Identification Of Mutagenic Progenies Of Trititrigia Alien Addition Line15 With In Situ Hybridization

Thinopyrum intermedium?2n=6x=42,JJJ^SJ^SStSt or E^eE^eE^bE^bStSt?is a perennial wild species in Triticeae.It has excellent resistance to drought,cold,salt,alkali and also to various wheat diseases,such as powdery mildew.It is a wild parent widely used in wheat genetic improvement at home and abroad.Our laboratory has created and identified the Trititrigia alien addition Line15 resistant to powdery mildew in the progenies of common wheat Yannong 15 and Th.intermedium.On this basis,Line15 was mutagenized,and M₃ plants were obtained.In this study,based on the identification of agronomic traits and powdery mildew resistance,GISH,FISH and molecular markers were used to identify their chromosome composition,thus laying a foundation for further research and utilization.The main results obtained are as follows:1.Using[AFA-3,AFA-4,pAs1-1,pAs1-3,pAs1-4,pAs1-6,pSc119.2-1 and?GAA?₁₀]as probes,the FISH patterns of Line15 showed that it contained a complete set of wheat chromosomes plus a pair of Th.intermedium chromosomes.Compared with the parent Yannong15,there were significant structural variations in wheat chromosomes 3A,1D,2D,4D and 5D.175 pairs of IT primers were used to amplify the polymorphism among Yannong 15,Th.intermedium and Line15,and 12 specific markers from the second partial homologous group were obtained,indicating that the alien chromosomes in Line15 were 2J of Th.intermedium.2.Agronomic traits.Compared to Line15,B18502-1-2 and B18502-3-1 had higher plant,longer spikes and more grains per ear.B18470-5 and B18470-6 had more spikelets with bigger spikes.The number of grains per spike of B18470-6 was up to 96.3.Resistance to powdery mildew.B18464-4,B18467-1-8,B18470-5,B18470-6,B18482-2,B18497-1-1,B18497-1-2,B18502-1-2,B18502-3-1 and B18490-1-3 showed high resistance to the isolate E20 at seedling stage and near immunity at adult stage.4.Identification of different substitution lines.GISH analysis,using Th.intermedium genomic DNA as probe and Yannong 15 genomic DNA as blocker,showed that B18497-1-1,B18502-1-2,B18502-3-1 contained 40 wheat chromosomes plus two 2J chromosomes of Th.intermedium,and that B18497-1-2 contained 41 wheat chromosomes and two 2J chromosomes of Th.intermedium.FISH based on the composite oligonucleotide probe set showed that a pair of 2D chromosomes in B18497-1-1,B18502-1-2,B18502-3-1 and a 2D chromosome in B18497-1-2 were absent.Therefore,B18497-1-1,B18502-1-2,B18502-3-1 were 2J?2D?disomic alien substitution lines,and B18497-1-2 was a two 2J addidion and one 2D substitution line.Compared with Line15,chromosomes 7A,1B,2B,6B and 7B of B18497-1-1,1A,2A,7A,2B and 3B of B18502-1-2,7A,1B,2B,6B and 4D of B18502-3-1,and 1A,2A,7A and 4D of B18497-1-2 had different structural changes,suggesting that they were different lines.5.Identification of addition lines.GISH analysis showed that B18490-1-3 carried 42 wheat chromosomes plus one 2J ditelosome of Th.intermedium.Compared to Line15,FISH revealed that structural variations had taken place in chromosomes 3A,4A,7A,4B,6B and 7B.6.Identification of introgression lines.GISH,probed with Th.intermedium genomic DNA and bloked with Yannong 15 genomic DNA,didn't detect alien hybridized signal in B18482-2,B18470-5 and B18470-6,indicating that they were introgression lines.FISH showed that the banding patterns of chromosomes 5A,1B,4B,7B of B18482-2,1A,2A,7A,5B,7B,1D,2D of B18470-5,5A,6A,7A,2B,5B,1D,2D,4D of B18470-6 had changed compared with Line15,indicating that chromosomal structural variations had occurred during their formation process.Differences in FISH patterns and agronomic traits suggested that they were different introgression lines.7.Other structural variations.B18467-1-8 and B18464-4 have more spikelets and grains per spike.B18467-1-8 is shorter with plump grains,while B18464-4 is taller with shriveled grains.No exogenous chromosome/fragment was detected in both of them by GISH,and chromosome deletion,breakage and reconnection were found in both of them by FISH.Chromosome fragments of 5B and 6B of B18467-1-8 and chromosomes 3A and 4A of B18464-4 were missing,and two D-genomic chromosome fragments were added.