Characterization of MT1 (128/Xyl, cf. Fig. S1e) transformants.

<p>(A) PCR analysis of 13 randomly chosen doubled-haploid T₁ seedlings of primary transformant MT1 B4. Lanes: 1 = 1 kb ladder; 2 =  plasmid DNA; 3 =  wild type wheat DNA; 4–16 = T₁ seeds. (B) Southern blot analysis of homozygous MT1 transformants from T₁ seedlings with the xylanase gene. Lanes: C1, C2, C4 respectively showing 2, 4 and 8 copies of 837 bp probe; M =  DNA ladder; lanes 1–10 = T₁ doubled-haploid seedlings of 6 different T₀ transformants (1 and 2 = MT1-1, 3 and 4 = MT1-2, 5 and 6 = MT1-3, 7 = MT1B5, 8 and 9 = MT1-6, and 10 = MT1-7); lanes 11–13 = T₁ doubled-haploid seedlings of MT1-B4; lanes 14–15 =  wild type DNA. (C) Zymogram assay for identification of transgenic wheat grains synthesizing recombinant 1,4-β-xylanase. Transgenic wheat grains (T₂ of MT1-B4) secrete the enzyme into the medium containing oat-spelt xylan that is stainable with Congo Red. De-polymerization of the xylan by the enzyme results in an unstained yellow ring around the seed. Wild type wheat grains lack the yellow ring (arrows).</p

Electron micrographs of wheat microspores after pretreatment using transmission electron microscopy.

<p>Figures a-c show differences in thickness of intine, number of cytoplasmic organelles and amount of starch accumulated in amyloplasts. (A) represents type I developmental pathway, (B) represents type II developmental pathway, and (C) represents type III developmental pathway. am =  amyloplast, ex =  exine wall, in =  intine layer, mt =  mitochondria, gl =  Golgi apparatus, p =  proplastid, rer =  rough endoplasmic reticulum, st =  starch.</p

Developmental pathways of pre-treated wheat microspores in culture, as determined by time-lapse tracking.

<p>According to the type of development, microspores are grouped into three classes: type I (A–H), type II (I–O) and type III (P–R). Microspores were stained with FM 4–64 (Molecular Probes Cat. # T-3166) to confirm their viability in culture. All pictures were taken at a 25× magnification and 6× optical zoom except for G, O and H, where the former two pictures were taken at the same magnification but at 3× optical zoom and the latter picture was taken at 10× magnification and 1.7× optical zoom.</p

Assessment of Genetic Diversity among Barley Cultivars and Breeding Lines Adapted to the US Pacific Northwest, and Its Implications in Breeding Barley for Imidazolinone-Resistance

<div><p>Extensive application of imidazolinone (IMI) herbicides had a significant impact on barley productivity contributing to a continuous decline in its acreage over the last two decades. A possible solution to this problem is to transfer IMI-resistance from a recently characterized mutation in the ‘Bob’ barley <i>AHAS</i> (<i>acetohydroxy acid synthase</i>) gene to other food, feed and malting barley cultivars. We focused our efforts on transferring IMI-resistance to barley varieties adapted to the US Pacific Northwest (PNW), since it comprises ∼23% (335,000 ha) of the US agricultural land under barley production. To effectively breed for IMI-resistance, we studied the genetic diversity among 13 two-rowed spring barley cultivars/breeding-lines from the PNW using 61 microsatellite markers, and selected six barley genotypes that showed medium to high genetic dissimilarity with the ‘Bob’ <i>AHAS</i> mutant. The six selected genotypes were used to make 29–53 crosses with the <i>AHAS</i> mutant and a range of 358–471 F₁ seeds were obtained. To make informed selection for the recovery of the recipient parent genome, the genetic location of the <i>AHAS</i> gene was determined and its genetic nature assessed. Large F₂ populations ranging in size from 2158–2846 individuals were evaluated for herbicide resistance and seedling vigor. Based on the results, F₃ lines from the six most vigorous F₂ genotypes per cross combination were evaluated for their genetic background. A range of 20%–90% recovery of the recipient parent genome for the carrier chromosome was observed. An effort was made to determine the critical dose of herbicide to distinguish between heterozygotes and homozygotes for the mutant allele. Results suggested that the mutant can survive up to the 10× field recommended dose of herbicide, and the 8× and 10× herbicide doses can distinguish between the two <i>AHAS</i> mutant genotypes. Finally, implications of this research in sustaining barley productivity in the PNW are discussed.</p></div

Microsatellite markers classified according to repeat element type.

<p>Number of simple sequence repeats (SSRs) or microsatellites falling in each category is listed and the range of alleles detected by SSRs in these categories and their average PIC (polymorphic information content) values are shown.</p

List of two-rowed spring barley varieties/breeding lines used in the study.

<p>List of two-rowed spring barley varieties/breeding lines used in the study.</p

Genetic linkage map of chromosome 6H showing the respective locations of 61 microsatellite markers used in the present study (left).

<p>Various alleles detected from six barley genotypes used for crossing with the Bob <i>AHAS</i> mutant are indicated by colored boxes (middle), where each color represents a unique allele and the white color represents the ‘Bob’-type allele. The total number of polymorphic markers identified per genotype pair with the Bob mutant is shown below. The PIC value calculated for each marker was plotted against its location on the genetic linkage map (right) to indicate the level of nucleotide diversity observed using 13 barley genotypes, and its distribution along the entire length of chromosome 6H.</p

A part of the DNA sequence of the <i>AHAS</i> gene showing the point mutation responsible for IMI-resistance (highlighted in blue).

<p>The DNA sequencing results clearly demonstrated the transfer of IMI-resistance to two feed barleys, 05WA-316.99 and 07WA-682.1, two food barleys, Clearwater and WAS4, and two malting barleys, Radiant and Conrad.</p

List of chromosome 6H specific microsatellite markers used for the genetic diversity analysis and marker-assisted background selection, their repeat elements, respective locations in the genetic-linkage map [17], number of alleles detected and their polymorphic information content (PIC).

<p>List of chromosome 6H specific microsatellite markers used for the genetic diversity analysis and marker-assisted background selection, their repeat elements, respective locations in the genetic-linkage map <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100998#pone.0100998-SaghaiMaroof1" target="_blank">[17]</a>, number of alleles detected and their polymorphic information content (PIC).</p

Diagrammatic representation of the results of the marker assisted background selection on the F₃ progeny of the selected F₂ lines.

<p>After foreground selection, the six F₃ lines per cross combination were evaluated for the recovery of recipient parent background by genotyping each line with 10 to 12 chromosome 6H-specific microsatellite markers. The markers were selected on the basis of polymorphism data obtained earlier during diversity analysis and their respective location on chromosome 6H. Map locations of selected markers are shown on the left. Each column in the picture represents a F₃ line and each row represents a DNA marker, whereas each cell represents the marker genotype in an individual. The marker genotype is represented by a color code: a) light green color denotes heterozygotes carrying marker alleles from both parents; b) dark green color denotes a marker allele similar to the recipient parent; and c) red color denotes the marker allele of the donor parent. Thus, a column with more dark and light green cells represent a genotype showing high percentage of the recipient parent genome, as observed for the 6^th F₃ individual in the Conrad×Bob mutant cross, which had 90% recovery of the Conrad alleles. In contrast, a column showing more red cells represents a donor-like carrier chromosome as examplified by the 6^th F₃ individual in the 05WA-316.99×Bob mutant cross, which had only 20% recovery of the 05WA-316.99 parent genome.</p