Molecular tagging of Thinopyrum distichum chromosomes involved in salt tolerance

[摘要] ENGLISH ABSTRACT: Much has been written about the effects of soil salinity on plant growth. Its devastating effects havealready been reported 2000 years BC. In the 21· century an alarming 80 million hectares ofcultivated land area are affected by salt (Munns, 2002a) and represent a growing threat toagriculture. Salt tolerance is a complex trait moderately expressed in only a few plant genotypes(Ruiz, 2001).An attempt to transfer salt tolerance genes from the wild grass, Thinopyrum distichum, to triticaleand éommon wheat was initiated by Marais and Marais (2003). A study of Th. distichum x ryehybrids enabled the authors to identify chromosomes 2Jld, 3Jld, 4Jld and SJld as being involved inthe determination of salt tolerance. Indirect (yet unconfirmed) evidence suggested that 7Jld mightalso have a role. A programme aiming to transfer regions of the critical chromosomes tohomoeologous triticale chromosomes, which relies heavily on the use of molecular markers, waslaunched. While an RFLP marker is available for each of the Thinopyrum chromosomes, these arenot suited for the screening of large numbers of segregates. This study therefore represents anattempt to convert the RFLP markers into less time consuming and cost-effective SCAR markers.The published DNA sequences of the RFLP probes in question were used as templates to designPCR primers. The PCR reactions were optimised using DNA of Th. distichum, rye and their FIhybrid. When Thinopyrum specific amplification products were obtained, the primers were alsotested on a panel of genotypes with and without the target chromosomes. Seemingly polymorphicbands were confirmed by Southern blotting and hybridisation with the corresponding RFLP probes.The primers were also tested on a panel of genotypes that included 'Rex' triticale to ensure that theywould also detect a difference in a triticale genetic background during transfer. Polymorphic bandswere then isolated and sequenced to further refine the markers. In certain eases, sequences of thesame fragment amplified in triticale ('Rex') and Thinopyrum were aligned in an attempt to designmore specific markers. Using this approach, it was possible to develop chromosome specificSCARs for Thinopyrum chromosomes 3Jld and 7J2d. Three and one set(s) of PCR markers,respectively, have been developed and can be used to unequivocally detect the Thinopyrumchromosomes involved in salt tolerance against a triticale background. A SCAR marker was alsofound for chromosome 6J. Thus, an attempt was made to convert thirteen RFLP probes to SCAR markers. Only three weresuccessfully converted. The main reason for the low success rate is the syntenic relationshipsbetween the genomes of the different cereals that made it an arduous- task to find discriminatingprimer sets. Based on the results obtained, an adapted procedure is suggested for future attempts todevelop chromosome specific markers utilizing published sequence information that was obtainedfor a different species.

[发布机构] Stellenbosch University