Recombination in the wheat stem rust pathogen mediated by an indigenous barberry species in Spain.

The comeback of wheat stem rust in Europe, caused by Puccinia graminis f. sp. tritici, and the prevalence of the alternate (sexual) host in local areas have recently regained attention as a potential threat to European wheat production. The aim of this study was to investigate a potential epidemiological link between the aecia found on an indigenous barberry species and stem rust infections on nearby cereals and grasses. Aecial infections collected from Berberis vulgaris subsp. seroi were inoculated on a panel of susceptible genotypes of major cereal crop species. In total, 67 stem rust progeny isolates were recovered from wheat (51), barley (7), and rye (9), but none from oat, indicating the potential of barberry derived isolates to infect multiple cereals. Molecular genotyping of the progeny isolates and 20 cereal and grass stem rust samples collected at the same locations and year, revealed a clear genetic relatedness between the progeny isolated from barberry and the stem rust infections found on nearby cereal and grass hosts. Analysis of Molecular Variance indicated that variation between the stem rust populations accounted for only 1%. A Principal Components Analysis using the 62 detected multilocus genotypes also demonstrated a low degree of genetic variation among isolates belonging to the two stem rust populations. Lastly, pairwise comparisons based on fixation index (Fst), Nei's genetic distances and number of effective migrants (Nm) revealed low genetic differentiation and high genetic exchange between the two populations. Our results demonstrated a direct epidemiological link and functionality of an indigenous barberry species as the sexual host of P. graminis in Spain, a factor that should be considered when designing future strategies to prevent stem rust in Europe and beyond.

Barberry plays an active role as an alternate host of Puccinia graminis in Spain.

Stem rust, caused by Puccinia graminis, is a destructive group of diseases. The pathogen uses Berberis species as alternate hosts to complete its life cycle. B. vulgaris and the endemic species B. hispanica and B. garciae are present in Spain. The objective of this study was to investigate the functionality of the indigenous barberry as alternate hosts. Field surveys were conducted in 2018 and 2019 in Huesca, Teruel and Albacete provinces of Spain. Aecial samples on barberry were analysed via infection assays and DNA analysis. B. garciae was predominant in Huesca and Teruel provinces, often found in the field margins of cereal crops. Aecial infections on B. garciae were observed in May and uredinial infections on cereal crops in June. Scattered B. hispanica bushes were occasionally found near cereal crops in Albacete, where aecial infections on B. hispanica were observed in June when most cereal crops were mature. Infection assays using aeciospores resulted in stem rust infections on susceptible genotypes of wheat, barley, rye and oat, indicating the presence of the sexual cycle for P. graminis f. sp. tritici, f. sp. secalis and f. sp. avenae. Sequence analyses from aecial samples supported this finding as well as the presence of Puccinia brachypodii. This study provides the first evidence that indigenous Berberis species play an active role in the sexual cycle of P. graminis under natural conditions in Spain.

Wheat Stem Rust Back in Europe: Diversity, Prevalence and Impact on Host Resistance.

The objective of this study was to investigate the re-emergence of a previously important crop pathogen in Europe, Puccinia graminis f.sp. tritici, causing wheat stem rust. The pathogen has been insignificant in Europe for more than 60 years, but since 2016 it has caused epidemics on both durum wheat and bread wheat in local areas in southern Europe, and additional outbreaks in Central- and West Europe. The prevalence of three distinct genotypes/races in many areas, Clade III-B (TTRTF), Clade IV-B (TKTTF) and Clade IV-F (TKKTF), suggested clonal reproduction and evolution by mutation within these. None of these genetic groups and races, which likely originated from exotic incursions, were detected in Europe prior to 2016. A fourth genetic group, Clade VIII, detected in Germany (2013), was observed in several years in Central- and East Europe. Tests of representative European wheat varieties with prevalent races revealed high level of susceptibility. In contrast, high diversity with respect to virulence and Simple Sequence Repeat (SSR) markers were detected in local populations on cereals and grasses in proximity to Berberis species in Spain and Sweden, indicating that the alternate host may return as functional component of the epidemiology of wheat stem rust in Europe. A geographically distant population from Omsk and Novosibirsk in western Siberia (Russia) also revealed high genetic diversity, but clearly different from current European populations. The presence of Sr31-virulence in multiple and highly diverse races in local populations in Spain and Siberia stress that virulence may emerge independently when large geographical areas and time spans are considered and that Sr31-virulence is not unique to Ug99. All isolates of the Spanish populations, collected from wheat, rye and grass species, were succesfully recovered on wheat, which underline the plasticity of host barriers within P. graminis. The study demonstrated successful alignment of two genotyping approaches and race phenotyping methodologies employed by different laboratories, which also allowed us to line up with previous European and international studies of wheat stem rust. Our results suggest new initiatives within disease surveillance, epidemiological research and resistance breeding to meet current and future challenges by wheat stem rust in Europe and beyond.

Agronomic, Physiological and Genetic Changes Associated With Evolution, Migration and Modern Breeding in Durum Wheat.

A panel of 172 Mediterranean durum wheat landraces and 200 modern cultivars was phenotyped during three years for 21 agronomic and physiological traits and genotyped with 46,161 DArTseq markers. Modern cultivars showed greater yield, number of grains per spike (NGS) and harvest index (HI), but similar number of spikes per unit area (NS) and grain weight than the landraces. Modern cultivars had earlier heading but longer heading-anthesis and grain-filling periods than the landraces. They had greater RUE (Radiation Use Efficiency) up to anthesis and lower canopy temperature at anthesis than the landraces, but the opposite was true during the grain-filling period. Landraces produced more biomass at both anthesis and maturity. The 120 genotypes with a membership coefficient q > 0.8 to the five genetic subpopulations (SP) that structured the panel were related with the geographic distribution and evolutionary history of durum wheat. SP1 included landraces from eastern countries, the domestication region of the "Fertile Crescent." SP2 and SP3 consisted of landraces from the north and the south Mediterranean shores, where durum wheat spread during its migration westward. Decreases in NS, grain-filling duration and HI, but increases in early soil coverage, days to heading, biomass at anthesis, grain-filling rate, plant height and peduncle length occurred during this migration. SP4 grouped modern cultivars gathering the CIMMYT/ICARDA genetic background, and SP5 contained modern north-American cultivars. SP4 was agronomically distant from the landraces, but SP5 was genetically and agronomically close to SP1. GWAS identified 2,046 marker-trait associations (MTA) and 144 QTL hotspots integrating 1,927 MTAs. Thirty-nine haplotype blocks (HB) with allelic differences among SPs and associated with 16 agronomic traits were identified within 13 QTL hotspots. Alleles in chromosomes 5A and 7A detected in landraces were associated with decreased yield. The late heading and short grain-filling period of SP2 and SP3 were associated with a hotspot on chromosome 7B. The heavy grains of SP3 were associated with hotspots on chromosomes 2A and 7A. The greater NGS and HI of modern cultivars were associated with allelic variants on chromosome 7A. A hotspot on chromosome 3A was associated with the high NGS, earliness and short stature of SP4.

The Effect of Photoperiod Genes and Flowering Time on Yield and Yield Stability in Durum Wheat.

This study analysed the effect of flowering time as influenced by photoperiod sensitivity genes on yield and yield stability in durum wheat. Twenty-three spring genotypes harbouring different allele combinations at Ppd-A1 and Ppd-B1 were grown in 15 field experiments at three sites at latitudes from 41° to 19° N (Spain, Mexico-North and Mexico-South). Low temperature and solar radiation before flowering and long day length during grain-filling characteristic for the Spanish site resulted in high grain number/m2 (GN) and yield (GY), while a moderate GN combined with high solar radiation during grain-filling at Mexico-North led to heavier grains. Allele combination GS100-Ppd-A1a/Ppd-B1a reduced the flowering time up to nine days when compared with Ppd-A1b/Ppd-B1a. Differences in flowering time caused by Ppd-A1/Ppd-B1 allele combinations did not affect yield. Combinations GS105-Ppd-A1a/Ppd-B1b and Ppd-A1b/Ppd-B1b resulted in the highest GN, linked to spikelets/spike, which was higher in GS105-Ppd-A1a/Ppd-B1b due to more grains/spikelet. Flowering time caused by Eps had a minor effect on GN, spikes/m2 and grains/spike, but late flowering resulted in reduced grain weight and GY. Allele combinations harbouring alleles conferring a similar photoperiod sensitivity response at Ppd-A1 and Ppd-B1 resulted in greater yield stability than combinations that carry alleles conferring a different response. Allele combination GS100-Ppd-A1a/Ppd-B1a was the most suitable in terms of yield and yield stability of durum wheat cultivated under irrigation within the studied latitudes.

Allelic Variation at the Vernalization Response (Vrn-1) and Photoperiod Sensitivity (Ppd-1) Genes and Their Association With the Development of Durum Wheat Landraces and Modern Cultivars.

Wheat adaptability to a wide range of environmental conditions is mostly determined by allelic diversity within genes controlling vernalization requirement (Vrn-1) and photoperiod sensitivity (Ppd-1). We characterized a panel of 151 durum wheat Mediterranean landraces and 20 representative locally adapted modern cultivars for their allelic composition at Vrn-1 and Ppd-1 gene using diagnostic molecular markers and studied their association with the time needed to reach six growth stages under field conditions over 6 years. Compared with the more diverse and representative landrace collection, the set of modern cultivars were characterized by a reduction of 50% in the number of allelic variants at the Vrn-A1 and Vrn-B1 genes, and the high frequency of mutant alleles conferring photoperiod insensitivity at Ppd-A1, which resulted on a shorter cycle length. Vrn-A1 played a greater role than Vrn-B1 in regulating crop development (Vrn-A1 > Vrn-B1). The results suggest that mutations in the Vrn-A1 gene may have been the most important in establishing the spring growth habit of Mediterranean landraces and modern durum cultivars. The allele Vrn-A1d, found in 10 landraces, delayed development. The relative effects of single Vrn-A1 alleles on delaying the development of the landraces were vrn-A1 = Vrn-A1d > Vrn-A1b > Vrn-A1c. Allele vrn-B1 was present in all except two landraces and in all modern cultivars. The null allele at Ppd-A1 (a deletion first observed in the French bread wheat cultivar 'Capelle-Desprez') was found for the first time in durum wheat in the present study that identified it in 30 landraces from 13 Mediterranean countries. Allele Ppd-A1a (GS105) was detected in both germplasm types, while the allele Ppd-A1a (GS100) was found only in modern North American and Spanish cultivars. The relative effect of single Ppd-A1 alleles on extending phenological development was Ppd-A1(DelCD) > Ppd-A1b > Ppd-A1a (GS105) > Ppd-A1a (GS100). Sixteen Vrn-1+Ppd-1 allelic combinations were found in landraces and six in modern cultivars, but only three were common to both panels. Differences in the number of days to reach anthesis were 10 days in landraces and 3 days in modern cultivars. Interactive effects between Vrn-1 and Ppd-1 genes were detected.

Phytoene synthase 1 (Psy-1) and lipoxygenase 1 (Lpx-1) Genes Influence on Semolina Yellowness in Wheat Mediterranean Germplasm.

Phytoene synthase 1 (Psy1) and lipoxygenase 1 (Lpx-1) are key genes involved in the synthesis and catalysis of carotenoid pigments in durum wheat, regulating the increase and decrease in these compounds, respectively, resulting in the distinct yellow color of semolina and pasta. Here, we reported new haplotype variants and/or allele combinations of these two genes significantly affecting yellow pigment content in grain and semolina through their effect on carotenoid pigments. To reach the purpose of this work, three complementary approaches were undertaken: the identification of QTLs associated to carotenoid content on a recombinant inbred line (RIL) population, the characterization of a Mediterranean panel of accessions for Psy1 and Lpx-1 genes, and monitoring the expression of Psy1 and Lpx-1 genes during grain filling on two genotypes with contrasting yellow pigments. Our data suggest that Psy1 plays a major role during grain development, contributing to semolina yellowness, and Lpx-1 appears to be more predominant at post-harvest stages and during pasta making.

Effect of allele combinations at Ppd-1 loci on durum wheat grain filling at contrasting latitudes.

Flowering time is the most critical developmental stage in wheat, as it determines environmental conditions during grain filling. Thirty-five spring durum genotypes carrying all known allele variants at Ppd-1 loci were evaluated in fully irrigated field experiments for three years at latitudes of 41°N (Spain), 27°N (northern Mexico) and 19°N (southern Mexico). Relationships between weight of central grains of main spikes (W) and thermal time from flowering to maturity were described by a logistic equation. Differences in flowering time between the allele combination causing the earliest (GS100/Ppd-B1a) and the latest (Ppd-A1b/Ppd-B1a) flowering were 7, 20 and 18 days in Spain, northern Mexico and southern Mexico, respectively. Flowering delay drastically reduced the mean grain filling rate (R) and W at all sites. At autumn-sowing sites, an increase of 1°C in mean temperature during the first half of the grain filling period decreased W by 5.2 mg per grain. At these sites, W was strongly dependent on R. At the spring-sowing site (southern Mexico), W depended on both R and grain filling duration. Our results suggest that incorporating the allele combinations GS100/Ppd-B1a and GS105/Ppd-B1a (alleles conferring photoperiod insensitivity) in newly released varieties can reduce the negative effects of climate change on grain filling at the studied latitudes.

Pasta-Making Quality QTLome From Mediterranean Durum Wheat Landraces.

In order to identify genome regions related to pasta-making quality traits, association mapping (AM) was performed in a set of 165 durum wheat landraces from 21 Mediterranean countries. The collection was genotyped using 1149 DArT markers and 872 of them with a known genetic position were used for AM. The collection was grown in north-east Spain during 3 years. Results of ANOVA showed that trait variation for quality traits, except for grain protein content (GPC), was mainly explained by genetic effects. Landraces showed higher GPC than modern cultivars but lower gluten strength (GS). Modern and eastern landraces showed the highest yellow color index (YI). Balkan landraces showed the lowest test weight (TW). A total of 92 marker-trait associations were detected, 20 corresponding to GS, 21 to GPC, 21 to YI and 30 to TW. With the aim of detecting new genomic regions involved in grain quality, the position of the associations was compared with previously mapped QTL by a meta-QTL analysis. A total of 249 QTLs were projected onto the same map used for AM, identifying 45 meta-QTL (MQTL) regions and the remaining 15 QTLs as singletons. The position of known genes involved in grain quality was also included, and gene annotation within the most significant regions detected by AM was carried out using the wheat genome sequence.

Effect of Ppd-A1 and Ppd-B1 Allelic Variants on Grain Number and Thousand Kernel Weight of Durum Wheat and Their Impact on Final Grain Yield.

The main yield components in durum wheat are grain number per unit area (GN) and thousand kernel weight (TKW), both of which are affected by environmental conditions. The most critical developmental stage for their determination is flowering time, which partly depends on photoperiod sensitivity genes at Ppd-1 loci. Fifteen field experiments, involving 23 spring durum wheat genotypes containing all known allelic variants at the PHOTOPERIOD RESPONSE LOCUS (Ppd-A1 and Ppd-B1) were carried out at three sites at latitudes ranging from 41° to 27° N (Spain, Mexico-north, and Mexico-south, the latter in spring planting). Allele GS100 at Ppd-A1, which causes photoperiod insensitivity and results in early-flowering genotypes, tended to increase TKW and yield, albeit not substantially. Allele Ppd-B1a, also causing photoperiod insensitivity, did not affect flowering time or grain yield. Genotypes carrying the Ppd-B1b allele conferring photoperiod sensitivity had consistently higher GN, which did not translate into higher yield due to under-compensation in TKW. This increased GN was due to a greater number of grains spike-1 as a result of a higher number of spikelets spike-1. Daylength from double ridge to terminal spikelet stage was strongly and positively associated with the number of spikelets spike-1 in Spain. This association was not found in the Mexico sites, thereby indicating that Ppd-B1b had an intrinsic effect on spikelets spike-1 independently of environmental cues. Our results suggest that, in environments where yield is limited by the incapacity to produce a high GN, selecting for Ppd-B1b may be advisable.

Durum Wheat Landraces from East and West Regions of the Mediterranean Basin Are Genetically Distinct for Yield Components and Phenology.

Genetic diversity of durum wheat landraces is a powerful tool for the introgression of new alleles of commercial interest in breeding programs. In a previous study, our team structured a collection of 172 durum wheat landraces from 21 Mediterranean countries in four genetic populations related to their geographical origin: east Mediterranean (17), east Balkan and Turkey (23), west Balkan and Egypt (25), and West Mediterranean (73), leaving 34 genotypes as admixed, and association mapping was carried out for important agronomic traits. Using a subset of this collection, the current study identified 23 marker alleles with a differential frequency in landraces from east and west regions of the Mediterranean Basin, which affected important agronomic traits. Eastern landraces had higher frequencies than the western ones of alleles increasing the number of spikes (wPt-5385 on chromosome 1B), grains per m2 (wPt-0841 on chromosome 7B), and grain filling duration (7 significant marker trait associations). Eastern landraces had higher frequencies of marker alleles located on chromosomes 4A, 5B, and 6B associated with reduced cycle length, and lighter grains than the western ones. Also for lower kernel weight, four marker alleles were located on chromosome 1A. Breeders may use the molecular markers identified in the current study for improving yield under specific Mediterranean environments.

Genetic Structure of Modern Durum Wheat Cultivars and Mediterranean Landraces Matches with Their Agronomic Performance.

A collection of 172 durum wheat landraces from 21 Mediterranean countries and 20 modern cultivars were phenotyped in 6 environments for 14 traits including phenology, biomass, yield and yield components. The genetic structure of the collection was ascertained with 44 simple sequence repeat markers that identified 448 alleles, 226 of them with a frequency lower than 5%, and 10 alleles per locus on average. In the modern cultivars all the alleles were fixed in 59% of the markers. Total genetic diversity was HT = 0.7080 and the genetic differentiation value was GST = 0.1730. STRUCTURE software allocated 90.1% of the accessions in five subpopulations, one including all modern cultivars, and the four containing landrace related to their geographic origin: eastern Mediterranean, eastern Balkans and Turkey, western Balkans and Egypt, and western Mediterranean. Mean yield of subpopulations ranged from 2.6 t ha-1 for the western Balkan and Egyptian landraces to 4.0 t ha-1 for modern cultivars, with the remaining three subpopulations showing similar values of 3.1 t ha-1. Modern cultivars had the highest number of grains m-2 and harvest index, and the shortest cycle length. The diversity was lowest in modern cultivars (HT = 0.4835) and highest in landraces from the western Balkans and Egypt (HT = 0.6979). Genetic diversity and AMOVA indicated that variability between subpopulations was much lower (17%) than variability within them (83%), though all subpopulations had similar biomass values in all growth stages. A dendrogram based on simple sequence repeat data matched with the clusters obtained by STRUCTURE, improving this classification for some accessions that have a large admixture. landraces included in the subpopulation from the eastern Balkans and Turkey were separated into two branches in the dendrogram drawn with phenotypic data, suggesting a different origin for the landraces collected in Serbia and Macedonia. The current study shows a reliable relationship between genetic and phenotypic population structures, and the connection of both with the geographic origin of the landraces.

Transcriptomic and proteomic analyses of a pale-green durum wheat mutant shows variations in photosystem components and metabolic deficiencies under drought stress.

BACKGROUND: Leaf pigment content is an important trait involved in environmental interactions. In order to determine its impact on drought tolerance in wheat, we characterized a pale-green durum wheat mutant (Triticum turgidum L. var. durum) under contrasting water availability conditions. RESULTS: The pale-green mutant was investigated by comparing pigment content and gene/protein expression profiles to wild-type plants at anthesis. Under well-watered (control) conditions the mutant had lower levels of chlorophylls and carotenoids, but higher levels of xanthophyll de-epoxidation compared to wild-type. Transcriptomic analysis under control conditions showed that defense genes (encoding e.g. pathogenesis-related proteins, peroxidases and chitinases) were upregulated in the mutant, suggesting the presence of mild oxidative stress that was compensated without altering the net rate of photosynthesis. Transcriptomic analysis under terminal water stress conditions, revealed the modulation of antioxidant enzymes, photosystem components, and enzymes representing carbohydrate metabolism and the tricarboxylic acid cycle, indicating that the mutant was exposed to greater oxidative stress than the wild-type plants, but had a limited capacity to respond. We also compared the two genotypes under irrigated and rain-fed field conditions over three years, finding that the greater oxidative stress and corresponding molecular changes in the pale-green mutant were associated to a yield reduction. CONCLUSIONS: This study provides insight on the effect of pigment content in the molecular response to drought. Identified genes differentially expressed under terminal water stress may be valuable for further studies addressing drought resistance in wheat.

Conventional digital cameras as a tool for assessing leaf area index and biomass for cereal breeding.

Affordable and easy-to-use methods for assessing biomass and leaf area index (LAI) would be of interest in most breeding programs. Here, we describe the evaluation of a protocol for photographic sampling and image analysis aimed at providing low-labor yet robust indicators of biomass and LAI. In this trial, two genotypes of triticale, two of bread wheat, and four of tritordeum were studied. At six dates during the growing cycle, biomass and LAI were measured destructively, and digital photography was taken on the same dates. Several vegetation indices were calculated from each image. The results showed that repeatable and consistent values of the indices were obtained in consecutive photographic samplings on the same plots. The photographic indices were highly correlated with the destructive measurements, though the magnitude of the correlation was lower after anthesis. This work shows that photographic assessment of biomass and LAI can be fast, affordable, have good repeatability, and can be used under bright and overcast skies. A practical vegetation index derived from pictures is the fraction of green pixels over the total pixels of the image, and as it shows good correlations with all biomass variables, is the most robust to lighting conditions and has easy interpretation.

Changes in duration of developmental phases of durum wheat caused by breeding in Spain and Italy during the 20th century and its impact on yield.

BACKGROUND AND AIMS: Although the apical development of wheat has been widely described, studies analysing how genetic breeding over the 20th century influenced the developmental phases and its consequences on yield generation are lacking, especially for durum wheat under field conditions in Mediterranean environments. The aims of this study were to analyse the effects of breeding in Spain and Italy on crop development during the last century, to determine whether or not breeding significantly altered the developmental phases between sowing and maturity, and to evaluate the importance of each phase in determining the number of grains per spike of durum wheat (Triticum durum) cultivars representing the germplasm grown throughout the 20th century in Spain and Italy. METHODS: Eight field experiments were carried out during 4 years in two contrasting latitudes (Lleida and Granada, Spain). Plant material consisted of 24 durum wheat cultivars (12 Italian and 12 Spanish) grown throughout the 20th century in Spain and Italy. KEY RESULTS: In Spanish materials, breeding reduced the duration of the period from sowing to anthesis, placing the grain-filling period in better conditions. In those cultivars, the sub-phase sowing-terminal spikelet formation was reduced while the duration of the period from booting to anthesis was increased. The number of grains per spike increased by 23 % from old to modern cultivars, by changes in the number of grains per spikelet in both Spanish and Italian cultivars. Floral abortion from booting to anthesis diminished by 24 % from old to modern cultivars, and grain setting increased by 13 %. CONCLUSIONS: The results suggest that breeding reduced not only plant height, but also the time to anthesis. By extending the duration of the phase from booting to anthesis, which was associated with an increase in spike dry weight and grains per spike, it suggests that future increases in spike fertility could be achieved by enlarging that phase.

Association mapping in durum wheat grown across a broad range of water regimes.

Association mapping was used to dissect the genetic basis of drought-adaptive traits and grain yield (GY) in a collection of 189 elite durum wheat accessions evaluated in 15 environments highly different for water availability during the crop cycle (from 146 to 711 mm) and GY (from 9.9 to 67.3 q ha(-1)). For highly heritable traits (e.g. heading date, kernel weight, etc.) several significant experiment-wise marker-trait associations were detected across five or more (up to 13 for kernel weight) environments, with R(2) values ranging from ca. 5 to 10%. As to GY, significant associations (R(2) from 2.5 to 4.2%) were mostly detected in one environment only (56 markers), while decreasing rapidly from two to five environments (from 20 to three markers, respectively) and with only one marker (Xbarc197 on chr. 5A) found significant in six environments (ranging from low- to high-yielding). These results are probably due to the complex genetic basis of GY and its interaction with environmental conditions. The number of markers significantly affecting GY decreased considerably under drought conditions, suggesting a limited effectiveness of association mapping to identify loci for GY under low-moisture conditions, most likely because different genotypes can attain similar phenotypes via different morpho-physiological traits and corresponding gene networks. Our study confirmed the role of major loci for phenology previously described in biparental mapping populations, highlighted a novel set of loci for drought-adaptive traits, and provided information on the agronomic value of the alleles at such loci across a broad range of soil moisture conditions.