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 approximately 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.

Thermo-photoperiod-sensitive genic male sterile (TPGMS) wheat is important in utilization of heterosis. To facilitate the use of such wheat line in agriculture, more knowledge about molecular mechanisms of TPGMS genes is required. In this study, we set up a normalized complementary DNA (cDNA) library based on the strategy of saturation hybridization with genomic DNA using TPGMS wheat line. This normalized cDNA library consists of cDNA from six directionally cloned cDNA libraries constructed with spike and anther tissues from spike developmental stages. From the normalized cDNA library, 3,264 single-pass expressed sequence tag (EST) were obtained. Exclusion of sequences shorter than 100 bp resulted in 3,223 vector-trimmed ESTs with a mean length of 926 bp. Clustering and assembly analysis resulted in 2,175 unique ESTs from 423 contigs and 1,752 singletons. Taking advantage of various tools and database, gene function classification showed that 60% of the ESTs were predicted to have putative gene function. Of the 2,175 unique ESTs, 264 (12%) displayed significant homology (BlastX E values <10 −5 ) to genes previously reported to be involved in cold-response related processes. Among these, sequences encoding activities related to primary metabolism, signal transduction, and transcriptional regulation were observed. Finally, in the total EST sequences, 108 potential SSRs were found. The unigene dataset will now be used to fabricate biochips carrying all identified genes for TPGMS wheat functional genomic research.