Thesis (MSc.)--University of Stellenbosch, 2000. / ENGLISH ABSTRACT: The detrimental effect of soil salinity on crop production is a growmg problem worldwide (Tanji, 1990b). The degree to which plants can tolerate high concentrations of salt in their rooting medium is under genetic control with different genetic and physiological mechanisms contributing to salt tolerance at different developmental stages (Epstein & Rains, 1987). Only limited variation exists for salt tolerance in the cultivated cereals. This has prompted attempts to select tolerant progeny following hybridisation of cultivated species and wild, salt-tolerant species. Thinopyrum distichum, an indigenous wheatgrass that is naturally adapted to saline environments (McGuire & Dvorak, 1981), was crossed with triticale (x Triticosecale) in an attempt to transfer its salt tolerance and other hardiness characteristics (Marais & Marais, 1998). The aims of this study were to (i) identify Thinopyrum chromosomes carrying genes for salt tolerance and to identify molecular markers for these chromosomes, (ii) identify a number of diverse monosomic and disomie addition plants. Bulked segregant analysis (BSA), in combination with AFLP, RAPD and DAF marker analysis was implemented to screen for polymorphisms associated with salt tolerance. Five putative AFLP markers and two RAPD markers were detected using bulks composed of salt tolerant plants and bulks composed of salt sensitive plants. The distribution of the markers in these bulks suggests that more than one Thinopyrum chromosome carry genes for salt tolerance. Salt tolerant monosomic and disomie addition plants were characterised for AFLP, RAPD and DAF polymorphisms in an attempt to find markers associated with the chromosome(s) conditioning salt tolerance. One salt tolerant monosomic and one disomie addition plant was identified. One AFLP and two RAPD markers were identified for the Thinopyrum chromosome( s) present in the monosomic addition plant, while three AFLP and three RAPD markers were identified for the disomie addition plant. An attempt was also made to identify diverse chromosome addition plants having complete or near complete triticale genomes plus an additional random Thinopyrum chromosome. Plants with 2n = 43 /44 were identified and characterised for molecular markers (AFLP and RAPD). Cluster analysis was used to group the putative monosomic or disomie addition plants according to the specific Thinopyrum chromosomes they retained. Seventeen AFLP and RAPD markers could be used to group the 24 putative addition plants into six broadly similar groups with different additional Thinopyrum chromosomes. While the members of each group are likely to carry the same additional Thinopyrum chromosomes, this may not necessarily be the case as the interpretation of the marker results is complicated by heterogeneity among plants with regard to the triticale background chromosomes they possess. It is also likely that chromosome translocations occurred during backerossing which may further complicate data. Nonetheless, it is now possible to select disomie addition plants from each group that are likely to represent different Thinopyrum chromosomes. The data will also be useful in future attempts to find further addition plants carrying the remaining Thinopyrum chromosomes. / AFRIKAANSE OPSOMMING: Die skadelike effek van grond versouting op gewasproduksie neem wêreldwyd toe (Tanji, 1990b). Die mate waartoe plante hoë konsentrasies sout in die wortelstelsel kan hanteer is onder genetiese beheer en verskillende genetiese en fisiologiese meganismes dra by tot die soutverdraagsaamheid tydens verskillende ontwikkelingstadia (Epstein & Rains, 1987). Slegs beperkte variasie bestaan vir soutverdraagsaamheid in verboude grane. Dit het aanleiding gegee tot pogings om soutverdraagsame nageslag te selekteer na hibridisasie van verboude spesies en wilde, soutverdraagsame spesies. Thinopyrum distichum, 'n inheemse koringgras, wat aangepas is by brak omgewings (McGuire & Dvorak, 1981), is met korog (x Triticosecale) gekruis in 'n poging om die gene vir soutverdraagsaamheid en ander gehardheidseienskappe oor te dra (Marais & Marais, 1998). Die oogmerke van hierdie studie was om (i) Thinopyrum chromosome te identifiseer wat gene bevat vir soutverdraagsaamheid en molekulêre merkers te vind vir hierdie chromosome, (ii) 'n aantal diverse monosomiese en disomiese addisieplante te identifiseer. Bulksegregaatanalise (BSA), gekombineer met AFLP-, RAPD- en DAF-merkeranalise, is gebruik om polimorfismes geassosieerd met soutverdraagsaamheid op te spoor. Vyf moontlike AFLPmerkers en twee RAPD-merkers is geïdentifiseer met gebruik van bulks bestaande uit soutverdraagsame plante en bulks bestaande uit soutgevoelige plante. Die verspreiding van die merkers in soutverdraagsame bulks dui daarop dat meer as een Thinopyrum chromosoom bydra tot soutverdraagsaamheid. Soutverdraagsame, monosomiese en disomiese addisieplante is gekarakteriseer vir AFLP- en RAPD-polimorfismes in 'n verdere poging om merkers te vind vir chromosome betrokke by soutverdraagsaamheid. Een soutverdraagsame monosomiese en een disomiese addisieplant is geïdentifiseer. Een AFLP- en twee RAPD-merkers is geïdentifiseer vir die Thinopyrum chromosoom(e) teenwoordig in die monosomiese addisieplant, terwyl drie AFLP- en drie RAPDmerkers geïdentifiseer is vir die disomiese addisieplant. 'n Poging is ook gemaak om diverse addisieplante te identifiseer met 'n volledige koroggenoom plus 'n addisionele Thinopyrum chromosoom. Plante met 2n = 43 / 44 is geïdentifiseer en gekarakteriseer met molekulêre merkers (AFLP en RAPD). Tros-analise is gebruik om die vermoedelik monosomiese of disomiese addisieplante te groepeer volgens die spesifieke Thinopyrum chromosome wat hulle behou het. Sewentien AFLP- en RAPD-merkers is gebruik om die 24 vermoedelike addisieplante in 6 groepe met verskillende Thinopyrum chromosome te groepeer. Alhoewel dit voorkom of die verskillende plante in 'n groep dieselfde addisionele Thinopyrum chromosoom het, is dit nie noodwendig die geval nie aangesien die interpretasie van die merkers bemoeilik word deur die heterogeniteit tussen die plante wat betref die agtergrond korogchromosome wat hulle besit. Dit is ook moontlik dat chromosoom herrangskikkings plaasgevind het gedurende die terugkruisings, wat die data verder kan bemoeilik. Nietemin, dit is nou moontlik om disomiese addisies te selekteer uit elke groep wat moontlik verskillende Thinopyrum chromosome bevat. Die data kan ook gebruik word om in die toekoms verdere addisieplante te identifiseer wat die oorblywende Thinopyrum chromosome bevat.

[Truncated abstract] Lophopyrum elongatum, a wild relative of wheat, can be used as a source of novel genes for improving the salt tolerance of bread wheat. Na+ `exclusion? is a major physiological mechanism for salt tolerance in the wheat L. elongatum amphiploid, and a large proportion (~50%) of the improved Na+ `exclusion? is contributed by a gene(s) on chromosome 3E. This study integrated physiological analysis with comparative genomics to identify gene orthologues that may regulate Na+ transport, and designed and implemented molecular markers for developing wheat L. elongatum recombinant lines with reduced portions of L. elongatum chromatin retaining the Na+ `exclusion? trait. Physiological analysis of leaf Na+ accumulation in wheat L. elongatum substitution lines confirmed that the 3E chromosome contributes a major effect on reduced leaf Na+ accumulation in wheat when grown at 200 mM NaCl. Candidate genes from the model plant, Arabidopsis thaliana, controlling Na+ transport into and from cells (SOS1, HKT1) or compartmentalisation within vacuoles (NHX1, NHX5, AVP1, AVP2) were targeted for comparative analysis in wheat. Wheat gene orthologues were identified by BLAST searching to identify either FL-cDNAs or ESTs, which were subsequently used to amplify genomic DNA, and orthologues confirmed by similar intron-exon structure between Arabidopsis and rice. Intron-exon comparisons showed the majority of exons were conserved between Arabidopsis, rice and wheat, but also indicated exon shuffling events since divergence from a common ancestor. Gene orthologues were assigned to homoeologous chromosomes and non-syntenic regions between wheat and L. elongatum, with the SOS1 orthologue located on group 3 chromosomes in wheat and L. elongatum. ... The recombinant line 524-568 contains a small introgression on the distal end of the long arm of wheat chromosome 3A and represents the most desirable line presently available for further germplasm development. The main outcomes of this thesis have been an increased understanding of the physiology and evolution of orthologues for Na+ transport in wheat and L. elongatum, improved methodologies for designing alien-specific PCR markers, and the development of overlapping recombinant lines that provide a source of novel genes for pyramiding into wheat and improving its tolerance to salt stress.