Efficient enrichment of synchronized mouse spermatocytes suitable for genome-wide analysis.

Chromatin-based mechanisms regulating developmental transitions during meiosis are fundamental but understudied aspects of male gametogenesis. Indeed, chromatin undergoes extensive remodeling dur-ing meiosis, leading to specific patterns of gene expression and chromosome organization, which ulti-mately controls fundamental meiotic processes such as recombination and homologous chromosome associations. Recent game-changing advances have been made by analysis of chromatin binding sites of meiotic specific proteins genome-wide in mouse spermatocytes. However, further progress is still highly dependent on the reliable isolation of sufficient quantities of spermatocytes at specific stages of prophase I. Here, we describe a combination of methodologies adapted for rapid and reliable isolation of synchronized fixed mouse spermatocytes. We show that chromatin isolated from these cells can be used to study chromatin binding sites by ChIP-seq. High quality data we obtained from INO80 ChIP-seq in zygotene cells was used for functional analysis of chromatin binding sites.

Linear Dose Response of Acrocentric Chromosome Associations (Aca) To Gamma Irradiation In Human Lymphocytes

Purpose: The frequency of acrocentric chromosome associations (ACA) was studied to determine the possible dose-response relation with low doses of gamma irradiation in lymphocytes. Methods: Peripheral blood collected from three healthy donors were irradiated with 0, 0.1, 0.25, 0.5, 0.75, and 1 Gy gamma radiation. Chromosomal preparations were made after 48 hrs culture as per the standard guidelines. Results: The average number of ACA and ACA % were increased significantly with an increase in a dose. The D-G and D-D type of association was most prominent and showed a dose-dependent increase. The ACA frequency in irradiated lymphocytes showed an increase concerning the dose. The fitted regression equation was y=0.4759x+0.1663 (R2=0.9635; p=0.0005). An assessment of dicentric chromosomes (DC) was carried for the same slides. The correlation curve was prepared for ACA frequencies versus DC frequencies, resulting in a regression equation as y=8.659x+0.2.37 (R2=0.8275; p=0.0119). Conclusion: Our results showed an increase in frequencies of ACA in irradiated lymphocytes with an increase in radiation dose and followed a similar linear trend with DC frequency, thus, ACA may serve as a candidate cytogenetic biomarker for radiation biodosimetry especially for low radiation doses.

Analysis of Chromosome Associations during Early Meiosis in Wheat Lines Carrying Chromosome Introgressions from Agropyron cristatum

Crested wheatgrass (Agropyron cristatum L. Gaertn., genome P), included in the Triticeae tribe (family Poaceae), is one of the most important grasses in temperate regions. It has been valued as a donor of important agronomic traits for wheat improvement, including tolerance to cold, drought, and high salinity, as well as resistance to leaf rust, stripe rust, and powdery mildew. For successful incorporation of beneficial alleles into wheat, it is essential that recombination between wheat and A. cristatum chromosomes occurs. In this work, we analysed chromosome associations during meiosis in wheat lines carrying chromosome introgressions from A. cristatum chromosomes 5P and 6P in the presence and absence of Ph1 locus using fluorescence in situ hybridisation. The results showed that the Ph1 locus does not affect chromosome associations between A. cristatum and wheat chromosomes because there were no interspecific chromosome associations; therefore, no recombination between chromosomes from wheat and Agropyron were observed in the absence of the Ph1 locus. The 5P and 6P A. cristatum chromosomes do not have a suppressor effect on the Ph1 locus. Wheat univalents in metaphase I suggest that Agropyron chromosomes might carry genes having a role in wheat homologous chromosome associations. Putative effect of the Agropyron genes on wheat chromosome associations does not interact with the Ph1 locus.

Sporogenesis in F1 hybrids from the crosses between common wheat and lines with introgressions from Amblyopyrum muticum

Hybridization of wheat lines comprising fragments of alien genetic material (introgression) with common wheat cultivars is effective and widespread means of transferring alien genes into genomes of modern wheat cultivars, and remains the main method of expending genetic pool of common wheat using genes of wild relatives. Success of such transfer depends on the processes of sporo- and gametogenesis in F1 hybrids, therefore cytological assessment of this processes is obligatory. Stages of meiosis and microgametogenesis were studied on cytological preparations of spikes of F1 hybrids from reciprocal crosses of common wheat cultivars and wheat lines of introgression origin with alien genetic material from wheat wild relative Amblyopyrum muticum. Sporogenesis in F1 hybrids occurs with disorders in both male and female sexual areas. Instead of 21 closed bivalents chromosome configurations in maximal association of chromosomes in M1 PMC could contain up to 8 open bivalents, up to 12 univalents, including three- and quadrivalents. In A1 lagging chromatids were observed, and up to 5 micronuclei per cell were registered in tetrads. Quantitative characteristics of chromosome associations in M1 PMC did not differ for hybrids obtained using introgression lines as female (direct crossing) and male (reverse crossing) cross components. The difference between reciprocal crosses was detected only for the quantity of cells in tetrads with different quantity of micronuclei. F1 hybrids from direct crosses had smaller portion of cells without micronuclei, and more cells with 1-3 micronuclei compared to hybrids of reverse crosses.

Telomeres and Subtelomeres Dynamics in the Context of Early Chromosome Interactions During Meiosis and Their Implications in Plant Breeding

Genomic architecture facilitates chromosome recognition, pairing, and recombination. Telomeres and subtelomeres play an important role at the beginning of meiosis in specific chromosome recognition and pairing, which are critical processes that allow chromosome recombination between homologs (equivalent chromosomes in the same genome) in later stages. In plant polyploids, these terminal regions are even more important in terms of homologous chromosome recognition, due to the presence of homoeologs (equivalent chromosomes from related genomes). Although telomeres interaction seems to assist homologous pairing and consequently, the progression of meiosis, other chromosome regions, such as subtelomeres, need to be considered, because the DNA sequence of telomeres is not chromosome-specific. In addition, recombination operates at subtelomeres and, as it happens in rye and wheat, homologous recognition and pairing is more often correlated with recombining regions than with crossover-poor regions. In a plant breeding context, the knowledge of how homologous chromosomes initiate pairing at the beginning of meiosis can contribute to chromosome manipulation in hybrids or interspecific genetic crosses. Thus, recombination in interspecific chromosome associations could be promoted with the aim of transferring desirable agronomic traits from related genetic donor species into crops. In this review, we summarize the importance of telomeres and subtelomeres on chromatin dynamics during early meiosis stages and their implications in recombination in a plant breeding framework.

Wheat, Rye, and Barley Genomes Can Associate during Meiosis in Newly Synthesized Trigeneric Hybrids

Polyploidization, or whole genome duplication (WGD), has an important role in evolution and speciation. One of the biggest challenges faced by a new polyploid is meiosis, in particular, discriminating between multiple related chromosomes so that only homologs recombine to ensure regular chromosome segregation and fertility. Here, we report the production of two new hybrids formed by the genomes of species from three different genera: a hybrid between Aegilops tauschii (DD), Hordeum chilense (HchHch), and Secale cereale (RR) with the haploid genomic constitution HchDR (n = 7× = 21); and a hybrid between Triticum turgidum spp. durum (AABB), H. chilense, and S. cereale with the constitution ABHchR (n = 7× = 28). We used genomic in situ hybridization and immunolocalization of key meiotic proteins to establish the chromosome composition of the new hybrids and to study their meiotic behavior. Interestingly, there were multiple chromosome associations at metaphase I in both hybrids. A high level of crossover (CO) formation was observed in HchDR, which shows the possibility of meiotic recombination between the different genomes. We succeeded in the duplication of the ABHchR genome, and several amphiploids, AABBHchHchRR, were obtained and characterized. These results indicate that recombination between the genera of three economically important crops is possible.

Chromosome Manipulation for Plant Breeding Purposes

The transfer of genetic variability from related species into crops has been a main objective for decades in breeding programs. Breeders have used interspecific genetic crosses and alien introgression lines to achieve this goal, but the success is always dependent on the interspecific chromosome associations between the alien chromosomes and those from the crop during early meiosis. In this Special Issue, the strength of chromosome manipulation in a breeding framework is revealed through research and review papers that combine molecular markers, cytogenetics tools and other traditional breeding techniques. The papers and reviews included in this Special Issue “Chromosome manipulation for plant breeding purposes” describe the development and/or characterization of new plant material carrying desirable traits and the study of chromosome associations and recombination during meiosis. New tools to facilitate the transfer of desired traits from a donor species into a crop can be developed by expanding the knowledge of chromosome associations during meiosis.

Identification of a meiosis-specific chromosome movement pattern induced by persistent DNA damage

ABSTRACTAs one of the main events occurring during meiotic prophase, the dynamics of meiotic chromosome movement is not yet well understood. Currently, although it is well-established that chromosome movement takes an important role during meiotic recombination promoting the pairing between homologous chromosomes and avoiding excessive chromosome associations, it is mostly unclear whether those movements follow a particular fixed pattern, or are stochastically distributed. Using Schizosaccharomyces pombe as a model organism, which exhibits dramatic meiotic nuclear oscillations, we have developed a computationally automatized statistical analysis of three-dimensional time-lapse fluorescence information in order to characterize nuclear trajectories and morphological patterns during meiotic prophase. This approach allowed us to identify a patterned oscillatory microvariation during the meiotic nuclear motion. Additionally, we showed evidence suggesting that this unexpected oscillatory motif might be due to the detection of persistent DNA damage during the nuclear movement, supporting how the nucleus also regulates its oscillations. Our computationally automatized tool will be useful for the identification of new patterns of nuclear oscillations during gametogenesis.

Derived alleles of two axis proteins affect meiotic traits in autotetraploid Arabidopsis arenosa

Polyploidy, which results from whole genome duplication (WGD), has shaped the long-term evolution of eukaryotic genomes in all kingdoms. Polyploidy is also implicated in adaptation, domestication, and speciation. Yet when WGD newly occurs, the resulting neopolyploids face numerous challenges. A particularly pernicious problem is the segregation of multiple chromosome copies in meiosis. Evolution can overcome this challenge, likely through modification of chromosome pairing and recombination to prevent deleterious multivalent chromosome associations, but the molecular basis of this remains mysterious. We study mechanisms underlying evolutionary stabilization of polyploid meiosis using Arabidopsis arenosa, a relative of A. thaliana with natural diploid and meiotically stable autotetraploid populations. Here we investigate the effects of ancestral (diploid) versus derived (tetraploid) alleles of two genes, ASY1 and ASY3, that were among several meiosis genes under selection in the tetraploid lineage. These genes encode interacting proteins critical for formation of meiotic chromosome axes, long linear multiprotein structures that form along sister chromatids in meiosis and are essential for recombination, chromosome segregation, and fertility. We show that derived alleles of both genes are associated with changes in meiosis, including reduced formation of multichromosome associations, reduced axis length, and a tendency to more rod-shaped bivalents in metaphase I. Thus, we conclude that ASY1 and ASY3 are components of a larger multigenic solution to polyploid meiosis in which individual genes have subtle effects. Our results are relevant for understanding polyploid evolution and more generally for understanding how meiotic traits can evolve when faced with challenges.

Relationship between meiotic behaviour and fertility in backcross-1 derivatives of the [(Gossypium hirsutum × G. thurberi)2 × G. longicalyx] trispecies hybrid

Wild cotton species are an important source of desirable genes for genetic improvement of cultivated cotton Gossypium hirsutum Linnaeus, 1763. For the success of such an improvement, chromosome pairings and recombinations in hybrids are fundamental. The wild African species G. longicalyx Hutchinson & Lee, 1958 could be used as donor of the desirable trait of fiber fineness. Twelve BC1 plants obtained from the backcrossing of [(G. hirsutum × G. thurberi Todaro, 1877)2 × G. longicalyx] (AhDhD1F1, 2n = 4x = 52) trispecies hybrid (HTL) by G. hirsutum (cv. C2) (AhAhDhDh, 2n = 4x = 52) were investigated for meiotic behaviour and plant fertility. Their chromosome associations varied as follows: (2.5 to 11.5) I + (17 to 22) II + (0.31 to 1.93) III + (0.09 to 1.93) IV + (0 to 0.07) V + (0 to 0.14) VI. Their pollen fertility ranged from 4.67 to 32.10 %. Only four BC1 plants produced a few seeds through self-pollination. The remaining BC1 were totally self-sterile and usually presented the highest number of univalents. All BC1 materials produced BC2 seeds (0.44 to 6.50 seeds per backcross) with the number of seeds negatively correlated with the number of univalents (R2 = 0.45, P < 0.05). Most BC1 plants gave significantly finer fiber compared to the cultivated G. hirsutum. SSR markers showed a segregation of wild alleles among the backcross derivatives and Genomic in situ hybridization (GISH) revealed presence of entire chromosomes of G. longicalyx as well as recombinant chromosomes in the backcross derivatives. The significance and details of these results are presented and the prospects of successfully exploiting these plant materials are discussed.