Molecular genetic study of wheat rusts affecting cereal production in the Western Cape

Le Maitre, Nicholas Carlyle (2010-03)

Thesis (MSc (Genetics))--University of Stellenbosch, 2010.


ENGLISH ABSTRACT: Microsatellites were used to differentiate Leaf (Puccinia triticina Eriks.) and Yellow rust (Puccinia striiformis Westend. f. sp. tritici Eriks.) pathotypes. There was sufficient diversity in the Leaf rust microsatellite markers to differentiate the pathotypes and create a phylogenetic tree of Leaf rust. Three of the microsatellite markers were sufficient to differentiate all the Leaf rust pathotypes. Sufficient diversity in the Yellow rust microsatellite markers was also observed which made it possible to differentiate the pathotypes. Only three pathotypes were used so no phylogenetic inference was made. Two microsatellite markers were sufficient to differentiate all the yellow rust pathotypes. Microsatellite and Amplified Fragment Length Polymorphisms (AFLP) markers were used to differentiate Stem rust (Puccinia graminis f. sp. tritici Eriks. and Henn.) pathotypes, and the data was combined for phylogenetic analysis. AFLP bands unique to each Stem rust pathotype were converted to Sequence Characterised Amplified Region (SCAR) markers. A single specific SCAR marker was created for UVPgt52. A second SCAR marker amplified four of the eight pathotypes. None of the other SCAR markers were specific. A 270 basepair fragment of the ITS1 region of the rDNA gene of all the Puccinia spp. was also sequenced in order to develop pathotype specific primers that could be used in a Real Time-PCR to determine relative levels of pathogen inoculum in a sample. Unfortunately insufficient diversity in the sequences of the ITS1 region of the rDNA gene did not allow unique primers to be designed for each pathotype making it impossible to proceed with the relative quantification using Real Time-PCR. Following marker development ninety one field isolates were collected from eleven sites in the Overberg and Swartland regions during 2008 and 2009. In the field isolates, four different Leaf rust pathotypes were identifiable. UVPgt13 and UVPgt10 were most prevalent. The most prevalent Stem rust pathotypes were UVPgt50, UVPgt52, UVPgt54 and UVPgt57. Only 6E16A- was identifiable in the Yellow rust isolates. There were no apparent patterns in the distribution of Leaf, Stem or Yellow rust. Leaf and Stem rust were widely distributed, while Yellow rust was confined to three sites in the central South Cape, the only sites where climatic conditions were favourable for its development during the sampling period. The low levels of diversity found in the rust population when compared to international populations are probably due to the relatively small population size, the lack of a host for sexual reproduction, the small sample size, the effective monoculture and the strong selective pressure created by artificial control methods.

AFRIKAANSE OPSOMMING: Mikrosatellietmerkers is gebruik om Blaar- (Puccinia triticina Eriks.) en Geelroes-( Puccinia striiformis Westend. f. sp. tritici Eriks.) patotipes te onderskei. Daar was genoeg diversiteit in die Blaarroesmerkers om verskillende patotipes te kon onderskei en om „n filogenetiese-boom te kon saamstel. Met drie van die mikrosatellietmerkers was dit moontlik om al die Blaarroespatotipes te kon onderskei. Daar was genoeg diversiteit in die Geelroesmerkers om al die patotipes te kon skei en met twee van die mikrosatellietmerkers kon al drie Geelroespatotipes van mekaar onderskei word. Mikrosatelliet- en ge-Amplifiseerde-Fragment-Lengte-Polimorfismes (AFLP) is gebruik om die Stamroes- (Puccinia graminis f. sp. tritici Eriks. and Henn.) patotipes te skei. AFLP-fragmente uniek aan „n spesifieke patotipe is omgeskakel na Volgorde-Spesifieke-ge-Amplifiseerde-Streek (SCAR) merkers. „n Spesifieke SCAR-merker is gemaak vir UVPgt52. „n Tweede SCAR-merker het vier van die patotipes geidentifiseer. Nie een van die ander SCAR-merkers was spesifiek t.o.v. „n spesifieke patotipe nie. Die volgorde van „n 270 basispaar fragment van die ITS1-streek van die rDNS-geen van al die Puccinia spp. is bepaal om patotipe spesifieke inleiers te kon ontwerp. Hierdie inleiers kan gebruik word om „n Intydse-Polimerase-Ketting-Reaksie (RT-PCR) te ontwerp om sodoende die relatiewe vlakke van die patogeen besmetting in „n monster te bepaal. Daar was nie genoeg diversiteit in die bepaalde volgordes om die spes1fieke inleiers te kon identifiseer nie en dus is RT-PCR laat vaar. Na die ontwikkeling van die merkers was een-en-negentig veldmonsters ingesamel afkomstig van elf lokaliteite in die Overberg en Swartland gedurende 2008 en 2009. Vier Blaarroespatotipes was uitkenbaar. Blaarroespatotipes UVPrt10 en UVPrt13 was die mees algemeenste. UVPgt50, UVPgt52, UVPgt54 en UVPgt57 was die mees algemene Stamroespatotipes. Net 6E16A- is geidentifiseer by die Geelroes-isolate. Daar was geen patroon in die verspreiding van Blaar-, Stam- of Geelroes patotipes. Blaar- en Stamroes was die wydste versprei, maar Geelroes het net by drie lokale in die sentrale Suid-Kaap voorgekom. Die lokaliteite is die enigste waar die weersomstandighede gunstig was vir Geelroes ontwikkeling gedurende die periode van monsterneming. Die lae vlakke van diversiteit wat in die roespopulasie gevind was is in teenstelling met internasionale populasies. Dit mag moontlik wees as gevolg van die relatief beperkte populasie grootte, die afwesigheid van „n gasheer vir seksuele voortplanting, die beperkte hoeveelheid monsters wat ingesamel is en die sterk selektiewe druk weens kunsmatige beheer.

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