Molecular Cytogenetic Analysis Of New Wheat-thinopyrum Intermedium Ssp. Trichophorum Introgression Lines

Thinopyrum intermedium?2n=6x=42,JJJ^sJ^sStSt?,a perennial species related to wheat?Triticum aestivum L.?in the Triticeae tribe,carries many resistance genes against salt,drought,winter hardiness,powdery mildew?Blumeria graminis f.sp.tritici?,stripe rust?Puccinia striiformis f.sp.tritici?,leaf rust?P.recondita f.sp.tritic?,stem rust?P.graminis f.sp.tritici?and Fusarium head blight,becoming a valuable source of germplasm for wheat distant hybridization and chromosome engineering.Th.intermedium ssp.trichophorum?2n=6x=42,JJJ^sJ^sStSt?,a subspecies of Th.intermedium,shows pubescence on the heads and leaves in contrast to Th.intermedium,and exhibits superior resistance against various diseases,making it a potential gene pool for wheat improvement.TE1508,a wheat-Th.intermedium ssp.trichophorum partial amphiploid,was developed in our laboratory,expressing immune or highly resistant to powdery mildew,stripe rust,leaf rust and sharp eyespot at the adult stage.A wheat-Th.intermedium 7J?7A?substitution line was originated from the progenies of the partial amphiploid TAF46 and the common wheat cultivar Vilmorin27,displaying purple coleoptile and resistance to stem rust?Ug99?.In order to transfer the excellent genes from Thinopyrum to cultivated wheat and enrich the genetic diversity of wheat,we conducted the following studies:?1?By crossing TE1508 with both hexaploid triticale line and common wheat,we obatined a series of new wheat-alien introgression lines from the progenies of the cross.Then we used non-denaturing fluorescence in situ hybridization?ND-FISH?,genomic in situ hybridization?GISH?,molecular markers,disease assessment to evaluate the potential value of those newly developed introgression lines for broadening the wheat genetic diversity,and map some elite genes transferred from Thinopyrum to wheat.?2?By using ND-FISH and molecular markers,we characterized multiple wheat-Th.intermedium 7J translocation lines and mapped a gene controlling the purple coleoptile trait at the seedling stage.The main results are as follows:1.By using sequential multicolor fluorescence in situ hybridization?mc-FISH?analysis with Oligo-pSc119.2,Oligo-pTa535,Oligo-pSt122,Oligo-?GAA?₇,Oligo-?CAA?₇,Oligo-3A1,Oligo-5SrDNA,Oligo-6H-2-100 and Oligo-pTa71,and GISH analysis with Pseudoroegneri stipifolia total genomic DNA as a probe,we identified the constitution of somatic mitotic cell of TE1508?2n=56?with 40W+6St+4J+6J^s,including 20 pairs of wheat chromosomes?missing one pair of wheat 4B chromosomes?+three pairs of St-genome chromosomes?one pair of 1St,one pair of6St and one pair of 7St?+three pairs of J^s-genome chromosomes?one pair of 2J^s,one pair of 4J^s and one pair of 5J^s?+two pairs of J-genome chromosomes?one pair of 3J and one pair of 4J?.We used FISH to analyze their chromosome compositions of F₄ to F₆ progenies derived from the trigeneric hybrids between TE1508 and Currency,the results showed that:?1?Th.intermedium chromosomes were more preferentially transmitted than rye chromosomes in the latter generations of selfed progenies;?2?Among the different groups of Thinopyrum chromosomes,the J^s-genome chromosomes exhibited higher transmission rates than the J-and St-genome chromosomes.2.By using mc-FISH analysis with Oligo-pSc119.2,Oligo-pTa535 and other oligo probes,and GISH analysis with Th.intermedium total genomic DNA as a probe,we identified the chromosome karyotypes of somatic mitotic cells of of the newly developed introgression lines,the results were as follows:?1?X24C-10 was a stable wheat-Th.intermedium ssp.trichophorum 4J?4B?disomic substitution line;?2?X24C-5was a stable wheat-Th.intermedium ssp.trichophorum 4J^s?4B?disomic substitution line;?3?X24C-14 was a stable wheat-Th.intermedium ssp.trichophorum 2J^s disomic addition line;?4?A1082 was a stable wheat-Th.intermedium ssp.trichophorum 3J disomic addition line;?5?X24C-1-1 was a stable wheat-Th.intermedium ssp.trichophorum T4BS·4JL disomic translocation line;?6?A5-5 is a stable wheat-Th.intermedium ssp.trichophorum T4BS·5J^sL Robertsonian translocation line.3.We used Australian pathotypes of the rusts?stripe rust,leaf rust and stem rust?and Chinese Pst pathotypes to conduct the disease assessment of the newly developed introgression lines,the results were as follows:?1?the 4J?4B?substitution line X24C-10 exhibited high levels of resistance to Pst pathotypes at the all stage,and highly resistant to Pgt pathotype at the seedling stage;?2?the 4J^s?4B?substitution line X24C-5 exhibited moderately resistant to Pgt pathotype at the seedling stage,and highly resistant to Pst pathotypes at the adult stage;?3?the 2J^s addition line X24C-14exhibited high levels of resistance to Pst pathotypes at the all stage,and moderately resistant to Pt pathotype and Pgt pathotypes at the seedling stage;?4?the 3J addition line A1082 was nearly immune to Pt pathotype and Pgt pathotypes at the seedling stage,and nearly immune to Pst pathotypes at the adult stage;?5?the T4BS·4JL translocation line X24C-1-1 exhibited high levels of resistance to Pst pathotypes at the all stage;?6?the Robertsonian translocation line T4BS·5J^sL displayed moderately resistant to Pst pathotypes at the adult stage,and immune to the widely virulent Pst pathotype 239E237 A-17+33+at the seedling stage,whereas highly susceptible to the low virulent race 108 E205 A+at the seedling stage.4.We used two different types of molecular markers,PCR-based landmark unique gene?PLUG?and intron targeting?IT?,to establish the homoeologous relationships between Th.intermedium and wheat chromosomes.We assigned 81,97,174,71,106 Th.intermedium-specific markers to 4J,4J^s,2J^s,3J,and 5J^sL,respectively.5.By analyzing the differences between the 4J?4B?substitution line X24C-10 and the T4BS·4JL translocation line X24C-1-1 in the agronomic trait,disease resistance and chromosome karyotypes,we assumed that a blue grain gene and a stripe rust resistance gene were located in 4JL bin 0.60-1.00,a stem rust resistance gene were located in the chromosome 4JS or 4JL bin 0.00-0.60.And one new R2R3-MYB transcription factor ThMYB4J was isolated from the 4JL bin 0.60-1.00,and might be related to the gene controlling the blue grain.6.We used ND-FISH and molecular markers to:?1?identify the FISH karyotype of chromosome 7J;?2?obtain 88 7J-specific markers;?3?characterize 12 wheat-Th.intermedium 7J translocation lines;?4?construct a physical map of chromosome 7J;?5?map a purple coleoptile gene to the 7JS bin 0.35-0.62;?6?design two new 7J chromosome-specific EST-PCR markers.In summary,the present study created a series of new wheat-Th.intermedium introgression lines carrying excellent agronomic traits,and initially mapped a stripe rust resistance gene,a stem rust resistance gene,a blue grain gene and a purple coleoptile gene,and cloned one R2R3-MYB transcription factor ThMYB4J related to the blue grain gene.To some extent,the newly developed wheat-alien introgression lines will make up for the limited availability of resistance genes in cultivated wheat,and be valuable in wheat resistance breeding and improvement program.