Sequence and variation analysis of the mtCOII gene fragment in M. enterolobi populations
A total of 19 homologous sequences of the mtCOII gene were amplified by PCR. Each sequences of 19 M enterolobii populations all were 831bp which used for genetic analysis. The accession numbers of 19 M enterolobii populations is shown in the Table2. There are 15 polymorphic loci (1.8% of the total number of bases analyzed), 6 S-singleton sites and 9 parsimony-informative sites, which accounted for 40% and 60% of the total polymorphisms identified, respectively. The S-singleton sites were located at positions 566, 647, 669, 675, 763, and 798 of the mtCOII gene fragments, and the parsimony-informative sites were located at positions 64, 65, 103, 465, 661, 678, 720, 727, 731, respectively. The contents of a, t, c and g were 48.99%, 36.37%, 10.68%, and 3.96%, respectively, and the content of a + t was 85.36%, showing a significant a/t bias. The total number of mutations detected in the M enterolobii populations was 25, and the conversion/transversion rate R was 0.9.
Nucleotide and haplotype diversity analysis of M enterolobii populations based on mtCOII gene
The number of variable sites, average number of nucleotide differences (K), haplotype diversity (Hd), and nucleotide diversity (π) of mtCOII gene in M enterolobii populations were 25, 3.563, 0.942 (> 0.5) and 0.00429. Tajima's D and the Fu's Fs values were-1.26569 and − 9.072, respectively, indicating that all populations underwent neutral selection, and the changes in population were not significant.
Among the 14 haplotypes identified (Table 3), Hap9 was found in 5 populations with a frequency of 26.36% (MN907173、MN9071715、MN907179、MN9071710、MN9071716); Hap14 appeared in 2 populations (MN9071712、MN9071717) with a frequency of 10.52%. Other haplotypes were identified only once in the tested populations.
Phylogenetic analysis of M enterolobii populations based on mtCOII gene
We selected sequences of M. enterolobii from different hosts in NCBI and 19 M. enterolobii sequences in this study for phylogenetic analysis. The phylogenetic tree constructed by the neighborhood-joining method showed that the 19 M enterolobii populations are on the same big branch with 96% bootstrap support and the other sequences selected on NCBI gather on another big branch with 98% bootstrap support. The out-group (C. elegans) is far away other branches (Fig. 1). The 19 M enterolobii populations were distributed in two evolutionary clades, among which Clade A with 12 populations took the main part while only 7 populations appeared in Clade B (Fig. 1). MN9071716, MN907179, MN9071710, MN9071715 and MN907173 lie in the same clade, in which there was no obvious grouping among populations in this clade, and the similarity among all the populations reached 100%. Additionally, MN9071712 and MN9071717 also lie in the same clade with the similarity of 100% among the populations. Other populations had their own independent haplotypes (Fig. 1). Based on the phylogenetic tree, it was found that the haplotypes of 19 populations were scattered free without any obvious geographical group distribution pattern. Therefore, it is demonstrated that M enterolobii populations in China have abundant mitochondria Hd and π, suggesting that the nematode populations have stronger adaptability to the changes in external environment.
Haplotype mediation network map of M enterolobii populations based on mtCOII gene
An intermediary network of M enterolobii populations was constructed by NETWORK 5.0 based on the Median-joining method. As shown in Fig. 2, Hap9 was shared by seven geographic populations (MN907173、MN9071715、MN907179、MN9071710、MN9071716). Hap14 occurred in two populations (MN9071712、MN9071717). The other haplotypes occur only once, corresponding to a single geographic population. This network could clearly explain the evolutionary relationships between each haplotype and the distribution of each geographical group, further supporting the phylogenetic tree (Fig. 2).
Genetic distance analysis of M.enterolobii populations based on mtCOII gene
The genetic distances among different M enterolobii populations were calculated based on mtCOII sequences using MEGA 7.0 (Appendix 1). The results showed that the genetic distances between various groups ranged from 0.000 to 0.011. YL and MM, QQ, SS, KB; MM and QQ, SS, KB; QQ and SS, KB; ZS and ZS1; and the genetic distance between the SS and KB populations had the smallest genetic distance (0.000), whereas the genetic distance between the YD and GB population was the greatest (0.011). However, the genetic distances between different populations varied little.
Correlation between geographic distance and genetic distance in M. enterolobi ipopulations based on mtCOII gene
The correlation between genetic distance (Appendix 1) and geographic distance were investigated (Appendix 2) based on mtCOII gene (Fig. 3). The results showed that there was no significant correlation between the genetic distance and the natural logarithm (LN km) matrix (r = 0.093, p=|-0.204|>0.05) of the geographic distance among samples collected, indicating that geographical distance is not the main factor leading to root-knot nematode populations differentiation.
Genetic variance of the M enterolobii groups based on mtCOII gene
Based on the AMOVA method, Arlequin software was used to analyze the genetic variation between M enterolobii groups in Guangzhou, Hunan and Guangxi Province in China. The intra-population differentiation parameter FST was 0.02004 (P < 0.0001) (Table 4). The variations within groups accounted for 98% of total variation, and the variations among groups accounted for 2% of total variation. These results indicated that the genetic differentiation of the root-knot nematode populations was mainly due to the variations within each group rather than those among different groups.
Nucleotide and haplotype diversity analysis of M enterolobii groups based on mtCOII gene
The number of haplotypes detected in the YB, YZ and YN groups was 4, 7, and 4 (Table 5), and the haplotype diversity Hd values among the YB, YZ, and YN groups were all close, 0.900, 0.867 and 1.000, respectively, indicating that the three groups are rich in haplotype diversity. The highest nucleotide diversity of the YN group is 0.0470, and the lowest nucleotide diversity of the YZ group is 0.00209. The sequence is YN > YZ > YB, indicating that there are differences in nucleotide diversity among the three groups. The values of the Tajima's D and Fu's Fs of the three groups conform to the law of neutrality, and the group changes are not significant.
Genetic differentiation and gene flow analysis of the M enterolobii groups based on mtCOII
The total Fst value and gene flow (Nm) value of 19 M enterolobii populations was 0.125 (P < 0.15) and 2.83 (P > 1). We observed the highest gene exchange rate between YB and YZ (Nm = 4.5) (Table 6), whereas the gene exchange rate between YZ and YN was the lowest (Nm = 1.94). The Kxy values (sum/individual number) between YZ and YB, YN groups are similar to 4.14743 and 4.04762, respectively; the minimum Kxy value between YB and YN groups is 2.93333.