RESULTS
Sequence characteristics and variation study of the mtCOI
gene fragment in M. graminicola populations: The COI gene fragments of
54 M. graminicola populations isolated in 10 provinces of China
were PCR-amplified and sequenced. The sequence fragment lengths of 787
bp were obtained by sequence splicing and multiple sequence alignment.
The accession numbers of the generated sequences are shown in Table 1.
Thirty-nine polymorphic loci were found (4.9% of the total number of
bases examine), with 12 S-singleton sites and 27 parsimony-informative
sites, accounting for 30.8% and 69.2% of the total polymorphisms,
respectively. The S-singleton sites on the mtCOI gene fragment
were found at positions of 205, 260, 285, 301, 305, 312, 315, 379, 418,
498, 546and 582, whereas the parsimony-informative sites were found at
positions of 51, 114, 182, 188, 195, 266, 289, 330, 333, 353, 381, 414,
441,446, 447, 468, 519, 523, 565, 577, 586, 609, 628, 634, 642, 651 and
681, respectively. The contents of a, t, c, and g were 28.1%, 46.3%,
7.4%, and 18.2%, respectively, and the content of a+t was 74.4%,
indicating a significant a/t bias. The conversion/transversion rate R
was 0.6.
Phylogenetic analysis of M. graminicola populations based
on COI genes: Phylogenetic suite software was used to perform Bayesian
interference analysis and construct a phylogenetic tree of COI genes inM. graminicola populations (Fig.1). M.enterolobii strains
from NCBI with the same mtCOI gene were selected as out groups
for the phylogenetic tree. Fifty-four populations of M.
graminicola were divided into three clades; the population from Henan
province, Jiangsu and Anhui Province in Yangtze valley (YP) were grouped
in Cluster 1; Cluster 2 comprises the populations from central region of
China (CR) (Hunan, Sichuan, Jiangxi provinces) and eight French strains
of M. graminicola selected from NCBI, Cluster 3 includes all
populations in southern China (SC) (Guangdong, Guangxi, Fujian and
Hainan provinces).
Nucleotide and haplotype diversity analysis of M.
graminicola populations: The haplotype diversity (Hd) and nucleotide
diversity (π) of mtCOI genes in M. graminicola populations
(Table 2) showed that the Hd was 0.646, indicating that the total
population was higher in haplotypes. The nucleotide diversity (π) and
nucleotide mean difference number (k) were 0.00682 and 5.370,
respectively. The Tajima’s D (-1.252) and Fu’s Fs values (-3.06764) of
the total population were less than zero (0), which indicated that the
entire population conformedto the law of neutrality.
Among the mtCOI gene sequences of the three groups, the highest
point of variation (31) was observed in the YP group (Table 2), and the
lowest mutation sites were observed in the CR group. The haplotype
diversity (Hd) of the three groups ranged from 0.143 to 0.772, while the
lowest (0.143) and the highest (0.804) Hd value was in CR group and YP
group, respectively. The π values of the three groups ranged from
0.00018-0.01127, the smallest π value was detected in the CR groups
(0.00018) and the largest π value was detected in the YP group
(0.01127). The highest mean number of nucleotide differences (k) among
groups was found in the YP group whilethe lowest was found in the CR
group. The neutral test results for the three groups were shown to be
less than zero (0) for Tajima’s D and Fu’s Fs values and were not
significantly different.
Haplotype frequency analysis of M. graminicola: There
are 15 different haplotypes (Hap 1-15) that have been discovered in 54M. graminicola populations (Table 3). Hap8 appeared significantly
more frequent among all individuals tested, accounting for 59.3%
(32/54). Hap10 was detected in four populations, accounting for 7.4%
(4/54). Hap1, Hap6, Hap7, Hap12, and Hap15 accounted for 3.7% (2/54) of
the populations examined. Among these 15 haplotypes identified, 8
haplotypes occurred only once and were found to be endemic in M.
graminicola populations. The haplotype results (Fig. 2) of the clusters
showed that only one haplotype (Hap8) was shared among the three
clusters. Nine haplotypes species were in the YP group, of which were
identified as endemic haplotypes and the highest haplotype frequency was
identified (60%). There were five haplotypes in the CR group, with a
haplotype frequency of 33.3%. Four haplotype species was found in SC
group with the lowest haplotype frequency of 26.7%.
Haplotype mediation network map of M. graminicola
populations: The M. graminicola populations’ mtCOI genes
were then used to construct the haplotype-mediated network map (Fig.3).
The haplotype-mediated network formed a circular topological
distribution pattern, indicating that the M. graminicolapopulations have historically undergone expansion. Hap8 occurred most
frequently and had the largest area distribution. The haplotype of the
three clusters in common atHap8. Therefore, Hap8 may be the original
haplotype of M. graminicola . However, the haplotypes in Henan
province were grouped separately. Hap5 (HENXX) was a transitional
haplotype linking the Henan haplotype to other haplotypes. Hence, this
network may clarify the evolutionary relationships between each
haplotype and the geographical distribution of each group, bolstering
the phylogenetic tree.
Genetic differentiation and gene flow analysis of the M.
graminicola populations: The genetic differentiation and gene flow of
the 54 populations of M. graminicola were analyzed based onmtCOI gene sequence data (Table 4). The overall genetic
differentiation coefficient Gst value, fixation coefficient Fst value,
and gene flow Nm value among the populations of M. graminicolawere 0.431, 0.593, and 0.33, respectively. These suggested that there
was a large genetic differentiation (Fst> 0.25) and a low
gene exchange (Nm < 1) among the studied populations. The
minimum Fst value observed between the population of Hainan (HAN) and
Fujian (FUJ)was 0.047, while the highest gene flow was 10.125, which
indicated that the gene exchange between these two populations was
frequent with low genetic differences. The gene flow between the
Guangdong (GUD) population and the Jiangsu (JIS) population was higher
(1.75), indicating that there was some gene exchange between
them.Similarly, the gene flow between populations of GUD, JIX, ANH, HAN,
and JIS were all 1,suggesting that there was low gene exchange between
these populations.
Genetic and geographic distance analysis of M. graminicola
populations: Using the Kimura2-Parameter model, the genetic distances
between different M. Graminicola populations were calculated
based on mtCOI gene sequences (Table 5). Results showed that the
genetic distances between different populations varied from0 to0.013.
Among them, the lowest genetic distances (0.000) was observed between
the populations of HAI and SIC, FUJ; GUD and JIX, ANH, JIS; JIX and ANH,
JIS; ANH and JIS; SIC and FUJ, while the highest genetic distance
(0.013) was observed between Henan, GUX, and Hunan.
Based on mtCOI gene, the relationship between genetic distance
and geographic distance of different M. graminicola populations
were examined using SPSS software (Fig.4). The findings revealed that
among the collected samples, there was no significant correlation
between genetic distance and the natural logarithm (LN km) matrix of
geographical distance (Table 5) (R<0.2), P>0.05),
suggesting that geographic distance was not the major factor
contributing to M. graminicola populations differentiation.
Analysis of molecular variance (AMOVA) of the M.graminicola groups: This analysis results of AMOVA based on
different M. graminicola populations were shown in Table 6. The
genetic differentiation (FST) among different
populations was low (FST= 0.17847 P < 0.0001),
96.3% of total variation was mainly occurred within populations whereas
only 3.7% of the total variation was within groups. These findings
suggested that the M. graminicola populations’ genetic variation
was basically from within the population.