Jose M. Carrillo

Analysis of Genetic Variability in a Sample of the Durum Wheat (Triticum durum Desf.) Spanish Collection Based on Gliadin Markers

In this work gliadin proteins were used to analyse the genetic variability in a sample of the durum wheat Spanish collection conserved at the CRF-INIA. In total 38 different alleles were identified at the loci Gli-A1, Gli-A3, Gli-B5, Gli-B1, Gli-A2 and Gli-B2. All the gliadin loci were polymorphic, possessed large genetic diversity and small and large differentiation within and between varieties, respectively. The Gli-A2 and Gli-B2 loci were the most polymorphic, the most fixed within varieties and the most useful to distinguish among varieties. Alternatively, Gli-B1 locus presented the least genetic variability out of the four main loci Gli-A1, Gli-B1, Gli-A2 and Gli-B2. The Gli-B1 alleles coding for the gliadin γ-45, associated with good quality, had an accumulated frequency of 69.7%, showing that the Spanish germplasm could be a good source for breeding quality. The Spanish landraces studied showed new gliadin alleles not catalogued so far. These new alleles might be associated with specific Spanish environment factors. The large number of new alleles identified also indicates that durum wheat Spanish germplasm is rather unique.

Validation of microsatellite markers for cytotype discrimination in the model grass Brachypodium distachyon

Brachypodium distachyon (L.) P. Beauv. (2n = 2x = 10) is a small annual grass species where the existence of three different cytotypes (10, 20, and 30 chromosomes) has long been regarded as a case of autopolyploid series with x = 5. However, it has been demonstrated that the cytotypes assumed to be polyploids represent two separate Brachypodium species recently named as Brachypodium stacei (2n = 2x = 20) and Brachypodium hybridum (2n = 4x = 30). The aim of this study was to find a PCR-based alternative approach that could replace standard cytotyping methods (i.e., chromosome counting and flow cytometry) to characterize each of the three Brachypodium species. We have analyzed with four microsatellite (SSR) markers 83 B. distachyon-type lines from varied locations in Spain, including the Balearic and Canary Islands. Within this set of lines, 64, 4, and 15 had 10, 20, and 30 chromosomes, respectively. The surveyed markers produced cytotype-specific SSR profiles. So, a single amplification product was generated in the diploid samples, with nonoverlapping allelic ranges between the 2n = 10 and 2n = 20 cytotypes, whereas two bands, one in the size range of each of the diploid cytotypes, were amplified in the 2n = 30 lines. Furthermore, the remarkable size difference obtained with the SSR ALB165 allowed the identification of the Brachypodium species by simple agarose gel electrophoresis.

Variation in B-LMW glutenin subunits in Einkorn wheats

A collection of 50 Einkorn wheat accessions (26 of T. monococcum, 14 of T. baeoticum and 10 T. urartu) was analyzed for low-molecular-weight (LMW) glutenin subunit composition. At the B-LMW glutenin region, T. monococcum possessed 1 or 2 subunits, and T. baeoticum and T. urartu from 1 to 3 subunits. Seven different electrophoretical patterns were detected among T. monococcum and six in both T. baeoticum and T. urartu. T. monococcum was shown to be the less polymorphic, followed by T. baeoticum and by T. urartu. The fact that more glutenin genes are expressed in Einkorn wheats than in polyploid cultivated wheats suggests their possible use in breeding for improving bread and durum wheat quality.

Polymorphism, variation and genetic identity of Spanish common wheat germplasm based on gliadin alleles

Gliadin analysis has shown that Spanish common wheat germplasm is highly polymorphic and rather unique. In total, 81 different alleles, 70 of them catalogued, were identified in the 52 Spanish landraces studied, and 25 new gliadin alleles, not present in germplasm from other countries, were found. Eleven alleles were identified at the Gli-A1 locus, 14 at the Gli-B1 ,9a t the Gli-D1, 17 at the Gli-A2, 14 at the Gli-B2 and 16 at the Gli-D2. Comparison of gliadin alleles among varieties released before and after 1966 revealed significant differences for 46 gliadin alleles. Forty-six alleles (36 of them catalogued) present in the material developed before 1966 were lost. In contrast, 25 alleles (23 of them catalogued) appear for the first time in the genepool bred after 1966. On the other hand, most of the Gli alleles frequent in recent Spanish cultivars were already present in Spanish landraces. In general, the results showed that Spanish varieties have suffered qualitative alterations of genetic variation during the last 35 years mainly due to changes in breeding goals and crosses with foreign germplasm. Seventy-five percent of the landraces analysed were shown to have maintained their genetic identity in the genebank although they have been regenerated several times during the last century. However, mistakes in three accessions modified 10 alleles (allele losses or presence of new alleles), and error in one suggests alteration of five alleles. These results demonstrate that great care during maintenance and handling of samples is an important requirement in conserving germplasm collections. Gliadin alleles allowed us to identify 48 different genotypes, some accession duplicates and one intermixing. Also, several mistakes could be resolved using gliadins as genetic markers. # 2002 Elsevier Science B.V. All rights reserved.

Assessment of storage protein variation in relation to some morphological characters in a sample of Spanish landraces of common wheat (Triticum aestivum L. ssp. aestivum)

Fifty nine Spanish landraces of common wheat belonging to differentagrotypes were evaluated with four morphological spike characters and 11 seedprotein loci in order to study the level of agreement between the agrotypeclassification and the morphological and biochemical variation of the wheats. Asa result of the work 22 new gliadin alleles were found and 12 of them werecatalogued. Multivariate and cluster analysis performed on the two sets of data(morphological plus biochemical and biochemical only) showed that there was aclear relationship between seed protein composition and the wheat classificationfor the agrotypes ‘Barbilla’, ‘Candeal’ and‘Negrete’. For the other groups, agrotype classification was notconsistent with the variation for biochemical characters although, some allelesin common were found except for the ‘Jeja’ type. Morphologicaltraits revealed that they could be useful as first step in discriminating amongagrotypes. In contrast, biochemical markers showed that genetic variation amongaccessions was higher than among agrotypes. The protein lociGli-B1,Gli-A2,Gli-D2 andGli-A1 were shown to be the mostpolymorphic and useful in discriminating between the accessions studied.

Polymorphism of high molecular weight glutenin subunits in three species of Aegilops

A collection of 136 accessions of Aegilops umbellulata (39), Ae. comosa (75) and Ae. markgrafii (22) was analysed for high-molecular-weight (HMW) glutenin subunits composition. The homogeneity of the accessions was studied and 55.1% of the collection was homogeneous for HMW glutenin subunits (29 Ae. umbellulata, 33 Ae. comosa and 14 Ae. markgrafii). The HMW glutenin subunits of Ae. umbellulata are encoded by the Glu-U1 locus; in Ae. comosa results showed that this proteins are encoded at the 1M chromosome, and the locus was named Glu-M1. In Ae. markgrafii it was assumed that HMW glutenin subunits were encoded by an homoeologous locus and it was named Glu-C1. All the accessions of Ae. umbellulata and Ae. markgrafii expressed both, x-type and y-type subunits. Among the Ae. comosa accessions, only one expressed an x-type subunit alone. All the accessions of Ae. umbellulata and some of Ae. comosa had x-type glutenins of higher molecular weights than those commonly present in bread wheat. A total of 8 alleles were detected at the Glu-U1 locus, 11 at the Glu-M1 and 4 at the Glu-C1. The new HMW glutenin variation found in this work suggests their possible utilisation in breeding for wheat quality.

Combined use of gliadins and SSRs to analyse the genetic variability of the Spanish collection of cultivated diploid wheat ( Triticum monococcum L. ssp. monococcum )

This work studied the combined use of gliadins and SSRs to analyse inter- and intra-accession variability of the Spanish collection of cultivated einkorn (Triticum monococcum L. ssp. monococcum) maintained at the CRF-INIA. In general, gliadin loci presented higher discrimination power than SSRs, reflecting the high variability of the gliadins. The loci on chromosome 6A were the most polymorphic with similar PIC values for both marker systems, showing that these markers are very useful for genetic variability studies in wheat. The gliadin results indicated that the Spanish einkorn collection possessed high genetic diversity, being the differentiation large between varieties and small within them. Some associations between gliadin alleles and geographical and agro-morphological data were found. Agro-morphological relations were also observed in the clusters of the SSRs dendrogram. A high concordance was found between gliadins and SSRs for genotype identification. In addition, both systems provide complementary information to resolve the different cases of intra-accession variability not detected at the agro-morphological level, and to identify separately all the genotypes analysed. The combined use of both genetic markers is an excellent tool for genetic resource evaluation in addition to agro-morphological evaluation.

Thermographic Imaging: Assessment of Drought and Heat Tolerance in Spanish Germplasm of Brachypodium Distachyon☆

The annual grass Brachypodium distachyon has been recently recognized as the model plant for functional genomics of temperate grasses, including cereals of economic relevance like wheat and barley. Sixty-two lines of B. distachyon were assessed for response to drought stress and heat tolerance. All these lines, except the reference genotype BD21, derive from specimens collected in 32 distinct locations of the Iberian Peninsula, covering a wide range of geo- climatic conditions. Sixteen lines of Brachypodium hybridum, an allotetraploid closely related to B. distachyon were used as reference of abiotic-stress well-adapted genotypes. Drought tolerance was assessed in a green-house trial. At the rosette-stage, no irrigation was applied to treated plants whereas their replicates at the control were maintained well watered during all the experiment. Thermographic images of treated and control plants were taken after 2 and 3 weeks of drought treatment, when stressed plants showed medium and extreme wilting symptoms. The mean leaf temperature of stressed (LTs) and control (LTc) plants was estimated based upon thermographic records from selected pixels (183 per image) that strictly correspond to leaf tissue. The response to drought was based on the analysis of two parameters: LTs and the thermal difference (TD) between stressed and control plants (LTs – LTc). The response to heat stress was based on LTc. Comparison of the mean values of these parameters showed that: 1) Genotypes better adapted to drought (B. hybridum lines) presented a higher LTs and TD than B. distachyon lines. 2) Under high temperature conditions, watered plants of B. hybridum lines maintained lower LTc than those of B. distachyon. Those results suggest that in these species adaptation to drought is linked to a more efficient stomata regulation: under water stress stomata are closed, increasing foliar temperature but also water use efficiency by reducing transpiration. With high temperature and water availability the results are less definite, but still seems that opening stomata allow plants to increase transpiration and therefore to diminish foliar temperature.

Genetic Diversity and Association Mapping for Agromorphological and Grain Quality Traits of a Structured Collection of Durum Wheat Landraces Including subsp. durum, turgidum and diccocon

Association mapping was performed for 18 agromorphological and grain quality traits in a set of 183 Spanish landraces, including subspecies durum, turgidum and dicoccon, genotyped with 749 DArT (Diversity Array Technology) markers. Large genetic and phenotypic variability was detected, being the level of diversity among the chromosomes and genomes heterogeneous, and sometimes complementary, among subspecies. Overall, 356 were monomorphic in at least one subspecies, mainly in dicoccon, and some of them coincidental between subspecies, especially between turgidum and dicoccon. Several of those fixed markers were associated to plant responses to environmental stresses or linked to genes subjected to selection during tetraploid wheat domestication process. A total of 85 stable MTAs (marker–trait associations) have been identified for the agromorphological and quality parameters, some of them common among subspecies and others subspecies-specific. For all the traits, we have found MTAs explaining more than 10% of the phenotypic variation in any of the three subspecies. The number of MTAs on the B genome exceeded that on the A genome in subsp. durum, equalled in turgidum and was below in dicoccon. The validation of several adaptive and quality trait MTAs by combining the association mapping with an analysis of the signature of selection, identifying the putative gene function of the marker, or by coincidences with previous reports, showed that our approach was successful for the detection of MTAs and the high potential of the collection to identify marker–trait associations. Novel MTAs not previously reported, some of them subspecies specific, have been described and provide new information about the genetic control of complex traits.

Use of thermographic imaging to screen for drought-tolerant genotypes in Brachypodium distachyon

Abstract. Thermal imaging has been used to evaluate the response to drought and warm temperatures in a collection of Brachypodium distachyon lines adapted to varied environmental conditions. Thermographic records were able to separate lines from contrasting rainfall regimes. Genotypes from dryer environments showed warmer leaves under water deficit, which suggested that decreased evapotranspiration was related to a more intense stomatal closure. When irrigated and under high temperature conditions, drought-adapted lines showed cooler leaves than lines from wetter zones. The consistent, inverse thermographic response of lines to water stress and heat validates the reliability of this method to assess drought tolerance in this model cereal. It additionally supports the hypothesis that stomatal-based mechanisms are involved in natural variation for drought tolerance in Brachypodium. The study further suggests that these mechanisms are not constitutive but likely related to a more efficient closing response to avoid dehydration in adapted genotypes. Higher leaf temperature under water deficit seems a dependable criterion of drought tolerance, not only in B. distachyon but also in the main cereal crops and related grasses where thermography can facilitate high-throughput preliminary screening of tolerant materials.