Plant-parasitic nematodes are an important limiting factor in vegetable production, and in many areas a major factor requiring extensive use of pesticides. Root-knot nematodes (RKNs) of the genus
Considering that morphological and morphometrical methods for the identification species of RKNs are considerably time consuming and there is a lot of ambiguity, using species-specific primers could be a rapid method for detection (these primers listed in Cunha et al., 2018).
The genetic diversity among
Molecular markers that have proved to be useful in these studies are mitochondrial gene cytochrome C oxidase subunit I (COI) (Ye et al., 2015; Powers et al., 2018), the small subunit 18 S rRNA gene (Tigano et al., 2005; Ye et al., 2015), D2-D3 expansion segment of the large subunit 28 S rRNA (Palomares-Rius et al., 2007; Ye et al., 2015) and ribosomal internal transcribed spacer (ITS1 and ITS2) (Powers et al., 1997; Ye et al., 2015). These markers were useful for separating species of RKNs; however, they could not separate main species including
Although ISSR technique has been used to resolve genetic diversity in certain species of RKNs such as
However, ISSR and RAPD markers are similar in some aspects, but there are a number of differences between them that make the two markers act as complementing of each other. RAPD primers are short (10 nucleotides) with a random sequence and markers are considered to be uniformly distributed along the genome, whereas ISSR primers is based on the amplification of regions flanked by repeating sequences which found only between microsatellite loci. In the other hands, annealing temperature in RAPD is lower than ISSR (Lax et al., 2007).
The aim of the present study is to infer the genetic intraspecific diversity among populations of
In total, 37 populations of
Populations of
Number | Sampling area | N | E | Sample | Host plant | Molecular code |
---|---|---|---|---|---|---|
1 | Boshehr province-Abdan | 28°11´00.38˝ | 51°74´07.94˝ | Soil and root | Tomato | MJ1 |
2 | Boshehr province-Abdan | 28°10´99.86˝ | 51°74´08.46˝ | Soil and root | Tomato | MJ2 |
3 | Boshehr province-Abdan | 28°11´00.58˝ | 51°74´07.97˝ | Soil and root | Tomato | MJ3 |
4 | Boshehr province-Abdan | 28°11´00.29˝ | 51°74´08.27˝ | Soil and root | Tomato | MJ4 |
5 | Boshehr province-Abdan | 28°11´00.31˝ | 51°74´08.28˝ | Soil and root | Tomato | MJ5 |
6 | Boshehr province-Abdan | 28°10´95.09˝ | 51°74´04.66˝ | Soil and root | Tomato | MJ6 |
7 | Boshehr province-Abdan | 28°10´94.93˝ | 51°74´04.44˝ | Soil and root | Tomato | MJ7 |
8 | Boshehr province-Abdan | 28°10´94.82˝ | 51°74´04.43˝ | Soil and root | Tomato | MJ8 |
9 | Boshehr province-Abdan | 28°10´94.56˝ | 51°74´03.85˝ | Soil and root | Tomato | MJ9 |
10 | Boshehr province-Abdan | 28°10´94.47˝ | 51°74´03.81˝ | Soil and root | Tomato | MJ10 |
11 | Boshehr province-Deir Port | 27°91´15.43˝ | 51°98´02.27˝ | Soil and root | Tomato | MJ11 |
12 | Boshehr province-Deir Port | 27°91´15.16˝ | 51°98´02.71˝ | Soil and root | Tomato | MJ12 |
13 | Boshehr province-Deir Port | 27°91´17.39˝ | 51°98´03.58˝ | Soil and root | Tomato | MJ13 |
14 | Boshehr province-Deir Port | 27°91´17.33˝ | 51°98´03.47˝ | Soil and root | Tomato | MJ14 |
15 | Boshehr province-Deir Port | 27°91´44.84˝ | 51°98´43.36˝ | Soil and root | Tomato | MJ15 |
16 | Boshehr province-Bardkhon | 28°07´54.24˝ | 51°48´89.26˝ | Soil and root | Tomato | MJ16 |
17 | Boshehr province-Bardkhon | 28°07´51.78˝ | 51°48´90.21˝ | Soil and root | Tomato | MJ17 |
18 | Boshehr province-Bardkhon | 28°36´13.93˝ | 51°48´90.22˝ | Soil and root | Tomato | MJ18 |
19 | Boshehr province-Bardkhon | 28°07´51.25˝ | 51°48´84.44˝ | Soil and root | Tomato | MJ19 |
20 | Boshehr province-Bardkhon | 28°07´51.92˝ | 51°48´82.48˝ | Soil and root | Tomato | MJ20 |
21 | Boshehr province-Bardkhon | 28°07´52.13˝ | 51°48´82.42˝ | Soil and root | Tomato | MJ21 |
22 | Boshehr province-Bardkhon | 28°07´52.15˝ | 51°48´82.19˝ | Soil and root | Tomato | MJ22 |
23 | Kerman province-Jiroft | 28°41´09.00˝ | 57°40´13.81˝ | Root | Mallow | MJ23 |
24 | Kerman province-Jiroft | 28°41´35.08˝ | 57°39´25.18˝ | Root | Goosefoot | MJ24 |
25 | Kerman province-Jiroft | 28°42´22.57˝ | 57°43´05.86˝ | Root | Eggplant | MJ25 |
26 | Fars province-Beyza | 29°55´30.53˝ | 52°26´28.41˝ | Soil and root | Cucumber | MJ26 |
27 | Fars province-Beyza | 29°55´51.85˝ | 52°26´44.88˝ | Soil and root | Cucumber | MJ27 |
28 | Fars province-Beyza | 29°55´59.49˝ | 52°26´51.00˝ | Soil and root | Cucumber | MJ28 |
29 | Fars province-Beyza | 29°55´16.00˝ | 52°27´20.15˝ | Soil and root | Cucumber | MJ29 |
30 | Fars province-Beyza | 29°55´11.72˝ | 52°27´27.93˝ | Soil and root | Cucumber | MJ30 |
31 | Fars province-khafr | 28°57´44.57˝ | 53°11´58.46˝ | Soil and root | Okra | MJ31 |
32 | Fars province-khafr | 28°57´35.80˝ | 53°12´32.31˝ | Soil and root | Okra | MJ32 |
33 | Fars province-khafr | 28°57´34.97˝ | 53°12´28.38˝ | Soil and root | Okra | MJ33 |
34 | Fars province-khafr | 28°58´56.08˝ | 53°11´47.87˝ | Soil and root | Okra | MJ34 |
35 | Fars province-Kazeron | 29°33´23.23˝ | 51°45´56.27˝ | Soil and root | Tomato | MJ35 |
36 | Fars province-Kazeron | 29°33´25.22˝ | 51°45´42.76˝ | Soil and root | Tomato | MJ36 |
37 | Fars province-Kazeron | 29°33´42.68˝ | 51°45´08.64˝ | Soil and root | Tomato | MJ37 |
38 | Fars province-Fasa | 29°00´46.56˝ | 53°37´42.27˝ | Soil and root | Cucumber | MI1 |
39 | Fars province-Fasa | 29°00´46.56˝ | 53°37´42.27˝ | Soil and root | Cucumber | MI2 |
40 | Fars province-Fasa | 29°00´46.56˝ | 53°37´42.27˝ | Soil and root | Cucumber | MI3 |
41 | Fars province-Fasa | 29°00´46.56˝ | 53°37´42.27˝ | Soil and root | Cucumber | MI4 |
One full egg mass was put in 16 µl ddH2O in a 0.2 ml tube. DNA was extracted according to Tanha Maafi et al. (2003) with some modifications: tubes were frozen at –80°C for at least 15 min and crushed by vortexing, and 20 µl worm lysis buffer (500 mM KCl, 100 mM Tris-Cl pH 8, 15 mM MgCl2, 0.05% Mercaptoethanol, and 4.5% Tween 20) and 4 µl proteinase K (600 µg/ml) were added, respectively. The samples were incubated at 65°C for 1 hr and at 95°C for 10 min. After incubation, the tubes were centrifuged for 2 min at 38,000 g and kept at –20°C for next uses.
PCR was performed containing 1 µl of DNA template, 8 µl mastermix (Amplicon Red), 1 µl of each primer (10 pmol) and 4 µl ddH2O to a final volume of 15 µl. Four set species-specific primers (Table 2) were used for PCR reactions (Zijlstra, 2000; Zijlstra et al., 2000). A negative control containing the PCR mixture without DNA template was also included.
All primers used for ISSR, RAPD and species-specific identification of
ISSR | RAPD | ||
---|---|---|---|
(CCA)5 | 5´-CCACCACCACCACCA-3' | OPA8 | 5’-GTGACGTAGG-3' |
(GTG)6 | 5'-GTGGTGGTGGTGGTGGTG-3' | OPA10 | 5'-GTGATCGCAG-3' |
(ATG)6 | 5'-ATGATGATGATGATGATG-3' | OPA13 | 5'-CAGCACCCAC-3' |
(GAG)4GC | 5'-GAGGAGGAGGAGGC-3' | OPAD10 | 5'-AAGAGGCCAG-3' |
(GACA)4 | 5'-GACAGACAGACAGACA-3' | OPE18 | 5'-GGACTGCAGA-3' |
(GA)8C | 5'-GAGAGAGAGAGAGAGAC-3' | OPP17 | 5'-TGACCCGCCT-3' |
(AC)8T | 5'-ACACACACACACACACT-3' | OPB11 | 5'-GTAGACCCGT-3' |
(CA)8G | 5'-CACACACACACACACAG-3' | OPB17 | 5'-AGGGAACGAG-3' |
(CTC)4GC | 5'-CTCCTCCTCCTCGC-3' | OPD8 | 5'-GTGTGCCCCA-3' |
(GAGA)4GG | 5'-GAGAGAGAGAGAGAGAGG-3' | OPC8 | 5'-TGGACCGGTG-3' |
|
|||
|
Fjav (5'-GGTGCGCGATTGAACTGAGC-3') |
|
Finc (5'-CTCTGCCCAATGAGCTGTCC-3') |
Rjav (5'-CAGGCCCTTCAGTGGAACTATAC-3') | Rinc (5'-CTCTGCCCTCACATTAAG-3') | ||
|
Far (5'-TCGGCGATAGAGGTAAATGAC-3') |
|
F (5'- TGACGGCGGTGAGTGCGA-3') |
Rar (5'-TCGGCGATAGACACTACAAACT-3') | R (5'-TGACGGCGGTACCTCATAG-3') |
The PCR amplification profile consisted of 5 min at 94°C, 35 cycles of 30 sec at 94°C, 30 sec at annealing temperature (64°C for
The ISSR-PCR reactions were performed in a 20 μl volume containing 2 µl of genomic DNA, 2 µl of primer, 8 µl mastermix (Amplicon Red), and 8 µl ddH2O. Ten primers including (CCA)5, (GTG)6, (ATG)6, (GAG)4GC, (GACA)4, (GA)8C, (AC)8T, (CA)8G, (CTC)4GC, and (GAGA)4GG (Table 2) were implemented (Lax et al., 2007).
PCR amplification reactions were programmed for an initial denaturation at 94°C for 3 min, followed by 35 cycles of 30 sec at 93°C, 90 sec at 48°C, 1 min at 72°C, and a final extension of 10 min at 72°C. PCR success was visualized by loading 7 µl of the PCR product into a 1.7% TBE buffered agarose gel (75 V, 120 min).
Amplification reactions were carried out in reactions of 20 µl total volume containing 2 µl of genomic DNA, 2 µl of primer, 8 µl mastermix (Amplicon Red), and 8 µl ddH2O. Ten primers (Table 2) were used in RAPD-PCR (Mokaram Hesar et al., 2011).
For RAPD amplifications, the thermos-cycle profile was 5 min at 94°C; 35 cycles of 1 min at 94°C, 1 min at 35°C and 2 min at 72°C, followed by a final step of 5 min at 72°C. PCR success was visualized loading 7 µl of the PCR product into a 1.7% TBE buffered agarose gel (75 V, 120 min).
For the ISSR and RAPD fragment analysis, data were scored for all primers: “1” for the presence of band and “0” for the absence of band. To be sure, all steps were repeated twice and smeared or weak bands were not counted. Distance matrixes were calculated using the Dice and Jaccard coefficients. Cluster analysis was performed with the Numerical Taxonomy Multivariate Analysis System (NTSYSPC V-2.02) software package (Rohlf, 2000). The genetic dissimilarity matrix and ultrametric distance matrix produced from UPGMA-based dendrogram with COPH module nested in the same software was compared using Mantel’s matrix correspondence test (Mantel, 1967). Also, we try to use Mantel test in linear shape for searching correlation between different matrix produced by RAPD and ISSR. Bootstrapping was done using the Winboot with 1000 replicates (Yap and Nelson, 1996) and measurements under 60 were excluded. Frequency analyses were performed to estimate the variance components and their significance levels of genetic variation within and among populations using GenALEx version 6.5 (Peakall and Smouse, 2006). We used P (percentage of polymorphic loci), H (the expected heterozygosity) and I (Shannon’s information index) to calculate genetic diversity. Also,
In total, 41 populations of RKNs were recovered including 34 populations from vegetable fields and nine populations from greenhouses in southern Iran. Two nematodes populations were obtained from weeds growing in vegetable fields. All the recovered populations in this study were tested with the species-specific molecular marker type Sequence Characterized Amplified Region (SCAR) for the four major species (Zijlstra, 2000; Zijlstra et al., 2000). Expected band for
Average of the morphometrics of females, J2s and males of five populations of
Females | Males | Second-stage juveniles (J2s)† | |||||||||
---|---|---|---|---|---|---|---|---|---|---|---|
Diagnostic characters | 15 | CV | 10 | CV | MJ1 | MJ4 | MJ8 | MJ14 | MJ15 | 50 | CV |
L (including neck) | 686±127 (506-1,010) | 18 | 1482±155 (1189-1,694) | 10 | 463 | 457 | 449 | 482 | 469 | 464±19 (429-497) | 4 |
Body width (W) | 420±99 (274-568) | 23 | 33±2.8 (29-38) | 9 | 14 | 14 | 15 | 15 | 16 | 15±1.5 (12-18) | 9 |
Neck length | 259±87 (153-540) | 33 | − | − | − | − | − | − | − | − | − |
Stylet | 15±2 (13-18) | 14 | 18±1.2 (16-19) | 6 | 9.7 | 10.4 | 10.5 | 10.1 | 10.2 | 10±0.5 (9-12) | 5 |
DGO | 3.1±0.6 (2.0-3.9) | 20 | 3.3±0.2 (3.1-3.5) | 5 | 2.7 | 2.1 | 2.2 | 1.9 | 2.0 | 2.2±0.5 (1.5-3.7) | 22 |
Excretory pore | 190±30 (145-270) | 16 | − | − | − | − | − | − | − | − | − |
Vulval slit length | 21±3 (15-27) | 15 | − | − | − | − | − | − | − | − | − |
Vulva-anus distance | 16±4 (12-28) | 25 | − | − | − | − | − | − | − | − | − |
Inter-phasmidial distance | 78 ± 16 (58-108) | 20 | − | − | − | − | − | − | − | − | − |
Tail length | − | − | 14±2.1 (10-17) | 15 | 51 | 55 | 55 | 58 | 57 | 55±5 (40-66) | 9 |
Hyaline length | − | − | − | − | 11 | 12 | 11 | 16 | 16 | 13±4 (7-20) | 27 |
Pharynx (glands end) | − | − | 473±50 (390-552) | 11 | 164 | 202 | 197 | 201 | 201 | 193±23 (90-216) | 12 |
Body length to width (a) | 1.7±0.4 (1.2-2.6) | 22 | 45±4.5 (37-51) | 10 | 38 | 32 | 29 | 33 | 30 | 31±3 (25-39) | 10 |
b | − | − | 6.6±1.3 (4.8-8.1) | 20 | 6.4 | 4.9 | 4.7 | 5.0 | 5.2 | 5.2±1.4 (4.3-12.4) | 27 |
b' | − | − | 3.1±0.6 (2.4-3.9) | 18 | 3.0 | 2.3 | 2.3 | 2.4 | 2.3 | 2.5±0.5 (2.0-5.2) | 22 |
c | − | − | 113±26 (87-169) | 23 | 9.4 | 8.4 | 8.2 | 8.3 | 8.2 | 8.5±0.8 (7.2-11.3) | 10 |
c' | − | − | 0.5±0.1 (0.3-1.0) | 19 | 4.6 | 5.3 | 5.5 | 5.9 | 5.2 | 5.7±0.5 (4.7-7.2) | 10 |
Spicules | − | − | 23±5.8 (15-29) | 24 | − | − | − | − | − | − | − |
Gubernaculum | − | − | 13±2.8 (8-17) | 20 | − | − | − | − | − | − | − |
With the 10 random primers used in the RAPD analysis, a total of 180 bands were produced. All primers resulted in amplification and different patterns were tested for population differences. The resulting gel electrophoresis for 19 populations for (CCA)5 primer of ISSR and OPAD10 primer of RAPD is presented in Figure 3. The number of reproducible amplified fragments varied from 14 to 23 per primer (average 18), their size ranging from 200 to 2500 bp. The percentage of polymorphic bands (P%) within each population ranged from 16.1 to 36.1%, (in populations of Khafr and Abdan, respectively) and I index ranged from 0.093 to 0.218 (same as P%). The highest value of H index was in populations of Abdan (0.150). The mean gene variations for the 27 populations were 0.103 and 0.148 by H and I, respectively (Table 4).
Genetic diversity estimates of
ISSR | RAPD | |||||
---|---|---|---|---|---|---|
Population | %P† | H | I | %P | H | I |
Abdan (10)†† | 25.61 | 0.110 | 0.158 | 36.11 | 0.150 | 0.218 |
Deir (5) | 20.73 | 0.092 | 0.130 | 30.00 | 0.132 | 0.189 |
Bardkhon (7) | 24.39 | 0.097 | 0.141 | 30.56 | 0.118 | 0.173 |
Jiroft (3) | 10.37 | 0.039 | 0.058 | 17.78 | 0.079 | 0.113 |
Beiza (5) | 21.34 | 0.088 | 0.128 | 22.78 | 0.095 | 0.137 |
Khafr (4) | 18.29 | 0.076 | 0.110 | 16.11 | 0.064 | 0.093 |
Kazeron (3) | 12.80 | 0.057 | 0.081 | 18.89 | 0.079 | 0.114 |
Mean | 19.08 | 0.080 | 0.115 | 24.60 | 0.103 | 0.148 |
In ISSR, a total of 164 loci, with average of 16.4 loci per primer, were scored. P, H, and I were used to estimate genetic diversity within the
The Nei’s (1978) genetic distances (D) between pairs of
Population† | Abdan | Deir | Bardkhon | Jiroft | Beiza | Khafr | Kazeron |
---|---|---|---|---|---|---|---|
Abdan | – | 0.032 | 0.070 | 0.074 | 0.091 | 0.084 | 0.088 |
Deir | 0.082 | – | 0.054 | 0.076 | 0.068 | 0.088 | 0.091 |
Bardkhon | 0.090 | 0.076 | – | 0.046 | 0.045 | 0.039 | 0.059 |
Jiroft | 0.069 | 0.127 | 0.119 | – | 0.071 | 0.040 | 0.025 |
Beiza | 0.043 | 0.080 | 0.119 | 0.108 | – | 0.073 | 0.071 |
Khafr | 0.080 | 0.055 | 0.143 | 0.134 | 0.075 | – | 0.051 |
Kazeron | 0.120 | 0.071 | 0.050 | 0.156 | 0.132 | 0.162 | – |
The UPGMA trees obtained with RAPD and ISSR data showed some differences in tree topologies between calculations based on Dice and Jaccard. The positions of some populations are different by ISSR and RAPD, although the number of clades was consistent as both methods revealed four clades. None of these clades grouped
In analyses of molecular variance analysis, percentages of molecular variance within and among populations were 11.36% and 2.62% for RAPD, and 8.26% and 1.33% for ISSR, respectively (Table 6).
Analysis of molecular variance (AMOVA) of
Marker | Source of variation | Degree of freedom | Sum of squares | Variance components | Percentage of variation | Fixation index ( |
Number of migrants (Nm) |
---|---|---|---|---|---|---|---|
Between populations | 6 | 148.88 | 2.63* | 19 | |||
RAPD | Within populations | 30 | 340.85 | 11.36* | 81 | 0.188 | 1.079 |
Total | 36 | 489.73 | 13.99 | ||||
Between populations | 6 | 90.68 | 1.34* | 14 | |||
ISSR | Within populations | 30 | 247.80 | 8.26* | 86 | 0.140 | 1.535 |
Total | 36 | 338.49 | 9.60 |
This is the first comprehensive study of RKN species in southern regions of Iran which demonstrated that
Accurate distinction of
On the whole, P, H, and I revealed a low level of genetic diversity within populations by incorporation of RAPD and ISSR data (Table 4). To our knowledge, this is the first study for the genetic structure of
The mean
The amount of genetic diversity of RAPD marker in this study is higher than that of the northern regions of Iran, which has previously reported by Mokaram hesar et al. (2011), although the host plants are almost identical. Moreover, it seems that genetic diversity revealed by ISSR marker, is slightly higher; however, there is not any other reference that can be compared with the present results. One possible source might be related to the presence of males in the studied populations. Genetic changes in nematode population are partially driven by the environment including the use of resistant varieties and pesticides. Exploring such genetic changes at the population level is crucial for understanding and predicting the behavior of nematodes in the field (Golden and Birchfield, 1978). When we compare warmer conditions in southern regions with those in northern regions of Iran, it may be concluded that the crop cultivation is carried out consecutively throughout the year, and it seems that activity of RKNs continuing during the year, which may be a cause of increased genetic diversity among the present populations.
It should also be pointed that the results of Mantel test revealed that there is a low correlation between the two genetic diversity sets of RAPD and ISSR (
In this paper, the species-specific SCAR primer technique proved to be a powerful tool to distinguish the populations of the two widely distributed RKNs (