory power (DP) ranged from 0.21 to 0.90. The 500 accessions were clustered into 15 distinct groups and the 180 accessions into four distinct groups in the Structure analysis. According to analysis of molecular variance, the most divergent accessions comprised 97.2% of the observed genetic variability present within the base collection, confirming the efficiency of the selection criterion. The 180 selected accessions will be used for association mapping in future studies and could be potentially used by breeders to direct new crosses and generate elite cultivars that meet current and future global market needs.     

Intragenomic sequence variation of the ITS-1 region within a single flow-cytometry-counted Cyclospora cayetanensis oocysts

Cyclospora cayetanensis, a protozoan of emerging concern, causes self-limiting gastroenteritis in immune-competent hosts. It has been established that sequence variability exists in the first internal transcribed spacer region (ITS-1) of the ribosomal DNA operon from collections of oocysts obtained from individual or pooled fecal samples. To determine if single oocysts also exhibited ITS-1 sequence variability, DNA was extracted from individually flow-cytometry-counted oocysts. We determined that ITS-1 sequence variability exists at an individual-genome level for C. cayetanensis and approached or exceeded the variability exhibited among oocyst collections. ITS-1 variability, at the genome level, reduces this region's utility for inferring relationships between strains.     

Mature male parr contribution to the effective size of an anadromous Atlantic salmon (Salmo salar) population over 30 years

We describe temporal changes in the genetic composition of a small anadromous Atlantic salmon (Salmo salar) population from South Newfoundland, an area where salmon populations are considered threatened (COSEWIC 2010). We examined the genetic variability (13 microsatellite loci) in 869 out‐migrating smolt and post‐spawning kelt samples, collected from 1985 to 2011 for a total of 22 annual collections and a 30 year span of assigned cohorts. We estimated the annual effective number of breeders (N_b) and the generational effective population size (N_e) through genetic methods and demographically using the adult sex ratio. Comparisons between genetic and demographic estimates show that the adult spawners inadequately explain the observed N_e estimates, suggesting that mature male parr are significantly increasing N_b and N_e over the study period. Spawning as parr appears to be a viable and important strategy in the near absence of adult males.     

Cryopreserved storage of clonal germplasm in the USDA National Plant Germplasm System

The US Department of Agriculture-Agricultural Research Service (USDA-ARS), National Plant Germplasm System (NPGS) plant collections are a critical source of genetic diversity for breeding and selection of improved crops, including vegetatively propagated plants. Information on these collections is readily accessible to breeders and researchers on the internet from the Germplasm Resources Information Network (GRIN). The clonal collections are at risk for loss due in part to their genetic diversity that makes growing them in one location a challenge, but also because it is difficult to have duplicate collections without incurring great expense. The development of cryopreservation techniques during the last two decades provides a low maintenance form of security backup for these collections. National plant collections for vegetatively propagated crop plants and their wild relatives are maintained by the USDA-ARS, NPGS at 15 sites across the country. These sites include various combinations of field, greenhouse, screenhouse, and in vitro collections. Cryopreserved backup collections in liquid nitrogen storage were instituted in the 1990s, increased greatly in the 2000s with the advent of new techniques, and are continuing today. Collections of dormant buds of temperate trees, shoot tips of in vitro cultures of many crops, and embryonic axes of some large seeded or recalcitrant seeded plants are all part of the clonal backup storage system.     

Methodological innovations for the study of irreplaceable samples reveal giardiasis in extinct animals (Nothrotherium maquinense and Palaeolama maior)

The use of diagnostic methods that prevent irreplaceable samples (from museum collections, archaeological and paleontological samples) of being consumed or that increase their yield is relevant. For museum collections, archaeological and paleontological samples it is essential to conserve samples, subsamples or portions for future research. We are addressing methods for conservation of irreplaceable samples that could be fully consumed. Innovations in methodologies that are used in studies of Paleoparasitology and Paleomicrobiology will contribute to the preservation of collections. Therefore, to the development of archaeology and paleontology in the future, we evaluated whether the discarded material of the immunochromatography test could be used for molecular diagnosis and vice versa. We used a genotyped experimental coprolite positive for Giardia duodenalis. The diagnosis was positive for giardiasis in both cases. This methodology can be corroborated with the coprolite of a Paleolama maior (extinct llama) previously diagnosed for G. duodenalis with an immunoenzymatic test. The residue of the pre-digestion step of the DNA extraction before adding Proteinase K was confirmed positive with the immunochromatographic test. Also, the DNA extraction residue from a coprolite of Nothrotherium maquinense (ground sloth) was tested positive with immunochromatographic test for G. duodenalis. These are the oldest findings for G. duodenalis confirming that this intestinal parasite occurred among Northeastern Brazilian Megafauna animals from the late Pleistocene period, correlated to human occupation. The relevance of these results will allow the study by different methodological approaches from a small amount of material, reusing discarded materials.     

Genetic and germ plasm stocks worth conserving

The relative costs and benefits of genetic stock collections and germ plasm collections are discussed. The status of national and international collections is compared with the needs of plant breeders and geneticists. There is an international need for germ plasm systems that emphasize the use and employment of materials rather than acquisition and storage. For base collections to function, they must provide for regeneration, characterization, documentation, and evaluation of their materials. The quality of a germ plasm system should be judged on the basis of the quality of the materials available to scientists. Adequate quantities of high-quality seed that are of known provenience, spanning the range of known genetic diversity, promptly delivered, and well described constitute the minimum that should be expected. All too often such minimal requirements are not met.     

Nondestructive DNA extraction method for mitochondrial DNA analyses of museum specimens

Museum specimens have provided the material for a large proportion of ancient DNA studies conducted during the last 20 years. However, a major drawback of the genetic analyses is that the specimens investigated are usually damaged, as parts of skin, bone, or a tooth have to be removed for DNA extraction. To get around these limitations, we have developed a nondestructive extraction method for bone, tooth, and skin samples. We found that it is possible to amplify mitochondrial DNA (mtDNA) sequences up to at least 414 bp long from samples up to 164 years old. Using this method, almost 90% (35 of 40) of the investigated samples yielded amplifiable mtDNA. Moreover, we found that repeated extractions of the same samples allowed amplifications of the expected length for all samples at least three times and for some samples up to at least five times. Thus this method opens up the possibility to repeatedly use museum collections for mtDNA analyses without damaging the specimens and thus without reducing the value of irreplaceable collections for morphological analyses.     

Effort to reconstruct past population history in the fern <Emphasis Type="Italic">Blechnum spicant</Emphasis>

Our aim was to evaluate the potential of existing herbarium collections in reconstructing the past population history of the rediscovered clone of the regionally extinct Blechnum spicant by comparing it with herbarium material from previous sites of occurrences in Finland and elsewhere in northern Europe, as well as to reveal the genetic and geographic relationship among the samples. We detected a total of nine polymorphic sites within the three sequenced regions, two SCAR markers developed now for B. spicant and the chloroplast trnL-trnF spacer, totalling 763 bp. Despite low variability, the phylogeographic analysis revealed the presence of some geographic pattern among the samples, of which all except one represented herbarium samples of up to 100 years of age. The rediscovered clone of B. spicant proved to represent the prevalent genotype occurring in northern Europe. The studied sequences were neutral in terms of evolution. It is apparent that existing herbarium collections are useful resources for a range of evolutionary and population studies.     

Analysis of host response to bacterial infection using error model based gene expression microarray experiments

A key step in the analysis of microarray data is the selection of genes that are differentially expressed. Ideally, such experiments should be properly replicated in order to infer both technical and biological variability, and the data should be subjected to rigorous hypothesis tests to identify the differentially expressed genes. However, in microarray experiments involving the analysis of very large numbers of biological samples, replication is not always practical. Therefore, there is a need for a method to select differentially expressed genes in a rational way from insufficiently replicated data. In this paper, we describe a simple method that uses bootstrapping to generate an error model from a replicated pilot study that can be used to identify differentially expressed genes in subsequent large-scale studies on the same platform, but in which there may be no replicated arrays. The method builds a stratified error model that includes array-to-array variability, feature-to-feature variability and the dependence of error on signal intensity. We apply this model to the characterization of the host response in a model of bacterial infection of human intestinal epithelial cells. We demonstrate the effectiveness of error model based microarray experiments and propose this as a general strategy for a microarray-based screening of large collections of biological samples.     

Developing an appropriate strategy to assess genetic variability in plant germplasm collections

 The value of molecular biology for monitoring the genetic status of germplasm collections is subject to practical limitations. The large number and variability of accessions held usually dictates the approach that can be employed. A quick, simple but reliable molecular protocol must be combined with an appropriate strategy for handling large sample sizes. In this study, ISSR-PCR was used to reveal genetic variability within and between accessions held in a collection of lupin germplasm. Pooling of DNA from individuals within accessions was found to be the most appropriate strategy for assessing large quantities of plant material. Band profiles generated from pools containing five individuals were fully representative of all constituent individuals used in the mix. Pools comprising 10 or 20 individuals, however, sometimes failed to contain minor bands that had been present only in the profile of one individual. Variation was observed between pools containing five different genotypes from the same accession. Routine large-scale screens are required to assess the genetic diversity and homogeneity of the lupin germplasm collection held in Reading. It is concluded that 2–3 pools of five genotypes may be sufficient to represent the genetic variability within and between accessions in the lupin and similar collections. Received: 10 August 1998 / Accepted: 13 November 1998     

Determination of the biological diatom index (IBD NF T 90–354): results of an intercomparison exercise

A diatom intercomparison exercise on sampling, slide preparation, countsand identification has been carried out in September 1999 with 24French-speaking diatomists on the River Loup (Alpes Maritimes, France). Fewoperators strictly respected the standard, especially for the number ofindividuals to be counted. It has however been demonstrated this had a very lowimpact on the index value and that old counts can be used for indexcalculation,if there are at least 300 frustules or valves. Counts higher than 400 did notlead to a better precision. The total variability largely depended on theoperator and was distributed unevenly between sampling, which appears to be acritical stage, slide preparation and counting. For those who strictlyrespectedthe standard procedure, the natural variability can be estimated to about oneunit value of the Biological Diatom Index. This intra-operator variability canbe considered as very low, as the IBD scale is from 0 to 20, and notsignificantin assessing water quality. Despite this, the inter-operator variability isgreater. When comparing operators' results, after excluding those who did notrespect the standard requirements, the main source of variability is due tomisidentifications, especially for small Achnanthes(A. minutissima and A. biasolettiana)and Cocconeis (C. placentula var.placentula, var. euglypta and var.lineata). Intercomparison exercises, internet exchangesforchecking and reference material collections should be encouraged both tosatisfythe internal and external quality controls and to acquaint biologists with theimportance of field and laboratory protocols, as well as to allow progress indiatom identification.     

Tomato genetic resources and activities in Poland

The vegetables germplasm conservation programme in Poland was started in 1982, activities under this program resulted up to this date in building up a tomato germplasm collection of 782 accessions that are available for distribution to breeders and researchers in Poland and abroad. Between 1990 and 1999 we distributed 651 tomato seed samples to breeders and scientists in Poland, and 95 samples to users abroad. During 1990 – 1999 the 652 tomato accessions were evaluated for 43 traits of plants and fruits. Work is in progress to expand the collection and identify useful characteristics of the collected material, and to make use of them in breeding and other research works.     

The role of RAPD markers in breeding for disease resistance in common bean

Diseases are regarded as the leading constraint to increased common bean (Phaseolus vulgaris L.) production worldwide. The range in variability and complexity among bean pathogens can be controlled with different single gene and quantitative resistance sources. Combining these resistance sources into commercial cultivars is a major challenge for bean breeders. To assist breeders, a major effort to identify RAPD markers tightly linked to different genes was undertaken. To date, 23 RAPD and five SCAR markers linked to 15 different resistance genes have been identified, in addition to QTL conditioning resistance to seven major pathogens of common bean. We review the feasibility of using marker-assisted selection (MAS) to incorporate disease resistance into common bean. Indirect selection of single resistance genes in the absence of the pathogen and the opportunity afforded breeders to pyramid these genes to improve their longevity and retain valuable hypostatic genes is discussed. The role of markers linked to the QTL controlling complex resistance and the potential to combine resistance sources using marker based selection is reviewed. Improving levels of selection efficiency using flanking markers, repulsion-phase linkages, co-dominant marker pairs, recombination-facilitated MAS and SCAR markers is demonstrated. Marker-assisted selection for disease resistance in common bean provides opportunities to breeders that were not feasible with traditional breeding methods.