Molecular tagging of Thinopyrum distichum chromosomes involved in salt tolerance

Loubser, Dalene (2004-03)

Thesis (MSc)--Stellenbosch University, 2004.

Thesis

ENGLISH ABSTRACT: Much has been written about the effects of soil salinity on plant growth. Its devastating effects have already been reported 2000 years BC. In the 21· century an alarming 80 million hectares of cultivated land area are affected by salt (Munns, 2002a) and represent a growing threat to agriculture. 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 triticale and éommon wheat was initiated by Marais and Marais (2003). A study of Th. distichum x rye hybrids enabled the authors to identify chromosomes 2Jld , 3Jld , 4Jld and SJld as being involved in the determination of salt tolerance. Indirect (yet unconfirmed) evidence suggested that 7Jld might also have a role. A programme aiming to transfer regions of the critical chromosomes to homoeologous triticale chromosomes, which relies heavily on the use of molecular markers, was launched. While an RFLP marker is available for each of the Thinopyrum chromosomes, these are not suited for the screening of large numbers of segregates. This study therefore represents an attempt 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 design PCR primers. The PCR reactions were optimised using DNA of Th. distichum, rye and their FI hybrid. When Thinopyrum specific amplification products were obtained, the primers were also tested on a panel of genotypes with and without the target chromosomes. Seemingly polymorphic bands 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 they would also detect a difference in a triticale genetic background during transfer. Polymorphic bands were then isolated and sequenced to further refine the markers. In certain eases, sequences of the same fragment amplified in triticale ('Rex') and Thinopyrum were aligned in an attempt to design more specific markers. Using this approach, it was possible to develop chromosome specific SCARs for Thinopyrum chromosomes 3Jld and 7J2 d . Three and one set(s) of PCR markers, respectively, have been developed and can be used to unequivocally detect the Thinopyrum chromosomes involved in salt tolerance against a triticale background. A SCAR marker was also found for chromosome 6J. Thus, an attempt was made to convert thirteen RFLP probes to SCAR markers. Only three were successfully converted. The main reason for the low success rate is the syntenic relationships between the genomes of the different cereals that made it an arduous- task to find discriminating primer sets. Based on the results obtained, an adapted procedure is suggested for future attempts to develop chromosome specific markers utilizing published sequence information that was obtained for a different species.

AFRIKAANSE OPSOMMING: Baie is al geskryf oor die uitwerking van grond versouting op plantproduksie. Die vernietigende gevolge van versouting is alreeds 2000 jaar VC gerapporteer. In die 21* eeu is 'n geraamde 80 miljoen hektaar (Munns, 2002a) bewerkte land-area sout-geaffekteerd. Die ontstellende verwikkelinge verteenwoordig 'n groeiende bedreiging vir die landbou. Soutverdraagsaamheid is 'n komplekse kenmerk en slegs enkele plantgenotipes met matige verdraagsaamheid kon nog ontwikkel word (Ruiz, 2001). 'n Poging om soutverdraagsaamheidsgene vanaf die wilde gras, Thinopyrum distichum, na triticale en gewone koring oor te dra, is deur Marais en Marais (2003) geïnisieer. 'n Studie van Th. distichum x rog hibriede het die skrywers in staat gestelom chromosome (2Jld, 3Jld, 4Jld en SJld) wat bydra to soutverdraagsaamheid te identifiseer. Indirekte (maar onbevestigde) aanduidings is gevind dat 7J1dook' n rol mag speel. 'n Program is daarna geloods om segmente van chromosome na homoeoloë triticale chromosome oor te dra, 'n onderneming wat swaar steun op die gebruik van molekulêre merkers. Alhoewel daar'n RFLP merker beskikbaar is vir elk van die Thinopyrum chromosome, is hierdie merkers nie geskik vir die sifting van groot getalle segregate nie. Hierdie studie verteenwoordig 'n poging om die RFLP merkers om te skakel na 'n minder tydrowende en meer koste-effektiewe SCAR merkers. Die gepubliseerde DNS-volgordes van die betrokke RFLP peilers is as templaat gebruik om PKR inleiers te ontwerp. Die PKR reaksies is geoptimiseer deur gebruik te maak van DNS van Th. distichum. rog en hulle FI hibried. In gevalle waar Thinopyrum spesifieke amplifikasie produkte verkry is, is die inleiers ook getoets op 'n paneel van genotipes met en sonder die teikenchromosoom. Skynbare polimorfiese bande is bevestig deur 'n 'Southern' klad te maak en te hibridiseer met die tersaaklike RFLP peiler. Die inleiers is ook getoets op 'n paneel van genotipes waarby 'Rex' triticale ingesluit was om te verseker dat dit ook verskille in 'n triticale genetiese agtergrond opspoor (nodig tydens oordrag). Polimorfiese bande is verder verfyn. Dit is geïsoleer en die DNS-volgorde daarvan is bepaal. Tn sekere gevalle is ooreenstemmende fragmente geamplifiseer in triticale ('Rex') en Thinopyrum. Die volgordes is dan bepaal en met mekaar vergelyk in 'n poging om meer spesifieke merkers te ontwerp. Met die gebruik van hierdie benadering was dit moontlik om chromosoom-spesifieke SCAR-merkers vir die Thinopyrum chromosome 3Jld en 7J2d te ontwikkel. Drie en een stel(le) PKR merkers is onderskeidelik ontwikkel en kan gebruik word om ondubbelsinnig te bepaal of die betrokke Thinopyrum chromosoom segregeer in 'n triticale kruising. 'n SCAR merker is ook gevind vir chromosoom 6J. Dus, daar is probeer om dertien RFLP peilers na SCAR merkers om te skakel. Slegs drie van die pogings was suksesvol. Die hoofrede vir die lae sukseskoers is die hoë graad van sintenie tussen die genome van die verskillende grane wat dit 'n moeilike taak gemaak het om diskriminerende inleierstelle te ontwerp. Op grond van die resultate word 'n ietwat gewysigde prosedure vir die toekomstige pogings om chromosoom-spesifieke merkers te ontwerp met gebruik van gepubliseerde volgorde inligting vanaf' n ander spesie, voorgestel.

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