Jiachen Sun

The Yr5 gene confers resistance to all races of the wheat stripe rust pathogen [Puccinia striiformis Westend. f. sp. tritici Eriks. ] identified so far in the USA. Cosegregating resistance gene analog polymorphism markers for Yr5 are available but their use requires skills in polyacrylamide gel electrophoresis and may not be polymorphic across various varieties. To develop better markers to be used in marker-assisted selection for the Yr5 resistance, sequence tagged site primers were designed on the basis of the sequences of RGAP markers Xwgp-18 from the spring wheat 'Avocet Susceptible' and Xwgp-17 from the Yr5 near isogenic line in the AVS background carrying the Yr5 gene from T. aestivum subsp. spelta Thell. cv. Album. Three sets of STS markers were developed to amplify a region including a polymorphic 6-base pairs insertion-deletion. The cosegregation of the STS markers with Yr5 was confirmed with 114 BC 7:F3 lines developed from the cross between AVS and TSA. The STS markers worked well in five out of 17 non-Yr5 wheat varieties, but the remaining varieties had a similar size of fragment to the Yr5 marker. Because the codominant STS markers were based on a 6-bp indel, they could not be separated by agarose gel electrophoresis. Cleaved amplified polymorphic sequence markers were then developed on the basis of a DpnII restriction site that is present in all non-Yr5 varieties and absent in the Yr5 NIL. The CAPS markers for the Yr5 NIL and non-Yr5 varieties can be separated by agarose gel electrophoresis. The codominant STS markers are easier to score than the original RGAP markers. The CAPS markers are not only easier to score, but also can be used in crosses of an Yr5 donor with a much wider range of wheat germplasms. These markers should be valuable tools to accelerate the introgression of Yr5 into commercial cultivars and to combine Yr5 with other genes for durable resistance to stripe rust.

© 2014 Elsevier Inc. All rights reserved.Sequence-programmable transcription activator-like effector proteins have emerged as a highly efficient tool for genome engineering. Recent crystal structures depict a transition between an open unbound solenoid and more compact DNA-bound solenoid formed by the 34 amino acid repeats. How TALEs switch conformation between these two forms without substantial energetic compensation, and how the repeat-variable di-residues discriminate between the cognate base and other bases still remain unclear. Computational analysis on these two aspects of TALE-DNA interaction mechanism has been conducted in order to achieve a better understanding of the energetics. High elasticity was observed in the molecular dynamics simulations of DNA-free TALE structure that started from the bound conformation where it sampled a wide range of conformations including the experimentally determined apo and bound conformations. This elastic feature was also observed in the simulations starting from the apo form which suggests low free energy barrier between the two conformations and small compensation required upon binding. To analyze binding specificity, we performed free energy calculations of various combinations of RVDs and bases using Poisson-Boltzmann surface area and other approaches. The PBSA calculations indicated that the native RVD-base structures had lower binding free energy than mismatched structures for most of the RVDs examined. Our theoretical analyses provided new insight on the dynamics and energetics of TALE-DNA binding mechanism.