Thesis (PhDAgric)--University of Stellenbosch, 2001. / ENGLISH ABSTRACT: Maize is undoubtedly South Africa's most important field crop. The identification of
markers and genes for traits of interest is important to sustain the improvement of
maize cultivation. Northern corn leaf blight (NClB) is a disease that occurs worldwide
and can dramatically reduce yield. A number of single dominant resistance
genes have been identified for NClB and some have been mapped. Currently there
are no simple PCR markers for any of these resistance genes, making markerassisted
selection (MAS) difficult.
The aim of this study was to develop PCR markers for the NClB resistance genes
Ht1, Ht2, Ht3 and Htn1 in maize. To accomplish this, the AFlP (amplified fragment
length polymorphism) technique was first optimised. The results indicated that the
Mlul/Msel restriction enzyme combination produces a higher percentage of
polymorph isms when compared to the PstllMsel enzyme combination. It was also
shown that the enzyme combination plays an important role in the percentage of
polymorphic fragments observed, whereas the number of restriction enzymes used in
AFlP analysis only significantly affects the total number of fragments scored.
Populations segregating for the different resistance genes were not available for this
study. Nearly-isogenic lines (Nils) were used in combination with AFlP technology
to identify markers that map close to the genes. AFlP markers common in at least
two resistant or susceptible lines were cloned and converted to PCR markers. Two
commercially available recombinant inbred line (Ril) populations were then used to
map the identified markers.
For Htn1 fifteen polymorphic fragments were present in both resistant lines. They
were selected for sequence specific marker conversion. Seven of the fifteen
sequence characterized amplified region (SCAR) markers were polymorphic on the
Nil pairs and five mapped to one region of maize chromosome 8.05/06. Twenty-one
AFlP markers were identified for Ht1 and four SCAR markers were polymorphic In
the Ht1 Nils. Three of these were mapped to chromosome 2.07. Three AFlP
markers were identified for Ht2 of which two were converted to SCAR markers. Both
SCAR markers were polymorphic on the Ht2 Nils and mapped to chromosome 8.05/06. On the Ht3 NILs, four AFLP markers were identified and two converted
SCAR markers and one microsatellite marker (bnlg1666) were polymorphic. One of
the SCAR markers and the microsatellite marker were mapped to chromosome 7.04
using a RIL population. This reports the first tentative mapping position for the Ht3
locus.
The next step was to determine if a set of marker alleles could be used in a number
of Htn 1 resistance lines to identify a common donor region selected by the breeders.
Nine markers consisting of five SCAR markers, three converted RFLP markers and
one microsatellite marker were used on 16 Htn1 resistant lines. The marker allele of
us3 was in 12 of the 16 lines in coupling with Htn1 resistance. Second was the
marker us5 in 11 of the 16 lines. Using this data 14 of the 16 lines shared a common
introgressed region between the markers us3 and us5. A further common
introgressed region between 11 of the inbred lines was found between the markers
us14 and asg17.
The last aim of this study was to propose a new marker technique that might be more
successful than the AFLP technique in the identification of markers closely linked to
genes. A new marker approach was identified where a MITE (Hbr) primer was used
as an anchor primer in combination with resistance gene analog primers. This was
found to be a highly polymorphic marker technique that could be used to identify
markers and possibly candidate genes. It is a robust technique, which is affordable
since amplifications occur from undigested genomic DNA and the primers mainly
amplify fragments from genic regions. / AFRIKAANSE OPSOMMING: Mielies (Zea mays) is ongetwyfeld Suid Afrika se belangrikste lanbou gewas. Vir
volgehoue opbrengs verbetering is die identifisering van merkers en gene vir
belangrike eienskappe noodsaaklik. Noordelike blaarskroei (NBS) kan opbrengs
wesenlik kan beïnvloed. Tans is daar reeds "n aantal enkel weerstandsgene
geïdentifiseer, maar geen PKR-merkers is beskikbaar vir merker gebaseerde
seleksie nie.
Die doelwit van hierdie studie was om PKR-merkers te ontwikkel vir vier enkel
weerstands gene (Ht1, Ht2, Ht3 en Htn1) teen NBS in mielies. Om die doelstelling te
bereik is die AFLP-tegniek eers geoptimiseer. Op grond van waargenome aantal
polimorfismes, was Mlul/Mse/"n beter restriksie ensiem kombinasie as Pstl/Msel. In
die studie is ook bewys dat die aantal (meer as twee) restriksie ensieme wat gebruik
word slegs die aantal fragmente, en nie die persentasie polimorfismes, wesenlik
beïnvloed nie.
Geen segregerende populasie was vir die verskillende gene beskikbaar nie. Naby
isogeniese lyne (NILe) is daarom in kombinasie met die AFLP-tegniek gebruik om
merkers te identifiseer wat naby die gene karteer. Alleenlik polimorfiese merkers wat
in ten minste twee weerstand biedende of vatbare lyne voorgekom het, is gekloneer
en omgeskakel na PKR-merkers. Daarna is twee kommersiële rekombinante
ingeteelde lyn populasies gebruik om die gene te karteer.
Vyftien fragmente is gevind wat gekoppel was met die Htn1 weerstand. Sewe van
hierdie merkers is omgeskakel in polimorfiese SCAR-merkers waarvan vyf gekarteer
is in een gebied op chromosoom 8.05/06. Een-en-twintig AFLP-merkers is
geïndentifiseer vir Ht1 en vier is omgeskakel na polimorfiese SCAR-merkers. Drie
hiervan is gekarteer op chromosoom 2.07. Drie AFLP-merkers is geïndetifiseer vir
Ht2 waarvan 2 omgeskakel is na polimorfiese SCAR-merkers. Altwee hierdie
merkers is gekarteer op chromosoom 8.05/06. Op die Ht3 lyne is vier AFLP-merkers
geïdentifiseer waarvan twee omgeskakel is na polimorfiese SCAR-merkers. Een
mikrosatelliet merker (bnlg1666) is ook gevind wat die selfde polimorfiese patroon
wys op die Ht3 lyne. Die mikrosateliet en een van die SCAR-merkers het gekarteer op chromosomale posisie 7.04. Hierdie is die eerste tentatiewe posisie vir die Ht3
lokus.
Die volgende stap was om te bepaal of "n stel polimorfiese merker-allele gebruik kan
word om die donor DNA-segment te identifiseer wat die plantteiers geselekteer het.
Nege PKR-merkers wat bestaan het uit vyf SCAR-merkers, 3 omgeskakelde RFLP
merkers en een mikrosateliet is gebruik op 16 Hnt1 weerstandslyne. Us3 was die
merker alleel wat in die meeste gevalle gekoppel was met die Htn1 weerstandslyne
(12/16). Tweede was die merker us5 (in 11 van die 16 lyne). Uit die data blyk dit dat
14 van die 16 lyne "n donor segment het wat beide merkers us3 en us5 bevat.
Merkers us14 en asg17 het in 11 van die 16 bestande lyne saam voorgekom.
Die laaste doelstelling van hierdie studie was om "n nuwe tegniek te ontwikkel wat
dalk meer suksesvol as AFLPs kan wees om merkers te identifiseer nabyaan gene.
"n Nuwe tegniek word voorgestel waar "n MITE (Hbr) inleier gebruik kan word in
kombinasie met weerstandgeen-analoog inleiers. Dit is gevind dat hierdie
kombinasie van inleiers "n hoogs polimorfiese band patroon gee en dat die merkers
ook dalk kandidaat-gene kan wees. Die tegniek is maklik uitvoerbaar, relatief
goedkoop en maak gebruik van onverteerde genomiese DNA. Die fragmente wat
geamplifiseer word is hoofsaaklik vanaf geenryke areas.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/52078 |
| Date | 12 1900 |
| Creators | Van Staden, Derick |
| 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 | Unknown |
| Type | Thesis |
| Format | xi, 103 p. : ill. |
| Rights | Stellenbosch University |
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