Thesis (MSc)--Stellenbosch University, 2004. / 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.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/53754 |
| Date | 03 1900 |
| Creators | Loubser, Dalene |
| Contributors | Marais, G. F., Stellenbosch University. Faculty of AgriSciences. Dept. of Genetics. |
| Publisher | Stellenbosch : Stellenbosch University |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | Unknown |
| Type | Thesis |
| Format | xii, 95 pages : illustrations |
| Rights | Stellenbosch University |
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