Thesis (PhD)--Stellenbosch University, 2001. / 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.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/52117 |
| Date | 12 1900 |
| Creators | Lambert, Carol-Ann |
| Contributors | Retief, A. E., Stellenbosch University. Faculty of AgriSciences. Dept. of Genetics. |
| Publisher | Stellenbosch : Stellenbosch University |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | English |
| Type | Thesis |
| Format | 169 pages : illustrations |
| Rights | Stellenbosch University |
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