Copy number variation at the HvCBF4–HvCBF2 genomic segment is a major component of frost resistance in barley

A family of CBF transcription factors plays a major role in reconfiguring the plant transcriptome in response to low-freezing temperature in temperate cereals. In barley, more than 13 HvCBF genes map coincident with the major QTL FR-H2 suggesting them as candidates to explain the function of the locus. Variation in copy number (CNV) of specific HvCBFs was assayed in a panel of 41 barley genotypes using RT-qPCR. Taking advantage of an accurate phenotyping that combined Fv/Fm and field survival, resistance-associated variants within FR-H2 were identified. Genotypes with an increased copy number of HvCBF4 and HvCBF2 (at least ten and eight copies, respectively) showed greater frost resistance. A CAPS marker able to distinguish the CBF2A, CBF2B and CBF2A/B forms was developed and showed that all the higher-ranking genotypes in term of resistance harbour only CBF2A, while other resistant winter genotypes harbour also CBF2B, although at a lower CNV. In addition to the major involvement of the HvCBF4-HvCBF2 genomic segment in the proximal cluster of CBF elements, a negative role of HvCBF3 in the distal cluster was identified. Multiple linear regression models taking into account allelic variation at FR-H1/VRN-H1 explained 0.434 and 0.550 (both at p < 0.001) of the phenotypic variation for Fv/Fm and field survival respectively, while no interaction effect between CNV at the HvCBFs and FR-H1/VRN-H1 was found. Altogether our data suggest a major involvement of the CBF genes located in the proximal cluster, with no apparent involvement of the central cluster contrary to what was reported for wheat

Determinants of barley grain yield in drought-prone Mediterranean environments

The determinants of barley grain yield in drought-prone Mediterranean environments have been studied in the Nure x Tremois (NT) population. A large set of yield and other morpho-physiological data were recorded in 118 doubled haploid (DH) lines of the population, in multi-environment field trials (18 site-year combination). Agrometeorological variables have been recorded and calculated at each site too. Four main periods of barley development were considered, vegetative, reproductive early and late grain filling phases, to dissect the effect on yield traits of the growth phases. Relationships between agrometeorological variables, grain yield (GY) and its main components (GN and GW) were also investigated by correlation. Results firstly gave a clear indication of the involvement of water consumption in determining GY and GW (r2=0.616, P=0.007 and r2=0.703, P=0.005, respectively) calculated from sowing to the early grain filling period, while GN showed its highest correlation with the total photothermal quotient (PQ) calculated for the same period (r2=0.646, P=0.013). With the only exception of total PQ calculated during the vegetative period, all significant correlations with GY were associated to water-dependent agrometeorological parameters. As a second result, the NT segregating population allowed us to weight the amount of interaction due to genotypes over environments or to environments in relation to genotypes by a GGE analysis; 47.67% of G+GE sum of squares was explained by the first two principal components. Then, the introduction of genomic information at major barley genes regulating the length of growth cycle allowed us to explain patterns of adaptation of different groups of NT lines according to the variants (alleles) harbored at venalization (Vrn-H1) in combination with earliness (Eam6) genes. The superiority of the lines carrying the Nure allele at Eam6 was confirmed by factorial ANOVA testing the four possible haplotypes obtained combining alternative alleles at Eam6 and Vrn-H1. Maximum yield potential and differentials among the NT genotypes was finally explored through Finlay-Wilkinson model to interpret grain yield of NT genotypes together with yield adaptability (Ya), as the regression coefficient bi; Ya ranged from 0.71 for NT77 to 1.20 for NT19. Lines simply harboring the Nure variants at the two genes behaved as highest yielding (3.04 t ha\u20131), and showed the highest yield adaptability (bi=1.05). The present study constitutes a starting point towards the introduction of genomic variables in agronomic models for barley grain yield in Mediterranean environments

Elevated CO2has concurrent effects on leaf and grain metabolism but minimal effects on yield in wheat

While the general effect of CO2 enrichment on photosynthesis, stomatal conductance, N content, and yield has been documented, there is still some uncertainty as to whether there are interactive effects between CO2 enrichment and other factors, such as temperature, geographical location, water availability, and cultivar. In addition, the metabolic coordination between leaves and grains, which is crucial for crop responsiveness to elevated CO2, has never been examined closely. Here, we address these two aspects by multi-level analyses of data from several free-air CO2 enrichment experiments conducted in five different countries. There was little effect of elevated CO2 on yield (except in the USA), likely due to photosynthetic capacity acclimation, as reflected by protein profiles. In addition, there was a significant decrease in leaf amino acids (threonine) and macroelements (e.g. K) at elevated CO2, while other elements, such as Mg or S, increased. Despite the non-significant effect of CO2 enrichment on yield, grains appeared to be significantly depleted in N (as expected), but also in threonine, the S-containing amino acid methionine, and Mg. Overall, our results suggest a strong detrimental effect of CO2 enrichment on nutrient availability and remobilization from leaves to grains.This work was supported by the Department of Industry, Energy and Innovation of the Government of Navarre (PI040 TRIGOCLIM). The technical support given by Inés Urretavizcaya, Petra Högy, and Jürgen Franzaring in harvesting and sample management is acknowledged. JC was supported by an Australia Awards PhD Scholarship. GT was supported by a Connect Talent Award from the Region Pays de la Loire – Angers Loire Metropole (France). Research at the Australian Grains Free Air CO2 Enrichment (AGFACE) facility was jointly run by the University of Melbourne and Agriculture Victoria with funding from the Grains Research and Development Corporation (under contract no. DAV00137) and the Australian Commonwealth Department of Agriculture and Water Resources (under contract no. FtRG 1193982-41). CAAS-FACE was supported by the National Key Research and Development Project (under contracts 2016YFD0300401 and 2019YFA0607403). The FACE experiment in Italy was supported by the AGER project ‘Durum wheat adaptation to global change: effect of elevated CO2 on yield and quality traits’ and by the collaboration CREA-CNR. Finally, the authors also acknowledge support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI)

Elevated CO2 has concurrent effects on leaf and grain metabolism but minimal effects on yield in wheat

While the general effect of CO2 enrichment on photosynthesis, stomatal conductance, N content, and yield has been documented, there is still some uncertainty as to whether there are interactive effects between CO2 enrichment and other factors, such as temperature, geographical location, water availability, and cultivar. In addition, the metabolic coordination between leaves and grains, which is crucial for crop responsiveness to elevated CO2, has never been examined closely. Here, we address these two aspects by multi-level analyses of data from several free-air CO2 enrichment experiments conducted in five different countries. There was little effect of elevated CO2 on yield (except in the USA), likely due to photosynthetic capacity acclimation, as reflected by protein profiles. In addition, there was a significant decrease in leaf amino acids (threonine) and macroelements (e.g. K) at elevated CO2, while other elements, such as Mg or S, increased. Despite the non-significant effect of CO2 enrichment on yield, grains appeared to be significantly depleted in N (as expected), but also in threonine, the S-containing amino acid methionine, and Mg. Overall, our results suggest a strong detrimental effect of CO2 enrichment on nutrient availability and remobilization from leaves to grains.This work was supported by the Department of Industry, Energy and Innovation of the Government of Navarre (PI040 TRIGOCLIM). The technical support given by Inés Urretavizcaya, Petra Högy, and Jürgen Franzaring in harvesting and sample management is acknowledged. JC was supported by an Australia Awards PhD Scholarship. GT was supported by a Connect Talent Award from the Region Pays de la Loire – Angers Loire Metropole (France). Research at the Australian Grains Free Air CO2 Enrichment (AGFACE) facility was jointly run by the University of Melbourne and Agriculture Victoria with funding from the Grains Research and Development Corporation (under contract no. DAV00137) and the Australian Commonwealth Department of Agriculture and Water Resources (under contract no. FtRG 1193982-41). CAAS-FACE was supported by the National Key Research and Development Project (under contracts 2016YFD0300401 and 2019YFA0607403). The FACE experiment in Italy was supported by the AGER project ‘Durum wheat adaptation to global change: effect of elevated CO2 on yield and quality traits’ and by the collaboration CREA-CNR. Finally, the authors also acknowledge support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI

Genetic variants of HvCbf14 are statistically associated with frost tolerance in a European germplasm collection of Hordeum vulgare

Two quantitative trait loci (Fr-H1 and Fr-H2) for frost tolerance (FT) have been discovered on the long arm of chromosome 5H in barley. Two tightly linked groups of CBF genes, known to play a key role in the FT regulatory network in A. thaliana, have been found to co-segregate with Fr-H2. Here, we investigate the allelic variations of four barley CBF genes (HvCbf3, HvCbf6, HvCbf9 and HvCbf14) in a panel of European cultivars, landraces and H. spontaneum accessions. In the cultivars a reduction of nucleotide and haplotype diversities in CBFs compared with the landraces and the wild ancestor H. spontaneum, was evident. In particular, in cultivars the loss of HvCbf9 genetic variants was higher compared to other sequences. In order to verify if the pattern of CBF genetic variants correlated with the level of FT, an association procedure was adopted. The pairwise analysis of linkage disequilibrium (LD) among the genetic variants in four CBF genes was computed to evaluate the resolution of the association procedure. The pairwise plotting revealed a low level of LD in cultivated varieties, despite the tight physical linkage of CBF genes analysed. A structured association procedure based on a general liner model was implemented, including the variants in CBFs, of Vrn-H1, and of two reference genes not involved in FT (α-Amy1 and Gapdh) and considering the phenotypic data for FT. Association analysis recovered two nucleotide variants of HvCbf14 and one nucleotide variant of Vrn-H1 as statistically associated to FT

Genetic diversity in Mediterranean Brassica vegetables: seed phenotyping could be useful for sustainable crop production

The European BrasExplor project aims to explore the genetic diversity present in two economically important Brassica crop species, Brassica oleracea and B. rapa, for sustainable crop production. This diversity is present in wild populations but also in cultivated landraces and has been shaped by contrasting environments. An international consortium of 11 partners has begun to collect and multiply wild populations extending from the French North Atlantic coast to the southern Algerian desert as well as local cultivars from 6 contributing countries in order to characterize the genetic diversity available over a wide soil-climate gradient. A total of 100 populations has been obtained for each species. Identifying the genetic variation and understanding the basis for it will allow the development of breeding strategies for a better adaptation of turnip (Brassica rapa) and cabbage (Brassica oleracea var. capitata) to climate change. One third of the collection has been already phenotyped for its germination traits of native seeds harvested in 2020 for wild populations or of local landraces provided by farmers and seed banks. In favourable conditions, a high diversity in germination capacity and germination rate was observed independently of seed age. The two species have a different germination profile: some turnip seeds can stand higher temperature and lower water potential than most cabbage seeds. Variation in flowering time has also been documented in these plants.We are grateful to Benjamin Foltran for his contribution to the project during his internship, Anne-Sophie Grenier for her input in communication and project website. We also thank Hakima Arrar, Fariza Boussad, Thouraya Rhim, Ilahy Riadh who are involved in field phenotyping. BrasExplor project is funded by the PRIMA programme supported under Horizon ? ? ? ? the European Union’s framework programme for research and innovation.Peer reviewe

A Combined Field/Laboratory Method for Assessment of Frost Tolerance with Freezing Tests and Chlorophyll Fluorescence

Recent progress in genotyping allows for studies of the molecular genetic basis of cold resistance in cereals. However, as in many other fields of molecular genetic analysis, phenotyping for high numbers of genotypes is still a major bottleneck. The use of chlorophyll fluorescence measurements as an indicator for freezing stress is a well established and rapid method for evaluation of frost tolerance. In order to extend the applicability of this technique beyond plants grown under controlled conditions in growth chambers and sacrificed for the test, here we study its applicability for leaves harvested from field trials during winter and subjected to freezing tests. Such an approach allows for simultaneous studies of the advancement of cold hardening and other components of winter survival apart from frost tolerance. It is shown that cutting or senescence of cut leaves does not have adverse effects on the outcome of subsequent freezing stress tests. The time requirements for field sampling and laboratory testing on high numbers of genotypes allow for the application of the proposed approach for genotyping/phenotyping studies

Evaluation of frost resistance in oat

Resistance to biotic and abiotic stress is a crucial aspect for cultivar adaptation to agriculture environments. Among winter cereals, oat is the most frost sensitive and its insufficient level of winterhardiness is the main factor limiting sowing of winter oat in Northern Italy and Central Europe. Frost resistance, the most important component of winterhardiness, is an inducible process promoted by cold exposure and associated with a number of biochemical and molecular changes. We employed field tests and physiological parameters in a set of cultivars released during the last 40 years to explore the genetic variability in frost resistance and identify resistant genotypes. Plants were hardened at +3/+1°C and exposed to freezing temperatures. The efficiency of the excitation capture by open Photosystyem II (PSII) reaction centres was evaluated after freezing, through the chlorophyll fluorescence parameter Fv/Fm, according to Rizza et al. (2001). Different hardening length and pre-hardening temperature (+12/+7°C) were also employed to investigate if a higher level of resistance is associated with the ability to induce early hardening. This capacity is of advantage under field conditions, where temperature falls progressively, because it enables resistant plants to prepare for cold before the susceptible ones. Some significant molecular traits associated to frost resistance such as the accumulation of cor14 (Cattivelli et al., 1995) were also investigated on selected cultivars. Fv/Fm analysis appears as an attractive test being rapid, non invasive and able to monitor a trait related to a crucial mechanism of the resistance acquisition. Significant differences in frost resistance were detected within winter cultivars. The most resistant showed a behaviour similar to that of winter barley, suggesting that it should be possible to improve the resistance of winter oat to a level similar to that of winter barley. Rizza, F. Pagani, D. Cattivelli, C. Stanca, A.M. 2001. Plant Breeding 120: 389-396; Cattivelli, L. Crosatti, C. Rizza, F. 1995 Journal of Genetics & Breeding 49:333-338.vokMyynti MTT tietopalvelu

Mendelizing Barley VRN-H1/FR-H1 and FR-H2 Quantitative Trait Loci in alternative backgrounds

Freezing temperatures is one of the major limitations among the abiotic stress in cereals. In Triticeae tribe, several genetic studies have revealed the phenotypic variation for frost tolerance (FT) is attributed to two QTLs located in the long arm of homoeologous group 5 chromosome: Frost Resistance 1 (FR-1) and Frost Resistance 2 (FR-2). Besides these QTLs, the genotype background might also play a role in the phenotype. Considering barley as a model specie for the Triticeae tribe, in order to study the impact of the FR-H1 (VRN-H1), FR-H2 (cluster of 13 CBFs) locus and the genotype background, a series of experiments in controlled environment (Votsch chamber) has been carried out to test the freezing resistance of the QTL-NILs (Nure or Tremois x DH/RIL), Nure (winter genotype, resistance FT) and Tremois (spring genotype, susceptible FT). Temperature stress ranges from -10°C to -12°C. Plants at seedling stage (1-2 leaves) were firstly acclimated at 3/1°C (200 uE, 8/16H light/dark regime) for 4 weeks. Frost resistance score was measured using chlorophyll fluorescence analysis (Fv/Fm parameter). A marker-assisted backcross scheme was used to develop four QTL-Near Isogenic Lines aiming at separating FR-H1 and FR-H2 genomic regions in different alternative backgrounds. PCR-based molecular markers for HvCBF3 (Fr-H2) and HvBM5 (Vrn-H1/Fr-H1) were used to select plant with the desired allelic state, and after at least BC4 generation, heterozygous plants multiple selfing generations were obtained. At early selfing generations, the genotypic status of the lines was tested using 19 polymorphic SSR markers scattered throughout the barley genome. Moreover, a Cod42 Nu-Fr-H2 in Tremois background and Cod44 Tr-Fr-H2 in Nure were genotyped with barley 50K chip (Trait genetics). The result of the freezing tests showed that putatively the Fr-H2 (CBF cluster) appears to have a greater effect compared to FR-H1 (VRN-H1). Moreover, the data showed a difference between Nure and the QTL-NILs with Nure background with at least one allele from the spring Tremois. It can be hypothesized that the spring allelic state at the locus negatively affected the phenotype even in a winter background. All the treatments showed that the role of the background is secondary compared to the two FR locus. To confirm these hypothesises, gene expression analysis will be carried out to evaluate VRN-H1, the impact of the whole cluster and each CBF gene on the FT phenotype. Additionally, further investigation will be assessed on open field trials to evaluate recombinants genotype coming from the cross between Nure (winter genotype, resistance FT) and Pamina (facultative genotype, highly resistance FT)