Development of non-transgenic glyphosate tolerant wheat by TILLING.

Glyphosate (N-phosphonomethyl-glycine) is the world's most widely used broad spectrum, post-emergence herbicide. It inhibits the chloroplast-targeted enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS; EC 2.5.1.19), a component of the plant and microorganism-specific shikimate pathway and a key catalyst in the production of aromatic amino acids. Variants of EPSPS that are not inhibited by glyphosate due to particular amino acid alterations in the active site of the enzyme are known. Some of these variants have been identified in weed species that have developed resistance to glyphosate because of the strong selective pressure of continuous, heavy glyphosate use. We have used TILLING (Targeting Induced Local Lesions in Genomes), a non-transgenic, target-selected, reverse genetics, mutation breeding technique, and conventional genetic crosses, to identify and combine, through two rounds of mutagenesis, wheat lines having both T102I and P106S (so-called TIPS enzyme) mutations in both the A and the D sub-genome homoeologous copies of the wheat EPSPS gene. The combined effects of the T102I and P106S mutations are known from previous work in multiple species to minimize the binding of the herbicide while maintaining the affinity of the catalytic site for its native substrates. These novel wheat lines exhibit substantial tolerance to commercially relevant levels of glyphosate.

Development of Decreased-Gluten Wheat Enabled by Determination of the Genetic Basis of lys3a Barley.

Celiac disease is the most common food-induced enteropathy in humans, with a prevalence of approximately 1% worldwide. It is induced by digestion-resistant, proline- and glutamine-rich seed storage proteins, collectively referred to as gluten, found in wheat (Triticum aestivum). Related prolamins are present in barley (Hordeum vulgare) and rye (Secale cereale). The incidence of both celiac disease and a related condition called nonceliac gluten sensitivity is increasing. This has prompted efforts to identify methods of lowering gluten in wheat, one of the most important cereal crops. Here, we used bulked segregant RNA sequencing and map-based cloning to identify the genetic lesion underlying a recessive, low-prolamin mutation (lys3a) in diploid barley. We confirmed the mutant identity by complementing the lys3a mutant with a transgenic copy of the wild-type barley gene and then used targeting-induced local lesions in genomes to identify induced single-nucleotide polymorphisms in the three homeologs of the corresponding wheat gene. Combining inactivating mutations in the three subgenomes of hexaploid bread wheat in a single wheat line lowered gliadin and low-molecular-weight glutenin accumulation by 50% to 60% and increased free and protein-bound lysine by 33%.

Development of high amylose wheat through TILLING.

BACKGROUND: Wheat (Triticum spp.) is an important source of food worldwide and the focus of considerable efforts to identify new combinations of genetic diversity for crop improvement. In particular, wheat starch composition is a major target for changes that could benefit human health. Starches with increased levels of amylose are of interest because of the correlation between higher amylose content and elevated levels of resistant starch, which has been shown to have beneficial effects on health for combating obesity and diabetes. TILLING (Targeting Induced Local Lesions in Genomes) is a means to identify novel genetic variation without the need for direct selection of phenotypes. RESULTS: Using TILLING to identify novel genetic variation in each of the A and B genomes in tetraploid durum wheat and the A, B and D genomes in hexaploid bread wheat, we have identified mutations in the form of single nucleotide polymorphisms (SNPs) in starch branching enzyme IIa genes (SBEIIa). Combining these new alleles of SBEIIa through breeding resulted in the development of high amylose durum and bread wheat varieties containing 47-55% amylose and having elevated resistant starch levels compared to wild-type wheat. High amylose lines also had reduced expression of SBEIIa RNA, changes in starch granule morphology and altered starch granule protein profiles as evaluated by mass spectrometry. CONCLUSIONS: We report the use of TILLING to develop new traits in crops with complex genomes without the use of transgenic modifications. Combined mutations in SBEIIa in durum and bread wheat varieties resulted in lines with significantly increased amylose and resistant starch contents.

Determination of the antimony valence state in Eu10Mn6Sb13.

The antimony-121 Mössbauer spectra of Eu10Mn6Sb13 have been measured between 2 and 295 K. Although the Zintl formalism indicates that the nine crystallographically distinct antimony sites in Eu10Mn6Sb13 should have formal valence states of -2, -1, 0, and +1, the Mössbauer spectral isomer shifts reveal that the valence states of the different sites are all quite similar and correspond to an average electronic configuration for antimony of 5s(1.7)5p(4.0). This configuration corresponds to an excess of negative charge on the antimony of 0.7 or an average valence of -0.7, a valence which is rather consistent with the average antimony valence of -0.61 obtained from the Zintl formalism for the nine antimony sites in Eu10Mn6Sb13. The spectra obtained between 90 and 295 K are more consistent with the absence rather than the presence of any transferred magnetic hyperfine field at the antimony. In contrast, the spectra obtained at 2 and 5 K reveal the presence of an average transferred magnetic hyperfine field of ca. 8 T, a field that arises from the ferromagnetic ordering of the near-neighbor manganese(II) ions.

Eu10Mn6Sb13: a new ternary rare-Earth transition-metal Zintl phase.

A new transition-metal-containing Zintl compound, Eu(10)Mn(6)Sb(13), was prepared by a high-temperature Sn-flux synthesis. The structure was determined by single-crystal X-ray diffraction. Eu(10)Mn(6)Sb(13) crystallizes in the monoclinic space group C2/m with a = 15.1791(6) A, b = 19.1919(7) A, c = 12.2679(4) A, beta = 108.078(1)*, Z = 4 (R1 = 0.0410, wR2 = 0.0920), and T = 90(2) K. The structure of Eu(10)Mn(6)Sb(13) is composed of double layers of Mn-centered tetrahedra separated by Eu(2+) cations. The double layers are composed of edge- and corner-sharing Mn-centered tetrahedra which form cavities occupied by Eu(2+) cations and [Sb(2)](4-) dumbbells. Linear [Sb(3)](5-) trimers bridging two tetrahedra across the cavity are also present. Bulk susceptibility data indicate paramagnetic behavior with a ferromagnetic component present below 60 K. Temperature-dependent electrical resistivity measurements show semiconducting behavior above 60 K (E(a)() = 0.115(2) eV), a large and unusually sharp maximum in the resistivity at approximately 40 K, and metallic behavior below 40 K. (151)Eu Mössbauer spectra confirm that the europium is divalent with an average isomer shift of -11.2(1) mm/s at 100 K; the spectra obtained below 40 K reveal magnetic ordering of six of the seven europium sublattices and, at 4.2 K, complete ordering of the seven europium sublattices.

The crystal structure and magnetic properties of a new ferrimagnetic semiconductor: Ca21Mn4Sb18.

Single crystals of the new transition metal Zintl phase, Ca(21)Mn(4)Sb(18), were prepared by high temperature melt synthesis. The crystal structure was determined by single crystal X-ray diffraction to be monoclinic in the space group C2/c. Crystal information was obtained at 90 K, and unit cell parameters were determined (a = 17.100(2) A, b = 17.073(2) A, c = 16.857(2) A, beta = 92.999(2) degrees, Z = 2, R1 = 0.0540, wR2 = 0.1437). The structure can be described as containing 4 discreet units per formula unit: 1 linear [Mn(4)Sb(10)](22-) anion, 2 dumbbell-shaped [Sb(2)](4-) anions, 4 individual Sb(3-) anions, and 21 Ca(2+) cations. The [Mn(4)Sb(10)](22-) anion contains four edge-shared MnSb(4) tetrahedra with distances between Mn ions of 3.388(4) A, 2.782(4) A, and 2.760(4) A. Electron counting suggests that the Mn are 2+. Temperature dependent magnetization shows a ferromagnetic-like transition temperature at approximately 52 K which is suppressed with increasing magnetic field. The paramagnetic regime is best fit to a ferrimagnetic model, providing a total effective moment of 4.04(2) mu(B), significantly less than that expected for 4 Mn(2+) ions (11.8 mu(B)). Temperature dependent resistivity shows that this compound is a semiconductor with an activation energy of 0.159(2) eV (100-300 K).

XMCD characterization of the ferromagnetic state of Yb14MnSb11.

X-ray magnetic circular dichroism (XMCD) measurements on Yb14MnSb11 provide experimental evidence of a moment of 5 microB on Mn, with partial cancellation by an opposing moment on the Sb4 cage surrounding each Mn ion. The compound is isostructural to Ca14AlSb11, with Mn occupying the Al site in the AlSb4(9-) discrete tetrahedral, anionic unit. Bulk magnetization measurements indicate a saturation moment of 3.90 +/- 0.02 microB/formula unit consistent with four unpaired spins and implying a Mn3+, high-spin d4 state. XMCD measurements reveal that there is strong dichroism in the Mn L23 edge, the Sb M45 edge shows a weak dichroism indicating antialignment to the Mn, and the Yb N45 edge shows no dichroism. Comparisons of the Mn spectra with theoretical models for Mn2+ show excellent agreement. The bulk magnetization can be understood as Mn with a moment of 5 microB and a 2+ configuration, with cancellation of one spin by an antialigned moment from the Sb 5p band of the Sb4 cage surrounding the Mn.