Genomic insights from the first chromosome-scale assemblies of oat (Avena spp.) diploid species

Background Cultivated hexaploid oat (Common oat; Avena sativa) has held a significant place within the global crop community for centuries; although its cultivation has decreased over the past century, its nutritional benefits have garnered increased interest for human consumption. We report the development of fully annotated, chromosome-scale assemblies for the extant progenitor species of the As- and Cp-subgenomes, Avena atlantica and Avena eriantha respectively. The diploid Avena species serve as important genetic resources for improving common oat’s adaptive and food quality characteristics. Results The A. atlantica and A. eriantha genome assemblies span 3.69 and 3.78 Gb with an N50 of 513 and 535 Mb, respectively. Annotation of the genomes, using sequenced transcriptomes, identified ~ 50,000 gene models in each species—including 2965 resistance gene analogs across both species. Analysis of these assemblies classified much of each genome as repetitive sequence (~ 83%), including species-specific, centromeric-specific, and telomeric-specific repeats. LTR retrotransposons make up most of the classified elements. Genome-wide syntenic comparisons with other members of the Pooideae revealed orthologous relationships, while comparisons with genetic maps from common oat clarified subgenome origins for each of the 21 hexaploid linkage groups. The utility of the diploid genomes was demonstrated by identifying putative candidate genes for flowering time (HD3A) and crown rust resistance (Pc91). We also investigate the phylogenetic relationships among other A- and C-genome Avena species. Conclusions The genomes we report here are the first chromosome-scale assemblies for the tribe Poeae, subtribe Aveninae. Our analyses provide important insight into the evolution and complexity of common hexaploid oat, including subgenome origin, homoeologous relationships, and major intra- and intergenomic rearrangements. They also provide the annotation framework needed to accelerate gene discovery and plant breeding.

The Effect of Chromosome Structure upon Meiotic Homologous and Homoeologous Recombinations in Triticeae

The tribe Triticeae contains about 500 diploid and polyploid taxa, among which are important crops, such as wheat, barley and rye. The phylogenetic relationships, genome compo-sition and chromosomal architecture, were already reported in the pioneer genetic studies on these species, given their implications in breeding-related programs. Hexaploid wheat, driven by its high capacity to develop cytogenetic stocks, has always been at the forefront of these studies. Cytogenetic stocks have been widely used in the identification of homoeologous relationships between the chromosomes of wheat and related species, which has provided valuable information on genome evolution with implications in the transfer of useful agronomical traits into crops. Meiotic recombination is non-randomly distributed in the Triticeae species, and crossovers are formed in the distal half of the chromosomes. Also of interest for crops improvement is the possibility of being able to modulate the intraspecific and interspecific recombination landscape to increase its frequency in crossover-poor regions. Structural changes may help in this task. In fact, chromosome truncation increases the recombination frequency in the adjacent intercalary region. However, structural changes also have a negative effect upon recombination. Gross chromosome rearrangements produced in the evolution usually suppress meiotic recombination between non-syntenic homoeologs. Thus, the chromosome structural organization of related genomes is of great interest in designing strategies of the introgression of useful genes into crops.

Molecular Cytogenetic Mapping of Satellite DNA Sequences in Aegilops geniculata and Wheat

Fluorescence in situ hybridization (FISH) provides an efficient system for cytogenetic analysis of wild relatives of wheat for individual chromosome identification, elucidation of homoeologous relationships, and for monitoring alien gene transfers into wheat. This study is aimed at developing cytogenetic markers for chromosome identification of wheat and Aegilops geniculata (2n = 4x = 28, UgUgMgMg) using satellite DNAs obtained from flow-sorted chromosome 5Mg. FISH was performed to localize the satellite DNAs on chromosomes of wheat and selected Aegilops species. The FISH signals for satellite DNAs on chromosome 5Mg were generally associated with constitutive heterochromatin regions corresponding to C-band-positive chromatin including telomeric, pericentromeric, centromeric, and interstitial regions of all the 14 chromosome pairs of Ae. geniculata. Most satellite DNAs also generated FISH signals on wheat chromosomes and provided diagnostic chromosome arm-specific cytogenetic markers that significantly improved chromosome identification in wheat. The newly identified satellite DNA CL36 produced localized Mg genome chromosome-specific FISH signals in Ae. geniculata and in the M genome of the putative diploid donor species Ae. comosa subsp. subventricosa but not in Ae. comosa subsp. comosa, suggesting that the Mg genome of Ae. geniculata was probably derived from subsp. subventricosa.

Development of oat-based markers from barley and wheat microsatellites

Although microsatellites are an efficient and reliable genetic marker system, availability is limited in cultivated oat ( Avena sativa L.). Previous research has suggested that microsatellites from related species may be adapted to oat. This study investigated the stability of existing oat microsatellites, sequenced polymorphic oat amplicons derived from wheat ( Triticum aestivum L.) and barley ( Hordeum vulgare L.) primers, and redesigned primers to develop oat-based markers. We evaluated 161 published oat microsatellites and identified 9 with polymorphism between mapping parents Ogle1040 and TAM O-301 (OT). We also studied 30 wheat, 1 Aegilops tauschii Coss., and 9 barley primers with reported oat polymorphism. Sixteen primers (1 A. tauschii, 10 wheat, 5 barley) amplified random oat sequences and were used to generate 28 new oat STS markers. Eight primers, 4 each from wheat and barley, amplified oat repetitive motifs, generating 10 new oat SSRs. Four additional SSRs were developed from characterization of thaumatin-like pathogenesis-related protein sequences formerly utilized as the Rast1–4 oat marker. These new markers, along with 9 existing oat SSRs and 6 previously identified disease resistance loci, were mapped in the OT population, joining 3 pairs of linkage groups. Map locations of multiallelic SSRs and disease-resistance QTL interactions suggested possible homoeologous relationships among the oat chromosomes.

EST-SNPs in bread wheat: discovery, validation, genotyping and haplotype structure

The present study involves discovery, validation and use of single-nucleotide polymorphisms (SNPs) in bread wheat utilizing 48 EST-contigs (individual contigs having 20-89 ESTs, derived from 2 to 11 different genotypes). In order to avoid a problem due to homoeologous relationships, the ESTs in each contig were classified into 175 sub-contigs (3.7 sub-contigs/EST-contig) using characteristic homoeologue sequence variants (HSVs), which had a density of 1 HSV every 136.7 bp. In silico analysis of sub-contigs led to the discovery of 230 candidate EST-SNPs with a density of 1SNP/273.9 bp. Locus specific primers (each primer pair flanking 1–18 SNPs) were designed utilizing one sub-contig each from 42 EST-contigs that contained SNPs, the remaining 6 contigs having no SNPs. To provide locus specificity to the PCR products, each primer was tagged with an HSV at its 3' end. Only 10 primer pairs, which gave each a characteristic solitary band, were utilized to validate EST-SNPs over 30 diverse bread wheat genotypes; 7 SNPs were validated through resequencing the PCR products. Allele specific primers were designed and utilized for genotyping of 50 diverse bread wheat accessions (including 30 bread wheat genotypes previously used for validation of SNPs), with an aim to test their utility in genotyping and map construction. The allele specific primers allowed the classification of 50 genotypes in two alternative allele groups for each SNP as expected, thus suggesting their utility for genotyping. Of the above 7 validated SNPs, 4 belonged to a solitary locus (PKS37); 7 haplotypes were available at this locus. Altogether, the results suggested that EST-SNPs constitute an important source of molecular markers for studies on wheat genomics.

Molecular cytogenetic identification of nullisomy 5B induced homoeologous recombination between wheat chromosome 5D and barley chromosome 5H

Chromosome 5H of Hordeum vulgare 'New Golden' (NG) carries a gene(s) that accelerates heading in a wheat background. To introduce the early heading gene(s) of NG barley into the wheat genome, we attempted to induce homoeologous recombination between wheat and NG 5H chromosomes by 5B nullisomy. A nullisomic 5B, trisomic 5A, monosomic 5H plant (2n = 42) was produced from systematic crosses between aneuploid stocks of wheat group 5 chromosomes. A total of 656 F2 plants produced by self-fertilization were screened for recombinants by a PCR assay with 3 5H-specific amplicon markers. Twelve plants (1.8%) were selected as putative wheat–barley 5H recombinants. Five of them were inviable or sterile and the remaining 7 were fertile and subjected to the progeny test. Cytological analyses using fluorescence in situ hybridization and C-banding revealed that 6 of the 7 progeny lines are true homoeologous recombinants between the long arms of chromosomes 5D and 5H, but that the other one was not a recombinant having an aberrant barley telosome. The 6 cytologically confirmed recombinant lines included only 2 types (3 lines each), which were reciprocal products derived from exchanges at the same distal interval defined by two flanking markers. One type had a small 5HL segment translocated to the 5DL terminal, and the other type had a small terminal 5DL segment translocated to the 5HL terminal. In the latter type, the physical length of translocated barley segments slightly differed among lines. Homoeologous recombinants obtained in this study should be useful for further chromosome manipulation to introgress a small interstitial 5HL chromosome segment with the early heading gene(s) to wheat. Preferential occurrence of restricted types of recombinants is discussed in relation to homoeologous relationships between wheat and barley chromosomes.Key words: genomic in situ hybridization, homoeologous pairing, Hordeum vulgare, introgression, recombinant, Triticum aestivum.