The authors have declared that no competing interests exist.
Apple scab caused by
Apple (
One sexual and multiple asexual cycles, of this pathogen annually causes noteworthy variations in
Samples were collected from 12 apple growing districts of Jammu and Kashmir India during the year 2017–18 as shown in
S.No | Sample Code | Location | District | Latitude | Longitude | Accession number |
---|---|---|---|---|---|---|
1 | M1 | Trehgam | Kupwara | 34.521°N | 74.184°E | MK478885.1 |
2 | M2 | Bankoot | Bandipora | 34.420°N | 74.650°E | MK359025.1 |
3 | M3 | Char-i-sharief | Budgam | 74.766°E | MK504436.1 | |
4 | M4 | Syedpora | Shopian | 74.83°E | MK478887.1 | |
5 | M5 | Beerva | Budgam | 34° 001°N | 74.5953°E | MK359026.1 |
6 | M6 | Chadoora | Budgam | 75.100°E | MK504428.1 | |
7 | M7 | Batpora | Srinagar | 74.464°E | MK504429.1 | |
8 | M8 | Hajin | Bandipora | 34.09°N | 74.79°E | MK359032.1 |
9 | M9 | Yaripora | Kulgam | 33.7°N | 75.0°E | MK530205.1 |
10 | M10 | Handwara | Kupwara | 34.40°N | 74.28°E | MK359027.1 |
11 | M11 | Kakapora | Pulwama | 33.88°N | 74.92°E | MK359028.1 |
12 | M12 | Wakura | Ganderbal | 34.05°N | 74.47°E | MK367580.1 |
13 | M13 | Gutlibagh | Ganderbal | 34.09°N | 74.09°E | MK504434.1 |
14 | M14 | Naidkhai | Bandipora | 34.09°N | 74.79°E | MK359029.1 |
15 | M15 | Gantmulla | Baramulla | 34.086°N | 74.033°E | MK504430.1 |
16 | M16 | Pinjura | Shopian | 33.72°N | 74.83°E | MK504437.1 |
17 | M17 | Pattan | Baramulla | 34.85°N | 74.37°E | MK504431.1 |
18 | M18 | Sogam | Budgam | 34.020°N | 74.780°E | MK504435.1 |
19 | M19 | Mattan | Anantnag | 33.701°N | 75.285°E | MK359031.1 |
25 | M25 | Uri | Baramula | 34.080°N | 74.05°E | MK532035.1 |
20 | M20 | PranuBaderwah | Doda | 32.58° N | 75.538° E. | MK359030.1 |
21 | M21 | NalthiBaderwah | Doda | 32.93°N | 75.712°E | MK583539.1 |
22 | M22 | Doda | Doda | 33.13°N | 75.57°E | MK504432.1 |
23 | M23 | Kishtwar | Kishtwar | 33.32°N | 75.77°E | MK504433.1 |
24 | M24 | Tund | Kishtwar | 33.32°N | 75.77°E | MK532037.1 |
26 | M26 | Faisal Abad | Kishtwar | 33.32°N | 75.77°E | MK532036.1 |
27 | M27 | Padder | Kishtwar | 33.13°N | 75.09°E | MK532034.1 |
28 | M28 | Padder | Kishtwar | 33.13°N | 75.220°E | MK532033.1 |
29 | M29 | KotiBaderwah | Doda | 33.145°N | 75.547°E | MK532032.1 |
30 | M30 | Baderwah | Doda | 33.13°N | 75.57°E | MK532031.1 |
The fungus from the infected samples was isolated and purified using monoconidial method by streaking out spores on plates containing 2% water agar, pure fungal cultures were obtained by transferring single germinated conidium on potato dextrose agarcontaining antibacterial chloromphenicol (50 μg/ ml) to avoid bacterial contaminations[
Cultured fungal isolates in 100ml of potato dextrose broth was kept in incubator-shaker at 19°C for about 25–30 days under continuous dark. The mycelia harvested was blotted dry between the tissue layers and immediately frozen in liquid nitrogen. After freeze-drying, DNA was extracted using Fungal DNA isolation kit (GCC Biotech India Pvt. Ltd). The DNA was quantitatively and qualitatively checked using a Nanodropspectrophotometer (Themoscientific) and was further diluted to a workingconcentration of 30ng/μl and stored at -20°C for further use.
All 30 isolates were amplified using polymerase chain reaction (PCR) in a thermal cycler (Takara Japan) using 30ng of genomic DNA in a final volume of 25 μl per reaction. The universal ITS primers with ITS 1 as forward and ITS4 as reverse primerwere used for PCR amplification (White et al., 1991). ThePCR was performed in a 0.2-ml tube containing 0.5μM forward and reverse primer, 200 μM eachdNTP, 1 unit kappa Taqpolymerase and 1ul of genomic DNA in a 10xkappa buffer and 5 mM MgCl2. The PCR was normalized after repetitive cycles till optimal amplification was achieved and consists of 35 cycles involving initial denaturation step at 94°C for 5 min, followed by 94°C for 30 s, annealing at 53°C for 45 s, extension at 72°C for 1min and final extension at 72°C for 15min[
For diversity and structure analysis of selected fungal samples, 28 published SSR primer pairs were used
Locus | Primer sequence 5′–3′ | Allele Size (bp) | Annealing Temp. |
---|---|---|---|
173–241 | 58 | ||
227–247 | 58 | ||
132–152 | 58 | ||
184–278 | 58 | ||
188–196 | 58 | ||
167–191 | 58 | ||
192–224 | 58 | ||
184–196 | 60 | ||
228–236 | 60 | ||
134–169 | 58 | ||
196–232 | 60 | ||
128–134 | 60 | ||
147–165 | 58 | ||
87–97 | 58 | ||
159–173 | 60 | ||
155–167 | 58 | ||
225–239 | 58 | ||
108–172 | 58 | ||
180–186 | 58 | ||
171–173 | 60 | ||
267–285 | 58 | ||
1tc1a | 109–187 | 54 | |
1tc1b | 149–210 | 54 | |
1tc1g | 111–185 | 57 | |
1aac3b | 118–174 | 55 | |
1aac4b | 166–177 | 58 | |
1aac4f | 96–116 | 58 | |
1aac4h | 198–201 | 56 |
Amplified PCR products were sequenced at Agri Genome Labs (Infopark Road, Kakkanad, Kerala, India). Primers for the sequencing PCR product were the same as for the PCR amplification. The sequences of PCR products were assembled using DNA baser V.4 program to produce complete contig. These were further aligned using CLUSTAL W method of Bio-Edit software and aligned sequences were deposited in NCBI GenBank. A database search of homologous sequences was performed by BLAST analysis at NCBI (
Analysis was carried out using POPGENE for gene frequency, allele number, effective allele number, polymorphic loci, gene diversity, Shannon index, gene flow, genetic distance. The GenAlEx version 6.5 for distance-based analysis like AMOVA (Analysis of molecular variance), and PCoA. (Principle coordinate analysis)[
Identification based on morphological characters from fungal culture (
2A. Conidia observed under 40 x resolutions. 2B. Pure culture of
The ITS based primers amplified ~550bp ampliconproducts after sequencing were run for BLASTn and all obtained sequences showed 96%-98% sequence homology with
In total 30
Locus | Ne | I | Ob_He | Exp_Ho | Exp_He | Nei | Obs_Hom | Ave_Het | Pic | Fst |
---|---|---|---|---|---|---|---|---|---|---|
1.25 | 0.36 | 0.23 | 0.79 | 0.20 | 0.20 | 0.76 | 0.20 | 0.20 | 0.15 | |
1.18 | 0.28 | 0.16 | 0.84 | 0.15 | 0.15 | 0.83 | 0.15 | 0.15 | 0.08 | |
1.92 | 0.67 | 0.66 | 0.51 | 0.48 | 0.48 | 0.33 | 0.48 | 0.10 | 0.32 | |
1.38 | 0.45 | 0.33 | 0.71 | 0.28 | 0.27 | 0.66 | 0.27 | 0.25 | 0.21 | |
1.99 | 0.69 | 0.83 | 0.49 | 0.50 | 0.49 | 0.16 | 0.49 | 0.16 | 0.64 | |
1.99 | 0.69 | 0.86 | 0.49 | 0.50 | 0.49 | 0.13 | 0.49 | 0.32 | 0.07 | |
2.00 | 0.69 | 1.00 | 0.49 | 0.50 | 0.50 | 0.00 | 0.50 | 0.52 | 0.00 | |
1.86 | 0.65 | 0.73 | 0.52 | 0.47 | 0.46 | 0.26 | 0.46 | 0.20 | 0.12 | |
1.94 | 0.67 | 0.76 | 0.50 | 0.49 | 0.48 | 0.23 | 0.48 | 0.16 | 0.00 | |
1.86 | 0.65 | 0.73 | 0.52 | 0.47 | 0.46 | 0.26 | 0.46 | 0.07 | 0.24 | |
1.99 | 0.69 | 0.96 | 0.49 | 0.50 | 0.49 | 0.03 | 0.49 | 0.16 | 0.00 | |
1.94 | 0.67 | 0.43 | 0.50 | 0.49 | 0.48 | 0.56 | 0.48 | 0.16 | 0.00 | |
1.98 | 0.68 | 0.63 | 0.49 | 0.50 | 0.49 | 0.36 | 0.49 | 0.16 | 0.15 | |
1.14 | 0.24 | 0.13 | 0.87 | 0.12 | 0.12 | 0.86 | 0.12 | 0.16 | 0.00 | |
1.96 | 0.68 | 0.86 | 0.50 | 0.49 | 0.49 | 0.13 | 0.49 | 0.16 | 0.00 | |
1.99 | 0.69 | 0.96 | 0.49 | 0.50 | 0.49 | 0.03 | 0.49 | 0.16 | 0.00 | |
1.94 | 0.67 | 0.83 | 0.50 | 0.49 | 0.48 | 0.16 | 0.48 | 0.16 | 0.04 | |
2.00 | 0.69 | 1.00 | 0.49 | 0.54 | 0.50 | 0.00 | 0.50 | 0.16 | 0.00 | |
1.96 | 0.68 | 0.86 | 0.50 | 0.48 | 0.49 | 0.13 | 0.49 | 0.30 | 0.00 | |
1.99 | 0.69 | 0.96 | 0.49 | 0.50 | 0.49 | 0.03 | 0.49 | 0.10 | 0.00 | |
1.64 | 0.57 | 0.53 | 0.60 | 0.39 | 0.39 | 0.46 | 0.39 | 0.25 | 0.00 | |
1tc1a | 1.00 | 0.00 | 0.00 | 1.00 | 0.00 | 0.00 | 1.00 | 0.00 | 0.15 | 0.00 |
1tc1b | 2.00 | 0.69 | 1.00 | 0.49 | 0.50 | 0.50 | 0.00 | 0.50 | 0.12 | 0.00 |
1tc1g | 1.30 | 0.39 | 0.26 | 0.76 | 0.23 | 0.23 | 0.73 | 0.23 | 0.10 | 0.00 |
1aac3b | 1.76 | 0.62 | 0.63 | 0.55 | 0.44 | 0.43 | 0.36 | 0.43 | 0.13 | 0.00 |
1aac4b | 1.86 | 0.65 | 0.73 | 0.52 | 0.47 | 0.46 | 0.26 | 0.46 | 0.15 | 0.00 |
1aac4f | 1.06 | 0.14 | 0.06 | 0.93 | 0.06 | 0.06 | 0.93 | 0.06 | 0.10 | 0.18 |
1aac4h | 1.96 | 0.68 | 0.73 | 0.50 | 0.49 | 0.49 | 0.26 | 0.49 | 0.15 | 0.00 |
(Exp_Ho) Expected homozygosty (Exp_he) heterozygosity were computed using Levene (1949), (Nei) Nei’s (1973) Na = Observed number of alleles, Ne = Effective number of alleles [Kimura and Crow (1964)], I = Shannon’s Information index [Lewontin (1972)], (Ob_He) observed heterozygosity, (Ob_Ho) homozygosity, Fst = genetic differentiation.
The cluster analysis of 30 isolates revealed a high genotypic diversity within
The bootstrap values are given on the nodes.
Structure analysis revealed that isolates of
The two populations along with admixture isolates generated from structure analysis were analyzed for genetic variation among and within populations using AMOVA (
Source | Df | SS | MS | Est. Var. | % |
---|---|---|---|---|---|
Among Pops | 2 | 21.720 | 10.860 | 0.554 | 25% |
Among Indiv | 27 | 50.980 | 1.888 | 0.211 | 9% |
Within Indiv | 30 | 44.000 | 1.467 | 1.467 | 66% |
Total | 59 | 116.700 | 2.231 | 100% | |
0.248 | 0.001 | ||||
0.126 | 0.040 | ||||
0.343 | 0.001 |
Df-Degree of freedom, SS Sum of squares, MS Mean sum of squares, Est. Var-Estimated variance
Apples are grown in high altitude areas of India particularly in J&K[
We used ITS ribotyping of 30 isolates collected from two distinct regions. The noncoding ribosomal DNA ITS sequences doesn’t change more rapidly than the coding sequences and may diverge between species and populations [
Microsatellites or SSRs are very useful markers for population genetics analysis because of their high specificity, polymorphism, and reproducibility. These are major advantages of using SSR markers over Random Amplification of Polymorphic DNA. The SSR markers used in this study were highly variable and results generally corresponded with previous population genetics studies conducted in Europe. However, in this study the markers1tc1a, 1tc1b, 1tc1g, 1aac3b, 1aac4b, 1aac4f, and 1aac4h showed only one allele compared to eight to ten alleles reported previously[
The distance between the Jammu region and the Kashmir region where we collected samples is approximately 400 km and allowed us to collect
The isolates in this study shared a high percentage of identical alleles, indicating considerable gene flow among all isolates of
Structure analyses divided the isolates into two populations (K = 2) with a clear differentiation between the two apple-growing regions. Pathogen distance seems to be the most significant factor in steep gene flow because it explains 50% of the variation among the
The genetic variation and population structure of scab causing
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