Genetic diversity, distribution and domestication history of the neglected GGAtAt genepool of wheat.

KEY MESSAGE: We present a comprehensive survey of cytogenetic and genomic diversity of the GGAtAt genepool of wheat, thereby unlocking these plant genetic resources for wheat improvement. Wheat yields are stagnating around the world and new sources of genes for resistance or tolerances to abiotic traits are required. In this context, the tetraploid wheat wild relatives are among the key candidates for wheat improvement. Despite its potential huge value for wheat breeding, the tetraploid GGAtAt genepool is largely neglected. Understanding the population structure, native distribution range, intraspecific variation of the entire tetraploid GGAtAt genepool and its domestication history would further its use for wheat improvement. The paper provides the first comprehensive survey of genomic and cytogenetic diversity sampling the full breadth and depth of the tetraploid GGAtAt genepool. According to the results obtained, the extant GGAtAt genepool consists of three distinct lineages. We provide detailed insights into the cytogenetic composition of GGAtAt wheats, revealed group- and population-specific markers and show that chromosomal rearrangements play an important role in intraspecific diversity of T. araraticum. The origin and domestication history of the GGAtAt lineages is discussed in the context of state-of-the-art archaeobotanical finds. We shed new light on the complex evolutionary history of the GGAtAt wheat genepool and provide the basis for an increased use of the GGAtAt wheat genepool for wheat improvement. The findings have implications for our understanding of the origins of agriculture in southwest Asia.

Tissue-Specific Transcriptome Analysis Reveals Candidate Transcripts Associated with the Process of Programmed B Chromosome Elimination in Aegilops speltoides.

Some eukaryotes exhibit dramatic genome size differences between cells of different organs, resulting from programmed elimination of chromosomes. Here, we present the first transcriptome analysis of programmed chromosome elimination using laser capture microdissection (LCM)-based isolation of the central meristematic region of Aegilops speltoides embryos where B chromosome (B) elimination occurs. The comparative RNA-seq analysis of meristematic cells of embryos with (Bplus) and without Bs (B0) allowed the identification of 14,578 transcript isoforms (35% out of 41,615 analyzed transcript isoforms) that are differentially expressed during the elimination of Bs. A total of 2908 annotated unigenes were found to be up-regulated in Bplus condition. These genes are either associated with the process of B chromosome elimination or with the presence of B chromosomes themselves. GO enrichment analysis categorized up-regulated transcript isoforms into 27 overrepresented terms related to the biological process, nine terms of the molecular function aspect and three terms of the cellular component category. A total of 2726 annotated unigenes were down-regulated in Bplus condition. Based on strict filtering criteria, 341 B-unique transcript isoforms could be identified in central meristematic cells, of which 70 were functionally annotated. Beside others, genes associated with chromosome segregation, kinetochore function and spindle checkpoint activity were retrieved as promising candidates involved in the process of B chromosome elimination.

Discovery of multi-megabase polymorphic inversions by chromosome conformation capture sequencing in large-genome plant species.

Chromosomal inversions occur in natural populations of many species, and may underlie reproductive isolation and local adaptation. Traditional methods of inversion discovery are labor-intensive and lack sensitivity. Here, we report the use of three-dimensional contact probabilities between genomic loci as assayed by chromosome-conformation capture sequencing (Hi-C) to detect multi-megabase polymorphic inversions in four barley genotypes. Inversions are validated by fluorescence in situ hybridization and Bionano optical mapping. We propose Hi-C as a generally applicable method for inversion discovery in natural populations.

Nondisjunction and unequal spindle organization accompany the drive of Aegilops speltoides B chromosomes.

B chromosomes (Bs) are supernumerary chromosomes, which are often preferentially inherited. When transmission rates of chromosomes are higher than 0.5, not obeying the Mendelian law of equal segregation, the resulting transmission advantage is collectively referred to as 'chromosome drive'. Here we analysed the drive mechanism of Aegilops speltoides Bs. The repeat AesTR-183 of A. speltoides Bs, which also can be detected on the Bs of Aegilops mutica and rye, was used to track Bs during pollen development. Nondisjunction of CENH3-positive, tubulin interacting B sister chromatids and an asymmetric spindle during first pollen grain mitosis are key for the accumulation process. A quantitative flow cytometric approach revealed that, independent of the number of Bs present in the mother plant, Bs accumulate in the generative nuclei to > 93%. Nine out of 11 tested (peri)centromeric repeats were shared by A and B chromosomes. Our findings provide new insights into the process of chromosome drive. Quantitative flow cytometry is a useful and reliable method to study the drive frequency of Bs. Nondisjunction and unequal spindle organization accompany during first pollen mitosis the drive of A. speltoides Bs. The prerequisites for the drive process seems to be common in Poaceae.

A Set of Cytogenetic Markers Allows the Precise Identification of All A-Genome Chromosomes in Diploid and Polyploid Wheat.

Karyotypes of 3 diploid wheat species containing different variants of the A-genome, Triticum boeoticum (A(b)), T. monococcum (A(b)), and T. urartu (A(u)), were examined using C-banding and FISH with DNA probes representing 5S and 45S rDNA families, the microsatellite sequences GAAn and GTTn, the already known satellite sequences pSc119.2, Spelt52, Fat, pAs1, and pTa535, and a newly identified repeat called Aesp_SAT86. The C-banding patterns of the 3 species in general were similar; differences were observed in chromosomes 4A and 6A. Besides 2 major 45S rDNA loci on chromosomes 1A and 5A, 2 minor polymorphic NORs were observed in the terminal part of 5AL and in the distal part of 6AS in all species. An additional minor locus was found in the distal part of 7A(b)L of T. boeoticum and T. monococcum, but not in T. urartu. Two 5S rDNA loci were observed in 1AS and 5AS. The pTa535 probe displayed species- and chromosome-specific hybridization patterns, allowing complete chromosome identification and species discrimination. The distribution of pTa535 on the A(u)-genome chromosomes was more similar to that on the A-genome chromosomes of T. dicoccoides and T. araraticum, thus confirming the origin of these genomes from T. urartu. The probe pAs1 allowed the identification of 4 chromosomes of T. urartu and 2 of T. boeoticum or T. monococcum. The Aesp_SAT86-derived patterns were polymorphic; main clusters were observed on chromosomes 1A(u )and 3A(u) of T. urartu and chromosomes 3A(b) and 6A(b) of T. boeoticum. Thus, a set of probes, pTa535, pAs1, GAAn and GTTn, pTa71, pTa794, and Aesp_SAT86, proved to be most informative for the analysis of A-genomes in diploid and polyploid wheat species.

Highly reactive chemicals meet haploidization.

Mutation of the sperm-specific phospholipase A and treatment of pollen with reactive oxygen species (ROS) reagents lead to the induction of maize haploids. ZmPOD65, a gene associated with sperm-specific ROS metabolism, also exhibits a haploidization effect.

B-A Chromosome Translocations Possessing an A Centromere Partly Overcome the Root-Restricted Process of Chromosome Elimination in Aegilops speltoides.

Some eukaryotes exhibit dramatic genome size differences between cells of different organs, resulting from the programmed elimination of chromosomes. Aegilops speltoides is an annual diploid species from the Poaceae family, with a maximum number of eight B chromosomes (Bs) in addition to its inherent seven pairs of standard A chromosomes (As). The Bs of this species undergo precise elimination in roots early in embryo development. In areal parts of the plant, the number of Bs is stable. To affect the root restricted process of B chromosome elimination, we employed X-ray mutagenesis, and different types of restructured Bs were identified. Standard Bs were observed in all analyzed shoots of mutagenized plants, while B-A translocations were only observed in 35.7% of F1 plants. In total 40 different B variants inconsistently escaped the elimination process in roots. As a result, mosaicism of B chromosome variants was found in roots. Only a small B chromosome fragment fused to an A chromosome was stably maintained in roots and shoots across F1 to F3 generations. The absence of B-A translocation chromosomes possessing a derived B centromere in root cells implies that the centromere of the B is a key component of the chromosome elimination process.

Analysis of Pollen Grains by Immunostaining and FISH in Triticeae Species.

In this chapter we describe protocols for immunolabeling and FISH of pollen grains undergoing postmeiotic mitosis using Aegilops speltoides, Secale cereale, and Hordeum vulgare as models. Tissue sectioning of pollen overcomes the problem of the pollen grain wall impermeability that interferes with immunolocalization and in situ hybridization. The crucial element of the protocol is the generation and immobilization of tissue sections. Our method facilitates the investigation of the microspore cell divisions and pollen grain maturation.

Supernumerary B chromosomes of Aegilops speltoides undergo precise elimination in roots early in embryo development.

Not necessarily all cells of an organism contain the same genome. Some eukaryotes exhibit dramatic differences between cells of different organs, resulting from programmed elimination of chromosomes or their fragments. Here, we present a detailed analysis of programmed B chromosome elimination in plants. Using goatgrass Aegilops speltoides as a model, we demonstrate that the elimination of B chromosomes is a strictly controlled and highly efficient root-specific process. At the onset of embryo differentiation B chromosomes undergo elimination in proto-root cells. Independent of centromere activity, B chromosomes demonstrate nondisjunction of chromatids and lagging in anaphase, leading to micronucleation. Chromatin structure and DNA replication differ between micronuclei and primary nuclei and degradation of micronucleated DNA is the final step of B chromosome elimination. This process might allow root tissues to survive the detrimental expression, or overexpression of B chromosome-located root-specific genes with paralogs located on standard chromosomes.

European maize genomes highlight intraspecies variation in repeat and gene content.

The diversity of maize (Zea mays) is the backbone of modern heterotic patterns and hybrid breeding. Historically, US farmers exploited this variability to establish today's highly productive Corn Belt inbred lines from blends of dent and flint germplasm pools. Here, we report de novo genome sequences of four European flint lines assembled to pseudomolecules with scaffold N50 ranging from 6.1 to 10.4 Mb. Comparative analyses with two US Corn Belt lines explains the pronounced differences between both germplasms. While overall syntenic order and consolidated gene annotations reveal only moderate pangenomic differences, whole-genome alignments delineating the core and dispensable genome, and the analysis of heterochromatic knobs and orthologous long terminal repeat retrotransposons unveil the dynamics of the maize genome. The high-quality genome sequences of the flint pool complement the maize pangenome and provide an important tool to study maize improvement at a genome scale and to enhance modern hybrid breeding.

Evolution of the S-Genomes in Triticum-Aegilops Alliance: Evidences From Chromosome Analysis.

Five diploid Aegilops species of the Sitopsis section: Ae. speltoides, Ae. longissima, Ae. sharonensis, Ae. searsii, and Ae. bicornis, two tetraploid species Ae. peregrina (= Ae. variabilis) and Ae. kotschyi (Aegilops section) and hexaploid Ae. vavilovii (Vertebrata section) carry the S-genomes. The B- and G-genomes of polyploid wheat are also the derivatives of the S-genome. Evolution of the S-genome species was studied using Giemsa C-banding and fluorescence in situ hybridization (FISH) with DNA probes representing 5S (pTa794) and 18S-5.8S-26S (pTa71) rDNAs as well as nine tandem repeats: pSc119.2, pAesp_SAT86, Spelt-1, Spelt-52, pAs1, pTa-535, and pTa-s53. To correlate the C-banding and FISH patterns we used the microsatellites (CTT)10 and (GTT)9, which are major components of the C-banding positive heterochromatin in wheat. According to the results obtained, diploid species split into two groups corresponding to Emarginata and Truncata sub-sections, which differ in the C-banding patterns, distribution of rDNA and other repeats. The B- and G-genomes of polyploid wheat are most closely related to the S-genome of Ae. speltoides. The genomes of allopolyploid wheat have been evolved as a result of different species-specific chromosome translocations, sequence amplification, elimination and re-patterning of repetitive DNA sequences. These events occurred independently in different wheat species and in Ae. speltoides . The 5S rDNA locus of chromosome 1S was probably lost in ancient Ae. speltoides prior to formation of Timopheevii wheat, but after the emergence of ancient emmer. Evolution of Emarginata species was associated with an increase of C-banding and (CTT)10-positive heterochromatin, amplification of Spelt-52, re-pattering of the pAesp_SAT86, and a gradual decrease in the amount of the D-genome-specific repeats pAs1, pTa-535, and pTa-s53. The emergence of Ae. peregrina and Ae. kotschyi did not lead to significant changes of the S*-genomes. However, partial elimination of 45S rDNA repeats from 5S* and 6S* chromosomes and alterations of C-banding and FISH-patterns have been detected. Similarity of the Sv-genome of Ae. vavilovii with the Ss genome of diploid Ae. searsii confirmed the origin of this hexaploid. A model of the S-genome evolution is suggested.

How Next-Generation Sequencing Has Aided Our Understanding of the Sequence Composition and Origin of B Chromosomes.

Accessory, supernumerary, or-most simply-B chromosomes, are found in many eukaryotic karyotypes. These small chromosomes do not follow the usual pattern of segregation, but rather are transmitted in a higher than expected frequency. As increasingly being demonstrated by next-generation sequencing (NGS), their structure comprises fragments of standard (A) chromosomes, although in some plant species, their sequence also includes contributions from organellar genomes. Transcriptomic analyses of various animal and plant species have revealed that, contrary to what used to be the common belief, some of the B chromosome DNA is protein-encoding. This review summarizes the progress in understanding B chromosome biology enabled by the application of next-generation sequencing technology and state-of-the-art bioinformatics. In particular, a contrast is drawn between a direct sequencing approach and a strategy based on a comparative genomics as alternative routes that can be taken towards the identification of B chromosome sequences.

Chromatin Ring Formation at Plant Centromeres.

We observed the formation of chromatin ring structures at centromeres of somatic rye and Arabidopsis chromosomes. To test whether this behavior is present also in other plant species and tissues we analyzed Arabidopsis, rye, wheat, Aegilops and barley centromeres during cell divisions and in interphase nuclei by immunostaining and FISH. Furthermore, structured illumination microscopy (super-resolution) was applied to investigate the ultrastructure of centromere chromatin beyond the classical refraction limit of light. It became obvious, that a ring formation at centromeres may appear during mitosis, meiosis and in interphase nuclei in all species analyzed. However, varying centromere structures, as ring formations or globular organized chromatin fibers, were identified in different tissues of one and the same species. In addition, we found that a chromatin ring formation may also be caused by subtelomeric repeats in barley. Thus, we conclude that the formation of chromatin rings may appear in different plant species and tissues, but that it is not specific for centromere function. Based on our findings we established a model describing the ultrastructure of plant centromeres and discuss it in comparison to previous models proposed for animals and plants.

B chromosomes of Aegilops speltoides are enriched in organelle genome-derived sequences.

B chromosomes (Bs) are dispensable components of the genome exhibiting non-Mendelian inheritance. Chromosome counts and flow cytometric analysis of the grass species Aegilops speltoides revealed a tissue-type specific distribution of the roughly 570 Mbp large B chromosomes. To address the question whether organelle-to-nucleus DNA transfer is a mechanism that drives the evolution of Bs, in situ hybridization was performed with labelled organellar DNA. The observed B-specific accumulation of chloroplast- and mitochondria-derived sequences suggests a reduced selection against the insertion of organellar DNA in supernumerary chromosomes. The distribution of B-localised organellar-derived sequences and other sequences differs between genotypes of different geographical origins.