Development and application of a STS marker for grain PPO gene located on chromosome 2D in common wheat

中国人. 开发出小麦籽粒PPO活性的分子标记，并对新标记的应用进行研究，为面制食品外观品质的改良提供参考。根据一条由小麦2D染色体上PPO基因编码的mRNA序列（GenBank：AY515506）设计引物，对7个高PPO活性和7个低PPO活性小麦品种进行PCR扩增，筛选出有差异的引物，对130份小麦品种资源进行检测，验证不同带型与PPO活性的相关性，并利用一套中国春缺体、四体及双端体对STS标记进行染色体定位。在此基础上，结合一个位于小麦2A染色体上的PPO基因分子标记（PPO18），对新开发的标记在小麦低PPO活性分子标记辅助选择中的作用进行评价。在AY515506的不同位置共设计了8对引物，其中一对引物（STS01）在高、低PPO活性材料中表现出多态性。该引物在7个低PPO活性的材料中能扩增出560 bp的目标片段，在7个高PPO活性的材料中没有扩增出目标片段，利用中国春缺体、四体及双端体最终将该标记定位在2D染色体长臂上。利用该标记检测130份小麦品种，结果表明有75个品种可以扩增出560 bp的目标片段，其PPO活性均值为221.08 A475/（min•g•103）；55个品种没有扩增出目标片段，PPO活性均值为309.98 A475/（min•g•103），方差分析表明两者的差异达极显著水平（P＜0.01）。利用STS01和PPO18共同检测后发现，130份品种中，有37个品种的双标记扩增均表现出高活性带型（H1H2），这些品种PPO活性的均值为337.82 A475/（min•g•103），显著高于其它几种扩增带型品种的PPO活性均值（P＜0.01）。32个双标记扩增均表现为低活性带型（L1L2）的小麦品种PPO活性值普遍较低，可作为改良面制食品外观品质的候选亲本。STS01是一个位于小麦2D染色体长臂上PPO基因分子标记，可以在小麦PPO活性分子标记辅助选择中加以应用。

英语. 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 of appearance. Molecular marker-assisted selection (MAS) is more effective and faster than conventional selection in breeding program. Based on a wheat grain PPO mRNA sequence (GenBank Accession Number AY515506), eight pairs of primers were designed. Seven wheat cultivars with high and seven with low PPO activity were used for screening the newly developed STS marker. A total of 130 cultivars were used to verify the association between PPO activities and the STS marker. A set of nulli-tetrasomic lines and ditelosomic line 2DL of Chinese Spring were used to verify the chromosomal location of the newly developed STS marker, which was used in combination with PPO 18, a STS marker for PPO gene on chromosome 2AL to evaluate their feasibility in wheat breeding programs aimed at low grain PPO activity. One pair of primer, designated as STS01, amplified a fragment of 560 bp in length 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 on 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 A475/(min•g•103)] was significantly (P＜0.01) lower than that of the latter [309.98 A475/(min•g•103)]. 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 A475/(min•g•103)] 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. STS01 is an efficient and reliable molecular marker for PPO activity, and can be used in wheat breeding programs aimed at low PPO activity.