Allelic variation of polyphenol oxidase (PPO) genes located on chromosomes 2A and 2D and development of functional markers for the PPO genes in common wheat
Allelic variation of polyphenol oxidase (PPO) genes located on chromosomes 2A and 2D and development of functional markers for the PPO genes in common wheat
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DOI：
10.1007/s00122-007-0539-8
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发表时间：
2007-06-01
影响因子：
5.4
通讯作者：
Xia, X. C.
中科院分区：
文献类型：
--
作者：
He, X. Y.;He, Z. H.;Xia, X. C.
多酚氧化酶(PPO)活性与小麦终端产品，特别是亚洲面条的褐变密切相关。PPO基因的鉴定及其功能标记的开发对小麦育种中的分子标记辅助选择具有重要意义。本研究通过电子克隆和实验验证的方法，对位于染色体2A和2D上的两个PPO基因及其等位基因变异体的全基因组DNA序列进行了分析。序列在DNA和蛋白质水平上都进行了比对。2D染色体上的两种单倍型在DNA水平上的序列同源性为95.2%，比2A染色体上的99.6%的序列同源性高得多。位于第2A和2D染色体上的PPO基因均含有1,731bp的开放阅读框，编码577个氨基酸的PPO前体多肽，预测的分子质量与KD相似。根据位于2D染色体上的PPO基因单倍型，开发了两个互补的显性STS标记PPO16和PPO29，它们分别在PPO活性低的品种中扩增出713bp的片段，在PPO活性高的品种中扩增出490bp的片段。利用来自杂交组合中优9507/CA9632的加倍单倍体群体和一组中国春的缺体-四体品系和二体品系2DS，将这两个标记定位在2DL染色体上。QTL分析表明，PPO基因与两个STS标记共分离，并与位于2DL染色体上的SSR标记Xwmc41紧密连锁，可解释三个环境中PPO活性表型变异的9.6%~24.4%。为了同时检测2A和2D染色体上的PPO基因座，开发了PPO33/PPO16复合标记组合，并在多个品种上获得了可区分的DNA图谱。位于第2A染色体上PPO基因的STS标记PPO33与我们之前报道的PPO18基因序列相同，可以从低PPO活性和高PPO活性的品种中分别扩增出481bp和290bp的片段。以217个中国小麦品种和优良品系为材料，验证了多态片段与籽粒PPO活性的相关性。结果表明，PPO33/PPO16标记组合能有效、可靠地评价PPO活性，可用于面条等终端产品品质改良的小麦育种。
Polyphenol oxidase (PPO) activity is highly related to the undesirable browning of wheat-based end products, especially Asian noodles. Characterization of PPO genes and the development of their functional markers are of great importance for marker-assisted selection in wheat breeding. In the present study, complete genomic DNA sequences of two PPO genes, one each located on chromosomes 2A and 2D and their allelic variants were characterized by means of in silico cloning and experimental validation. Sequences were aligned at both DNA and protein levels. Two haplotypes on chromosome 2D showed 95.2% sequence identity at the DNA level, indicating much more sequence diversity than those on chromosome 2A with 99.6% sequence identity. Both of the PPO genes on chromosomes 2A and 2D contain an open reading frame (ORF) of 1,731 bp, encoding a PPO precursor peptide of 577 amino acids with a predicted molecular mass of similar to 64 kD. Two complementary dominant STS markers, PPO16 and PPO29, were developed based on the PPO gene haplotypes located on chromosome 2D; they amplify a 713-bp fragment in cultivars with low PPO activity and a 490-bp fragment in those with high PPO activity, respectively. The two markers were mapped on chromosome 2DL using a doubled haploid population derived from the cross Zhongyou 9507/CA9632, and a set of nullisomic-tetrasomic lines and ditelosomic line 2DS of Chinese Spring. QTL analysis indicated that the PPO gene co-segregated with the two STS markers and was closely linked to SSR marker Xwmc41 on chromosome 2DL, explaining from 9.6 to 24.4% of the phenotypic variance for PPO activity across three environments. In order to simultaneously detect PPO loci on chromosomes 2A and 2D, a multiplexed marker combination PPO33/PPO16 was developed and yielded distinguishable DNA patterns in a number of cultivars. The STS marker PPO33 for the PPO gene on chromosome 2A was developed from the same gene sequences as PPO18 that we reported previously, and can amplify a 481-bp and a 290-bp fragment from cultivars with low and high PPO activity, respectively. A total of 217 Chinese wheat cultivars and advanced lines were used to validate the association between the polymorphic fragments and grain PPO activity. The results showed that the marker combination PPO33/PPO16 is efficient and reliable for evaluating PPO activity and can be used in wheat breeding programs aimed for noodle and other end product quality improvement.