Orthogonal design in the optimization of a start codon targeted (SCoT) PCR system in Roegneria kamoji Ohwi.

Roegneria kamoji Ohwi is an excellent forage grass due to its high feeding value and high resistance to some biotic and abiotic stresses. However, the start codon targeted (SCoT) polymorphism has not been conducted on R. kamoji. In this study, an orthogonal L16 (45) design was employed to investigate the effects of five factors (Mg2+, dNTPs, Taq DNA polymerase, primer, and template DNA) on the polymerase chain reaction (PCR) to determine the optimal SCoT-PCR system for R. kamoji. The results showed that the most suitable conditions for SCoT-PCR in R. kamoji included 1.5 mM Mg2+, 0.15 mM dNTPs, 1.0 U Taq DNA polymerase, 0.4 pM primer, and 40 ng template DNA. SCoT primers 39 and 41 were used to verify the stability of the optimal reaction system, and amplification bands obtained from diverse samples were found to be clear, rich, and stable in polymorphisms, indicating that this reaction system can be used for SCoT-PCR analysis of R. kamoji. We have developed a simple and rapid way to study the mutual effects of factors and to obtain positive results through the use of an orthogonal design L16 (45) to optimize the SCoT-PCR system. This method may provide basic information for molecular marker-assisted breeding and analyses of genetic diversity in R. kamoji.

Nucleotide variation in the Toxoplasma gondii micronemal protein 8 gene.

Toxoplasma gondii is a successful opportunistic protozoan distributed worldwide, which can infect all vertebrates, leading to serious infection, blindness, and abortion. Micronemal (MIC) proteins are critically important for T. gondii infection, as they participate in various stages of the Toxoplasma life cycle, including invasion and attachment to host cells. MIC8 secretion relies on the concentration of intracellular calcium, and can mediate the invasion of T. gondii by interacting with soluble MIC3. To investigate genetic diversity of the MIC8 gene, 16 T. gondii strains from different hosts and geographical locations, and two reference isolates (ToxoDB: TGME49_245490 and TGVEG_245490) were examined in this study. The results showed that all the examined MIC8 genes are 2055 bp, with an A+T content ranging from 50.2 to 50.6%. Conversely, lower levels of variation were detected within their nucleotide and amino acid sequences. Phylogenetic analyses indicated that three classical genotypes of T. gondii and the ToxoDB#9 genotype did not group exclusively via Bayesian inference, maximum parsimony, neighbor joining, and/or maximum likelihood assays based on the nucleotide and amino acid sequences of the MIC8 gene. In summary, the T. gondii MIC8 gene is not a suitable marker for population genetic studies of this parasite.

Sequence variation in ROP8 gene among Toxoplasma gondii isolates from different hosts and geographical localities.

The protozoan parasite Toxoplasma gondii has a worldwide distribution; it can cause serious diseases in humans and almost all other warm-blooded animals. Different genotypes of T. gondii result in different lesions in the same host. T. gondii rhoptry protein 8 (TgROP8) is a major factor of T. gondii acute virulence. We examined sequence variation in the TgROP8 gene among T. gondii isolates from different hosts and geographical localities. The TgROP8 gene was amplified from individual isolates and sequenced. A phylogenetic tree was constructed using Bayesian inference, maximum parsimony, and maximum likelihood based on the sequences obtained plus TgME49 from the ToxoDB database. The TgROP8 gene was 1728 bp in length for all the examined T. gondii strains, and their A+T contents were 45.37-45.95%. Sequence analysis detected 140 (0.06-5.56%) variable nucleotide positions resulting in 96 (0-10.78%) amino acid substitutions. Sequence variations in the TgROP8 gene resulted in polymorphic restriction sites for endonucleases BstBI, BsaI, and XhoI, which allowed the differentiation of the three classical genotype strains (types I, II, and III) by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). However, phylogenetic analyses indicated that the TgROP8 gene is not a suitable genetic marker for population studies of T. gondii.