Association Analysis Among Variation Of PPO Gene, Ppo-A1Gene Polymorphism And Its Flour Whiteness In Wheat

The flour whiteness of wheat is a very important index parameter in wheat qualityresearch and breeding, it also a key sensory index for quality evaluation of the wheat flourand food made by flour. The level of polyphenol oxidase(PPO) of wheat grain has animportant influence on wheat flour and flour food whiteness, and the PPO activity controledby genes. Therefore, to study the variation of the wheat PPO gene Ppo-A1and itspolymorphism, the correlation with flour whiteness has a great significance for wheatimprovement of whiteness in China.In this study,173wheat cultivars were used to detected the variation of PPO genes bymolecular markers, and the flour whiteness and PPO activity variation were analysedcombined with the relevant physiochemical method, in addition, a system analysis to find therelationship of wheat flour whiteness, PPO activity and PPO variants. Ppo-A1specificprimers were used to detect the Ppo-A1gene polymorphism of26wheat varieties (AABBDD)and eight of wheat diploid related species rye (RR) by the method of direct cloning andsequencing, and analysed of the correlation with PPO activity and flour whiteness, results areas follows:1. The PPO active functional marker which located on the2A,2D chromosome wereused for gene variants analysis and classification in173wheat cultivars. The results showedthat four kinds of gene combination including Ppo-A1b/Ppo-D1a, Ppo-A1b/Ppo-D1b,Ppo-A1a/Ppo-D1a and Ppo-A1a/Ppo-D1b173were detected in173wheat cultivars, and thefrequency of each type of gene combination were38.7%,13.9%,35.8%and11.6%.The PPOallele frequency had great differences in different sites of the wheat cultivars. The frequencyof allele Ppo-A1a were similar to Ppo-A1b which located on the same chromosome of2A,however, the frequency of allele Ppo-D1a were3times as Ppo-D1b on2D chromosome.2. The analysis of wheat flour whiteness and PPO activity test showed that the meanflour whiteness were low, only73%, however, the variation range of different cultivars wassignificantly and38varieties of flour whiteness had reached the national standard. The ratioof cultivars with PPO activity is less than100AU min-1g-1was high(45.7%), and coefficientof flour whiteness with PPO activity was-0.337**, indicated that feasible to improve the whiteness of wheat flour by decreased the PPO activity through genetic improvement, highwhiteness and low PPO activity resources presented in domestic wheat varieties in wheatbreeding.3. The analysis of gene combination types and the relationship between PPO activity andflour whiteness showed that the order of four gene combination types was Ppo-A1a/Ppo-D1bï¹¥Ppo-A1a/Ppo-D1aï¹¥Ppo-A1b/Ppo-D1bï¹¥Ppo-A1b/Ppo-D1a, the differences between eachother have reached a significant level (P <0.05), the combination of Ppo-A1b/Ppo-D1a withlow PPO activity had a high frequency(38.7%), the mean flour whiteness of combinationPpo-A1a/Ppo-D1b was significantly lower than other combinations.4. With the comprehensive analysis of flour whiteness, PPO activity, molecular markersdetected of PPO activity allele combination (Ppo-A1a/Ppo-D1b),23cultivars with a stable ofhigh whiteness and low PPO activity were screened and can be used as parent materials inwheat breeding.5. There34wheat Ppo-A1gene were cloned, including12Ppo-A1a,17Ppo-A1b and5types of new alleles, named as Ppo-A1s. Three types of alleles have the typical exon/intronstructure of wheat PPO gene, which the three exons separated by two introns. The gene havecomplete coding frame, the whole gene length (defined as starting codon to stop codonsequence) were1961bp,2152bp and2100bp,577,577and431length of the amino-acidprotein sequence derivated corresponding to the encoding.6. Ppo-A1a llele analysis in34cultivars respectively showed that12cultivars weredetected to38SNP loci, not find the InDel loci, the allele SNP frequency was1SNP/519bp.17cultivars of Ppo-A1b were detected to53polymorphisms (SNPs), including51SNPs andtwo of InDel, frequencies were1SNP/717bp and1SNP/18291bp.18polymorphic lociwere detected in5cultivars of Ppo-A1s sequences, including17SNP and1InDel, the twofrequencies were1SNP/518bp and1SNP/10499bp.7.34of the cloned Ppo-A1gene sequence were divided into11haplotypes, of which H5and H11were typical low PPO activity haplotypes, it was a obvious correspondingrelationship between the polymorphism of Ppo-A1gene and PPO activity, but only for thegene polymorphisms cannot yet fully explain the activity of PPO variation.There was noobvious correlation between the polymorphism and flour whiteness.