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Petr Smykal

Palacky University, Olomouc, Department of Botany, Faculty Member

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I am interested in study of legume seed dormancy (physical type of dormancy) one of the two key domestication traits using combination of anatomical, genetic, transcriptomics and analytical chemistry tools. Seed dormancy is also studied as adaptive trait in context of ecological genomics combining next generation sequencing, seed testing and geoinformatics using wild pea and Medicago truncatula HapMap resources and models. Transcriptomic analysis is applied to identify candicate genes in wild pea, along with Medicago truncatula mutants to study the respective gene function.
Knowledge on Pisum genus diversity is applied to broaden the pea crop genetic diversity, the existing Crop Wild Relatives are used in series of introgression lines.

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Marcelo Cervini

Università Politecnica delle Marche, Italy

University of Idaho

London School of Hygiene & Tropical Medicine

Université de Liège

Madawalabu University

Institut National des Sciences Appliquées et de Technologie

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Papers by Petr Smykal

TAG_2012-European pea collections.pdf

The distinctness of, and overlap between, pea genotypes held in several Pisum germplasm collectio... more The distinctness of, and overlap between, pea genotypes held in several Pisum germplasm collections has been used to determine their relatedness and to test previous ideas about the genetic diversity of Pisum. Our characterisation of genetic diversity among 4,538 Pisum accessions held in 7 European Genebanks has identified sources of novel genetic variation, and both reinforces and refines previous interpretations of the overall structure of genetic diversity in Pisum. Molecular marker analysis was based upon the presence/absence of polymorphism of retrotransposon insertions scored by a high-throughput microarray and SSAP approaches. We conclude that the diversity of Pisum constitutes a broad continuum, with graded differentiation into sub-populations which display various degrees of distinctness. The most distinct genetic groups correspond to the named taxa while the cultivars and landraces of Pisum sativum can be divided into two broad types, one of which is strongly enriched for modern cultivars. The addition of germplasm sets from six European Genebanks, chosen to represent high diversity, to a single collection previously studied with these markers resulted in modest additions to the overall diversity observed, suggesting that the great majority of the total genetic diversity collected for the Pisum genus has now been described. Two interesting sources of novel genetic variation have been identified. Finally, we have proposed reference sets of core accessions with a range of sample Communicated by I. Mackay.

TAG_From Mendel’s discovery on pea to today’s.pdf

PTOC-Vavilovia_2016.pdf

sustained division. Subsequently, protoplasts isolated from internode callus proliferated and als... more sustained division. Subsequently, protoplasts isolated from internode callus proliferated and also underwent organogenesis coupled with whole plant recovery at a low frequency, while protoplasts from leaf callus origin followed both organogenesis and embryogenesis simultaneously but failed to yield viable plants. Flow cytometry assessments permitted to ascertain the genetic idelity of both propagated and regenerated plants irrespectively of the source tissue from which they were derived (i.e., either callus from explants or from protoplasts). Finally, low cytometry also permitted us to provide the irst record on the relative nuclear DNA content and genome size for Vavilovia formosa.

Vavilovia-PlantSystEvol_2017.pdf

Vavilovia formosa is one of five genera in tribe Fabeae, (Fabaceae, Leguminosae) with close phylo... more Vavilovia formosa is one of five genera in tribe Fabeae, (Fabaceae, Leguminosae) with close phylogenetic relationships to Pisum. It grows in subalpine and alpine levels in Armenia, Azerbaijan, Georgia, Iran, Iraq, Lebanon, Russia and Turkey and is recognized as an endangered and protected plant. This study was conducted to reveal its intraspecific variability, as well as to predict the past, extant and future species distribution range. We analysed 51 accessions with common phylogenetic markers (trnF-trnL, trnS-trnG, matK, rbcL, psbA-trnH and ITS). These represent in total up to 2551 bp of chloroplast and 664 bp of nuclear sequences per sample. Two populations from Turkey and Armenia were analysed for genetic diversity by AFLP. Leaf morphometry was conducted on 1457 leaflets from 43 specimens. Extracted bioclimatic parameters were used for niche-modelling approach. Analysis of cpDNA revealed two haplotypes, 12 samples from Armenia, Daghestan, Nakhichevan and Iran belonged to H1 group, while 39 samples of all Turkish and part of Armenian were in H2 group. The mean intrapopulation diversity based on AFLP was low (H E = 0.088) indicating limited outcrossing rate. A significantly positive correlation between geographical latitude and leaf area (q = 0.527, p \ 0.05) was found. Niche modelling has shown temporal variation of predicted occurrence across the projected time periods. Vavilovia formosa has suffered a range reduction following climate warming after last glacial maximum, which classify this species as cold-adapted among the Fabeae species as well as a glacial relict.

Eco-geographic distribution of Cicer isauricum P.H. Davis-2017.pdf

Wild Cicer species are considered as useful genetic resources for resistance to biotic and abioti... more

Frontiers_combinatorial analysis of seed coat_2017.pdf

The origin of the agriculture was one of the turning points in human history, and a central part ... more The origin of the agriculture was one of the turning points in human history, and a central part of this was the evolution of new plant forms, domesticated crops. Seed dispersal and germination are two key traits which have been selected to facilitate cultivation and harvesting of crops. The objective of this study was to analyze anatomical structure of seed coat and pod, identify metabolic compounds associated with water-impermeable seed coat and differentially expressed genes involved in pea seed dormancy and pod dehiscence. Comparative anatomical, metabolomics, and transcriptomic analyses were carried out on wild dormant, dehiscent Pisum elatius (JI64, VIR320) and cultivated, indehiscent Pisum sativum non-dormant (JI92, Cameor) and recombinant inbred lines (RILs). Considerable differences were found in texture of testa surface, length of macrosclereids, and seed coat thickness. Histochemical and biochemical analyses indicated genotype related variation in composition and heterogeneity of seed coat cell walls within macrosclereids. Liquid chromatography-electrospray ionization/mass spectrometry and Laser desorption/ionization-mass spectrometry of separated seed coats revealed significantly higher contents of proanthocyanidins (dimer and trimer of gallocatechin), quercetin, and myricetin rhamnosides and hydroxylated fatty acids in dormant compared to non-dormant genotypes. Bulk Segregant Analysis coupled to high throughput RNA sequencing resulted in identification of 770 and 148 differentially expressed genes between dormant and non-dormant seeds or dehiscent and indehiscent pods, respectively. The expression of 14 selected dormancy-related genes was studied by qRT-PCR. Of these, expression pattern of four genes: porin (MACE-S082), peroxisomal membrane PEX14-like protein (MACE-S108), 4-coumarate CoA ligase (MACE-S131), and UDP-glucosyl transferase (MACE-S139) was in Hradilová et al.

Scientific Reports_2017_Pea genomic diversity and macroecology.pdf

There is growing interest in the conservation and utilization of crop wild relatives (CWR) in int... more There is growing interest in the conservation and utilization of crop wild relatives (CWR) in international food security policy and research. Legumes play an important role in human health, sustainable food production, global food security, and the resilience of current agricultural systems. Pea belongs to the ancient set of cultivated plants of the Near East domestication center and remains an important crop today. Based on genome-wide analysis, P. fulvum was identified as a well-supported species, while the diversity of wild P. sativum subsp. elatius was structured into 5 partly geographically positioned clusters. We explored the spatial and environmental patterns of two progenitor species of domesticated pea in the Mediterranean Basin and in the Fertile Crescent in relation to the past and current climate. This study revealed that isolation by distance does not explain the genetic structure of P. sativum subsp. elatius in its westward expansion from its center of origin. The genetic diversity of wild pea may be driven by Miocene-Pliocene events, while the phylogenetic diversity centers may reflect Pleisto-Holocene climatic changes. These findings help set research and discussion priorities and provide geographical and ecological information for germplasm-collecting missions, as well as for the preservation of extant diversity in ex-situ collections.

PLOS One_2018_Smýkal-Wild pea in nature.pdf

Agronomy-2018_Domestication review.pdf

Humans have domesticated hundreds of plant and animal species as sources of food, fiber, forage, ... more Humans have domesticated hundreds of plant and animal species as sources of food, fiber, forage, and tools over the past 12,000 years, with manifold effects on both human society and the genetic structure of the domesticated species. The outcomes of crop domestication were shaped by selection driven by human preferences, cultivation practices, and agricultural environments, as well as other population genetic processes flowing from the ensuing reduction in effective population size. It is obvious that any selection imposes a reduction of diversity, favoring preferred genotypes, such as nonshattering seeds or increased palatability. Furthermore, agricultural practices greatly reduced effective population sizes of crops, allowing genetic drift to alter genotype frequencies. Current advances in molecular technologies, particularly of genome sequencing, provide evidence of human selection acting on numerous loci during and after crop domestication. Population-level molecular analyses also enable us to clarify the demographic histories of the domestication process itself, which, together with expanded archaeological studies, can illuminate the origins of crops. Domesticated plant species are found in 160 taxonomic families. Approximately 2500 species have undergone some degree of domestication, and 250 species are considered to be fully domesticated. The evolutionary trajectory from wild to crop species is a complex process. Archaeological records suggest that there was a period of predomestication cultivation while humans first began the deliberate planting of wild stands that had favorable traits. Later, crops likely diversified as they were grown in new areas, sometimes beyond the climatic niche of their wild relatives. However, the speed and level of human intentionality during domestication remains a topic of active discussion. These processes led to the so-called domestication syndrome, that is, a group of traits that can arise through human preferences for ease of harvest and growth advantages under human propagation. These traits included reduced dispersal ability of seeds and fruits, changes to plant structure, and changes to plant defensive characteristics and palatability. Domestication implies the action of selective sweeps on standing genetic variation, as well as new genetic variation introduced via mutation or introgression. Furthermore, genetic bottlenecks during domestication or during founding events as crops moved away from their centers of origin may have further altered gene pools. To date, a few hundred genes and loci have been identified by classical genetic and association mapping as targets of domestication and postdomestication divergence. However, only a few of these have been characterized, and for even fewer is the role of the wild-type allele in natural populations understood. After domestication, only favorable haplotypes are retained around selected genes, which creates a genetic valley with extremely low genetic diversity. These "selective sweeps" can allow mildly deleterious alleles to come to fixation and may create a genetic load in the cultivated gene pool. Although the population-wide genomic consequences of domestication offer several predictions for levels of the genetic diversity in crops, our understanding of how this diversity corresponds to nutritional aspects of crops is not well understood. Many studies have found that modern cultivars have lower levels of key micronutrients and vitamins. We suspect that selection for palatability and increased yield at domestication and during postdomestication divergence exacerbated the low nutrient levels of many crops, although relatively little work has examined this question. Lack of diversity in modern germplasm may further limit our capacity to breed for higher nutrient levels, although little effort has gone into this beyond a handful of staple crops. This is an area where an understanding of domestication across many crop taxa may provide the necessary insight for breeding more nutritious crops in a rapidly changing world.

G3-Patterns of Genetic Structure and Linkage Disequilibrium in a Large Collection of Pea Germplasm.pdf

Pea (Pisum sativum, L.) is a major pulse crop used both for animal and human alimentation. Owing ... more Pea (Pisum sativum, L.) is a major pulse crop used both for animal and human alimentation. Owing to its association with nitrogen-fixing bacteria, it is also a valuable component for low-input cropping systems. To evaluate the genetic diversity and the scale of linkage disequilibrium (LD) decay in pea, we genotyped a collection of 917 accessions, gathering elite cultivars, landraces, and wild relatives using an array of 13,000 single nucleotide polymorphisms (SNP). Genetic diversity is broadly distributed across three groups corresponding to wild/landraces peas, winter types, and spring types. At a finer subdivision level, genetic groups relate to local breeding programs and type usage. LD decreases steeply as genetic distance increases. When considering subsets of the data, LD values can be higher, even if the steep decay remains. We looked for genomic regions exhibiting high level of differentiation between wild/landraces, winter, and spring pea, respectively. Two regions on linkage groups 5 and 6 containing 33 SNPs exhibit stronger differentiation between winter and spring peas than would be expected under neutrality. Interestingly, QTL for resistance to cold acclimation and frost resistance have been identified previously in the same regions.

Genes-Pea domestication 2018.pdf

Pea, one of the founder crops from the Near East, has two wild species: Pisum sativum subsp. elat... more Pea, one of the founder crops from the Near East, has two wild species: Pisum sativum subsp. elatius, with a wide distribution centered in the Mediterranean, and P. fulvum, which is restricted to Syria, Lebanon, Israel, Palestine and Jordan. Using genome wide analysis of 11,343 polymorphic single nucleotide polymorphisms (SNPs) on a set of wild P. elatius (134) and P. fulvum (20) and 74 domesticated accessions (64 P. sativum landraces and 10 P. abyssinicum), we demonstrated that domesticated P. sativum and the Ethiopian pea (P. abyssinicum) were derived from different P. elatius genepools. Therefore, pea has at least two domestication events. The analysis does not support a hybrid origin of P. abyssinicum, which was likely introduced into Ethiopia and Yemen followed by eco-geographic adaptation. Both P. sativum and P. abyssinicum share traits that are typical of domestication, such as non-dormant seeds. Non-dormant seeds were also found in several wild P. elatius accessions which could be the result of crop to wild introgression or natural variation that may have been present during pea domestication. A sub-group of P. elatius overlaps with P. sativum landraces. This may be a consequence of bidirectional gene-flow or may suggest that this group of P. elatius is the closest extant wild relative of P. sativum. Genes 2018, 9, 535 2 of 20 use of wild cereals and pulses considerably predate their domestication ; similarly, the gathering of pea from 'gardens' by pre-agricultural peoples has been discussed .

Janska_AoB_2018-Role of testa- anatomy.pdf

Background A water-impermeable testa acts as a barrier to a seed's imbibition, thereby imposing d... more Background A water-impermeable testa acts as a barrier to a seed's imbibition, thereby imposing dormancy. The physical and functional properties of the macrosclereids are thought to be critical determinants of dormancy; however, the mechanisms underlying the maintenance of and release from dormancy in pea are not well understood.

Hradilova_Pea seed dormancy_PeerJ 2019.pdf

Background. Seed germination is one of the earliest key events in the plant life cycle. The timin... more Background. Seed germination is one of the earliest key events in the plant life cycle. The timing of transition from seed to seedling is an important developmental stage determining the survival of individuals that influences the status of populations and species. Because of wide geographical distribution and occurrence in diverse habitats, wild pea (Pisum sativum subsp. elatius) offers an excellent model to study physical type of seed dormancy in an ecological context. This study addresses the gap in knowledge of association between the seed dormancy, seed properties and environmental factors, experimentally testing oscillating temperature as dormancy release clue. Methods. Seeds of 97 pea accessions were subjected to two germination treatments (oscillating temperatures of 25/15 • C and 35/15 • C) over 28 days. Germination pattern was described using B-spline coefficients that aggregate both final germination and germination speed. Relationships between germination pattern and environmental conditions at the site of origin (soil and bioclimatic variables extracted from World-Clim 2.0 and SoilGrids databases) were studied using principal component analysis, redundancy analysis and ecological niche modelling. Seeds were analyzed for the seed coat thickness, seed morphology, weight and content of proanthocyanidins (PA). Results. Seed total germination ranged from 0% to 100%. Cluster analysis of germination patterns of seeds under two temperature treatments differentiated the accessions into three groups: (1) non-dormant (28 accessions, mean germination of 92%), (2) dormant at both treatments (29 acc., 15%) and (3) responsive to increasing temperature range (41 acc., with germination change from 15 to 80%). Seed coat thickness differed between groups with dormant and responsive accessions having thicker testa (median 138 and 140 µm) than non-dormant ones (median 84 mm). The total PA content showed to be higher in the seed coat of dormant (mean 2.18 mg g −1 ) than those of non-dormant (mean 1.77 mg g −1 ) and responsive accessions (mean 1.87 mg g −1 ). Each soil and bioclimatic variable and also germination responsivity (representing How to cite this article Hradilová I, Duchoslav M, Brus J, Pechanec V, Hýbl M, Kopecký P, Smržová L, Štefelová N, Vaclávek T, Michael B, Machalová J Hron K, Pirintsos S and Smýkal P. 2019. Variation in wild pea (Pisum sativum subsp. elatius) seed dormancy and its relationship to the environment and seed coat traits. PeerJ 7:e6263 http://doi.org/10.7717/peerj.6263

Cechová_AnBiochem_2019.pdf

Your article is protected by copyright and all rights are held exclusively by Springer-Verlag Gmb... more Your article is protected by copyright and all rights are held exclusively by Springer-Verlag GmbH Germany, part of Springer Nature. This e-offprint is for personal use only and shall not be self-archived in electronic repositories. If you wish to self-archive your article, please use the accepted manuscript version for posting on your own website. You may further deposit the accepted manuscript version in any repository, provided it is only made publicly available 12 months after official publication or later and provided acknowledgement is given to the original source of publication and a link is inserted to the published article on Springer's website. The link must be accompanied by the following text: "The final publication is available at link.springer.com".

Janska_AoB_2018-comprimed.pdf

Background A water-impermeable testa acts as a barrier to a seed's imbibition, thereby imposing d... more Background A water-impermeable testa acts as a barrier to a seed's imbibition, thereby imposing dormancy. The physical and functional properties of the macrosclereids are thought to be critical determinants of dormancy; however, the mechanisms underlying the maintenance of and release from dormancy in pea are not well understood.

Genetic Diversity of Albanian Pea (Pisum sativum L.) Landraces Assessed by Morphological Traits and Molecular Markers

by Gixhari Belul, Belul Gixhari, Hekuran Vrapi, Hairi Ismaili, Michaela Ludvikova, and Petr Smykal

Gixhari B., Pavelková M., Ismaili H., Vrapi H., Jaupi A., Smýkal P. (2014): Genetic diversity of ... more Gixhari B., Pavelková M., Ismaili H., Vrapi H., Jaupi A., Smýkal P. (2014): Genetic diversity of Albanian pea (Pisum sativum L.) landraces assessed by morphological traits and molecular markers. Czech J. Genet. Plant Breed., 50: 177–184. In order to investigate the genetic diversity present in the pea germplasm stored in the Albanian genebank, we analyzed 28 local pea genotypes of Albanian origins for 23 quantitative morphological traits, as well as 14 retrotransposon-based insertion polymorphism (RBIP) molecular markers. The study of morphological characters carried out during three growing seasons (2010, 2011 and 2012) had the objective of characterization of traits useful in breeding programs. RBIP marker analysis revealed the genetic similarity in range from 0.06 to 0.45. ANOVA, principal component analysis (PCA) and cluster analysis was used to visualize the association among different traits. Most of the quantitative morphological traits showed significant differences. PCA and cluster analysis (Ward's method) carried out for morphological traits divided the local pea genotypes into three clusters. Finally, the study identified the agronomicaly important traits which will facilitate the maintenance and agronomic evaluation of the collections.

The legume manifesto: (Net)workers on Fabaceae, unite!

by Frederick Stoddard and Petr Smykal

Ratarstvo i povrtarstvo, 2011

Legumes have been an important part of cropping systems since the dawn of agriculture. The shift ... more Legumes have been an important part of cropping systems since the dawn of agriculture. The shift in Europe from draught animals to meat animals coincided with the increasing availability of soybean meal from North and South America, and the Common Agricultural Policy of the European Union promoted the growing of cereals and oilseeds at the expense of other crops, so legumes fell out of favour with farmers and decision-makers. Continental concerns about food and feed security, high prices of oil and soybean meal, and advances in the application of fundamental molecular genetics to crop species, all mean that now is a good opportunity to promote the return of legumes to European cropping systems by enhancing the ef ciency of research and development on this family. Hence we propose the establishment of a Legume Society that will promote information exchange and scienti c productivity by uniting the various legume research communities.

Modulation of flowering responses in different Nicotiana varieties

Plant Molecular Biology, 2004

We have identified and characterized a FLOWERING PROMOTING FACTOR 1(FPF1) gene from tobacco (NtFP... more We have identified and characterized a FLOWERING PROMOTING FACTOR 1(FPF1) gene from tobacco (NtFPF1). Over-expression of NtFPF1 leads to early flowering in the day-neutral tobacco Nicotiana tabacum cv. Hicks, and under inductive photoperiods also in the short-day Nicotiana tabacum cv. Hicks Maryland Mammoth (MM) tobacco and the long-day plant Nicotiana sylvestris. N. sylvestris wild-type plants remained in the rosette stage and never flowered under non-inductive short-days, whereas 35S::NtFPF1 transgenic plants bolted but did not flower. However, if treated with gibberellins, transgenic N. sylvestrisplants flowered much faster under non-inductive short days than corresponding wild type plants, indicating an additive effect of gibberellins and the NtFPF1 protein in flowering time control. The day-neutral wild type cv. Hicks and the short-day cv. Hicks MM plants exhibit an initial rosette stage, both under short- and long-days. In the transgenic lines, this rosette stage was completely abolished. Wild-type plants of cv. Hicks MM never flowered under long days; however, all transgenic lines over-expressing NtFPF1 flowered under this otherwise non-inductive photoperiod.

Effect of Environmental and Genetic Factors on the Stability of Pea (Pisum sativum L.) Isozyme and DNA Markers

Environmental (geographic location, year-to-year effect) and genetic (intravarietal variation) ef... more Environmental (geographic location, year-to-year effect) and genetic (intravarietal variation) effects on the stability of the isozyme patterns of esterase (EST), acid phosphatase (ACP), alcohol dehydrogenase (ADH), leucine aminopeptidase (LAP), shikimate dehydrogenase (SDH) and glucose-6-phosphate isomerase (PGI) used for pea cultivar genotyping were studied. In addition, selected DNA markers (RAPD, SSR, ISSR, IRAP) were used to study intravarietal genetic homogeneity/variation at a DNA level. Five commercial dry-seed pea (Pisum sativum L.) cultivars Canis, Gotik, Komet, Sonet and Zekon were grown during four years (2001 -2004) in three locations in the Czech Republic (Šumperk, Čáslav, Uherský Ostroh) that differed in soil and climatic characteristics. Mature dry seeds were used as a standard input sample for analyses. No variation in isozyme spectra was found within particular cultivars between years, locations and fruiting nodes in enzymes providing in general a low polymorphism in pea cultivars (ACP, ADH, LAP, SDH, PGI); similarly, these enzymes also exhibited high stability as related to intravarietal variation. In contrast, EST -highly polymorphic in pea cultivars -showed certain qualitative variation within particular cultivars as related to both environmental and genetic factors. The intravarietal variation detected by selected DNA markers was negligible and mostly quantitative. Possible reasons for the instability/variation of isozyme markers are discussed from the aspect of cultivar genotyping used in pea breeding and seed production.

Estimation of pea ( Pisum sativum L.) microsatellite mutation rate based on pedigree and single-seed descent analyses

Journal of Applied Genetics

Microsatellites, or simple sequence repeats (SSRs) are widespread class of repetitive DNA sequenc... more Microsatellites, or simple sequence repeats (SSRs) are widespread class of repetitive DNA sequences, used in population genetics, genetic diversity and mapping studies. In spite of the SSR utility, the genetic and evolutionary mechanisms are not fully understood. We have investigated three microsatellite loci with different position in the pea (Pisum sativum L.) genome, the A9 locus residing in LTR region of abundant retrotransposon, AD270 as intergenic and AF016458 located in 5′untranslated region of expressed gene. Comparative analysis of a 35 pair samples from seven pea varieties propagated by single-seed descent for ten generations, revealed single 4 bp mutation in 10th generation sample at AD270 locus corresponding to stepwise increase in one additional ATCT repeat unit. The estimated mutation rate was 4.76 × 10-3 per locus per generation, with a 95% confidence interval of 1.2 × 10-4 to 2.7 × 10-2. The comparison of cv. Bohatýr accessions retrieved from different collections, showed intra-, inter-accession variation and differences in flanking and repeat sequences. Fragment size and sequence alternations were also found in long term in vitro organogenic culture, established at 1983, indicative of somatic mutation process. The evidence of homoplasy was detected across of unrelated pea genotypes, which adversaly affects the reliability of diversity estimates not only for diverse germplasm but also highly bred material. The findings of this study have important implications for Pisum phylogeny studies, variety identification and registration process in pea breeding where mutation rate influences the genetic diversity and the effective population size estimates.

TAG_2012-European pea collections.pdf

The distinctness of, and overlap between, pea genotypes held in several Pisum germplasm collectio... more The distinctness of, and overlap between, pea genotypes held in several Pisum germplasm collections has been used to determine their relatedness and to test previous ideas about the genetic diversity of Pisum. Our characterisation of genetic diversity among 4,538 Pisum accessions held in 7 European Genebanks has identified sources of novel genetic variation, and both reinforces and refines previous interpretations of the overall structure of genetic diversity in Pisum. Molecular marker analysis was based upon the presence/absence of polymorphism of retrotransposon insertions scored by a high-throughput microarray and SSAP approaches. We conclude that the diversity of Pisum constitutes a broad continuum, with graded differentiation into sub-populations which display various degrees of distinctness. The most distinct genetic groups correspond to the named taxa while the cultivars and landraces of Pisum sativum can be divided into two broad types, one of which is strongly enriched for modern cultivars. The addition of germplasm sets from six European Genebanks, chosen to represent high diversity, to a single collection previously studied with these markers resulted in modest additions to the overall diversity observed, suggesting that the great majority of the total genetic diversity collected for the Pisum genus has now been described. Two interesting sources of novel genetic variation have been identified. Finally, we have proposed reference sets of core accessions with a range of sample Communicated by I. Mackay.

TAG_From Mendel’s discovery on pea to today’s.pdf

PTOC-Vavilovia_2016.pdf

sustained division. Subsequently, protoplasts isolated from internode callus proliferated and als... more sustained division. Subsequently, protoplasts isolated from internode callus proliferated and also underwent organogenesis coupled with whole plant recovery at a low frequency, while protoplasts from leaf callus origin followed both organogenesis and embryogenesis simultaneously but failed to yield viable plants. Flow cytometry assessments permitted to ascertain the genetic idelity of both propagated and regenerated plants irrespectively of the source tissue from which they were derived (i.e., either callus from explants or from protoplasts). Finally, low cytometry also permitted us to provide the irst record on the relative nuclear DNA content and genome size for Vavilovia formosa.

Vavilovia-PlantSystEvol_2017.pdf

Vavilovia formosa is one of five genera in tribe Fabeae, (Fabaceae, Leguminosae) with close phylo... more Vavilovia formosa is one of five genera in tribe Fabeae, (Fabaceae, Leguminosae) with close phylogenetic relationships to Pisum. It grows in subalpine and alpine levels in Armenia, Azerbaijan, Georgia, Iran, Iraq, Lebanon, Russia and Turkey and is recognized as an endangered and protected plant. This study was conducted to reveal its intraspecific variability, as well as to predict the past, extant and future species distribution range. We analysed 51 accessions with common phylogenetic markers (trnF-trnL, trnS-trnG, matK, rbcL, psbA-trnH and ITS). These represent in total up to 2551 bp of chloroplast and 664 bp of nuclear sequences per sample. Two populations from Turkey and Armenia were analysed for genetic diversity by AFLP. Leaf morphometry was conducted on 1457 leaflets from 43 specimens. Extracted bioclimatic parameters were used for niche-modelling approach. Analysis of cpDNA revealed two haplotypes, 12 samples from Armenia, Daghestan, Nakhichevan and Iran belonged to H1 group, while 39 samples of all Turkish and part of Armenian were in H2 group. The mean intrapopulation diversity based on AFLP was low (H E = 0.088) indicating limited outcrossing rate. A significantly positive correlation between geographical latitude and leaf area (q = 0.527, p \ 0.05) was found. Niche modelling has shown temporal variation of predicted occurrence across the projected time periods. Vavilovia formosa has suffered a range reduction following climate warming after last glacial maximum, which classify this species as cold-adapted among the Fabeae species as well as a glacial relict.

Eco-geographic distribution of Cicer isauricum P.H. Davis-2017.pdf

Wild Cicer species are considered as useful genetic resources for resistance to biotic and abioti... more

Frontiers_combinatorial analysis of seed coat_2017.pdf

The origin of the agriculture was one of the turning points in human history, and a central part ... more The origin of the agriculture was one of the turning points in human history, and a central part of this was the evolution of new plant forms, domesticated crops. Seed dispersal and germination are two key traits which have been selected to facilitate cultivation and harvesting of crops. The objective of this study was to analyze anatomical structure of seed coat and pod, identify metabolic compounds associated with water-impermeable seed coat and differentially expressed genes involved in pea seed dormancy and pod dehiscence. Comparative anatomical, metabolomics, and transcriptomic analyses were carried out on wild dormant, dehiscent Pisum elatius (JI64, VIR320) and cultivated, indehiscent Pisum sativum non-dormant (JI92, Cameor) and recombinant inbred lines (RILs). Considerable differences were found in texture of testa surface, length of macrosclereids, and seed coat thickness. Histochemical and biochemical analyses indicated genotype related variation in composition and heterogeneity of seed coat cell walls within macrosclereids. Liquid chromatography-electrospray ionization/mass spectrometry and Laser desorption/ionization-mass spectrometry of separated seed coats revealed significantly higher contents of proanthocyanidins (dimer and trimer of gallocatechin), quercetin, and myricetin rhamnosides and hydroxylated fatty acids in dormant compared to non-dormant genotypes. Bulk Segregant Analysis coupled to high throughput RNA sequencing resulted in identification of 770 and 148 differentially expressed genes between dormant and non-dormant seeds or dehiscent and indehiscent pods, respectively. The expression of 14 selected dormancy-related genes was studied by qRT-PCR. Of these, expression pattern of four genes: porin (MACE-S082), peroxisomal membrane PEX14-like protein (MACE-S108), 4-coumarate CoA ligase (MACE-S131), and UDP-glucosyl transferase (MACE-S139) was in Hradilová et al.

Scientific Reports_2017_Pea genomic diversity and macroecology.pdf

There is growing interest in the conservation and utilization of crop wild relatives (CWR) in int... more There is growing interest in the conservation and utilization of crop wild relatives (CWR) in international food security policy and research. Legumes play an important role in human health, sustainable food production, global food security, and the resilience of current agricultural systems. Pea belongs to the ancient set of cultivated plants of the Near East domestication center and remains an important crop today. Based on genome-wide analysis, P. fulvum was identified as a well-supported species, while the diversity of wild P. sativum subsp. elatius was structured into 5 partly geographically positioned clusters. We explored the spatial and environmental patterns of two progenitor species of domesticated pea in the Mediterranean Basin and in the Fertile Crescent in relation to the past and current climate. This study revealed that isolation by distance does not explain the genetic structure of P. sativum subsp. elatius in its westward expansion from its center of origin. The genetic diversity of wild pea may be driven by Miocene-Pliocene events, while the phylogenetic diversity centers may reflect Pleisto-Holocene climatic changes. These findings help set research and discussion priorities and provide geographical and ecological information for germplasm-collecting missions, as well as for the preservation of extant diversity in ex-situ collections.

PLOS One_2018_Smýkal-Wild pea in nature.pdf

Agronomy-2018_Domestication review.pdf

Humans have domesticated hundreds of plant and animal species as sources of food, fiber, forage, ... more Humans have domesticated hundreds of plant and animal species as sources of food, fiber, forage, and tools over the past 12,000 years, with manifold effects on both human society and the genetic structure of the domesticated species. The outcomes of crop domestication were shaped by selection driven by human preferences, cultivation practices, and agricultural environments, as well as other population genetic processes flowing from the ensuing reduction in effective population size. It is obvious that any selection imposes a reduction of diversity, favoring preferred genotypes, such as nonshattering seeds or increased palatability. Furthermore, agricultural practices greatly reduced effective population sizes of crops, allowing genetic drift to alter genotype frequencies. Current advances in molecular technologies, particularly of genome sequencing, provide evidence of human selection acting on numerous loci during and after crop domestication. Population-level molecular analyses also enable us to clarify the demographic histories of the domestication process itself, which, together with expanded archaeological studies, can illuminate the origins of crops. Domesticated plant species are found in 160 taxonomic families. Approximately 2500 species have undergone some degree of domestication, and 250 species are considered to be fully domesticated. The evolutionary trajectory from wild to crop species is a complex process. Archaeological records suggest that there was a period of predomestication cultivation while humans first began the deliberate planting of wild stands that had favorable traits. Later, crops likely diversified as they were grown in new areas, sometimes beyond the climatic niche of their wild relatives. However, the speed and level of human intentionality during domestication remains a topic of active discussion. These processes led to the so-called domestication syndrome, that is, a group of traits that can arise through human preferences for ease of harvest and growth advantages under human propagation. These traits included reduced dispersal ability of seeds and fruits, changes to plant structure, and changes to plant defensive characteristics and palatability. Domestication implies the action of selective sweeps on standing genetic variation, as well as new genetic variation introduced via mutation or introgression. Furthermore, genetic bottlenecks during domestication or during founding events as crops moved away from their centers of origin may have further altered gene pools. To date, a few hundred genes and loci have been identified by classical genetic and association mapping as targets of domestication and postdomestication divergence. However, only a few of these have been characterized, and for even fewer is the role of the wild-type allele in natural populations understood. After domestication, only favorable haplotypes are retained around selected genes, which creates a genetic valley with extremely low genetic diversity. These "selective sweeps" can allow mildly deleterious alleles to come to fixation and may create a genetic load in the cultivated gene pool. Although the population-wide genomic consequences of domestication offer several predictions for levels of the genetic diversity in crops, our understanding of how this diversity corresponds to nutritional aspects of crops is not well understood. Many studies have found that modern cultivars have lower levels of key micronutrients and vitamins. We suspect that selection for palatability and increased yield at domestication and during postdomestication divergence exacerbated the low nutrient levels of many crops, although relatively little work has examined this question. Lack of diversity in modern germplasm may further limit our capacity to breed for higher nutrient levels, although little effort has gone into this beyond a handful of staple crops. This is an area where an understanding of domestication across many crop taxa may provide the necessary insight for breeding more nutritious crops in a rapidly changing world.

G3-Patterns of Genetic Structure and Linkage Disequilibrium in a Large Collection of Pea Germplasm.pdf

Pea (Pisum sativum, L.) is a major pulse crop used both for animal and human alimentation. Owing ... more Pea (Pisum sativum, L.) is a major pulse crop used both for animal and human alimentation. Owing to its association with nitrogen-fixing bacteria, it is also a valuable component for low-input cropping systems. To evaluate the genetic diversity and the scale of linkage disequilibrium (LD) decay in pea, we genotyped a collection of 917 accessions, gathering elite cultivars, landraces, and wild relatives using an array of 13,000 single nucleotide polymorphisms (SNP). Genetic diversity is broadly distributed across three groups corresponding to wild/landraces peas, winter types, and spring types. At a finer subdivision level, genetic groups relate to local breeding programs and type usage. LD decreases steeply as genetic distance increases. When considering subsets of the data, LD values can be higher, even if the steep decay remains. We looked for genomic regions exhibiting high level of differentiation between wild/landraces, winter, and spring pea, respectively. Two regions on linkage groups 5 and 6 containing 33 SNPs exhibit stronger differentiation between winter and spring peas than would be expected under neutrality. Interestingly, QTL for resistance to cold acclimation and frost resistance have been identified previously in the same regions.

Genes-Pea domestication 2018.pdf

Pea, one of the founder crops from the Near East, has two wild species: Pisum sativum subsp. elat... more Pea, one of the founder crops from the Near East, has two wild species: Pisum sativum subsp. elatius, with a wide distribution centered in the Mediterranean, and P. fulvum, which is restricted to Syria, Lebanon, Israel, Palestine and Jordan. Using genome wide analysis of 11,343 polymorphic single nucleotide polymorphisms (SNPs) on a set of wild P. elatius (134) and P. fulvum (20) and 74 domesticated accessions (64 P. sativum landraces and 10 P. abyssinicum), we demonstrated that domesticated P. sativum and the Ethiopian pea (P. abyssinicum) were derived from different P. elatius genepools. Therefore, pea has at least two domestication events. The analysis does not support a hybrid origin of P. abyssinicum, which was likely introduced into Ethiopia and Yemen followed by eco-geographic adaptation. Both P. sativum and P. abyssinicum share traits that are typical of domestication, such as non-dormant seeds. Non-dormant seeds were also found in several wild P. elatius accessions which could be the result of crop to wild introgression or natural variation that may have been present during pea domestication. A sub-group of P. elatius overlaps with P. sativum landraces. This may be a consequence of bidirectional gene-flow or may suggest that this group of P. elatius is the closest extant wild relative of P. sativum. Genes 2018, 9, 535 2 of 20 use of wild cereals and pulses considerably predate their domestication ; similarly, the gathering of pea from 'gardens' by pre-agricultural peoples has been discussed .

Janska_AoB_2018-Role of testa- anatomy.pdf

Background A water-impermeable testa acts as a barrier to a seed's imbibition, thereby imposing d... more Background A water-impermeable testa acts as a barrier to a seed's imbibition, thereby imposing dormancy. The physical and functional properties of the macrosclereids are thought to be critical determinants of dormancy; however, the mechanisms underlying the maintenance of and release from dormancy in pea are not well understood.

Hradilova_Pea seed dormancy_PeerJ 2019.pdf

Background. Seed germination is one of the earliest key events in the plant life cycle. The timin... more Background. Seed germination is one of the earliest key events in the plant life cycle. The timing of transition from seed to seedling is an important developmental stage determining the survival of individuals that influences the status of populations and species. Because of wide geographical distribution and occurrence in diverse habitats, wild pea (Pisum sativum subsp. elatius) offers an excellent model to study physical type of seed dormancy in an ecological context. This study addresses the gap in knowledge of association between the seed dormancy, seed properties and environmental factors, experimentally testing oscillating temperature as dormancy release clue. Methods. Seeds of 97 pea accessions were subjected to two germination treatments (oscillating temperatures of 25/15 • C and 35/15 • C) over 28 days. Germination pattern was described using B-spline coefficients that aggregate both final germination and germination speed. Relationships between germination pattern and environmental conditions at the site of origin (soil and bioclimatic variables extracted from World-Clim 2.0 and SoilGrids databases) were studied using principal component analysis, redundancy analysis and ecological niche modelling. Seeds were analyzed for the seed coat thickness, seed morphology, weight and content of proanthocyanidins (PA). Results. Seed total germination ranged from 0% to 100%. Cluster analysis of germination patterns of seeds under two temperature treatments differentiated the accessions into three groups: (1) non-dormant (28 accessions, mean germination of 92%), (2) dormant at both treatments (29 acc., 15%) and (3) responsive to increasing temperature range (41 acc., with germination change from 15 to 80%). Seed coat thickness differed between groups with dormant and responsive accessions having thicker testa (median 138 and 140 µm) than non-dormant ones (median 84 mm). The total PA content showed to be higher in the seed coat of dormant (mean 2.18 mg g −1 ) than those of non-dormant (mean 1.77 mg g −1 ) and responsive accessions (mean 1.87 mg g −1 ). Each soil and bioclimatic variable and also germination responsivity (representing How to cite this article Hradilová I, Duchoslav M, Brus J, Pechanec V, Hýbl M, Kopecký P, Smržová L, Štefelová N, Vaclávek T, Michael B, Machalová J Hron K, Pirintsos S and Smýkal P. 2019. Variation in wild pea (Pisum sativum subsp. elatius) seed dormancy and its relationship to the environment and seed coat traits. PeerJ 7:e6263 http://doi.org/10.7717/peerj.6263

Cechová_AnBiochem_2019.pdf

Your article is protected by copyright and all rights are held exclusively by Springer-Verlag Gmb... more Your article is protected by copyright and all rights are held exclusively by Springer-Verlag GmbH Germany, part of Springer Nature. This e-offprint is for personal use only and shall not be self-archived in electronic repositories. If you wish to self-archive your article, please use the accepted manuscript version for posting on your own website. You may further deposit the accepted manuscript version in any repository, provided it is only made publicly available 12 months after official publication or later and provided acknowledgement is given to the original source of publication and a link is inserted to the published article on Springer's website. The link must be accompanied by the following text: "The final publication is available at link.springer.com".

Janska_AoB_2018-comprimed.pdf

Background A water-impermeable testa acts as a barrier to a seed's imbibition, thereby imposing d... more Background A water-impermeable testa acts as a barrier to a seed's imbibition, thereby imposing dormancy. The physical and functional properties of the macrosclereids are thought to be critical determinants of dormancy; however, the mechanisms underlying the maintenance of and release from dormancy in pea are not well understood.

Genetic Diversity of Albanian Pea (Pisum sativum L.) Landraces Assessed by Morphological Traits and Molecular Markers

by Gixhari Belul, Belul Gixhari, Hekuran Vrapi, Hairi Ismaili, Michaela Ludvikova, and Petr Smykal

Gixhari B., Pavelková M., Ismaili H., Vrapi H., Jaupi A., Smýkal P. (2014): Genetic diversity of ... more Gixhari B., Pavelková M., Ismaili H., Vrapi H., Jaupi A., Smýkal P. (2014): Genetic diversity of Albanian pea (Pisum sativum L.) landraces assessed by morphological traits and molecular markers. Czech J. Genet. Plant Breed., 50: 177–184. In order to investigate the genetic diversity present in the pea germplasm stored in the Albanian genebank, we analyzed 28 local pea genotypes of Albanian origins for 23 quantitative morphological traits, as well as 14 retrotransposon-based insertion polymorphism (RBIP) molecular markers. The study of morphological characters carried out during three growing seasons (2010, 2011 and 2012) had the objective of characterization of traits useful in breeding programs. RBIP marker analysis revealed the genetic similarity in range from 0.06 to 0.45. ANOVA, principal component analysis (PCA) and cluster analysis was used to visualize the association among different traits. Most of the quantitative morphological traits showed significant differences. PCA and cluster analysis (Ward's method) carried out for morphological traits divided the local pea genotypes into three clusters. Finally, the study identified the agronomicaly important traits which will facilitate the maintenance and agronomic evaluation of the collections.

The legume manifesto: (Net)workers on Fabaceae, unite!

by Frederick Stoddard and Petr Smykal

Ratarstvo i povrtarstvo, 2011

Legumes have been an important part of cropping systems since the dawn of agriculture. The shift ... more Legumes have been an important part of cropping systems since the dawn of agriculture. The shift in Europe from draught animals to meat animals coincided with the increasing availability of soybean meal from North and South America, and the Common Agricultural Policy of the European Union promoted the growing of cereals and oilseeds at the expense of other crops, so legumes fell out of favour with farmers and decision-makers. Continental concerns about food and feed security, high prices of oil and soybean meal, and advances in the application of fundamental molecular genetics to crop species, all mean that now is a good opportunity to promote the return of legumes to European cropping systems by enhancing the ef ciency of research and development on this family. Hence we propose the establishment of a Legume Society that will promote information exchange and scienti c productivity by uniting the various legume research communities.

Modulation of flowering responses in different Nicotiana varieties

Plant Molecular Biology, 2004

We have identified and characterized a FLOWERING PROMOTING FACTOR 1(FPF1) gene from tobacco (NtFP... more We have identified and characterized a FLOWERING PROMOTING FACTOR 1(FPF1) gene from tobacco (NtFPF1). Over-expression of NtFPF1 leads to early flowering in the day-neutral tobacco Nicotiana tabacum cv. Hicks, and under inductive photoperiods also in the short-day Nicotiana tabacum cv. Hicks Maryland Mammoth (MM) tobacco and the long-day plant Nicotiana sylvestris. N. sylvestris wild-type plants remained in the rosette stage and never flowered under non-inductive short-days, whereas 35S::NtFPF1 transgenic plants bolted but did not flower. However, if treated with gibberellins, transgenic N. sylvestrisplants flowered much faster under non-inductive short days than corresponding wild type plants, indicating an additive effect of gibberellins and the NtFPF1 protein in flowering time control. The day-neutral wild type cv. Hicks and the short-day cv. Hicks MM plants exhibit an initial rosette stage, both under short- and long-days. In the transgenic lines, this rosette stage was completely abolished. Wild-type plants of cv. Hicks MM never flowered under long days; however, all transgenic lines over-expressing NtFPF1 flowered under this otherwise non-inductive photoperiod.

Effect of Environmental and Genetic Factors on the Stability of Pea (Pisum sativum L.) Isozyme and DNA Markers

Environmental (geographic location, year-to-year effect) and genetic (intravarietal variation) ef... more Environmental (geographic location, year-to-year effect) and genetic (intravarietal variation) effects on the stability of the isozyme patterns of esterase (EST), acid phosphatase (ACP), alcohol dehydrogenase (ADH), leucine aminopeptidase (LAP), shikimate dehydrogenase (SDH) and glucose-6-phosphate isomerase (PGI) used for pea cultivar genotyping were studied. In addition, selected DNA markers (RAPD, SSR, ISSR, IRAP) were used to study intravarietal genetic homogeneity/variation at a DNA level. Five commercial dry-seed pea (Pisum sativum L.) cultivars Canis, Gotik, Komet, Sonet and Zekon were grown during four years (2001 -2004) in three locations in the Czech Republic (Šumperk, Čáslav, Uherský Ostroh) that differed in soil and climatic characteristics. Mature dry seeds were used as a standard input sample for analyses. No variation in isozyme spectra was found within particular cultivars between years, locations and fruiting nodes in enzymes providing in general a low polymorphism in pea cultivars (ACP, ADH, LAP, SDH, PGI); similarly, these enzymes also exhibited high stability as related to intravarietal variation. In contrast, EST -highly polymorphic in pea cultivars -showed certain qualitative variation within particular cultivars as related to both environmental and genetic factors. The intravarietal variation detected by selected DNA markers was negligible and mostly quantitative. Possible reasons for the instability/variation of isozyme markers are discussed from the aspect of cultivar genotyping used in pea breeding and seed production.

Estimation of pea ( Pisum sativum L.) microsatellite mutation rate based on pedigree and single-seed descent analyses

Journal of Applied Genetics

Microsatellites, or simple sequence repeats (SSRs) are widespread class of repetitive DNA sequenc... more Microsatellites, or simple sequence repeats (SSRs) are widespread class of repetitive DNA sequences, used in population genetics, genetic diversity and mapping studies. In spite of the SSR utility, the genetic and evolutionary mechanisms are not fully understood. We have investigated three microsatellite loci with different position in the pea (Pisum sativum L.) genome, the A9 locus residing in LTR region of abundant retrotransposon, AD270 as intergenic and AF016458 located in 5′untranslated region of expressed gene. Comparative analysis of a 35 pair samples from seven pea varieties propagated by single-seed descent for ten generations, revealed single 4 bp mutation in 10th generation sample at AD270 locus corresponding to stepwise increase in one additional ATCT repeat unit. The estimated mutation rate was 4.76 × 10-3 per locus per generation, with a 95% confidence interval of 1.2 × 10-4 to 2.7 × 10-2. The comparison of cv. Bohatýr accessions retrieved from different collections, showed intra-, inter-accession variation and differences in flanking and repeat sequences. Fragment size and sequence alternations were also found in long term in vitro organogenic culture, established at 1983, indicative of somatic mutation process. The evidence of homoplasy was detected across of unrelated pea genotypes, which adversaly affects the reliability of diversity estimates not only for diverse germplasm but also highly bred material. The findings of this study have important implications for Pisum phylogeny studies, variety identification and registration process in pea breeding where mutation rate influences the genetic diversity and the effective population size estimates.