Seed-mediated gene flow promotes genetic diversity of weedy rice within populations: implications for weed management.

Increased infestation of weedy rice-a noxious agricultural pest has caused significant reduction of grain yield of cultivated rice (Oryza sativa) worldwide. Knowledge on genetic diversity and structure of weedy rice populations will facilitate the design of effective methods to control this weed by tracing its origins and dispersal patterns in a given region. To generate such knowledge, we studied genetic diversity and structure of 21 weedy rice populations from Sri Lanka based on 23 selected microsatellite (SSR) loci. Results indicated an exceptionally high level of within-population genetic diversity (He = 0.62) and limited among-population differentiation (Fst = 0.17) for this predominantly self-pollinating weed. UPGMA analysis showed a loose genetic affinity of the weedy rice populations in relation to their geographical locations, and no obvious genetic structure among populations across the country. This phenomenon was associated with the considerable amount of gene flow between populations. Limited admixture from STRUCTURE analyses suggested a very low level of hybridization (pollen-mediated gene flow) between populations. The abundant within-population genetic diversity coupled with limited population genetic structure and differentiation is likely caused by the considerable seed-mediated gene flow of weedy rice along with the long-distance exchange of farmer-saved rice seeds between weedy-rice contaminated regions in Sri Lanka. In addition to other effective weed management strategies, promoting the application of certified rice seeds with no weedy rice contamination should be the immediate action to significantly reduce the proliferation and infestation of this weed in rice ecosystems in countries with similar rice farming styles as in Sri Lanka

The expression of SPATA12 was induced by UV-C radiation.

<p>A: The protein levels of SPATA12, CHD2 and p53 in UV-C radiated cells were detected by western blot, respectively. β-actin was used as the normalization control. B: The protein levels of p53 and p-p53 (Ser 15 and Ser 33) in cells were detected 3 hours post UV-C-irradiation, respectively. β-actin was used as normalization control. </p

SPATA12 and Its Possible Role in DNA Damage Induced by Ultraviolet-C

<div><p>Our previous studies indicated that SPATA12, a novel spermatogenesis-associated gene, might be an inhibitor involved in spermatogenesis and tumorigenesis. To obtain a better understanding of the functions of SPATA12, a yeast two-hybrid screening system was used to search for interacting proteins, and chromodomain helicase DNA binding protein 2 (CHD2) was successfully identified. Bimolecular fluorescence complementation (BiFC) and subcellular co-localization assays further suggested a possible interaction between SPATA12 and CHD2 in the nuclei. CHD2 is known to be involved in the later stage of the DNA damage response pathway by influencing the transcriptional activity of p53. Thus, our hypothesis is that SPATA12 might play a role in DNA damage signaling. Western blotting results showed that SPATA12 expression could be induced in ultraviolet-C (UV-C) irradiated cells. Through reporter gene assays and the activator protein-1 (AP-1) decoy oligodeoxynucleotide method, we demonstrated that <i>SPATA12</i> promoter activity could be up-regulated in response to UV-C radiation exposure and an AP-1 binding site in the <i>SPATA12</i> promoter may have a role in transcriptional regulation of <i>SPATA12</i>. Using colony formation and host cell reactivation assays, it was demonstrated that SPATA12 might lead to inhibition of cellular proliferation in UV-C-irradiated DNA damage. Furthermore, SPATA12 was transfected into H1299, MCF-7 and HeLa cells, and flow cytometry (FCM) results suggested that there are some biological association between SPATA12 and p53 in UV-C-irradiated DNA damage. In addition, we investigated whether SPATA12 could up-regulate the expression of p53. Taken together, these findings indicate that SPATA12 could be induced under UV-C stress. During DNA damage process, AP-1 involves in the transcriptional up-regulation of <i>SPATA12</i> in response to UV-C radiation and p53 involves in growth inhibitory effects of SPATA12 on UV-C irradiated cells.</p> </div

SPATA12 may affect cell cycle progression in a p53-dependent manner after UV-C irradiation.

<p>pRevTRE-SPATA12 and the empty vector pRevTRE were transfected into H1299 (p53-null), HeLa (p53 functional deletion) and MCF-7 (p53 wild type) cells. Cell lines were irradiated with 600 J/m² UV-C and harvested for FCM analysis. </p

Genetic relationships of 21 weedy rice populations (Am-1 to Pu-2) and rice varieties (CV) from STRUCTURE analysis.

<p>Each individual is represented by a vertical bar assigned into three colors, with height proportional to each of the three inferred genetic components. The numbers beside vertical axis represent probability of assignment.</p

Analysis of molecular variance (AMOVA) of 21 weedy rice populations based on 23 SSR primer pairs.

<p>Df: degree of freedom; SS: sum of squared deviations; Var. comp.: variance component estimates; %: percentage of total variation.</p><p>Analysis of molecular variance (AMOVA) of 21 weedy rice populations based on 23 SSR primer pairs.</p

Number of pair-wise migrants per generation (<i>Nm</i>) between 21 weedy rice populations and cultivated rice group (CV) calculated based on the formula proposed by Slatkin and Barton [28].

<p>Number of pair-wise migrants per generation (<i>Nm</i>) between 21 weedy rice populations and cultivated rice group (CV) calculated based on the formula proposed by Slatkin and Barton <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112778#pone.0112778-Slatkin1" target="_blank">[28]</a>.</p

AP-1 involves in the transcriptional up-regulation of <i>SPATA12</i> in response to UV-C radiation.

<p>A: A schematic representation of the 5'-regulated region of SPATA12 gene. B: The AP-1 binding site involves in transcriptional regulation of SPATA12 in response to UV-C radiation. Renilla and firefly luciferase reporter genes combined with the wild-type, point mutation and deletion mutation versions of the SPATA12 promoter construct were co-transfected into cells prior to UV-C irradiation. The data are the means ± SD (n=3). **<i>p</i><0.01, *<i>p</i><0.05 versus the control groups by <i>t</i>-test. Top: MCF-7 cell lines. Bottom: HeLa cell lines. C: Relative luciferase activity analysis of SPATA12 promoter construct (pGL3-136/302, wild-type) using the dual luciferase reporter gene system after AP-1 decoy ODN treatment in cells. Renilla and firefly luciferase reporter genes, combined with AP-1 decoy ODNs or mismatched decoy ODNs, respectively, were co-transfected into cells prior to UV-C irradiation.The mismatched decoy ODNs served as negative controls. The data are the means ± SD (n=3). In blank control groups (cells without decoy ODN), **<i>p</i><0.01, *<i>p</i><0.05 versus the control groups by <i>t</i>-test. M indicates mismatched AP-1 decoy ODN; D indicates AP-1 decoy ODN. Top: MCF-7 cell lines. Bottom: HeLa cell lines.</p

The interaction of SPATA12 and CHD2 verified by bimolecular fluorescence complementation (BiFC) assay.

<p>A: Cells co-transfected with pcDNA3.1(+)-SPATA12-YC and pcDNA3.1(+)-YN, pcDNA3.1(+)-CHD2-YN and pcDNA3.1-YC were served as negative controls (×400). B: Cells were co-transfected with pcDNA3.1(+)-CHD2-YN and pcDNA3.1(+)-SPATA12-YC plasmids; yellow signal represents interaction of SPATA12 and CHD2 in nuclei (×400). </p

The analysis of chromosomal DNA integrity after UV-C irradiation.

<p>DNA damage (tail moment) induced by various does of UV-C radiation was detected by the neutral comet assay in HeLa cells. A: Representative images from the neutral comet assay (×200). B: UV-C-induced DNA damage as indicated by tail moment. **<i>p</i><0.01 versus control group by <i>t</i>-test. </p