AFLP and PCR markers for the Ht1, Ht2, Ht3 and Htn1 resistance genes in maize

Van Staden, Derick (2001-12)

Thesis (PhDAgric)--University of Stellenbosch, 2001.

Thesis

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.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/52078
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