Recessive resistance against beet chlorosis virus is conferred by the eukaryotic translation initiation factor (iso)4E in Beta vulgaris.

Eukaryotic translation initiation factors (eIFs) are important for mRNA translation but also pivotal for plant-virus interaction. Most of these plant-virus interactions were found between plant eIFs and the viral protein genome-linked (VPg) of potyviruses. In case of lost interaction due to mutation or deletion of eIFs, the viral translation and subsequent replication within its host is negatively affected, resulting in a recessive resistance. Here we report the identification of the Beta vulgaris Bv-eIF(iso)4E as a susceptibility factor towards the VPg-carrying beet chlorosis virus (genus Polerovirus). Using yeast two-hybrid and bimolecular fluorescence complementation assays, the physical interaction between Bv-eIF(iso)4E and the putative BChV-VPg was detected, while the VPg of the closely related beet mild yellowing virus (BMYV) was found to interact with the two isoforms Bv-eIF4E and Bv-eIF(iso)4E. These VPg-eIF interactions within the polerovirus-beet pathosystem were demonstrated to be highly specific, as single mutations within the predicted cap-binding pocket of Bv-eIF(iso)4E resulted in a loss of interaction. To investigate the suitability of eIFs as a resistance resource against beet infecting poleroviruses, B. vulgaris plants were genome edited by CRISPR/Cas9 resulting in knockouts of different eIFs. A simultaneous knockout of the identified BMYV-interaction partners Bv-eIF4E and Bv-eIF(iso)4E was not achieved, but Bv-eIF(iso)4EKO plants showed a significantly lowered BChV accumulation and decrease in infection rate from 100% to 28.86%, while no influence on BMYV accumulation was observed. Still, these observations support that eIFs are promising candidate genes for polerovirus resistance breeding in sugar beet.

The effect of heat stress on sugar beet recombination.

Meiotic recombination plays a crucial role in plant breeding through the creation of new allelic combinations. Therefore, lack of recombination in some genomic regions constitutes a constraint for breeding programmes. In sugar beet, one of the major crops in Europe, recombination occurs mainly in the distal portions of the chromosomes, and so the development of simple approaches to change this pattern is of considerable interest for future breeding and genetics. In the present study, the effect of heat stress on recombination in sugar beet was studied by treating F1 plants at 28 °C/25 °C (day/night) and genotyping the progeny. F1 plants were reciprocally backcrossed allowing the study of male and female meiosis separately. Genotypic data indicated an overall increase in crossover frequency of approximately one extra crossover per meiosis, with an associated increase in pericentromeric recombination under heat treatment. Our data indicate that the changes were mainly induced by alterations in female meiosis only, showing that heterochiasmy in sugar beet is reduced under heat stress. Overall, despite the associated decrease in fertility, these data support the potential use of heat stress to foster recombination in sugar beet breeding programmes.

Discovery of interesting new polymorphisms in a sugar beet (elite [Formula: see text] exotic) progeny by comparison with an elite panel.

KEY MESSAGE: The comparison of QTL detection performed on an elite panel and an (elite [Formula: see text] exotic) progeny shows that introducing exotic germplasm into breeding programs can bring new interesting allelic diversity. Selection of stable varieties producing the highest amount of extractable sugar per hectare (ha), resistant to diseases, and respecting environmental criteria is undoubtedly the main target for sugar beet breeding. As sodium, potassium, and [Formula: see text]-amino nitrogen in sugar beets are the impurities that have the biggest negative impact on white sugar extraction, it is interesting to reduce their concentration in further varieties. However, domestication history and strong selection pressures have affected the genetic diversity needed to achieve this goal. In this study, quantitative trait locus (QTL) detection was performed on two populations, an (elite [Formula: see text] exotic) sugar beet progeny and an elite panel, to find potentially new interesting regions brought by the exotic accession. The three traits linked with impurities content were studied. Some QTLs were detected in both populations, the majority in the elite panel because of most statistical power. Some of the QTLs were colocated and had favorable effect in the progeny since the exotic allele was linked with a decrease in the impurity content. A few number of favorable QTLs were detected in the progeny, only. Consequently, introgressing exotic genetic material into sugar beet breeding programs can allow the incorporation of new interesting alleles.

The genetic architecture of freezing tolerance varies across the range of Arabidopsis thaliana.

The capacity to tolerate freezing temperatures limits the geographical distribution of many plants, including several species of agricultural importance. However, the genes involved in freezing tolerance remain largely unknown. Here, we describe the variation in constitutive freezing tolerance that occurs among worldwide accessions of Arabidopsis thaliana. We found that although plants from high latitudes tend to be more freezing tolerant than plants from low latitudes, the environmental factors that shape cold adaptation differ across the species range. Consistent with this, we found that the genetic architecture of freezing tolerance also differs across its range. Conventional genome-wide association studies helped identify a priori and other promising candidate genes. However, simultaneously modelling climate variables and freezing tolerance together pinpointed other excellent a priori candidate genes. This suggests that if the selective factor underlying phenotypic variation is known, multi-trait mixed models may aid in identifying the genes that underlie adaptation.

Epigenomics and bolting tolerance in sugar beet genotypes.

In sugar beet (Beta vulgaris altissima), bolting tolerance is an essential agronomic trait reflecting the bolting response of genotypes after vernalization. Genes involved in induction of sugar beet bolting have now been identified, and evidence suggests that epigenetic factors are involved in their control. Indeed, the time course and amplitude of DNA methylation variations in the shoot apical meristem have been shown to be critical in inducing sugar beet bolting, and a few functional targets of DNA methylation during vernalization have been identified. However, molecular mechanisms controlling bolting tolerance levels among genotypes are still poorly understood. Here, gene expression and DNA methylation profiles were compared in shoot apical meristems of three bolting-resistant and three bolting-sensitive genotypes after vernalization. Using Cot fractionation followed by 454 sequencing of the isolated low-copy DNA, 6231 contigs were obtained that were used along with public sugar beet DNA sequences to design custom Agilent microarrays for expression (56k) and methylation (244k) analyses. A total of 169 differentially expressed genes and 111 differentially methylated regions were identified between resistant and sensitive vernalized genotypes. Fourteen sequences were both differentially expressed and differentially methylated, with a negative correlation between their methylation and expression levels. Genes involved in cold perception, phytohormone signalling, and flowering induction were over-represented and collectively represent an integrative gene network from environmental perception to bolting induction. Altogether, the data suggest that the genotype-dependent control of DNA methylation and expression of an integrative gene network participate in bolting tolerance in sugar beet, opening up perspectives for crop improvement.

Genome-wide association study of 107 phenotypes in Arabidopsis thaliana inbred lines.

Although pioneered by human geneticists as a potential solution to the challenging problem of finding the genetic basis of common human diseases, genome-wide association (GWA) studies have, owing to advances in genotyping and sequencing technology, become an obvious general approach for studying the genetics of natural variation and traits of agricultural importance. They are particularly useful when inbred lines are available, because once these lines have been genotyped they can be phenotyped multiple times, making it possible (as well as extremely cost effective) to study many different traits in many different environments, while replicating the phenotypic measurements to reduce environmental noise. Here we demonstrate the power of this approach by carrying out a GWA study of 107 phenotypes in Arabidopsis thaliana, a widely distributed, predominantly self-fertilizing model plant known to harbour considerable genetic variation for many adaptively important traits. Our results are dramatically different from those of human GWA studies, in that we identify many common alleles of major effect, but they are also, in many cases, harder to interpret because confounding by complex genetics and population structure make it difficult to distinguish true associations from false. However, a-priori candidates are significantly over-represented among these associations as well, making many of them excellent candidates for follow-up experiments. Our study demonstrates the feasibility of GWA studies in A. thaliana and suggests that the approach will be appropriate for many other organisms.

Breeding patterns and cultivated beets origins by genetic diversity and linkage disequilibrium analyses.

KEY MESSAGE: Genetic diversity in worldwide population of beets is strongly affected by the domestication history, and the comparison of linkage disequilibrium in worldwide and elite populations highlights strong selection pressure. Genetic relationships and linkage disequilibrium (LD) were evaluated in a set of 2035 worldwide beet accessions and in another of 1338 elite sugar beet lines, using 320 and 769 single nucleotide polymorphisms, respectively. The structures of the populations were analyzed using four different approaches. Within the worldwide population, three of the methods gave a very coherent picture of the population structure. Fodder beet and sugar beet accessions were grouped together, separated from garden beets and sea beets, reflecting well the origins of beet domestication. The structure of the elite panel, however, was less stable between clustering methods, which was probably because of the high level of genetic mixing in breeding programs. For the linkage disequilibrium analysis, the usual measure (r (2)) was used, and compared with others that correct for population structure and relatedness (r S (2) , r V (2) , r VS (2)). The LD as measured by r (2) persisted beyond 10 cM within the elite panel and fell below 0.1 after less than 2 cM in the worldwide population, for almost all chromosomes. With correction for relatedness, LD decreased under 0.1 by 1 cM for almost all chromosomes in both populations, except for chromosomes 3 and 9 within the elite panel. In these regions, the larger extent of LD could be explained by strong selection pressure.

Towards the discovery of novel genetic component involved in stress resistance in Arabidopsis thaliana.

The exposure of plants to high concentrations of trace metallic elements such as copper involves a remodeling of the root system, characterized by a primary root growth inhibition and an increase in the lateral root density. These characteristics constitute easy and suitable markers for screening mutants altered in their response to copper excess. A forward genetic approach was undertaken in order to discover novel genetic factors involved in the response to copper excess. A Cu(2+) -sensitive mutant named copper modified resistance1 (cmr1) was isolated and a causative mutation in the CMR1 gene was identified by using positional cloning and next-generation sequencing. CMR1 encodes a plant-specific protein of unknown function. The analysis of the cmr1 mutant indicates that the CMR1 protein is required for optimal growth under normal conditions and has an essential role in the stress response. Impairment of the CMR1 activity alters root growth through aberrant activity of the root meristem, and modifies potassium concentration and hormonal balance (ethylene production and auxin accumulation). Our data support a putative role for CMR1 in cell division regulation and meristem maintenance. Research on the role of CMR1 will contribute to the understanding of the plasticity of plants in response to changing environments.

Genetic diversity and linkage disequilibrium analysis in elite sugar beet breeding lines and wild beet accessions.

Linkage disequilibrium decay in sugar beet is strongly affected by the breeding history, and varies extensively between and along chromosomes, allowing identification of known and unknown signatures of selection. Genetic diversity and linkage disequilibrium (LD) patterns were investigated in 233 elite sugar beet breeding lines and 91 wild beet accessions, using 454 single nucleotide polymorphisms (SNPs) and 418 SNPs, respectively. Principal coordinate analysis suggested the existence of three groups of germplasm, corresponding to the wild beets, the seed parent and the pollen parent breeding pool. LD was investigated in each of these groups, with and without correction for genetic relatedness. Without correction for genetic relatedness, in the pollen as well as the seed parent pool, LD persisted beyond 50 centiMorgan (cM) on four (2, 3, 4 and 5) and three chromosomes (2, 4 and 6), respectively; after correction for genetic relatedness, LD decayed after <6 cM on all chromosomes in both pools. In the wild beet accessions, there was a strong LD decay: on average LD disappeared after 1 cM when LD was calculated with a correction for genetic relatedness. Persistence of LD was not only observed between distant SNPs on the same chromosome, but also between SNPs on different chromosomes. Regions on chromosomes 3 and 4 that harbor disease resistance and monogermy loci showed strong genetic differentiation between the pollen and seed parent pools. Other regions, on chromosomes 8 and 9, for which no a priori information was available with respect to their contribution to the phenotype, still contributed to clustering of lines in the elite breeding material.

A contiguous de novo genome assembly of sugar beet EL10 (Beta vulgaris L.).

A contiguous assembly of the inbred 'EL10' sugar beet (Beta vulgaris ssp. vulgaris) genome was constructed using PacBio long-read sequencing, BioNano optical mapping, Hi-C scaffolding, and Illumina short-read error correction. The EL10.1 assembly was 540 Mb, of which 96.2% was contained in nine chromosome-sized pseudomolecules with lengths from 52 to 65 Mb, and 31 contigs with a median size of 282 kb that remained unassembled. Gene annotation incorporating RNA-seq data and curated sequences via the MAKER annotation pipeline generated 24,255 gene models. Results indicated that the EL10.1 genome assembly is a contiguous genome assembly highly congruent with the published sugar beet reference genome. Gross duplicate gene analyses of EL10.1 revealed little large-scale intra-genome duplication. Reduced gene copy number for well-annotated gene families relative to other core eudicots was observed, especially for transcription factors. Variation in genome size in B. vulgaris was investigated by flow cytometry among 50 individuals producing estimates from 633 to 875 Mb/1C. Read-depth mapping with short-read whole-genome sequences from other sugar beet germplasm suggested that relatively few regions of the sugar beet genome appeared associated with high-copy number variation.

Quantitative trait loci analysis of mineral element concentrations in an Arabidopsis halleri x Arabidopsis lyrata petraea F2 progeny grown on cadmium-contaminated soil.

SUMMARY: This study describes the quantitative trait locus (QTL) analysis of cadmium (Cd), zinc (Zn), iron (Fe), potassium (K), magnesium (Mg) and calcium (Ca) accumulation in the pseudometallophyte Arabidopsis halleri under conditions of Cd excess using an interspecific A. halleri x Arabidopsis lyrata F(2) population. *Our data provide evidence for the implication of one major QTL in Cd hyperaccumulation in A. halleri, and suggests that Cd tolerance and accumulation are not independent in A. halleri. Moreover, the major loci responsible for Zn hyperaccumulation in the absence of Cd appear to be the same when Cd is present at high concentrations. *More than twofold higher Fe concentrations were measured in A. halleri shoots than in A. lyrata, suggesting a different regulation of Fe accumulation in the hyperaccumulator. *With the exception of Ca, the accumulation of Cd was significantly correlated with the accumulation of all elements measured in the F(2) progeny, suggesting pleiotropic gene action. However, QTL analysis identified pleiotropic QTLs only for Cd, Zn and Fe. Mg accumulation was negatively correlated with Cd accumulation, as well as with dry shoot biomass, suggesting that it might indicate cellular damage.

Genetic architecture of zinc hyperaccumulation in Arabidopsis halleri: the essential role of QTL x environment interactions.

SUMMARY: This study sought to determine the main genomic regions that control zinc (Zn) hyperaccumulation in Arabidopsis halleri and to examine genotype x environment effects on phenotypic variance. To do so, quantitative trait loci (QTLs) were mapped using an interspecific A. halleri x Arabidopsis lyrata petraea F(2) population. *The F(2) progeny as well as representatives of the parental populations were cultivated on soils at two different Zn concentrations. A linkage map was constructed using 70 markers. *In both low and high pollution treatments, zinc hyperaccumulation showed high broad-sense heritability (81.9 and 74.7%, respectively). Five significant QTLs were detected: two QTLs specific to the low pollution treatment (chromosomes 1 and 4), and three QTLs identified at both treatments (chromosomes 3, 6 and 7). These QTLs explained 50.1 and 36.5% of the phenotypic variance in low and high pollution treatments, respectively. Two QTLs identified at both treatments (chromosomes 3 and 6) showed significant QTL x environment interactions. *The QTL on chromosome 3 largely colocalized with a major QTL previously identified for Zn and cadmium (Cd) tolerance. This suggests that Zn tolerance and hyperaccumulation share, at least partially, a common genetic basis and may have simultaneously evolved on heavy metal-contaminated soils.

Using Arabidopsis to explore zinc tolerance and hyperaccumulation.

Identifying the particular gene or genes underlying a specific adaptation is a major challenge in modern biology. Currently, the study of naturally occurring variation in Arabidopsis thaliana provides a bridge between functional genetics and evolutionary analyses. Nevertheless, the use of A. thaliana to study adaptation is limited to those traits that have undergone selection. Therefore, to understand fully the genetics of adaptation, the vast arsenal of genetic resources developed in A. thaliana must be extended to other species that display traits absent in this model species. Here, we discuss how A. thaliana resources can significantly enhance the study of heavy-metal tolerance and hyperaccumulation in the wild species Arabidopsis halleri.

Methicillin-resistant Staphylococcus aureus ST398 in swine farm personnel, Belgium.

We assessed methicillin-resistant Staphylococcus aureus (MRSA) in persons on 49 swine farms in Belgium. Surveys showed that 48 (37.8%) persons carried MRSA ST398 and 1 (0.8%) had concurrent skin infection. Risk factors for carriage were MRSA carriage by pigs, regular contact with pigs and companion animals, and use of protective clothing.

Zinc distribution and speciation in Arabidopsis halleri x Arabidopsis lyrata progenies presenting various zinc accumulation capacities.

The purpose of this study was to investigate the relationship between the chemical form and localization of zinc (Zn) in plant leaves and their Zn accumulation capacity. An interspecific cross between Arabidopsis halleri sp. halleri and Arabidopsis lyrata sp. petrea segregating for Zn accumulation was used. Zinc (Zn) speciation and Zn distribution in the leaves of the parent plants and of selected F(1) and F(2) progenies were investigated by spectroscopic and microscopic techniques and chemical analyses. A correlation was observed between the proportion of Zn being in octahedral coordination complexed to organic acids and free in solution (Zn-OAs + Zn(aq)) and Zn content in the leaves. This pool varied between 40% and 80% of total leaf Zn depending on the plant studied. Elemental mapping of the leaves revealed different Zn partitioning between the veins and the leaf tissue. The vein : tissue fluorescence ratio was negatively correlated with Zn accumulation. The higher proportion of Zn-OAs + Zn(aq) and the depletion of the veins in the stronger accumulators are attributed to a higher xylem unloading and vacuolar sequestration in the leaf cells. Elemental distributions in the trichomes were also investigated, and results support the role of carboxyl and/or hydroxyl groups as major Zn ligands in these cells.

The genetic basis of zinc tolerance in the metallophyte Arabidopsis halleri ssp. halleri (Brassicaceae): an analysis of quantitative trait loci.

The species Arabidopsis halleri, an emerging model for the study of heavy metal tolerance and accumulation in plants, has evolved a high level of constitutive zinc tolerance. Mapping of quantitative trait loci (QTL) was used to investigate the genetic architecture of zinc tolerance in this species. A first-generation backcross progeny of A. halleri ssp. halleri from a highly contaminated industrial site and its nontolerant relative A. lyrata ssp. petraea was produced and used for QTL mapping of zinc tolerance. A genetic map covering most of the A. halleri genome was constructed using 85 markers. Among these markers, 65 were anchored in A. thaliana and revealed high synteny with other Arabidopsis genomes. Three QTL of comparable magnitude on three different linkage groups were identified. At all QTL positions zinc tolerance was enhanced by A. halleri alleles, indicating directional selection for higher zinc tolerance in this species. The two-LOD support intervals associated with these QTL cover 24, 4, and 13 cM. The importance of each of these three regions is emphasized by their colocalization with HMA4, MTP1-A, and MTP1-B, respectively, three genes well known to be involved in metal homeostasis and tolerance in plants.

Prevalence and antimicrobial susceptibility of methicillin-resistant Staphylococcus aureus among pigs in Belgium.

The prevalence, distribution, and antimicrobial susceptibility of methicillin-resistant Staphylococcus aureus (MRSA) in Belgian pig farms has been investigated. To that end, nasal samples were collected from 1,500 pigs on 50 farms randomly selected over Belgium. Both closed (breeding or farrow-to-finish) and open (fattening) farms were included. Within closed farms different age groups were investigated. A total number of 663 (44%) pigs belonging to 34 (68%) farms carried MRSA. According to their management practice, MRSA was detected on 94% of the open farms and 56% of the closed farms. Focusing on the in-herd prevalence among fattening pigs for both management systems, a significantly higher rate was found in open farms (72%) compared to closed farms (26%). Within the closed farms, piglets (41%) showed a higher MRSA prevalence than sows (26%) and fattening pigs (26%). All strains tested were ST398 and showed mainly spa-type t011, as commonly found on pig herds in Europe. Less dominating spa-types were t034, t567, and t2970. The MRSA strains carried two SCCmec-types, type IVa or V. All 643 MRSA strains were resistant to tetracycline and additional resistances to trimethoprim (97%), lincosamides (73%), macrolides (56%), aminoglycosides (48%), and fluoroquinolones (32%) were found. Multiresistance (defined as resistance to four or more non-ß-lactam antimicrobial classes) was found in 63% of the tested strains. In conclusion, a high prevalence of MRSA was found in Belgian pig farms, with the highest prevalence in open farms. In accordance with other European countries, age-related and management-related differences in MRSA prevalence were observed that should be considered when control strategies are outlined.

Merging methods in molecular and ecological genetics to study the adaptation of plants to anthropogenic metal-polluted sites: implications for phytoremediation.

Metallophyte species that occur naturally on metal-enriched soils represent major biological resources for the improvement of phytoremediation, a benign and cost-effective technology that uses plants to clean up anthropogenic metal-polluted soils. Within the last decade, molecular genetic studies carried out on several model organisms (including Arabidopsis halleri) have considerably enhanced our understanding of metal tolerance and hyperaccumulation in plants, but the identification of the genes of interest for phytoremediation purposes remains a challenge. To meet this challenge, we propose to combine '-omics' with molecular ecology methods. Using A. halleri, we confronted molecular genetic results with: (i) within-species polymorphism and large-scale population differentiation for zinc tolerance; (ii) the demographical context (e.g. migration pattern) of the species for zinc tolerance evolution; (iii) the Quantitative Trait Loci (QTL) analysis of the genetic architecture for zinc tolerance; and (iv) the fine-scale dissection of identified QTL regions, to discuss more precisely the nature of the genes potentially involved in the adaptation to zinc-polluted soils.

A major quantitative trait locus for cadmium tolerance in Arabidopsis halleri colocalizes with HMA4, a gene encoding a heavy metal ATPase.

Cadmium (Cd) tolerance seems to be a constitutive species-level trait in Arabidopsis halleri sp. halleri. Therefore, an interspecific cross was made between A. halleri and its closest nontolerant interfertile relative, Arabidopsis lyrata sp. petraea, and a first-generation backcross population (BC1) was used to map quantitative trait loci (QTL) for Cd tolerance. Three QTL were identified, which explained 43%, 24%, and 16% of the phenotypic variation in the mapping population. Heavy metal transporting ATPases4 (HMA4), encoding a predicted heavy metal ATPase, colocalized with the peak of the major QTL Cdtol-1 and was consequently further studied. HMA4 transcripts levels were higher in the roots and the shoots of A. halleri than in A. lyrata sp. petraea. Furthermore, HMA4 was also more highly expressed in all BC1 genotypes harboring the HMA4 A. halleri allele at the QTL Cdtol-1, independently of the presence of an A. halleri allele at the two other QTL. Overexpression of AhHMA4 in yeast (Saccharomyces cerevisiae) supported a role of HMA4 in zinc (Zn) and Cd transport by reducing the Cd and Zn contents of the yeast cells. In epidermal tobacco (Nicotiana tabacum) cells, AhHMA4:green fluorescent protein was clearly localized in the plasma membrane. Taken together, all available data point to the elevated expression of HMA4 P(1B)-type ATPase as an efficient mechanism for improving Cd/Zn tolerance in plants under conditions of Cd/Zn excess by maintaining low cellular Cd(2+) and Zn(2+) concentrations in the cytoplasm.