37 research outputs found

    Combination of SFs after renaturation.

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    <p>SF 1–4 (<b>pOKCA construction, </b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030267#pone-0030267-g004" target="_blank"><b>Fig 4A</b></a><b>and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030267#pone.0030267.s001" target="_blank">Fig. S1</a></b>) were mixed (resulting M), denatured and renatured (resulting M&H). They were added to agarose gel in a volume ratio of 1∶1∶1∶1∶4∶4. From the comparison between M and M&H, we can find that, the bands corresponding to SF 1–4 weakened while at least two new bands emerged after the hybridization treatment. The new bands are around 4 and 5 kb, about twice bigger than the four SFs, suggesting that they are resulted from inter-fragment hybridization. M&H was further transformed into <i>E. coli</i> DH5α and generated plasmid pOKCA.</p

    The impact of overlap length on assembly efficiency.

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    <p>Five reconstruction of pOKCA were designed, in which the overlap between SF1 (red) and SF4X (blue, stands for SF4, SF41, SF42, SF43, or SF44) varied from 0 bp to 1019 bp. SF1 and SF3 were prepared as described in the text (and <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030267#pone.0030267.s001" target="_blank">Fig. S1</a></b>). SF4X and SF2X (SF2, SF21, SF22, SF23, or SF24) were amplified from pOKC2μUA with corresponding primers (<b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030267#pone.0030267.s005" target="_blank">Table S2</a></b>). Transformations were selected on Cm plates. Assembly efficiencies are given as colony forming units per microgram of SFs.</p

    General methods for SF preparation.

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    <p>(<b>A</b>) Two elements are joined by OE-PCR. (B) One short element is added to the 5′-end of a primer; the long element is PCR amplified. (C) SFs are synthesized chemically. As extra oligos are not required, the cost of two SFs is not much higher than the total cost of the three elements.</p

    Scheme of SHA (take a 6-SF assembling for example).

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    <p>To construct the final plasmid from the six starting materials (gene 1–4, replication origin and a selectable marker), SFs are first prepared by linking every two materials together, usually suing overlap-extension PCR (OE-PCR). So as shown in this figure, every SF has its 3′-half overlapped with the 5′-half of the next SF and the 5′-half of the first SF overlaps with the 3′-half of the last SF. A mixture of these SFs was denatured at 100°C to free all single strands. When it cools back down to room temperature, annealing between the overlaps would assemble the single strands one after another into a cycle which can be further repaired into double-stranded, closed circular molecule after transformation into the cells.</p

    Plasmids constructed in this article and their assembly efficiencies.

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    <p>(<b>A</b>) The outer cycle is a plasmid map showing the main genetic elements; two inner green cycles show the overlapped SFs, along with the overlap lengths (bp); the center is plasmid name and size (bp). Two examples are provided to show how nicks or gaps form. In pOKCA construction, the antisense primer for SF1 (SF1Wa) and the sense primer for SF3 (SF3Ws) were designed to be back to back, such that a nick formed between SF1 and SF3. In pAcetone construction, primers SFTAWs and SFHBWa were designed to be apart, so a 110 bp gap formed between SFTA and SFHB. (B) <sup>a</sup>Colony forming units per microgram DNA. <sup>b</sup>The proportion of transformants caused by undigested PCR template, determined by colony PCR. <sup>c</sup>The proportion of unwanted recombinants, determined by restriction mapping (<b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030267#pone.0030267.s002" target="_blank">Fig. S2</a></b>) and DNA sequencing. <sup>d</sup>This plasmid was difficult to construct inherently (see text for details).</p

    QTL for KW and GY detected in a DH population derived from a TX9425/Naso Nijo cross grown in different environments.

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    <p>The position is that of the nearest marker; R<sup>2</sup> means percentage genetic variance explained by the nearest marker.</p><p>*X: No significant QTL was detected.</p

    A New QTL for Plant Height in Barley (<i>Hordeum vulgare</i> L.) Showing No Negative Effects on Grain Yield

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    <div><p>Introduction</p><p>Reducing plant height has played an important role in improving crop yields. The success of a breeding program relies on the source of dwarfing genes. For a dwarfing or semi-dwarfing gene to be successfully used in a breeding program, the gene should have minimal negative effects on yield and perform consistently in different environments.</p><p>Methods</p><p>In this study, 182 doubled haploid lines, generated from a cross between TX9425 and Naso Nijo, were grown in six different environments to identify quantitative trait loci (QTL) controlling plant height and investigate QTL × environments interaction.</p><p>Results</p><p>A QTL for plant was identified on 7H. This QTL showed no significant effects on other agronomic traits and yield components and consistently expressed in the six environments. A sufficient allelic effect makes it possible for this QTL to be successfully used in breeding programs.</p></div

    The correlation between actual grain yields and yields predicted using makers linked to QTL for PH on 1H (QPh.NaTx-1H), 2H (QPh.NaTx-2H), 3H(QPh3H) and 7H (QPh7H) (Table S1).

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    <p>The correlation between actual grain yields and yields predicted using makers linked to QTL for PH on 1H (QPh.NaTx-1H), 2H (QPh.NaTx-2H), 3H(QPh3H) and 7H (QPh7H) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090144#pone.0090144.s003" target="_blank">Table S1</a>).</p
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