High-resolution mapping of the pericentromeric region on wheat chromosome arm 5AS harbouring the Fusarium head blight resistance QTL Qfhs.ifa-5A.

The Qfhs.ifa-5A allele, contributing to enhanced Fusarium head blight resistance in wheat, resides in a low-recombinogenic region of chromosome 5A close to the centromere. A near-isogenic RIL population segregating for the Qfhs.ifa-5A resistance allele was developed and among 3650 lines as few as four recombined within the pericentromeric C-5AS1-0.40 bin, yielding only a single recombination point. Genetic mapping of the pericentromeric region using a recombination-dependent approach was thus not successful. To facilitate fine-mapping the physically large Qfhs.ifa-5A interval, two gamma-irradiated deletion panels were generated: (i) seeds of line NIL3 carrying the Qfhs.ifa-5A resistance allele in an otherwise susceptible background were irradiated and plants thereof were selfed to obtain deletions in homozygous state and (ii) a radiation hybrid panel was produced using irradiated pollen of the wheat line Chinese Spring (CS) for pollinating the CS-nullisomic5Atetrasomic5B. In total, 5157 radiation selfing and 276 radiation hybrid plants were screened for deletions on 5AS and plants containing deletions were analysed using 102 5AS-specific markers. Combining genotypic information of both panels yielded an 817-fold map improvement (cR/cM) for the centromeric bin and was 389-fold increased across the Qfhs.ifa-5A interval compared to the genetic map, with an average map resolution of 0.77 Mb/cR. We successfully proved that the RH mapping technique can effectively resolve marker order in low-recombining regions, including pericentromeric intervals, and simultaneously allow developing an in vivo panel of sister lines differing for induced deletions across the Qfhs.ifa-5A interval that can be used for phenotyping.

Oscheius tipulae in Italy: Evidence of an Alien Isolate in the Integral Natural Reserve of Montecristo Island (Tuscany).

Montecristo Island is an integral natural reserve of the Tuscan Archipelago National Park (Central Italy), characterized by a peculiar assemblage of flora and fauna, with several endemic taxa, and also with a high number of alien species. During a soil survey, we found an alien Oscheius tipulae Lam & Webster, 1971 isolate, phylogenetically close to others from South America. In this article, we examined the possible pathways of introduction of this nematode. Because of the high number of alien plants in this protected area and the low desiccation survival ability of O. tipulae, we hypothesized that the presence of this alien nematode isolate may be related to the soil of introduced plants, although historical association with plant-associated invertebrates is also possible. Further studies with more populations and marker molecules are necessary to investigate the distribution of O. tipulae and the possible impact on this natural reserve.

The barley Frost resistance-H2 locus.

Frost resistance-H2 (Fr-H2) is a major QTL affecting freezing tolerance in barley, yet its molecular basis is still not clearly understood. To gain a better insight into the structural characterization of the locus, a high-resolution linkage map developed from the Nure × Tremois cross was initially implemented to map 13 loci which divided the 0.602 cM total genetic distance into ten recombination segments. A PCR-based screening was then applied to identify positive bacterial artificial chromosome (BAC) clones from two genomic libraries of the reference genotype Morex. Twenty-six overlapping BACs from the integrated physical-genetic map were 454 sequenced. Reads assembled in contigs were subsequently ordered, aligned and manually curated in 42 scaffolds. In a total of 1.47 Mbp, 58 protein-coding sequences were identified, 33 of which classified according to similarity with sequences in public databases. As three complete barley C-repeat Binding Factors (HvCBF) genes were newly identified, the locus contained13 full-length HvCBFs, four Related to AP2 Triticeae (RAPT) genes, and at least five CBF pseudogenes. The final overall assembly of Fr-H2 includes more than 90 % of target region: all genes were identified along the locus, and a general survey of Repetitive Elements obtained. We believe that this gold-standard sequence for the Morex Fr-H2 will be a useful genomic tool for structural and evolutionary comparisons with Fr-H2 in winter-hardy cultivars along with Fr-2 of other Triticeae crops.

A new wild emmer wheat panel allows to map new loci associated with resistance to stem rust at seedling stage.

Wheat stem rust, caused by Puccinia graminis f. sp. tritici (Pgt), is a major wheat disease worldwide. A collection of 283 wild emmer wheat [Triticum turgidum L. subsp. dicoccoides (Körn. ex Asch. & Graebn.) Thell] accessions, representative of the entire Fertile Crescent region where wild emmer naturally occurs, was assembled, genotyped, and characterized for population structure, genetic diversity, and rate of linkage disequilibrium (LD) decay. Then, the collection was employed for mapping Pgt resistance genes, as a proof of concept of the effectiveness of genome-wide association studies in wild emmer. The collection was evaluated in controlled conditions for reaction to six common Pgt pathotypes (TPMKC, TTTTF, JRCQC, TRTTF, TTKSK/Ug99, and TKTTF). Most resistant accessions originated from the Southern Levant wild emmer lineage, with some showing a resistance reaction toward three to six tested races. Association analysis was conducted considering a 12K polymorphic single-nucleotide polymorphisms dataset, kinship relatedness between accessions, and population structure. Eleven significant marker-trait associations (MTA) were identified across the genome, which explained from 17% to up to 49% of phenotypic variance with an average 1.5 additive effect (based on the 1-9 scoring scale). The identified loci were either effective against single or multiple races. Some MTAs colocalized with known Pgt resistance genes, while others represent novel resistance loci useful for durum and bread wheat prebreeding. Candidate genes with an annotated function related to plant response to pathogens were identified at the regions linked to the resistance and defined according to the estimated small LD (about 126 kb), as typical of wild species.

Genome-wide association study for morphological, phenological, quality, and yield traits in einkorn (Triticum monococcum L. subsp. monococcum).

Einkorn (Triticum monococcum L. subsp. monococcum, 2n = 2× = 14, AmAm) is a diploid wheat whose cultivation was widespread in the Mediterranean and European area till the Bronze Age, before it was replaced by the more productive durum and bread wheats. Although scarcely cultivated nowadays, it has gained renewed interest due to its relevant nutritional properties and as source of genetic diversity for crop breeding. However, the molecular basis of many traits of interest in einkorn remain still unknown. A panel of 160 einkorn landraces, from different parts of the distribution area, was characterized for several phenotypic traits related to morphology, phenology, quality, and yield for 4 years in two locations. An approach based on co-linearity with the A genome of bread wheat, supported also by that with Triticum urartu genome, was exploited to perform association mapping, even without an einkorn anchored genome. The association mapping approach uncovered numerous marker-trait associations; for 37 of these, a physical position was inferred by homology with the bread wheat genome. Moreover, numerous associated regions were also assigned to the available T. monococcum contigs. Among the intervals detected in this work, three overlapped with regions previously described as involved in the same trait, while four other regions were localized in proximity of loci previously described and presumably refer to the same gene/QTL. The remaining associated regions identified in this work could represent a novel and useful starting point for breeding approaches to improve the investigated traits in this neglected species.

Next generation breeding.

The genomic revolution of the past decade has greatly improved our understanding of the genetic make-up of living organisms. The sequencing of crop genomes has completely changed our vision and interpretation of genome organization and evolution. Re-sequencing allows the identification of an unlimited number of markers as well as the analysis of germplasm allelic diversity based on allele mining approaches. High throughput marker technologies coupled with advanced phenotyping platforms provide new opportunities for discovering marker-trait associations which can sustain genomic-assisted breeding. The availability of genome sequencing information is enabling genome editing (site-specific mutagenesis), to obtain gene sequences desired by breeders. This review illustrates how next generation sequencing-derived information can be used to tailor genomic tools for different breeders' needs to revolutionize crop improvement.

Physical Mapping of Bread Wheat Chromosome 5A: An Integrated Approach.

The huge size, redundancy, and highly repetitive nature of the bread wheat [Triticum aestivum (L.)] genome, makes it among the most difficult species to be sequenced. To overcome these limitations, a strategy based on the separation of individual chromosomes or chromosome arms and the subsequent production of physical maps was established within the frame of the International Wheat Genome Sequence Consortium (IWGSC). A total of 95,812 bacterial artificial chromosome (BAC) clones of short-arm chromosome 5A (5AS) and long-arm chromosome 5A (5AL) arm-specific BAC libraries were fingerprinted and assembled into contigs by complementary analytical approaches based on the FingerPrinted Contig (FPC) and Linear Topological Contig (LTC) tools. Combined anchoring approaches based on polymerase chain reaction (PCR) marker screening, microarray, and sequence homology searches applied to several genomic tools (i.e., genetic maps, deletion bin map, neighbor maps, BAC end sequences (BESs), genome zipper, and chromosome survey sequences) allowed the development of a high-quality physical map with an anchored physical coverage of 75% for 5AS and 53% for 5AL with high portions (64 and 48%, respectively) of contigs ordered along the chromosome. In the genome of grasses, Brachypodium [Brachypodium distachyon (L.) Beauv.], rice (Oryza sativa L.), and sorghum [Sorghum bicolor (L.) Moench] homologs of genes on wheat chromosome 5A were separated into syntenic blocks on different chromosomes as a result of translocations and inversions during evolution. The physical map presented represents an essential resource for fine genetic mapping and map-based cloning of agronomically relevant traits and a reference for the 5A sequencing projects.

Emerging knowledge from genome sequencing of crop species.

Extensive insights into the genome composition, organization, and evolution have been gained from the plant genome sequencing and annotation ongoing projects. The analysis of crop genomes provided surprising evidences with important implications in plant origin and evolution: genome duplication, ancestral re-arrangements and unexpected polyploidization events opened new doors to address fundamental questions related to species proliferation, adaptation, and functional modulations. Detailed paleogenomic analysis led to many speculation on how chromosomes have been shaped over time in terms of gene content and order. The completion of the genome sequences of several major crops, prompted to a detailed identification and annotation of transposable elements: new hypothesis related to their composition, chromosomal distribution, insertion models, amplification rate, and evolution patterns are coming up. Availability of full genome sequence of several crop species as well as from many accessions within species is providing new keys for biodiversity exploitation and interpretation. Re-sequencing is enabling high-throughput genotyping to identify a wealth of SNP and afterward to produce haplotype maps necessary to accurately associate molecular variation to phenotype. Conservation genomics is emerging as a powerful tool to explain adaptation, genetic drift, natural selection, hybridization and to estimate genetic variation, fitness and population's viability.

Inside the CBF locus in Poaceae.

Several molecular evidences have been gathered in Poaceae that point out a central role of the CBF/DREB1 transcription factors in the signal transduction pathways leading to low-temperature tolerance, although to a quite different extent between crops originating from either temperate or tropical climates. A common feature of the CBF/DREB1 genes in Poaceae is their structural organization at the genome level in clusters of tandemly duplicated genes. In temperate cereals such as barley and wheat, expansion of specific multigene phylogenetic clades of CBFs that map at the Frost Resistance-2 locus has been exclusively observed. In addition, copy number variants of CBF genes between frost resistant and frost sensitive genotypes raise the question if multiple copies of the CBF/DREB1s are required to ensure freezing tolerance. On the other hand, in crops of tropical origin such as rice and maize, a smaller or less-responsive CBF regulon may have evolved, and different mechanisms might determine chilling tolerance. In this review, recent advances on the organization and diversity at the CBF cluster locus in the grasses are provided and discussed.