Genetic mapping of gray leaf spot resistance genes in maize

Lehmensiek, Anke (2000-12)

Thesis (PhD)--Stellenbosch University, 2000.

Thesis

ENGLISH ABSTRACT: Gray leaf spot (GLS) of maize, caused by the fungus Cercospora zeae-maydis, can reduce grain yields by up to 60% and it is now recognized as one of the most significant yield-limiting diseases of maize in many parts of the world. The most sustainable and long-term management strategy for GLS will rely heavily on the development of high-yielding, locally adapted GLS resistant hybrids. Molecular markers could be useful to plant breeders to indirectly select for genes affecting GLS resistance and to identify resistance genes without inoculation and at an early stage of plant development. Only two studies in the USA have examined quantitative trait loci (QTL) association with GLS resistance. The aim of this study was to map GLS resistance genes in a resistant Seed Co LTD, Zimbabwean inbred line. Molecular markers linked to the GLS resistance QTL were identified by using the amplified fragment length polymorphism (AFLP) technique together with bulked segregant analysis. Eleven polymorphic AFLP fragments were identified and converted to sequence-specific PCR (polymerase chain reaction) markers. Eight of the 11 converted AFLP markers were added to the maize marker database of the University of Stellenbosch. Five of the 8 converted AFLP markers were polymorphic between the resistant and the susceptible parent. They were amplified on the DNA of 230 plants of a segregating F2 population and linkage analysis was performed with MAPMAKER/EXP. Two linkage groups consisting of two markers each, with a linkage distance of 10.4 cM (LOD 22.83) and 8.2 cM (LOD 55.41) between the two markers, were identified. QTL mapping with MAPMAKER/QTL confirmed the presence of QTL in both linkage groups. Two publicly available recombinant inbred families (Burr et a/., 1988) were used to localize the converted AFLP markers on the genetic map of maize. The QTL, which were identified with the AFLP markers, were mapped to chromosomes 1 and 5. Another AFLP marker was mapped to chromosome 2 and a further to chromosome 3. To obtain more precise localizations of the QTL on chromosomes 1 and 5, sequence-tagged site markers and microsatellite markers were used. The markers were amplified on the DNA of the 230 plants of the F2 population and linkage analysis was performed with MAPMAKER/EXP. The order of the markers was in agreement with the UMC map of the Maize Genome Database. Interval mapping using MAPMAKERlQTL and composite interval mapping using QTL Cartographer were performed. The QTL on chromosome 1 had a LOD score of 21 and was localized in bin 1.05/06. A variance of 37% was explained by the QTL. Two peaks were visible for the QTL on chromosome 5, one was localized in bin 5.03/04 and the other in bin 5.05/06. Both peaks had a LOD score of 5 and 11% of the variance was explained by the QTL. To test the consistency of the detected QTL, the markers flanking each QTL were amplified on selected plants of two F2 populations planted in consecutive years and regression analysis was performed. Both the QTL on chromosome 1 and the QTL on chromosome 5 were detected in these populations. Furthermore, the presence of a QTL on chromosome 3 was confirmed with these populations. A variance of 8 -10% was explained by the QTL on chromosome 3. In this study, a major GLS resistance QTL was thus mapped on chromosomes 1 and two minor GLS resistance QTL were mapped on chromosomes 3 and 5 using a resistant Seed Co LTD, Zimbabwean inbred line. Markers were identified which could be used in a marker-assisted selection program to select for the GLS resistance QTL.

AFRIKAANSE OPSOMMING: Grys blaarvlek (GBV) van mielies, veroorsaak deur die swam Cercospora zeaemaydis, kan graanopbrengs met tot 60% verlaag en word beskou as een van die vernaamste opbrengs-beperkende siektes wêreldwyd. Die toepaslikste langtermyn stragtegie vir GBV beheer sal wees om plaaslike mieliebasters met hoë opbrengs en GBV weerstand te ontwikkel. Molekulêre merkers kan nuttig deur plantetelers gebruik word om weerstandsgene te selekteer. Seleksie is moontlik in die afwesigheid van inokolum en op 'n vroeë stadium van plant ontwikkeling. Slegs twee vorige studies (in die VSA) het kwantitatiewe-kenmerk-Iokusse (KKL), vir GBVweerstand ondersoek. Die doel van hierdie studie was om die GBV weerstandsgene in 'n weerstandbiedende ingeteelde lyn (Seed Co BPK, Zimbabwe) te karteer. Molekulêre merkers gekoppel aan die GBV weerstands KKL is geïdentifiseer deur gebruik te maak van die geamplifiseerde-fragmentlengte-polimorfisme- (AFLP-) tegniek en gebulkte-segregaat-analise. Elf polimorfiese merkers is geïdentifiseer en omgeskakel na volgorde-spesifieke PKR (polimerase kettingreaksie) merkers. Agt van die elf omgeskakelde AFLP-merkers is by die mieliemerker databasis van die Universiteit van Stellenbosch gevoeg. Vyf van die 8 omgeskakelde AFLP-merkers was polimorfies tussen die bestande en vatbare ouers. Hulle is geamplifiseer op die DNA van 230 plante van 'n segregerende F2-populasie en is gebruik in 'n koppelingstudie met MAPMAKER/EXP. Twee koppelingsgroepe, elk bestaande uit twee merkers, met onderskeidelik koppelingsafstande van 10.4 eM (LOD 22.83) en 8.2 eM (LOD 55.41) tussen die merkers, is geïdentifiseer. KKL-kartering het getoon dat KKL in albei koppelingsgroepe aanwesig is. Twee kommersieël beskikbare, rekombinant-ingeteelde families (Burr et aI., 1988) is gebruik om die omgeskakelde AFLP-merkers op die mielie genetiese kaart te plaas. Die KKL wat met die AFLP-merkers geïdentifiseer is, is gekarteer op chromosome 1 en 5. 'n Verdere AFLP-merker is op chromosoom 2 gekarteer en 'n ander op chromosoom 3. Ten einde die KKL op chromosome 1 en 5 meer akkuraat te karteer, is volgordege- etikeerde en mikrosatelliet merkers gebruik. Die merkers is geamplifiseer op die DNA van die 230 plante van die F2-populasie en koppelings-analises is uitgevoer. Die volgorde van die merkers was dieselfde as die van die UMC-kaart in die Mielie Genoom Databasis. Interval kartering met MAPMAKER/QTL en komposiet interval kartering met QTL Cartographer is uitgevoer. Die KKL op chromosoom 1 het 'n LOD-telling van 21 gehad en is in bin 1.05/06 geplaas. Die KKL was verantwoordelik vir 37% van die variansie. Twee pieke was onderskeibaar vir die KKL op chromosoom 5, een in bin 5.03/04 geleë en die ander in bin 5.05/06. Elke piek het 'n LOD-telling van 5 gehad en die twee KKL was verantwoordelik vir 11% van die variansie. Om die herhaalbaarheid van die effek van die KKL te toets is die merkers naaste aan elke KKL geamplifiseer op geselekteerde plante van twee F2-populasies wat in opeenvolgende jare geplant is. Regressie analise is op die data gedoen. Beide die KKL op chromosoom 1 en die KKL op chromosoom 5 kon in hierdie populasies geïdentifiseer word. Verder kon die aanwesigheid van 'n verdere KKL op chromosoom 3 in hierdie populasies bevestig word. Laasgenoemde KKL was verantwoordelik vir 8-10% van die totale variansie. In hierdie studie is daar dus 'n hoof GBV-weerstands KKL gekarteer op chromosoom 1 en twee kleiner GBV-weerstands KKL gekarteer op chromosome 3 en 5. Merkers is geïdentifiseer wat moontlik in merker-gebaseerdetelingsprogramme gebruik kan word om plante te selekteer wat die GBVweerstands KKL het.

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