Genetic Diversity of the root‐knot nematode Meloidogyne enterolobii in Mulberry Based on the Mitochondrial COI Gene

Abstract This study explores the genetic diversity and structure of Meloidogyne enterolobii in mulberry in China. The COI mitochondrial gene (mtCOI) in M.enterolobii populations in Guangdong, Guangxi, and Hunan Provinces was PCR‐amplified, sequenced, and analyzed for genetic diversity. The total number of variations, haplotypes (Hap), the average number of nucleotide differences (k), haplotype diversity (H), and nucleotide diversity (π) of mtCOI were 25, 11, 4.248, 0.900, and 0.00596, respectively. Insignificant differences in Fst value (0.0169) and a high level of gene flow (7.02) were detected among the 19‐mulberry root‐knot nematode populations, and high genetic variation within each population and a small genetic distance among populations were observed. Both phylogenetic analyses and network mapping of the 11 haplotypes revealed a dispersed distribution pattern of 19 mulberry root‐knot nematode populations and an absence of branches strictly corresponding to the 19 range sampling sites. The neutrality test and mismatch analysis indicated that mulberry root‐knot nematode populations experienced a population expansion in the past. The analysis of molecular variance (AMOVA) revealed that the genetic differentiation of M. enterolobii was mainly contributed by the variation within each group. No significant correlation was found between the genetic distance and geographical distance of M. enterolobii populations. The findings of this study provide a profound understanding of the M. enterolobii population and will inform the development of strategies to combat and manage root‐knot nematodes in mulberry.

Ribosomal DNA (rDNA) has been used as a molecular marker to identify and phylogenetically characterize different nematodes by recent studies. For example, the rDNA-PCR technology has been employed to identify Bursaphelenchus spp. (Jiang, Liang, & Zhen, 2005) and Pratylenchus spp. (Mizukubo, Sugimura, & Uesugi, 2007). Mitochondrial DNA (mtDNA) has emerged as another useful tool for the genetic and taxonomic studies of various parasitic nematodes due to its small molecular weight, simple and stable structure, and relatively conserved gene composition (Duan, 2013). For example, Sun, Liao, and Li (2005) used the mitochondrial COII-LrRNA gene fragment to distinguish between different Meloidogyne spp. populations. Wang (2015) determined the genetic diversity of the mitochondrial mtCOI gene in 318 soybean cyst nematode individuals from 16 populations in China. The results showed that there was a certain genetic differentiation among various groups, but the level of gene exchange was also high. Tu, Gao, and Zhou (2007) used the COI gene to study the genetic diversity of 6 local chicken breeds in China. The results showed that there were 22 mutation sites in the gene sequence, and the COI gene sequence of the 6 local chicken breeds had high genetic diversity. It has been reported already using GBS method by Rashidifard et al. (2018) and AFLP, ISSR, and RAPD methods by Tiago, Siqueira, and Castagnone-Sereno (2010), but they have not used COI mtDNA. Thus, this research aims to explore the structure and genetic differentiation of M. enterolobii populations by integrating bioinformatic and the DNA barcoding technologies. Therefore, this research, by applying PCR and DNA bar codes techniques, combining with means of bioinformatics analyzes and compares population genetics indexes of M. enterolobii, and lays the foundation for mulberry breeding and the prevention and cure of M. enterolobii disease in the future.

| Nematode collection
The nematode samples used in this experiment were collected from the main sericulture areas in Guangdong, Guangxi, and Hunan Provinces, China (Table 1).

| PCR and sequencing
Based on previous experimental results, rDNA-ITS was used to identify the 19 populations of Mulberry root-knot nematode as M.
enterolobii. Mature female (ten females for each population) was identified (the female body is white, pear-shaped to globular, with a prominent neck of variable size) and placed into 5 μL of worm lysis buffer (WLB) containing proteinase K for DNA extraction (Williams, Schrank, & Huynh, 1992 subunit ribosomal RNA gene, partial sequence) was amplified by primers LCO1490 (5′-GGTCAACAAAT-CTAAAGATATTGG-3′) and HCO2198 (5′-TAAACTTCAGGGTGACCAAAAAATCA-3′) described by Boehme, Amendt, and Zehner (2012). The 25 μL PCR mixture contained 12.5 μL 2 × PCR buffer for KOD FX (TOYOBO), 5 μL 2 mM dNTPs, 1 μL of each primer, 2 μL of the isolated DNA, and distilled water. The PCR was carried out in a lab cycler (Applied Biosystems) under the following conditions: initial denaturation at 94°C for 4 min; followed by 35 cycles of denaturation at 94°C for 1 min, annealing at 50°C for 1 min, and elongation at 72°C for 2 min, and a final extension at 72°C for 10 min.
All amplicons were separated by electrophoresis on a 1% TBE agarose gel. The amplified products were sequenced (BGI Genomics, BGI-Shenzhen), and the haplotypes were calculated using DNASP 5.0. The sequences obtained were submitted to GenBank and get its number.

| Genetic diversity analysis
The sequencing chromatography was analyzed by Chromas 2.3 (https://chrom as.updat estar.com/), and the DNA sequences were analyzed using the Seq Man program in DNA star 5.0 (DNA STAR, Madison USA).

| Sequence analysis
The sequence within the mtCOI gene was viewed using ClustalX

| Neutrality test
According to Kimura (1979) and Tajima (1989), the DNA fragments were subjected to Tajima's D and Fu's Fs neutrality tests at the population and group levels using DNASP 5.0 (Kimura, 1979;Tajima, 1989).

| Haplotype analysis
Base content and polymorphic loci were analyzed by MEGA 7.0 according to a previously reported method (Librado P & Rozas J, 2009). The variability between sequences was calculated based on the Kimura 2-Parameter C K2P model, and a neighbor-joining (NJ) phylogenetic tree was constructed using MEGA 7.0 (Tamura, Dudley, Nei, & Kumar, 2007). The haplotype network diagram was drawn by NETWORK 5.0 based on the median-joining method.

| Molecular variation analysis
The genetic distance between populations was calculated using the MEGA 7.0 software (Fu, 2016), and the AMOVA molecular variance components and haplotype frequencies were analyzed using the Arlequin 3.5 software (Excoffier & Lischer, 2010). The correlation between genetic distance and geographic distance was calculated using SPSS (22.0). total polymorphisms were identified, respectively. The S-singleton sites were located at positions 17,188,296,501,504,587,646,647,648,650,662, and 691 of the mtCOI fragment, and the parsimony-informative sites were located at positions 189,314,328,702,703,705,706,707,708,709,710,712,713. The contents of a, t, c, and g were 45.79%, 28.31%, 16.60%, and 9.26%, respectively: And the content of a + t was 74.10%, showing a significant a/t bias. And the conversion/ transversion rate R was 0.5.

| Nucleotide and haplotype diversity analysis of mtCOI
The number of variable sites, average number of nucleotide differences (k), haplotype diversity (H), and nucleotide diversity(π) of mtCOI in M. enterolobii populations (Table 3) Table 1.
The number of haplotypes detected in the YB, YZ, and YN groups was 3, 6, and 3 (Table 3), and the Hd values of the haplotype diversity in the YB and YN groups were close, which were 0.700 and 0.733, respectively. The haplotype diversity of YZ had the highest Hd value, which was 0.952, indicating that the haplotype diversity of the three groups was rich. The nucleotide diversity of the YB and YZ groups was relatively close, being 0.00785 and 0.00708, respectively, and the nucleotide diversity of the YN group was the lowest, being 0.00355. The average number of nucleotide differences (k) is in the order of YB > YZ>YN. After testing, the values of the Tajima's D and Fu's Fs of the three groups all conform to the law of neutrality, and the group changes are not significant.

| Phylogenetic analysis of mtCOI haplotypes in M. enterolobii
Phylogenetic tree of 10 aforementioned mtCOI haplotypes was constructed using neighbor-joining (NJ) method under Kimura 2-parameter model in MEGA 7.0 (Fig. 1). The results showed that haplotype topology did not correlate with geography. There was a correlation between locations, and the 10 haplotypes could not be divided into single-line groups corresponding to different geographic regions.

| Haplotype mediation network map of mtCOI
An intermediary network of mtCOI haplotypes of the root-knot nematode populations was then constructed. As shown in Figure 2

| Genetic differentiation and gene flow analysis of the M. enterolobii populations based on mtCOI
The three geographic groups, YB, YN, and YZ, had a Fst value of 0.0169 (p < .05) and a Nm value of 7.02 (p > 4) (

| Genetic distance analysis based on mtCOI
The genetic distances among different M.enterolobii groups were calculated based on mtCOI sequences using MEGA 7.0 (

| Correlation between geographic distance and genetic distance
The correlation between genetic distance and geographic distance based on mtCOI was investigated (Fig. 3). The results showed that there was no significant correlation between the genetic distance and the natural logarithm (LN km) matrix (r = −0.123, p = |−0.155|>0.05) of the geographic distance among samples collected, indicating that geographical distance is not the main factor leading to root-knot nematode population differentiation (Table 7).

| Genetic variance of the M. enterolobii populations
Based on the AMOVA method, the Arlequin software was used to analyze the genetic variation among YB (the Yuebei group), YZ (the Yuezhong group), and YN (the Yuenan group). The intrapopulation differentiation parameter FST was 0.04498 (p < .0001). The variations within a population accounted for 95.5% of total variation, and the variations among populations accounted for 4.50% of total variation (Table 8). 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.

| CON CLUS I ON AND D ISCUSS I ON
In the present study, we carried out an in-depth investigation of the genetic diversity, population structure, as well as the association between the geographic distribution and genetic distance of M.
enterolobii populations based on the mtCOI gene for the first time. and there was no significant correlation between genetic distance and geographic distribution.
In recent years, molecular tools have found their applications in the research of genetic diversity, population differentiation, and evolutionary or taxonomic relationships between closely-related species. So, the genetic diversity study of parasite populations has gained increasing interests. Mitochondrial DNA markers have emerged as a useful tool due to a relatively higher Fst value compared with nuclear sequences, and the mtDNA of nematodes was reported to evolve more quickly than that of other parasites (Anderson, Blouin, & Beech, 1998;Blouin, Yowell, & Courtney, 1995;Dantas et al., 2013). Mitochondrial DNA genes (such as mtCytb) have been widely used to resolve the phylogenetic relationships between nematodes at the subspecies, species, genus, and order levels (Liu et al., 2013;Plantard, Picard, & Valette, 2008;Zhao, Li, & Ryan, 2012). Here, the mtCOI gene was sequenced in 19 M. enterolobii populations to determine the genetic diversity of M. enterolobii. The results of this study are similar to those of Wang (2015) who used the COI gene to study the genetic diversity of soybean cyst nematode population. However, its overall fixation coefficient Fst value is 0.27442 and its overall gene flow Nm is 1.322. The results showed that there was certain genetic differentiation among various populations, but the level of gene exchange was also high.
The Fst value of the total fixed coefficient and the Nm value of the total gene flow in this study are 0.0169 and 7.02, respectively. The results show that the total population has little genetic differentiation, sufficient gene exchange, and differences. It may be caused by the fact that different nematodes come from different regions.
There is no correlation between genetic distance and geographical distance between the two. The results of Li, Zhang, and Tang (2015) analysis of the genetic differentiation level of the wild population of Corbicula flumina in Hongze Lake are also different. Gene by Zhu, Jian, and Wang (2015) and his team. Both research results indicate that the population has experienced expansion, but without no obvious population differentiation, and no evident geographic genetic structure has been formed. The topological structure of the phylogenetic tree shows that the phylogenetic relationship ( Figure. 1)  based on rDNA, mtDNA, ITS and IGS sequences, and satellite DNA probes, this method is time-saving and low-cost, and does not need multiple PCR analysis. As a novel and rapid molecular genotyping tool, GBS can obtain more detailed information about genetic diversity among nematode species. In the future, it is planned to analyze the genetic diversity of M. enterolobii in China using GBS, so as to obtain more detailed genetic information.

ACK N OWLED G M ENTS
The author would like to acknowledge all the teachers and students from the laboratory of the Agricultural College of Zhongkai Agricultural Engineering College for their help. We thank Topedit (www.toped itsci.com) for its linguistic assistance during the preparation of this manuscript.

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.