Chloroplast genome analyses and genomic resource development for epilithic sister genera Oresitrophe and Mukdenia (Saxifragaceae), using genome skimming data.

BACKGROUND: Epilithic sister genera Oresitrophe and Mukdenia (Saxifragaceae) have an epilithic habitat (rocky slopes) and a parapatric distribution in East Asia, which makes them an ideal model for a more comprehensive understanding of the demographic and divergence history and the influence of climate changes in East Asia. However, the genetic background and resources for these two genera are scarce. RESULTS: The complete chloroplast (cp) genomes of two Oresitrophe rupifraga and one Mukdenia rossii individuals were reconstructed and comparative analyses were conducted to examine the evolutionary pattern of chloroplast genomes in Saxifragaceae. The cp genomes ranged from 156,738 bp to 156,960 bp in length and had a typical quadripartite structure with a conserved genome arrangement. Comparative analysis revealed the intron of rpl2 has been lost in Heuchera parviflora, Tiarella polyphylla, M. rossii and O. rupifraga but presents in the reference genome of Penthorum chinense. Seven cp hotspot regions (trnH-psbA, trnR-atpA, atpI-rps2, rps2-rpoC2, petN-psbM, rps4-trnT and rpl33-rps18) were identified between Oresitrophe and Mukdenia, while four hotspots (trnQ-psbK, trnR-atpA, trnS-psbZ and rpl33-rps18) were identified within Oresitrophe. In addition, 24 polymorphic cpSSR loci were found between Oresitrophe and Mukdenia. Most importantly, we successfully developed 126 intergeneric polymorphic gSSR markers between Oresitrophe and Mukdenia, as well as 452 intrageneric ones within Oresitrophe. Twelve randomly selected intergeneric gSSRs have shown that these two genera exhibit a significant genetic structure. CONCLUSIONS: In this study, we conducted genome skimming for Oresitrophe rupifraga and Mukdenia rossii. Using these data, we were able to not only assemble their complete chloroplast genomes, but also develop abundant genetic resources (cp hotspots, cpSSRs, polymorphic gSSRs). The genomic patterns and genetic resources presented here will contribute to further studies on population genetics, phylogeny and conservation biology in Saxifragaceae.

Detection of residual E. coli host cell DNA by 23S ribosomal RNA gene-targeted quantitative polymerase chain reactions.

Among the many systems available for heterologous protein production gram-negative bacterium Escherichia coli (E. coli) has long been widely used because of its ability to grow rapidly with a high density on inexpensive substrates. The use of E. coli as the host system has many regulatory issues, one of which is the residual host cell DNA. Residual DNA carried by biological products may lead to carcinogenicity and immunomodulation risks. The World Health Organization (WHO) for the acceptable amounts of residual host cell DNA is less than 10 ng per dose. Therefore, it is important to keep an extremely low level of residual host DNA in the biological products derived from E. coli. In this study, we designed primer/probe sets targeting E. coli 23S ribosomal RNA gene to quantify the residual DNA of E. coli by quantitative polymerase chain reactions (qPCR). Result showed that this primer/probe has high species specificity. The limit of detection (LOD) in this method is 0.01 pg/µl and this allowed for detection of residual host DNA of much lower concentrations. We assessed accuracy by calculating the recovery (92.1∼140.1 %) of the spiked DNA in plasmids which were produced from E. coli. We also checked intra-assay precision (9.8∼15.1 %) and inter-assay precision (10.9∼18.3 %) by repeatedly measuring the four different concentration standards. In addition, the robustness assay was performed by generating standard curve using short length E. coli DNA. The result showed that appropriate degree of DNA fragmentation will not affect tests. These validation studies demonstrated that our method has excellent specificity, linearity, accuracy, precision and robustness.