Molekulere karakterisering van 'n Aegilops speltoides verhaalde translokasie en verkorte vorms

Bekker, Tamrin Annelie (2009-03)

Thesis (MSc (Genetics))--University of Stellenbosch, 2009.


Gene transfer from wild gras species to wheat is complicated by the simultaneous integration of large amounts of alien chromatin. The alien chromatin containing the target gene is inherited as a linkage block and the phenomenon is known as linkage drag. The degree of linkage drag depends on whether, and how readily, recombination occurs between the foreign and wheat chromatin. The S13 translocation line was developed by the department of Genetics, US. A cross was made between Chinese Spring and a leaf rust resistant Aegilops speltoides accession. Resistant backcross F1 was backcrossed to Chinese Spring and W84-17. S13 was selected from the backcross progeny and found to carry three rust resistance genes temporarily named LrS13, SrS13 and YrS13. Unfortunately, the resistance genes were completely linked to gametocidal (Gc) genes that were co-transferred from the wild parent. In wheat Gc genes cause reduced fertility, poor plant phenotype and hybrid necrosis. In order to use employ the rust resistance genes commercially they need to be separated from the Gc genes. At the onset of this study four putative shortened forms of the S13 translocation were provided. The four lines were identified in a homoeologous paring induction experiment (involving the test cross 04M127). This study aimed to achieve the following: (i) characterize the four recombinants with the use of molecular markers, (ii) use the knowledge gained to identify further recombinants in the 04M127 cross, (iii) identify the shortest (most useful) recombinant, and (iv) attempt to shorten the shortest recombinant form still further and thereby remove as many of the Gc genes as possible. In total, seven recombinants of the S13 translocation (04M127-1, -2, -3, -4, -7, -11 and -12; referred to as recombinant group A) were identified and characterised with microsatellite and SCAR markers. These recombinants have exchanged different amounts of foreign chromatin for wheat chromatin, but were still associated with Gc genes, showing hybrid necrosis and seed shrivelling. Some of the recombinants have lost the undesirable „brittle rachis‟ phenotype which occurs in Ae. speltoides and the S13 translocation line. In plants VII having this trait, the rachis spontaneously disarticulates after the third spikelet upon ripening of the ear. Recombinant 3 appeared to be least affected by Gc genes and was therefore used in further attempts to shorten the translocation. Recombinant 3 was crossed with wheat (W84-17) and resistant F1 (heterozygous for the translocation) were test crossed with Chinese Spring nullisomic 3A tetrasomic 3B/D plants. Thirty five resistant testcross F1 plants were identified (named recombinant group B). The resistant group B recombinants as well as nine susceptible test cross F1 (which also appeared to be recombinant) were characterised making use of microsatellites and a SCAR marker. From the results it appeared that each of the 35 resistant plants exchanged substantial amounts of Ae. speltoides chromatin for wheat chromatin. The species chromatin that remained (and which contains LrS13) is probably located either close to the 3AS telomere or within the proximal regions of 3AS and 3AL. A SCAR marker that has been developed specifically for the S13 translocation provided useful confirmation of the presence of Ae. speltoides chromatin in the 35 recombinants. If the SCAR marker proves to be tightly linked to LrS13 it may eventually be used for marker assisted selection of the resistance or it may be employed in continued attempts to reduce the amount of foreign chromatin. Seedling rust resistance tests showed that the recombinants have lost SrS13 and YrS1 during recombination. An attempt was also made to develop additional markers that specifically detect the translocation in order to further characterise the group B recombinants. Published information on Ae. speltoides specific repeated and transposon sequences were obtained and used for primer design. Unfortunately, no suitable markers could be found and the primers that were designed tended to amplify the same fragments in both the wheat and species genomes. DArT markers were also employed in an attempt to characterise the 35 group B recombinants and controls. The DArT results provided an independent verification of the results obtained with the microsatellite markers. The DArT results confirmed that the group B recombinants exchanged large amounts of species chromatin for wheat chromatin. Even though the 35 resistant group B recombinants have undergone extensive recombination they still show signs of residual Gc effects. It is believed these effects can be removed by continued backcrossing to wheat accompanied by selection against Gc symptoms. While the effects of Gc genes per se were not studied, their properties were reminiscent of those of transposable elements. Indications were that complex interactions involving the Gc genes themselves as well as genetic factors in the wheat genome may have a drastic effect on the selective survival of recombinant gametes.

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