Aegilops caudata is an important gene source for wheat breeding. Intensive evaluation of its utilization value is an essential first step prior to its application in breeding. In this research, the agronomical and quality traits of Triticum aestivum-Ae. caudata additions B–G (homoeologous groups not identified) were analyzed and evaluated. Disease resistance tests showed that chromosome D of Ae. caudata might possess leaf rust resistance, and chromosome E might carry stem rust and powdery mildew resistance genes. Investigations into agronomical traits suggested that the introduction of the Ae. caudata chromosome in addition line F could reduce plant height. Grain quality tests showed that the introduction of chromosomes E or F into wheat could increase its protein and wet gluten content. Therefore, wheat-Ae. caudata additions D–F are all potentially useful candidates for chromosome engineering activities to create useful wheat-alien chromosome introgressions. A total of 55 EST-based molecular markers were developed and then used to identify the chromosome homoeologous group of each of the Ae. caudata B–G chromosomes. Marker analysis indicated that the Ae. caudata chromosomes in addition lines B to G were structurally altered, therefore, a large population combined with intensive screening pressure should be taken into consideration when inducing and screening for wheat-Ae. caudata compensating translocations. Marker data also indicated that the Ae. caudata chromosomes in addition lines C–F were 5C, 6C, 7C, and 3C, respectively, while the homoeologous group of chromosomes B and G of Ae. caudata are as yet undetermined and need further research.

This study characterized and evaluated a set of wheat-Aegilops comosa introgression lines, including six additions and one substitution. A total of 47 PLUG markers and a set of cytogenetic markers specific for Ae. comosa chromosomes were established after screening 526 PLUG primer pairs and performing FISH using oligonucleotides as probes. Marker analysis confirmed that these lines were wheat-Ae. comosa 2M–7M addition lines and a 6M(6A) substitution line. The molecular and cytogenetic markers developed herein could be used to trace Ae. comosa chromatin in wheat background. In order to evaluate the breeding value of the material, disease resistance tests and agronomical trait investigations were carried out on these alien chromosome introgression lines. Disease resistance tests showed that chromosomes 2M and 7M of Ae. comosa might harbor new stripe rust and powdery mildew resistance genes, respectively, therefore, they could be used as resistance sources for wheat breeding. Investigations into agronomical traits showed that all chromosomes 2M to 7M had detrimental effects on the agronomic performance of wheat, therefore, the selection of plants with relatively negative effects should be avoided when inducing wheat-A. comosa chromosome translocations using chromosome engineering procedures.

Polyphenol oxidase (PPO) activity is a major cause of the undesirable brown color of wheat-based products. Ppo1, a major gene for PPO activity, was cloned based on sequence homology in previous studies; however, its function and regulation mechanism remain unclear. In this study, the function and genetic regulation of Ppo1 were analyzed using RNA interference (RNAi) and Targeting Induced Local Lesions IN Genomes (TILLING) technology, and superior mutants were identified. Compared with the control, the level of Ppo1 transcript in RNAi transgenic lines was drastically decreased by 15.5%–60.9% during grain development, and PPO activity was significantly reduced by 12.9%–20.4%, confirming the role of Ppo1 in PPO activity. Thirty-two Ppo1 mutants were identified in the ethyl methanesulfonate (EMS)-mutagenized population, including eight missense mutations, 16 synonymous mutations, and eight intron mutations. The expression of Ppo1 was reduced significantly by 6.7%–37.1% and 10.1%–54.4% in mutants M092141 (G311S) and M091098 (G299R), respectively, in which PPO activity was decreased by 29.7% and 28.8%, respectively, indicating that mutation sites of two mutants have important effects on PPO1 function. Sequence and structure analysis revealed that the two sites were highly conserved among 74 plant species, where the frequency of glycine was 94.6% and 100%, respectively, and adjacent to the entrance of the hydrophobic pocket of the active site. The M092141 and M091098 mutants can be used as important germplasms to develop wheat cultivars with low grain PPO activity. This study provided important insights into the molecular mechanism of Ppo1 and the genetic improvement of wheat PPO activity.

Apart from starch content, structure is also vital for the eating and nutritional qualities of final products. The study investigated how the starch structure changed during grain filling period using two wheat cultivars with different filling rate. The starch biosynthesis during grain filling was not at uniform speed and most of starches were synthesized before 19 DAF. The starch structure establishment was latter than starch accumulation, but the structure formed at early stage of grain filling was highly related to the final one. The process of starch biosynthesis was closely accompanied by the elongation of amylose chains, increase on branch numbers, B-type granule number and expansion of A-type granule. Further analysis on expression profiles of key genes involved in starch biosynthesis demonstrated that different genes exhibited varying responsiveness to the filling rate, and the resulting imbalance in gene expression may underlie the observed structural differences. Faster filling promoted earlier activation of starch-biosynthesis genes, particularly GBSSI, leading to preferential elongation of amylose chains, higher crystallinity, and more B-type granules. The results will provide new insights to understanding the formation starch structure during grain filling, which may benefit future research on management and breeding.

Nuclear proteins play critical roles in regulating mRNA transcription and processing, DNA replication, and epigenetic genome modification. Therefore, the ability to monitor changes in nuclear proteins is helpful not only to identify important regulatory proteins but also to study the mechanisms of actions of nuclear proteins. However, no effective methods have been developed yet. Rye is strongly resistant to various biotic and abiotic stresses; however, few genes have been functionally characterized to date due to the complexity of its genome and a lack of genomic sequence information. We developed an integrative Nuclear Transportation Trap (iNTT) system that includes an improved nuclear transportation trap and utilizes the “after suppression subtraction” method. Oligonucleotides encoding a nuclear localization signal (NLS) or a transcription factor, GmAREB, were inserted into pLexAD or pLexAD-NES, respectively, and then transformed into yeast cells (EGY48). We showed that the pLexAD vector expressing a cDNA library in the iNTT system was more efficient for screening than the vector pLexAD-NES, which has previously been used in an NTT system. We used the iNTT system to screen a cDNA library of cold-treated rye. A total of 241 unique genes were identified, including 169 differentially expressed proteins; of these, 106 were of known and 63 were of unknown function. Moreover, 82 genes (49 %) among the 169 differentially expressed genes were predicted to contain an NLS domain. Thirty-three (31 %) of the 106 functionally known proteins have DNA-binding activity. To test the specificity of the nuclear proteins identified using the iNTT screen, four of the proteins differentially expressed in response to temperature stress, ScT1 (a heat shock protein), ScT36 (a MYB-like transcription factor), ScT133 (an ERF-like transcription factor) and ScT196 (a protein of unknown function), were studied in more depth. These proteins were shown to exclusively localize to the nucleus, and their expression levels were increased in response to low-temperature stress. To identify the function of these screened nuclear proteins, ScT1- and ScT36-transgenic Arabidopsis plants were constructed, and ScT1 or ScT36 overexpression was found to enhance tolerance to high-temperature or freezing stresses, respectively. The newly developed iNTT system provides an effective method for identifying nuclear-targeted proteins and monitoring induced expression levels. ScT1 and ScT36 might be good candidate genes for improving the stress tolerance of plants by genetic transformation.