Average glucan chain length and polydispersity index (PDI).

<p>(A) Comparison of the average glucan chain length among RILs. Each column contains multiple lines homozygous for the parental alleles (W64A<i>o2</i> or QPM) of <i>Zpu1</i> or <i>SSIII</i>. Each column represents mean glucan chain length of all RILs of the corresponding genotype with three independent ears of each individual RIL. (B) Positive correlation between average glucan chain length and PDI. The correlations are significant, tested by ANOVA of the slope at <i>p</i><0.05. Each data point represents mean glucan chain length of three independent ears of each individual RIL. (C) Comparison of PDI between parental lines (W64A+, W64A<i>o2</i> and K0326Y). Each column represents the mean PDI value of three independent ears of corresponding lines. The letters above each column of (A) and (C) represent statistically significant differences among the lines for <i>p</i><0.05 by each pair t test. Columns sharing the same letter are not significantly different from one another. The error bars represent corresponding standard error. (D) Nonlinear regression analysis between PDI and kernel vitreousness among RILs, test statistics are listed below. The relationship was significantly positive, because 0 was excluded from the 95% confidence interval of Growth Rate, indicating that the Growth Rate was greater than 0 at <i>p</i>-value = 0.05 level. Each data point represents mean PDI of three independent ears of each individual RIL. (E-G) Glucan chain length distribution between opaque lines (dash curves) and vitreous lines (solid curves).</p

Sequence analysis of maize Zpu1 and <i>SSIII</i> genes.

<p>(A) Multiple alignments of Zpu1 gene sequences between W64A+, W64A<i>o2</i> and K0326Y. The highlighted positions showed four single nucleotide polymorphisms in K0326Y. (B) Multiple alignments of translated amino acid sequences of pullulanase. One amino acid difference (highlighted) was found in K0326Y due to the substitution of nucleotide from A to C at position 2864. (C) Restriction analysis of a 3’ end fragment (position 2632–2965) of the Zpu1 gene. The gel bands represent the size of fragments before (uncut) and after digestion (dig) with BslI. (D) Multiple alignments of <i>SSIII</i> gene sequences between W64A+, W64A<i>o2</i> and K0326Y. Hash marks on each sequence showed nucleotide polymorphisms. Prior studies identified three regions of the nucleotide sequences: N-terminal region (base 1–2304), homology region (base 2305–3679), and catalytic region (base 3680–5025) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130856#pone.0130856.ref028" target="_blank">28</a>]. Full nucleotide sequence alignments are provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130856#pone.0130856.s001" target="_blank">S1 Fig</a>. (E) Multiple alignments of translated amino acid sequences of SSIII, and hash marks on each sequence showed amino acid changes. Sequence annotation showed three domains of the amino acid sequences: N-terminal domain (amino acid 1–768) and homology domain (amino acid 769–1226), and catalytic domain (amino acid 1227–1674). Full protein sequence alignments are provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130856#pone.0130856.s002" target="_blank">S2 Fig</a>.</p

Pullulanase activity and SSIII abundance in parental lines and RILs.

<p>(A) Pullulanase activity of W64A+, W64A<i>o2</i>,K0326Y and RILs with four Zpu1-SSIII genotypes. Pullulanase activity was measured by the spectrophotometer at 490 nm absorbance (y-axis). For the first three columns, each represents mean pullulanase activity of three independent ears of the corresponding lines; and for the remaining four columns, each represents mean pullulanase activity of all RILs with the corresponding genotype from three independent ears of each individual RIL. The letters above each column represent statistically significant differences among the lines with <i>p</i><0.05 by pairwise two-tailed t-test. Columns with the same letter are not significantly different from one another. The error bars represent standard error. (B) Positive correlation between pullulanase activity and kernel vitreousness. The significance level of the correlation was tested by ANOVA of the slope at <i>p</i><0.05. Each data point represents mean pullulanase activity of three independent ears of each individual RIL. (C) SSIII abundance and SSIII activity of W64A+, W64A<i>o2</i>, K0326Y and <i>du</i>1M4. SSIII abundance was tested by SDS-PAGE followed by Western blot (Images of the whole blots and SDS-PAGE gels are provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130856#pone.0130856.s004" target="_blank">S4</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130856#pone.0130856.s006" target="_blank">S6</a> Figs). SSIII activity was tested by native PAGE followed by a zymogram for the RILs. (Image of whole zymogram gel is provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130856#pone.0130856.s004" target="_blank">S4 Fig</a>) (D) Western blot of SSIII among RILs homologous for W64Ao2 (W) or QPM (Q)–derived <i>Zpu1</i> or <i>SSIII</i> alleles. The order of samples corresponded to their kernel vitreousness, from most opaque to most vitreous (left to right; images of whole blots and SDS-PAGE gels are provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130856#pone.0130856.s005" target="_blank">S5</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130856#pone.0130856.s007" target="_blank">S7</a> Figs).</p

Thermal properties of starch.

<p>(A-C) Comparison of starch thermal properties, including onset temperature, maximum temperature and enthalpy, between W64A<i>o2</i> and K0326Y. Significant level indicated by asterisks at p<0.05 by two-tailed <i>t</i> test (D-F) Comparison of the starch thermal properties among RILs. For parental lines, each column represents mean thermal property values of three independent ears of corresponding lines; and for RILs, each column represents mean thermal property values of all RILs of the corresponding genotype with three independent ears for each individual RIL. The letters above each column represent statistically significant differences among the lines for <i>p</i><0.05 by pairwise two-tailed t-test. Columns sharing the same letter are not significantly different from one another. The error bars represent standard error.</p

Scanning electron microscopy of starch granules from parental lines and RILs.

<p>(A) Smooth starch granules in W64A<i>o2</i> endosperm with a little matrix surrounding them. (B) Contacts (arrows) and interconnections (asterisks) formed between adjacent starch granules in K0326Y endosperm. (C) Starch granules in the endosperm of the opaque RIL 217 showed similar smooth surface as in W64A<i>o2</i>. (D) Starch granules in the endosperm of RIL 27 showed similar contact and interconnection structure as in K0326Y. (E-F) Starch granules in the endosperm of RIL 209 (E) and RIL 112 (F) showed similar smooth surface as in W64A<i>o2</i>.</p

Tales of diversity: Genomic and morphological characteristics of forty-six <i>Arthrobacter</i> phages

<div><p>The vast bacteriophage population harbors an immense reservoir of genetic information. Almost 2000 phage genomes have been sequenced from phages infecting hosts in the phylum Actinobacteria, and analysis of these genomes reveals substantial diversity, pervasive mosaicism, and novel mechanisms for phage replication and lysogeny. Here, we describe the isolation and genomic characterization of 46 phages from environmental samples at various geographic locations in the U.S. infecting a single <i>Arthrobacter</i> sp. strain. These phages include representatives of all three virion morphologies, and Jasmine is the first sequenced podovirus of an actinobacterial host. The phages also span considerable sequence diversity, and can be grouped into 10 clusters according to their nucleotide diversity, and two singletons each with no close relatives. However, the clusters/singletons appear to be genomically well separated from each other, and relatively few genes are shared between clusters. Genome size varies from among the smallest of siphoviral phages (15,319 bp) to over 70 kbp, and G+C contents range from 45–68%, compared to 63.4% for the host genome. Although temperate phages are common among other actinobacterial hosts, these <i>Arthrobacter</i> phages are primarily lytic, and only the singleton Galaxy is likely temperate.</p></div

Genome organization of <i>Arthrobacter</i> phage Jawnski, Cluster AO.

<p>See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0180517#pone.0180517.g005" target="_blank">Fig 5</a> for details.</p

Genome organization of <i>Arthrobacter</i> phage Maggie, Cluster AN.

<p>See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0180517#pone.0180517.g005" target="_blank">Fig 5</a> for details.</p

Genome organization of <i>Arthrobacter</i> phage Amigo, Cluster AQ.

<p>See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0180517#pone.0180517.g005" target="_blank">Fig 5</a> for details.</p

Genome organization of <i>Arthrobacter</i> phage Circum, Cluster AM.

<p>See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0180517#pone.0180517.g005" target="_blank">Fig 5</a> for details.</p