Polymorphisms of Mitochondrial COII and16S rRNA Gene in Meloidogyne enterolobii on Mulberry


 This study explores the genetic diversity and polymorphisms of Meloidogdyne enterolobii (M enterolobii) on mulberry in China. The sequence of cytochrome oxidase subunit II (COII) and 16S rRNA gene in M enterolobii populations in Guangdong, Guangxi, and Hunan Provinces were PCR-amplified, sequenced, and analyzed for genetic diversity. The haplotypes (Hap) numbers, the total number of mutations, the average number of nucleotide differences (K), haplotype diversity (Hd), and nucleotide diversity (π) of mtCOII gene were 14, 25, 3.563, 0.942 and 0.00429, respectively. The significant differences in Fst value (0.125) and a high level of gene flow (2.83) were detected among the 19 M enterolobii populations. High genetic variation within each population and a small genetic distance among populations was observed. Both phylogenetic analyses and network mapping of the 14 haplotypes revealed a dispersed distribution pattern of the 19 M enterolobii populations. There was an absence of branches strictly corresponding to the 19 range sampling sites. The analysis of molecular variance (AMOVA) revealed that the genetic differentiation of M enterolobii populations was mainly contributed by the variation within each of the defined geographical groups. No significant correlation was found between the genetic distance and geographical distance of 19 M enterolobii populations. This study provides theoretical basis for the future control of M enterolobii and also provides a guarantee for the production of other hosts of M enterolobii.

In recent years, M enterolobii has gradually spread from the south to the north of China (Wu et al., 2019). It has also been found in Africa, America, and Europe (Onkendi et al., 2013). Currently, M enterolobii is considered one of the most threatening pathogenic nematodes in tropical and subtropical regions throughout the world, with an estimated potential yield loss of 20% (Zhuo et al., 2008). Chemical treatments remain the most commonly used measured to control M enterolobii infections. However, the overuse of such chemicals has led to the development of pesticide resistance. Additionally, pesticides residues pose potential health risks to humans (Kaplan, 2004). Therefore, the better understanding of the genetic characteristics of M enterolobii may identify potential resistance genes and novel approaches to control M enterolobii infections.
Ribosomal DNA (rDNA) is widely used as a molecular marker to identify and phylogenetically characterise different nematodes. Notably, rDNA PCR-based approaches have been employed to identify Bursaphelenchus spp (Jiang et al., 2005). and Pratylenchus spp. (Mizukubo et al., 2007). Mitochondrial DNA (mtDNA) is another emerging tool for the genetic and taxonomic characterisation of plant parasitic nematodes due to its small molecular weight, high stability, and relatively conserved gene composition (Duan, 2013). Sun et al. (2005) used the mitochondrial cytochrome oxidase subunit II (COII)-LrRNA gene fragment to distinguish between different Meloidogyne spp. Deng et al. (2016) used the COII gene to analyse the genetic diversity of the Rotylenchulus reniformis population. Janssen et al (2016) performed a haplotype-based mtDNA analysis in Meloidogyne and found that certain mitochondrial haplotypes were associated with speci c esterase isozyme patterns, suggesting that different parthenogenetic lineages can be identi ed using mitochondrial haplotypes. Rashidifard (2019) analysed the molecular characteristics of 37 Meloidogyne populations from four provinces in South Africa and found that COII-16S could accurately identify different M enterolobii populations. Additionally, COII and 16S rRNA characterisation has been proven useful for the identi cation of different Meloidogyne species from different geographic regions (Onkendi & Moleleki, 2013). Ye et al. (2007) used the sequence of cytochrome oxidase subunit I (COI) and other genes to construct 19 phylogenetic trees of Bursaphelenchus spp., and analysed the phylogenetic relationships among the species of the genus. Therefore, mitochondrial genes can be used to analyse the evolution and genetic diversity of plant nematodes.
In this study, we analysed the genetic diversity of M enterolobii collected from mulberry trees in southern China, Guangdong, Hunan, and Guangxi. Phylogenetic analysis was performed based on the cytochrome oxidase subunit II (COII) gene, partial cds; tRNA-His gene, complete sequence; and 16S ribosomal RNA gene, partial sequence; mitochondrial.

Materials And Methods
Nematode collection: The nematode samples in this experiment were collected from the main sericulture areas in Guangdong, Guangxi, and Hunan Provinces, China ( Table 1). The 19 populations were classi ed into three groups: YB (Northern Region of Guangdong Province, China), YN (Southern Region of Guangdong Province, China) and YZ (Central Region of Guangdong Province, China). This distribution rule is based on the geographical distribution and climatic zone characteristics of the M enterolobii populations in Guangdong Province.

Nematode extraction
Female nematode was extracted from root-knot samples using the method described in Liu et al. (2020).

PCR and sequencing
A total of 19 populations, previously identify as M enterolobii based on rDNA-ITS sequencing, and maintained in the lab were used in this study. Ten females of each population were placed into 5 µL of worm lysis buffer (WLB) containing proteinase K for DNA extraction (Williams et al., 1992). The DNA samples were stored at -20℃.
All PCR products were separated by electrophoresis on a 1% TBE agarose gel. The ampli ed 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 accession number.
(2) Neutrality test According to Tajima (1989) and Kimura (1983), the DNA fragments were subjected to Tajima's D and Fu's Fs neutrality test in the population and group levels using Dna SP 6.0 (Kimura, 1983;Tajima, 1989).

(3) Haplotype analysis
Base content and polymorphic loci were analyzed by MEGA 7.0 according to a previously reported method (Librado & Rozas, 2009). The variability between sequences was calculated based on the Kimura-2-Parameter (K2P) model, and a Neighbor-Joining (NJ) phylogenetic tree was constructed using MEGA 7.0. NETWORK 5.0 based on the Median-joining method drew the haplotype network diagram.

(4) Molecular variation analysis
The genetic distance between populations was gured up using the MEGA 7.0, and the AMOVA molecular variance components and haplotype frequencies were analyzed using the Arlequin 3.5. The correlation between genetic distance and geographic distance was calculated using SPSS (22.0).

Results
Sequence and variation analysis of the mtCOII gene fragment in M. enterolobi populations A total of 19 homologous sequences of the mtCOII gene were ampli ed 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 identi ed, 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 signi cant 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 signi cant.
Among the 14 haplotypes identi ed (Table 3) 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 signi cant 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 signi cant.
Genetic differentiation and gene ow analysis of the M enterolobii groups based on mtCOII The total Fst value and gene ow (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) (

Discussion
In this study, we assessed the genetic diversity of M enterolobii from different geographic regions in China by analysing the sequence of the mtDNA gene COII. We observed a moderate genetic diversity among the populations from different collection sites (Fst = 0.125, Nm = 2.83), suggesting gene exchange between these populations. The overall genetic variation in M enterolobii was primarily caused by variation within rather than between different geographic groups. We found no signi cant correlation between genetic distance and geographic distribution. This study enriches the phylogenetic information of M enterolobii, and provides the basic evidence for the inherent genetic factors of damage from M enterolobii.
Genetic diversity is not only the basis of biodiversity but also a driving force for the evolution of species. The reduction or loss of genetic diversity is a great threat to a population or species living in a changing environment. Haplotype polymorphisms (Hd) and nucleotide polymorphisms (π) are commonly used to measure the genetic diversity of species or populations (Hao et al., 2014). The 19 nematode population isolates used in this study had a total of 14 haplotypes with an Hd of 0.942, indicating high haplotype diversity in M enterolobii populations in Guangdong Province. In contrast, the total nucleotide diversity was very low (π = 0.00429). The high haplotype diversity and low nucleotide diversity suggests that a bottleneck of M enterolobii populations occurred, followed by rapid population expansion. The accumulation of mutations could have led to the high haplotype diversity observed among these populations, which maintained a high nucleotide similarity. High haplotype diversity and low nucleotide diversity is very common in invertebrates with large maternal effective populations and strong reproductive abilities (Grant & Bowen, 1998;Lavery et al., 2008). Tajima's D and Fu's FS analyses con rmed that the all M enterolobii populations population might have undergone a population expansion event during evolution.
From the phylogenetic results (Fig. 1), the 19 M enterolobii populations (Clade A and Clade B) in this study and the sequence selected on NCBI (Clade C) are not on the same branch. The M enterolobii populations in this study were all isolated from the mulberry root, other M enterolobii were parasitic on Ipomoea aquatica, Zingiber o cinale, Daucus carota, Psidium guajava, and came from different regions. The 19 M enterolobii populations in this study were clustered on the same branch. The M enterolobii selected on NCBI gathered on another branch. It further shows that these 19 M enterolobii populations have no geographical barriers and had su cient genetic exchanges. At the same time, it also shows that the genetic differentiation of M enterolobii from different hosts is different. However, whether M enterolobii will have a biased active host selection, and whether M enterolobii of the same host will have genetic changes due to infecting different host plants, this will require further research. From the evolutionary perspective of 19 M enterolobii populations in a single cluster, most of the M enterolobii populations are clustered in Clade A, and the genetic relationship is inconsistent with their geographical distribution. It can be seen that the M enterolobii populations parasitizing on mulberry may have slowly evolved from a population in a certain area.
In this study, we evaluated the genetic diversity of M enterolobii from different regions using mitochondrial genes. Rashidifard  of different M enterolobii isolates using intergenic regions (IGS) of the rDNA, COII, and 16S rRNA; they found a low level of diversity among the isolates tested, suggesting that M enterolobii is a genetically homogeneous root-knot nematode. However, in this study, we found evidence of extensive gene exchange among different populations. These discrepancies could be explained by the fact that, in contrast to previous studies, the host of the M enterolobii isolates used in this study was mulberry. The second instar larvae and the male had limited mobility because the M enterolobii colonized mainly in the host roots. Therefore, we hypothesized that the egg and larvae remaining in the soil after the formation of the egg were separated from the plant root. They will be the result of long-distance passive propagation of human activities such as agricultural operations or wind, rain and water currents. Additionally, all the nematode populations used in this study were isolated from different regions of the south of China, and geographical factors may have caused nucleotide polymorphisms. The use of different genes and research methodologies are also possible causes of discrepancies. Deng et al. (2016) analysed the genetic diversity of Rotylenchulus reniformis from different geographic regions in China based on the sequence of COII; they found a high variation in COII-LrRNA sequence, suggesting the high genetic variation among different Rotylenchulus reniformis populations, contributing to their ability to adapt to environmental changes. Consistent with our ndings, Newton et al. (2003) used random ampli cation of polymorphic DNA (RAPD) markers to analyse the genetic diversity of soybean cyst nematodes and found that phylogenetic clusters were not associated with geographic proximity. Consistently, we found that the phylogenetic relationship of different M enterolobii populations was not related to the geographic location of the isolates.
The haplotype analysis revealed that the haplotype Hap9 is shared among mulberry root-knot nematode species in Guangdong Province and that the other haplotypes have evolved from Hap9. Furthermore, the low genetic variation among the populations and the absence of a relationship between haplotype and the geographical region further support that the different mulberry root-knot nematode populations isolated from Guangdong may have originated from the same population (Yu, 2009). The topological structure of the phylogenetic tree also suggested that the absence of phylogenetic relationship among the different M enterolobii populations from Guangdong Province. The haplotype Hap9 diverged into three large groups, while no clustering was observed between different haplotypes from the same geographical region; these ndings further support the genetic ow among different mulberry root-knot nematodes in Guangdong Province, leading to a low genetic diversity. It is likely that during the genetic ow, genes involved in drug resistance are also transferred, enabling the transmission of mulberry root-knot nematodes.

DATA AVAILABILITY STATEMENT
The assembled gene sequences of M. enterolobii are available in NCBI (MN907170-MN9071718, respectively). The authors a rm that all data necessary for con rming the conclusions of the article are present within the article, gures, and tables.      Correlation between genetic distance(Appendix 1) and geographical distance(Appendix 2) in the 19 M enterolobii populations based on mtCOII gene using SPSS soft. Relationship between genetic and geographic distance matrices for M enterolobii (r=0.093, p=|-0.204|>0.05).