Analysis Of Structural Variation And Histone Modification Of Alien Chromosomes In Wheat Background

Within a long history of domestication,wheat germplasm has been improved by introducing genetic resource of wheat related species.Thinopyrum species,which are perennial species related to wheat,have been hybridized with wheat to develop various of wheat-Thinopyrum introgression lines possessing novel disease resistance and excellent agronomic traits.Introgression and structural variations including wheat-alien chromosome translocations often remain in offsprings’genetic bacgkground after multiple crosses and backcrosses,which can affect the expression of related genes and change the traits of plants.Several studies suggested that transmission and structural variation of alien chromosomes in wheat background are related to the plant centromere function protein CENH3,which plays an important role in cell division.The hybrids between two wheat-Thinopyrum introgression lines and common wheat MY11 were used as the present research materials to carry out molecular and cytologenetic identification of alien chromosomes and their structural variations.The wheat-Thinopyrum introgression lines consist of A155（6Ae）,a wheat-Th.ponticum introgression line carrying genes Sr B and Sr26 for resistance to stem rust,and X479（1St/1B,4St S-4J^SS.4J^SL/4B）,a wheat-Th.intermedium introgression line with excellent resistance to powdery mildew and stripe rust.The immunofluorescence experiments of centromeric histone CENH3 and transcriptional active marker H3K36me3 histone modification on alien chromosomes were characterized,while RNA-seq was performed on structural variation materials invloved homologous group 1 chromosomes to explore differential gene expressions（DGEs）and their functions.The main results are as follows:1.By using non-denaturing FISH（ND-FISH）and Oligo-FISH Paints,the homologous group of St-J^S chromosome in line X479 was precisely identified,the chromosome composition was thus determined to be 4St S-4J^SS.4J^SL.Combined with Thi-specific makers analysis,the 4St part of the recombined chromosome was predicted about 53.7 Mb with the proportion 21%length of chromsome 4St.A new stable translocation 1BS.1St L was identified in F₅ lines from the cross of X479/MY11.A new stable deletion 6Ae S-and a new 6Ae-wheat translocation 1DS.6Ae were also identified in F₃of A155/MY11 hybrid.2.Through field stripe rust resistance observation,agronomic traits survey and quality analysis tested by near infrared grain detector,the stripe rust resistance,agronomic traits and seed quality of F₅ population from X479/MY11 and F₃ population from A155/MY11 were preliminarily analyzed.Chromosome 6Ae and 4J^SL showed resistance to stripe rust by investigating resistance reaction for individual plants with translocation chromosomes.Grain quality analysis showed that the wheat-6Ae introgression lines showed the seed protein content and grain hardness to increase by 22%and 9%compared those to MY11.The seed protein content of 4St S-4J^SS.4J^SL intrgression lines was 13%higher than that of 1St intrgression lines.3.By immunofluorescence staining observation,it was found that the wheat CENH3antibody can produce distinct signals in centromeric region of 6Ae,1St,4St S-4J^SS.4J^SL.Using the CENH3 mean fluorescence intensity of chromosome 3B as a standard control,it was found that the mean fluorescence intensity of 1St S.1BL was 98%stronger than that of 1BS.1St L,and CENH3 protein content was significantly increased,which explained the phenomenon that the transmission rate of 1St S.1BL was much higher than that of1BS.1St L with respect to CENH3 histone function in cell divison.At the same time,it was found that the H3K36me3 modification signal of chromosome 1St is enriched in centromeric region,the signal is stronger than that of 6B,and the signal at telomeric regions is much weaker.The H3K36me3 signal of the 1St part of the translocation lines1St S.1BL and 1BS.1St L was stronger than that of the 1B part,which confirmed that the epigenetic modification occurred in centromeric region of the translocation lines.4.RNA-seq analysis of 1St（1B）substitution line,1St S.1BL,1BS.1St L translocation lines showed that the genes from wheat and wheatgrass genomes were differentially expressed in the three tested materials.The numbers and physical locations of differentially expressed genes in the centromeric region of chromosome 1B were analyzed in above materials.The number of down-regulated 1BL-centromeric genes in1St material was 60%higher than that in 1St S.1BL material,and the number of down-regulated 1BS-cetromeric genes in 1St material was 72%higher than that in the 1BL.1St L material,which reflects the excellent complementarity of structure and function between1St and 1B translocations.Through the functional enrichment of the differentially expressed genes in the 1B centromeric region in the 1St（1B）substitution line,it was found that they are mainly related to cellular process,metabolic process,molecular binding functions and catalytic activities.In conclusion,this study combined structural variation indentification by FISH,centromeric histone distribution and histone modification levels by immino-staining,functions of differentially expressed genes by RNA-seq,as well stripe rust resistance and agronomic traits observation,to determine the genetic and epi-genetic effect of Thinopyrum chromosomes in wheat background.The structural variation and histone modification caused by the Thinopyrum were systematically discussed,which laid a potential theoritical foundation for the research of CENH3 centromere protein,histone modification level and the discovery of new resistant materials.