Conserved Ortholog Set Research Articles

Development of clubroot resistant cabbage line through introgressing six CR loci from Chinese cabbage via interspecific hybridization and embryo rescue

Multiple R-gene is required to achieve strong resistance against clubroot, a disease caused by Plasmodiophora brassicae in Cabbage (Brassica oleracea, 2n=18, CC). Most of the R-genes were identified in Chinese cabbage (B. rapa, 2n=20, AA), which could be transferred to cabbage via inter interspecific hybridization. We present a breeding scheme to transfer six major CR-loci namely, Crr1, Crr2, Crr3, CRa, CRb and CRc from Chinese cabbage line, LCR36 to cabbage line, Plimio. Approximately, 48% of the interspecific hybrid embryos were rescued, of which chromosomes of 48.15% colchiploid plants were successfully doubled. The hybridity of the colchiploid interspecific hybrids were confirmed by diagnostic sub-genome specific Conserved Ortholog Set (COS) markers, ploidy level, pollen fertility and meiotic chromosome number analyses. Backcross generations are characterized by very low seed set (0.57-1.33% success rate) in backcrossed generations (using Plimio as the recurrent parent). Marker assisted selection using the six CR-loci specific markers identified the resistant plants in F1 and backcross generations. MAS and bioassay test of BC2F2 plants revealed that plants having all six R-loci are resistant to clubroot. Development of subsequent backcross generations are in progress. More number of back-crosses and/or embryo rescue will be performed to ensure sufficient seed set. The success so far is the first report of successful introgression of multiple R-genes via interspecific hybridization in the initial backcrossed generations, indicating that an elite cabbage line having strong resistance to clubroot can be produced upon the completion of the breeding scheme.

Phylogenomic data reveal four major clades of Australian Gnaphalieae (Asteraceae)

AbstractWithin tribe Gnaphalieae (Asteraceae), the Australasian clade is one of the four major clades. In Australia, the Gnaphalieae account for 488 species or approximately half of the native Asteraceae, encompassing wide ecological and morphological diversity including shrubs, everlasting paper daisies, cudweeds, alpine cushion plants, and ephemeral herbs in the arid zone. The evolution of the Australasian clade is still poorly understood. The most detailed previous infratribal classification of Gnaphalieae has recently been revised, resulting in the recognition of two subtribes, with all Australian species placed in subtribe Gnaphaliinae. The most comprehensive previous phylogeny of Australian Gnaphalieae used high‐copy ribosomal and chloroplast markers but showed limited resolution and branch support. We used conserved ortholog set data produced with sequence capture and 53 chloroplast genes to infer nuclear and chloroplast likelihood phylogenies for Australian Gnaphalieae, generating data for at least one species each from 80 of the 86 native genera. Four major clades were resolved: the Euchiton clade of cudweed‐like and alpine perennial species; the shrubby Cassinia clade; the predominantly perennial and eastern Australian Waitzia clade; and the predominantly ephemeral and western Australian to Eremaean Angianthus clade. The Cassinia, Waitzia, and Angianthus clades are largely congruent with “groups” in a previous morphological analysis and classification of Gnaphalieae. Analysis of ancestral ranges implied the temperate Southeast of Australia as the most likely area of origin for the Australian Gnaphalieae as a whole and for three of the four major clades. The Angianthus clade was implied to be ancestrally Eremaean, with a major secondary radiation originating in southwestern Australia. Our broadly sampled phylogeny provides a framework to inform sampling and design of future studies to test the circumscription of genera.

Development and Characterization of Wheat-Agropyron cristatum Introgression Lines Induced by Gametocidal Genes and Wheat ph1b Mutant

The P genome of Agropyron cristatum Gaertn. contains many desirable genes that can be utilized as genetic resources to improve wheat. In this research, we used both the gametocidal chromosome 2Cc and the pairing homologous gene (Ph1b) mutant to induce structural aberrations and translocations between wheat and the 4P, 5P, and 6P genome chromosomes. By using the two approaches, a total of 19 wheat-A. cristatum translocations have been identified, in which 13 were induced by the Triticum aestivum cv. Chinese Spring (CS) ph1b mutant (CS ph1b) and six were induced by gametocidal chromosome 2Cc from Aegilops cylindrica Host. The wheat-4P, -5P and -6P A. cristatum translocations were characterized by in situ hybridization and by a set of conserved orthologous set (COS) molecular markers. The aberrations included centromeric translocations, terminal translocations, dicentric translocations, and deletions. The average induction frequency of chromosome structural aberrations was 10.9% using gametocidal 2Cc chromosome and 8.8% using ph1b mutant. The highest frequency obtained was for chromosome 4P using both approaches. All the wheat-A. cristatum translocation lines obtained were valuable for identifying A. cristatum chromosome 4P, 5P, and 6P related genes. In addition, these lines provided genetic resources and new germplasm accessions for the genetic improvement of wheat.

Production and characterization of a disomic 1M/1D Triticum aestivum-Aegilops comosa substitution line.

The online version contains supplementary material available at 10.1007/s11032-021-01207-2.

Hexaploid sweetpotato (Ipomoea batatas (L.) Lam.) may not be a true type to either auto- or allopolyploid.

To better define the sweetpotato polyploidy, we sought to reconstruct phylogenies of its subgenomes based on hybridization networks that could trace reticulate lineages of differentiated homoeolog triplets of multiple single-copy genes. In search of such homoeolog triplets, we distinguished cDNA variants of 811 single-copy Conserved Ortholog Set II (COSII) genes from two sweetpotato clones into variation partitions specified by corresponding homologs from two I. trifida lines, I. tenuissima and I. littoralis using a phylogenetic partition method, and amplicon variants of the COSII-marker regions from 729 of these genes from two sweetpotato clones into putative homoeoallele groups using haplotype tree and the partition methods referenced by corresponding homologs from I. tenuissima. These analyses revealed partly or completely differentiated expressed-homoeologs and homoeologs from a majority of these genes with three important features. 1. Two variation types: the predominant interspecific variations (homoeoalleles), which are non-randomly clustered, differentially interspecifically conserved or sweetpotato-specific, and the minor intraspecific ones (alleles), which are randomly distributed mostly at non-interspecifically variable sites, and usually sweetpotato-specific. 2. A clear differentiation of cDNA variants of many COSII genes into the variation partition specified by I. tenuissima or I. littoralis from that by I. trifida. 3. Three species-homolog-specified and one sweetpotato-specific variation partitions among 293 different COSII cDNAs, and two or three out of the four partitions among cDNA variants of 306 COSII genes. We then constructed hybridization networks from two concatenations of 16 and 4 alignments of 8 homologous COSII cDNA regions each, which included three taxa of expressed homoeologs in a triple-partition combination from the 16 or 4 sweetpotato COSII genes and 5 taxa each of respective cDNA homologs from the three sweetpotato relatives and I. nil, and inferred a species tree embodying both networks. The species tree predicted close-relative origins of three partly differentiated sweetpotato subgenomes.

Development of wheat—Hordeum chilense Chromosome 2Hch Introgression Lines Potentially Useful for Improving Grain Quality Traits

The chromosome 2Hch of Hordeum chilense. has the potential to improve seed carotenoid content in wheat as it carries a set of endosperm carotenoid-related genes. We have obtained structural changes in chromosome 2Hch in a common wheat (Triticum aestivum L. “Chinese Spring”) background by crossing a wheat double disomic substitution 2Hch(2D) and 7Hch(7D) line with a disomic addition line carrying chromosome 2Cc from Aegilops cylindrica Host.. Seven introgressions of chromosome 2Hch into wheat were characterized by fluorescence in situ hybridization (FISH) and DNA markers. Chromosome-specific simple sequence repeats (SSRs) were used for identifying wheat chromosomes. In addition, we tested 82 conserved orthologous set (COS) markers for homoeologous group 2, of which 65 amplified targets in H. chilense and 26 showed polymorphism between H. chilense and wheat. A total of 24 markers were assigned to chromosome 2Hch with eight allocated to 2HchS and 16 to 2HchL. Among the seven introgressions there was a disomic substitution line 2Hch(2D), a ditelosomic addition line for the 2HchL arm, an isochromosome for the 2HchL arm, a homozygous centromeric 2HchS·2DL translocation, a double monosomic 2HchS·2DL plus 7HchS·D translocation, a homozygous centromeric 7HchS·2HchL translocation and, finally, a 2HchL·7HchL translocation. Wheat—H. chilense macrosyntenic comparisons using COS markers revealed that H. chilense chromosome 2Hch exhibits synteny to wheat homoeologous group 2 chromosomes, and the COS markers assigned to this chromosome will facilitate alien gene introgression into wheat. The genetic stocks developed here include new wheat—H. chilense recombinations which are useful for studying the effect of chromosome 2Hch on grain quality traits.

Introgression of a Novel Ug99-Effective Stem Rust Resistance Gene into Wheat and Development of Dasypyrum villosum Chromosome-Specific Markers via Genotyping-by-Sequencing (GBS).

Dasypyrum villosum is a wild relative of common wheat (Triticum aestivum L.) with resistance to Puccinia graminis f. tritici, the causal agent of stem rust, including the highly virulent race TTKSK (Ug99). In order to transfer resistance, T. durum-D. villosum amphiploids were initially developed and used as a bridge to create wheat-D. villosum introgression lines. Conserved ortholog set (COS) markers were used to identify D. villosum chromosome introgression lines, which were then subjected to seedling P. graminis f. tritici resistance screening with race TTKSK. A COS marker-verified line carrying chromosome 2V with TTKSK resistance was further characterized by combined genomic in situ and fluorescent in situ analyses to confirm a monosomic substitution line MS2V(2D) (20″ + 1' 2V[2D]). This is the first report of stem rust resistance on 2V, which was temporarily designated as SrTA10276-2V. To facilitate the use of this gene in wheat improvement, a complete set of previously developed wheat-D. villosum disomic addition lines was subjected to genotyping-by-sequencing analysis to develop D. villosum chromosome-specific markers. On average, 350 markers per chromosome were identified. These markers can be used to develop diagnostic markers for D. villosum-derived genes of interest in wheat improvement.

Identification of COS markers specific for Thinopyrum elongatum chromosomes preliminary revealed high level of macrosyntenic relationship between the wheat and Th. elongatum genomes.

Thinopyrum elongatum (Host) D.R. Dewey has served as an important gene source for wheat breeding improvement for many years. The exact characterization of its chromosomes is important for the detailed analysis of prebreeding materials produced with this species. The major aim of this study was to identify and characterize new molecular markers to be used for the rapid analysis of E genome chromatin in wheat background. Sixty of the 169 conserved orthologous set (COS) markers tested on diverse wheat-Th. elongatum disomic/ditelosomic addition lines were assigned to various Th. elongatum chromosomes and will be used for marker-assisted selection. The macrosyntenic relationship between the wheat and Th. elongatum genomes was investigated using EST sequences. Several rearrangements were revealed in homoeologous chromosome groups 2, 5, 6 and 7, while chromosomes 1 and 4 were conserved. Molecular cytogenetic and marker analysis showed the presence of rearranged chromosome involved in 6ES and 2EL arms in the 6E disomic addition line. The selected chromosome arm-specific COS markers will make it possible to identify gene introgressions in breeding programmes and will also be useful in the development of new chromosome-specific markers, evolutionary analysis and gene mapping.

Comparison of taxon-specific versus general locus sets for targeted sequence capture in plant phylogenomics.

Premise of the StudyTargeted sequence capture can be used to efficiently gather sequence data for large numbers of loci, such as single‐copy nuclear loci. Most published studies in plants have used taxon‐specific locus sets developed individually for a clade using multiple genomic and transcriptomic resources. General locus sets can also be developed from loci that have been identified as single‐copy and have orthologs in large clades of plants.MethodsWe identify and compare a taxon‐specific locus set and three general locus sets (conserved ortholog set [COSII], shared single‐copy nuclear [APVO SSC] genes, and pentatricopeptide repeat [PPR] genes) for targeted sequence capture in Buddleja (Scrophulariaceae) and outgroups. We evaluate their performance in terms of assembly success, sequence variability, and resolution and support of inferred phylogenetic trees.ResultsThe taxon‐specific locus set had the most target loci. Assembly success was high for all locus sets in Buddleja samples. For outgroups, general locus sets had greater assembly success. Taxon‐specific and PPR loci had the highest average variability. The taxon‐specific data set produced the best‐supported tree, but all data sets showed improved resolution over previous non‐sequence capture data sets.DiscussionGeneral locus sets can be a useful source of sequence capture targets, especially if multiple genomic resources are not available for a taxon.

Primers for Castilleja and their utility across Orobanchaceae: II. Single-copy nuclear loci.

Premise of the study:We developed primers targeting nuclear loci in Castilleja with the goal of reconstructing the evolutionary history of this challenging clade. These primers were tested across other major clades in Orobanchaceae to assess their broader utility.Methods and Results:We assembled low-coverage genomes for three taxa in Castilleja and developed primer combinations for the single-copy conserved ortholog set (COSII) and the pentatricopeptide repeat (PPR) gene family. These primer combinations were designed to take advantage of the Fluidigm microfluidic PCR platform and are well suited for high-throughput sequencing applications. Eighty-seven primers were designed for Castilleja, and 27 were found to have broader utility in Orobanchaceae.Conclusions:These results demonstrate the utility of these primers, not only across Castilleja, but for other lineages within Orobanchaceae as well. This expanded molecular toolkit will be an asset to future phylogenetic studies in Castilleja and throughout Orobanchaceae.

Chromosomal location of genes for resistance to powdery mildew in Agropyron cristatum and mapping of conserved orthologous set molecular markers

Agropyron cristatum exhibits resistance to Blumeria graminis f. sp. tritici. Disomic and ditelosomic chromosome addition lines of A. cristatum in ‘Chinese Spring’ wheat were utilized to determine which A. cristatum chromosomes carry resistance gene(s). Resistance is conferred by gene(s) on chromosome arms 2PL and 6PL. The availability of molecular markers capable of detecting these chromosome arms in a wheat background would be very useful for marker-assisted introgression of 2PL and 6PL chromatin into common wheat. With this aim, 170 wheat conserved orthologous set (COS) markers (92 and 78 from wheat homoeologous groups 2 and 6 respectively) were assessed for their utility in A. cristatum. A total of 116 (68.2%) COS markers successfully amplified product in A. cristatum and 46 (40.0%) of these markers were polymorphic between A. cristatum and common wheat. From marker loci mapping on wheat homoeologous group 2 chromosomes, 23 markers (34.9%) were polymorphic between A. cristatum and common wheat and from them 13 markers were assigned to chromosome arm 2PL and six markers were mapped to chromosome 4P of A. cristatum showing that this chromosome is related to wheat homoeologous group 2. From marker loci mapping on wheat homoeologous group 6 chromosomes, 23 (46.0%) markers were polymorphic between A. cristatum and common wheat and from them 17 markers were located on chromosome 6P, six of them were mapped to chromosome arm 6PS and five to chromosome arm 6PL, respectively. The specific COS markers allocated on the long arms of chromosomes 2P and 6P may have a role in marker-assisted screening in wheat breeding for powdery mildew disease resistance.

QTL mapping and comparative genome analysis of agronomic traits including grain yield in winter rye.

Genetic diversity in elite rye germplasm as well as F 2:3 testcross design enables fast QTL mapping to approach genes controlling grain yield, grain weight, tiller number and heading date in rye hybrids. Winter rye (Secale cereale L.) is a multipurpose cereal crop closely related to wheat, which offers the opportunity for a sustainable production of food and feed and which continues to emerge as a renewable energy source for the production of bioethanol and biomethane. Rye contributes to increase agricultural crop species diversity particularly in Central and Eastern Europe. In contrast to other small grain cereals, knowledge on the genetic architecture of complex inherited, agronomic important traits is yet limited for the outbreeding rye. We have performed a QTL analysis based on a F2:3 design and testcross performance of 258 experimental hybrids in multi-environmental field trials. A genetic linkage map covering 964.9cM based on SSR, conserved-orthologous set (COS), and mixed-phase dominant DArT markers allowed to describe 22 QTL with significant effects for grain yield, heading date, tiller number, and thousand grain weight across seven environments. Using rye COS markers, orthologous segments for these traits have been identified in the rice genome, which carry cloned and functionally characterized rice genes. The initial genome scan described here together with the existing knowledge on candidate genes provides the basis for subsequent analyses of the genetic and molecular mechanisms underlying agronomic important traits in rye.

Development of gene-based identification markers for Phalaenopsis ‘KS Little Gem’ based on comparative genome analysis

Gene-based markers are useful tools for genetic background surveys and comparative genome analysis due to their ease of application across closely related species. We developed gene-based DNA markers for Phalaenopsis ‘KS Little Gem’, a new Phalaenopsis variety with excellent ornamental traits, based on a genome-wide comparison of chloroplast and nuclear sequences with those of orchids as well as monocot species, including rice and banana, to establish a molecular basis for species identification. Sequence comparisons of chloroplast DNA from P. ‘KS Little Gem’ with those of P. aphrodite and P. equestris identified six variable genic regions of the chloroplast genome in which an intron of rpl16 was the most polymorphic between related orchid species. To develop conserved ortholog set (COS) markers, we compared transcriptome unigenes of P. ‘KS Little Gem’ with the P. equestris, rice, and banana genomes and identified 582 Phalaenopsis COS candidates with at least two exons. PCR application using primer sets targeting six variable regions of the chloroplast genome and introns of 45 randomly selected COS candidate genes showed 92-98% amplification in three Phalaenopsis species. Among the candidate genes, we developed rpl16 and three COS genes as diagnostic cross-species markers for P. ‘KS Little Gem’ and diverse, related orchids. The gene-based molecular markers developed in this study will play an important role in species identification to facilitate the protection of P. ‘KS Little Gem’ variety rights, as well as breeding and genetic studies of Phalaenopsis orchids.

Fine mapping of the restorer gene Rfp3 from an Iranian primitive rye (Secale cereale L.).

A comparative genetics approach allowed to precisely determine the map position of the restorer gene Rfp3 in rye and revealed that Rfp3 and the restorer gene Rfm1 in barley reside at different positions in a syntenic 4RL/6HS segment. Cytoplasmic male sterility (CMS) is a reliable and striking genetic mechanism for hybrid seed production. Breeding of CMS-based hybrids in cereals requires the use of effective restorer genes as an indispensable pre-requisite. We report on the fine mapping of a restorer gene for the Pampa cytoplasm in winter rye that has been tapped from the Iranian primitive rye population Altevogt 14160. For this purpose, we have mapped 41 gene-derived markers to a 38.8cM segment in the distal part of the long arm of chromosome 4R, which carries the restorer gene. Male fertility restoration was comprehensively analyzed in progenies of crosses between a male-sterile tester genotype and 21 recombinant as well as six non-recombinant BC4S2 lines. This approach allowed us to validate the position of this restorer gene, which we have designated Rfp3, on chromosome 4RL. Rfp3 was mapped within a 2.5cM interval and cosegregated with the EST-derived marker c28385. The gene-derived conserved ortholog set (COS) markers enabled us to investigate the orthology of restorer genes originating from different genetic resources of rye as well as barley. The observed localization of Rfp3 and Rfm1 in a syntenic 4RL/6HS segment asks for further efforts towards cloning of both restorer genes as an option to study the mechanisms of male sterility and fertility restoration in cereals.

Dissecting the U, M, S and C genomes of wild relatives of bread wheat (Aegilops spp.) into chromosomes and exploring their synteny with wheat.

Goat grasses (Aegilops spp.) contributed to the evolution of bread wheat and are important sources of genes and alleles for modern wheat improvement. However, their use in alien introgression breeding is hindered by poor knowledge of their genome structure and a lack of molecular tools. The analysis of large and complex genomes may be simplified by dissecting them into single chromosomes via flow cytometric sorting. In some species this is not possible due to similarities in relative DNA content among chromosomes within a karyotype. This work describes the distribution of GAA and ACG microsatellite repeats on chromosomes of the U, M, S and C genomes of Aegilops, and the use of microsatellite probes to label the chromosomes in suspension by fluorescence insitu hybridization (FISHIS). Bivariate flow cytometric analysis of chromosome DAPI fluorescence and fluorescence of FITC-labelled microsatellites made it possible to discriminate all chromosomes and sort them with negligible contamination by other chromosomes. DNA of purified chromosomes was used as a template for polymerase chain reation (PCR) using Conserved Orthologous Set (COS) markers with known positions on wheat A, B and D genomes. Wheat-Aegilops macrosyntenic comparisons using COS markers revealed significant rearrangements in the U and C genomes, while the M and S genomes exhibited structure similar to wheat. Purified chromosome fractions provided an attractive resource to investigate the structure and evolution of the Aegilops genomes, and the COS markers assigned to Aegilops chromosomes will facilitate alien gene introgression into wheat.

Identification of Conserved Orthologous Set Markers in Cultivated Vigna radiata (L.) Wilczek

Identification of Conserved Orthologous Set Markers in Cultivated Vigna radiata (L.) Wilczek With an aim to develop widely applicable conserved gene markers to identify and tag orthologous genes from related Vigna species to reveal phylogenetic relationships and the nature of genes conserved in genus Vigna across the evolution, low copy nuclear Conserved Ortholog Set (COS) genes were tested to explore the interspecific genetic relationship of Vigna radiata (L.) Wilczek with other species such as V. mungo (L.), V. umbellata Thunb., V. angularis Wild and V. unguiculata (L.) Walp. To detect COS regions, computational approach was followed utilizing the available expressed sequence tags (EST) database of Vigna radiata and its related species. The ESTs were processed to eliminate sequence repeats, contaminants and low-complexity sequences. Upon alignment with Soybean genome, only high quality ESTs were grouped into clusters from which consensus sequences representing putative genes are generated for each Vigna species. From 6443 ESTs, 2550 contigs were acquired to develop 230 primer pairs to amplify conserved orthologous sequences across Vigna species. Among 14 primer pairs used for validation with genotypes of V. radiata, 3 gave double bands (paralogs) and 9 produced single bands indicating single copy orthologs. This infers that DNA sequences identified by this comparative genomics approach would be of great use in analyzing genomic information of related, unexplored crop genomes to augment gene discovery and plant breeding in other legumes.

Combined Use of Molecular Markers and High-Resolution Melting (HRM) to Assess Chromosome Dosage in Potato Hybrids.

In plants, the most widely used cytological techniques to assess parental genome contributions are based on in situ hybridization (FISH and GISH), but they are time-consuming and need specific expertise and equipment. Recent advances in genomics and molecular biology have made PCR-based markers a straightforward, affordable technique for chromosome typing. Here, we describe the development of a molecular assay that uses single-copy conserved ortholog set II (COSII)-based single nucleotide polymorphisms (SNPs) and the high-resolution melting (HRM) technique to assess the chromosome dosage of interspecific hybrids between a Solanum phureja-S. tuberosum diploid (2n = 2x = 24) hybrid and its wild relative S. commersonii. Screening and analysis of 45 COSII marker sequences allowed S. commersonii-specific SNPs to be identified for all 12 chromosomes. Combining the HRM technique with the establishment of synthetic DNA hybrids, SNP markers were successfully used to predict the expected parental chromosome ratio of 5 interspecific triploid hybrids. These results demonstrate the ability of this strategy to distinguish diverged genomes from each other, and to estimate chromosome dosage. The method could potentially be applied to any species as a tool to assess paternal to maternal ratios in the framework of a breeding program or following transformation techniques.

Flow sorting of C-genome chromosomes from wild relatives of wheat Aegilops markgrafii, Ae. triuncialis and Ae. cylindrica, and their molecular organization.

Aegilops markgrafii (CC) and its natural hybrids Ae. triuncialis (U(t)U(t)C(t)C(t)) and Ae. cylindrica (D(c)D(c)C(c)C(c)) represent a rich reservoir of useful genes for improvement of bread wheat (Triticum aestivum), but the limited information available on their genome structure and the shortage of molecular (cyto-) genetic tools hamper the utilization of the extant genetic diversity. This study provides the complete karyotypes in the three species obtained after fluorescent in situ hybridization (FISH) with repetitive DNA probes, and evaluates the potential of flow cytometric chromosome sorting. The flow karyotypes obtained after the analysis of 4',6-diamidino-2-phenylindole (DAPI)-stained chromosomes were characterized and the chromosome content of the peaks on the flow karyotypes was determined by FISH. Twenty-nine conserved orthologous set (COS) markers covering all seven wheat homoeologous chromosome groups were used for PCR with DNA amplified from flow-sorted chromosomes and genomic DNA. FISH with repetitive DNA probes revealed that chromosomes 4C, 5C, 7C(t), T6U(t)S.6U(t)L-5C(t)L, 1C(c) and 5D(c) could be sorted with purities ranging from 66 to 91%, while the remaining chromosomes could be sorted in groups of 2-5. This identified a partial wheat-C-genome homology for group 4 and 5 chromosomes. In addition, 1C chromosomes were homologous with group 1 of wheat; a small segment from group 2 indicated 1C-2C rearrangement. An extensively rearranged structure of chromosome 7C relative to wheat was also detected. The possibility of purifying Aegilops chromosomes provides an attractive opportunity to investigate the structure and evolution of the Aegilops C genome and to develop molecular tools to facilitate the identification of alien chromatin and support alien introgression breeding in bread wheat.

Identification of a new molecular marker C2-25 linked to the Fusarium oxysporum f.sp. radicis-lycopersici resistance Frl gene in tomato

Fusarium oxysporum Schlecht. f.sp. radicis-lycopersici Jarvis & Schoemaker (FORL) is a saprophytic fungus, responsible for the fusarium crown and root rot disease in tomato (Solanum lycopersicum L.). This is one of the most destructive pathogens of this species. A new cleaved amplified polymorphic sequence (CAPS) marker C2-25 was developed for the detection of the dominant gene Frl, which confers tomato resistance to FORL. C2-25 was amplified from a conserved ortholog set II (COSII) sequence C2_At2g38025. The XapI-derived restriction product of 700 bp was informative for the identification of FORL resistant tomato genotypes and can be used as a diagnostic marker in tomato breeding programmes and hybrid seed production.

Cytological and molecular characterization of wheat-Hordeum chilense chromosome 7Hch introgression lines

Chromosome 7Hch of Hordeum chilense carries the Phytoene synthase 1 (Psy1) gene encoding the first step in the carotenoid biosynthetic pathway. As such it can be used in the improvement of seed carotenoid content in wheat. Four introgressions of chromosome 7Hch into wheat have been characterized by in situ hybridization of labeled DNA probes and by several sets of DNA markers. Chromosome-specific SSR were used for the identification of wheat chromosomes. Besides 113 conserved orthologous set (COS) markers were tested for homoeologous group 7, of which 97 amplified in H. chilense and 32 were polymorphic between H. chilense and wheat, and 28 expressed sequence tag (EST) barley markers previously allocated to chromosome 7. A total of 60 markers (32 COS and 28 EST) were allocated to chromosome 7Hch with 28 assigned to 7HchS and 22 to 7HchL. A combination of in situ probing and marker genotyping have shown that among the four introgressions there was a substitution line 7Hch (7D), a ditelosomic addition line for the long arm of 7Hch and two homozygous centric translocations 7HchS·2DS and 7HchS·5AL. The Psy1 gene was localized on the short arm of 7Hch. The positions of markers from the international barley consortium map (IBSC2012) were determined and the comparative arm location between H. chilense and H. vulgare is discussed. The genetic stocks characterized here include new wheat-H. chilense recombinations useful for genetic studies and with a potential for breeding.