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.