Coping with salinity stress: segmental group 7 chromosome introgressions from halophytic Thinopyrum species greatly enhance tolerance of recipient durum wheat.

Increased soil salinization, tightly related to global warming and drought and exacerbated by intensified irrigation supply, implies highly detrimental effects on staple food crops such as wheat. The situation is particularly alarming for durum wheat (DW), better adapted to arid/semi-arid environments yet more sensitive to salt stress than bread wheat (BW). To enhance DW salinity tolerance, we resorted to chromosomally engineered materials with introgressions from allied halophytic Thinopyrum species. "Primary" recombinant lines (RLs), having portions of their 7AL arms distally replaced by 7el1L Th. ponticum segments, and "secondary" RLs, harboring Th. elongatum 7EL insertions "nested" into 7el1L segments, in addition to near-isogenic lines lacking any alien segment (CLs), cv. Om Rabia (OR) as salt tolerant control, and BW introgression lines with either most of 7el1 or the complete 7E chromosome substitution as additional CLs, were subjected to moderate (100 mM) and intense (200 mM) salt (NaCl) stress at early growth stages. The applied stress altered cell cycle progression, determining a general increase of cells in G1 and a reduction in S phase. Assessment of morpho-physiological and biochemical traits overall showed that the presence of Thinopyrum spp. segments was associated with considerably increased salinity tolerance versus its absence. For relative water content, Na+ accumulation and K+ retention in roots and leaves, oxidative stress indicators (malondialdehyde and hydrogen peroxide) and antioxidant enzyme activities, the observed differences between stressed and unstressed RLs versus CLs was of similar magnitude in "primary" and "secondary" types, suggesting that tolerance factors might reside in defined 7el1L shared portion(s). Nonetheless, the incremental contribution of 7EL segments emerged in various instances, greatly mitigating the effects of salt stress on root and leaf growth and on the quantity of photosynthetic pigments, boosting accumulation of compatible solutes and minimizing the decrease of a powerful antioxidant like ascorbate. The seemingly synergistic effect of 7el1L + 7EL segments/genes made "secondary" RLs able to often exceed cv. OR and equal or better perform than BW lines. Thus, transfer of a suite of genes from halophytic germplasm by use of fine chromosome engineering strategies may well be the way forward to enhance salinity tolerance of glycophytes, even the sensitive DW.

Relaunching a Traditional Durum Wheat Product: New Cultivars and Introgression Lines Identified for Frike Making in Turkey.

Frike is an ancient and traditional food product prepared from early harvested whole wheat grain, particularly durum wheat (DW). Due to its many health beneficial effects, frike is considered a functional food. It is also a lucrative commodity, produced in various West Asian and North African countries and typically in Southeastern Turkey. However, no systematic assessment of the most-suitable genotypes for frike production in the region is available. This study aimed to carry out such an evaluation, based on frike yield, quality traits, marketing price, and profitability, on a set of 20 DW cultivars and DW-Thinopyrum ponticum introgression lines (ILs). The results based on a field trial performed in Gölbasi (Adiyaman, Turkey) in the 2021-2022 season revealed the Turkish varieties Tüten-2002, Edessa, Artuklu, and Perre, together with the R5 IL to have the highest frike yields measured on 3 kg of roasted fresh spikes. The highest marketing prices were obtained by Turkish varieties Sariçanak-98, Burgos, Sümerli, and Artuklu, along with the R112 IL, excelling in quality traits. Considering all parameters, the Turkish cultivars Artuklu, Firat-93, and Sariçanak-98, besides the R112 IL, resulted in being the most-convenient genotypes for frike making, thus representing good candidates for maintaining cultural and genetic diversity in food production from a staple crop such as DW.

Untargeted Metabolomics Reveals a Multi-Faceted Resistance Response to Fusarium Head Blight Mediated by the Thinopyrum elongatum Fhb7E Locus Transferred via Chromosome Engineering into Wheat.

The Thinopyrum elongatum Fhb7E locus has been proven to confer outstanding resistance to Fusarium Head Blight (FHB) when transferred into wheat, minimizing yield loss and mycotoxin accumulation in grains. Despite their biological relevance and breeding implications, the molecular mechanisms underlying the resistant phenotype associated with Fhb7E have not been fully uncovered. To gain a broader understanding of processes involved in this complex plant-pathogen interaction, we analysed via untargeted metabolomics durum wheat (DW) rachises and grains upon spike inoculation with Fusarium graminearum (Fg) and water. The employment of DW near-isogenic recombinant lines carrying or lacking the Th. elongatum chromosome 7E region including Fhb7E on their 7AL arm, allowed clear-cut distinction between differentially accumulated disease-related metabolites. Besides confirming the rachis as key site of the main metabolic shift in plant response to FHB, and the upregulation of defence pathways (aromatic amino acid, phenylpropanoid, terpenoid) leading to antioxidants and lignin accumulation, novel insights were revealed. Fhb7E conferred constitutive and early-induced defence response, in which specific importance of polyamine biosynthesis, glutathione and vitamin B6 metabolisms, along with presence of multiple routes for deoxynivalenol detoxification, was highlighted. The results suggested Fhb7E to correspond to a compound locus, triggering a multi-faceted plant response to Fg, effectively limiting Fg growth and mycotoxin production.

The Response of Chromosomally Engineered Durum Wheat-Thinopyrum ponticum Recombinant Lines to the Application of Heat and Water-Deficit Stresses: Effects on Physiological, Biochemical and Yield-Related Traits.

Abiotic stress occurrence and magnitude are alarmingly intensifying worldwide. In the Mediterranean basin, heat waves and precipitation scarcity heavily affect major crops such as durum wheat (DW). In the search for tolerant genotypes, the identification of genes/QTL in wild wheat relatives, naturally adapted to harsh environments, represents a useful strategy. We tested three DW-Thinopyrum ponticum recombinant lines (R5+, R112+, R23+), their control sibs lacking any alien introgression, and the heat-tolerant cv. Margherita for their physiological, biochemical and yield response to heat stress (HS) application at anthesis, also in combination with water-deficit stress applied from booting until maturity. Under HS, R5+ and R112+ (23%- and 28%-long 7el1L Th. ponticum chromosome segment distally inserted on DW 7AL, respectively) showed remarkable stability of the yield-related traits; in turn, R23+ (40%-long 7el1L segment), despite a decreased grain yield, exhibited a greater spike fertility index and proline content in spike than its control sib. Under water-deficit + HS, R5+ showed the highest increment in water use efficiency and in flag leaf proline content, accompanied by the lowest yield penalty even vs. Margherita. This research confirms the value of harnessing wild gene pools to enhance DW stress tolerance and represents a starting point for elucidating the mechanisms of Thinopyrum spp. contribution to this relevant breeding target.

Small "Nested" Introgressions from Wild Thinopyrum Species, Conferring Effective Resistance to Fusarium Diseases, Positively Impact Durum Wheat Yield Potential.

Today wheat cultivation is facing rapidly changing climate scenarios and yield instability, aggravated by the spreading of severe diseases such as Fusarium head blight (FHB) and Fusarium crown rot (FCR). To obtain productive genotypes resilient to stress pressure, smart breeding approaches must be envisaged, including the exploitation of wild relatives. Here we report on the assessment of the breeding potential of six durum wheat-Thinopyrum spp. recombinant lines (RLs) obtained through chromosome engineering. They are characterized by having 23% or 28% of their 7AL chromosome arm replaced by a "nested" alien segment, composed of homoeologous group 7 chromosome fractions from Th. ponticum and Th. elongatum (=7el1L + 7EL) or from different Th. ponticum accessions (=7el1L + 7el2L). In addition to the 7el1L genes Lr19 + Yp (leaf rust resistance, and yellow pigment content, respectively), these recombinant lines (RLs) possess a highly effective QTL for resistance to FHB and FCR within their 7el2L or 7EL portion. The RLs, their null segregants and well-adapted and productive durum wheat cultivars were evaluated for 16 yield-related traits over two seasons under rainfed and irrigated conditions. The absence of yield penalties and excellent genetic stability of RLs was revealed in the presence of all the alien segment combinations. Both 7el2L and 7EL stacked introgressions had positive impacts on source and sink yield traits, as well as on the overall performance of RLs in conditions of reduced water availability. The four "nested" RLs tested in 2020 were among the top five yielders, overall representing good candidates to be employed in breeding programs to enhance crop security and safety.

Equipping Durum Wheat-Thinopyrum ponticum Recombinant Lines With a Thinopyrum elongatum Major QTL for Resistance to Fusarium Diseases Through a Cytogenetic Strategy.

Prompted by recent changes in climate trends, cropping areas, and management practices, Fusarium head blight (FHB), a threatening disease of cereals worldwide, is also spreading in unusual environments, where bread wheat (BW) and durum wheat (DW) are largely cultivated. The scarcity of efficient resistance sources within adapted germplasm is particularly alarming for DW, mainly utilized for human consumption, which is therefore at high risk of kernel contamination by health-dangerous mycotoxins (e.g., deoxynivalenol = DON). To cope with this scenario, we looked outside the wheat primary gene pool and recently transferred an exceptionally effective FHB resistance QTL (Fhb-7EL) from Thinopyrum elongatum 7EL chromosome arm onto a Thinopyrum ponticum 7el1L arm segment, containing additional valuable genes (including Lr19 for leaf rust resistance and Yp for yellow pigment content), distally inserted onto 7DL of BW lines. Two such lines were crossed with two previously developed DW-Th. ponticum recombinants, having 7el1L distal portions on 7AL arms. Genomic in situ hybridization (GISH) analysis showed homologous pairing, which is enabled by 7el1L segments common to the BW and DW recombinant chromosomes, to occur with 42-78% frequency, depending on the shared 7el1L amount. Aided by 7EL/7el1L-linked markers, 7EL+7el1L tetraploid recombinant types were isolated in BC1 progenies to DW of all cross combinations. Homozygous 7EL+7el1L recombinant plants and null segregates selected in BC2F2 progenies were challenged by Fusarium graminearum spike inoculation to verify the Fhb-7EL efficacy in DW. Infection outcomes confirmed previous observations in BW, with >90% reduction of disease severity associated with Fhb-7EL presence vs. its absence. The same differential effect was detected on seed set and weight of inoculated spikes, with genotypes lacking Fhb-7EL having ∼80% reduction compared with unaffected values of Fhb-7EL carriers. In parallel, DON content in flour extracts of resistant recombinants averaged 0.67 ppm, a value >800 times lower than that of susceptible controls. Furthermore, as observed in BW, the same Fhb-7EL also provided the novel DW recombinants with resistance to Fusarium crown rot (∼60% symptom reduction) as from seedling infection with Fusarium culmorum. Through alien segment stacking, we succeeded in equipping DW with a very effective barrier against different Fusarium diseases and other positive attributes for crop security and safety.

Cytogenetic mapping of a major locus for resistance to Fusarium head blight and crown rot of wheat on Thinopyrum elongatum 7EL and its pyramiding with valuable genes from a Th. ponticum homoeologous arm onto bread wheat 7DL.

KEY MESSAGE: A major locus for resistance to different Fusarium diseases was mapped to the most distal end of Th. elongatum 7EL and pyramided with Th. ponticum beneficial genes onto wheat 7DL. Perennial Triticeae species of the Thinopyrum genus are among the richest sources of valuable genes/QTL for wheat improvement. One notable and yet unexploited attribute is the exceptionally effective resistance to a major wheat disease worldwide, Fusarium head blight, associated with the long arm of Thinopyrum elongatum chromosome 7E (7EL). We targeted the transfer of the temporarily designated Fhb-7EL locus into bread wheat, pyramiding it with a Th. ponticum 7el1L segment stably inserted into the 7DL arm of wheat line T4. Desirable genes/QTL mapped along the T4 7el1L segment determine resistance to wheat rusts (Lr19, Sr25) and enhancement of yield-related traits. Mapping of the Fhb-7EL QTL, prerequisite for successful pyramiding, was established here on the basis of a bioassay with Fusarium graminearum of different 7EL-7el1L bread wheat recombinant lines. These were obtained without resorting to any genetic pairing promotion, but relying on the close 7EL-7el1L homoeology, resulting in 20% pairing frequency between the two arms. Fhb-7EL resided in the telomeric portion and resistant recombinants could be isolated with useful combinations of more proximally located 7el1L genes/QTL. The transferred Fhb-7EL locus was shown to reduce disease severity and fungal biomass in grains of infected recombinants by over 95%. The same Fhb-7EL was, for the first time, proved to be effective also against F. culmorum and F. pseudograminearum, predominant agents of crown rot. Prebreeding lines possessing a suitable 7EL-7el1L gene/QTL assembly showed very promising yield performance in preliminary field tests.

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.