Development and marker-trait relationships of functional markers for glutamine synthetase GS1 and GS2 homoeogenes in bread wheat.

GS1 and GS2 genes encode, respectively, the main cytosolic and the plastidic isoforms of glutamine synthetase (GS). In the present study, the wheat GS1 and GS2 homoeogenes located in the A, B and D genome chromosomes have been sequenced in a group of 15 bread wheat varieties including landraces, old commercial varieties and modern cultivars. Phenotypic characterization by multi-environment field trials detected significant effects of specific GS homoeogenes on three of the seven agronomic and grain quality traits analyzed. Based on the gene sequence polymorphisms found, biallelic molecular markers that could facilitate marker-assisted breeding were developed for genes GS1A, GS2A and GS2D. The remaining genes encoding main wheat GS were excluded because of being monomorphic (GS1D) or too polymorphic (GS1B and GS2B) in the sequencing panel varieties. A collection of 187 Spanish bread wheat landraces was genotyped for these gene-based molecular markers. Data analyses conducted with phenotypic records reported for this germplasm collection in López-Fernández et al. (Plants-Basel 10: 620, 2021) have revealed the beneficial influence of some individual alleles on thousand-kernel weight (TKW), kernels per spike (KS) and grain protein content. Furthermore, genetic interactions between GS1A, a cytosolic GS isoform coding gene, and GS2A or GS2D, plastidic GS enzyme coding genes, were found to affect TKW and KS. The finding that some alleles at one locus may mask the effect of positive alleles at hypostatic GS loci should be kept in mind if gene pyramiding strategies are attempted for the improvement of N-use efficiency-related traits. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-022-01354-0.

Genomic analysis of Spanish wheat landraces reveals their variability and potential for breeding.

BACKGROUND: One of the main goals of the plant breeding in the twenty-first century is the development of crop cultivars that can maintain current yields in unfavorable environments. Landraces that have been grown under varying local conditions include genetic diversity that will be essential to achieve this objective. The Center of Plant Genetic Resources of the Spanish Institute for Agriculture Research maintains a broad collection of wheat landraces. These accessions, which are locally adapted to diverse eco-climatic conditions, represent highly valuable materials for breeding. However, their efficient use requires an exhaustive genetic characterization. The overall aim of this study was to assess the diversity and population structure of a selected set of 380 Spanish landraces and 52 reference varieties of bread and durum wheat by high-throughput genotyping. RESULTS: The DArTseq GBS approach generated 10 K SNPs and 40 K high-quality DArT markers, which were located against the currently available bread and durum wheat reference genomes. The markers with known locations were distributed across all chromosomes with relatively well-balanced genome-wide coverage. The genetic analysis showed that the Spanish wheat landraces were clustered in different groups, thus representing genetic pools providing a range of allelic variation. The subspecies had a major impact on the population structure of the durum wheat landraces, with three distinct clusters that corresponded to subsp. durum, turgidum and dicoccon being identified. The population structure of bread wheat landraces was mainly biased by geographic origin. CONCLUSIONS: The results showed broader genetic diversity in the landraces compared to a reference set that included commercial varieties, and higher divergence between the landraces and the reference set in durum wheat than in bread wheat. The analyses revealed genomic regions whose patterns of variation were markedly different in the landraces and reference varieties, indicating loci that have been under selection during crop improvement, which could help to target breeding efforts. The results obtained from this work will provide a basis for future genome-wide association studies.

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.

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.

Exploring the End-Use Quality Potential of a Collection of Spanish Bread Wheat Landraces.

Modern plant-breeding practices have narrowed the genetic base of wheat, such that there is a need to introduce new germplasms with underexploited diversity into breeding programs. Wheat landraces are a very valuable resource when searching for genetic variation, which not only possess increased adaptability, but also quality-related traits. Several studies have shown a wide genetic diversity in Spanish wheat landraces compared to other germplasm collections; therefore, the main objective of this study is to analyze the variability in a collection of 189 landraces from the Spanish National Plant Genetic Resources Centre (Centro de Recursos Fitogenéticos, CRF-INIA, Alcalá de Henares), in relation to end-use quality traits. We characterized the whole collection for high-molecular-weight glutenin and puroindoline allelic composition, and for gluten strength. In addition, grain protein content, grains per spike, and thousand kernel weight were evaluated in samples from four-year field trials. The relationship between glutenin composition and quality was evaluated, and some alleles strongly associated with high quality were identified in the collection, some of them specific for Iberian landraces. The results also show the presence of novel variability within high-molecular-weight glutenin and puroindolines, which needs to be characterized further in order to assess its influence on wheat quality. In addition, a set of landraces showing outstanding values for gluten quality and a good agronomic performance was selected for testing in field trials in order to evaluate the suitability of their direct use in cropping systems.

An F2 Barley Population as a Tool for Teaching Mendelian Genetics.

In the context of a general genetics course, mathematical descriptions of Mendelian inheritance and population genetics are sometimes discouraging and students often have serious misconceptions. Innovative strategies in expositive classes can clearly encourage student's motivation and participation, but laboratories and practical classes are generally the students' favourite academic activities. The design of lab practices focused on learning abstract concepts such as genetic interaction, genetic linkage, genetic recombination, gene mapping, or molecular markers is a complex task that requires suitable segregant materials. The optimal population for pedagogical purposes is an F2 population, which is extremely useful not only in explaining different key concepts of genetics (as dominance, epistasis, and linkage) but also in introducing additional curricular tools, particularly concerning statistical analysis. Among various model organisms available, barley possesses several unique features for demonstrating genetic principles. Therefore, we generated a barley F2 population from the parental lines of the Oregon Wolfe Barley collection. The objective of this work is to present this F2 population as a model to teach Mendelian genetics in a medium-high-level genetics course. We provide an exhaustive phenotypic and genotypic description of this plant material that, together with a description of the specific methodologies and practical exercises, can be helpful for transferring our fruitful experience to anyone interested in implementing this educational resource in his/her teaching.

Complete characterization of wheat-alien metaphase I pairing in interspecific hybrids between durum wheat (Triticum turgidum L.) and jointed goatgrass (Aegilops cylindrica Host).

The pattern of homoeologous metaphase I (MI) pairing has been fully characterized in durum wheat x Aegilops cylindrica hybrids (2n = 4x = 28, ABC(c)D(c)) by an in situ hybridization procedure that has permitted individual discrimination of every wheat and wild constituent genome. One of the three hybrid genotypes examined carried the ph1c mutation. In all cases, MI associations between chromosomes of both species represented around two-third of total. Main results from the analysis are as follows (a) the A genome chromosomes are involved in wheat-wild MI pairing more frequently than the B genome partners, irrespective of the alien genome considered; (b) both durum wheat genomes pair preferentially with the D(c) genome of jointed goatgrass. These findings are discussed in relation to the potential of genetic transference between wheat crops and this weedy relative. It can also be highlighted that inactivation of Ph1 provoked a relatively higher promotion of MI associations involving B genome.

Wheat-alien metaphase I pairing of individual wheat genomes and D genome chromosomes in interspecific hybrids between Triticum aestivum L. and Aegilops geniculata Roth.

Homoeologous metaphase I (MI) pairing of Triticum aestivum x Aegilops geniculata hybrids (2n = 5x = 35, ABDU(g)M(g)) has been examined by an in situ hybridization procedure permitting simultaneous discrimination of A, B, D and wild genomes. The seven D genome chromosomes (and their arms, except for 6D and 7D) plus some additional wheat chromosomes were also identified. Wheat-wild MI associations represented more than 60% of total, with an average ratio of 5:1:12 for those involving the A, B and D genomes, respectively. A remarkable between-chromosome variation for the level of wheat-wild genetic exchange is expected within each wheat genome. However, it can be concluded that 3DL and 5DL are the crop genome locations with the highest probability of being transferred to Ae. geniculata. Hybrids derived from the ph2b wheat mutant line showed increased MI pairing but identical pattern of homoeologous associations than those with active Ph2.

Neutral molecular markers support common origin of aluminium tolerance in three congeneric grass species growing in acidic soils.

Aluminium (Al) toxicity is the main abiotic stress limiting plant productivity in acidic soils that are widely distributed among arable lands. Plant species differ in the level of Al resistance showing intraspecific and interspecific variation in many crop species. However, the origin of Al-tolerance is not well known. Three annual species, difficult to distinguish phenotypically and that were until recently misinterpreted as a single complex species under Brachypodium distachyon, have been recently separated into three distinct species: the diploids B. distachyon (2n = 10) and B. stacei (2n = 20), and B. hybridum (2n = 30), the allotetraploid derived from the two diploid species. The aims of this work were to know the origin of Al-tolerance in acidic soil conditions within these three Brachypodium species and to develop new DNA markers for species discrimination. Two multiplex SSR-PCRs allowed to genotype a group of 94 accessions for 17 pentanucleotide microsatellite (SSRs) loci. The variability for 139 inter-microsatellite (ISSRs) markers was also examined. The genetic relationships obtained using those neutral molecular markers (SSRs and ISSRs) support that all Al-tolerant allotetraploid accessions of B. hybridum have a common origin that is related with both geographic location and acidic soils. The possibility that the adaptation to acidic soils caused the isolation of the tolerant B. hybridum populations from the others is discussed. We finally describe a new, easy, DNA barcoding method based in the upstream-intron 1 region of the ALMT1 gene, a tool that is 100 % effective to distinguish among these three Brachypodium species.

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

The small, annual grass (L.) Beauv., a close relative of wheat ( L.) and barley ( L.), is a powerful model system for cereals and bioenergy grasses. Genome-wide association studies (GWAS) of natural variation can elucidate the genetic basis of complex traits but have been so far limited in by the lack of large numbers of well-characterized and sufficiently diverse accessions. Here, we report on genotyping-by-sequencing (GBS) of 84 , seven , and three accessions with diverse geographic origins including Albania, Armenia, Georgia, Italy, Spain, and Turkey. Over 90,000 high-quality single-nucleotide polymorphisms (SNPs) distributed across the Bd21 reference genome were identified. Our results confirm the hybrid nature of the genome, which appears as a mosaic of -like and -like sequences. Analysis of more than 50,000 SNPs for the accessions revealed three distinct, genetically defined populations. Surprisingly, these genomic profiles are associated with differences in flowering time rather than with broad geographic origin. High levels of differentiation in loci associated with floral development support the differences in flowering phenology between populations. Genome-wide association studies combining genotypic and phenotypic data also suggest the presence of one or more photoperiodism, circadian clock, and vernalization genes in loci associated with flowering time variation within populations. Our characterization elucidates genes underlying population differences, expands the germplasm resources available for , and illustrates the feasibility and limitations of GWAS in this model grass.

Detection of intergenomic chromosome rearrangements in irradiated Triticum aestivum--Aegilops biuncialis amphiploids by multicolour genomic in situ hybridization.

The frequency and pattern of irradiation-induced intergenomic chromosome rearrangements were analysed in the mutagenized (M0) and the first selfed (M1) generations of Triticum aestivum L. - Aegilops biuncialis Vis. amphiploids (2n = 70, AABBDDUbUbMbMb) by multicolour genomic in situ hybridization (mcGISH). mcGISH allowed the simultaneous discrimination of individual Ae. biuncialis genomes and wheat chromosomes. Dicentric chromosomes, fragments, and terminal translocations were most frequently induced by gamma-irradiation, but centric fusions and internal exchanges were also more abundant in the treated plants than in control amphiploids. Rearrangements involving the Ub genome (Ub-type aberrations) were more frequent than those involving the Mb genome (Mb-type aberrations). This irradiation sensitivity of the Ub chromosomes was attributed to their centromeric or near-centromeric regions, since Ub-type centric fusions were significantly more abundant than Mb-type centric fusions at all irradiation doses. Dicentrics completely disappeared, but centric fusions and translocations were well transmitted from M0 to M1. Identification of specific chromosomes involved in some rearrangements was attempted by sequential fluorescence in situ hybridization with a mix of repeated DNA probes and GISH on the same slide. The irradiated amphiploids formed fewer seeds than untreated plants, but normal levels of fertility were recovered in their offspring. The irradiation-induced wheat - Ae. biuncialis intergenomic translocations will facilitate the successful introgression of drought tolerance and other alien traits into bread wheat.

A cytomolecular approach to assess the potential of gene transfer from a crop (Triticum turgidum L.) to a wild relative (Aegilops geniculata Roth.).

When a crop hybridizes with a wild relative, the potential for stable transmission to the wild of any crop gene is directly related to the frequency of crop-wild homoeologous pairing for the chromosomal region where it is located within the crop genome. Pairing pattern at metaphase I (MI) has been examined in durum wheat x Aegilops geniculata interspecific hybrids (2n=4x=ABUgMg) by means of a genomic in-situ hybridization procedure that resulted in simultaneous discrimination of A, B and wild genomes. The level of MI pairing in the hybrids varied greatly depending on the crop genotype. However, their pattern of homoeologous association was very similar, with a frequency of wheat-wild association close to 60% in all genotype combinations. A-wild represented 80-85% of wheat-wild associations which supports that, on average, A genome sequences are much more likely to be transferred to this wild relative following interspecific hybridization and backcrossing. Combination of genomic DNA probes and the ribosomal pTa71 probe has allowed to determine the MI pairing behaviour of the major NOR-bearing chromosomes in these hybrids (1 B, 6B, 1 Ug and 5 Ug), in addition to wheat chromosome 4A which could be identified with the sole use of genomic probes. The MI pairing pattern of the wild chromosome arms individually examined has confirmed a higher chance of gene escape from the wheat A genome. However, a wide variation regarding the amount of wheat-wild MI pairing among the specific wheat chromosome regions under analysis suggests that the study should be extended to other homoeologous groups.