The study area, Changsha, China.

<p>The study area, Changsha, China.</p

Summary of model performance and the estimated coefficients.

<p>Abbreviations: ARIMA = Autoregressive Integrated Moving Average; S = Seasonal; X = with explanatory variables; LST = Land Surface Temperature; AP = Accumulative Precipitation; NDVI = Normalized Difference Vegetation Index; TVDI = Temperature Vegetation Dryness Index; MRPE = Mean Relative Prediction Error; AIC = Akaike’s Information Criterion; AR = Autoregressive coefficients; MA = Moving Average Coefficients; Est = Estimated values through conditional least square method.</p

Observed versus predicted rodent density in Changsha.

<p><b>(a) Temporal dynamics and (b) scatterplot.</b></p

Environmental variables in Changsha.

<p><b>(a) monthly average of temperature, (b) monthly accumulated rainfall and (c) monthly NVDI and TVDI for rice paddies.</b></p

Cross-correlations between pre-whitened series and the rodent density.

*<p><i>p</i><0.05,</p>**<p><i>p</i><0.01.</p

High-Throughput Discovery of Chloroplast and Mitochondrial DNA Polymorphisms in Brassicaceae Species by ORG-EcoTILLING

<div><h3>Background</h3><p>Information on polymorphic DNA in organelle genomes is essential for evolutionary and ecological studies. However, it is challenging to perform high-throughput investigations of chloroplast and mitochondrial DNA polymorphisms. In recent years, EcoTILLING stands out as one of the most universal, low-cost, and high-throughput reverse genetic methods, and the identification of natural genetic variants can provide much information about gene function, association mapping and linkage disequilibrium analysis and species evolution. Until now, no report exists on whether this method is applicable to organelle genomes and to what extent it can be used.</p> <h3>Methodology/Principal Findings</h3><p>To address this problem, we adapted the CEL I-based heteroduplex cleavage strategy used in Targeting Induced Local Lesions in Genomes (TILLING) for the discovery of nucleotide polymorphisms in organelle genomes. To assess the applicability and accuracy of this technology, designated ORG-EcoTILLING, at different taxonomic levels, we sampled two sets of taxa representing accessions from the Brassicaceae with three chloroplast genes (<em>accD</em>, <em>matK</em> and <em>rbcL</em>) and one mitochondrial gene (<em>atp6</em>). The method successfully detected nine, six and one mutation sites in the <em>accD</em>, <em>matK</em> and <em>rbcL</em> genes, respectively, in 96 <em>Brassica</em> accessions. These mutations were confirmed by DNA sequencing, with 100% accuracy at both inter- and intraspecific levels. We also detected 44 putative mutations in <em>accD</em> in 91 accessions from 45 species and 29 genera of seven tribes. Compared with DNA sequencing results, the false negative rate was 36%. However, 17 SNPs detected in <em>atp6</em> were completely identical to the sequencing results.</p> <h3>Conclusions/Significance</h3><p>These results suggest that ORG-EcoTILLING is a powerful and cost-effective alternative method for high-throughput genome-wide assessment of inter- and intraspecific chloroplast and mitochondrial DNA polymorphisms. It will play an important role in evolutionary and ecological biology studies, in identification of related genes associated with agronomic importance such as high yield and improved cytoplasmic quality, and for identifying mitochondrial point mutations responsible for diseases in humans and other animals.</p> </div

Overview of mutation detections in Brassicaceae samples by ORG- EcoTILLING Electrophoresis on a 6.5% KBplus Gel of CEL I-digested products of APCR fragment of <i>accD</i> gene, 827 bp in ALI-COR 4300 DNA Analyzer were displayed here.

<p>The sample in each lane from one to six was A64, A65, A61, A67, A68 and A69, respectively. CEL I-cleaved heteroduplexes appeared as intense bands (circled in different color geometric figures of IRD 700 channel and corresponding same color ones of IRD 800 channel). The fragment sizes indicated the position of the mutation present in the locus <i>accD</i> gene of the collected samples. The reference sample in this gene was A64.</p

Detection of amplified fragments of selectcd chloroplast genes by agarose gel electrophoresis.

<p>a. <i>accD</i> gene, the size of PCR products in <i>accD</i>-1 and <i>accD</i>-2 amplified by two pairs of primers were 974 and 789 bp, respectively. b. <i>matK</i> gene, the size of PCR products in <i>matK</i>-1 and <i>matK</i>-2 amplified by two pairs of primers were 959 and 948 bp, respectively. c. <i>rbcL</i> gene, the size of PCR products in <i>rbcL</i>-1 and <i>rbcL</i>-2 amplified by two pairs of primers were 974 and 1081 bp, respectively. The sizes of the DNA fragments produced for the three genes corresponded with what we expected, and the abundance of DNA was fit for ORG-EcoTILLING analysis. The samples from 1 to 5 were B12, B20, B26, B85 and B94 in <i>accD</i>-1 gene fragment, and the same in other gene fragments. The molecular size of DNA is shown at the left with arrowheads. M represented the DNA ladder DL 2000 (Promega. Inc).</p

SNP mutation types and band analysis of <i>B. napus</i> varieties identified by ORG- EcoTILLING and DNA sequencing.

<p>Note: The sample codes were corresponding with <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047284#pone-0047284-t002" target="_blank">Table 2</a>, and mutation position was located from initiation site (ATG) of a gene. The length of <i>accD</i>, <i>matK</i> and <i>rbcL</i> were 1470 bp, 1575 bp and 1440 bp, respectively. “+” indicated that there was an intense band visualized by denaturing polyacrylamide gel electrophoresis with the LI-COR DNA analyzer.</p>*<p>The base pairs changes in round bracket were detected by DNA sequencing and G to T change, while “AAAGTG” meant deletion of base pairs. The reference sample of <i>accD</i>, <i>matK</i> and <i>rbcL</i> were B26, B26 and B85, respectively.</p

Comparison of efficiency in different types of SNPs in <i>accD</i> and <i>atp6</i> gene detected by ORG-EcoTILLING and DNA sequencing.

*<p>indicated that the reference sample was A64 and A24 in <i>accD</i> gene and <i>atp6</i> gene, respectively. “+” indicated that there was an intense band visualized by ORG-EcoTIILLING. “−” indicated that there was no intense band detected by ORG-EcoTIILLING. “NP” indicated no polymorphism.</p