MdaB and NfrA, Two Novel Reductases Important in the Survival and Persistence of the Major Enteropathogen Campylobacter jejuni.

The paralogues RrpA and RrpB, which are members of the MarR family of DNA binding proteins, are important for the survival of the global bacterial foodborne pathogen Campylobacter jejuni under redox stress. We report that RrpA is a positive regulator of mdaB, encoding a flavin-dependent quinone reductase that contributes to the protection from redox stress mediated by structurally diverse quinones, while RrpB negatively regulates the expression of cj1555c (renamed nfrA for NADPH-flavin reductase A), encoding a flavin reductase. NfrA reduces riboflavin at a greater rate than its derivatives, suggesting that exogenous free flavins are the natural substrate. MdaB and NfrA both prefer NADPH as an electron donor. Cysteine substitution and posttranslational modification analyses indicated that RrpA and RrpB employ a cysteine-based redox switch. Complete genome sequence analyses revealed that mdaB is frequently found in Campylobacter and related Helicobacter spp., while nfrA is predominant in C. jejuni strains. Quinones and flavins are redox cycling agents secreted by a wide range of cell types that can form damaging superoxide by one-electron reactions. We propose a model for stress adaptation where MdaB and NfrA facilitate a two-electron reduction mechanism to the less toxic hydroquinones, thus aiding survival and persistence of this major pathogen. IMPORTANCE Changes in cellular redox potential result in alteration in the oxidation state of intracellular metabolites and enzymes; consequently, cells make adjustments that favor growth and survival. The work we present here answers some of the many questions that have remained elusive over the years of investigation into the enigmatic microaerophile bacterium Campylobacter jejuni. We employed molecular approaches to understand the regulation mechanisms and functional analyses to reveal the roles of two novel quinone and flavin reductases; both serve as major pools of cellular redox-active molecules. This work extends our knowledge on bacterial redox sensing mechanisms and the significance of hemostasis.

Genomic Rearrangements in Arabidopsis Considered as Quantitative Traits.

To understand the population genetics of structural variants and their effects on phenotypes, we developed an approach to mapping structural variants that segregate in a population sequenced at low coverage. We avoid calling structural variants directly. Instead, the evidence for a potential structural variant at a locus is indicated by variation in the counts of short-reads that map anomalously to that locus. These structural variant traits are treated as quantitative traits and mapped genetically, analogously to a gene expression study. Association between a structural variant trait at one locus, and genotypes at a distant locus indicate the origin and target of a transposition. Using ultra-low-coverage (0.3×) population sequence data from 488 recombinant inbred Arabidopsis thaliana genomes, we identified 6502 segregating structural variants. Remarkably, 25% of these were transpositions. While many structural variants cannot be delineated precisely, we validated 83% of 44 predicted transposition breakpoints by polymerase chain reaction. We show that specific structural variants may be causative for quantitative trait loci for germination and resistance to infection by the fungus Albugo laibachii, isolate Nc14. Further we show that the phenotypic heritability attributable to read-mapping anomalies differs from, and, in the case of time to germination and bolting, exceeds that due to standard genetic variation. Genes within structural variants are also more likely to be silenced or dysregulated. This approach complements the prevalent strategy of structural variant discovery in fewer individuals sequenced at high coverage. It is generally applicable to large populations sequenced at low-coverage, and is particularly suited to mapping transpositions.

Wheat Landrace Genome Diversity.

Understanding the genomic complexity of bread wheat (Triticum aestivum L.) is a cornerstone in the quest to unravel the processes of domestication and the following adaptation of domesticated wheat to a wide variety of environments across the globe. Additionally, it is of importance for future improvement of the crop, particularly in the light of climate change. Focusing on the adaptation after domestication, a nested association mapping (NAM) panel of 60 segregating biparental populations was developed, mainly involving landrace accessions from the core set of the Watkins hexaploid wheat collection optimized for genetic diversity. A modern spring elite variety, "Paragon," was used as common reference parent. Genetic maps were constructed following identical rules to make them comparable. In total, 1611 linkage groups were identified, based on recombination from an estimated 126,300 crossover events over the whole NAM panel. A consensus map, named landrace consensus map (LRC), was constructed and contained 2498 genetic loci. These newly developed genetics tools were used to investigate the rules underlying genome fluidity or rigidity, e.g., by comparing marker distances and marker orders. In general, marker order was highly correlated, which provides support for strong synteny between bread wheat accessions. However, many exceptional cases of incongruent linkage groups and increased marker distances were also found. Segregation distortion was detected for many markers, sometimes as hot spots present in different populations. Furthermore, evidence for translocations in at least 36 of the maps was found. These translocations fell, in general, into many different translocation classes, but a few translocation classes were found in several accessions, the most frequent one being the well-known T5B:7B translocation. Loci involved in recombination rate, which is an interesting trait for plant breeding, were identified by QTL analyses using the crossover counts as a trait. In total, 114 significant QTL were detected, nearly half of them with increasing effect from the nonreference parents.

Application of a library of near isogenic lines to understand context dependent expression of QTL for grain yield and adaptive traits in bread wheat.

BACKGROUND: Previous quantitative trait loci (QTLs) studies using the Avalon × Cadenza doubled haploid (DH) population identified eleven QTLs determining plant height, heading date and grain yield. The objectives of this study were: (i) to provide insight into the effects of these QTLs using reciprocal multiple near isogenic lines (NILs) with each pair of alleles compared in both parental backgrounds (Avalon or Cadenza), (ii) quantifying epistasis by looking at the background effects and (iii) predict favourable allelic combinations to develop superior genotypes adapted to a target environment. RESULTS: To this aim, a library of 553 BC2 NILs and their recurrent parents were tested over two growing seasons (2012/2013 and 2013/2014). The results obtained in the present study validated the plant height, heading date and grain yield QTLs previously identified. Epistatic interactions were detected for the 6B QTL for plant height and heading date, 3A QTL for heading date and grain yield and 2A QTL for grain yield. CONCLUSION: The marker assisted backcrossing strategy used provided an efficient method of resolving QTL for key agronomic traits in wheat as Mendelian factors determining possible epistatic interactions. The study shows that these QTLs are amenable to marker assisted selection, fine mapping, future positional cloning, and physiological trait dissection.

Validation of a 1DL earliness per se (eps) flowering QTL in bread wheat (Triticum aestivum).

Vernalization, photoperiod and the relatively poorly defined earliness per se (eps) genes regulate flowering in plants. We report here the validation of a major eps quantitative trait locus (QTL) located on wheat 1DL using near isogenic lines (NILs). We used four independent pairs of NILs derived from a cross between Spark and Rialto winter wheat varieties, grown in both the field and controlled environments. NILs carrying the Spark allele, defined by QTL flanking markers Xgdm111 and Xbarc62, consistently flowered 3-5 days earlier when fully vernalized relative to those with the Rialto. The effect was independent of photoperiod under field conditions, short days (10-h light), long days (16-h light) and very long days (20-h light). These results validate our original QTL identified using doubled haploid (DH) populations. This QTL represents variation maintained in elite north-western European winter wheat germplasm. The two DH lines used to develop the NILs, SR9 and SR23 enabled us to define the location of the 1DL QTL downstream of marker Xgdm111. SR9 has the Spark 1DL arm while SR23 has a recombinant 1DL arm with the Spark allele from Xgdm111 to the distal end. Our work suggests that marker assisted selection of eps effects is feasible and useful even before the genes are cloned. This means eps genes can be defined and positionally cloned in the same way as the photoperiod and vernalization genes have been. This validation study is a first step towards fine mapping and eventually cloning the gene directly in hexaploid wheat.

Establishing the A. E. Watkins landrace cultivar collection as a resource for systematic gene discovery in bread wheat.

KEY MESSAGE: A high level of genetic diversity was found in the A. E. Watkins bread wheat landrace collection. Genotypic information was used to determine the population structure and to develop germplasm resources. In the 1930s A. E. Watkins acquired landrace cultivars of bread wheat (Triticum aestivum L.) from official channels of the board of Trade in London, many of which originated from local markets in 32 countries. The geographic distribution of the 826 landrace cultivars of the current collection, here called the Watkins collection, covers many Asian and European countries and some from Africa. The cultivars were genotyped with 41 microsatellite markers in order to investigate the genetic diversity and population structure of the collection. A high level of genetic diversity was found, higher than in a collection of modern European winter bread wheat varieties from 1945 to 2000. Furthermore, although weak, the population structure of the Watkins collection reveals nine ancestral geographical groupings. An exchange of genetic material between ancestral groups before commercial wheat-breeding started would be a possible explanation for this. The increased knowledge regarding the diversity of the Watkins collection was used to develop resources for wheat research and breeding, one of them a core set, which captures the majority of the genetic diversity detected. The understanding of genetic diversity and population structure together with the availability of breeding resources should help to accelerate the detection of new alleles in the Watkins collection.

Syntenic relationships between the U and M genomes of Aegilops, wheat and the model species Brachypodium and rice as revealed by COS markers.

Diploid Aegilops umbellulata and Ae. comosa and their natural allotetraploid hybrids Ae. biuncialis and Ae. geniculata are important wild gene sources for wheat. With the aim of assisting in alien gene transfer, this study provides gene-based conserved orthologous set (COS) markers for the U and M genome chromosomes. Out of the 140 markers tested on a series of wheat-Aegilops chromosome introgression lines and flow-sorted subgenomic chromosome fractions, 100 were assigned to Aegilops chromosomes and six and seven duplications were identified in the U and M genomes, respectively. The marker-specific EST sequences were BLAST-ed to Brachypodium and rice genomic sequences to investigate macrosyntenic relationships between the U and M genomes of Aegilops, wheat and the model species. Five syntenic regions of Brachypodium identified genome rearrangements differentiating the U genome from the M genome and from the D genome of wheat. All of them seem to have evolved at the diploid level and to have been modified differentially in the polyploid species Ae. biuncialis and Ae. geniculata. A certain level of wheat-Aegilops homology was detected for group 1, 2, 3 and 5 chromosomes, while a clearly rearranged structure was showed for the group 4, 6 and 7 Aegilops chromosomes relative to wheat. The conserved orthologous set markers assigned to Aegilops chromosomes promise to accelerate gene introgression by facilitating the identification of alien chromatin. The syntenic relationships between the Aegilops species, wheat and model species will facilitate the targeted development of new markers specific for U and M genomic regions and will contribute to the understanding of molecular processes related to allopolyploidization.