Mapping Quantitative Trait Loci For Peroxidase Activity,cloning Peroxidase Genes And Developing Functional Markers

Flour color is an important trait in the assessment of flour quality, and peroxidase（POD） has browning and bleaching effects on the color of flour and end-use products. Characterization of POD genes and development of functional markers are important for molecular marker-assisted selection in wheat quality breeding. In the present study, QTL for POD activity in common wheat were mapped using a RIL population derived from the Doumai/Shi 4185 cross grown in four environments and the wheat 90 K i Select assay. The POD genes were cloned by the method of homologous cloning and PCR verification, and the functional markers were developed for the genes based on allelic variants among different wheat cultivars. The functional markers were verified on 281 wheat cultivars and advanced lines from three wheat regions in China and 214 RILs of Doumai/Shi 4185 population. The main results were summarized as follows:1. POD activity of a RIL population derived from the Doumai/Shi 4185 cross was significantly affected by genotypes, environments and their interaction. Heritability of POD activity across four environments was 0.76, indicating that POD activity is mainly affected by genetic factors and that selection for POD activity can be effectively applied at the early generation in wheat breeding program.2. QTL for POD activity in common wheat were mapped using a RIL population derived from the Doumai/Shi 4185 cross grown in four environments and the RIL population was finally genotyped with 7,391 SNP markers and a new STS marker developed in this study. Three QTL for POD activity were detected in the RIL population across the four environments and the averaged data, designated QPod.caas-3AL, QPod.caas-4BS and QPod.caas-5AS, on chromosomes 3AL, 4BS and 5AS, respectively. QPod.caas-3AL was flanked by Excalibur_c6906₁167 and POD-3A1/POD-3A2 with a genetic distance of 2.3 c M; QPod.caas-4BS was flanked by BS00026143₅1 and Excalibur_c17607₅42 with a genetic distance of 9.8 c M and QPod.caas-5AS was flanked by wsnp_Ex_c16551₂5061517 and CAP8_s9855₁65 with a genetic distance of 10.8 c M. QPod.caas-3AL, QPod.caas-4BS and QPod.caas-5AS explained 5.3-9.2%、9.3-21.2% and 5.8-11.7% of phenotypic variation, respectively.3. The full-length genomic DNA sequence of Ta Pod-A1, Ta Pod-B1, Ta Pod-A2, Ta Pod-A3 and Ta Pod-D2 on chromosomes 3AL, 3B, 7A, 4AL and 7D, respectively, were characterized by homolog cloning and experimental validation. The coding sequences（CDS） were 1107, 1110, 1089, 1083 and 1077 bp for Ta Pod-A1, Ta Pod-B1, Ta Pod-A2, Ta Pod-A3 and Ta Pod-D2, respectively. Similar to rice prx23, there was one intron in Ta Pod-A1 and Ta Pod-B1 genes. However, no introns were found in Ta Pod-A2, Ta Pod-A3 and Ta Pod-D2.4. Two alleles, designated Ta Pod-A1 a and Ta Pod-A1 b, were amplified from cultivars with lower and higher POD activities, respectively. Five single nucleotide polymorphisms were found between two alleles. Based on the first, second and fifth SNP in Ta Pod-A1 a and Ta Pod-A1 b, two complementary dominant markers, designated POD-3A1 and POD-3A2, were developed. POD-3A1 designed from the Ta Pod-A1 a sequence, amplified a 291 bp PCR fragment in cultivars with lower POD activities, but produced no PCR products in those with higher POD activities. In contrast, POD-3A2, developed from Ta Pod-A1 b, yielded a 766 bp fragment in cultivars with higher POD activities, but no PCR products in those with lower POD activities. Thus POD-3A1 and POD-3A2 were complementary markers; they were located on chromosome 3AL using a set of CS nulli-tetrasomics and ditelosomic 3AL and 3AS lines, in agreement with the linkage analysis based on SNP markers. POD-3A1 and POD-3A2 were verified on 281 wheat cultivars and advanced lines, and showed highly significant（P < 0.05） associations with POD activities. Morever, ANOVA also indicated that lines with Ta Pod-A1 a from Doumai/Shi 4185 RIL population had significantly lower POD activities than those with Ta Pod-A1b（P < 0.01） in all four environments and the averaged data. Therefore, POD-3A1 and POD-3A2, co-segregating with Ta Pod-A1, could serve as gene-specific markers for improving color attributes in wheat breeding programs.This study first reported the allelic variations of Ta Pod-A1, and the functional markers POD-3A1 and POD-3A2 were of high value in wheat quality breeding.