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Th. J. L. van Hintum C. C. M. van de Wiel D. L. Visser R. van Treuren B. Vosman
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1 Theor Appl Genet (7) 114: DOI 1.17/s ORIGINAL PAPER The distribution of genetic diversity in a Brassica oleracea gene bank collection related to the evects on diversity of regeneration, as measured with AFLPs Th. J. L. van Hintum C. C. M. van de Wiel D. L. Visser R. van Treuren B. Vosman Received: June 6 / Accepted: 4 November 6 / Published online: 2 February 7 Springer-Verlag 7 Abstract The ex situ conservation of plant genetic resources in gene banks involves the selection of accessions to be conserved and the maintenance of these accessions for current and future users. Decisions concerning both these issues require knowledge about the distribution of genetic diversity within and between accessions sampled from the gene pool, but also about the changes in variation of these samples as a result of regenerations. These issues were studied in an existing gene bank collection of a cross-pollinating crop using a selection of groups of very similar Dutch white cabbage accessions, and additional groups of reference material representing the Dutch, and the global white cabbage gene pool. Six accessions were sampled both before and after a standard regeneration. plants of each of accessions plus 6 regeneration populations included in the study were characterised with AFLPs, using scores for 13 polymorphic bands. It was shown that the genetic changes as a result of standard gene bank regenerations, as measured by AFLPs, are of a comparable magnitude as the diverences between some of the more similar accessions. The observed changes are mainly due to highly signiwcant changes in Communicated by H. H. Geiger. Th. J. L. van Hintum (&) R. van Treuren Centre for Genetic Resources, The Netherlands (CGN), DLO Foundation, PO Box 16, 67 AA Wageningen, The Netherlands C. C. M. van de Wiel D. L. Visser B. Vosman Plant Research International, PO Box 16, 67 AA Wageningen, The Netherlands allele frequencies for a few fragments, whereas for the majority of fragments the alleles occur in similar frequencies before and after regeneration. It is argued that, given the changes of accessions over generations, accessions that display similar levels of diverentiation may be combined safely. Introduction Ex situ conservation of plant genetic resources in gene banks involves selection of accessions to be conserved and the maintenance of these accessions for current and future users. The Wrst element, the choice of accessions, used to be taken rather lightly in the past. It was considered easy to collect or buy seeds and store these in a freezer. This resulted in more than six million accessions conserved ex situ worldwide (FAO 1996) of which over 1, Brassica oleracea accessions in Europe (Boukema and Hintum 1998). However, when it became apparent that all this material had to be characterised and regenerated to be useful, limiting the number of accessions gradually became a new target in plant genetic resources conservation (e.g. McGregor et al. 2). Especially for cross-pollinating crops this appeared an important concern, since regenerating cross-pollinating accessions is far more costly in comparison to self-pollinating crops due to precautions that need to be taken to avoid pollen contamination. Besides reducing the number of accessions, alternative options for managing costs are valid, such as structuring the collection and selecting a core collection (Hintum et al. ). In general, selecting accessions involves criteria such as the threat of genetic erosion, the potential usefulness in breeding and research, the
2 778 Theor Appl Genet (7) 114: representation in other collections, but also the genetic diversity in the accession as related to the other accessions. The second element of ex situ conservation is keeping them available. When germinability starts to decrease, or when seeds run out of stock, accessions need to be regenerated. It is of highest importance that the genetic integrity of the gene bank accessions is maintained. Some change of the genetic composition is generally unavoidable, due to hardly controllable processes such as genetic drift and natural selection. In addition, contamination and unintended human selection occur. Therefore, the dangerous step of regeneration needs to be avoided as much as possible by improving the storage condition to increase lifespan of the stored seeds. EVects of regeneration on the genetic composition of gene bank accessions have been studied previously for self-pollinating species (Börner et al. ; Parzies et al. ), and cross-pollinating species (Chebotar et al. 3; Diaz et al. 1997; Reedy et al. 199). These studies have shown that genetic changes resulting from regeneration can be quite substantial. Diaz et al. (1997) showed considerable genetic shift as a result of regeneration of Brassica accessions at diverent European institutions, Chebotar et al. (3) found large changes in allele frequencies and inxux of up to % of new alleles in the course of 7 13 subsequent regenerations of rye, Parzies et al. () found considerable genetic drift in regenerated barley samples. In the studies of Reedy et al. (199) and Börner et al. (), who studied regenerations of maize and wheat, respectively, the evects of drift and shift were much smaller and certainly not alarming. Both elements, initial selection and regeneration, contain a clear genetic aspect and are inter-related. Questions such as how diverent should accessions be to justify inclusion in the collection or how similar should they be to be considered a duplicate, or a candidate for bulking are related to how much genetic shift and drift is acceptable during regeneration. In the case of the cross-pollinator B. oleracea it has been shown that bulking of similar accessions can be justiwable (Hintum et al. 1996; Phippen et al. 1997) without losing the genetic or historical identity of the accessions. These studies used the conservative approach that only very similar accessions were combined. However, the question is whether one could go further, taking into consideration the changes over regenerations. To study these issues in an existing gene bank collection a selection of groups of very similar Dutch white cabbage accessions ( duplication groups ) was made based on the names and origin of these accessions. Based on historical data, these would be the groups within which bulking would be most feasible. To put this selection into perspective, an additional group representing the entire diversity in Dutch white cabbages, and of white cabbages in the world was made. This resulted in a total of accessions. Six of these accessions were sampled both before and after a regular regeneration, to quantify the evects thereof. All accessions were characterised using AFLP. With the help of the AFLP data the following questions were addressed: 1. What is the magnitude of genetic change occurring during regeneration relative to the diversity in the collection, and does it avect all alleles or just a few? 2. To what extent would bulking be justiwable without changing the genetic identity of the accessions, i.e., how unique are the accessions within their duplication group, relative to the changes during regeneration, how unique are the duplication groups in the context of the total genetic diversity of Dutch cabbages and Wnally how unique are Dutch white cabbages in a global perspective? The analysis focussed on diverences between accessions in duplication groups related to diverences over regenerations and visa versa. The data that were generated also allow an analysis of diverences between accessions in duplication groups related to diverences in the global or Dutch collection. The latter analysis is not discussed here and will be the topic of a subsequent paper. Materials and methods Material A selection was made of seven groups of very similar Dutch white cabbage accessions ( duplication groups ). The accessions were expected to be similar based on their names and origin. It concerned the duplication groups Brunswijker, Gouden Akker, Roem van Enkhuizen, Delikatesse, Amager Kortstronk, Herfstdeen and Langedijker. All CGN accessions in these groups were characterised, except in the group Langedijker. This group was too large, and a selection of 14 out of 34 accessions was made from this group. To put these accessions into perspective, an additional group of nine accessions representing the diversity in Dutch white cabbages and another group of nine accessions representing white cabbages in the rest of the world was made by selecting as diverse material as possible, based on our knowledge of the material. This resulted in a
3 Theor Appl Genet (7) 114: total number of accessions. From six of these accessions samples from before and after a regular gene bank regeneration were included (Table 1). The accessions have been referred to by their CGN number. If regenerations are concerned these numbers are followed by an o (original) or a n (new) indicating the accession before or after regeneration, respectively. At CGN the following procedures are used for regenerating cabbage accessions: (1) Seeds are sown in seed trays at a temperature of 16 C in February. (2) After Table 1 The most relevant passport data of the accessions included in the study. The accessions presented in bold font were included with samples from before and after regeneration Group CGN-nr Name Country P Year Brunswijker CGN11 Brunswijker-Group 1 Netherlands B 1987 CGN1129 Brunswijker Netherlands B 18 CGN732 Brunsviger-Ladu Denmark B Gouden Akker CGN737 Ditmarsker-Primax; Gouden Akker Denmark B CGN738 Gouden Akker Netherlands B 196 CGN739 Gouden Akker Netherlands B 1923 CGN111 Gouden Akker-Goldack Netherlands B 197 CGN149 Gouden Akker-Group 1 Netherlands B 1988 CGN1491 Gouden Akker-Group 2 Netherlands B 1988 Roem van Enkhuizen CGN762 Roem van Enkhuizen Netherlands B 192 CGN1131 Roem van Enkhuizen-Group 1 Netherlands B 1987 Delikatesse CGN1116 Delikatesse Denmark B CGN1128 Delikatesse-Group 1 Netherlands B 1987 Amager Kortstronk CGN114 Amager Lav-Amlaro; Amager Kortstronk Denmark B CGN1134 Amager Kortstronk-Group 1 Netherlands B 1987 CGN7 Deense Kortpoot; Amager Kortstronk Netherlands B 196 Herfstdeen CGN79 Late Herfstdeen-Group 1 Netherlands B 1987 CGN74 Herfstdeen; Grote Herfstdeen Netherlands B 1936 Langedijker CGN73 Langedijker Bewaar-Graag Group 1 Netherlands B 1987 CGN1121 Langedijker Bewaar-Graag Group 2 Netherlands B 1987 CGN71 Langedijker Bewaar-Gewoon Group 1 Netherlands B CGN1166 Langedijker Bewaar-selectie Veldboer Netherlands B CGN7 Langedijker Bewaar-selectie Overtoom Netherlands B 197 CGN748 Langedijker Bewaar-selectie Koopman Netherlands B 1967 CGN74 Langedijker Bewaar-Taai Origineel; Netherlands B Langedijker Bewaar-van Loenen CGN743 Langedijker Bewaar-Harwin Netherlands B CGN11 Langedijker Vroege Witte-Junia Netherlands B 196 CGN1123 Langedijker Vroege Witte-Group 2 Netherlands B 1987 CGN1122 Langedijker Vroege Witte-Group 1 Netherlands B 1987 CGN1132 Langedijker Vroege Herfstwitte-Group 1 Netherlands B 1987 CGN78 Langedijker Vroege Herfstwitte-Hark Netherlands B CGN76 Langedijker Vinter-Hekla Denmark B Dutch genepool CGN763 Succes Netherlands B 194 CGN76 Nora Netherlands B 19 CGN761 Quintal d Alsace Netherlands B 196 CGN1124 Kopenhagen Markt-Group 1 Netherlands B 1987 CGN116 Vezufa Netherlands B 19 CGN118 Blankora Netherlands B CGN742 Kopenhagen Markt Coverno Netherlands B 196 CGN734 Capata Netherlands B CGN224 Septa Netherlands B World genepool CGN17277 Indonesia L 1989 CGN17273 Tai-on no.3 China L 198 CGN177 Nepal L 198 CGN17247 USSR L 1991 CGN778 Fuji Early Japan CGN769 Pazardzhishko; Padobreno Bulgaria B CGN726 Balady improved Egypt L 198 CGN727 Sabeini Egypt L 1986 CGN78 Hungary L Synonyms in the Name column are separated by ;, the column P indicates the population type: B is a variety, L is a landrace
4 78 Theor Appl Genet (7) 114: germination seedlings are transplanted to peat pots in a greenhouse with a temperature of 12 C. (3) In spring the plants are transplanted to the Weld. (4) In the autumn the plants are uprooted from the Welds, potted and overwintered in a greenhouse during which period they receive a vernalization treatment at temperatures between 1 C. () In spring, when Xowering shoots start to appear, 8 plants or more are placed in isolation rooms (gauze cages) in a non-heated greenhouse. Blow- Xies are used for pollination. (6) Accessions are harvested when the majority of plants have ripe seeds. Each accession was characterised on the basis of at least plants. Reproducibility of AFLP bands was monitored by sampling two plants twice for each population. This resulted in 1,832 samples to be characterised. Additionally, on each gel one sample of a standard genotype (plant 31 of accession CGN74) was included for reference. DNA extraction Young leaves from seedlings were collected in 2 ml Eppendorf tubes, frozen in liquid nitrogen, lyophilized and ground with 7 glass pearls in a Retch shaking mill prior to the DNA extraction. DNA extraction was performed according to Fulton et al. (199). AFLP protocol The AFLP method was performed essentially as described by Vos et al. (199). Primary template DNA was prepared in a one-step restiction-ligation reaction. Total genomic DNA ( ng) was digested with U EcoRI and U MseI (Life Technologies) in 4 μl 1 mm Tris HCl buver, 1 mm magnesium acetate, mm potassium acetate, ph 7. for 1 h at 37 C. Subsequently, 1 μl of a ligation mixture containing pmol EcoRI adapter, pmol MseI adapter (Isogen) and 2 U T4 DNA ligase (Life Technologies) in the same solution as before but with.4 mm ATP, was added. This restriction/ligation reaction was incubated for 3 h at 37 C. The resulting primary template was diluted to μl with 1 mm Tris HCl,.1 mm EDTA ph 8. and stored at C until use. AFLP Wngerprints were made using a two-step PCR ampliwcation. The Wrst step (preampliwcation) was performed on primary template using a primer pair based on the sequences of the EcoRI and MseI adapter with one additional selective nucleotide at the 3 end. Ampli- Wcation reactions ( μl) contained μl primary template, ng of each primer,.4 U Taq polymerase,.2 mm of all four dntps, 1 mm Tris HCl ph 8.3, mm KCl, 1. mm MgCl 2 and.1% gelatine. PreampliWcation products were diluted -fold with 1 mm Tris HCl,.1 mm EDTA ph 8. and used as template in the second ampliwcation reaction. This second step (selective ampliwcation) was performed with primers having three selective nucleotides each. AmpliWcation conditions were as described above except that only ng of 33 P-labeled EcoRI primer was used. Standard cycling conditions were: 1 cycle 94 C for s, 6 C for s and 72 C for 1 min. The 6 C annealing temperature was subsequently reduced by.7 C for the next 12 cycles, and continued at 6 C for a remaining 23 cycles. Reaction products were loaded on a 6% polyacrylamide gel (Sequagel-6, Biozym) in 1 TBE electrophoresis buver using a SequiGen 38 cm gel apparatus (BioRad Laboratories). Gels were dried on Whatmann 3MM paper and X-ray Wlms were exposed for 2 days at room temperature. In order to identify primer combinations that yielded well scorable polymorphisms approximately 1 EcoRI/MseI primer combinations were tested on six samples. Suitable combinations were selected based on the number of unambiguously scorable polymorphic bands. Finally two primer combinations were selected for analysis, E39/M36 and E39/M38. Using these two primer combinations, bands could be scored consistently (77 for the Wrst, 73 for the second primer combination). Bands were scored by two persons subsequently. Reproducibility of bands over gels was, apart from running the reference sample, tested by repeating PCR reactions from preampliwcation reactions as well as from the original DNA samples. These tests showed good reproducibility (data not shown). Scoring Out of the bands scored, 36 were monomorphic, 19 for the E39/M36 and 17 for the E39/M38 combination. During the analysis, an additional 11 bands showed not to be reliable for analysis. All these were removed from the data set, resulting in a total of 13 polymorphic bands scored. Since in each population samples 1 and 2, and samples 3 and 4 were respectively sampled from the same individual, the AFLP prowles could be checked for reliability. 1,131 comparisons could be made without missing values, of which 17 appeared diverent in the two plants (.%). Plants, number 2 and 4 were removed from the data set, except if the number of missing values was higher in the 1 or 3. The resulting number of plants included in the analysis was 1,714, scored for 13 bands. Since the percentage of missing values was 4.7%, the resulting number
5 Theor Appl Genet (7) 114: of data points was 16,82, an average of 3, per accession, and 98 per plant. Statistical analysis Since AFLP bands are dominant, allele-frequencies were estimated from the band frequencies, by using the frequency (q) of absent bands as an estimate of the squared frequency of the recessive allele, assuming Hardy Weinburg equilibrium in the population. Gene diversity (H), calculated as 2q(1 q), is used as a measure of the genetic diversity in accessions or groups of accessions (for discussions, see Crow 1986). To correct for small sample sizes, an unbiased estimate of H is obtained by multiplying the value by the factor n/(n 1) where n is the number of individuals measured (Nei 1987). These marker diversities are averaged over all markers to calculate the genetic diversity within a group (H T ) or within an accession (H S ). G ST was used as a measure of genetic diverentiation between accessions (see Nei 197 for a discussion). It is the proportion of the total genetic diversity that can be attributed to diverences among accessions: (H T H S )/H T in which H T is the total genetic diversity in a set of accessions and H S is the average diversity within these accessions. The probability of the realised G ST of two populations (an accession before and after regeneration) occurring by chance was determined by simulating the division in groups using the actual Wngerprints of the accessions before and after regeneration, and calculating the G ST of the simulated groups. This was repeated 1, times. The probability of changes of individual allele frequencies was estimated using the chi-square (χ 2 ) analysis of the 2 2 contingency table with the number of present and absent alleles before and after regeneration (one degree of freedom). The Bonferroni (1936) correction was considered but not applied given the lack of independence between the tests. For the clustering, an agglomerative approach was used in which the genetic distance between clusters was calculated using Roger s distance (Rogers 1972; Wright 1978). This distance in this two-state case is basically the sum of absolute band frequency diverences. The clustering algorithm used the cluster size as weight. This implied that after each clustering step the frequencies of bands in the newly formed cluster were determined on the basis of all individuals in that cluster and distances of the new cluster with all others were calculated. All data analyses were performed using software written in MS Visual Basic, interfacing with MS-Excel. Results Diversity within and between accessions and regenerations The genetic diversity (H S ) within the accessions varied from. in CGN111, member of the Gouden Akker group, to.18 in CGN732 of the Brunswijker group. The average genetic diversity within single accessions was.13 and the total diversity (H T ) was.24 (Table 2). The genetic diverentiation (G ST ) in the complete set was thus.4. The genetic distances between individual accessions, including the regenerated material, as measured with Rogers distance, were the smallest between the populations before and after regeneration, in the range.4.. The smallest distance between two diverent accessions was. between CGN739 and CGN1491, both of the Gouden Akker group. This implied that the distance between the most similar accessions were of the same order of magnitude as the changes caused by regeneration. The largest distance was. between CGN1116 of the Delikatesse group and CGN727, an Egyptian landrace. Weighted clustering of accessions and regenerations based on Rogers distance resulted in Fig. 1, where the genetic distances between the accessions before and after regeneration are shown, together with the diversity in the most homogeneous duplication group Gouden Akker and the most diverse group Langedijker. EVects of regeneration Comparison of the accessions before and after regeneration showed only minor changes in the diversity within the accessions (Table 3). However, due to the diverences between the levels of diversity within the Table 2 The groups with the number of accessions included in the study (N acc ), the average genetic diversity within accessions (H S ), the total genetic diversity in the group (H T ), and the smallest and largest genetic distance between accessions (D low and D high ) Group N acc H S H T D low D high Brunswijker Gouden Akker Roem van Enkhuizen Delikatesse Amager Kortstronk Herfstdeen Langedijker Dutch genepool World genepool Overall
6 782 Theor Appl Genet (7) 114: Fig. 1 Dendrograms showing the six regenerations, and groups Gouden Akker and Langedijker, based on Roger s distance and weighted clustering Table 3 The regenerated accessions with their diversity before regeneration (H S -o) after regeneration (H S -n), the genetic diverentiation between regeneration (G ST ) and the probability of these values resulting from sampling errors (Prob) CGNnr H S -o H S -n G ST Prob CGN CGN CGN CGN CGN CGN accessions, the genetic diverentiation (G ST ) over regeneration divered considerably, from. to.. The probability of such diverentiation being only due to sampling evects was in two out of the six cases below %, so most of the changes were not signiwcant. The changes over regenerations were also examined by comparing the allele frequencies of individual loci. This showed that most of the allele frequencies did not change signiwcantly, but in every population there were a few that did. These changes were highly signiwcant (Fig. 2), between 2 and 21% of the polymorphic bands were signiwcantly diverent at the 1% level. A Bonferroni correction was considered to avoid the chance of erroneously considering diverences signiwcant as a direct result of the high number of tests performed (Bonferroni 1936), but this was not considered appropriate here since the observations could not be assumed to be uncorrelated (Bland and Altman 199; Perneger 1998). However, even if the Bonferroni correction would have been applied most of the highest chi-squares would still have been signiwcant since the % signiwcance level with 6 independent tests (the highest number of polymorphic bands in a regeneration) would correspond to a chi-square of 11.. A closer examination of all loci with chi-squares over 4., i.e., signiwcant at %, did not reveal any consistent changes. Only a few alleles had changed signiwcantly in more than one regeneration. One allele increased in frequency in three regenerations signiwcantly, in two other it increased slightly, and in the sixth regeneration the allele was already Wxed before regeneration. Discussion When studying the evects of regeneration of a crosspollinating accession, the question whether or not the change by one regeneration cycle is signiwcant depends on the methodology applied. There will be changes, in the best case only small and only due to sampling
7 Theor Appl Genet (7) 114: regeneration CGN74 4 regeneration CGN regeneration CGN114 4 regeneration CGN regeneration CGN769 4 regeneration CGN CGN739-CGN CGN727-CGN1116o 1 1 Fig. 2 Distribution of χ 2 values of the frequencies of polymorphic AFLP bands, before and after regeneration (Wrst six histograms) and in the two most similar and the two most distinct accessions. The X-axis presents subsequent intervals of χ 2 values ( 1 to >), the Y-axis the absolute frequency thereof. These values have 1 degree of freedom, and values above 3.84 are signiwcant at the % level, and values above 6.64 are signiwcant at the 1% level
8 784 Theor Appl Genet (7) 114: evects. But even small changes may become signiwcant by characterising many plants and using exact measurements. A more relevant question is what the magnitude of the change occurring during regeneration is, related to the diverences between the accessions in the collection. If diverences resulting from regeneration exceed those between related accessions, either the improvement of the regeneration protocol or a reduction of the number of accessions should be considered; if a gene bank is not able to maintain the accessions as separate entities over regenerations because the diverences between accessions are too small relative to the changes over regenerations, bulking the most similar accessions should be considered (Sackville Hamilton et al. 2; Hintum et al. 2). Regeneration protocol The regeneration of white cabbage is a rather complicated procedure (see material section). In this protocol there are a few aspects that will have an inxuence on the genetic composition of the accession. First there is infra-accession competition, the plants with many Xowers will produce more pollen and therefore more seed. Secondly, natural selection will occur, for example, there are seeds that do not germinate and plants that do not survive under these conditions. But also unintentional selection can take place. For example, the seeds are harvested once. Seeds that are not ripe yet will not contribute to the next generation, resulting in a selection for earlyness. The next disturbing factor is genetic drift, although the minimum number of 8 plants per accession should be suycient to maintain nearly all alleles in the accession. The loss of variation that may be expected after a single regeneration equals 1/2N e, in which N e is the evective population size (Falconer 1981). Assuming that N e = 8, the loss of variation due to random processes is.6% per regeneration. Finally, there can be contamination, either of pollen, which is prevented by regenerating in isolation rooms, or of seeds during harvesting, drying, cleaning and packing. Changes over regenerations at the allelic level Considering the complexity of the procedure and the threats to the genetic integrity of the accessions, the changes that were observed can be considered small. There was no apparent loss of diversity, and the genetic diverentiation was only in two out of six cases signiwcant, even with thirty plants measured before and thirty after regeneration (Table 3). These diverences between the accessions before and after regeneration are thus mainly due to sampling evects. This is supported by the dendrograms showing the individual plants in the regenerations (not shown) in which, in all six cases, plants did not cluster according to generation. The changes over regenerations were hardly signiwcant at the population level, but at the allelic level there were some very clear frequency changes (Fig. 2). These changes were similar to the diverences between similar accessions within a group such as the diverences between the two most similar accessions found in the study CGN739 and CGN1491 (Fig. 2). Three causes of these diverences should be considered, i.e., methodological errors, random processes and evects of selection. Concerning the methodological errors, the reproducibility was shown to be very high. Occasional errors due to contamination of plants with fungi or other organisms or occasional irreproducibility of PCR product formation in AFLP analysis are possible, but given the consistency of the occurrence of signiwcantly diverent bands not very probable. Concerning the random processes, both genetic sampling evects during the regeneration and statistical sampling evects during the analysis should be considered. The observed diverences were too extreme, however, to be explained by either of those. Genetic sampling evects result in a continuous distribution of χ 2 values, and do not explain the high values that were observed in all regenerations. Statistical sampling evects, due to the high number of χ 2 values calculated, could have been corrected with the Bonferroni correction but this would not change the results as was described earlier. What remains is the third possible cause: the diverences might be the result of close genetic linkage of these loci to QTL s or qualitative traits that were selected during regeneration. However, the fact that the signiwcant diverences are caused by diverent bands in diverent regenerations does not support this hypothesis. In the regeneration of CGN1116, an additional explanation is possible, which is related to the relatively high level of homogeneity in this accession (cf. Table 3). Inbreeding resulting in reduced heterogeneity within the accession, might have reached a level at which problems in subsequent multiplication occur, i.e. matings leading to successful seed set are no longer random due to incompatibility factors. As a consequence, signiwcant shifts in band frequencies can occur in the next generation. In Fig. 2, this is not only observed in the occurrence of band frequency shifts with relatively high χ 2 values, but also in a relative lack of band frequency shifts with a χ 2 of 1 or lower compared to the distribution of χ 2 values in the other regenerations presented in Fig. 2. This pattern is also seen in the CGN comparison (Fig. 2), where the same explanation
9 Theor Appl Genet (7) 114: could apply because of a similar level of genetic diversity in these accessions (Hs of.86 and.89, respectively). Changes over regenerations versus diverences between accessions When the genetic diversity between accessions in the collection is studied, some diverences between individual accessions are surprisingly small (Table 2, Fig. 1). The accessions within the duplication groups Gouden Akker and Herfstdeen (latter not shown) are so closely related that one regeneration would result in comparable genetic diverences, also if the amount of diversity within the accessions is taken into consideration. Some other groups, such as the Langedijker group, which is shown as reference in Fig. 1, show considerable larger diverences, even comparable to the diverences in the Dutch genepool or the world genepool groups. These groups should therefore not be considered as duplication groups. The small diverences between accessions as related to the diverences over regenerations can be interpreted as an indication that the regeneration protocols should be improved. This improvement should be aimed at reduction of selection, since the important changes that were observed were not caused by drift, but only by shift. Selection can be reduced, for example, by harvesting equal amounts of seeds per plant, or by controlled pollination. Such measures, however, will generally be quite costly, and might not be feasible economically. The small diverences between accessions as related to the diverences over regenerations can also be interpreted as an indication that these similar accessions should be combined in a new bulk accession. Some of the accessions in the CGN Brassica collection are already the result of such combination of very similar samples prior to inclusion in the collection (Hintum et al. 1996). Based on the advice of crop experts, who studied the passport data and who looked at the material in the Weld diverent selections from common landraces were merged in one or more groups. This resulted in a considerable reduction of the number of (candidate) accessions. This evort of merging material was supported by a study using isozymes, which concluded that the groups might even have been larger (Hintum et al. 1996). This conclusion is supported by the current study: material of groups such as the Gouden Akker could have been further merged based on the observed diversity. Phippen et al. (1997) also concluded, in their case on the basis of a RAPD study on the Golden Acre group (same as Gouden Akker ), that the number of accessions could be reduced without signiwcant loss of diversity. It should, however, be taken into account that neutral markers such as AFLPs, as used in the current study and RAPDs, as used by Phippen et al. (1997), show mainly neutral diversity whereas germplasm users are interested in functional diversity. The decisions of not combining certain accessions have always been based on the functional diversity they possessed. Based on expert advice it was often decided to keep the distinct morphological or phenological types separate: the early, the Xat or the white. The question how diycult it would be to reconstruct such types via simple mass selection in the combined accession is relevant in this respect. If gene banks would be able to reduce costs without losing genetic diversity by combining genetically highly similar accessions of cross-pollinating crops, distinct phenotypes of morphological or phenological traits might be lost. One could recover these phenotypes by one or two cycles of mass selection, but this would have to be done by the user of the material. This implies that quality, in the form of phenotypes, would be sacriwced to save money. However, this money could be invested with a higher return in other quality aspects of gene bank management. Conclusion The genetic changes over normal gene bank regenerations, as measured by AFLPs, are of a comparable magnitude as the diverences between some of the more similar accessions. These changes and diverences are mainly due to some highly signiwcant diverences in allele frequencies, whereas the majority of alleles occur in similar frequencies. This suggests that selection is taking place. This implies that either regeneration protocols should be improved or the composition of the collection should be changed either by merging, removing or replacing gene bank accession. Acknowledgments The authors would like to thank Ietje Boukema and Liesbeth de Groot for their collaboration in selecting and supplying the material analysed in this experiment, and Rene Smulders and Bert Visser for their detailed comments that greatly improved the quality of the manuscript. The authors are also grateful for the valuable suggestions for improvement given by the reviewers of the paper. References Bland JM, Altman DG (199) Multiple signiwcance tests: the Bonferroni method. Br Med J 31:17
10 786 Theor Appl Genet (7) 114: Bonferroni CE (1936) Teoria statistica delle classi e calcolo delle probabilita. Pubblicazioni del R Istituto Superiore di Scienze Economiche e Commerciali di Firenze 8:3 62 Borner A, Chebotar S, Korzun V () Molecular characterization of the genetic integrity of wheat (Triticum aestivum L.) germplasm after long-term maintenance. Theor Appl Genet 1: Boukema IW, Hintum TJL van (1998) The European Brassica database. Proceedings of an international symposium on Brassicas. Acta Hort 49:249 4 Chebotar S, Röder MS, Korzum V, Saal B, Weber WE, Borner A (3) Molecular studies on genetic integrity of open-pollinating species rye (Secale cereale L.) after long-term gene bank maintenance. Theor Appl Genet 17: Crow JF (1986) Basic concepts in population, quantitative and evolutionary genetics. W.H. Freeman, New York, p 273 Diaz O, Gustafsson M, Astley D (1997) EVect of regeneration procedures on genetic diversity in Brassica napus and B. rapa as estimated by isozyme analysis. Genet Resour Crop Evol 44:23 32 FAO (1996) FAO State of the World s plant genetic resources for food and agriculture. Rome Falconer DS (1981) Introduction to quantitative genetics 2nd edn. Longman, London Fulton TM, Chunwongse J, Tanksley SD (199) Microprep protocol for extraction of DNA from tomato and other herbaceous plants. Plant Mol Biol Rep 13:7 9 Hintum TJL van, Boukema IW, Visser DL (1996) Reduction of duplication in a Brassica oleracea germplasm collection. Genet Res Crop Evol 43: Hintum TJL van, Brown AHD, Spillane C, Hodgkin T () Core collections of plant genetic resources. IPGRI Technical Bulletin No. 3. International Plant Genetic Resources Institute, Rome, p 48 Hintum TJL van, Sackville Hamilton NR, Engels JMM, Treuren R van (2) Accession management strategies: splitting and lumping. In: Managing plant genetic resources. CAB International 2, pp McGregor CE, Treuren R van, Hoekstra R, Hintum TJL van (2) Analysis of the wild potato germplasm of the series Acaulia with AFLPs: implications for ex situ conservation. Theor Appl Genet 14:146 6 Nei M (197) Molecular population genetics and evolution. Elsevier, New York, p 6 Nei M (1987) Molecular evolutionary genetics. Columbia University Press, New York Parzies HK, Spoor W, Ennos RA () Genetic diversity of barley landrace accessions (Hordeum vulgare ssp. vulgare) conserved for diverent lengths of time in ex situ gene banks. Heredity 84: Perneger TV (1998) What is wrong with Bonferroni adjustments. Br Med J 136:6 8 Phippen WB, Kresovich S, Candelas FG, McFerson JR (1997) Molecular characterization can quantify and partition variation among gene bank holdings: a case study with phenotypically similar accessions of Brassica oleracea var. capitata L. (cabbage) Golden Acre. Theor Appl Genet 94: Reedy ME, Knapp AD, Lamkey KR (199) Isozyme allelic frequency changes following maize (Zea mays L.) germplasm regeneration. Maydica 4: Rogers JS (1972) Measures of genetic similarity and genetic distance. In: Studies in Genetics VII. University of Texas Publication 7213, Austin Sackville Hamilton NR, Engels JMM, Hintum TJL van, Koo B, Smale M (2) Accession management. Combining or splitting accessions as a tool to improve germplasm management eyciency. IPGRI Technical Bulletin No.. International Plant Genetic Resources Institute, Rome Vos P, Hogers R, Bleeker M, Reijans M, van de Lee T, Hornes M, Frijters A, Pot J, Peleman J, Kuiper M, Zabeau M (199) AFLP: a new technique for DNA Wngerprinting. Nucleic Acids Res 23: Wright S (1978) Evolution and the genetics of populations, vol. 4: Variability within and among natural populations. University of Chicago Press, Chicago
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Join Brenda Culbertson, NASA/JPL Solar System ambassador, and other astronomers to observe Jupiter and the Galilean satellites in the night sky. See what Galileo saw, but do... details>>
On display: beautiful Harris-Kearney House quilts from the 19th century. The featured exhibitor is Mrs. Tammy Reid, the wife of Andy Reid, the head coach of the Kansas City... details>>
Da Vinci was a dreamer and an inventor. Math, science and art are highlighted in this interactive, hands-on exhibit. Catapults, parachutes, and a hang glider are some of the... details>>
This summer Kansas, Historical Society sites are Blue Star Museums honoring active duty military personnel and their families. Presented by the National Endowment for the Arts and Blue Star Families, this program provides free admission to active military, guard, and reserve personnel and up to five family members at the following state sites. • Kansas Museum of History · Topeka
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PointsBet Announces Early Payouts for All James Harden MVP Bets
Online bookmaker PointsBet has announced that it has issued Early Payouts on all bets for James Harden as MVP of this year's NBA season. The Early Payout comes nearly four months before the winner is officially announced in late June. "Our number one goal right now is to provide unmatched value to our customers"
All PointsBet customers who wagered on Harden to win the 2019 MVP award have received payouts at the odds they placed their bets on those specific markets.
"Our number one goal right now is to provide unmatched value to our customers," said PointsBet US CEO, Johnny Aitken. "There are many operators in the New Jersey space, but PointsBet is the only one offering unique promotions like these. We want to prove that no one has the bettor's back like we do, and this is just another example of that philosophy in action."
This bettor-first initiative is just one of many seen by PointsBet customers since their launch in January, including their most recent NBA Early Payout, where all MoneyLine NBA wagers were paid out early if that team led at Halftime, even if they went on to lose the game. The promotion produced many "Double Payouts," or scenarios where the team that is leading at halftime goes on to lose the game. In each of those instances, both sides of MoneyLine bets on the game are ultimately graded as winners for bettors.


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Manufacturer:Ametek / Lloyd Instruments
Types:Single Column Testing Systems up to 5kN
Industries:Aerospace & Aviation, Chemical & Petrochemical, Food & Beverage, Laboratory & R&D, Manufacturing, Medical & Pharmaceutical, Plastics & Polymers, Transportation & Logistics
Applications:Young's Modulus Testing, Puncture Testing, Polymer Testing Solutions, Plastics Testing Solutions, Pharmaceutical Testing Solutions, Peel Strength Testing, Paper and Cardboard Testing Solutions, Packaging Testing Solutions, Modulus of Elasticity Testing, Metals Testing Solutions, Medical Device Testing Solutions, International Standards – Wire, Yarn and Thread, International Standards – Textiles and Fabrics, International Standards – Plastics and Rubber, Quality Control Testing, Research & Development Testing, Wood Testing Solutions, Toughness Testing, Textile Testing Solutions, Testing by Standard, Tension Testing, Tensile Testing, Tear Strength Testing, Tear Resistance Testing, Stress Relaxation Testing, Spring Testing Solutions, Shear Strength Testing, Rupture Testing, Rubber Testing Solutions, International Standards – Paper and Cardboard, International Standards – Packaging, Durability Testing, Ductility Testing, Delamination Strength Testing, Deformation Testing, Crush Resistance Testing, Creep Testing, Compression Testing, Coefficient of Friction Testing, Building Material Testing Solutions, Break Strength Testing, Bond Strength Testing, Bending Flexural Testing, Automotive Components Testing Solutions, Elastic Limit Testing, Electronics Testing Solutions, International Standards – Metals, International Standards – Medical Devices, International Standards – Glass, International Standards – Footware, International Standards – Foams, International Standards – Electrical Components, International Standards – Composites, International Standards – Ceramics, International Standards – Building Products and Construction, International Standards – Adhesives and Sealants, Friction Testing, Flexural Strength Testing, Elongation Testing, Adhesion Testing
Tag:LS Series
Lloyd Instruments/Ametek LS5 Single Column Bench Mounted
The LS5 Single Column Bench Mounted testing machine is an affordable, easy to use system, ideal for material testing applications up to 5 kN (1124 lbf). The machine uses high accuracy interchangeable YLC Series load cells for tension, compression and cycling through zero force measurements.
The LS5 features an integral control console with membrance multi-function keypad and easy to read backlit LCD to display load and extension information. The control console displays prompts and menus, which guide the user through machine operations. The system is capable of storing up to 600 test results from a choice of 10 programmable test set-ups. Alternatively, machine performance can be optimized using our innovative, easy to use NEXYGENPlus material test and data analysis software. Test information can easily be exported to Microsoft products such as Word, Excel and Outlook.
Single column force testers today represent the largest single market segment of the force testing marketplace for a number of reasons. Capacities now range from a couple of ounces up to 500 Lbf, with a few models offering capacities up to 1,000 Lbf. Read more about single column force testers here.Read More...
- Load Cells
- Grips and Fixtures
- Extensometers
- Thermal Cabinets
- NEXYGENPlus Material Test and Data Analysis Software
- Load Cells
- Grips and Fixtures
- Extensometers
- Thermal Cabinets
- NEXYGENPlus material test and data analysis software
- Simple to set up, operate and maintain
- Load measuring system exceeds the requirements of all recognised international standards
- Uses high accuracy interchangeable YLC load cells (±0.5% from 1% to 100% of load cell value)
- Crosshead travel 800 mm (31.5 in)
- Extended version available with travel up to 1400 mm (55 in)
- Speed range 0.01 – 1016 mm/min (0.0004 to 40 in/min)
- Load rate control
- Data sampling rate 8 kHz
- Saves up to 600 test results
- 10 programmable test set-ups
- High resolution backlit LCD display with intuitive operator interface
- Multi-lingual and multi-unit display options as standard
- Flash memory upgradeable
- Full PC integration with NEXYGENPlus materials test and data analysis software.
|5kN (1124 lbf) Testing Machine
|YLC-0005-A1 0.5% accuracy 5N loadcell
|5 N, 1.1 lbf
|YLC-0010-A1 0.5% accuracy 10N loadcell
|10 N, 2.2 lbf
|YLC-0020-A1 0.5% accuracy 20N loadcell
|20 N, 4.5 lbf
|YLC-0050-A1 0.5% accuracy 50N loadcell
|50 N, 11 lbf
|YLC-0100-A1 0.5% accuracy 100N loadcell
|100 N, 22 lbf
|YLC-0250-A1 0.5% accuracy 250N loadcell
|250 N, 56 lbf
|YLC-0500-A1 0.5% accuracy 500N loadcell
|500 N, 112 lbf
|YLC-1000-A1 0.5% accuracy 1000N loadcell
|1 kN, 225 lbf
|YLC-2500-A1 0.5% accuracy 2500N loadcell
|2.5 kN, 562 lbf
|YLC-5000-A1 0.5% accuracy 5000N loadcell
|5 kN, 1124 lbf


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Seattle Seahawks
How convenient: Deep draft for running backs as Seahawks vow to fix run game
So much for that old belief drafting running backs is as passe in the NFL as the "Ickey Shuffle."
No fewer than 30 running backs got drafted into this supposedly all-passing league across seven rounds last year. Two backs went in the first eight picks, and the success Leonard Fournette, Christian McCaffrey, Alvin Kamara and Kareem Hunt had showed all those backs still have big-time value in the NFL.
This week's draft is deep in running backs, just as coach Pete Carroll makes improving the rushing offense his Seahawks' top priority in 2018. Penn State running back Saquon Barkley is so good he may be the first- or- second-overall pick Thursday.
Carroll and general manager John Schneider have not drafted a running back in the first round in any of their eight drafts leading Seattle. Actually, they've haven't used their initial first-round pick on anybody since 2011. The Seahawks have traded it in six consecutive drafts.
This could be the seventh consecutive year for a first-round trade. Seattle doesn't have a second- nor a third-round choice, the result of trading for defensive tackle Sheldon Richardson and for left tackle Duane Brown during last season. Trading down from 18 in round one could get back that second- and/or third-round selection.
The highest running back Carroll and Schneider have drafted is Christine Michael, in the second round in 2013 at 62nd overall. That didn't work out so well.
The Seahawks have Chris Carson, their surprise lead back to begin last season until the rookie broke his leg on Oct. 1. Carson's returning to Carroll's ongoing raves about him. The team re-signed Mike Davis, who started at the end of last season. Carroll said last month Davis and Carson will enter the preseason competing for the top running-back job.
But Carson has four games of NFL experience, and part of two injury-affected seasons at the major-college level before that, at Oklahoma State. Davis was stuck on Seattle's practice squad for the first 10 weeks of last season, after a two-win San Francisco team waived him following 2016.
Seattle's running backs produced the fewest yards in the NFL in 2017 The days of quarterback Russell Wilson leading the team in rushing by hundreds of yards need to end. So it stands to reason the Seahawks would draft new running backs to accelerate that change.
Here are the top ones in this year's class, and later-round options for the Seahawks. Six of Seattle's eight scheduled picks are in rounds five (four selections) and seven on Saturday.
1. Saquon Barkley, Penn State: The only Nittany Lion with 3,000 rushing yards and 1,000 yards receiving was a complete stud at the combine. It cemented his status as a do-it-all pick at the top of this draft.
2. Derrius Guice, LSU: Says he wants to emulate "Beast Mode," and runs with the power and punishment of Marshawn Lynch. Has endured his father being murdered when he was a child. Will Lynch's old team take Guice in round one?
3. Sony Michel, Georgia: Many have USC's Ronald Jones and Auburn's Kerryon Johnson going higher. But 279 yards in the national semifinal and championship a few months ago show how good Michel is big games. Skilled receiver for every-down NFL work.
Possible later-round options for Seahawks:
1. Royce Freeman, Oregon: Has the power and size (5-11, 234) Carroll loves. Oregon's all-time rushing leader got a ton of carries in college. Question will be whether he's around when Seahawks pick in round four.
2. Kalen Ballage, Arizona State: Huge (6-2, 227), but didn't always run that way at ASU. Then again, there was that eight—8!—touchdown day as a junior against Texas Tech.
3. Lavon Coleman, Washington: Overshadowed at UW by Myles Gaskin. Another bigger back (223 pounds) the Seahawks know well.


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Heavy rust treatment
Operating principle
Removing rust through a chemical process is a very simple and efficient alternative to sanding and blasting. Rust on steel in different alloys turns out well to dissolve in acid.
Benefits of using Ranex:
- Requires little manual power
- Works fast, thorough and efficient
Ranex Rustbuster is based on phosphoric acid and the great advantage of this acid is, that it has no aggressive reactions on material and coating, but it will only react with rust and the mill scale (during a rolling process, iron oxides are pressed in the steel surface). The specific gravity of Ranex Rustbuster is 1.2 and the surface tension is lower that of water. This makes it to penetrate deeply into the surface to destroy the pollution (rust). It is also pushes out moisture that was still present within the rust. This causes the rust to be made inactive up to its core. It also enters hard to reach places.
The detrimental oxide (rust) is destroyed pore deep. The rest product is a beneficial iron phosphate, an inactive substance, that provides good adhesion to the new coating. During the drying process of about 24 hours, the rust is converted and a temporary protective layer is formed. For some weeks, the treated surface is protected, provided in dry environment. For long term protection a coating must be applied.
Required surface condition
- Remove oil, grease, chemicals, loose paint and loose rust
- In old and thick rust, the surface could be phosphated, then it is helpful to first open the surface with coarse sandpaper or a wire brush.
- The area to be treated must be dry
Application method for destroying rust
- Apply Ranex Rustbuster on the dry surface with (block) brush, roller or brush
- Remove overflowing Ranex Rustbuster on the surrounding material (coating or metal) with a damp cloth
- Allow it to dry for at least 24 hours
- in case the surface becomes wet during the drying process, a whitish, powdery substance that occurs is phosphate. Removal of this powder is not necessary, because it will resolve in the coating to be applied for long term conservation
- After drying, the surface is suitable to be coated with a primer (1-or 2-component)
- It is not required to use anti-corrosion paints
- Waterborne paints and zinc-rich paints are not recommended
- Average consumption is about 1ltr. for 8 m2 heavy rust or for 30 m2 of superficial rust and rust stains
[carousel_slide id="785″]


text: + Go to wp console, then click to Accessibility spring in sidebar and enable options that you need to start to work. (Français) Mentions légales | Irancy Saint-Vincent tournante de Bourgogne 2016 Home Saint-Vincent 2016 The Producers Photo Gallery Infos pratiques Latest News Press Sponsors Volunteers (Français) Mentions légales Home(Français) Mentions légales Sorry, this entry is only available in French. search Navigation Home Saint Vincent 2016 Latest News The Producers Portfolio Contacts Language: Français English Archives February 2016 January 2016 December 2015 November 2015 October 2015 September 2015 June 2015 April 2015 March 2015 January 2015 Meta Log in


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Leopard Silk Top
€29.95 €14.50
This silk leopard print blouse is one of our key pieces for the season. We love its rich silk texture and mock neck line. We pair this top with black jeans, heels, and a clutch for drinks while we pair it with high waisted leather crop trousers and white boots for a more editorial look.
Product Code: C19986


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Bazi Suanming Introduction
Traditional Chinese Astrology:
Bazi Suanming "The Calculation of Life According to the Eight Signs"
Bazi Suanming, the "calculation of fate according to the eight signs", respectively the "calculation of life according to the Eight Signs", is a very detailed calculation method for the individual qi and its development during the whole life.
Because the calculations of Bazi Suanming are carried out by means of the birth date of a person and with relation to the Chinese calendar system (wannianli), the technique is also known in the West as "Four pillar astrology" (sizhu suanming), or simply as "Traditional Chinese Astrology" (TCA).
Bazi Suanming starts from the assumption based on the foundations of the Chinese calendar system, that the flow of time is subject to a continuous change of qualities which affects the life of an individual. If the functions of the quality changes of time are known, it is possible to calculate the development of the qi of an individual for his/her whole life span.
The results of the calculation can be evaluated and interpreted psychologically and for personality diagnosis, as well as biologically-medically and in relation to the diagnosis of the function of organs. Since the 10th century C.E. Bazi Suanming has developed into the central psychological system of Chinese culture, in which overarching theories for the categorization of human personality have matured and which has experienced an uninterrupted development up to this day. It prospers especially in Taiwan R.O.C. where a rich expert literature on the topic has been produced. It also finds lively application in Hong Kong, Japan, Singapore and in every other society committed to the classical Chinese traditions.
The Bazi Suanming expert usually assumes the position of a psychological or medical coach. The main applications are in the following areas:
Psychological and pedagogical applications
Children's education:
Promotion of predispositions of the child
Personality analysis:
Analysis of the personality and its changes during life and the detection of life episodes in which the qi force of the client is especially favourable in the sense of effective life planning.
Human ressources:
Choice of suitable staff for the filling of jobs with specific requirements.
Partnership counselling:
Analysis of the compatibility and incompatibility between people, e.g., for bringing together matrimonial partners, or for the fine-tuning of teams.
Crisis management:
Analysis and support of persons in a personal crisis and in need of psychological counsel, but also partnership counseling in cases of matrimonial conflict.
Behavioral training:
Practising of certain behavioral aspects of the client, in order to promote the development of his/her effectivity and life circumstances according to the needs of his/her basic disposition and his/her expectations.
Applications in Traditional Chinese Medicine (TCM)
Complementary medical diagnostics:
Bazi Suanming is particularly called upon in case of chronic illness as a complentary diagnostic instrument to determine the organ disposition of the client and to describe the organ's changes over a long time.
Preventive medical diagnostics:
Bazi Suanming calculates the organ disposition and its long-term development during life, which makes it possible, within the systematic correspondences of TCM, to give dietetic and preventive medical recommendations for specific life phases. This is done according to the well-known motto of TCM, "Curing the illness before it breaks out".
Elaboration of client-centered therapeutic strategies:
Through its theoretical background, Bazi Suanming is closely connected with TCM, and its calculations play a considerable role for instance in the determination of individual chrono-acupuncture. However, the adjustment of Chinese pharmacological therapy to the findings of Bazi Suanming is also possible. This very special combination of TCM and Bazi Suanming has found its theoretical expression in the medical discipline of the "five cycles and six qi" (Wuyun liuqi).


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FLSA Status: Exempt
Reports to: Director of Production
Employment Status: Full-time
The Associate Designer collaborates with the design, marketing, and editorial departments to produce creative solutions to address the marketing needs of the publishing units. This includes designing promotional materials as well as assisting Senior Designers with mechanical jacket/cover set-up and interior corrections.
Responsibilities and Duties:
How to Apply
Candidates are asked to submit a portfolio, in addition to a cover letter and resume, to Jessie Jonik at firstname.lastname@example.com
About the Company
IPG is the original independent distributor of books, ebooks, and audiobooks from publishers around the world, and a top 10 supplier of book content to Amazon.com, Barnes & Noble, independent bookstores, libraries, and specialty markets, in addition to thousands of retailers globally.
Established in 1971, Independent Publishers Group (IPG) was the first organization specifically created for the purpose of marketing titles from independent presses to the book trade. IPG was acquired by Chicago Review Press in 1987. In the fall of 2006, Chicago Review Press, Inc., the parent company of IPG, bought Trafalgar Square Publishing, Inc., the leading North American distributor of UK publishers. Trafalgar Square Publishing was founded in 1973 and as a division of IPG, now distributes products from clients such as Penguin Random House UK, Australia, New Zealand, and China; Allen & Unwin; The History Press UK; Amberley Publishing; HarperCollins UK; Pan Macmillan; and John Blake. IPG acquired International Specialized Book Services (ISBS) a premier distributor of over sixty publishers of academic, scholarly and professional publishers in April of 2018. Continuing its trajectory of growth in August of 2018, IPG acquired Midpoint Trade Books, a full-service book distribution company that was founded in 1996, located in the Flatiron District of New York City, Midpoint's distribution clients are made up of independent book publishers from across the US, the UK, Australia and Canada.
With consistent growth each year, IPG's success has come from supporting and encouraging the growth of its client publishers in the United States and worldwide. IPG has a wide reach in the market with its distribution of publishers with academic, Spanish-language, children's, and general nonfiction and fiction titles.


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August 06, 2017
The Single Cask is proud to present our latest range of expressions, which are available for purchase by the bottle, flight or dram from the bar!
The range includes a number of whiskies as well as our very first Rum and American Sour Mash expressions and are as follows:
- The Single Cask American Sour Mash 2011 5 Years Old (Tennessee, 59.7%)
- The Single Cask Diamond Rum 2004 12 Years Old (Guyana, 57%)
- The Single Cask Glenrothes 1997 19 Years Old (Speyside, Refill Sherry Butt, 58.5%)
- The Single Cask Glenrothes 1997 19 Years Old (Speyside, Dark Sherry Butt, 58%)
- The Single Cask Macduff 1997 19 Years Old (Speyside, 53.5%)
- The Single Cask Glenburgie 1995 21 Years Old (Speyside, 53.1%, not pictured)
- The Single Cask Braeval 1994 22 Years Old (Speyside, 55%)
- The Single Cask Tobermory 1994 22 Years Old (Highland/Island, 59.1%)
- The Single Cask Tomintoul 1995 22 Years Old (Speyside, 53.2%)
Most of these expressions will also be available as a part of a new range of whisky flights, which can be purchased from the bar from next week onwards.
See you at the bar!
Written By Brendan Pillai for The Single Cask Whisky Bar Singapore
UK Bar https://www.thesinglecask.co.uk/
UK - Online Shop
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May 19, 2019
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Photos from the Reflector
A great shot from the early 1960's of the lunch line and the walls which in 20 years would be lined with murals.
Before the Kraft Memorial Stadium in the 1960's the football field ran East and West. Restrooms would be used inside the building because there was not outdoor sports facility and the teams would use the basketball locker rooms.
Theres seems to be some controversy on which class of Elida is the best. Watch this video clip from "Reflections of Elida"
They don't build things like the use to. Coach Dally tells how he learned the hard way how thick the walls of 1917 section of the High School are
The final day the school was open to the public we take a look at what kind of bricks made up the old section of Elida.
Before the building was torn down, Documentary Producer John Ondo got up on the roof to give us a close up look at the bell tower.