Results of PCR amplification of nuclear ITS1-5.8S-ITS2 in C. argus
In this study we amplified the ITS1-5.8S-ITS2-28S fragment from seven C. argus individuals. A total of 29 isolated clones were randomly selected and determined (GenBank accession numbers ON667943 ~ ON667971). The complete ITS1-5.8S-ITS2 sequences of this study and five sequences of C. argus (WL1 ~ 5) from other study were extremely conservative in length and nucleotide composition (Table 2). The sequence polymorphism indicated that full length ITS1-5.8S-ITS2 exhibited 22 haplotypes and 32 variable sites, The most polymorphism is from ITS2 with 17 haplotypes and 18 variable sites, The least polymorphism is from 5.8S with 4 haplotypes and 3 variable sites. However, the full length GC content, ITS1, 5.8S and ITS2 shows inconsistency, with GC content of 68%, 72%, 56-57% and 70-71%, manifested in the higher GC content of ITS. Polymorphism of the sequences is also indicated by Pair distances with Full length, ITS1, 5.8S and ITS2 of 0.000 - 0.004, 0.000 - 0.007, 0.000 - 0.013 and 0.000 - 0.014 respectively.
Detailed variant sites of the the alignment of ITS1-5.8S-ITS2 sequences in C. argus
The twenty-four ITS1-5.8S-ITS2 sequences in the six white C. argus (GOF1.1~ 3.4 and GRF1.1~ 3.4) have 23 variable sites. Those sequences have two identical variation sites (T→C, C→G), and other twenty one variation sites were randomly distributed in GOF1 .2 (C→T, T→C), GOF1 .3 (C→T), GOF2 .1 (G→A, C→T), GOF2 .2 (C→T, T→C,), GOF2 .4 (T→C), GOF3 .1 (C→T), GOF3 .2 (T→C, T→C), GOF3 .4 (G→A ), GRF1 .1 (C→T), GRF2 .1 (T→C), GRF2 .3 (A→G, G→C), GRF2 .4 (T→C, T→C), GRF2 .5 (G→T, G→T), and GRF3 .2 (T→C). The GRF2 individual has the most variation sites (7 sites), but GOF group has more variation sites than GRF group. The transitions and transversions sites are 18 and 5 (Supplement Figure. 1). Comparing the variation sites of ten ITS1-5.8S-ITS2 sequences in the C. argus of Neijiang (BW1.1~1.5) and Jining (WL1~WL5) can be seen, except two same variation sites (T→C, C→G) from BW1.1 to others C. argus, other seven variation sites randomly distributed in BW1.4 (C→T), WL2 (T→C), WL3 (A→G, A→G), WL4 (A→G), WL5 (T→C, G→A). The transitions and transversions sites are 8 and 1 (Supplement Figure 1).
The prediction of the secondary structure of 159 bp nuclear 5.8S sequences in C. argus showed the corresponding minimum free energy of -59.8 kcal/mol. The structures formed include six perfect loops and six perfect stems (Figure. 2).
Phylogenetic analysis of C. argus
The clustering of C. argus from different geographical distributions and in different body colors was studied in the phylogenetic analysis by using the ML method. Based on the tree topology, the C. argus from the same geographical distributions clustered into one clade, C. argus of the Neijiang group and C. argus of the Jining group were sister-group with high support values of 95% and 97% respectively (Figure. 3), suggesting that the phylogenetic relationships based on these nuclear ITS1-5.8S-ITS2 sequences can separate C. argus that differ in geographic distribution. Consistent body color in C. argus of BW, GOF or GRF from Neijiang does not form a monophyletic cluster (Figure. 3), indicating that these sequences cannot separate fish with different body color traits from the same region.
The haplotype network of the 28 haplotypes (labelled H1 to H28) were examined for the presence of population structure (Figure. 4). The constructed haplotype network showed that the outgroup and C. argus formed two groups, respectively, and 22 haplotype types of C. argus were in the same clade. This further supported the fact that the samples of C. argus were from one genetically homogenous population.