Elena Benavente

Population Structure in the Model Grass Brachypodium distachyon Is Highly Correlated with Flowering Differences across Broad Geographic Areas

Genotyping diverse Brachypodium accessions expands research tools for grasses. The B. hybridum genome is a mosaic of B. distachyon‐ and B. stacei‐like sequences. Three distinct, genetically defined populations of B. distachyon were identified. Flowering time, more than geography, distinguishes B. distachyon populations. Results support the feasibility of genome‐wide association studies in a model grass.

Analysis of karyotypic stability of homoeologous-pairing (ph) mutants in allopolyploid wheats.

Abstract. Karyotypic analysis of wheat lines with different genotypes for the homoeologous-pairing loci Ph1 and Ph2 was carried out by means of a genomic in situ hybridization method that allowed unequivocal identification of the A, B and D genomes. Chromosomal rearrangements mainly affecting the A and D genomes were found in all plants of allohexaploid wheat (AABBDD) lacking Ph1 activity. The frequency of intergenomic exchanges per plant in ph1b mutant and nulli-5B lines was 4.31 and 3.40, respectively. In addition, an unbalanced genomic constitution was found in a few plants, some even showing a euploid chromosomal number. By contrast, rearranged karyotypes were detected neither in the ph1 mutant line (ph1c) of allotetraploid wheat (AABB) nor in the allohexaploid wheat lines lacking Ph2 activity, namely ph2b mutant and nulli-3D lines. These results were compared with the chromosomal pairing behaviour displayed by mutant lines ph1c, ph1b and ph2b at first meiotic metaphase. Despite the finding of standard, non-rearranged karyotypes in the ph1c tetraploid mutant, the frequency of A-B homoeologous metaphase I association was similar to that observed in the ph1b hexaploid mutant. The results presented clearly demonstrate that inactivity of the Ph1 locus induces karyotypic instability in wheat. Intergenomic exchanges have probably been accumulating since the original ph1 mutant and aneuploid lines were obtained, which should be taken into account when it is planned to use these lines for basic research on Ph1 function or in applied wheat breeding programmes.

Association between simple sequence repeat-rich chromosome regions and intergenomic translocation breakpoints in natural populations of allopolyploid wild wheats

BACKGROUND AND AIMS Repetitive DNA sequences are thought to be involved in the formation of chromosomal rearrangements. The aim of this study was to analyse the distribution of microsatellite clusters in Aegilops biuncialis and Aegilops geniculata, and its relationship with the intergenomic translocations in these allotetraploid species, wild genetic resources for wheat improvement. METHODS The chromosomal localization of (ACG)(n) and (GAA)(n) microsatellite sequences in Ae. biuncialis and Ae. geniculata and in their diploid progenitors Aegilops comosa and Aegilops umbellulata was investigated by sequential in situ hybridization with simple sequence repeat (SSR) probes and repeated DNA probes (pSc119·2, Afa family and pTa71) and by dual-colour genomic in situ hybridization (GISH). Thirty-two Ae. biuncialis and 19 Ae. geniculata accessions were screened by GISH for intergenomic translocations, which were further characterized by fluorescence in situ hybridization and GISH. KEY RESULTS Single pericentromeric (ACG)(n) signals were localized on most U and on some M genome chromosomes, whereas strong pericentromeric and several intercalary and telomeric (GAA)(n) sites were observed on the Aegilops chromosomes. Three Ae. biuncialis accessions carried 7U(b)-7M(b) reciprocal translocations and one had a 7U(b)-1M(b) rearrangement, while two Ae. geniculata accessions carried 7U(g)-1M(g) or 5U(g)-5M(g) translocations. Conspicuous (ACG)(n) and/or (GAA)(n) clusters were located near the translocation breakpoints in eight of the ten translocated chromosomes analysed, SSR bands and breakpoints being statistically located at the same chromosomal site in six of them. CONCLUSIONS Intergenomic translocation breakpoints are frequently mapped to SSR-rich chromosomal regions in the allopolyploid species examined, suggesting that microsatellite repeated DNA sequences might facilitate the formation of those chromosomal rearrangements. The (ACG)(n) and (GAA)(n) SSR motifs serve as additional chromosome markers for the karyotypic analysis of UM genome Aegilops species.

Relationship between the levels of wheat-rye metaphase I chromosomal pairing and recombination revealed by GISH

The metaphase I and anaphase I stages of meiosis of wheatxrye hybrids carrying theph1b mutation were analyzed by genomic in situ hybridization. This technique allows distinction between three different types of wheat-rye associations in metaphase I configurations as well as detection of wheat-rye recombinant chromosomes in anaphase I cells. The frequency of associations between wheat and rye chromosomes greatly exceeded the level of wheat-rye recombination found in the three hybrids examined. Extremely distal associations, which account for about 50% of the total wheat-rye metaphase I chromosomal pairing, can explain such a discrepancy between metaphase I and anaphase I data. It is further discussed whether these associations reflect very distally located chiasmata or nonchiasmatic pairing. The sizes of the segments exchanged in wheat-rye recombinant chromosomes provide cytological evidence that wheat-rye recombination is restricted to the distal chromosomal regions.

Meiotic pairing in wheat-rye derivatives detected by genomic in situ hybridization and C-banding — A comparative analysis

Meiosis of triticalextetraploid rye hybrids (genome constitution ABRRR) was analysed by genomic in situ hybridization (GISH) and C-banding. The results obtained reveal a considerable difference between these techniques with regard to their efficiency in detecting any type of pairing, either homologous or homoeologous. Thus the percentage of pollen mother cells containing wheat/rye homoeologous associations determined by C-banding and GISH was 2.5 and 9.2, respectively. Such a discrepancy can be ascribed to a certain proportion of wheat/rye associations not being identified by C-banding. The potential and limitations of the two techniques for meiotic analysis are discussed.

Environmental niche variation and evolutionary diversification of the Brachypodium distachyon grass complex species in their native circum-Mediterranean range.

UNLABELLED • PREMISE OF THE STUDY We conducted environmental niche modeling (ENM) of the Brachypodium distachyon s.l. complex, a model group of two diploid annual grasses (B. distachyon, B. stacei) and their derived allotetraploid (B. hybridum), native to the circum-Mediterranean region. We (1) investigated the ENMs of the three species in their native range based on present and past climate data; (2) identified potential overlapping niches of the diploids and their hybrid across four Quaternary windows; (3) tested whether speciation was associated with niche divergence/conservatism in the complex species; and (4) tested for the potential of the polyploid outperforming the diploids in the native range.• METHODS Geo-referenced data, altitude, and 19 climatic variables were used to construct the ENMs. We used paleoclimate niche models to trace the potential existence of ancestral gene flow among the hybridizing species of the complex.• KEY RESULTS Brachypodium distachyon grows in higher, cooler, and wetter places, B. stacei in lower, warmer, and drier places, and B. hybridum in places with intermediate climatic features. Brachypodium hybridum had the largest niche overlap with its parent niches, but a similar distribution range and niche breadth.• CONCLUSIONS Each species had a unique environmental niche though there were multiple niche overlapping areas for the diploids across time, suggesting the potential existence of several hybrid zones during the Pleistocene and the Holocene. No evidence of niche divergence was found, suggesting that species diversification was not driven by ecological speciation but by evolutionary history, though it could be associated to distinct environmental adaptations.

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.

Comparative analysis of the meiotic effects of wheat ph1b and ph2b mutations in wheat×rye hybrids

Abstract Wheat-wheat and wheat-rye homoeologous pairing at metaphase I and wheat-rye recombination at anaphase I were examined by genomic in situ hybridization (GISH) in wild-type (Ph1Ph2) and mutant ph1b and ph2b wheat×rye hybrids. The metaphase-I analysis revealed that the relative contribution of wheat-rye chromosome associations in ph2b wheat×rye was similar to that of the wild-type hybrid genotype but differed from the effect of the ph1b mutation. The greater pairing promotion effect of the ph1b mutation appears to be relatively more on distant homoeologous partner metaphase-I associations, whereas the lower promoting effect of ph2b is evenly distributed among all types of homoeologous associations. This finding reveals that distinct mechanisms are involved in the control of wheat homoeologous pairing by the two Ph genes. The frequency of wheat-rye recombination calculated from anaphase-I analysis was lower than expected from the metaphase-I data. A greater discrepancy was found in ph2b than in ph1b wheat×rye hybrids, which may suggest a more distal chiasma localization in the former hybrid genotype.

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.

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.