Molecular identification and distribution of the polyphenol oxidase genes in Chinese winter wheat cultivars

Chino. 利用分子标记研究中国冬小麦品种PPO基因的等位变异及其与PPO活性的关系，为小麦PPO活性的分子标记辅助选择（MAS）奠定基础。利用PPO基因的功能标记PPO18、PPO29和PPO16对中国4个麦区的冬小麦品种（系）（试验Ⅰ）和山东省常用亲本（试验Ⅱ）共311份进行2A和2D染色体上PPO等位基因Ppo-A1a、Ppo-A1b、Ppo-D1a和Ppo-D1b的检测。PPO18在等位基因Ppo-A1a（高PPO）和Ppo-A1b（低PPO）中分别扩增685 bp和876 bp的片段，PPO16和PPO29在Ppo-D1a（低PPO）和Ppo-D1b（高PPO）两个等位基因中分别扩增713 bp和490 bp的片段。311份品种（系）中Ppo-A1a、Ppo-A1b、Ppo-D1a和Ppo-D1b等位基因的频率分别为41.8%、58.2%、59.8%和40.2%，PPO活性在同一基因的两个等位基因间差异均达显著水平（P＜0.05）；两个PPO基因的等位基因组合类型分布频率为：Ppo-A1a/Ppo-D1a（27.3%）、Ppo-A1a/Ppo-D1b（14.5%）、Ppo-A1b/Ppo-D1a（32.5%）和Ppo-A1b/Ppo-D1b（25.7%），彼此差异均达显著水平（P＜0.05）。各麦区间PPO活性基因分布存在明显差异，Ppo-A1a基因在长江中下游冬麦区和西南冬麦区分布较多，频率分别为60.7%和56.0%；Ppo-A1b基因在北部冬麦区和黄淮冬麦区分布较高，频率分别为65.6%和60.3%；而Ppo-D1a基因在北部冬麦区、黄淮冬麦区、长江中下游冬麦区和西南冬麦区分布明显高于Ppo-D1b基因频率，分别为65.6%、53.6%、75.0%和76.0%。PPO18、PPO16和PPO29标记的检测方法简单、稳定性好，所检测的等位变异类型能有效反映品种的PPO活性值。中国冬麦区品种低PPO活性基因分布频率相对较多。因此，根据PPO基因类型组配亲本，并利用PPO基因功能标记在育种早代筛选低PPO活性的基因型，可促进面制品色泽的改良。

Inglés. The aims of this study were to characterize the allelic distribution of PPO genes in Chinese winter wheat cultivars with molecular markers and analyze the association between PPO activity and different PPO alleles for the improvement of PPO activity in marker-assisted selection (MAS). A total of 311 Chinese wheat cultivars and advanced lines collected from four winter wheat regions (Experiment 1) and common parents in Shandong province (Experiment 2) were tested with the functional markers PPO18, PPO16 and PPO29 for the identification of four allelic variants Ppo-A1a, Ppo-A1b, Ppo-D1a and Ppo-D1b on chromosomes 2A and 2D. PPO18 could amplify a 685-bp and an 876-bp fragments in the cultivars with the allele Ppo-A1a (high PPO) and Ppo-A1b (low PPO), respectively. Two complementary dominant markers, PPO16 and PPO29, amplified a 713-bp and a 490-bp fragment in the genotypes with Ppo-D1a (low PPO) and Ppo-D1b (high PPO), respectively. The frequencies for Ppo-A1a, Ppo-A1b, Ppo-D1a and Ppo-D1b were 41.8%, 58.2%, 59.8% and 40.2%, in the 311 cultivars and advanced lines, respectively. Significant differences in PPO activities were detected between the genotypes with Ppo-A1a and those with Ppo-A1b (P＜0.05), and between the genotypes for Ppo-D1a and those for Ppo-D1b. Frequencies of PPO allelic combinations for Ppo-A1a/Ppo-D1a, Ppo-A1a/Ppo-D1b, Ppo-A1b/Ppo-D1a and Ppo-A1b/Ppo-D1b were 27.3%, 14.5%, 32.5% and 25.7%, and highly significant differences in PPO activity were found among the four genotypes (P＜0.05). Ppo-A1a was present as a major allele in middle and low Yangtze Valley winter wheat region and southwestern winter wheat region, with frequencies of 60.7% and 56.0%, respectively, whereas Ppo-A1b was found as a major allele in northern winter wheat region and Yellow and huai facultative winter wheat region, with frequencies of 65.6% and 60.3%, respectively. Ppo-D1a was dominant in northern winter wheat region, Yellow and Huai facultative winter wheat region, middle and low Yangtze Valley winter wheat region and southwestern winter wheat region, with frequencies of 65.6%, 53.6%, 75.0% and 76.0%, respectively. PPO18, PPO16 and PPO29 were simple and stable functional markers that can accurately discriminate different alleles of PPO genes on chromosomes 2A and 2D, exhibiting high correlation between grain PPO activity and PPO genotypes. The alleles of low PPO activity were dominant in Chinese winter wheat regions. Therefore, employing the PPO markers to test parents and separating generations descendants in wheat breeding programs would help breeder to select the individuals with low PPO activity, accelerating the progress for the improvement of undesirable darkening of end-use product.