Genetic Characterization And Molecular Marker For Fusarium Head Blight Resistance In Wheat

Fusarium Head Blight (FHB), caused mainly by Fusarium graminearum Schwabe, is a destructive disease of wheat (Triticum aestivum) worldwide. FHB is an important factor which affected yield and quality in wheat. In China, One quarter of the wheat growing area (about 6.7 million hectares) is being jeopardized by wheat FHB. The yield loss is about 2 million ton ³ million ton every year. Fusarium head blight causes not only severe yield loss but also decreased grain quality. The diseased grain that is contaminated with mycotoxins produced by the fungus is a major safety concern to the heath of human beings and livestock. Developing new wheat varieties with a high level of FHB resistance is the most effective measure for the disease control. FHB is controlled by multiple genes and its genetic mechanism is complicated. The FHB resistance of Sumai 3 and its derivatives was verified by different research groups. The Chinese variety Ning7840 bred by Jiangsu Academy of Agriculture Science was a derivative of Sumai 3. The FHB resistance of Ning7840 was higher than that of Sumai 3. The FHB resistance genetic mechanism and QTL of Ning7840 was little studied. Especially in the middle and lower reaches of Yangtse Rive there was no research. In order to accelerate the use of FHB resistance of Ning7840 in the FHB resistance breeding research, the FHB resistance genetic mechanism and QTL of Ning7840 need further studies. Although the FHB resistance QTL on chromosome 3BS of Sumai 3 and its derivatives was verified by different research groups in the world, this resistance QTL was still located in a region with large genetic distance. The number of simple and repeatable markers was few and couldn't meet the need of marker assisted selection (MAS). The exploitation of more simple and repeatable markers in this resistance QTL region is needed.This study focuses on three aspects. The first is the study on the FHB resistant genetic mechanism and FHB resistant QTL in Ning7840 with the identified data in Ning7840/Clark RIL (recombinant inbred line) population in the Nanjing district. The second is built of SSCP (Single Strand Conformation Polymorphism) technique and optimization of the conditions affecting SSCP technique in wheat. The third is exploitation of molecular markers in the Qfhb₃BS region in Ning7840 using the sequences of the ESTs (expressed sequence tags) located in 3BS8 0.78-1.0 in wheat and the ESTs whose sequences are homologue to the BAC(bacterial artificial chromosome) or PAC(Phage-derived artificial chromosome) sequences in the distal region of the chromosome 1S in rice to design primers.The main results from this study are as follows:1) The FHB evaluation in Ning7840/Clark RIL population was carried out using the floret-inoculation method. The distribution of the percentage of scabbed spikelets in this population had the characteristics of major genes. Inheritance of FHB resistance in Ning7840/Clark RIL population was analyzed by the mixed major gene plus poly-gene inheritance model of quantitative traits. The results showed that FHB resistance in the cross Ning7840/Clark was controlled by three major genes (the G-1 model) under the floret-inoculation method. Heritability value of the major genes was high. The additive genetic effect of the first major gene is relatively big and can cause 10% decease of the percentage of scabbed spikelets. Map Manager QTXb20 software was used to analyze the FHB resistant QTL in Ning7840/Clark RIL population. FHB resistant QTLs were identified on chromosome 3BS, 3AS and 1AL respectively. This result is coincided with the genetic model analysis. The effect of the resistant QTL on 3BS is the biggest and could explain 36%～61% of the phenotypic variation for FHB resistance.2) The FHB evaluation in Ning7840/Clark RIL population was also carried out using the spray -inoculation method. The distribution of the percentage of scabbed spikelets in one point in this population had the characteristics of major genes. Map Manager QTXb20 software was used to analyze the FHB resistant QTL under spray-inoculation method in Ning7840/Clark RIL population. The resistant QTL on 3BS could also be identified. Moreover, two QTLs on 3BL were identified. These two QTL maybe be mainly associated with the resistance to the fungal penetration.3) The influences of the loading buffer, the denaturalization temperature and time , the polyacrylamide gel components (the concentration of the polyacrylamide gel, the Acr/Bis ratio and the additives in the gel) and the electrophoresis factors (the electrophoresis temperature, the electrophoresis buffer and the electrophoresis power) on SSCP technique were studied. The results were as follows: when the volume of the loading buffer without glycerol was 1.5-3 times of the volume of the PCR(Polymerase Chain Reaction) product; the denaturalization temperature was 98℃; the denaturalization time was 10-15 minutes; the gel component was 12% polyacrylamide gel (Acr:Bis 29:1) without sucrose and glycerol; the electrophoresis buffer was 0.5×TBE; the electrophoresis temperature was about 25℃; The electrophoresis power was 80W (2.67W/ cm); the bands of the Single Strand DNA were clear and easy to read.4) 250 primer pairs were designed from the sequences of the ESTs located in 3BS8 0.78-1.0 in wheat. Through polymorphic analysis between Ning7840 and Clark and the localization analysis using Chinese Spring and its nulli-tetrasomic lines Nulli (N) 3B Tetra (T) 3A, N3AT3B and N3DT3A, 5 STS (Sequence tagged site) markers and 5 SSCP markers were obtained and localized on chromosome 3B. These markers were used for genotyping of all the RILs from the population of Ning 7840/Clark. These 10 DNA markers could be located in the Qfhb₃BS region in Ning7840. Among these 10 DNA markers, 5 DNA markers Xsts4,Xsscp1,Xsscp2,Xsscp3 and Xsscp4 were located between SSR marker Xgwm389 and Xgwm493. The R2 of these 5 DNA markers was higher than that of Xgwm389 and Xgwm493 both in floret-inoculation method and spray-inoculation method in 2006 and 2007. Among these 5 DNA markers, The R2 of SSCP marker Xsscp1 and Xsscp2 was higher than that of Xgwm533 both in floret-inoculation method and spray-inoculation method in 2006 and 2007.5) 286 primer pairs were designed from the sequences of the ESTs whose sequences were homologue to the BAC or PAC sequences in the distal region of the chromosome 1S in rice. Through polymorphic analysis between Ning7840 and Clark and the localization analysis using Chinese Spring and its nulli-tetrasomic lines N3BT3A, N3AT3B and N3DT3A, 3 STS markers and 2 SSCP markers were obtained and localized on chromosome 3B. Through genotyping of all the RILs from the population of Ning 7840/Clark, these 5 DNA markers could be located in the Qfhb₃BS region in Ning7840. Among these 5 DNA markers, SSCP marker Xsscp7 was located between SSR marker Xgwm389 and Xgwm493. The R2 of Xsscp7 was higher than that of Xgwm389 and Xgwm493 and lower than that of Xgwm533 both in floret-inoculation method and spray-inoculation method in 2006 and 2007.6) The primers of the 35 STS markers located on chromosome 3BS that were reported by Liu (2003a, 2003b, 2005) were used to develop DNA markers in the Qfhb₃BS region in Ning7840. Through polymorphic analysis between Ning7840 and Clark and the localization analysis using Chinese Spring and its nulli-tetrasomic lines N3BT3A, N3AT3B and N3DT3A, one STS marker Xsts1 and one SSCP marker Xsscp8 were obtained and localized on chromosome 3B. Two SSR markers XCS-SSR7 and XCS-SSR20 reported by Shen et al(2005)were located in the Qfhb₃BS region. Through polymorphic analysis between Ning7840 and Clark and the localization analysis using Chinese Spring and its nulli-tetrasomic lines N3BT3A, N3AT3B and N3DT3A, Only XCS-SSR7 was polymorphic between Ning7840 and Clark and located on chromosome 3B.Through genotyping of all the RILs from the population of Ning 7840/Clark, Xsts1, Xsscp8 and XCS-SSR7 could be located in the Qfhb₃BS region in Ning7840, but all outside Xgwm493. The R2 of Xsts1and Xsscp8 was significantly lower than that of Xgwm493 both in floret-inoculation method and spray-inoculation method in 2006 and 2007.7) 571 primer pairs were used to exploit CAPS (Cleaved Amplified Polymorphic Sequence) markers in the Qfhb₃BS region. 2 CAPS markers were obtained and localized on chromosome 3B. Through genotyping of all the RILs from the population of Ning 7840/Clark, these 2 CAPS markers could be located in the Qfhb₃BS region in Ning7840. Xcaps1 is outside Xgwm389, while Xcaps2 is outside Xgwm493. The R2 of these two CAPS markers was lower than that of Xgwm389, Xgwm493 and Xgwm533 in almost all cases. 8) A genetic linkage map in the Qfhb₃BS region in Ning7840 was constructed with 27 simple and economical markers that consisted of 8 SSR markers, 9 STS markers, 8 SSCP markers and 2 CAPS markers. The smallest distance between markers reaches 0.2 cM.In summary, FHB evaluation in Ning7840/Clark RIL population was carried out in Nanjing district. Analyzed by the mixed major gene plus poly-gene inheritance model of quantitative traits, FHB resistance in the cross Ning7840/Clark was controlled by three major genes under floret-inoculation method. Analyzed by Map Manager QTXb20 software, three FHB resistant QTLs was identified in this population which is coincided with the genetic model analysis. The effect of the QTL on 3BS was the biggest among the three QTLs. This QTL also had the biggest effect under spray-inoculation method. Using STS, SSCP and CAPS technique to exploit DNA markers in the Qfhb₃BS region in Ning7840, 18 new DNA markers were obtained and located in this region. A regional linkage map was constructed with relative high simple marker density. 6 new DNA markers were located between Xgwm389 and Xgwm493. These new exploited DNA markers could be used in marker-assisted selection for FHB resistant breeding and map-based cloning of the 3BS QTL. Moreover, SSCP technique in wheat was built and optimized, which provide a simple method for detecting minor difference and mutation in wheat genome.