From Transgenesis to Genome Editing in Crop Improvement: Applications, Marketing, and Legal Issues.

The biotechnological approaches of transgenesis and the more recent eco-friendly new breeding techniques (NBTs), in particular, genome editing, offer useful strategies for genetic improvement of crops, and therefore, recently, they have been receiving increasingly more attention. The number of traits improved through transgenesis and genome editing technologies is growing, ranging from resistance to herbicides and insects to traits capable of coping with human population growth and climate change, such as nutritional quality or resistance to climatic stress and diseases. Research on both technologies has reached an advanced stage of development and, for many biotech crops, phenotypic evaluations in the open field are already underway. In addition, many approvals regarding main crops have been granted. Over time, there has been an increase in the areas cultivated with crops that have been improved through both approaches, but their use in various countries has been limited by legislative restrictions according to the different regulations applied which affect their cultivation, marketing, and use in human and animal nutrition. In the absence of specific legislation, there is an on-going public debate with favorable and unfavorable positions. This review offers an updated and in-depth discussion on these issues.

Genomic Approaches to Identify Molecular Bases of Crop Resistance to Diseases and to Develop Future Breeding Strategies.

Plant diseases are responsible for substantial crop losses each year and affect food security and agricultural sustainability. The improvement of crop resistance to pathogens through breeding represents an environmentally sound method for managing disease and minimizing these losses. The challenge is to breed varieties with a stable and broad-spectrum resistance. Different approaches, from markers to recent genomic and 'post-genomic era' technologies, will be reviewed in order to contribute to a better understanding of the complexity of host-pathogen interactions and genes, including those with small phenotypic effects and mechanisms that underlie resistance. An efficient combination of these approaches is herein proposed as the basis to develop a successful breeding strategy to obtain resistant crop varieties that yield higher in increasing disease scenarios.

Sustainable Use of Bioactive Compounds from Solanum Tuberosum and Brassicaceae Wastes and by-Products for Crop Protection-A Review.

Defatted seed meals of oleaginous Brassicaceae, such as Eruca sativa, and potato peel are excellent plant matrices to recover potentially useful biomolecules from industrial processes in a circular strategy perspective aiming at crop protection. These biomolecules, mainly glycoalkaloids and phenols for potato and glucosinolates for Brassicaceae, have been proven to be effective against microbes, fungi, nematodes, insects, and even parasitic plants. Their role in plant protection is overviewed, together with the molecular basis of their synthesis in plant, and the description of their mechanisms of action. Possible genetic and biotechnological strategies are presented to increase their content in plants. Genetic mapping and identification of closely linked molecular markers are useful to identify the loci/genes responsible for their accumulation and transfer them to elite cultivars in breeding programs. Biotechnological approaches can be used to modify their allelic sequence and enhance the accumulation of the bioactive compounds. How the global challenges, such as reducing agri-food waste and increasing sustainability and food safety, could be addressed through bioprotector applications are discussed here.

Genome-Wide Association Mapping of Prostrate/Erect Growth Habit in Winter Durum Wheat.

By selecting for prostrate growth habit of the juvenile phase of the cycle, durum wheat cultivars could be developed with improved competitive ability against weeds, and better soil coverage to reduce the soil water lost by evaporation. A panel of 184 durum wheat (Triticum turgidum subsp. durum) genotypes, previously genotyped with DArT-seq markers, was used to perform association mapping analysis of prostrate/erect growth habit trait and to identify candidate genes. Phenotypic data of plant growth habit were recorded during three consecutive growing seasons (2014-2016), two different growth conditions (field trial and greenhouse) and two sowing periods (autumn and spring). Genome-wide association study revealed significant marker-trait associations, twelve of which were specific for a single environment/year, 4 consistent in two environments, and two MTAs for the LSmeans were identified across all environments, on chromosomes 2B and 5A. The co-localization of some MTAs identified in this study with known vernalization and photoperiod genes demonstrated that the sensitivity to vernalization and photoperiod response are actually not only key components of spring/winter growth habit, but they play also an important role in defining the magnitude of the tiller angle during the tillering stage. Many zinc-finger transcription factors, such as C2H2 or CCCH-domain zinc finger proteins, known to be involved in plant growth habit and in leaf angle regulation were found as among the most likely candidate genes. The highest numbers of candidate genes putatively related to the trait were found on chromosomes 3A, 4B, 5A and 6A. Moreover, a bioinformatic approach has been considered to search for functional ortholog genes in wheat by using the sequence of rice and barley tiller angle-related genes. The information generated could be used to improve the understanding of the mechanisms that regulate the prostrate/erect growth habit in wheat and the adaptive potential of durum wheat under resource-limited environmental conditions.

Regulation and Evolution of NLR Genes: A Close Interconnection for Plant Immunity.

NLR (NOD-like receptor) genes belong to one of the largest gene families in plants. Their role in plants' resistance to pathogens has been clearly described for many members of this gene family, and dysregulation or overexpression of some of these genes has been shown to induce an autoimmunity state that strongly affects plant growth and yield. For this reason, these genes have to be tightly regulated in their expression and activity, and several regulatory mechanisms are described here that tune their gene expression and protein levels. This gene family is subjected to rapid evolution, and to maintain diversity at NLRs, a plethora of genetic mechanisms have been identified as sources of variation. Interestingly, regulation of gene expression and evolution of this gene family are two strictly interconnected aspects. Indeed, some examples have been reported in which mechanisms of gene expression regulation have roles in promotion of the evolution of this gene family. Moreover, co-evolution of the NLR gene family and other gene families devoted to their control has been recently demonstrated, as in the case of miRNAs.

Genetic Mapping of Loci for Resistance to Stem Rust in a Tetraploid Wheat Collection.

Stem rust, caused by Puccinia graminis f. sp. tritici (Pgt), is a major biotic constraint to wheat production worldwide. Disease resistant cultivars are a sustainable means for the efficient control of this disease. To identify quantitative trait loci (QTLs) conferring resistance to stem rust at the seedling stage, an association mapping panel consisting of 230 tetraploid wheat accessions were evaluated for reaction to five Pgt races under greenhouse conditions. A high level of phenotypic variation was observed in the panel in response to all of the races, allowing for genome-wide association mapping of resistance QTLs in wild, landrace, and cultivated tetraploid wheats. Twenty-two resistance QTLs were identified, which were characterized by at least two marker-trait associations. Most of the identified resistance loci were coincident with previously identified rust resistance genes/QTLs; however, six regions detected on chromosomes 1B, 5A, 5B, 6B, and 7B may be novel. Availability of the reference genome sequence of wild emmer wheat accession Zavitan facilitated the search for candidate resistance genes in the regions where QTLs were identified, and many of them were annotated as NOD (nucleotide binding oligomerization domain)-like receptor (NLR) genes or genes related to broad spectrum resistance.

Genetic analysis of root morphological traits in wheat.

Traits related to root architecture are of great importance for yield performance of crop species, although they remain poorly understood. The present study is aimed at identifying the genomic regions involved in the control of root morphological traits in durum wheat (Triticum durum Desf.). A set of 123 recombinant inbred lines derived from the durum wheat cross of cvs. 'Creso' × 'Pedroso' were grown hydroponically to two growth stages, and were phenotypically evaluated for a number of root traits. In addition, meta-(M)QTL analysis was performed that considered the results of other root traits studies in wheat, to compare with the 'Creso' × 'Pedroso' cross and to increase the QTL detection power. Eight quantitative trait loci (QTL) for traits related to root morphology were identified on chromosomes 1A, 1B, 2A, 3A, 6A and 6B in the 'Creso' × 'Pedroso' segregating population. Twenty-two MQTL that comprised from two to six individual QTL that had widely varying confidence intervals were found on 14 chromosomes. The data from the present study provide a detailed analysis of the genetic basis of morphological root traits in wheat. This study of the 'Creso' × 'Pedroso' durum-wheat population has revealed some QTL that had not been previously identified.

Genetic basis of qualitative and quantitative resistance to powdery mildew in wheat: from consensus regions to candidate genes.

BACKGROUND: Powdery mildew (Blumeria graminis f. sp. tritici) is one of the most damaging diseases of wheat. The objective of this study was to identify the wheat genomic regions that are involved in the control of powdery mildew resistance through a quantitative trait loci (QTL) meta-analysis approach. This meta-analysis allows the use of collected QTL data from different published studies to obtain consensus QTL across different genetic backgrounds, thus providing a better definition of the regions responsible for the trait, and the possibility to obtain molecular markers that will be suitable for marker-assisted selection. RESULTS: Five QTL for resistance to powdery mildew were identified under field conditions in the durum-wheat segregating population Creso × Pedroso. An integrated map was developed for the projection of resistance genes/ alleles and the QTL from the present study and the literature, and to investigate their distribution in the wheat genome. Molecular markers that correspond to candidate genes for plant responses to pathogens were also projected onto the map, particularly considering NBS-LRR and receptor-like protein kinases. More than 80 independent QTL and 51 resistance genes from 62 different mapping populations were projected onto the consensus map using the Biomercator statistical software. Twenty-four MQTL that comprised 2-6 initial QTL that had widely varying confidence intervals were found on 15 chromosomes. The co-location of the resistance QTL and genes was investigated. Moreover, from analysis of the sequences of DArT markers, 28 DArT clones mapped on wheat chromosomes have been shown to be associated with the NBS-LRR genes and positioned in the same regions as the MQTL for powdery mildew resistance. CONCLUSIONS: The results from the present study provide a detailed analysis of the genetic basis of resistance to powdery mildew in wheat. The study of the Creso × Pedroso durum-wheat population has revealed some QTL that had not been previously identified. Furthermore, the analysis of the co-localization of resistance loci and functional markers provides a large list of candidate genes and opens up a new perspective for the fine mapping and isolation of resistance genes, and for the marker-assisted improvement of resistance in wheat.

Different stress responsive strategies to drought and heat in two durum wheat cultivars with contrasting water use efficiency.

BACKGROUND: Durum wheat often faces water scarcity and high temperatures, two events that usually occur simultaneously in the fields. Here we report on the stress responsive strategy of two durum wheat cultivars, characterized by different water use efficiency, subjected to drought, heat and a combination of both stresses. RESULTS: The cv Ofanto (lower water use efficiency) activated a large set of well-known drought-related genes after drought treatment, while Cappelli (higher water use efficiency) showed the constitutive expression of several genes induced by drought in Ofanto and a modulation of a limited number of genes in response to stress. At molecular level the two cvs differed for the activation of molecular messengers, genes involved in the regulation of chromatin condensation, nuclear speckles and stomatal closure. Noteworthy, the heat response in Cappelli involved also the up-regulation of genes belonging to fatty acid ß-oxidation pathway, glyoxylate cycle and senescence, suggesting an early activation of senescence in this cv. A gene of unknown function having the greatest expression difference between the two cultivars was selected and used for expression QTL analysis, the corresponding QTL was mapped on chromosome 6B. CONCLUSION: Ofanto and Cappelli are characterized by two opposite stress-responsive strategies. In Ofanto the combination of drought and heat stress led to an increased number of modulated genes, exceeding the simple cumulative effects of the two single stresses, whereas in Cappelli the same treatment triggered a number of differentially expressed genes lower than those altered in response to heat stress alone. This work provides clear evidences that the genetic system based on Cappelli and Ofanto represents an ideal tool for the genetic dissection of the molecular response to drought and other abiotic stresses.

Plant nucleotide binding site-leucine-rich repeat (NBS-LRR) genes: active guardians in host defense responses.

The most represented group of resistance genes are those of the nucleotide binding site-leucine-rich repeat (NBS-LRR) class. These genes are very numerous in the plant genome, and they often occur in clusters at specific loci following gene duplication and amplification events. To date, hundreds of resistance genes and relatively few quantitative trait loci for plant resistance to pathogens have been mapped in different species, with some also cloned. When these NBS-LRR genes have been physically or genetically mapped, many cases have shown co-localization between resistance loci and NBS-LRR genes. This has allowed the identification of candidate genes for resistance, and the development of molecular markers linked to R genes. This review is focused on recent genomics studies that have described the abundance, distribution and evolution of NBS-LRR genes in plant genomes. Furthermore, in terms of their expression, NBS-LRR genes are under fine regulation by cis- and trans-acting elements. Recent findings have provided insights into the roles of alternative splicing, the ubiquitin/ proteasome system, and miRNAs and secondary siRNAs in the regulation of NBS-LRR gene expression at the post-transcriptional, post-translational and epigenetic levels. The possibility to use this knowledge for genetic improvement of plant resistance to pathogens is discussed.

Genome-wide association study of common resistance to rust species in tetraploid wheat.

Rusts of the genus Puccinia are wheat pathogens. Stem (black; Sr), leaf (brown; Lr), and stripe (yellow; Yr) rust, caused by Puccinia graminis f. sp. tritici (Pgt), Puccinia triticina (Pt), and Puccinia striiformis f. sp. tritici (Pst), can occur singularly or in mixed infections and pose a threat to wheat production globally in terms of the wide dispersal of their urediniospores. The development of durable resistant cultivars is the most sustainable method for controlling them. Many resistance genes have been identified, characterized, genetically mapped, and cloned; several quantitative trait loci (QTLs) for resistance have also been described. However, few studies have considered resistance to all three rust pathogens in a given germplasm. A genome-wide association study (GWAS) was carried out to identify loci associated with resistance to the three rusts in a collection of 230 inbred lines of tetraploid wheat (128 of which were Triticum turgidum ssp. durum) genotyped with SNPs. The wheat panel was phenotyped in the field and subjected to growth chamber experiments across different countries (USA, Mexico, Morocco, Italy, and Spain); then, a mixed linear model (MLM) GWAS was performed. In total, 9, 34, and 5 QTLs were identified in the A and B genomes for resistance to Pgt, Pt, and Pst, respectively, at both the seedling and adult plant stages. Only one QTL on chromosome 4A was found to be effective against all three rusts at the seedling stage. Six QTLs conferring resistance to two rust species at the adult plant stage were mapped: three on chromosome 1B and one each on 5B, 7A, and 7B. Fifteen QTLs conferring seedling resistance to two rusts were mapped: five on chromosome 2B, three on 7B, two each on 5B and 6A, and one each on 1B, 2A, and 7A. Most of the QTLs identified were specific for a single rust species or race of a species. Candidate genes were identified within the confidence intervals of a QTL conferring resistance against at least two rust species by using the annotations of the durum (cv. 'Svevo') and wild emmer wheat ('Zavitan') reference genomes. The 22 identified loci conferring resistance to two or three rust species may be useful for breeding new and potentially durable resistant wheat cultivars.

Characterization of wheat DArT markers: genetic and functional features.

Diversity array technology (DArT) markers are largely used for mapping, genetic diversity, and association mapping studies. For years, they have been used as anonymous genomic markers, as their sequences were not known. As the sequences of 2,000 wheat DArT clones are now available, this study was designed to analyze these sequences with bioinformatic approaches, and to study the genetic features of a subset of 291 markers positioned on the A and B genomes in three durum wheat genetic maps. A set of 1,757 non-redundant sequences was identified, and used as queries for similarity searches. Analysis of the genetic positions of markers corresponding to nearly identical sequences indicates that redundancy of sequences is one of the factors that explains the clustering of these markers in specific genomic regions. Of a total of 1,124 DArT clones (64 %) that represent putatively expressed sequences, putative functions are proposed for more than 700 of them. Of note, many clones correspond to genes that are related to disease resistance, as characterized by leucine-rich repeat domains, and 40 of these clones are positioned in the three genetic maps presented in this study. Finally, DArT markers have been used to find syntenic regions in the Brachypodium and rice genomes. In conclusion, the analyses herein presented contribute to explain the main features of DArT markers observed in genetic maps, as clustering in short chromosome regions. Moreover, the attribution of putative gene functions for more than 700 sequences makes these markers an optimal tool for collinearity studies or for the identification of candidate genes.

A high-density consensus map of A and B wheat genomes.

A durum wheat consensus linkage map was developed by combining segregation data from six mapping populations. All of the crosses were derived from durum wheat cultivars, except for one accession of T. ssp. dicoccoides. The consensus map was composed of 1,898 loci arranged into 27 linkage groups covering all 14 chromosomes. The length of the integrated map and the average marker distance were 3,058.6 and 1.6 cM, respectively. The order of the loci was generally in agreement with respect to the individual maps and with previously published maps. When the consensus map was aligned to the deletion bin map, 493 markers were assigned to specific bins. Segregation distortion was found across many durum wheat chromosomes, with a higher frequency for the B genome. This high-density consensus map allowed the scanning of the genome for chromosomal rearrangements occurring during the wheat evolution. Translocations and inversions that were already known in literature were confirmed, and new putative rearrangements are proposed. The consensus map herein described provides a more complete coverage of the durum wheat genome compared with previously developed maps. It also represents a step forward in durum wheat genomics and an essential tool for further research and studies on evolution of the wheat genome.

Specialized metabolites: Physiological and biochemical role in stress resistance, strategies to improve their accumulation, and new applications in crop breeding and management.

Specialized plant metabolites (SPMs), traditionally referred to as 'secondary metabolites', are chemical compounds involved in a broad range of biological functions, including plant responses to abiotic and biotic stresses. Moreover, some of them have a role in end-product quality with potential health benefits in humans. For this reason, they became an important target of studies focusing on their mechanisms of action and use in crop breeding and management. In this review we summarize the specific role of SPMs in physiological processes and in plant resistance to abiotic and biotic stresses, and the different strategies to enhance their production/accumulation in plant tissues under stress, including genetic approaches (marker-assisted selection and biotechnological tools) and agronomic management (fertilizer applications, cultivation method and beneficial microorganisms). New crop management strategies based on the direct application of the most promising compounds in form of plant residuals or liquid formulations are also described.

QTL Mapping of Stem Rust Resistance in Populations of Durum Wheat.

Stem rinfectionust, caused by the fungus Puccinia graminis f. sp. tritici (Pgt), is one of the most devastating fungal diseases of durum and common wheat worldwide. The identification of sources of resistance and the validation of QTLs identified through genome-wide association studies is of paramount importance for reducing the losses caused by this disease to wheat grain yield and quality. Four segregating populations whose parents showed contrasting reactions to some Pgt races were assessed in the present study, and 14 QTLs were identified on chromosomes 3A, 4A, 6A, and 6B, with some regions in common between different segregating populations. Several QTLs were mapped to chromosomal regions coincident with previously mapped stem rust resistance loci; however, their reaction to different Pgt races suggest that novel genes or alleles could be present on chromosomes 3A and 6B. Putative candidate genes with a disease-related functional annotation have been identified in the QTL regions based on information available from the reference genome of durum cv. 'Svevo'.

Importance of Landraces in Cereal Breeding for Stress Tolerance.

The renewed focus on cereal landraces is a response to some negative consequences of modern agriculture and conventional breeding which led to a reduction of genetic diversity. Cereal landraces are still cultivated on marginal lands due to their adaptability to unfavourable conditions, constituting an important source of genetic diversity usable in modern plant breeding to improve the adaptation to abiotic or biotic stresses, yield performance and quality traits in limiting environments. Traditional agricultural production systems have played an important role in the evolution and conservation of wide variability in gene pools within species. Today, on-farm and ex situ conservation in gene bank collections, together with data sharing among researchers and breeders, will greatly benefit cereal improvement. Many efforts are usually made to collect, organize and phenotypically and genotypically analyse cereal landrace collections, which also utilize genomic approaches. Their use in breeding programs based on genomic selection, and the discovery of beneficial untapped QTL/genes/alleles which could be introgressed into modern varieties by MAS, pyramiding or biotechnological tools, increase the potential for their better deployment and exploitation in breeding for a more sustainable agricultural production, particularly enhancing adaptation and productivity in stress-prone environments to cope with current climate changes.

The Global Durum Wheat Panel (GDP): An International Platform to Identify and Exchange Beneficial Alleles.

Representative, broad and diverse collections are a primary resource to dissect genetic diversity and meet pre-breeding and breeding goals through the identification of beneficial alleles for target traits. From 2,500 tetraploid wheat accessions obtained through an international collaborative effort, a Global Durum wheat Panel (GDP) of 1,011 genotypes was assembled that captured 94-97% of the original diversity. The GDP consists of a wide representation of Triticum turgidum ssp. durum modern germplasm and landraces, along with a selection of emmer and primitive tetraploid wheats to maximize diversity. GDP accessions were genotyped using the wheat iSelect 90K SNP array. Among modern durum accessions, breeding programs from Italy, France and Central Asia provided the highest level of genetic diversity, with only a moderate decrease in genetic diversity observed across nearly 50 years of breeding (1970-2018). Further, the breeding programs from Europe had the largest sets of unique alleles. LD was lower in the landraces (0.4 Mbp) than in modern germplasm (1.8 Mbp) at r 2 = 0.5. ADMIXTURE analysis of modern germplasm defined a minimum of 13 distinct genetic clusters (k), which could be traced to the breeding program of origin. Chromosome regions putatively subjected to strong selection pressure were identified from fixation index (F st ) and diversity reduction index (DRI) metrics in pairwise comparisons among decades of release and breeding programs. Clusters of putative selection sweeps (PSW) were identified as co-localized with major loci controlling phenology (Ppd and Vrn), plant height (Rht) and quality (gliadins and glutenins), underlining the role of the corresponding genes as driving elements in modern breeding. Public seed availability and deep genetic characterization of the GDP make this collection a unique and ideal resource to identify and map useful genetic diversity at loci of interest to any breeding program.

Durum wheat genome highlights past domestication signatures and future improvement targets.

The domestication of wild emmer wheat led to the selection of modern durum wheat, grown mainly for pasta production. We describe the 10.45 gigabase (Gb) assembly of the genome of durum wheat cultivar Svevo. The assembly enabled genome-wide genetic diversity analyses revealing the changes imposed by thousands of years of empirical selection and breeding. Regions exhibiting strong signatures of genetic divergence associated with domestication and breeding were widespread in the genome with several major diversity losses in the pericentromeric regions. A locus on chromosome 5B carries a gene encoding a metal transporter (TdHMA3-B1) with a non-functional variant causing high accumulation of cadmium in grain. The high-cadmium allele, widespread among durum cultivars but undetected in wild emmer accessions, increased in frequency from domesticated emmer to modern durum wheat. The rapid cloning of TdHMA3-B1 rescues a wild beneficial allele and demonstrates the practical use of the Svevo genome for wheat improvement.

Genetic markers associated to arbuscular mycorrhizal colonization in durum wheat.

In this work we investigated the variability and the genetic basis of susceptibility to arbuscular mycorrhizal (AM) colonization of wheat roots. The mycorrhizal status of wild, domesticated and cultivated tetraploid wheat accessions, inoculated with the AM species Funneliformis mosseae, was evaluated. In addition, to detect genetic markers in linkage with chromosome regions involved in AM root colonization, a genome wide association analysis was carried out on 108 durum wheat varieties and two AM fungal species (F. mosseae and Rhizoglomus irregulare). Our findings showed that a century of breeding on durum wheat and the introgression of Reduced height (Rht) genes associated with increased grain yields did not select against AM symbiosis in durum wheat. Seven putative Quantitative Trait Loci (QTLs) linked with durum wheat mycorrhizal susceptibility in both experiments, located on chromosomes 1A, 2B, 5A, 6A, 7A and 7B, were detected. The individual QTL effects (r2) ranged from 7 to 16%, suggesting a genetic basis for this trait. Marker functional analysis identified predicted proteins with potential roles in host-parasite interactions, degradation of cellular proteins, homeostasis regulation, plant growth and disease/defence. The results of this work emphasize the potential for further enhancement of root colonization exploiting the genetic variability present in wheat.

Mapping QTL for Root and Shoot Morphological Traits in a Durum Wheat × T. dicoccum Segregating Population at Seedling Stage.

A segregating population of 136 recombinant inbred lines derived from a cross between the durum wheat cv. "Simeto" and the T. dicoccum accession "Molise Colli" was grown in soil and evaluated for a number of shoot and root morphological traits. A total of 17 quantitative trait loci (QTL) were identified for shoot dry weight, number of culms, and plant height and for root dry weight, volume, length, surface area, and number of forks and tips, on chromosomes 1B, 2A, 3A, 4B, 5B, 6A, 6B, and 7B. LODs were 2.1 to 21.6, with percent of explained phenotypic variability between 0.07 and 52. Three QTL were mapped to chromosome 4B, one of which corresponds to the Rht-B1 locus and has a large impact on both shoot and root traits (LOD 21.6). Other QTL that have specific effects on root morphological traits were also identified. Moreover, meta-QTL analysis was performed to compare the QTL identified in the "Simeto" × "Molise Colli" segregating population with those described in previous studies in wheat, with three novel QTL defined. Due to the complexity of phenotyping for root traits, further studies will be helpful to validate these regions as targets for breeding programs for optimization of root function for field performance.