The k13 gene of P. ovale is located in the 404824-407001 rt region of chromosome 12, with a coding region in full length of 2178 bp . Its encoded kelch protein has a skeletal region near the N-terminus, and a propeller domain near the C-terminus consisting of about 290 amino acids from 440th aa-725th aa . Studies have shown that amino acid substitutions in the propeller domain of the kelch protein in P. falciparum are genetically related to the formation of artemisinin resistance [27, 29]. Moreover, there are very few bases with more than two substitution loci in the entire coding region, which demonstrates [27, 30, 31] high conservation. Therefore, k13 gene can be used as a stable molecular marker to predict the artemisinin resistance in P. falciparum [32, 33, 34].
In this study, the polymorphism of the entire propeller domain and a fraction of the upstream skeletal domain in k13 gene of the P. ovale isolates imported into Yunnan Province from Myanmar and some African countries were analyzed. Of the 15 CDS sequences analyzed, we found base substitutions at 38 loci, such as c.711 ~ c.2118 (Table 2), showed the inter-type dimorphism of curtisi subtype and wallikeri subtype, as well as the complete intra-type monomorphism (Fig.2). The finding of such stable monomorphism and dimorphism characteristics at each locus is consistent with the results of polymorphism analysis conducted by Sutherland et al., , Fueher et al. , Chavatte et al.  on rbp2 (Reticulocyte binding protein 2), g3p (glyceraldehyde-3-phosphatase gene) and so on. All the above mentioned researches found the dimorphism of different genes in P. ovale, such as at 22 loci in rbp2 gene with the approximately 793 bp fragment and at 20 loci in g3p gene with 662 bp fragment between curtisi subtype and wallikeri subtype sequences. Moreover, the loci showed highly monomorphic within curtisi subtype and wallikeri subtype sequences. These findings suggest that k13 gene polymorphism in P. ovale is similar to the differentiation of other members in the genome, resulting in the distinction between curtisi subtype and wallikeri subtype. However, it is noted that the degree of k13 gene differentiation is weaker than ctrp (circumsporozoite protein / thromspondin-related anonymous-related protein), csp (circumsporozoite surface protein), and msp1 (merozoite surface protein 1), which were reported by Saralamba et al . The Pi value of these three genes was predicted to be between 0.12 and 0.11, which is greater than 0.0095 in this study. Of course, whether the observed dimorphism of k13 gene also exists in other genes in these P. ovale isolates as well as its consistency with other studies [35, 36, 37] requires further study.
Evidence indicated that P. ovale originated from Southeast Asian countries is mostly curtisi subtype, while Africa showed a sympatric distribution of P. ovale curtisi and P. ovalewallikeri [37, 7, 38, 39, 12], and the mutation type is mainly restrained in Western Africa . In this study, the distribution pattern of similar P. ovale subspecies was almost restored. The sequences of k13 propeller domain in five Myanmar isolates were all identified as curtisi subtype, while the ten African isolates included six curtisi subtype, three wallikeri subtype and one mutation type (Table 1). This result serves as a constant reminder that the population structure of P. ovale isolates imported into Yunnan province maybe are more complicated than those of the original population [36, 40]. Therefore, greater discretion and accuracy are needed in the diagnosis and antimalarial treatment of these P. ovale infections. To our knowledge, the current study is the first one to ascertain that the infected isolates in malaria cases officially reported in Yunnan Province include the two sub-species of P. ovalecurtisi and wallikeri and further providing a favorable basis for the control of ovale malaria epidemic in Myanmar . In addition, although amino acid substitution variation in the skeleton region of kelch protein was detected in only one sample, but the same amino acid variation has also detected and demonstrated by Jin’s study on the samples from Hangzhou city, China (being published). Therefore, it is reasonable to cast off the doubt of sequencing errors.
Although this study was not dedicated to exploring the genetic correlation between k13 gene mutations and artemisinin resistance in P. ovale, our spatial structure prediction on the peptide chain near the C-terminus from 224th aa to 725th aa in k13 gene found that curtisi subtype peptide chains and wallikeri subtype peptide chains share almost analogous monomeric crystal structures (Fig 3A, B). Moreover, with one amino acid variation in the skeleton region, yet the homology model has dramatically changed into a dimeric structure (Fig.3C). The finding is completely different from that of Choowongkomon et al.  in terms of the spatial structure prediction of dhfr (dihydrofolate reductase) gene in P. ovale. Their results showed the identities of dhfr peptide chain in P. ovale were merely 67.4%, 64.7% and 75.4% in comparison with P. vivax, P. falciparum, and P. malariae, respectively. However, the crystal structures of the four dhfr peptide chains are similar in regard to subunit composition and the tendency of overall folding. All display monomeric and α-helix structure, which are folded on the surface of the homology model . This pattern might be related to the different proportions and intensities of α-helix and β-helix structures in the two peptide chains of k13 gene and dhfr gene. In the current study, β-helix structures accounted for 75.1% (377 aa / 505 aa) in the k13 peptide chain, and were mainly located in the C-terminus of the peptide chain to fold into a "propeller" shape. In addition, Bayih et al.  had proposed the substitution from basic-to-aliphatic residue at the kelch 13 propeller domain, especially β-helix structures region, may impact the protein function. However, further studies should be carried out to investigate whether the predicted structural change in skeletal region of the kelch protein in P. ovale, just like the mutation of the propeller domain, is related to the artemisinin-resistant phenotype [29, 27].
In this study, we broaden the understanding that there are numerous dimorphism in the genome of P. ovalecurtisi and P. ovalewallikeri. By using the multi-loci dimorphism of the k13 gene, it might be possible to establish a stable and accurate genotyping method of distinguishing different subtypes of P. ovale. Nevertheless, this study is not without limitations. Firstly, the sample size is small, and the lack of indigenous P. ovale isolates from Yunnan province obstructs the researches on the sympatric distribution of the different subtypes of P. ovale; Secondly, given the difficulty to accurately calculate the parasitemia of P. ovale in some blood slides, it is impracticable to explore the correlation between the density of the parasites and the copy number of k13 gene; Thirdly, the polymorphic analysis of the full sequence of the k13 gene has not been performed, and the incomplete identification of the dimeric loci in the skeleton region of kelch protein and the DNA sequence of P. ovalecurtisi and P. ovalewallikeri might not be conducive to assess of the degree of k13 gene differentiation more accurately.