MATERIALS AND METHODS
Nematode collection: The nematode samples used in this study were randomly collected from the main rice areas in Guangdong, Guangxi, Fujian, Hainan, Anhui, Jiangsu, Henan, Sichuan, Jiangxi, and Hunan Provinces, China (Table 1). One sample was collected in each site and 4-9 different geographical samples were collected in each province. Single nematode at the second stage (J2) was picked separately from single root-knot after hatching from eggs. Each site was replicated 3 times. All three J2s in each site were defined as a population.
DNA extraction, PCR amplification, and sequencing: DNA extraction was described as Liao et al. (2001). The species identities were determined by ribosomal DNA sequencing of the ITS region. The universal primers for root knot nematode identification were 26S (TTTCACTCGCCGTTACTAAGG) and V5367(TTGATTACGTCCCTGCCTTT) as described in Vrain et al. (1992). Single J2 of M. graminicola was cut into two pieces with a scalpel under a dissecting microscope and placed into 8μL of worm lysis buffer (WLB) solution containing1μL protein K (20 mg/mL) in a PCR tube (Zhuo et al., 2008). The PCR tube was incubated for 30 min at 65℃, then 15 min at 95℃. The final suspension was used as a DNA template for PCR amplification (Zhang et al. 2001).
The primer pairs COI-F (5’-ATCAGGAGTGAGATCTATTTCTAG-3’) and COI-R (5’- CGAGGTTGCCCTTGTCCAAA-3’) which designed using Primer 5 based on the 1-1500bp sequence of the accession number KJ139963 were used for the amplification of the mtCOI gene region. The 25μL PCR mixture contained 12.5 μL 2× PCR buffer for KOD FX (Toyobo Life Science Co., Ltd.), 5 μL 2 mM dNTPs, 1 μL of each primer (10 μM), 2μL (20ng) DNA, and 4μL distilled water. The PCR amplification was carried out in a lab cycler (Applied Biosystems) as described in Shao et al. (2020).
All PCR products were separated by electrophoresis on a 1% TBE agarose gel and purified by Tiangen Gel Extraction Kit (Tiangen Biotech Co., Ltd.), then cloned into the pMD19-T Vector (Takara Bio Inc.), and transformed into DH5 alpha Competent Cells. The amplified products were sequenced (BGI Genomics, BGI-Shenzhen) and the haplotypes were calculated using DNASP 5.0 (Librado and Rozas, 2009). After sequencing, the sequences obtained were submitted to GenBank and get its accession numbers.
Genetic diversity analysis: The original sequence was retrieved and the flanking sequences at both ends were deleted for further data analysis. Haplotypes were analysed using Alignment Transformation EnviRonment (http://sing.ei.uvigo.es/ALTER/). The percentage of variant loci in the sequence, parsimony informative loci, nucleotide diversity index(π), haplotype diversity (Hd), population genetic differentiation index, Fst values, Gst values, and the average number of nucleotide changes (K) of the 54 M. graminicolapopulations were calculated by Tajima (1989) and Fu (1997). Gene flow (Nm) between populations was calculated based on the mitochondrial-specific gene formula Fst=1/(1+2Nm) (Takahata and Palumbi 1985). The gene fragments were tested for neutrality using Tajima’s D and Fu’s Fs neutrality tests at the population and group levels. Phylogenetic trees were constructed using PhyloSuite software based on a Bayesian approach (GTR model) (Zhang et al., 2019). To study the genetic relationship between haplotypes, Network v.4.6.1 software (www.Fluxusengineering.com) was used to draw the mediation network between haplotypes (Bandelt et al., 1999). The correlation between genetic distance and geographic distance was calculated using SPSS (22.0) based on the mantel test method. Molecular analysis of variation (AMOVA) was performed using Arlequin 3.1 software (Excoffer et al., 1992) to estimate genetic variation among and within populations.