A novel marker technique : using miniature inverted-repeat transposable elements (MITEs) in combination with resistant gene analogues (RGAs)

Lambert, Carol-Ann (2001-12)

Thesis (PhD)--Stellenbosch University, 2001.

Thesis

ENGLISH ABSTRACT: Given the organisation of the maize genome as well as demands placed on the saturation of molecular linkage maps it would be desirable to identify informative molecular markers that is located or linked to genic rich areas. Sequences of gene products from different gene classes were investigated. Proteins containing a nucleotide binding site (NBS) and leucine-rich repeat (LRR) region comprise the largest class of disease resistance proteins. Resistant gene analogue (RGA) primers belonging to this specific class were derived from previous published literature studies. By means of similarity studies of short stretches of conserved amino acid and DNA sequences, primers were developed that belonged to the peroxidase and reductase gene classes. A novel class of transposable element was identified, that occurred in the gene rich areas of a diverse range of grass genomes. Of all the MITE families described so far, the Heartbreaker (Hbr) and Hb2 family elements were of particular interest. The unique properties of MITEs, especially their high copy number, polymorphism, stability and preference for genic areas together with the RGA primers, were exploited to develop a new marker technique for the isolation of a class of molecular marker with a strong preference for genic areas. Using the publicly available recombinant inbred population, Tx303 x C0159, 196 MITE/RGA markers were added to the existing recombinant inbred linkage map consisting of ±1033 already established markers. It became apparent that just like loci for disease resistance, the 196 MITE/RGA fragments were not randomly distributed across the maize genome but occurred in clusters spread across the ten maize chromosomes. Ninety-two (92) of the MITE/RGA fragments showed significant correlation to previously mapped maize resistance genes. To establish the conservation and specificity of both the Hbr and Hb2 elements, sequences of 19 MITE/RGA fragments were ascertained. When comparing the partial MITE element sequences from these fragments, a high degree of element conservation was observed. One fragment showed good sequence correlation to a NADPH He Toxin reductase protein product and mapped to the same chromosomal location as the hm1 gene locus in maize. This fragment can be considered a candidate gene for resistance against the pathogen, Helminthosporium carbonum. The Hbr primer used proved to be very specific for the Heartbreaker MITE element, this was in contrast to the non-specificity of the Hb2 primer. The applicability of this technique was tested on two maize diseases that cause immense damage in the maize production industries in South Africa. Fourteen MITE/RGA markers were used to fine map the putative chromosomal locations for the HtN1, Ht1, Ht2 and Ht3 genes that confer resistance. against Setosphaeria turcica, the northern corn leaf blight (NelS) pathogen in maize. Three MITE/RGA fragments were identified that aided in the saturation of the linkage map for quantitative trait resistance (QTl) against gray leaf spot (GlS) in maize. This novel MITE/RGA technique presented a unique opportunity to search for additional candidate genes by using polymerase chain reaction (peR) analysis. When compared to the conventional amplified fragment length polymorphism (AFLP) technique, the MITE/RGA technique proved to be just as efficient but was more cost effective and less time consuming.

AFRIKAANSE OPSOMMING: Die organisasie van die mielie genoom as ook die vereistes wat daar geplaas word op die versadiging van koppelingskaarte, vereis dat daar meer klem geplaas word op die ontwikkeling van molekulêre tegnieke wat merkers in geenryke areas identifiseer. Die volgordes van geenprodukte, wat behoort tot verskillende geenklasse, is deeglik bestudeer. Proteïenprodukte wat bestaan uit 'n nukleotiedbindingsarea (NBA) en 'n leusienryke herhalende (LRH) area is een van die grootste klasse waaronder siekteweerstandsproteïene sorteer. Polimerase kettingreaksie (PKR) inleiers wat behoort tot hierdie spesifieke klas, is verkry vanuit vorige publikasies. Deur kort gekonserveerde aminosuur en DNS volgordes te vergelyk is inleiers ontwikkel wat behoort tot die peroksidase en reduktase gene klasse. 'n Nuwe klas transponeerbare elemente wat voorkom in die geenryke areas van diverse gras genome, is geïdentifiseer. Van al die miniatuur inversie herhalende transponeerbare elemente (MITE) wat al geïdentifiseer is, is die twee elemente, Heartbreaker (Hbr) en Hb2, van groot belang. Unieke eienskappe van die MITEs, veral hul hoë kopie aantal, polimorfiese-indeks, stabiliteit asook voorkeur vir geenryke areas, tesame met die weerstandsgeen analoë (WGA) inleiers, is gebruik om 'n nuwe merker tegniek te ontwikkel. Hierdie nuwe tegniek identifiseer 'n klas merker wat 'n sterk voorkeur het vir geenryke areas. Deur gebruik te maak van die openbare beskikbare rekombinante ingeteelde (RI) populasie, Tx303 x C0159, is 196 MITE/WGA-merkers gekarteer op die bestaande RIL koppelingskaart, wat alreeds bestaan uit ±1033 gevestigde merkers. Net soos die lokusse vir siekteweerstand het dit geblyk dat hierdie 196 merkers in groepe voorkom wat verspreid is oor die tien mielie chromosome. Twee-en-negentig (92) van die 196 gekarteerde MITE/WGA-merkers het betekenisvolle korrelasie gewys met reeds gekarteerde mielie weerstandsgene. Die volgordes van 19 MITE/WGAfragmente is bepaal om sodoende die spesifisiteit en mate van konservering van die Hbr and Hb2 elemente te bereken. 'n Hoë mate van element konservering is waargeneem. Een fragment het In baie goeie volgorde korrelasie gewys met In NADPH HG toksien reduktase proteïen produk en karteer op dieselfde chromosomale posisie as die hm1 geen lokus. Hierdie fragment kan gesien word as In kandidaatgeen vir weerstand teen die mielie patogeen, Helminthosporium carbonum. Die toepasbaarheid van hierdie tegniek is getoets op twee siekte toestande, wat lei tot groot verliese in die mielie industrie, in Suid-Afrika. Veertien van die MITE/WGAmerkers is gebruik om die waarskynlike chromosomale posisies van die HtN1, Ht1, Ht2 en Ht3 gene, wat weerstand bied teen Setosphaeria turcica, die noordelike mielie blaarvlek (NMBV) patogeen, fyner te karteer. Drie MITE/WGA fragmente is geïdentifiseer wat gehelp het in die versadiging van die koppelingskaart vir die kwantitatiewe kenmerk weerstandbiedenheid (KKW) teen grys blaarvlek (GBV) in mielies. Deur gebruik te maak van polimerase kettingreaksie (PKR) analise, verskaf hierdie tegniek die moontlikheid om te soek vir addisionele kandidaatgene. Hierdie tegniek is ook vergelyk met die konvensionele geamplifiseerde fragment lengte polimorfisme (AFLP) tegniek. Daar is gevind dat die nuwe tegniek net so informatief is, maar wel meer koste effektief en tyd besparend.

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