Development And Validation Of A STS Marker Set Specific To Thinopyrum Intermedium Genome

The narrow genetic basis caused by long-term domestication and breeding practices has become a bottleneck limiting the further improvement of wheat(Triticum aestivum L.,2n=6x=42)as the staple food crop.Hence,there is an immediate demand to increase genetic diversity of wheat to ensure biotic stress(such as rust and powdery mildew)or abiotic stress(eg.drought,salinity and high-temperature)adaptability and food security in future.Wild relatives of wheat provide the opportunity to introduce novel genes via wide hybridization and thus represent a valuable source of genetic variation.Thinopyrum intermedium(2n=6x=42,JJSSt),as one of the most successful wild relatives for wheat genetic improvement,is a promising reservoirs of useful genes including tolerance to abiotic stresses,perenniality and disease resistance not available in the wheat.In order to successfully introduce the superior genes of Thinopyrum,a key step is to quickly and accurately identify the alien chromosomes or fragments containing the target genes in wheat.The current evaluation of recombinant lines was generally performed manually through intensive and time-consuming cytogenetic methods and could not be analysed by high-throughput techniques.The challenge presented by the detection of introgressions requires the development of high-throughput chromosome-specific molecular markers covering the entire wild relative genome,which is of vital importance for the introduction of external genes in wheat,accelerating the process of wheat-Thinopyrum distant hybridization and chromosome engineering breeding.In this study,a total of 5,877,409 contigs were assembled from the published genotyping-by-sequencing probe database of Th.intermedium,and then 5,452 non-redundant sequences with less than 80%similarity to the wheat genome were obtained.Based on this,a total of 2019 STS(sequence-tagged site)markers distributing on all the 21 pairs of Th.intermedium chromosome was developed.By testing in wheat landraces and Th.intermedium(accession Z1141),852 STS markers specific to Th.intermedium genome were obtained.Subsequently,these markers were verified in Th.elongatum(2n=2x=14,Js),Th.bessarabicum(2n=2x=14,Jb),Pseudoroegneria strigosa(2n=2x=14,St)and Dasypyrum villosum(2n=2x=14,V),and 472 STS markers found to be chromosome-specific and polymorphic between wheat and Th.intermedium or the putative progenitor species of its subgenome or the donor species of V genome.Testing of the developed 852 STS markers in the progenitor species of Th.intermedium subgenome and V genome demonstrated that the relationship between Th.intermedium and Ps.strigosa is much closer than the tested other species.Next is V genome,supposing that D.villosum played an important role in the early evolution of Th.intermedium genome.These Th.intermedium-specific STS markers were also assigned to the subgenome chromosome and their homologous groups(HG)by examining the substitution and/or addition line sets of wheat-Th.intermedium,wheat-Thinopyrum and wheat-Th.elongatum.Except HG 2,to which the markers were only assigned the St-chromosome,all the HGs and their subgenome chromosomes could be distinguished with these specific markers.Among 852 STS markers specific to the subgenome chromosome,HG 1 had 98 including 36 on 1J,22 on 1JS and 40 on 1St;HG 2 had 33,and only on 2St;HG 3 had 120 including 38 on 3J,22 on 3JS and 60 3St;HG 4 had 82 including 21 on 4J,24 on 4JS and 37 4St;HG 5 had 156 including 39 on 5J,56 on 5JS,61 on 5St;HG 6 had 154 including 43 on 6J,55 on 6JS and 56 on 6St;and HG 7 had 183 including 54 on 7J,67 on 7JS and 62 on 7St.In addition,the Th.intermedium-specific STS marker set was used for determining the chromosome-alien constitution in partial wheat-Th.intermedium amphiploids(2n=8x=56)to confirm the reliability of this specific marker set.Of the tested partial amphiploids,the alien genome in TAI7044 consisted of 1J,2St,3St,4St,5J and 7J chromosome pairs,and one translocated chromosome pair of 6JS-6St;in TAI7045,the donor of powdery mildew resistance gene Pm43,the alien genome constitution includes 1St,2St,3 St,4St,5St and 7JS chromosome pairs,and one translocated chromosome pair involved the 6JS and 6J;in TAI7047,the donor of Pm40 and stripe rust resistance gene Yr50,the exotic genome was composed of three chromosome pairs including 1St,2St and 7JS,and four translocated chromosome pairs of 3JS-3St,4J-4St,5J-5St and 6J-6JS;and in TAI8047,the alien genome consisted of five chromosomes pairs,namely 1St,2St,3JS,4J and 7JS,and two pairs of translocated chromosomes of 5J-5St and 6J-6JS.Moreover,these findings largely matched with the results from genomic in situ hybridization experiment carried out in our laboratory.Therefore,the STS marker set presented in this study should be useful in increasing the amount of genetic diversity via wide hybridization,offering a significant speed-up in wheat breeding and making it possible to deal with the transfer of the full genetic potential of Th.intermedium into wheat.