Andrea Gennaro

FISHIS: fluorescence in situ hybridization in suspension and chromosome flow sorting made easy.

The large size and complex polyploid nature of many genomes has often hampered genomics development, as is the case for several plants of high agronomic value. Isolating single chromosomes or chromosome arms via flow sorting offers a clue to resolve such complexity by focusing sequencing to a discrete and self-consistent part of the whole genome. The occurrence of sufficient differences in the size and or base-pair composition of the individual chromosomes, which is uncommon in plants, is critical for the success of flow sorting. We overcome this limitation by developing a robust method for labeling isolated chromosomes, named Fluorescent In situ Hybridization In suspension (FISHIS). FISHIS employs fluorescently labeled synthetic repetitive DNA probes, which are hybridized, in a wash-less procedure, to chromosomes in suspension following DNA alkaline denaturation. All typical A, B and D genomes of wheat, as well as individual chromosomes from pasta (T. durum L.) and bread (T. aestivum L.) wheat, were flow-sorted, after FISHIS, at high purity. For the first time in eukaryotes, each individual chromosome of a diploid organism, Dasypyrum villosum (L.) Candargy, was flow-sorted regardless of its size or base-pair related content. FISHIS-based chromosome sorting is a powerful and innovative flow cytogenetic tool which can develop new genomic resources from each plant species, where microsatellite DNA probes are available and high quality chromosome suspensions could be produced. The joining of FISHIS labeling and flow sorting with the Next Generation Sequencing methodology will enforce genomics for more species, and by this mightier chromosome approach it will be possible to increase our knowledge about structure, evolution and function of plant genome to be used for crop improvement. It is also anticipated that this technique could contribute to analyze and sort animal chromosomes with peculiar cytogenetic abnormalities, such as copy number variations or cytogenetic aberrations.

A candidate for Lr19, an exotic gene conditioning leaf rust resistance in wheat

Lr19, one of the few widely effective genes conferring resistance to leaf rust in wheat, was transferred from the wild relative Thinopyrum ponticum to durum wheat. Since Lr19 confers a hypersensitive response to the pathogen, it was considered likely that the gene would be a member of the major nucleotide-binding site (NBS)-leucine-rich repeat (LRR) plant R gene family. NBS profiling, based on PCR amplification of conserved NBS motifs, was applied to durum wheat–Th. ponticum recombinant lines involving different segments of the alien 7AgL chromosome arm, carrying or lacking Lr19. Differential PCR products were isolated and sequenced. From one such sequence (AG15), tightly linked to Lr19, a 4,121-bp full-length cDNA was obtained. Its deduced 1,258 amino acid sequence has the characteristic NBS-LRR domains of plant R gene products and includes a coiled-coil (CC) region typical of monocots. The genomic DNA sequence showed the presence of two exons and a short intron upstream of the predicted stop codon. Homology searches revealed considerable identity of AG15 with the cloned wheat resistance gene Pm3a and a lower similarity with wheat Lr1, Lr21, and Lr10. Quantitative PCR on leaf-rust-infected and non-infected Lr19 carriers proved AG15 to be constitutively expressed, as is common for R genes.

Structural–functional dissection and characterization of yield-contributing traits originating from a group 7 chromosome of the wheatgrass species Thinopyrum ponticum after transfer into durum wheat

For the first time, using chromosome engineering of durum wheat, the underlying genetic determinants of a yield-improving segment from Thinopyrum ponticum (7AgL) were dissected. Three durum wheat–Th. ponticum near-isogenic recombinant lines (NIRLs), with distal portions of their 7AL arm (fractional lengths 0.77, 0.72, and 0.60) replaced by alien chromatin, were field-tested for two seasons under rainfed conditions. Yield traits and other agronomic characteristics of the main shoot and whole plant were measured. Loci for seed number per ear and per spikelet were detected in the proximal 7AgL segment (0.60–0.72). Loci determining considerable increases of flag leaf width and area, productive tiller number per plant, biomass per plant, and grain yield per plant were located in the distally adjacent 0.72–0.77 7AgL segment, while in the most distal portion (0.77–1.00) genetic effects on spikelet number per ear were identified. Contrary to previous reports, trials with the bread wheat T4 translocation line, carrying on 7DL a sizeable 7AgL segment of which those present in the durum wheat-Th. ponticum NIRLs represent fractions, gave no yield advantage. The hypothesis that ABA might be a factor contributing to the 7AgL effects was tested by analysing endogenous ABA contents of the NIRLs and their responses to exogenous ABA application. The 7AgL yield-related loci were shown to be ABA-independent. This study highlights the value of wheat–alien recombinant lines for dissecting the genetic and physiological basis of complex traits present in wild germplasm, and provides a basis for their targeted exploitation in wheat breeding.

Flow Sorting and Molecular Cytogenetic Identification of Individual Chromosomes of Dasypyrum villosum L. (H. villosa) by a Single DNA Probe

Dasypyrum villosum (L.) Candargy (sin. Haynaldia villosa) is an annual wild diploid grass species (2n = 2x = 14; genome VV) belonging to the Poaceae family, which is considered to be an important source of biotic and abiotic stress resistance genes for wheat breeding. Enhanced characterization of D. villosum chromosomes can facilitate exploitation of its gene pool and its use in wheat breeding programs. Here we present the cytogenetic identification of D. villosum chromosomes on slide by fluorescent in situ hybridization (FISH), with the GAA simple sequence repeat (SSR) as a probe. We also describe the isolation and the flow cytometric analysis of D. villosum chromosomes in suspension, resulting in a distinguished flow karyotype. Chromosomes were flow sorted into three fractions, according their DNA content, one of which was composed of a single type of chromosome, namely 6 V, sorted with over 85% purity. Chromosome 6 V is known to carry genes to code for important resistance and seed storage characteristics, and its isolation represents a new source of genetic traits and specific markers useful for wheat improvement.

Pyramiding different alien chromosome segments in durum wheat: Feasibility and breeding potential

Wheat chromosome engineering, i.e., the transfer of alien chromosome segments from various Triticeae species into cultivated wheats, is greatly benefiting from the recent advancements in molecular genetics, cytogenetics, and genomics. Powerful tools are currently available that make selection of desired genotypes far more pre- cise and effective than in the past, thus giving this transfer strategy considerable po- tential for meaningful practical achievements. Such tools were successfully applied to engineer the durum wheat genome with small alien segments containing genes for disease resistance and quality traits, including Lr19 (leaf rust resistance) and Yp (yellow endosperm pigmentation) from Thinopyrumponticum, Pm13 (powdery mildew resistance) from Aegilops longissima, and the Glu-D3 and Glu-D1 glutenin subunit genes from Triticumaestivum. Targeted segments with such genes, all being of minimal size, were first separately incorporated into durum wheat by ph1-induced homoeologous recombination. The positive performance of selected recombinant lines from each transfer project encouraged attempts to develop multiple alien seg- ment combinations. The results obtained with double and also triple recombinants indicate a good tolerance of the durum wheat genome even toward such complex manipulations, which thus appear to offer good prospects for simultaneously enrich- ing durum wheat germplasm with several valuable traits from related Triticeae.

RNAi‐based GM plants: food for thought for risk assessors

RNA interference (RNAi) is an emerging technology that offers new opportunities for the generation of new traits in genetically modified (GM) plants. Potential risks associated with RNAi-based GM plants and issues specific to their risk assessment were discussed during an international scientific workshop (June 2014) organized by the European Food Safety Authority (EFSA). Selected key outcomes of the workshop are reported here.

Genomes, Chromosomes and Genes of the Wheatgrass Genus Thinopyrum: the Value of their Transfer into Wheat for Gains in Cytogenomic Knowledge and Sustainable Breeding

Perennial wheatgrass species of the genus Thinopyrum possess several appealing attributes for wheat improvement, contributing to tolerance to biotic and abiotic stresses, as well as to quality and even to yield increase. Major genes or QTLs underlying such traits have been identified on numerous chromosomes of both diploid (Th. elongatum and Th. bessarabicum) and polyploid (mainly Th. intermedium and Th. ponticum) representatives of the genus, having different genome origin (E, J, St/S) and involving several homoeologous groups. Thinopyrum chromosomes sharing homoeology with wheat group 7 chromosomes turned to be particularly rich in beneficial genes; among them, a Th. ponticum group 7 chromosome referred to as 7Ag or 7el has been extensively targeted in various successful attempts of harnessing its attractive gene content. A survey of the several wheat translocation/recombinant lines involving this chromosome in the background of both bread and durum wheat is given. Such lines are described as highly valuable tools for a variety of studies, from development of integrated genetic and physical maps, to the analysis of structural and functional characteristics associated with defined alien chromosome subregions. The validity of Th. ponticum group 7 transfers as breeding materials (notable genes and traits including Lr19, Sr25, Fusarium head blight resistance, yellow pigment content, and even yield) is also highlighted. Finally, examples are given of pyramiding of group 7 Thinopyrum genes through ‘precision’ breeding strategies of chromosome engineering, which, efficiently aided by current genetic, cytogenetic and genomic (or, collectively, ‘cytogenomic’) technologies, enable a multifaceted and sustainable improvement of the wheat crop based on the use of the wealth of natural genetic resources of its related gene pools.

Targeted exploitation of gene pools of alien Triticeae species for sustainable and multi-faceted improvement of the durum wheat crop

Abstract. Enlarging the genetic basis of essential crop species such as the polyploid wheats is a priority in breeding outlooks for the new millennium. To this end, one feasible approach to exploit the wide and largely untapped variation present in the gene pools of alien Triticeae species is chromosome engineering, which enables the transfer of alien chromosomal segments carrying targeted genes to wheat chromosomes. Recent progress in molecular marker technology, molecular cytogenetic techniques, and in genome knowledge has greatly enhanced the ability of chromosome engineering to contribute breeder-friendly germplasm, even in the case of durum wheat, considered more sensitive to genome manipulations than bread wheat. Using finely tuned chromosome engineering, stable incorporation into durum has been achieved for various alien segments containing genes for disease resistance, quality attributes, and even yield-related traits, both separately and in combination. The state of the art and the breeding potential of such transfers are reviewed and updated.

First detailed karyo-morphological analysis and molecular cytological study of leafy cardoon and globe artichoke, two multi-use Asteraceae crops

Abstract Traditionally globe artichoke and leafy cardoon have been cultivated for use as vegetables but these crops are now finding multiple new roles in applications ranging from paper production to cheese preparation and biofuel use, with interest in their functional food potential. So far, their chromosome complements have been poorly investigated and a well-defined karyotype was not available. In this paper, a detailed karyo-morphological analysis and molecular cytogenetic studies were conducted on globe artichoke (Cynara cardunculus Linnaeus, 1753 var. scolymus Fiori, 1904) and leafy cardoon (Cynara cardunculus Linneaus, 1753 var. altilis De Candolle, 1838). Fluorescent In Situ Hybridization In Suspension (FISHIS) was applied to nuclei suspensions as a fast method for screening of labelling probes, before metaphase spread hybridization. Classic Fluorescent In Situ Hybridization (FISH) on slide, using repetitive telomeric and ribosomal sequences and Simple Sequence Repeats (SSRs) oligonucleotide as probes, identified homologous chromosome relationships and allowed development of molecular karyotypes for both varieties. The close phylogenetic relationship between globe artichoke and cardoon was supported by the very similar karyotypes but clear chromosomal structural variation was detected. In the light of the recent release of the globe artichoke genome sequencing, these results are relevant for future anchoring of the pseudomolecule sequence assemblies to specific chromosomes. In addition, the DNA content of the two crops has been determined by flow cytometry and a fast method for standard FISH on slide and methodological improvements for nuclei isolation are described.

First production of wild hemmer ( Triticum turgidum ssp. dicoccoides) transgenic plants

Plant genetic transformation and regeneration has become a valuable research tool for functional genomics. A successful transformation event involves the transfer of the target gene into a suitable explant, the integration and expression of the transgene into the host genome and the regeneration of the fertile transgenic plants from the transformed tissues. Wheat is considered as a recalcitrant species even if many efforts have been done in recent years to improve transformation efficiency. The transformation of its progenitors has never been attempted, even though the possibility to transform wild hemmer represents a valuable tool to evaluate structural and functional variability occurring in wild hemmer and explaining its higher adaptation to abiotic stresses. In this paper we report, as far as we know, for the first time, the microparticle transformation of immature embryos of the wild hemmer Triticum dicoccoides with the Tapgip1 gene. The transformation method was successfully transferred from durum wheat and several transgenic lines were obtained. Its application for the exploitation of wheat progenitors for molecular breeding is of great relevance for genomic and functional genomics studies. This result, indeed, opens new perspectives in complementation studies for the comprehension of durum and bread wheat adaptation mechanisms to stresses.