Construction And Application Of St-Chromosome FISH Karyotypes Of Thinopyrum Intermedium

The Thinopyrum species are vital source of resistance genes with being tolerant of numerous biotic and abiotic stresses.Thinopyrum intermedium（2n=6x=42）and Thinopyrum ponticum（2n=10x=70）have been used in wheat chromosome engineering breeding programs since the 1950s.Due to the successful application practice for several decades,it has been proved that the Thinopyrum species makes an outstanding contribution to wheat disease resistance breeding.However,no chromosome-based draft sequence of Th.intermedium and Th.ponticum can be acquired,and even the FISH karyotypes have yet to be fully characterized,which makes it difficult to improve the efficiency of germplasm identification and breeding material screening.In this study,13 wheat–Thinopyrum disomic alien lines were identified,including six wheat–Th.intermedium disomic addition lines（DALs）,as well as 7 wheat–Th.intermedium disomic substitution lines（DSLs）.Based on in situ hybridization（mc-GISH and sequential FISH–GISH）and molecular marker（PLUG marker and SNP array）analysis,combined with morphological trait evaluation,the chromosomal composition of all the plant materials were characterized and analyzed clearly.The main results are as follows:（1）Pseudoroegneria spicata（St）genomic DNA and Thinopyrum bessarabicum（J）DNA were used as mc-GISH probes,simultaneously.Sequential FISH–GISH analysis was performed on the same tip cell by using Oligo-p Ta535 and Oligo-p Sc119.2 as FISH probes,and P.spicata（St）genomic DNA as GISH probe.The FISH karyotypes of Th.intermedium St genome-chromosome were obtained accurately.Based on the FISH karyotypes of Th.bessarabicum,P.spicata,and Dasypyrum villosum,the individual FISH karyotype of Th.intermedium was constructed by comparative FISH analysis.Three different genomes of Th.intermedium could be expressed as:J^rJ^rJ^vsJ^vsSt St.（2）Based on FISH analysis,19 Abbondanza aneuploidy lines were characterized,including 11 ditelosomic lines,Dt1AS,Dt1BL,Dt3DL,Dt4BS,Dt4AS,Dt5DL,Dt6AS,Dt6BS,Dt6DL,Dt7AS and Dt7DS,as well as,8 nullisomic lines,N1A,N1B,N4B,N4D,N6A,N6B,N6D and N7B.The morphology traits of the wheat chromosome engineering basic materials were determined by evaluation of agronomic traits and disease resistance,which was of vital importance to use the aneuploids as parent lines to create germplasm materials.（3）Based on in situ hybridization and PLUG molecular marker analysis,ES-01（DA1St）,ES-02（DA2St）,ES-03（DA3St）,ES-05（DA5St-W）,ES-06(DA6J^vs-W)and ES-07(DA7J^vs)was identified.And the chromosomal composition of Zhong4 was 14A+16B+14D+2（1St）+2（2St）+2（3St）+2（5St-W）+2(6J^vs-W)+2(7J^vs)+2（t）.Sequential FISH–GISH analysis of octoploid TAF46 and its two derived lines,L4（DA4St）and L7（DA6St）,were carried out to finish the construction of St-chromosome FISH karyotypes of Th.intermedium.In addition,the chromosomal composition of Xiaoyan784（a wheat–Th.ponticum partial amphiploid）was determined as:14A+14B+15D+2（1St）+2（2St）+2（3St）+2（4J）+2（5St-W）+2(6J^vs-W)+2(7J^vsS).（4）Based on in situ hybridization and 15K SNP array analysis,5 wheat–Th.intermedium DSLs derived from Zhong4,ES-20(DS6J^vs-W（6A）),ES-23（DS2St（2A））,ES-24（DS3St（3D））,ES-25（DS2St（2A））and ES-26（DS2St（2A））,as well as 2 wheat–Th.ponticum DSLs derived from Xiaoyan784,ES-09（DS2St（2A））and ES-10（DS3St（3D））,were characterized.And the FISH karyotypes of the alien chromosomes originated from Th.intermedium or Th.ponticum were obtained,which further validated the chromosomal composition of Zhong4 and Xiaoyan784,as well as,the constructed FISH karyotype of Th.intermedium.（5）The specific germplasm materials,ES-23 and ES-09,as well as,ES-24 and ES-10,were characterized to confer the same chromosome composition with the alien chromosomes derived from different donors.According to FISH karyotype and PLUG marker analysis,agronomic traits and stripe rust resistance evaluation,the close homoeologous relationship of the alien chromosomes was further validated by chromosome pairing and genomic polymorphism analysis.Based on SLAF-seq,4 transferable St-chromosome-specific markers were developed,including two 2St-chromosome-specific markers（PTH-005 and PTH-013）and two 3St-chromosome-specific markers（PTH-113 and PTH-135）.（6）ES-24 was highly resistant to wheat stripe rust at seedling stage.Huixianhong was used as the recurrent parent to create a BC₁F₂ population of ES-24.According to the FISH analysis combined with stripe rust evaluation of the BC₁F₂ population,all the resistant individuals carried chromosome 3St.It was suggested that Th.intermedium chromosome 3St carried the novel stripe rust resistant gene（s）.In addition,the utility of PTH-113 and PTH-135was further confirmed by PCR analyses of the BC₁F₂ individuals.The individual plants conferring remarkable stripe rust resistance also carried 3St chromosome-specific markers.（7）According to cytogenetic identification of the four wheat–Th.intermedium alien lines,ES-02（DA2St）,ES-23（DS2St（2A））,ES-25（DS2St（2B））,and ES-26（DS2St（2D））,different introduction forms of Th.intermedium chromosome 2St could be stably inherited.The agronomic traits evaluation result showed that ES-26 was the most similar to the parent line Abbondanza.Based on SLAF-seq and RNA-seq,bioinformatic analysis results further suggested that the replacement of chromosome 2D by alien chromosome 2St may have relatively minimal adverse effects on plant growth and development,because of the homoeologous compensation of chromosome 2St.In this study,13 novel wheat–Thinopyrum alien lines carrying remarkable agronomic traits were characterized and could sever as valuable germplasm materials for wheat chromosome engineering breeding;The FISH karyotype of Th.intermedium and the newly developed chromosome-specific molecular markers were able to rapidly trace Thinopyrum St-chromosome in a common wheat background,and it was valuable to improve the efficiency of germplasm material identification;Analyzing the effects of Th.intermedium chromosome 2St on common wheat provided a theoretical basis for creating alien DSLs for wheat chromosome engineering programs.