Identification Of Chromosomal Rearrangement And Analysis Of Grain Hardness Gene In Novel Wheat-thinopyrum Intermedium Addition Lines

Thinopyrum intermedium?2n=42,StStJ^sJ^sJJ?,a wild relative of wheat,is native to Europe and Western Asia.It obtained the ability of adptation to the adversity during the long-term evolution progresses due to the complex growth environment.Th.intermedium has provided abundant gene resources for high-yield,disease resistance or stress tolerance to wheat genetic breeding.The potential value of these resources can be achieved by transferring them into the wheat background.The research of Th.intermedium,Triticum aestivum-Th.intermedium partial amphiploid and wheat-Th.intermedium derivatives obtained from wheat-Thinopyrum hybridization is essential for the continuous and utilization of genetic resources from Th.intermedium.In this study,we obtained two Chinese Spring?CS?-Th.intermedium addition lines Hy36 and Hy37 through the BC₁F₆ generation from the hybridization between CS and wheat-Th.intermedium partial amphiploids Zhong 2 and Zhong 5,respectively.Th.intermedium,wheat-Th.intermedium partial amphiploids and two novel CS-Th.intermedium addition lines were analyzed through sequential non-denaturing fluorescence in situ hybridization?ND-FISH?using multiple Thinopyrum specific oligonucleotide?oligo?probes,molecular marker analysis and 90K SNP assay.The chromosomal constitution of them were characterized precisely.In addition,the Pina genes from Th.intermedium was sequenced by homology cloning strategy,and were subsequently used for a phylogenetic analysis.Field characters and grain quality of two alien addition lines were also investigated.The main results are as follows:?1?The karyotype of hexaploid Th.intermedium,wheat-Th.intermedium partial amphiploids Zhong 5 and Zhong 2,and two novel alien addition lines were identified by sequential ND-FISH with multiple new specific oligo-probes.We found that Zhong 5?2n=56?contains fourteen Th.intermedium chromosomes:6 St,2 J^s and 6 St-J^s translocation chromosomes.Zhong 2?2n=54?includes 42 wheat chromosomes and twelve alien chromosomes:6 J^s,2 St and 4 St-J^s translocation chromosomes.A pair of J^s-St translocation chromosomes were added to Hy36,and a pair of J-St translocation chromosomes were added to Hy37.We established an updated FISH banding patterns of Th.intermedium chromosomes by multicolor ND-FISH,which was helpful for the further studies and applications of Th.intermedium genomes.?2?Identification of Thinoprym chroatin in Hy36 and Hy37.The homoeologous groups of the added alien translocation chromosomes were determined in Hy36 and Hy37 based on 90K SNP array,PLUG?PCR-based landmark unique gene?molecular markers,IT?Intron targeting?molecular markers and Non denaturing polyacrylamide gel electrophoresis?Native-PAGE?analysis.The added chromosomes of Hy36 and Hy37 are 5J^sS.3StS and 5JS.3StS,respectively.?3?We successfully obtained the full sequence of Pina gene from Th.intermedium by homology cloning strategy.The gene is located on the short arm of chromosome 5J^s of Hy36.The protein sequence encoded by the gene has a conserved Ser-rich region?WRWWKWWK?,which located between the second and the third Cys residues.Also,we found that an amino acid substitution?Lys-to-Arg?occurred in the seventieth amino acid residue for the first time.?4?The agronomic traits of CS,Hy36 and Hy37 were measured at the adult stage in the field.Compared to Hy37,Hy36 showed a higher spikelet number and a longer grain length,but a shorter spike length.The plant height and tiller number of Hy36 and Hy37 are significantly lower than those of CS.The grain quality of Hy36,Hy37 and CS,were examined using near-infrared spectrometer DA7250.We found that the special Pina gene from Th.intermedium can soften seeds in wheat background due to lower hardness values were observed in Hy36 seeds.In addition,the grain protein contents of them were significantly higher than CS,which indicated some excellent gene resources from Th.intermedium could be used for the improvement of wheat quality.In the present study,we identified two novel wheat-Th.intermedium derivatives by molecular cytogenetic analysis.Chromosome rearrangements are ubiquitous in wheat-Th.intermedium progenies obtained by the processes of wheat-alien introgression.The identification of these rearrangement events are contributed to the evolutionary studies of the three sub-genomes of Th.intermedium.Also,these novel wheat-Th.intermedium derivatives can increase the diversity of wheat breeding resources and have great potential value for the practice of wheat breeding.