SNP Diversity and Genetic Structure of "Rogosija", an Old Western Balkan Durum Wheat Collection.

Durum wheat landraces represent a genetic resource for the identification and isolation of new valuable genes and alleles, useful to increase the crop adaptability to climate change. Several durum wheat landraces, all denominated "Rogosija", were extensively cultivated in the Western Balkan Peninsula until the first half of the 20th century. Within the conservation program of the Montenegro Plant Gene Bank, these landraces were collected, but without being characterized. The main goal of this study was to estimate the genetic diversity of the "Rogosija collection" consisting of 89 durum accessions, using 17 morphological descriptors and the 25K Illumina single nucleotide polymorphism (SNP) array. The genetic structure analysis of the Rogosija collection showed two distinguished clusters localized in two different Montenegro eco-geographic micro-areas, characterized by continental Mediterranean climate and maritime Mediterranean climate. Data suggest that these clusters could be composed of two different Balkan durum landrace collections evolved in two different eco-geographic micro-areas. Moreover, the origin of Balkan durum landraces is discussed.

A Survey of the Transcriptomic Resources in Durum Wheat: Stress Responses, Data Integration and Exploitation.

Durum wheat (Triticum turgidum subsp. durum (Desf.) Husn.) is an allotetraploid cereal crop of worldwide importance, given its use for making pasta, couscous, and bulgur. Under climate change scenarios, abiotic (e.g., high and low temperatures, salinity, drought) and biotic (mainly exemplified by fungal pathogens) stresses represent a significant limit for durum cultivation because they can severely affect yield and grain quality. The advent of next-generation sequencing technologies has brought a huge development in transcriptomic resources with many relevant datasets now available for durum wheat, at various anatomical levels, also focusing on phenological phases and environmental conditions. In this review, we cover all the transcriptomic resources generated on durum wheat to date and focus on the corresponding scientific insights gained into abiotic and biotic stress responses. We describe relevant databases, tools and approaches, including connections with other "omics" that could assist data integration for candidate gene discovery for bio-agronomical traits. The biological knowledge summarized here will ultimately help in accelerating durum wheat breeding.

Comparative Genetic Analysis of Durum Wheat Landraces and Cultivars Widespread in Tunisia.

The durum wheat (Triticum turgidum L. ssp. durum Desf.) landraces constitute a useful natural germplasm to increase the genetic diversity in the modern durum cultivars. The Tunisian durum germplasm constitutes 28 accessions conserved in Genebank of Tunisia, which are still unexplored. In this study, a comparative genetic analysis was performed to investigate the relationships between the Tunisian durum lines and the modern cultivars and detect divergent loci involved in breeding history. The genetic diversity analyses carried out using nine morphological descriptors and the 25K single-nucleotide polymorphism (SNP) array allowed us to distinguish two groups of Tunisian landraces and one of durum cultivars. The analysis of molecular variance and diversity indices confirmed the genetic variability among the groups. A total of 529 SNP loci were divergent between Tunisian durum landraces and modern cultivars. Candidate genes related to plant and spike architecture, including FLOWERING LOCUS T (FT-B1), zinc finger CONSTANS, and AP2/EREBPs transcription factors, were identified. In addition, divergent genes involved in grain composition and biotic stress nucleotide-binding site and leucine-reach repeats proteins and disease resistance proteins (NBS-LRR and RPM) were found, suggesting that the Tunisian durum germplasm may represent an important source of favorable alleles to be used in future durum breeding programs for developing well-adapted and resilient cultivars.

Polyphenol oxidase genes as integral part of the evolutionary history of domesticated tetraploid wheat.

Studying and understanding the genetic basis of polyphenol oxidases (PPO)-related traits plays a crucial role in genetic improvement of crops. A tetraploid wheat collection (T. turgidum ssp., TWC) was analyzed using the 90K wheat SNP iSelect assay and phenotyped for PPO activity. A total of 21,347 polymorphic SNPs were used to perform genome-wide association analysis (GWA) in TWC and durum wheat sub-groups, detecting 23 and 85 marker-trait associations (MTA). In addition, candidate genes responsible for PPO activity were predicted. Based on the 23 MTAs detected in TWC, two haplotypes associated with low and high PPO activity were identified. Four SNPs were developed and validated providing one reliable marker (IWB75732) for marker assisted selection. The 23 MTAs were used to evaluate the genetic divergence (FST > 0.25) between the T. turgidum subspecies, providing new information important for understanding the domestication process of Triticum turgidum ssp. and in particular of ssp. carthlicum.

Candidate Genes and Quantitative Trait Loci for Grain Yield and Seed Size in Durum Wheat.

Grain yield (YLD) is affected by thousand kernel weight (TKW) which reflects the combination of grain length (GL), grain width (GW) and grain area (AREA). Grain weight is also influenced by heading time (HT) and plant height (PH). To detect candidate genes and quantitative trait loci (QTL) of yield components, a durum wheat recombinant inbred line (RIL) population was evaluated in three field trials. The RIL was genotyped with a 90K single nucleotide polymorphism (SNP) array and a high-density genetic linkage map with 5134 markers was obtained. A total of 30 QTL were detected including 23 QTL grouped in clusters on 1B, 2A, 3A, 4B and 6B chromosomes. A QTL cluster on 2A chromosome included a major QTL for HT co-located with QTL for YLD, TKW, GL, GW and AREA, respectively. The photoperiod sensitivity (Ppd-A1) gene was found in the physical position of this cluster. Serine carboxypeptidase, Big grain 1 and ß-fructofuranosidase candidate genes were mapped in clusters containing QTL for seed size. This study showed that yield components and phenological traits had higher inheritances than grain yield, allowing an accurate QTL cluster detection. This was a requisite to physically map QTL on durum genome and to identify candidate genes affecting grain yield.

The Potential of Apulian Olive Biodiversity: The Case of Oliva Rossa Virgin Olive Oil.

In this study, the drupes and virgin olive oils extracted from the Oliva Rossa landrace are characterized. Oliva Rossa is an old landrace part of the autochthonous Apulian olive germplasm for which only few data have been reported till now. During the study, the maturity patterns of the drupes had been followed. Four samplings per year were planned, one every 14 days starting from the middle of October. The pigmentation index, the oil content and the total phenolic content of the drupes were measured. Simultaneously, virgin olive oils were extracted at the lab scale and analyzed for the fatty acid composition, the basic quality parameters and the content of minor compounds. The pigmentation pattern of the drupes was different among the years and, despite this trend, at the third sampling time the stage of maximum oil accumulation was always over. The extracted virgin olive oils had a medium to high level of oleic acid. With colder temperatures, a higher level of monounsaturated fatty acids, oleic/linoleic ratio and antioxidants was observed. The phenolic profile was dominated by 3,4-DPHEA-EDA and p-HPEA-EDA while the volatile profile by (E)-2-hexenal and 3-ethyl-1,5-octadiene.

Mapping Powdery Mildew (Blumeria graminis f. sp. tritici) Resistance in Wild and Cultivated Tetraploid Wheats.

Wheat is the most widely grown crop and represents the staple food for one third of the world's population. Wheat is attacked by a large variety of pathogens and the use of resistant cultivars is an effective and environmentally safe strategy for controlling diseases and eliminating the use of fungicides. In this study, a collection of wild and cultivated tetraploid wheats (Triticum turgidum) were evaluated for seedling resistance (SR) and adult plant resistance (APR) to powdery mildew (Blumeria graminis) and genotyped with a 90K single nucleotide polymorphism (SNP) array to identify new sources of resistance genes. The genome-wide association mapping detected 18 quantitative trait loci (QTL) for APR and 8 QTL for SR, four of which were identical or at least closely linked to four QTL for APR. Thirteen candidate genes, containing nucleotide binding sites and leucine-rich repeats, were localized in the confidence intervals of the QTL-tagging SNPs. The marker IWB6155, associated to QPm.mgb-1AS, was located within the gene TRITD1Av1G004560 coding for a disease resistance protein. While most of the identified QTL were described previously, five QTL for APR (QPm.mgb-1AS, QPm.mgb-2BS, QPm.mgb-3BL.1, QPm.mgb-4BL, QPm.mgb-7BS.1) and three QTL for SR (QPm.mgb-3BL.3, QPm.mgb-5AL.2, QPm.mgb-7BS.2) were mapped on chromosome regions where no resistance gene was reported before. The novel QTL/genes can contribute to enriching the resistance sources available to breeders.

Recovery, Assessment, and Molecular Characterization of Minor Olive Genotypes in Tunisia.

Olive is one of the oldest cultivated species in the Mediterranean Basin, including Tunisia, where it has a wide diversity, with more than 200 cultivars, of both wild and feral forms. Many minor cultivars are still present in marginal areas of Tunisia, where they are maintained by farmers in small local groves, but they are poorly characterized and evaluated. In order to recover this neglected germplasm, surveys were conducted in different areas, and 31 genotypes were collected, molecularly characterized with 12 nuclear microsatellite (simple sequence repeat (SSR)) markers, and compared with 26 reference cultivars present in the Tunisian National Olive collection. The analysis revealed an overall high genetic diversity of this olive's germplasm, but also discovered the presence of synonymies and homonymies among the commercialized varieties. The structure analysis showed the presence of different gene pools in the analyzed germplasm. In particular, the marginal germplasm from Ras Jbal and Azmour is characterized by gene pools not present in commercial (Nurseries) varieties, pointing out the very narrow genetic base of the commercialized olive material in Tunisia, and the need to broaden it to avoid the risk of genetic erosion of this species in this country.

Serendipitous In Situ Conservation of Faba Bean Landraces in Tunisia: A Case Study.

Cultivation of faba bean (Vicia faba L.) in Tunisia is largely based on improved varieties of the crop. However, a few farmers continue to produce local cultivars or landraces. The National Gene Bank of Tunisia (NGBT) recently launched a collection project for faba bean landraces, with special focus on the regions of the North West, traditionally devoted to cultivating grain legumes, and where around 80% of the total national faba bean cultivation area is located. The seed phenotypic features of the collected samples were studied, and the genetic diversity and population structure analyzed using simple sequence repeat markers. The genetic constitution of the present samples was compared to that of faba bean samples collected by teams of the International Center for Agricultural Research in the Dry Areas (ICARDA) in the 1970s in the same region, and stored at the ICARDA gene bank. The results of the diversity analysis demonstrate that the recently collected samples and those stored at ICARDA largely overlap, thus demonstrating that over the past 50 years, little genetic change has occurred to the local faba bean populations examined. These findings suggest that farmers serendipitously applied international best practices for in situ conservation of agricultural crops.

Genotyping by Sequencing of Cultivated Lentil (Lens culinaris Medik.) Highlights Population Structure in the Mediterranean Gene Pool Associated With Geographic Patterns and Phenotypic Variables.

Cultivated lentil (Lens culinaris Medik.) is one of the oldest domesticated crops and one of the most important grain legumes worldwide. The Mediterranean Basin holds large part of lentil biodiversity; however, no genetic structure was defined within the Mediterranean gene pool. In this study, we used high-throughput genotyping by sequencing to resolve the genetic structure of the Mediterranean ex situ lentil collection held at the Italian National Research Council. Sequencing of a 188-plex genotyping-by-sequencing library and bioinformatics treatment of data yielded 6,693 single nucleotide polymorphisms. Analysis of nonredundant genotypes with nonparametric and parametric methods highlighted the occurrence of five highly differentiated genetic clusters. Clustering could be related to geographic patterns and phenotypic traits, indicating that post-domestication routes introducing cultivation in Mediterranean countries and selection were major forces shaping lentil population structure. The estimation of the fixation index FST at individual single nucleotide polymorphism loci allowed the identification of distinctive alleles across clusters, suggesting the possibility to set up molecular keys for the assignment of lentil germplasm to specific genetic groups. Finally, significant associations between markers and phenotypic data were identified. Overall, the results of this study are of major importance for lentil conservation genetics and breeding and provide insights on the lentil evolutionary history.

Genetic Structure Analysis of a Collection of Tunisian Durum Wheat Germplasm.

The Tunisian durum wheat germplasm includes modern cultivars and traditional varieties that are still cultivated in areas where elite cultivars or intensive cultivation systems are not suitable. Within the frame of a collection program of the National Gene Bank of Tunisia (NGBT), durum wheat germplasm was collected from different Tunisian agro-ecological zones. The collected samples were studied using simple sequence repeats (SSRs) markers to explore the genetic diversity and evaluate the genetic structure in Tunisian germplasm. The results demonstrated significant diversity in the Tunisian durum wheat germplasm, with clear differentiation between traditional varieties and modern cultivars. The population structure analysis allowed the identification of five subpopulations, two of which appear to be more strongly represented in germplasm collected in central and southern Tunisia, where environmental conditions at critical development phases of the plant are harsher. Moreover these subpopulations are underrepresented in modern varieties, suggesting that traits of adaptation useful for breeding more resilient varieties might be present in central and southern germplasm. Moreover, our results will support, the activity of in situ on farm conservation of Tunisian durum wheat germplasm started by the National Gene Bank of Tunisia along with the ex situ approach.

GBS-derived SNP catalogue unveiled wide genetic variability and geographical relationships of Italian olive cultivars.

Information on the distribution of genetic variation is essential to preserve olive germplasm from erosion and to recover alleles lost through selective breeding. In addition, knowledge on population structure and genotype-phenotype associations is crucial to support modern olive breeding programs that must respond to new environmental conditions imposed by climate change and novel biotic/abiotic stressors. To further our understanding of genetic variation in the olive, we performed genotype-by-sequencing on a panel of 94 Italian olive cultivars. A reference-based and a reference-independent SNP calling pipeline generated 22,088 and 8,088 high-quality SNPs, respectively. Both datasets were used to model population structure via parametric and non parametric clustering. Although the two pipelines yielded a 3-fold difference in the number of SNPs, both described wide genetic variability among our study panel and allowed individuals to be grouped based on fruit weight and the geographical area of cultivation. Multidimensional scaling analysis on identity-by-state allele-sharing values as well as inference of population mixtures from genome-wide allele frequency data corroborated the clustering pattern we observed. These findings allowed us to formulate hypotheses about geographical relationships of Italian olive cultivars and to confirm known and uncover novel cases of synonymy.

Genetic dissection of the relationships between grain yield components by genome-wide association mapping in a collection of tetraploid wheats.

Increasing grain yield potential in wheat has been a major target of most breeding programs. Genetic advance has been frequently hindered by negative correlations among yield components that have been often observed in segregant populations and germplasm collections. A tetraploid wheat collection was evaluated in seven environments and genotyped with a 90K SNP assay to identify major and stable quantitative trait loci (QTL) for grain yield per spike (GYS), kernel number per spike (KNS) and thousand-kernel weight (TKW), and to analyse the genetic relationships between the yield components at QTL level. The genome-wide association analysis detected eight, eleven and ten QTL for KNS, TKW and GYS, respectively, significant in at least three environments or two environments and the mean across environments. Most of the QTL for TKW and KNS were found located in different marker intervals, indicating that they are genetically controlled independently by each other. Out of eight KNS QTL, three were associated to significant increases of GYS, while the increased grain number of five additional QTL was completely or partially compensated by decreases in grain weight, thus producing no or reduced effects on GYS. Similarly, four consistent and five suggestive TKW QTL resulted in visible increase of GYS, while seven additional QTL were associated to reduced effects in grain number and no effects on GYS. Our results showed that QTL analysis for detecting TKW or KNS alleles useful for improving grain yield potential should consider the pleiotropic effects of the QTL or the association to other QTLs.

Development of a High-Density SNP-Based Linkage Map and Detection of QTL for ß-Glucans, Protein Content, Grain Yield per Spike and Heading Time in Durum Wheat.

High-density genetic linkage maps of crop species are particularly useful in detecting qualitative and quantitative trait loci for important agronomic traits and in improving the power of classical approaches to identify candidate genes. The aim of this study was to develop a high-density genetic linkage map in a durum wheat recombinant inbred lines population (RIL) derived from two elite wheat cultivars and to identify, characterize and correlate Quantitative Trait Loci (QTL) for ß-glucan, protein content, grain yield per spike and heading time. A dense map constructed by genotyping the RIL population with the wheat 90K iSelect array included 5444 single nucleotide polymorphism (SNP) markers distributed in 36 linkage groups. Data for ß-glucan and protein content, grain yield per spike and heading time were obtained from replicated trials conducted at two locations in southern Italy. A total of 19 QTL were detected in different chromosome regions. In particular, three QTL for ß-glucan content were detected on chromosomes 2A and 2B (two loci); eight QTL controlling grain protein content were detected on chromosomes 1B, 2B, 3B (two loci), 4A, 5A, 7A and 7B; seven QTL for grain yield per spike were identified on chromosomes 1A, 2B, 3A (two loci), 3B (two loci) and 6B; and one marker-trait association was detected on chromosome 2A for heading time. The last was co-located with a ß-glucan QTL, and the two QTL appeared to be negatively correlated. A genome scan for genomic regions controlling the traits and SNP annotated sequences identified five putative candidate genes involved in different biosynthesis pathways (ß-glucosidase, GLU1a; APETALA2, TaAP2; gigantea3, TaGI3; 14-3-3 protein, Ta14A; and photoperiod sensitivity, Ppd-A1). This study provides additional information on QTL for important agronomic traits that could be useful for marker-assisted breeding to obtain new genotypes with commercial and nutritional relevance.

Polyphenol Oxidases in Crops: Biochemical, Physiological and Genetic Aspects.

Enzymatic browning is a colour reaction occurring in plants, including cereals, fruit and horticultural crops, due to oxidation during postharvest processing and storage. This has a negative impact on the colour, flavour, nutritional properties and shelf life of food products. Browning is usually caused by polyphenol oxidases (PPOs), following cell damage caused by senescence, wounding and the attack of pests and pathogens. Several studies indicated that PPOs play a role in plant immunity, and emerging evidence suggested that PPOs might also be involved in other physiological processes. Genomic investigations ultimately led to the isolation of PPO homologs in several crops, which will be possibly characterized at the functional level in the near future. Here, focusing on the botanic families of Poaceae and Solanaceae, we provide an overview on available scientific literature on PPOs, resulting in useful information on biochemical, physiological and genetic aspects.

The carotenoid biosynthetic and catabolic genes in wheat and their association with yellow pigments.

BACKGROUND: In plants carotenoids play an important role in the photosynthetic process and photo-oxidative protection, and are the substrate for the synthesis of abscisic acid and strigolactones. In addition to their protective role as antioxidants and precursors of vitamin A, in wheat carotenoids are important as they influence the colour (whiteness vs. yellowness) of the grain. Understanding the genetic basis of grain yellow pigments, and identifying associated markers provide the basis for improving wheat quality by molecular breeding. RESULTS: Twenty-four candidate genes involved in the biosynthesis and catabolism of carotenoid compounds have been identified in wheat by comparative genomics. Single nucleotide polymorphisms (SNPs) found in the coding sequences of 19 candidate genes allowed their chromosomal location and accurate map position on two reference consensus maps to be determined. The genome-wide association study based on genotyping a tetraploid wheat collection with 81,587 gene-associated SNPs validated quantitative trait loci (QTLs) previously detected in biparental populations and discovered new QTLs for grain colour-related traits. Ten carotenoid genes mapped in chromosome regions underlying pigment content QTLs indicating possible functional relationships between candidate genes and the trait. CONCLUSIONS: The availability of linked, candidate gene-based markers can facilitate breeding wheat cultivars with desirable levels of carotenoids. Identifying QTLs linked to carotenoid pigmentation can contribute to understanding genes underlying carotenoid accumulation in the wheat kernels. Together these outputs can be combined to exploit the genetic variability of colour-related traits for the nutritional and commercial improvement of wheat products.

Identification and Characterization of the Sucrose Synthase 2 Gene (Sus2) in Durum Wheat.

Sucrose transport is the central system for the allocation of carbon resources in vascular plants. Sucrose synthase (SUS), which reversibly catalyzes sucrose synthesis and cleavage, represents a key enzyme in the control of the flow of carbon into starch biosynthesis. In the present study the genomic identification and characterization of the Sus2-2A and Sus2-2B genes coding for SUS in durum wheat (cultivars Ciccio and Svevo) is reported. The genes were analyzed for their expression in different tissues and at different seed maturation stages, in four tetraploid wheat genotypes (Svevo, Ciccio, Primadur, and 5-BIL42). The activity of the encoded proteins was evaluated by specific activity assays on endosperm extracts and their structure established by modeling approaches. The combined results of sucrose synthase 2 expression and activity levels were then considered in the light of their possible involvement in starch yield.

Genetic Diversity and Population Structure of Tetraploid Wheats (Triticum turgidum L.) Estimated by SSR, DArT and Pedigree Data.

Levels of genetic diversity and population genetic structure of a collection of 230 accessions of seven tetraploid Triticum turgidum L. subspecies were investigated using six morphological, nine seed storage protein loci, 26 SSRs and 970 DArT markers. The genetic diversity of the morphological traits and seed storage proteins was always lower in the durum wheat compared to the wild and domesticated emmer. Using Bayesian clustering (K = 2), both of the sets of molecular markers distinguished the durum wheat cultivars from the other tetraploid subspecies, and two distinct subgroups were detected within the durum wheat subspecies, which is in agreement with their origin and year of release. The genetic diversity of morphological traits and seed storage proteins was always lower in the improved durum cultivars registered after 1990, than in the intermediate and older ones. This marked effect on diversity was not observed for molecular markers, where there was only a weak reduction. At K >2, the SSR markers showed a greater degree of resolution than for DArT, with their identification of a greater number of groups within each subspecies. Analysis of DArT marker differentiation between the wheat subspecies indicated outlier loci that are potentially linked to genes controlling some important agronomic traits. Among the 211 loci identified under selection, 109 markers were recently mapped, and some of these markers were clustered into specific regions on chromosome arms 2BL, 3BS and 4AL, where several genes/quantitative trait loci (QTLs) are involved in the domestication of tetraploid wheats, such as the tenacious glumes (Tg) and brittle rachis (Br) characteristics. On the basis of these results, it can be assumed that the population structure of the tetraploid wheat collection partially reflects the evolutionary history of Triticum turgidum L. subspecies and the genetic potential of landraces and wild accessions for the detection of unexplored alleles.

Fad7 gene identification and fatty acids phenotypic variation in an olive collection by EcoTILLING and sequencing approaches.

The ω-3 fatty acid desaturases (FADs) are enzymes responsible for catalyzing the conversion of linoleic acid to α-linolenic acid localized in the plastid or in the endoplasmic reticulum. In this research we report the genotypic and phenotypic variation of Italian Olea europaea L. germoplasm for the fatty acid composition. The phenotypic oil characterization was followed by the molecular analysis of the plastidial-type ω-3 FAD gene (fad7) (EC 1.14.19), whose full-length sequence has been here identified in cultivar Leccino. The gene consisted of 2635 bp with 8 exons and 5'- and 3'-UTRs of 336 and 282 bp respectively, and showed a high level of heterozygousity (1/110 bp). The natural allelic variation was investigated both by a LiCOR EcoTILLING assay and the PCR product direct sequencing. Only three haplotypes were identified among the 96 analysed cultivars, highlighting the strong degree of conservation of this gene.