Development Of Molecular Marker For Grain PPO Activity And Detection Of Flour Yellow Pigment Candidate Genes In Bread Wheat

Breeding wheat cultivars with low PPO activity is the best approach to reduce the undesirable darkening. Molecular markers could greatly improve selection efficiency in breeding programs. This paper include two parts: (1) development and identification of molecular markers associated with wheat grain polyphenol oxidase (PPO) activity which has been related to undersirable brown discoloration of bread wheat (Triticum aestivum L.) based end-products, particularly for Asian noodles. (2) detection of wheat PSY ( phytoene synthase ) and LOX ( lipoxygenase ) genes which may be related to wheat flour yellow pigment. 1. Validation of SSR marker assisted selection for polyphenol oxidase activities in bread wheat A total of 203 Chinese winter wheat cultivars and lines were analyzed to investigate the correlation of PPO activities with a SSR (simple sequence repeat) marker Xgwm312. The results indicated that a fragment with 198 bp is apparently correlated with high PPO activities in wheat grain. The marker Xgwm312 can be used for MAS for PPO activities. Nevertheless, the detection of PCR fragment amplified with SSR marker Xgwm312 is based on the technique of polyacrylamide gel, which is complicated and expensive. 2. A novel STS marker for PPO activity in bread wheat Based on the sequences of PPO genes (EST) conditioning PPO activity during kernel development, 28 pairs of primers were designed. One of the markers designated as PPO18, can amplify a 685-bp and a 876-bp fragment in the cultivars with high and low PPO activity, respectively. The STS marker PPO18 was mapped to chromosome 2AL using a DH population derived from a cross Zhongyou 9507 ×CA9632, and a set of nulli-tetrasomic lines and ditelosomic line 2AS of Chinese Spring. QTL analysis indicated that the PPO gene co-segregated with the STS marker PPO18 and is closely linked to Xgwm312 and Xgwm294 on chromosome 2AL, explaining 44% of phenotypic variance for PPO activity. A total of 233 Chinese wheat cultivars and advanced lines were used to validate the correlation between the polymorphic fragments of PPO18 and grain PPO activity. The results showed that PPO18 is a co-dominant, efficient and reliable molecular marker for PPO activity and can be used in wheat breeding programs targeted for noodle quality improvement. The sequence alignments revealed that at the position of intron I, the PPO gene in cultivars with low PPO activity has 191 bases more than that in cultivars with high PPO activity where PPO gene is expressed. The additional 191-bp insertion sequence might influence the splicing of premature mRNA, which could cause the PPO gene unable to express. In addition, a intron with 'GC-AG'characteristics which was not reported previously in plant genes has also been found. 3. Detection of phytoene synthase (PSY) and lipoxygenase (LOX) genes Based on the sequences of maize PSY gene and barley LOX gene, 2 pairs of primers were designed. The amplified fragments sequence alignments revealed that the number of identical nucleic acids of wheat and maize PSY gene is 90% and identical amino acids is 97%, respectively. Wheat LOX is similar to the other cereal crop LOX. Using a set of nulli-tetrasomic lines of Chinese Spring, PSY gene were preliminarily designed to 1D chromosome and LOX gene to 4BS chromosome.