| Grain polyphenol oxidase (PPO) activity is highly related to the brown discoloration of wheat based end-products, particularly for noodles and steam bread. Breeding wheat cultivars with low PPO activity is the best way to improve their quality in appearance. Molecular marker-assisted selection (MAS) is more effective and faster than conventional selection in breeding program. Studying on the variation of PPO activity and the distribution of allelic genes in Chinese wheat micro-core collections could take good advantage of germplasm and improve appearance quality of wheat flour food.1. Based on three wheat grain PPO gene sequences (GenBank Accession Number AY515506, AY596270 and AF507945), primers were designed to screen wheat cultivars seven with high and seven with low PPO activity. One pair of primers, designated as STS01, amplified a 560 bp fragment in the cultivars with low PPO activity, while no PCR product was detected in the cultivars with high PPO activity. The marker STS01 was located to chromosome 2DL using a set of nulli-tetrasomic lines and ditelosomic line 2DL of Chinese Spring. In a test of 130 cultivars using STS01, the 560-bp fragment was detected in 75 genotypes, but not in the remaining 55 genotypes, and the mean value of PPO activity of the former cultivars (221.08) was significantly (p<0.01) lower than that of the latter (309.98). The two STS markers, STS01 and PPO18, located on chromosomes 2D and 2A respectively, were evaluated for their feasibility in wheat breeding programs. The results showed that the 37 cultivars with the genotype H1H2 exhibited a significantly higher mean value of grain PPO activity (337.82), than those of other genotypes (P<0.01). Most of the 32 cultivars with the genotype L1L2 showed very low grain PPO activity, which can be used as parents for low grain PPO activity in wheat breeding. Thus STS01 is an efficient and reliable molecular marker for PPO activity, and can be used in wheat breeding programs aimed for low PPO activity.2. Wheat PPO sequences (mRNA) were searched/blasted in NCBI and aligned using DNAMAN software. The results showed that wheat PPO genes could be divided into 2 clusters (I and II) and 3 genes ('i') of the'II'cluster seemed not to be located on chromosomes 2A and 2D. Ninety-four single nucleotide polymorphisms (SNP) were detected between 2 haplotypes of PPO gene on chromosome 2D. Eighty SNPs of them were found in the coding region (coding SNP) and 36 were non-synonymous cSNPs, which could affect the PPO amino acid sequence. Primers were designed as STS-H at some non-synonymous cSNPs sites and used to research the correlations between allelic variants and PPO activity of seeds, which valued, in two years, a total of 130 common wheat varieties. The results showed that STS-H could amplify a 460 bp DNA fragment in most cultivars with high PPO activity, while no PCR product was detected in most cultivars with low PPO activity. To improve the selection efficiency of single dominance molecular marker, the multiplex PCR system of STS-H and STS01 markers was also studied, based on the complementary between them.3. The variation of PPO activity and the distribution of PPO allelic genes were detected by method of biochemistry and gene-based markers in 251 Chinese wheat micro-core collections growning in Hefei and Fengyang in 2004-2005 crop seasons. The results showed that most cultivars are with low PPO activity and the rate of varieties with PPO activity below 200 was 47.01% and the variation of PPO activity was affected by genotype and locations and interaction between them. Four genotypes PPO-2Aa1/PPO-2Da2 (a1a2), PPO-2Aa1/PPO-2Db1 (a1b1), PPO-2Aa1/PPO-2Db2 (a1b2), and PPO-2Ab1/PPO-2Db2 (b1b2) were detected in Chinese wheat micro-core collections. Cultivars with the genotype a1a2 gave the lowest PPO activity while the genotypes b1b2 were the highest, with genotype a1b2 and genotype b1a2 intermediate. The effect of interaction between marker genotypes and locations on PPO activity was not significant (P<0.05). The frequency of PPO allelic genes on two wheat chromosomes 2A and 2D was different. The frequency of allelic gene 2Aa1 and 2Ab1 on chromosome 2A was most alike, while the frequency of 2Da2 was almost 4 times than that of 2Db2 on chromosome 2D. The effect of haplotype of PPO genes on the same chromosome on PPO activity were PPO-2AaKPPO-2Ab1 and PPO-2Da2 |
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