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ValSten: a new wild species derived allotetraploid for increasing genetic diversity of the peanut crop (Arachis hypogaea L.)
Dongying Gao1,2,
Ana C. G. Araujo3,
Eliza F. M. B. Nascimento3,4,
M. Carolina Chavarro1,2,
Han Xia1 nAff5,
Scott A. Jackson ORCID: orcid.org/0000-0002-3172-16071,
David J. Bertioli ORCID: orcid.org/0000-0003-0294-72841,2 &
Soraya C. M. Leal-Bertioli ORCID: orcid.org/0000-0002-9683-53571,6
Genetic Resources and Crop Evolution (2021)Cite this article
Introgression of desirable traits from wild relatives plays an important role in crop improvement, as wild species have important characters such as high resistance to pests and pathogens. However, use of wild peanut relatives is challenging because almost all wild species are diploid and sexually incompatible with cultivated peanut, which is tetraploid (AABB genome type; 2n = 4x = 40). To overcome the ploidy barrier, we used 2 wild species to make a tetraploid with the same allotetraploid genome composition as cultivated peanut. Crosses were made between 2 diploid wild species, Arachis valida Krapov. and W.C. Greg. (BB genome; 2n = 2x = 20) and Arachis stenosperma Krapov. and W.C. Greg. (AA genome; 2n = 2x = 20). Cuttings from the diploid F1 AB hybrid were treated with colchicine to induce chromosome doubling thus generating an induced allotetraploid. Chromosome counts confirmed polyploidy (AABB genome; 2n = 4x = 40). We named the new allotetraploid ValSten. Plants had well-developed fertile pollen, produced abundant seed and were sexually compatible with cultivated peanut. ValSten exhibits the same high resistance to early and late leaf spot and rust as its diploid parents. Notably, we observed morphological variations, including flower width and branch angles in the earliest generation (S0) of allotetraploids. A SNP array was used to genotype 47 S0 allotetraploids. The great majority of markers showed the additive allelic state from both parents (AABB). However, some loci were AAAA or BBBB, indicating homeologous recombination. ValSten provides a new, vigorous, highly fertile, disease resistant germplasm for peanut research and improvement.
Peanut or groundnut (Arachis hypogaea L., AABB genome type, 2n = 4x = 40) is an important oil and food crop worldwide. Its production is frequently challenged by pests and pathogens including bacteria, fungi, nematodes and viruses that can cause severe yield loss. For example, early and late leaf spot diseases caused by the fungi Passalora arachidicola (syn. Cersospora arachidicola, teleomorph Mycosphaerella arachidis) and Northopassalora personata (syn. Cercosporidium personatum, teleomorph Mycosphaerella berkeleyi) and rust, caused by Puccinia arachidis, can cause over 50% pod yield losses in some areas where fungicides are used rarely or inappropriately (Waliyar 1990; Ouedraogo et al. 1994). In Georgia, the major peanut producing state in USA, more than 35 million dollars was spent on leaf spot control in 2015. Nevertheless, the losses caused by leaf spots alone was about 8.6 million dollars (https://secure.caes.uga.edu/extension). A reduction in reliance on fungicides and other plant protection products is also strategic because, in addition to cost and any real environmental impact, they are the subject of activist campaigns, and consequently very widespread public misconceptions and ever-tightening regulatory restrictions (EPRS 2019; Hill Cks 2012). Additionally, long-term use of fungicides leads to the emergence of resistant strains; in fact, some leaf spot strains already show resistance to benzimidazole fungicides, such as benomyl and thiophanate methyl (Kemerait R, personal communication). Development of disease resistant cultivars is one of the most efficient, environmentally friendly and economical methods to reduce disease losses in peanut (Holbrook and Culbreath 2007; Ouedraogo et al. 1994). Identification of new sources of resistance is vital, as new races or strains of pathogens may emerge and overcome any current resistance (Chen et al. 2017).
The genus Arachis is divided into 9 sections based on morphology, chromosomal characteristics, and cross-compatibility (Krapovickas and Gregory 1994). Whilst cultivated peanut is tetraploid, almost all other species are diploid. The wild species are only found in some regions of South America, are adapted to different growing conditions, and many of them harbor valuable traits, such as disease and pest resistance (Johnson et al. 1977; Leal-Bertioli et al. 2010; Mallikarjuna et al. 2011), which can be used for peanut improvement. Notably, some traits were only found in wild peanuts but absent in the cultivated peanut gene pool. For instance, no cultivated peanut variety shows strong resistance to root-knot nematode (Meloidogyne arenaria) that can cause substantial yield losses in infested fields (Starr et al. 2002). Furthermore, all root-knot nematode resistant peanut cultivars developed thus far harbor the same source of resistance, which was originally transferred from a single wild species, Arachis cardenasii Krapov. and W.C. Greg. (AA, 2n = 2x = 20) (Simpson and Starr 2001). Recently a strong new source of resistance, A. stenosperma Krapov. and W.C. Greg. (AA, 2n = 2x = 20), has also been found to be transferrable into the tetraploid genetic background (Ballén-Taborda et al. 2019).
Despite their great value for peanut improvement and other studies, direct introgression of traits from wild species into cultivated peanut by conventional hybridization is very challenging because interspecific hybrids between diploid wild species and tetraploid cultivated peanut are triploid and sterile. To overcome this ploidy barrier, the production of allotetraploids through genome duplication of the sterile AB diploid hybrids has proven to be a successful strategy for plant breeding programs (reviewed by Stalker 2017). Here we developed a new allotetraploid by inducing whole genome duplication of the hybrid between the two diploid wild species Arachis valida Krapov. and W.C. Greg. (BB genome, 2n = 2x = 20) and A. stenosperma (AA genome, 2n = 2x = 20). We find that the new induced allotetraploid shows strong resistance to early and late leaf spots and rust. We also observed phenotypic variation despite relatively high genomic stability in the neoallotetraploid individuals. The new synthetic allotetraploid provides a valuable genetic resource for breaking the ploidy barrier and speeding up the introgression of desirable traits from wild species into cultivated peanut.
Seeds of A. valida (accession PI 468,154 = GK 30,011) and A. stenosperma (accession PI666100 = V10309) were obtained from USDA seed bank. Seeds of the commercial peanut cultivars, including Tifrunner, TifNV-High O/L, GA06G, GA09B, GA12Y and NC3033 were obtained from the breeders.
Production of induced allotetraploids
Interspecific hybridization
Seeds of the 2 diploid wild species were treated overnight with 0.5% of Monterey Florel brand growth regulator to break dormancy and then maintained in a growth chamber for 2 weeks. Seedlings were transplanted into pots in greenhouse at the University of Georgia, GA, USA. Arachis species are autogamous, therefore, to avoid self-pollination and maximize cross efficiency, flower buds of A. valida (female parent) were emasculated after 4 PM of the day prior to pollination using fresh pollen of A. stenosperma (male parent), next morning, before 10 AM.
Identification of hybrid plants
Hybrid plants were identified by their aggregated pollen masses and low pollen fertility. Pollen fertility was determined by counting stained (fertile) and unstained (sterile) pollen grains as described by Fishman and Willis (2001). Flowers from different genotypes were taken between 9 and 11 AM and stored at 4 °C for subsequent analysis. To detect the pollen viability, anthers were squashed with tweezers and pollen grains detached from anthers were stained with 2% acetocarmine solution (Tokyo Chemical Industry Co., Ltd. Tokyo, Japan) for 30 s. The numbers of stained and non-stained pollen grains were determined using the Olympus BX 51 microscope. For each individual hybrid plant, 3 slides were prepared from three different anthers and at least 240 pollen grains were observed for each slide.
Induction of whole genome duplication
Chromosome doubling was performed essentially as previously described (Leal-Bertioli et al. 2015a). Briefly, approximately 20 cm sections of lateral branches (cuttings) of confirmed diploid hybrids were collected and immersed in 0.2% (50 mM) colchicine (Sigma-Aldrich, MO) for 18 h. Cuttings were then rinsed 3 times with for 15 min and transplanted into pots filled with sand and kept in the greenhouse. The survival rate was recorded after 30 days, with only the cuttings with new shoots counted and maintained in the same pots until pegs emerged and developed into mature pods. All pods harvested from the treated branches were considered as containing putative induced allotetraploid seed, used for cytological and phenotypic analysis.
Morphological variations of induced allotetraploid plants
Three characteristics were used to evaluate morphological variations in the induced allotetraploids (A. valida x A. stenosperma)4x: flower width, branch angle and pod weight. Flower width was determined using a 0–200 mm Digital caliper (Ted Pella. INC, Redding, CA). At least 8 flowers were measured for each individual and the data was collected on more than 3 different days. The angles of branches were measured for each individual, when the first pair of branches were fully developed, and the second pair of branches had emerged. The pod and seed weights were determined by weighing 5 pods or seeds, and analysis of 3 replicates (15 pods or seeds) were conducted for each sample. To observe variations in architecture/plant growth of ValSten, 24 S1 plants were observed in the field. Seed were germinated and grown for 4 weeks before transplanting to the UGA Southeast Georgia Research and Education Center experimental field in Midville, GA.
Preparation of chromosome spreads and fluorescence in situ hybridization (FISH)
Seeds of the putative induced allotetraploid were germinated on moist filter papers, in Petri dishes and maintained at 25 °C for 5 days. Plantlets were then grown in the greenhouse, for 4 weeks. After that, root tips (5–10 mm long) were isolated from 5 different individuals and the apical meristem was treated with 2 mM 8-hydroxyquinoline for 3 h, at 25 °C. Subsequently, the samples were incubated in a fixative solution containing absolute ethanol: glacial acetic acid (3:1, v/v), at 4 °C for 12 h and then treated with proteolytic enzymes (2% cellulase and 20% pectinase in sodium citrate buffer, pH7.0), at 37 °C for 2 h. Meristematic cells of each root were spread on drop of 45% acetic acid on slides and chromosome spreads were obtained using mechanical pressure onto the coverslip (Schwarzacher and Heslop-Harrison 2000). Slides containing well spread metaphases were selected using the phase contrast mode in the AxiosKop microscope (Zeiss, Oberkochen, Germany).
To observe and count chromosomes, selected slides were mounted with the Vectashild media (Vector Laboratories, Burlingame, California-USA), added to 4′, 6-diamidino-2-phenylindole (DAPI) fluorophore solution (2 µg/ml McIlvaine buffer pH 7.0) and visualized in the epifluorescence Zeiss AxioPhot Microscope (Zeiss, Oberkochen, Germany).
Ribosomal DNA sequences coding for the 5S (rDNA 5S) of Lotus japonicus (Pedrosa et al. 2002) and 18S-5.8S-25S sequences (rDNA 45S) of Arabidopsis thaliana (Wanzenböck et al. 1997) were used to obtain the DNA probes for Flourescent In Situ Hybridization (FISH). DNA sequences were labelled with, either digoxigenin-11-dUTP or Cy3-dUTP, by Nick Translation using the Nick Translation Mix kit from Roche (Basel, Switzerland). FISH was performed according to the established methods (Schwarzacher and Heslop-Harrison 2000). Chromosomes were observed in the epifluorescence microscope and the images captured using the Axiocam MRc digital camera, with the Axiovision Rel. 4.8 software. Images were treated using the Adobe Photoshop CS software.
Evaluation of disease resistance
Six different events originated from the same cross (A. valida x A. stenosperma)4× were evaluated for resistance to rust (Puccinia arachidis), early and late leaf spot (Passalora arachidicola syn. Cercospora arachidicola) and Northopassalora personata syn. Cercosporidium personatum). The method of detached leaf assays was used, as described in Leal-Bertioli et al. (2015b). In short, spores were collected from infested peanut plants in Tifton, GA. Rust inoculum was collected from the cultivars Tifguard, Tifrunner and NC3033 and LLS and ELS were collected from a population C1799 (cross between Tifrunner and NC3033). Leaves were inoculated with 105 spores/ml of 0.005% Tween 20 and incubated at 20–24 °C (ELS and LLS) or 26–28 °C (rust) and photoperiod of 10 h light and 14 h dark. Each experiment was conducted twice.
DNA extraction and genotyping
Genomic DNA was extracted from young leaves using the DNeasy Plant Mini Kit from QIAGEN (Hilden, Germany). DNA concentrations were measured using Qbit Fluorometric Quantification (Waltham, MA), and ~ 50 ng of DNA of each sample was used for genotyping.
In order to observe structural genomic variation in different events of polyploidization, a total of 47 synthetic allotetraploids (A. valida × A. stenosperma)4x individuals here obtained were genotypsed. Genotyping was conducted with the version 02 of the Affymetrix microarray (Thermo Fisher Scientific, Waltham, MA) containing 47,836 single nucleotides polymorphisms (Korani et al. 2019). SNP calling was performed with Axiom Analysis Suite software (Version1.1) provided by Affymetrix. Genotyping data was manually curated. For genomic analyses, only SNPs that had the following characteristics were used: (1) SNPs that showed polymorphic alleles between the diploid wild parents (2) SNPs that showed consistent calling between replicates. SNPs that were monomorphic and/or inconsistent between the replicates were discarded.
In 2015, between July 2 and August 7, 185 crosses were made between A. valida (female) and A. stenosperma (male). No reciprocal crosses were attempted as flowers of AA genome species tend not to support artificial pollination (not published). Thirty-one putative hybrid pods were harvested on october 28, 2015. Eleven seedlings prematurely germinated in the same pot before harvest, a characteristic sign of hybridity. Ten out of the 11 seedlings exhibited yellow flower, a dominant character present in the male parent, therefore, this phenotypic marker confirmed hybridity (Online Resource 1A). Anthers of F1 hybrids were thin and shriveled, compared the diploid parents (Online Resource 1B). Pollen grains of both A. valida and A. stenosperma were mostly stained, indicating high levels of viability. However, pollen from the hybrid seedlings were smaller and lacked staining, indicating high infertility (Online Resource 1C).
Eighty cuttings from diploid hybrids were treated with 0.2% colchicine solution to induce chromosome doubling (summer of 2016). Fifty-three cuttings survived and developed new branches and flowers. Some flowers developed into budding ovaries (pegs), which penetrated the sand and developed into pods (Fig. 1). A total of 47 pods and 44 seedlings (totaling 91 individuals) were obtained from the colchicine-treated cuttings. The pods and seedlings produced from the treated cuttings imply successful whole chromosome duplication in the diploid cuttings. We refer to these pods and seedlings as ValSten hereafter. Untreated diploid hybrids did not produce pods even after two years being maintained in the vegetative state.
The process of generation of an induced allotetraploid peanut via the tetraploid route. Branches (cuttings) are collected from the diploid F1 hybrid plant (AB genome) a, treated with colchicine solution and maintained in pots filled with wet sand b; surviving cuttings can grow and develop new branches, leaves and flowers c, pegs d and pods e. All the process, from cutting treatment to pod harvest, takes over seven months
Morphological variations of the induced allotetraploid
We investigated the morphological characters of the diploid wild parents and 48 synthetic tetraploid plants at S0, which is the generation immediately after chromosome doubling. The ValSten had a prominent main stem, which is similar to A. valida, and different from A. stenosperma (Fig. 2a). All 48 ValSten produced yellow flowers, which were as same as that of the male parent, A. stenosperma and the diploid hybrid. Figure 2b, Online Resource 1A). We also investigated the viability of mature pollen grains in the diploid parents and ValSten allotetraploids, with all of them showing high number of stained pollen, indicating high fertility (Fig. 2c, Table 1). ValSten plants produced pods similar in shape and size as A. valida (Figs. 2d, 3e).
Table 1 Summary of pollen staining in different genotypes
Comparison of whole plants (a), flowers (b), pollens (c), pods (d) and seeds (e) between diploid wild parents, induced allotetraploid ValSten and cultivated peanut cultivar TifNV high O/L
As all the induced allotetraploid were derived from a same F1 hybrid combination and could have the same expected genome composition, and therefore, similar phenotypes. However, the 48 induced allotetraploids plants from different events of tetraploidization varied. For instance, the average flower width of A. valida and A. stenosperma was 16.32 ± 1.28 and 15.54 ± 0.9 mm, respectively. Flower width of the 48 tetraploids varied from 14.1 to 20.9 mm; most of them (83%, 40/48) produced larger flowers than TifNV-High O/L (17.21 ± 0.86 mm) (Fig. 3a). Most flowers produced by the 48 ValSten individuals showed regular development and morphology: they consisted of a large banner and 2 lateral wings (Fig. 3b). However, some flowers with morphological irregularities were occasionally observed, including double or anomalously developed layers of banner (Fig. 3c) or with chimeric color patterns (white/yellow) (Fig. 3d). The angle between basal branches is an important trait of peanut, since it defines plant architecture and is one of the traits that distinguish the market types (Kayam et al 2017). The first pair of branches of both A. valida and A. stenosperma are spread and nearly parallel to the soil (Figs. 4a, b). Most of the induced allotetraploids had more upright branches with angles lower than the parents and of TifNV-High O/L, with the smallest angle corresponding to 40° (Fig. 4c–e, Online Resource 2). Pods and seeds of six events of ValSten were also compared. All were significantly heavier (t test, p < 0.003) than that of A. stenosperma. Three were generally similar to those of A. valida, with three ValSten-2, ValSten-3 and ValSten-9 producing heavier pods and seeds than that of A. valida (t test, p < 0.01) although, as expected significantly lighter (T test, p < 0.007) than those of TifNV-High O/L (Online Resource 3). Dramatic phenotypic differences were also observed among the S1 ValSten plants in the field. Some plants exhibited more spreading growth habits (Fig. 5a–c), while others were more compact (Fig. 5d–g). Large variations were also observed in plant sizes (Fig. 5a–k).
Flowers from Valsten plants a histogram showing the range of flower width of different ValSten plants: 14.3 to 20.9 mm. Arrows show flower width averages of A. stenosperma (Sten, 15.5 mm), A. valida (Val, 16.3) and A. hypogaea TifNV-H/O (TifNV, 17.2); b a regular yellow flower, with two wings and a single banner; c two abnormal flowers with unusual shape and 2 layers of banners; d a flower with mosaic white and yellow patterns
Angles between basal branches of the diploid parents, A. valida, A. stenosperma and some S0-generation ValSten plants. The angles were measured when the first pair of branches had fully emerged
Cytogenetic analyses
We examined the chromosome numbers from root apical meristem cells of ValSten. Forty mitotic chromosomes, including 38 metacentric and 2 submetacentric chromosomes were observed (Fig. 6a), which are equal to the sum of its diploid parents, A. stenosperma (AA, 2n = 2x = 20) and A. valida (BB, 2n = 2x = 20) (Robledo and Seijo 2010; Robledo et al. 2009). Ten pairs of chromosomes contained DAPI+ bands on their centromeric regions, which correspond to the A subgenome chromosomes from A. stenosperma. The other ten pairs lacked detectable DAPI+ bands, indicating that they were derived from A. valida. It is worth to note that cyt-B3 of ValSten presented distal DAPI+ bands (Fig. 6a), despite being a metacentric chromosome in A. valida (Robledo and Seijo 2010) (Because most correspondences between cytogenetic and sequenced chromosome assignments are unknown we will distinguish cytogenetic numbering by using the prefix "cyt- "). The small pair of chromosomes cyt-A9 and the submetacentric satellite (SAT) chromosomes, the cyt-A10, were also observed (Fig. 6a).
Growth habits of 11 three-month-old S1 ValSte plants in the field in July 2019. Distance between rows and plants was 6 foot. All plants were managed under the same condition, and no fungicides were applied during the growth season
The number and localization of the 5S rDNA and 45S rDNA loci can be used as additive characters to distinguish different chromosomes in the diploid species. The 5S rDNA loci were found on the proximal regions of chromosomes cyt-A3 and cyt-B3 of ValSten (Fig. 6b), consistent with the corresponding chromosomes in the diploid progenitors (Robledo and Seijo 2010; Robledo et al. 2009). Seven 45S rDNA loci were identified in ValSten (Fig. 6b), including the strong signals at the proximal regions of cyt-A2, cyt-A7, cyt-B4 and cyt-B7 chromosomes. The cyt-B10 chromosome contained one 45S rDNA locus on the proximal and another on the terminal region of the arm. However, the 45S rDNA locus on cyt-A10 was situated near the secondary constriction of the SAT chromosome (Fig. 6b), forming a thread-like region between the SAT and the long arm of the chromosome. This thread-like region is typically observed whenever DNA is under transcription, a characteristic of nucleolus organizer regions (NORs). Following earlier description by Robledo et al. (2009) for the corresponding chromosomes of A. stenosperma, here we also conclude that cyt-A10 has a NOR.
Cytogenetic analysis of ValSten. DAPI bands (bright white) on centromere regions of the chromosomes of subgenome A (red arrows) and subgenome B (green arrows) a. FISH using DNAr 5S and 45S probes and sites detected in green and red (arrows), respectively b. A10: short arm and proximal segment of the long arm (A10*) and the satellite (A10°). Barra: 5 μm
Disease evaluation
Resistance to early leaf spot (ELS), late leaf spot (LLS) and rust in ValSten and eight peanut cultivars was evaluated. Susceptibility to leaf spot was determined based on the number of lesions (incidence) and percentage of leaf area affected by the symptoms of the disease (DLA, severity). Five control peanut cultivars were common to all experiments. These controls were all infected by these pathogens, showing varying numbers of lesions. All 5 cultivars were highly susceptible to rust (Online Resource 4). No ELS, LLS or rust lesions were detected on the leaves of ValSten (Online Resource 4, Fig. 7a). All other cultivars, including Bailey, that presents partial resistance to ELS, showed lesions (Figs. 7b, c).
Leaves of ValSten a, A. hypogaea cv. Bailey b and A. hypogaea cv. NC3033 c twenty-one days after inoculation with Passalora arachidicola (syn. Cercospora arachidicola). ValSten leaves are completely clean while all leaves of both peanut cultivars exhibit symptoms of early leaf spot
Genome-wide SNP analysis of genome stability of synthetic allotetraploids
Since phenotypic variations were detected among the induced allotetraploid individuals, we hypothesized that there may have been genomic changes during polyploidization. To test this, DNAs of A. stenosperma (AA genome), A. valida (BB genome) and 47 induced allotetraploids (AABB genome) were genotyped with the 48 K Affymetrix chip (Korani et al. 2019). After filtering out all loci which or detected no polymorphisms between of A. stenosperma and A. valida, 1,312 loci were kept for genome-wide comparison. All ValSten presented SNPs from both parents on most loci (99.36%), having the expected genome configuration: AABB. This is consistent with the cytogenetic analysis (Fig. 6). A small number of loci deviated from this configuration, they were either AAAA (169, 0.27%) or BBBB (222, 0.36%), indicating some type of homeologous recombination or gene conversion, and a slight bias towards conversion to A. valida alleles BBBB (Fig. 8, Online Resource 5). These loci were present all chromosomes and tended to occur in short stretches of adjacent markers, representing short chromosome segments. Chromosome A06/B06 (1.47%), A03/B03 (0.98%) and A02/B02 (0.81%) had the highest level of homeologous recombination (Figs. 8a, b, Online Resource 5). Estimated genetic similarities between different ValSten plants ranged between 0.86 and 0.96 (Online Resource 6), these estimates being certainly inflated by noise. Estimated genetic similarities between the virtually genetically identical sibling seeds from the same accessions ranged between 0.91 and 0.97.
a Distribution of percentage of tetrasomic/nullisomic alleles on all chromosomes of 47 different ValSten plants; b Representation of the tetrasomic/nullisomic allele sites on the three "Hot spots" for tetrasomic recombination: chromosomes 02, 03 and 06 of S0 individuals ValSten7, 10, 37 and 53
A new source of allelic variation for peanut improvement
Modern plant breeding has made great progress in improving the yield and nutritional qualities of crops and continues making enormous impacts on food security worldwide. However, a key vulnerability is relative lack of genetic variation among commercial cultivars of important species (Fu 2015). This provides a fundamental limitation to the genetic gains that can be made, and also increases the risk of crop disease epidemics (Keneni et al. 2012). Wild crop relatives play pivotal roles in crop improvement and sustainable agriculture. They represent a large pool of genetic diversity, a valuable resource for improving yield, and adaptation to changing disease pressures, and other traits (Dempewolf et al. 2017). Thus far, many desirable traits from wild crop relatives have been successfully used for crop improvement, including improved yield traits, drought tolerance, disease resistance and nutritional value in legumes (reviewed by Zhang et al. 2019. In the case of peanut, introgression of new alleles from wild species is hindered by the ploidy differences. In this work, from the wild species A. valida and A. stenosperma, we created a new induced allotetraploid (ValSten) that is sexually compatible with cultivated peanut. This neo allotetraploid is being used to transfer wild alleles from the wild species into cultivated peanut, by crossing and backcrossing. Notably, ValSten is very vigorous, produces large biomass and is prolific in seed production. The male parent A. stenosperma V10309 is highly resistant to root-knot nematode (Proite et al. 2008) and this resistance is genetically mapped and transferrable to tetraploid peanut (Ballén-Taborda et al. 2019). Furthermore, ValSten showed complete resistance to three peanut major diseases: early and late leaf spot and rust.
The creation of ValSten (and others being developed in our research program) are intended for use as a source of valuable alleles in breeding. They have the advantage of being directly sexually compatible with cultivated peanut, and of having identified DNA markers linked to a subset of their alleles. These alleles are valuable once incorporated into the genetic background of cultivated peanut, but as outlined by Tanksley and Nelson (1996) and Tanksley and McCouch (1997), their value is mostly 'masked' whilst in a wild genetic background. In peanut, a clear example of that is the increase in seed size and biomass by introgressing alleles from the neo allotetraploid IpaDur1 that has small seed and low biomass (Faye et al. 2016; Leal-Bertioli et al. 2018; Suassuna et al. 2015). This indicates that ValSten has the potential to contribute useful alleles besides the ones that confer resistance to fungal pathogens.
Cytological characterization of the synthetic allotetraploid
In this study, the chromosomes of the allotetraploid ValSten were characterized by the visualization of heterochromatic regions of DNA rich in A-T using DAPI staining, and in situ localization of 5S and 45S rDNA loci. Karyotypes were compared to the progenitor species and other allotetraploids. Our data confirmed that ValSten has the expected 40 chromosomes and karyotype almost identical to the sum of its diploid parents. The majority of chromosomes were metacentric, except for 2 pairs of chromosomes that are submetacentric. The metaphase chromosomes from 2 different genomes, A and B, could be distinguished by their staining with DAPI. Chromosomes of the subgenome A of ValSten (A. stenosperma) having bands on the centromeric and pericentromeric regions (as is well established for the A genome species; Seijo et al. 2004; Robledo et al. 2009; Nascimento et al. 2018). Notably, however, although no DAPI band is detected in the chromosomes of the diploid B genome species, A. valida, A. ipaensis and A. magna (Robledo and Seijo 2010), we found distal DAPI+ bands on cyt-B3 of ValSten. Ribosomal DNA (rDNA) locations and number in ValSten were consistent with the sum of the diploid parents. The ValSten putative Nuclear Organizing Region (NOR) shows A genome nucleolar dominance, being present only on cyt-A10, and not cyt-B10.
These results can be compared to allotetraploids derived from A. ipaënsis and A. duranensis, the spontaneous allotetraploids A. hypogaea and A. monticola (Seijo et al. 2004; Robledo and Seijo 2010; Robledo et al. 2009), and induced allotetraploids, IpaDur1 (Nascimento et al. 2018). At least in the initial generations studied here, ValSten seems more similar in behavior to the more karyotypically stable spontaneous allotetraploids A. hypogaea and A. monticola, than the induced IpaDur1. All of these allotetraploids show the sum of DAPI bands essentially being the sum of their parental diploid species. All show A genome nucleolar dominance. However, in the IpaDur1 the rDNAs are unexpected, they are not additive of the parental species.
Phenotypic and genomic variations in synthetic allotetraploids
Polyploidy plays a critical role in the evolution and diversification of flowering plants it is thought to increase biological complexity, generate evolutionary novelties and improve adaption to changing environmental stresses (Van de Peer et al. 2009). It is now known that virtually all angiosperm lineages have undergone polyploidy at some stage in their evolutionary pasts. Indeed, most lineages have undergone multiple rounds of polyploidy. Species that have undergone recent whole genome duplications are easily identifiable as polyploid. Species whose most recent whole genome duplication is more ancient have undergone evolutionary processes, such as gene loss, that return them to a "diploid" state (Soltis et al. 2015). Cultivated peanut is an allotetraploid that arose very recently, less than 10,000 years ago. The interspecific hybridization between the diploid ancestors, A. duranensis and A. ipaensis was enabled by the human movement of A. ipaensis into the range of A. duranensis by proto-agriculturalists in prehistory (Bertioli et al. 2016). Although multiple diploid wild species were cultivated before the polyploid A. hypogaea, it was the polyploid species that became fully domesticated—compelling evidence that there was some "polyploid advantage" for domestication (Bertioli et al. 2019). By the observation of new induced allotetraploids, like ValSten, it is possible to begin to glimpse at what this advantage may have been. Here we observed that ValSten is exceptionally vigorous. It has larger flowers (Online Resource 2), larger leaves and more biomass (data not shown). However, the sizes of pods and seeds were only slightly larger than that of the diploid parents (Fig. 2d, e, Online Resource 3). This result indicates that artificial selection, more than the immediate effect of polyploidy has been the main cause of larger seed and pod sizes in cultivated peanut, as previously found by the observation of other induced allotetraploids (Leal-Bertioli et al., 2017).
The production of Arachis synthetic allotetraploids is a time-consuming task, starting with the generation of diploid hybrids. Once produced, they must be allowed to grow sufficiently to produce cuttings for treatment with colchicine. Survival rate of these cuttings is generally very low as is the recovery of fertility and the production of tetraploid seeds. In the case of ValSten, the recovery of fertility and production of seeds was the highest we have ever observed during more than ten years of our work producing induced allotetraploids (Leal-Bertioli et al. 2015a, 2017 and unpublished data). More than 90 S0 ValSten plants were obtained, allowing us to compare many different events at early generations. The naïve expectation, for plants of pure allotetraploid genetic behavior would be that all plants would be the same phenotypically. This was certainly not the case: extensive morphological differences were found among the S0 and S1 tetraploids, including flower width and color, angle between side branches, pod weight, plant architecture among others (These morphological changes are similar to those observed in smaller number in other allotetraploid plants; unpublished). Nevertheless, in spite of this morphological variability, as was consistent with karyotypic characterization, genotyping revealed relative genome stability. Most SNP loci had the expected AABB composition and that there was little variation between different ValSten genotypes. Only 0.64% of markers had unexpected tetrasomic/nullisomic composition (AAAA or BBBB).
An alternative approach for wild species conservation
Increasing anthropic action has destroyed the natural habitats of many crop wild relatives (Schoen and Brown 2001) many are now are endangered (Humphreys et al. 2019). Indeed, the location of the collection of the A. stenosperma (V10309; 16°28′14.54"S 54°39′19.30"W) used to make ValSten has been overtaken by urban sprawl, and the population is now extinct in the wild. Seed banks are of profound importance for species conservation and maintenance of genetic diversity (Schoen and Brown 2001; Hay and Probert 2013). However, the climates and photoperiods of the banks are often different from the native habitats. Thus, some wild species, do not grow well and/or produce only few or no seeds in the places where the seedbanks are located.
sThe genus Arachis is native to South America (Krapovickas and Gregory, 1994), the USDA seedbank holds around 500 accessions of wild peanut species. However, some accessions such as Grif 14,535 (A. simpsonii) can develop flowers, but don't produce seeds in greenhouse, therefore they are maintained and exchanged via vegetative tissue. Arachis stenosperma V10309 (PI666100), grows vigorously in Brazil but it grows weakly in the USA, even in greenhouse conditions and produces only about 20 seeds per plant. In contrast, the induced allotetraploid ValSten grows well and often produces over 100 seeds per plant. We propose that the allotetraploids derived from wild relatives of peanut can serve as an additional option for the maintenance and distribution of genetic diversity. Induced allotetraploids often produce abundant seed, harbor essentially the genetic information from both parents, and critically, are directly compatible with peanut, thus being directly usable in breeding programs seeking to increase genetic diversity and introgress desirable traits from wild species into the peanut crop.
All data is in the Online Resources. The genotypes are being release as germplasm and will be deposited at the USDA-PGRCU (Plant Genetic Resources Conservation Unit at the United States Department of Agriculture).
Ballén-Taborda C, Chu Y, Ozias-Akins P, Timper P, Holbrook CC, Jackson SA, Bertioli DJ, Leal-Bertioli SCM (2019) A new source of root-knot nematode resistance from Arachis stenosperma incorporated into allotetraploid peanut (Arachis hypogaea). Sci Rep 9:17702. https://doi.org/10.1038/s41598-019-54183-1
Bertioli DJ, Cannon SB, Froenicke L, Huang G, Farmer AD, Cannon EK, Liu X, Gao D, Clevenger J, Dash S, Ren L, Moretzsohn MC, Shirasawa K, Huang W, Vidigal B, Abernathy B, Chu Y, Niederhuth CE, Umale P, Araújo AC, Kozik A, Kim KD, Burow MD, Varshney RK, Wang X, Zhang X, Barkley N, Guimarães PM, Isobe S, Guo B, Liao B, Stalker HT, Schmitz RJ, Scheffler BE, Leal-Bertioli SC, Xun X, Jackson SA, Michelmore R, Ozias-Akins P (2016) The genome sequences of Arachis duranensis and Arachis ipaensis, the diploid ancestors of cultivated peanut. Nat Genet 48:438–446. https://doi.org/10.1038/ng.3517
Bertioli DJ, Jenkins J, Clevenger J, Dudchenko O, Gao D, Seijo G, Leal-Bertioli SCM, Ren L, Farmer AD, Pandey MK, Samoluk SS, Abernathy B, Agarwal G, Ballén-Taborda C, Cameron C, Campbell J, Chavarro C, Chitikineni A, Chu Y, Dash S, El Baidouri M, Guo B, Huang W, Kim KD, Korani W, Lanciano S, Lui CG, Mirouze M, Moretzsohn MC, Pham M, Shin JH, Shirasawa K, Sinharoy S, Sreedasyam A, Weeks NT, Zhang X, Zheng Z, Sun Z, Froenicke L, Aiden EL, Michelmore R, Varshney RK, Holbrook CC, Cannon EKS, Scheffler BE, Grimwood J, Ozias-Akins P, Cannon SB, Jackson SA, Schmutz J (2019) The genome sequence of segmental allotetraploid peanut Arachis hypogaea. Nat Genet 51(5):877–884. https://doi.org/10.1038/s41588-019-0405-z
Chen J, Upadhyaya NM, Ortiz D, Sperschneider J, Li F et al (2017) Loss of AvrSr50 by somatic exchange in stem rust leads to virulence for Sr50 resistance in wheat. Science 358:1607–1610
Dempewolf H, Baute G, Anderson J, Kilian B, Smith C, Guarino L (2017) Past and future use of wild relatives in crop breeding. Crop Sci 57:1070–1108
EPRS | European Parliamentary Research Service. Farming without plant protection products, Can we grown without using herbicides, fungicides and insecticides (2019) In-depth analysis, Panel for the Future of Science and Technology. https://www.europarl.europa.eu/RegData/etudes/IDAN/2019/634416/EPRS_IDA(2019)634416_EN.pdf
Faye I, Hodo-Abalo T, Ndoye O, Fonceka D (2016) Dossier technique d'homologation de nouvelles variétés d'arachide. In Institut Sénegalais de Rescherches Agricoles (ISRA) [ed.], Senegal, 34. ISRA, Dakar, Senegal
Fishman L, Willis JH (2001) Evidence for Dobzhansky-Muller incompatibilities contributing to the sterility of hybrids between Mimulus guttatus and M. nasutus. Evolution 55:1932–1942
Fu YB (2015) Understanding crop genetic diversity under modern plant breeding. Theor Appl Genet 128(11):2131–2142. https://doi.org/10.1007/s00122-015-2585-y
Hay FR, Probert RJ (2013) Advances in seed conservation of wild plant species: a review of recent research. Conserv Physiol 1(1):030
Hillocks RJ (2012) Farming with fewer pesticides: EU pesticide review and resulting challenges for UK agriculture. Crop Prot 31:85–93
Holbrook CC, Culbreath AK (2007) Registration of 'Tifrunner' Peanut. J Plant Reg 1:124
Humphreys AM, Govaerts R, Ficinski SZ, Nic Lughadha E, Vorontsova MS (2019) Global dataset shows geography and life form predict modern plant extinction and rediscovery. Nat Ecol Evol 3:1043–1047. https://doi.org/10.1038/s41559-019-0906-2
Johnson DR, Wynne JC, Campbell WV (1977) Resistance of wild species of Arachis to the two spotted spider mite, Tetranychus urticae. Peanut Sci 4:9–11
Kayam G, Brand Y, Faigenboim-Doron A, Patil A, Hedvat I, Hovav R (2017) Fine-mapping the branching habit trait in cultivated peanut by combining bulked segregant analysis and high-throughput sequencing. Front Plant Sci 8:467
Keneni G, Bekele E, Imtiaz M, Dagne K (2012) Genetic vulnerability of modern crop cultivars: causes, mechanism and remedies. Int J Plant Res 2:69–79
Korani W, Clevenger JP, Chu Y, Ozias-Akins P (2019) Machine learning as an effective method for identifying true Single Nucleotide Polymorphisms in polyploid plants. Plant Genome 12. https://doi.org/10.3835/plantgenome2018.05.0023
Krapovickas A, Gregory WC (1994) Taxonomía del género Arachis (Leguminosae). Bonplandia 8:1–186
Leal-Bertioli SCM, de Farias MP, Silva PIT, Guimarães PM, Brasileiro ACM, Bertioli DJ, Guerra de Araújo AC (2010) Ultrastructure of the initial interaction of Puccinia arachidis and Cercosporidium personatum with leaves of Arachis hypogaea and Arachis stenosperma. J Phytopathol 158:792–796
Leal-Bertioli SCM, Cavalcante U, Gouveia EG, Ballen-Taborda C, Shirasawa K, Guimaraes PM, Jackson SA, Bertioli DJ, Moretzsohn MC (2015a) Identification of QTLs for rust resistance in the peanut wild species Arachis magna and the development of KASP markers for Marker Assisted Selection. Genes/Genomes/Genet 5(7):1403–1413. https://doi.org/10.1534/g3.115.018796
Leal-Bertioli SCM, Santos SP, Dantas KM, Inglis PW, Nielen S, Araujo ACG, Silva JP, Cavalcante U, Guimarães PM, Brasileiro ACM, Carrasquilla-Garcia N, Penmetsa RV, Cook D, Moretzsohn MC, Bertioli DJ (2015b) Arachis batizocoi: a study of its relationship to cultivated peanut (A. hypogaea) and its potential for introgression of wild genes into the peanut crop using induced allotetraploids. Ann Bot 115:237–249. https://doi.org/10.1093/aob/mcu237
Leal-Bertioli SCM, Moretzsohn MC, Santos SP, Brasileiro ACM, Guimarães PM, Bertioli DJ, Araujo ACG (2017) Phenotypic effects of allotetraploidization of wild Arachis and their implications for peanut domestication. Am J Bot 104:379–388. https://doi.org/10.3732/ajb.1600402
Leal-Bertioli SCM, Godoy IJ, Santos JF, Doyle JJ, Guimarães PM, Abernathy BL, Jackson SA, Moretzsohn MC, Bertioli DJ (2018) Segmental allopolyploidy in action: Increasing diversity through polyploid hybridization and homoeologous recombination. Am J Bot 105:1–14
Mallikarjuna N, Senthilvel S, Hoisington D (2011) Development of new sources of tetraploid Arachis to broaden the genetic base of cultivated groundnut (Arachis hypogaea L.). Genet Resour Crop Evol 58:889–907
Nascimento EFDM, Santos BV, Marques LO, Guimarães PM, Brasileiro AC, Leal-Bertioli SC, Bertioli DJ, Araujo AC (2018) The genome structure of Arachis hypogaea (Linnaeus, 1753) and an induced Arachis allotetraploid revealed by molecular cytogenetics. Comp cytogenet 12:111–140
Ouedraogo M, Smith OD, Simpson CE (1994) Smith DH (1994) Early and late leaf spot resistance and agronomic performance of nineteen interspecific derived peanut lines. Peanut Science 21:99–104
Pedrosa A, Sandal N, Stougaard J, Schweizer D, Bachmair A (2002) Chromosomal map of the model legume Lotus japonicus. Genet 161:1661–1672
Proite P, Carneiro R, Falcão R, Gomes A, Leal-Bertioli S, GuimarãesBertioli PD (2008) Post-infection development and histopathology of Meloidogyne arenaria race 1 on Arachis spp. Plant Pathol on line. https://doi.org/10.1111/j.1365-3059.2008.01861.x
Robledo G, Seijo G (2010) Species relationships among the wild B genome of Arachis species (section Arachis) based on FISH mapping of rDNA loci and heterochromatin detection: A new proposal for genome arrangement. Theor Appl Genet 121:1033–1046. https://doi.org/10.1007/s00122-010-1369-7
Robledo G, Lavia GI, Seijo G (2009) Species relations among wild Arachis species with the A genome as revealed by FISH mapping of rDNA loci and heterochromatin detection. Theor Appl Genet 118:1295–1307. https://doi.org/10.1007/s00122-009-0981-x
Schwarzacher T, Heslop-Harrison J (2000) Practical in situ hybridization. BIOS Scientific Publishers Ltd., Oxford
Schoen DJ, Brown AHD (2001) The Conservation of wild plant Species in seed banks: attention to both taxonomic coverage and population biology will improve the role of seed banks as conservation tools. Bioscience 51:960–966
Seijo JG, Lavia GI, Fernández A, Krapovickas A, Ducasse D, Moscone EA (2004) Physical mapping of the 5S and 18S–25S rRNA genes by FISH as evidence that Arachis duranensis and A. ipaënsis are the wild diploid progenitors of A. hypogaea (Leguminosae). Am J Bot 91:1294–1303. https://doi.org/10.3732/ajb.91.9.1294
Simpson CE, Starr JL (2001) Registration of COAN peanut. Crop Sci 41:918
Soltis PS, Marchant DB, Van de Peer Y, Soltis DE (2015) Polyploidy and genome evolution in plants. Curr Opin Genet Dev 35:119–125
Stalker HT (2017) Utilizing wild species for peanut improvement. Crop Sci 57:1102–1120
Starr JL, Morgan ER, Simpson CE (2002) Management of the peanut root-knot nematode, Meloidogyne arenaria, with host resistance. Plant Health Progr. https://doi.org/10.1094/PHP-2002-1121-01-HM
Suassuna TMF, Suassuna ND, Moretzsohn MC, Leal-Bertioli SCM, Bertioli DJ, Medeiros EP (2015) Yield, market quality, and leaf spots partial resistance of interspecific peanut progenies. Crop Breed Applied Biotechnol 15:175–180
Tanksley SD, McCouch SR (1997) Seed banks and molecular maps: unlocking genetic potential from the wild. Science 277:1063–1066
Tanksley SD, Nelson JC (1996) Advanced backcross QTL analysis: a method for the simultaneous discovery and transfer of valuable QTLs from unadapted germplasm into elite breeding lines. Theor Appl Genet 92:191–203
Van de Peer Y, Maere S, Meyer A (2009) The evolutionary significance of ancient genome duplications. Nat Rev Genet 10:725–732
Waliyar F (1990) Evaluation of yield losses due to groundnut leaf diseases in West Africa. In: Nduguru BJ, Waliyar F, Ntare BR (eds) Summary Proceedings of the Second ICRISAT Regional Groundnut Meeting for West Africa. Niger, Niamey, pp 32–33
Wanzenböck EM, Schöfer C, Schweizer D, Bachmair A (1997) Ribosomal transcription units integrated via T-DNA transformation associate with the nucleolus and do not require upstream repeat sequences for activity in Arabidopsis thaliana. Plant J 11:1007–1016. https://doi.org/10.1046/j.1365-313X.1997.11051007.x
Zhang H, Yasmin F, Song BH (2019) Neglected treasures in the wild-legume wild relatives in food security and human health. Curr Opin Plant Biol 49:17–26
The authors appreciate Jenny Leveret, Carlos Ruiz and Rida Osman for their technical assistance. This work was supported by Agriculture and Food Research Initiative Competitive Grant no. 2018-67013-28139 co-funded by the USDA National Institute of Food and Agriculture and the National Peanut Board, grants from National Science Foundation (grant # MCB-1543922), Georgia Peanut Commission, Peanut Research Foundation and Mars Wrigley.
This work was supported by Agriculture and Food Research Initiative Competitive Grant no. 2018–67013-28139 co-funded by the USDA National Institute of Food and Agriculture and the National Peanut Board, grants from National Science Foundation (grant # MCB-1543922), Georgia Peanut Commission, Peanut Research Foundation and Mars Wrigley.
Han Xia
Present address: Biotechnology Research Center, Shandong Academy of Agricultural Sciences, Jinan, Shandong, China
Center for Applied Genetic Technologies and Institute of Plant Breeding, Genetics and Genomics, University of Georgia, 111 Riverbend Road, Athens, GA, 30602, USA
Dongying Gao, M. Carolina Chavarro, Han Xia, Scott A. Jackson, David J. Bertioli & Soraya C. M. Leal-Bertioli
Department of Crop and Soil Science, University of Georgia, Athens, GA, USA
Dongying Gao, M. Carolina Chavarro & David J. Bertioli
Embrapa Genetic Resources and Biotechnology, PqEB W5 Norte Final, CEP 70.770-917, Brasília, DF, Brazil
Ana C. G. Araujo & Eliza F. M. B. Nascimento
Institute of Biological Sciences, University of Brasilia, Campus Darcy Ribeiro, CEP 70.910-900, Brasília, DF, Brazil
Eliza F. M. B. Nascimento
Department of Plant Pathology, University of Georgia, Athens, GA, USA
Soraya C. M. Leal-Bertioli
Dongying Gao
Ana C. G. Araujo
M. Carolina Chavarro
Scott A. Jackson
David J. Bertioli
DG, SLB, DJB and SJ conceived and designed the study. DG and HX made cross. DG generated synthetic allotetraploids. EN and AA conducted cytogenetic analysis. SB, CC, DG and DJB performed SNP array analysis. SB conducted in vitro disease evaluation. DG and SLB drafted the manuscript. All authors were involved in manuscript revision and approved the submitted version.
Correspondence to Soraya C. M. Leal-Bertioli.
Human or Animal rights
This research does not involve human participants or animals.
Below is the link to the electronic supplementary information.
Online Resource 1. Comparison of flowers (A), anthers (B) and carmin acetic stained pollens (C) of two diploid wild parents A. valida and A. stenosperma and their F1 hybrid. (PPTX 290 kb)
Online Resource 2. Angle between basal branches of 46 S0 ValsSten individuals (blue), A. stenosperma (red), A. valida (yellow) and A. hypogaea TifNV-H/O (green). (PPTX 47kb)
Online Resource 3. Seed weight of six S0 ValsSten individuals (blue), A. stenosperma (red), A. valida (yellow) and A. hypogaea TifNV-H/O (green). Note that seeds of three individuals (ValSten2, 3 and 9) are significantly heavier than those of either parent. (PPTX 46 kb)
Online Resource 4. Data of in vitro bioassays of early and late leaf spot, and rust on the induced allotetraploid ValSten and seven different peanut cultivars. (XLSX 13kb)
Online Resource 5. Filtered genotyping data of 47 S0 ValsSten individuals and the diploid parents A. stenosperma V10309 (in duplicate) and A. valida PI468154 (in triplicate) obtained using the version 02 of the Affymetrix microarray (Thermo Fisher Scientific, Waltham, MA). (XLSX 271kb)
Online Resource 6. Similarity matrix constructed with 1,312 loci from SNP genotyping data (Online Resource 5). Similarity between 47 different S0 ValSten individuals ranged between 86 and 96%.(XLSX 31kb)
Gao, D., Araujo, A.C.G., Nascimento, E.F.M.B. et al. ValSten: a new wild species derived allotetraploid for increasing genetic diversity of the peanut crop (Arachis hypogaea L.). Genet Resour Crop Evol (2021). https://doi.org/10.1007/s10722-020-01076-2
Polyploidy


One of the largest foes of your roof is ice. When gutters again up and freeze up, they can push the shingles and even wooden up off the highest of your home. As soon as that ice begins to soften, it will leak inside and rot out your home. Watch for ice build up and fix the issue quickly. Only hire people who are professionals at each step. A contractor ought to be able to offer you a reliable quite, for instance. If the method is skilled, likelihood is the work is simply too.
Those who are engaged on their roof, could be wise to all the time watch where they step. Whereas it may be tempting to look out over the huge view that your roof offers, you can simply journey over one thing and fall. All the time keep your eyes forward of where you will be stepping. Only ever hire a roofer who has a license and is totally insured for the job. If anything happens whereas they're working, be it a worker who falls off the roof and is injured or a worker who falls by means of your roof and destroys your valuables, you will be pleased you held out for the correct firm!
Individuals typically test the roof and skylights for any leaks. Nonetheless, many neglect to test the rubber boots. Each time they dry, leaks can happen. Thankfully, it is very easy to switch them. A new roof boot can be bought at your native hardware store for a low worth. Merely take away the outdated one, take away some shingles if obligatory, and set up the brand new one. When taking on a roofing job, make sure that you understand what you are doing, before starting the mission. Taking a category at your native hardware store can put together you for the job. Not understanding what you might be doing can result in the job costing greater than you ever supposed.
In order for you a novel wanting roof and could be pondering of having turrets put in or even having a metallic roof placed on, then make sure your speak with the roofer about their expertise with those supplies. If the company has very little expertise with it, find someone else that does. It isn't a good idea to gamble on one thing as vital as your roof. There is no shame in having another person do roofing work for you. If you happen to're afraid of heights, don't have any stability or simply plain intimidated by your roof, rent another person to do the work for you. Roofers might be surprisingly inexpensive, and the fee is value the advantages to your mental well being.
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Just be sure you take the right security precautions before trying to go up in your roof. Generally, 5 bids is a good quantity. Any less than that, and you're most likely selling yourself quick.


text:
import socket, random, sys
import time

sensorFrequency = 1
sensorsCount = 1000

def createSensorData(sensorID ):

  #message =   + str(sensorID) + "'to='" + str(destination) + "'>"
  #+ "<fields xmlns='urn:xmpp:iot:sensordata' seqnr='1' done='true'>"+ "<node nodeId='"
  #+str(sensorID) + "'>"+  "<timestamp value='" + str(int(time.time())) +"'>"
  #+    "<numeric name='Temperature' momentary='true' automaticReadout='true' value='"
  #+ str(random.randint(10,100)) + "' unit='C'/>"+ "</timestamp>"+ "</node>"
  #+  "</fields>"+ "</message>"

  message = ''.join(["<message from='",  str(sensorID), "'to='" , str("spark") , "'>"
  , "<fields xmlns='urn:xmpp:iot:sensordata' seqnr='1' done='true'>", "<node nodeId='"
  ,str(sensorID) , "'>",  "<timestamp value='" , str(int(time.time())) ,"'>"
  ,    "<numeric name='Temperature' momentary='true' automaticReadout='true' value='"
  , str(random.randint(10,100)) , "' unit='C'/>", "</timestamp>", "</node>"
  ,  "</fields>", "</message>"] )
  return message

if __name__=='__main__':

# create a socket object
  serversocket = socket.socket(
                socket.AF_INET, socket.SOCK_STREAM)

# get local machine name
  host = "localhost"

  port = 9999

  args = sys.argv;
  if (len(args) > 1):  sensorsCount=int(args[1])
  if (len(args) > 2):  port=int(args[2])
  if (len(args) > 3):  host=str(args[3])
  print str(sensorsCount) + " on " + host + " : " + str(port)

# bind to the port
  serversocket.bind((host, port))

# queue up to 5 requests
  serversocket.listen(5)
  sensors = []
  #generate sensors
  for i in range(0,sensorsCount):
      sensors.append("Sensor_" + str(i))

  while True:
    # establish a connection
    clientsocket,addr = serversocket.accept()
    data = []
    for sensor in sensors:
        data.append(createSensorData(sensor))

    print("Got a connection from %s" % str(addr))
    currentTime = time.ctime(time.time()) + "\r\n"
    clientsocket.send("".join(data))
    clientsocket.close()

redpajama_set_name: RedPajamaGithub


Blu By the Sea is a world-class drug rehab facility that is neat, clean and comfortable. The food is home cooked and always fresh. We offer independent and private rooms, along with bunk style housing. Our 90 percent success rate means that there is hope and a solution for you or your loved one's drug addiction or alcohol addiction problem. With the Blu By the Sea way, you can be confident that you'll have the support and tools that you need to beat addiction and start a new life.
Begin the journey to sobriety here today and start work on a new life that you control. Our addiction therapists, world-class facility (view it here) and the support we give our students sets us apart from the rest. For the best in addiction treatment, come to Blu By the Sea in Florida.


text:
Our Relationship with McDonald's
Pop Tabs
Third Party Event Fundraising Application
Taxes and Your Donation
Red Shoe Shindig
Governor's Steer Show
Flights & Fries
You are here: Home » Pop Tabs
Pulling For The Families
Pop Tab Collecting
Since 1994, Ronald McDonald House of Siouxland has provided a place to stay for many families seeking medical care for a child. Although each family is asked for a $5 donation per night of their stay, many families cannot afford this. That is where pop tabs make their impact. The money received from pop tab recycling not only helps to cover those families who cannot afford the donation, but also helps offset the cost for every family staying at our House. Families are never turned away for inability to pay.
Therefore, this is a great and easy fundraiser for anyone to participate in! Everyone can collect pop tabs for RMHC-Siouxland. Collecting pop tabs is a way to support a community resource, teach kids about philanthropy and the importance of recycling, and know that you are helping families in need. It is also a great way for us to raise much needed funds to help keep families close while seeking medical care for their child.
Why only the tabs?
The Ronald McDonald House collects pop tabs rather than cans for several reasons. The tab is worth more because it is a different type of aluminum alloy than the can. The tab takes up less space and can be cleaned and stored easily.
What about soup and pet food can tabs?
For recycling purposes, we only accept tabs that are made of the non-magnetic aluminum alloy. Therefore, a great way to check your tabs is to hold a magnet to them. If they are not attracted, then you can donate them to us. We have recently found that some soup cans have the non-magnetic aluminum alloy that we can recycle.
Who can collect?
Any individual, service organization, business, school, or church group can collect pop tabs for RMHC-Siouxland. Thousands of dollars are raised each year from simply collecting pop tabs.
Collecting pop tabs is extremely easy! Pick a container that you want to fill and when it is filled, drop it off at the Ronald McDonald House at 2500 Nebraska St. Sioux City
Some tab collection placement ideas:
Eating areas at company picnic
Tables at a celebration event (Birthday, Weddings, New Years, Super Bowl, 4th of July)
School faculty lounge
Church fellowship hall or by pop machine
On the bar at your restaurant
In the break room at your work
In your can collection area at home
Any container will work, but if you would like one of the official pop tab collection houses, you can always stop by the House during business hours (8 a.m. - 8 p.m.) and pick one up, or you can contact RMHC-Siouxland at 712-255-4084 or info@rmhc-siouxland.org. We will be happy to get one to you!
What do I do with the collected pop tabs?
Just drop the tabs off at RMHC-Siouxland (2500 Nebraska St. Sioux City, IA 51104) anytime between 8 am and 8 pm. If you have a large amount, you are welcome to call in advance and we will have someone help you with them.
Just drop the tabs off at the Ronald McDonald House located at 2500 Nebraska Street in Sioux City anytime between 8 a.m. and 8 p.m. They are then weighed to determine their value and the recycling center will send the RMHC-Siouxland a check for the total value.
To learn more about starting your own pop tab collection, contact the Ronald McDonald House of Siouxland at 712-255-4084 or info@rmhc-siouxland.org.
Thank you to our 25th anniversary sponsors
© RMHC 2017
RMHC is recognized as a public charity under Internal Revenue Code section 509(a) and has 501(c)(3) status. Donations to RMHC are deductible. Donors should consult their tax advisor for questions regarding deductibility. The RMHC of Siouxland EIN is 42-1369988. A copy of the RMHC determination letter is available upon request.

redpajama_set_name: RedPajamaCommonCrawl


text:
Umeox X5 out to prove 'world's thinnest' smartphone tag isn't wearing thin
By Marc Chacksfield published 12 August 13
Chinese manufacturer looks to break records
Umeox X5 - coming to IFA soon
IFA 2013 is just around the corner which means the yearly tech jostle by minor companies for headlines is in full force.
This time it is Chinese company Umeox hoping to wow with the fact that its latest smartphone is set to be the "world's thinnest".
Originally rearing its head back in June, the Umeox X5 is a mere 5.6mm, 0.24mm thinner than its nearest rival the Huawei Ascend P6.
This means that it is thinner than a pencil – so us tech scribblers will have to come up with a brand-new object to measure the phone with.
Or we could just keep saying it's thinner than a pencil but that's not exactly scientific, is it?
Thompson talk
Given that IFA's halls are more spacious than Uwe Boll's film award cabinet, we expect there will be more companies clamoring for the world's thinnest crown.
But, for the moment, the Umeox X5 is the slimmest Jim around with its rumoured 4-inch display, MediaTek chip and 5MP camera.
Interestingly, when whispers first emerged about the device it had a 5.3-inch display but it seems that the smaller 4-inch variant is what will be unveiled in Berlin.
The phone is set to be shown off at IFA 2013 under its European Thompson Mobile guise and will be running Android Jelly Bean.
Want to know what else will be launched at IFA this year? Click here to magically find out.
Marc Chacksfield
Marc Chacksfield is the Editor In Chief, Shortlist.com at DC Thomson. He started out life as a movie writer for numerous (now defunct) magazines and soon found himself online - editing a gaggle of gadget sites, including TechRadar, Digital Camera World and Tom's Guide UK. At Shortlist you'll find him mostly writing about movies and tech, so no change there then.
See more Mobile phones news


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"The Family Unit in Holguín Is Very Damaged" / 14ymedio, Fernando Donate Ochoa
Marcos Pirán Gómez, parish priest of San José Church in Holguín (Photo Fernando Donate/14ymedio)
14ymedio, Fernando Donate Ochoa, Holguín. 21 September 2015 — He shares both his faith and Argentine citizenship with Pope Francis, yet Father Marcos Pirán Gómez is not on Cuban soil for just a few days. He has been living on the island for fifteen years, and since 2012 has been the parish priest of San José (Saint Joseph's) Church in Holguín.
A few hours before the Bishop of Rome travelled to this land of mountains, heat, and seas, Father Marcos met with 14ymedio to discuss his parishioners' expectations, the difficulties besetting his community, and the role of the Church in finding solutions.
14ymedio: What did you feel when you heard that Pope Francis was coming to Holguín?
Marcos Pirán Gómez: I felt an enormous thrill because of the joy another Papal visit would mean to our people; the third one in just 17 years, such a short period of time. This is a significant event not only for the life of the Church, but also for the Cuban people. Each one of the previous Papal visits have left its mark.
Pope Francis is an exemplary and admirable person for the way he thinks, for what he does, and for his beliefs, which are consistent with how he lived in Argentina. I remember what he was able to generate around him, especially in Buenos Aires where we were neighbors living in the same area, and where we had more contact with each other. I know a lot of things from back then that are now known in the public square.
14ymedio: How will this Papal visit to the island be different?
Marcos Pirán Gómez: This third visit is also linked to the resumption of talks between the Cuban and American governments. This time stands out because it hopes to keep alive the first step taken on December 17th of last year.
Pope Francis has closely followed the history of the Cuban people. He wrote a book about John Paul II's trip to Cuba, which undeniably signaled a before and after. It not only did so as far as the relationship between the Church and the Cuban government is concerned, but the relationship between religion and the government as well.
14ymedio: Is there special interest for Cuba in the Vatican?
Marcos Pirán Gómez: Interest and worry for these people. Cuba has undergone a political and social experiment unlike that experienced by most of Latin America's people. It's different in the fact that Communist ideology takes precedence in Cuba, so theres a very different attitude towards religion here than in most other countries.
Starting with John Paul's visit, an effort was made to initiate a new type of relationship (between the Church and the State) in which there would be an official recognition of the of religiosity of individuals and of our people as a whole.
14ymedio: What do believers in Holguín expect from this visit?
Marcos Pirán Gómez: On the one hand, the people of Holguín hope this visit will help them regain their enthusiasm and hope. There're many people who due to different difficulties, such as the frustrations of life, the breakdown of families, the scarcities, have lost their will to forge ahead. The Pope can help reverse this because his message aims to break apathy and indifference.
14ymedio: Do you sense a lot of apathy?
Marcos Pirán Gómez: Yes. People are apathetic because of their incapacity to react to situations they don't agree with. When an individual stops demanding or voicing his concerns, whether it is out of fear or because he thinks it would be useless, that is worrisome, because it shows an attitude of apathy and indifference. I hope, and many others do as well, that the presence of Pope Francis will help bring about a reawakening.
14ymedio: So you are excited about the visit?
Marcos Pirán Gómez: Very excited. I've been especially impressed by people who don't practice any religion but who see the (Papal) visit as a very positive thing. People are hopeful this visit will bring something that'll make them better persons.
14ymedio: The authorities have conceded that violence, drug addiction, suicide, and other social ills have increased in Holguín. Is there a spiritual crisis in Holguín?
Marcos Pirán Gómez: The family unit in Holguín is very damaged and divided. There're a lot of difficult situations, and that affects the social order.
Family units are fragmented because of financial difficulties, and because a lot of people emigrate, and that brings suffering. People don't know how to discuses issues, how to accept one another, how to collaborate, or how to promote solidarity within the family setting. When this starts happening to the family unit, it resonates throughout society at large, while adding to the already existing personal crises in each individual's life.
14ymedio: The Cuban government pardoned 3,522 before the Pope's arrival. What can you say about this?
Marcos Pirán Gómez: Some have reacted happily, while others were disappointed because they thought they met all the conditions for a pardon but weren't. The announcement (of the prisoner release) states that those convicted of "crimes against State security" would remain incarcerated. That's why in this case it is a matter of opinion if those still being held are political prisoners or not.
14ymedio: Are there political prisoners in Cuba?
Marcos Pirán Gómez: This isn't conjecture. The government itself has admitted there are political prisoners. Several years back, the President mentioned them. I don't know how many there are, because there's a lot of information I don't know or have any access to. That's why I can't say for certain how many political prisoners there are, or where they are.
14ymedio: Has the Church in Holguín, or you, received a request from the opposition to meet with Pope Francis?
Marcos Pirán Gómez: Up until now, that hasn't happened. However, we have received letters from people directed to the Pope, asking him to intercede in support of freedom for their relatives serving prison sentences. Still, I don't know if these cases are political prisoners.
14ymedio: Has the relationship between the Church and the State in Cuba been strengthened?
Marcos Pirán Gómez: The preparations for the Pope's visit have opened the way for some dialog. Catholics in the Papal visit's organizing committee note there have been important changes when compared to eighteen years ago while preparing for John Paul II's visit.
Also, for some time now we have been noticing that the government has somewhat stopped pressuring mission houses or small (parish) communities from opening. Today, these houses do exist, but there was a time when the government wouldn't allow people to gather in homes to celebrate the Word, to pray together, or to exchange ideas. The result of this is an increase in the social ills we're now facing. When you keep something from growing over a long period of time, that has negative repercussions.
14ymedio: The Catholic Church in Cuba does not have at its disposal a radio station or a television channel. Doesn't that limit your pastoral work?
Marcos Pirán Gómez: Nowadays, having access to the media is very important. I don't like things that are just Catholic. I'd like more diversity in the Cuban media. I don't need to have my own radio station, TV channel, or newspaper, because that in itself is exclusionary. I'm not interested in that way of thinking. What I do wish is that there be space for other voices, other ways of thinking, and other messages that contribute to the common good, within the media that already exists.
Translated by José Badué
Author 14ymedioPosted on October 4, 2015 October 4, 2015 Categories 14ymedio, Fernando Donate Ochoa, Translator: José Badué
Previous Previous post: General Raul Castro's Plastic Bag at the Papal Mass / Juan Juan Almeida
Next Next post: Cuban Phone Company Authorizes Internet Refills From Abroad / 14ymedio


Lou and I hit up Grindcore House for some vegan cake.
And I was finally able to give him his gift — I picked up these Empire Strikes Back glasses at Bric-A-Brac in Chicago and had to get 'em for him.
New patch for the collection. More on that later.
My pal Ian and I went to see Blue Jasmine on the Philly opening night. We accidentally dressed alike. Whoops.
The stoop sale was a huge success! Thanks for comin' out!


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56771Cape to Kalahari: 5 stops every traveller should make
Cape to Kalahari: 5 stops every traveller should make
On October 19 2016
The Kalahari is a sprawling arid area which spans somewhere around 900,000 square kilometres through Botswana, areas of Namibia and certain areas within South Africa.
The name for which this vast, desolate and inspiring area is a combination of two words within the Tswana language roughly translating to 'The great thirst Kgala and 'a waterless place Kgalagadi. There are many reasons people travel to this iconic area and some even choose it as a great road trip route from arguably South Africa's most famous city, Cape Town.
Cape Town has captured the imagination of travellers for what would seem like centuries now a city made from stories and legends. A legacy of diversity and rich culture and with all the first world amenities any traveller could ever want or need it's a great place to start your road trip through the breathtaking Kalahari Desert. Here are some great places to consider on your next road trip from Cape Town to the Kalahari.
Side note
These are only a few of the places for which you can visit on a road trip from the Cape to the Kalahari, do some research to get a better understanding on which routes you would be suitable based on your time and budget. Car hire Cape Town is your best bet of achieving this.
Iconic rock art as found in the Cederberg area - -By Valroe at English Wikipedia [Public domain], via Wikimedia Commons
There really is a lot to experience in this part of the world and it's a great place to travel to whilst starting your journey as it isn't far from your departure destination of Cape Town. From great waves in Lamberts or Elands Bay and an assortment of towns, villages littered with great boutique hotels and seafood restaurants in abundance it's no wonder the world has become a sought after destination on any road trip from Cape Town to the Kalahari.
During certain times of year this is what people travelling to Namaqualand will be treated to.
The Namaqua National Park is widely considered as one of the best places to see flowers in bloom throughout the whole of South Africa. There is even a separate reserve within it dedicated entirely to flowers entitled the Skilpad Wildflower Reserve. Other highlights to the area include the 'Roof of Africa's panoramic views and dedicated 4×4, mountain biking and walking trails as well as self-catering chalets and exclusive luxury tented camps.
Augrabies Falls National Park
Interesting terrain in the Augrabies Falls National Park - By NJR ZA (Own work) [CC BY-SA 3.0 (httpcreativecommons.orglicensesby-sa3.0) or GFDL (httpwww.gnu.orgcopyleftfdl.html)], via Wikimedia Commons
Loosely translated to 'place of Great Noise in the Khoi language and culture, this is also the location of animals and plant life of sheer resilience. This is the area of South Africa where the Nama people are indigenous and besides for the breathtaking falls, other noteworthy scenes and sights to explore include Moon Rock, Ararat and Echo Corner. The park changes with the seasons so do some research beforehand depending on what you wish to see and enjoy.
Majestic wildlife in the Kgalagadi Transfrontier Park - By PanBK at en.wikipedia (Transferred from en.wikipedia) [GFDL (httpwww.gnu.orgcopyleftfdl.html) or CC-BY-SA-3.0 (httpcreativecommons.orglicensesby-sa3.0)], from Wikimedia Commons
The Kgalagadi Transfrontier Park mandate it conserve the fragile ecosystem of the great Kalahari Desert and is a leading wilderness area for the wildlife enriched Botswana. Animals found in the park include wildebeest, springbok and black-maned Kalahari lions. The park is blessed with bush camps and lodges which offer patrons various ways to enjoy the abundant nature of the area.
A scene of splendour in Upington - By paffy (Upington Orange river) [CC BY-SA 2.0 (httpcreativecommons.orglicensesby-sa2.0)], via Wikimedia Commons
This iconic South African town established in 1884 in the Northern Cape is situated picturesquely on the banks of the Orange River. Formerly titled Olyfenhoudtsdrif translated in English to 'Olive Wood Drift&rsquo. The town was later named after Sir Thomas Upington, Attorney-General of the Cape. The town was also a mission station from 1875 and today one of its main attraction is the Kalahari Orange Museum. One of the main attractions of the museum is a statue of a donkey an animal which has great significance to the Kalahari area.


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Divided En Banc Fourth DCA On Reciprocity Of Attorneys' Fees
In Florida Hurricane Protection and Awning, Inc. v. Pastina (4D08-4641), a divided en banc Fourth District issued an opinion of the reciprocal application of section 57.105(7), Florida Statutes (2008). The majority opinion was written by Judge May who was joined by Chief Judge Gross, Judge Warner, Judge Damoorgian, Judge Gerber and Judge Levine. Judge Taylor wrote a dissent and was joined by Judge Farmer, Judge Stevenson, Judge Hazouri, and Judge Ciklin. Judge Farmer wrote a dissent and was joined by Judge Hazouri. Judge Polen was recused.
The court described the dispute as follows:
The contractor failed to complete the installation, forcing the homeowner to hire a replacement contractor to finish the job. She then filed a complaint against the contractor for breach of contract. She sought damages for the cost to complete the work and consequential damages for replacement of a window and frame damaged during a later hurricane. She also requested attorney's fees. The homeowner prevailed on her breach of contract claim. She then moved for attorney's fees. Her motion asserted that she was reciprocally entitled to attorney's fees based on the fee provision in the contract. The contractor opposed the request for fees. It argued the fee provision was narrow, was triggered only in a collection action, and was inapplicable to any other type of dispute between the parties. The contractor relied on our opinion in Subway.
The majority concluded:
Simply put, the statute means what it says and says what it means; nothing more, nothing less. Our court and others have consistently read the statute in the same way. Our latest pronouncement can be found in Subway Restaurants, Inc. v. Thomas, 860 So. 2d 462 (Fla. 4th DCA 2003). There, Subway filed a breach of contract suit against its franchisee. The franchisee correspondingly filed suit against Subway for wrongful eviction, violation of civil rights, and breach of contract under the lease and franchise agreement. The franchisee requested attorney's fees.
The franchisee prevailed. Following an appeal, the franchisee sought attorney's fees, relying on a provision of the contract that provided for fees for collection "on any part of said rental that may be collected by suit . . . ." Id. at 463. The trial court awarded fees based upon section 57.105(6), Florida Statutes, the predecessor of section 57.105(7). We reversed. Because the franchisee's claim was for wrongful eviction and breach of contract, it "never triggered Subway's limited entitlement to attorney's fees." Id. at 464.
Similarly, here the contract provided fees for the contractor in the event of a collection action. Section 57.105(7) requires reciprocity. Reciprocity would allow for the homeowner to receive fees if she prevailed in a collection action brought by the contractor. That is mutuality; that is reciprocity. To rule otherwise would be tantamount to re-writing the contract between the parties. This we will not do.
Labels: Fees, Fourth DCA


Cristiano Ronaldo shook his head as he walked off the field, while the Ajax players and staff celebrated with their fans long after the final whistle. The youth-filled Ajax team knocked off another one of Europe's giants on Tuesday, beating Juventus 2-1 in Turin to reach the Champions League semifinals for the first time since 1997. It is the first time since 2010 that Ronaldo has failed to reach the last four, having won the competition the last three years in a row with Madrid.
Even going behind to a goal from Ronaldo in the first half didn't faze Ajax's young players as Donny van de Beek levelled shortly after the 19-year-old captain Matthijs de Ligt scored the winner in the second half.
Ronaldo scored a hat trick in the previous round against Atletico Madrid to almost single-handedly overturn a two-goal deficit and had also found the back of the net in Amsterdam. The 34-year-old Portugal forward added his fifth goal in three Champions League matches in the 28th minute on Tuesday, heading in a Miralem Pjanic corner.


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Q: gnuplot: multiplot with "pause mouse close" skips a plot I've noticed that multiplot in gnuplot, when using "pause mouse close" to display a plot and then quit, only plots the second plot.  (Thisis gnuplot 5.0 patchlevel 3 on Ubuntu 16.04.)  Compare this
cat <<EOF | gnuplot
set multiplot
set size 0.4,0.4
set origin 0.1,0.1
plot sin(x)
set size 0.2,0.2
set origin 0.5,0.5
plot cos(x)
unset multiplot
pause mouse close
EOF

with this
cat <<EOF | gnuplot -persist
set multiplot
set size 0.4,0.4
set origin 0.1,0.1
plot sin(x)
set size 0.2,0.2
set origin 0.5,0.5
plot cos(x)
unset multiplot
EOF

I've not found anything relevant in the docs or on the Net.  Anyone have any pointers if this is my error or a bug?

redpajama_set_name: RedPajamaStackExchange


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Home > Net News > Andy Rooney Dead At 92
Andy Rooney Dead At 92
November 7, 2011 at 3:58 AM (PT)
Longtime CBS NEWS correspondent and "60 MINUTES" commentator ANDY ROONEY died FRIDAY (11/4) of complications after surgery at 92. ROONEY had retired after his final "60 MINUTES" commentary on OCTOBER 2nd.
Best known for his television work and curmudgeonly segments on everyday observations, ROONEY launched his journalism career while serving in WORLD WAR II as a writer for STARS AND STRIPES. He joined CBS in 1949, writing for radio and TV series "ARTHUR GODFREY'S TALENT SCOUTS," later joining "THE GARRY MOORE SHOW" and CBS NEWS shows. A series of critically acclaimed TV essay specials, starting with 1964's "AN ESSAY ON DOORS," aired on CBS, written by ROONEY and narrated by HARRY REASONER, during the 1960's, but when the network declined to air "AN ESSAY ON WAR" in 1970, ROONEY jumped to PBS and did his first on-camera special.
He returned to CBS for a series of prime-time specials, winning a PEABODY Award for "MR. ROONEY GOES TO WASHINGTON." He began "THREE MINUTES OR SO WITH ANDY ROONEY," later renamed "A FEW MINUTES WITH ANDY ROONEY," on "60 MINUTES" in 1978.


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Bonsu reaches a milestone
Bonsu Elite Athletics celebrated its 10th anniversary earlier this month. Located in Ceres, the sports performance training business is owned and operated by 33-year-old Les Bonsu.
Bonsu Elite Athletics celebrated its 10th anniversary on April 13.
The 9,000-square-foot sports performance training facility is located at 3427 Railroad Avenue in Ceres.
"I love what I do," said Les Bonsu, owner and trainer. "It doesn't feel like a job."
Bonsu Elite Athletics (BEA) specializes in speed, agility and strength training.
"We have kids come in because they want to get better for their sport and prepare for the transition to college," he said.
The majority of Bonsu's clients are in high school.
He also offers sessions for college, middle school and youth athletes.
BEA trains about 60 people a day.
Sessions are offered Monday through Saturday.
"Every hour and a half, there's a change of group of athletes that come in," Bonsu said. "We train athletes from every sport. I haven't expanded to the general population yet. In the future, that's something I might do."
Bonsu has been the go-to trainer for many of the top athletes in the area, including Kendall Wesenberg, Johnny Mundt, Ja'Quan Gardner, Cade Cowell, Nicole Bates and Madilyn Nickles.
Wesenberg finished 17th overall while representing Team USA in the women's skeleton competition at the 2018 Winter Olympics in South Korea.
"I first started training with him (Bonsu) in 2013 at his facility on Bangs in Modesto," said Wesenberg, a 2008 Central Catholic High School grad who completed her college education at the University of Colorado. "I wasn't great at Olympic lifting and he coached me. He also helped me with my sprint forms and mechanics. That helped me move more efficiently."
"The environment he creates is so positive. He's really constructive. He knows what he's talking about and gives good feedback."
Kendall Wesenberg
"Les is awesome," she added. "The environment he creates is so positive. He's really constructive. He knows what he's talking about and gives good feedback."
Mundt, a 2013 Central Catholic grad who played tight end for the University Oregon, suited up for the Los Angeles Rams in Super Bowl LIII in February. He made one tackle on special teams.
Gardner trained six times a week at BEA leading up to the 2018 NFL Draft.
The 2014 Central Valley High School grad/former Humboldt State star running back signed a contract as a rookie free agent with the San Francisco 49ers in August. He starred at running back with the San Diego Fleet in the Alliance of American Football league.
"I appreciate everything he's done for me," said Gardner, who started training with Bonsu during his sophomore year of high school. "There was never a time he wasn't willing to work with me. He helped prepare me for the next level. He's more than a coach. He's a leader and role model for the community."
Cowell, 15, signed five-year contract with the San Jose Earthquakes of Major League Soccer in January. He recently logged playing time in two preseason matches.
"Bonsu had a huge impact on me," said Cowell, who started training at BEA when he was in the seventh grade at Cesar Chavez Jr. High. "He helped me become a great explosive athlete and be able to hang with the big boys strength-wise. Bonsu is the most positive trainer I've ever been around. He is always super easy to talk to and is very funny. He has a great relationship with any athlete that goes in there, no matter who they are. He also knows his stuff, is always really good with any workout, and if you need extra help with anything, he knows exactly what to do. I couldn't thank Bonsu enough for what he did for me."
Bates (Ceres High) and Nickles (Merced) are standout softball players at Washington and UCLA, respectively. They played for the USA Softball Women's National Team during the summertime.
BEA has had more than 150 athletes earn scholarships to college.
"It's very satisfying having kids come in as eighth graders, progress and move on to the next level," Bonsu said. "I love those stories."
"When a kid gets a scholarship, all of the other kids are excited," he added. "They started that culture."
Coleby Garrett started training with Bonsu when he was in the fifth grade at Whitmore Charter School in Ceres.
The 2018 Central Catholic High School grad played fullback and special teams for the Cal Lutheran football team as a true freshman this past fall.
"Bonsu's a great guy," Garrett said. "He's always been there for me. He went to my youth and high school games. He made me a better all-round athlete. He's made me quicker and stronger. He improved my speed and strength."
"Coleby's dad was one of my groomsmen in my wedding," Bonsu said. "I met him through training his son."
In January, Bonsu attended a workshop at Notre Dame that focused on the latest stretching and recovery techniques.
"I still go to seminars every year," he said. "I don't know it all. I want to constantly learn."
BEA operated out of a 900-square-foot studio at 3025 McHenry Avenue in Modesto during its first two years of existence.
Business has been booming since Bonsu moved BEA from a 2,500-square-foot center at 401 Bangs Avenue in Modesto to Ceres six years ago.
"This is something I've always envisioned," Bonsu said. "I didn't think I'd grow as fast as I did. It's based on results. I don't really advertise. It's by word of mouth. Parents talk to each other."
"The growth is incredible," Wesenberg said. "It all goes back to him. Les is a great person. He creates an environment that's very positive. That's a major draw."
"When he started, it was me and 10 other people," Garrett said. "I'm not surprised by the growth that he's had. He's really knowledgeable about what he does. He's been doing it for a lot of years. He encourages you. He doesn't yell at you. He's really respectful. He wants the best for you. Every time I go in there, I'm seeing new faces. Everyone wants to be trained by the top guy."
"He's super humble," Gardner said. "He knows how to get the best out of his athletes. He has relationships with each and every one of his athletes. That's important."
"I'd still have the same passion if I had five kids," Bonsu said. "I love every day I'm there. I enjoy seeing kids getting better at their craft. It's very rewarding."
The facility is open Monday through Friday from 2-9 p.m. and on Saturday from 8 a.m. to 2 p.m.
For more info about BEA, call 247-2695.
Whitmore Charter School eighth-grader Hudson Wyatt works out during a recent training session at Bonsu Elite Athletics.


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mdigital » Jazz » Oscar Peterson - Oscar Peterson Plays The Duke Ellington Songbook
Oscar Peterson - Oscar Peterson Plays The Duke Ellington Songbook download mp3 album
Oscar_Peterson Swing 1999 US
Oscar Peterson Plays The Duke Ellington Songbook
MPC MIDI AU MMF APE ADX VOC
Oscar Peterson Plays the Duke Ellington Song book is a 1959 album by pianist Oscar Peterson of compositions written or performed by Duke Ellington. Peterson had recorded many of the pieces for his 1952 album Oscar Peterson Plays Duke Ellington, and had recently performed with Ella Fitzgerald on her 1957 album, Ella Fitzgerald Sings the Duke Ellington Song book. Oscar Peterson – piano. Ray Brown – double bass.
Producer Norman Granz invited Oscar Peterson and his Trio to record a set of Ellington songs in 1952. Seven years later, they did it again-this time in stereo and with different interpretations and personnel. This CD combines two LPs devoted to the same Duke Ellington material recorded by different versions of the Oscar Peterson trio. The first is of particular interest. This is a double album that combines an early album titled Oscar Peterson Plays Duke Ellington that was recorded in Los Angeles for Mercury, and a later 1959 album titled Oscar Peterson Plays The Duke Ellington Song Book that was recorded in Chicago for Verve.
The first of Oscar Petersons two 50's Duke Ellington songbook albums, originally released on a 10" album. The line-up on these december 1952 recordings are Oscar Peterson (Piano), Barney Kessel (Guitar) and Ray Brown (Double bass). Not to be mistaken for Petersons 1959 release "Oscar Peterson Plays the Duke Ellington Songbook" recorded with a different trio. This came out on Mercury prior to Clef.
Oscar Peterson Plays the George Gershwin Songbook (1959). Years given are for the recording(s), not first release. Note: All-Star albums feature sideman who are not necessarily listed while titles which include "Oscar Peterson" or the OP Trio are usually shortened. As leader or co-leader.
The Phenomenal Oscar Peterson, Vol. 2. Oscar Peterson. 2011 Original Album Mit Bonus Tracks. The Best Of Oscar Peterson. 2015 All Tracks Remastered. Plays the Duke Ellington Songbook & Plays the Count Basie Songbook.
No songs for Oscar Peterson Plays the Duke Ellington Songbook yet. embed
1 Don't Get Around Much Anymore
2 Sophisticated Lady
3 Rockin' In Rhythm
4 Prelude To A Kiss
5 In A Mellow Tone
6 Cottontail
7 Just A Sittin' And A Rockin'
8 Things Ain't What They Used To Be
9 Take The "A" Train
10 I've Got It Bad And That Ain't Good
11 Do Nothin' Till You Hear From Me
12 John Hardy's Wife
Record Company – Polydor K.K.
Distributed By – PolyGram K.K.
Art Direction – Sheldon Marks
Bass – Ray Brown
Cover [Illustration] – John Altoon
Drums – Ed Thigpen
Liner Notes – Lawrence D. Stewart
Piano – Oscar Peterson
Comes in mini LP replica with OBI & Japanese insert.
Barcode: 4 988005 233653.
MG V-2055 Oscar Peterson Oscar Peterson Plays The Duke Ellington Songbook (LP, Album) Verve Records MG V-2055 US 1959
C 06230 Oscar Peterson Oscar Peterson Plays The Duke Ellington Songbook (Cass, Album, Club) World Cassette Club C 06230 Australia 1979
MGC-2055 Oscar Peterson Oscar Peterson Plays The Duke Ellington Songbook (LP, Album) Verve Records MGC-2055 Australia 1959
2332 090 Oscar Peterson The Duke Ellington Songbook (LP, Album, RE) Verve Records 2332 090 Germany Unknown
UCCV-9393 Oscar Peterson Oscar Peterson Plays The Duke Ellington Songbook (CD, Album, Ltd, RE) Verve Records UCCV-9393 Japan 2010
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Oscar Peterson - The Song Is You (Best Of The Verve Songbooks) album mp3
Oscar Peterson - Oscar Peterson Plays George Gershwin (No. 2) album mp3
Oscar Peterson - Oscar Peterson At Carnegie album mp3
Oscar Peterson - Oscar Peterson In Paris album mp3