Identification And Chromosome Constitution Of Thinopyrum Intermedium And Octoploid Trititrigia

As one of the important relative species of Triticum aestivum,Th.intermedium?2n=6x=42,E^eE^eE^bE^bStSt or JJJ^SJ^SStSt?possessed many valuable traits such as large and multiflorous spikes,growingluxuriantly,resist to different diseases and tolerat to salt and drought,so it became one of the frequently used wild species in wheat improvement.Wheat-Th.intermedium chromosome lines derived from the cross of the common wheat and Th.intermedium,including octoploid Trititrigia?wheat-Th.intermedium partial amphiploid?,wheat-Th.intermedium addition line,wheat-Th.intermedium substitution line and so on.These germplasms showedcopositive excellent traits from their parents and they were the bridge materials used for transferring beneficial genes from Th.intermedium into common wheat.The genome composition and molecular evolution of Th.intermedium were analyzed in this study by using molecular cytogentics,comparative transcriptomics and molecular markers techniques.The agronomic trait,chromosome constitution and structural variation of Shannong TE series octoploid Trititrigia.Main results were as follows:1.Long terminal repeat?LTR?-pDb12H of Dasypyrum Villosum?2n=14,VV?and the genomic DNA of Pseudoroegneria strigosa?2n=14,StSt?were used as the probes while the genomic DNA of Th.bessarabicum?2n=14,JJ or E^bE^b?and common wheat YN15 were used as the block to hybrid with the root tip cell of Th.intermedium.Results showed that 42chromosomes of Th.intermedium could be divided into 3 genomes.Based on the results of studies on cytogenetics,the genome formula of Th.intermedium could beexpressed as JJJ^vsJ^vsStSt.Multiple probes including oligonucleotides of pAs1-4,pSc119.2-1,?GAA?₁₀,?AAC?₆ and pTa71 were used as probes to analysis the karyotype of Th.intermedium,seven pairs of homologous chromosomes could be identified in 3 different genomes of Th.intermedium.2.Tender leaves of Th.intermedium,Th.elongatum?2n=14,EE or E^eE^e?,Th.bessarabicum,Ps.strigosa and Th.ponticum?2n=10x=70,E^eE^eE^bE^bE^xE^xStStStSt or JJJJJJJ^SJ^SJ^SJ^S?were used for RNA-sequencing,the data were used to screen for orthologous genes and then used to constructphylogenetic trees of Th.intermedium and its relatives.The Ka/Ks value,GO?Gene Ontology?and KEGG?Kyoto Encyclopedia of Genes and Genomes?were also analysized among these orthologous genes.The result showed that Th.elongatum and Th.bessarabicum had close relationship but it also had highest differentiation degree,Ps.strigosa had closer relationships to Th.intermedium than Th.elongatum and Th.bessarabicum.Among the orthologous genes which were from several relatives,neutral gene?0.1<Ka/Ks<1?accounted for 64.07%,conserved genes?Ka/Ks<0.1?accounted for 32.19%,divergence gene?Ka/Ks>1?accounted for 3.73%;There are 6 divergence genesrelated to metal ion binding,the conserved genes which were related to organic substance metabolic process,ion binding and organic cyclic compound binding had a large number,besides,the conserved genes enriched to the metabolic pathway of the ribosome obviously.3.Based on the RNA-seq data of Th.intermedium,Th.elongatum,Th.bessarabicum and Ps.strigosa,a set of EST-SSR markerwere designed and its genome and chromosome location were analysised.In total,101,88,50 and 96 specific molecular markers belonged to Th.intermedium,E?E^e?genome,J?E^b?genome and St genome were screened and identified,respectively.Chromosome location of these specific molecular markers were analysised by using disomic alien addition line of Chinese Spring-Th.elongatum disomic alien addition lines?1-7E?and Chinese Spring-Th.bessarabicum disomic alien addition lines?1-7J?.Finally,1,1,3,5,3 and 1 chromosome specific markers belong to 1E to 7E?except 5E?,and 1,1,2,2and 3 chromosome specific markers belongs to 1J,2J,5J,6J and 7J were identified,respectively.4.Characters of octoploid Trititrigiawere studied,including powdery mildew and stripe rust resistance,aphid resistantance,cold tolerance and lodging traits.Most octoploid Trititrigia were immune or high resistance to stripe rust and nearly immune to powdery mildew in adult-plant stage.Besides,it had higher resistance to aphid and stronger ability to avoid lodging.5.The alien chromosome constitution of eighteen octoploid Trititrigia were analyzed by GISH,the results indicated that alien chromosomes in TE256 are 2St+8J^S+2J+2J-St,TE257 are 2St+8J^S+2J+2J-St,TE257-1 are 10J^S+2J+2J-St,0184-1 are 4St+6J^S+2J+2J-St,TE259 are 10J^S+4J,TE260 are 10J^S+4J,TE261 are 2St+8J^S+2J+2J-St,TE261-1 are2St+6J^S+2a-J^S?acrocentric chromosome?+2J+2J-St,TE262 are 2St+8J^S+4J,TE263 are2St+8J^S+2J+2J-St,TE265 are 10J^S+4J,TE266 are 4St+6J^S+2J+2J-St,TE267 are 4St+6J^S+4J,TE274 are 10J^S+2J+2J-St,TE346 are 2St+8J^S+2J+2J-St,TE347 are 10J^S+4J.The number of alien chromosomes in TE270,TE273 are not stable.The alien chromosome?E and J subgenome?homologous group constitution of eighteen octoploid Trititrigia were also analyzed by chromosome specific molecular markers,the results indicated that TE256 had2E+4E+6E+7E+1J+4J+7J,TE257 had 2E+4E+6E+7E+4J+7J,TE257-1 had 2E+4E+6E+7E+1J+4J+7J,0184-1 had 2E+4E+6E+7E+1J+4J+7J,TE259 had 2E+4E+6E+7E+1J+4J+7J,TE260 had 2E+4E+6E+7E+1J+4J,TE261 had 2E+4E+6E+7E+4J,TE261-1 had 2E+4E+6E+1J+4J+5J,TE262 had 2E+4E+6E+7E+1J+4J+5J,TE263 had 2E+4E+6E+7E+1J+4J+5J,TE265 had 2E+4E+7E+4J,TE266 had 2E+4E+6E+7E+4J+5J,TE267 had 2E+4E+6E+7E+4J+5J,TE270 had 2E+4E+6E+4J,TE273 had 2E+4E+6E+7E+4J,TE274 had 2E+4E+6E+7E+4J,TE346 had 2E+4E+7E+4J+7J,TE347 had 2E+4E+6E+7E+4J.All of octoploid Trititrigia had 2E,4E and 4J chromosomes,indicated that preferentially transferred of special chromosomes during the hybriding process of common wheat and Th.intermedium.6.The structural variations of common wheat chromosome in octoploid Trititrigia were detected by fluorescence in situ hybridization?FISH?,Multicolor Genomic in situ hybridization?McGISH?and wheat chromosome specific molecular markers.The results showed that the mutation sites of repetitive sequences were mainly located at the terminal of1AS arm and 1AL arm,the middle of 6AL arm,the terminal of 6BL arm,the near terminal of7DL arm.All wheat 7D chromosomes transferred into octoploid Trititrigia,whose terminal of long arms were replaced by the group-A chromosomes of wheat.There are also some other special variants,such as 2D chromosomes in TE257 and TE347 are replaced by A-D translocation chromosomes.7.Precise identification of alien chromosome composition and chromosomal structure variation in octoploid Trititrigia were made by using the developed multiple probes.The results indicated that the karyotype of the alien Chromosome in octoploid Trititrigia has been greatly changed compared with that of Th.intermedium,which resulted in the unidentified of some alien chromosomes.Compared with common used FISH probes,more repetitive sequence variation sites can be detected by the multiple probes,such as the centromere position in 2A,the middle of the 5AL,the terminal site of 1BL and 2BL,3BL.In addition,other severe structural changes were also detected,such as the absent of the terminal site of5BS in TE257-2,and the 4D chromosome of TE262*is replaced by the translocation chromosome of 4BS-6BL.8.Four whea-Th.intermedium addition lines?0655-5,0655-6,0655-7,0655-8?,which possessed good resistance to powdery mildew were characterized.The additional chromosomes in the four addition lines are 2E,but one chromosome of 0655-6 is acrocentric chromosome.