Molekulere merking van Thinopyrum distichum chromosome betrokke by soutverdraagsaamheid en die karakterisering van trigeneriese (Triticum/Secale/Thinopyrum) sekondêre hibriede

Visser, Hendrik Johannes (2008-12)

Thesis (MSc (Genetics))--Stellenbosch University, 2008.


Thinopyrum distichum (2n = 4x = 28; J1dJ1dJ2dJ2d) is a hardy, salt-tolerant maritime wheatgrass indigenous to southern Africa. In order to transfer its salt-tolerance to cultivated cereals, the Thinopyrum chromosomes involved must first be characterized with molecular markers. Thinopyrum distichum chromosomes 2J1d, 3J1d, 4J1d and 5J1d have previously been found to be major determinants of salt-tolerance. A genotype panel consisting of two triticale/Th. distichum allopolyploids, two Th. distichum/2*triticale doubled-haploids, eight triticale addition-lines (for chromosomes 2J1d; 2J1dβ; 3J1d; 3J1dL; 4J1d; 4J2d; 5J1d and 7J2d, respectively) and two triticale translocation-lines (involving chromosome arms 3J1dS and 3J1dL, respectively) were used for fluorescence-based, semi-automated AFLP-analyses and to a lesser extent for EST-SSR microsatellite marker-development, to identify molecular markers specific to the critical Th. distichum chromosomes. Thirteen EST-SSR primer pairs produced four putative Th. distichum-specific microsatellite-markers, one of which was specific for critical chromosome 5J1d. AFLP-analysis with 60 selective EcoRI/MseI and 18 Sse8387I/MseI primer combinations produced 159 AFLP-fragments specific for Th. distichum. These included seven putative markers for chromosome 2J1d, 15 for 3J1d, one marker for 4J1d and two for 5J1d. A salt-tolerance experiment was done to determine which chromosome 2J1d and 3J1d regions may carry genes for salt-tolerance. Plants were selected that had a monosomic addition of a chromosome 2J1d variant (either the complete chromosome or a modified version referred to as 2J1dβ) in addition to one of four chromosome 3J1d variants (the complete 3J1d chromosome; a 3J1dL-telosome; a 3J1dS-translocation or a 3J1dL-translocation). The results suggested that Th. distichum chromosome-arms 2J1dL and 3J1dS are probably involved in salt-tolerance. A group of 93 trigeneric (Triticum/Secale/Thinopyrum) F2 secondary hybrids were then analyzed in order to: (i) Evaluate some (ten) of the newly developed putative AFLP-markers; and (ii) attempt to find translocations, telosomes or substitutions involving the critical Thinopyrum chromosomes. Five (50 %) of the ten putative AFLP-markers could be reproduced, but only four proved to be chromosome-specific. It was also possible to assign hese four markers to chromosome arms: E32M49.118 (2J1dS); E41M49.103 (2J1dS); E35M49.137 (3J1d); and E41M49.188 (3J1dL). The selective primer combination that produced marker E41M49.103 (2J1dS), also amplified a fragment of the same size on chromosome 4J1d. These markers will be useful for further mapping and selection of the salt-tolerance genes. The fact that only four of the ten putative AFLP-markers evaluated proved to be repeatable implies that the remaining untested markers need to be confirmed against larger genotype panels as well. Probable reasons for the relatively low frequency of markers that turned out to be reliable are discussed. The marker-association study also revealed that visual examination of all electropherograms produced by AFLP-fragment analysis is necessary to correctly identify all AFLP-fragments. Use of the AFLP- and STS-/SCAR-markers in conjunction with the group of 93 F2 secondary hybrids showed that 18 of these probably carried a 3J1dL-translocation. Several hybrids possibly had translocations involving the 4J1d and 5J1d chromosomes. However, these results need to be confirmed. Various hybrids also appeared to have critical Th. distichum substitutions, although this still requires further confirmation. The identified plant material could prove useful for further characterization of salt-tolerance in Thinopyrum, and its eventual utilization in cereal crops.

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