Final Report Summary - ALIENC (Introgression of alien chromosomes from barley into wheat breeding lines to increase genetic variation: epigenetic control of centromere function stability and transmission) Wheat wild relatives carry valuable characters that are missing from current wheat varieties and these are required now to enhance European and global food security. However, one of the fundamental challenges of wheat improvement is the production of stable basic materials carrying the full range of the alien genetic variability. One part of the alien genome cannot be transmitted in modern pre-breeding programs, due to the instability of the alien chromosomes.The present project aimed to reveal mechanisms underlying selective chromosome elimination in wheat-barley introgression lines, to find new possibilities for effective gene transfer into wheat. According to our hypothesis, the elimination of the alien chromosomes can be explained by the behaviour of the centromere, the specific chromosomal regions responsible for poleward movement during mitosis and meiosis. We combined up-to-date molecular cytogenetics and cell biology approaches with confocal microscopy and next generation sequencing to study the organisation of the alien centromere, its epigenetic control and behaviour during cell division. The prerequisite for accurate centromere function is the presence of a centromere specific histon H3 variant within the centromeric nucleosomes. Wheat-barley hybrid lines, carrying an additional barley chromosome pair in the wheat genetic background were used in the present work to analyse the interaction of the barley centromeric DNA with wheat CENH3 protein and to test whether an insuficient CENH3 loading causes chromosome instability. We directly visualised barley chromatin and wheat and barley centromeric DNA (retrotransposons and repeats) on chromosomes of the addition lines by using genomic in situ hybridisation (GISH) and fluorescence in situ hybridisation (FISH) and detected no major differences in their organisation compared to the published data on parental cultivars (Figure 1a, b). We then combined in situ hybridisation with immunolabelling we observed that CENH3 protein can be loaded onto the alien centromeric sequences so barley centromeres are able to function as active centromeres in wheat background (Figure1c, d). To obtain a more detailed analysis of the alien centromeric sequences we carried out next generation sequencing including the wheat-barley addition lines and parental cultivars. After extracting centromeric DNA of plant tissue by chromatin immunoprecipitacion (ChIP) we used ChIP-sequencing with the help of Illumina MiSeq platform (Figure 2a). We detected a reorganisation of the centromeric DNA within the addition lines compared to the parental lines (Figure 2b), suggesting major evolutionary changes occuring at the centromeric regions following the hybridisation event. We aimed to study epigenetic mechanisms involved in chromosome retention by monitoring changes in the DNA cytosine methylation within the host and alien genome. Importantly, we revealed that the complete barley chromatin is hypomethylated in both stable and unstable addition lines (ie. the 7H or 3H chromosomes, respectively) suggesting an epigenetic reprogramming for the alien genome. On the other hand, wheat chromosomes showed uneven methylation intermitted with hypermethylated clusters while the centromeric regions tended to be hypomethylated (Figure 3a, b). Hypomethylation is known to be linked to chromosome instability and transposon reactivation, and can thus explain the frequent elimination of the alien barley chromosomes. Hypomethylation of the centromeric regions might be involved in the high evolutionary rate of the centromeric DNA by transposon reactivation.Finally, we investigated whether chromosome elimination is caused by a cell cycle asynchrony between the parental cultivars leading to chromosome loss. We carried out the first detailed cell cycle timing of CENH3 dynamics in hexaploid wheat. Our analyses detected stable CENH3 signal throughout mitosis while meiotic cells exhibited dramatically brighter CENH3 fluorescence intensity compared to the mitotic ones and showed a highly dynamic centromere movement at early meiosis. Despite substantial efforts, it prouved to be challenging to set up an accurate chronology of CENH3 dynamism based on chromatin morphology alone. Meiotic prophase one is marked by the alignement and pairing of homologous chromosomes facilitated by the formation of a proteinaceous structure, the synaptonemal complex (SC) between them. Axial- and central element proteins of the SC can be detected as early as meiotic interphase and early prophase one, respectively, and their specific progression provides a well defined staging. The present work used an original approach to define the correct timing of centromere dynamism by performing dual immunolabelling of CENH3- and SC proteins. Dual immunolabelling detected with high resolution confocal laser scanning microscopy (CLSM) enabled to correlate centromere movement to chromosome arm synapsis reporting the first direct evidence on how centromere associations progress to homologous pairing (Figure 4, 5). CENH3 timing in wild type wheat served as a reference to study CENH3 dynamism and SC formation in the addition lines. In order to determine where and when chromosome elimination occurs, we followed CENH3 signal of the barley chromosomes during meiosis. We showed an atypical CENH3 signal and SC axial element formation within the 3H addition line (Figure 6a, b) and detected abnormal tetrade formation at late meiosis of the 7H addition line. We detected lagging chromosomes with yet active centromeres, but in some of the cases the lagging chromosomes belonged to the wheat genome, showing that chromosome instability is not limited to the alien genome and is rather a result of meiotic errors in this specific line Figure 7a, b). These findings represent a substantial progress in the study of chromosome stability and open up new scientific questions. During wheat-barley hybrid production viable F1 plants were regenerated from tissue culture, which is known to result in a large array of genetic mutations. Genetic mutations can be overcame by backcrossing the deficient plants to wild type wheat. Plants carrying the desired alien chromosomes can be selected by modern cytogenetic methods (In situ hybridisation, molecular markers) and chromosome stability and fertility can be restored.
