Genetic diversity of Venturia inaequalis isolates from the scabs in apple trees in Gansu Province, China, using AFLP markers

Isolation of samples

Samples collected from Gansu Province between June to September 2021 as appeared in Table 1. Figure 1 gives the areas where tests were collected. The monoconidial strategy was utilized for sample isolation. The spore suspension was included into water agar (WA), and air-dried. Single spores were marked under the microscope after 24 h of incubation at 25 °C. A sterile needle was used to collect the marked areas on the WA, which were then placed in potato dextrose agar (PDA) for incubation at 25 °C in dark environment. The isolate was purified to obtain the target isolate when after 40 d of culture.

DNA extraction

The isolates were incubated for 40 days at 25 °C in dark environment. Approximately 100 mg of fungal mycelia was then collected and ground into powder after being frozen with liquid nitrogen. The Fungal Genomic DNA Extraction Kit (Omega Bio-tek, Sangon Company, Shanghai, China) was used to extract nucleic acid per the operating instructions of the kit, and was then stored at −20 °C.

AFLP markers

The AFLP molecular marker technique was modified from Vos et al. (1995). Mse I and EcoR I were used for double digestion. Approximately 500 ng of DNA, 5 U of Mse I, 5 U of EcoR I (Takara-Bio, Beijing, China) and 4 µl of H Buffer (Takara-Bio, Beijing, China) were used in a reaction. ddH₂O was added to obtain a final volume of 40 µl. The digestion protocol involved operation at 37 °C for 3 h followed by 80 °C for 20 min. The product of the double digestion was ligated to the adapters EcoR I (10 µM) and Mse I (10 µM) via a reaction containing: 40 µl of the double digestion product, 1 µl of (35 U) T4 DNA ligase (Takara-Bio, Beijing, China), 1 µl of 10 ×T4 DNA ligase Buffer (Takara-Bio, Beijing, China) and 1 µl of EcoR I and Mse I adapters. ddH₂O was used to attain a final volume of 50 µl. The protocol involved operation at 16 °C for 12 h followed by 80 °C for 20 min. Digestion-ligation products were visualized via patterning on 1.2% agarose gel. After ligation, the product was diluted using a 1:9 ratio with ultrapure water.

Pre-selective amplification was performed using 10 µl of Taq PCR MasterMix (Sangon Company, Shanghai, China), 2 µl of a pre-selective primer mixture E-TTC/M-TAA (10 µM) (Fu et al., 2010b), 5 µl of the ligated product and 8 µl of ddH₂O. Pre-selective amplification cycling parameters were 94 °C for 5 min followed by 31 cycles at 94 °C for 45 s, 45 °C for 40 s and an extension at 72 °C for 60 s, and 72 °C for 10 min and the product was then stored at −20 °C for further use. After pre-selective amplification, the PCR product was diluted using a 1:9 ratio with ultrapure water.

The 25 most common polymorphic combinations were chosen as molecular markers (Fu et al., 2010b) and selectively amplified. Selective amplification was performed using 10 µl of Taq PCR MasterMix (Sangon Company, Shanghai, China), 2 µl of the selective primer mixture EcoR I and Mse I (10 µM), 5 µl of the pre-selection amplification product and 8 µl of ddH₂O. Selective amplification cycling parameters were 94 °C for 5 min followed by 15 cycles at 94 °C for 40 s and 65 °C for 30 s. This was followed by an extension at 72 °C for 60 s where the temperature decreased by 0.7 °C after each cycle. These cycling parameters were then followed by 30 cycles at 94 °C for 30 s, 51 °C for 30 s, and 72 °C for 60 s and a final extension at 72 °C for 10 min, followed by storage at −20 °C for further use.

6% Polyacrylamide gel electrophoresis

The SDS-PAGE Preparation kit (Sangon Company, Shanghai, China) was used for polyacrylamide gel electrophoresis. The electrophoresis buffer was prepared by diluting 10 ×TBE (100 ml) in 900 ml of distilled water. The electrophoresis program began with 40 min of pre-electrophoresis followed by 70 min of electrophoresis after sample loading. The sample loading volume was 10 µl. The gel silver staining method was modified from Borzooeian et al. (2018). The method comprises of settling with 100 ml/L acetic acid (CH₃COOH) for 20 min, washing with ddH₂O for 1 min and silver staining with 1 g/L of silver nitrate solution (AgNO₃) for 20 min. The sample was then washed with ddH₂O for 10 s, developed using 16 g/L sodium hydroxide (NaOH), 8 ml/L formaldehyde (37%) solution for 5 min, a wash with ddH₂O for 10 s and a quenching reaction via the use of 7.5 g/L sodium carbonate (Na₂CO₃) for 5 min. The AFLP molecular marker bands were counted regardless of their presence or absence at the same position, with their presence marked as ‘1’ and absence marked as ‘0’. A binary data ‘0-1’ matrix was then established.

Data analysis

Matrix data were imported to NTSYS2.1 to analyze genetic distances (Weir & Ott, 1997). Nei’s genetic distance among the populations was estimated using POPGENE1.32 and GenAlEx 6.5 (Kimura & Crow, 1964; Lewontin, 1972; Nei, 1973; Yeh et al., 1999). GenAlEx 6.5 was used for analysis of molecular variance (AMOVA) and analysis of principal co-ordinates (PCoA) (Peakall & Smouse, 2006). Individual genotypes were analyzed in Structure 2.3.4 (Pritchard, Stephens & Donnelly, 2000). The optimal K value was determined by uploading the analysis data to the platform Structure Harvester (http://taylor0.biology.ucla.edu/structureHarvester/) (Earl & VonHoldt, 2012).

Analysis of genetic diversity

Analysis of genetic diversity for V. inaequalis using 6% SDS-PAGE (Fig. 2). The results of Popgene1.32 are shown in Table 2. In the 108 isolates, the number of polymorphic loci (Np) was 301, the percentage of polymorphic loci (P) was 99.67%, and the effective number of alleles (Ne) was 1.4398. Nei’s gene diversity (H) was 0.2712 and Shannon’s information index (I) was 0.4241, which shown that there were a few genetic diversities within the populace of V. inaequalis in Gansu Province. The analysis results for V. inaequalis based on 6 geographic regions show that Np values ranged from 224 to 283, P values ranged from 74.17% to 93.17% while Ne values ranged from 1.3586 to 1.5506. H values ranged from 0.2206 to 0.3147 while I value ranged from 0.3432 to 0.4703. Based on the analysis of H and I, Jingning isolates had the highest level of genetic diversity, Maiji, Jingchuan, Xifeng, Lingtai isolates had the second highest level while Ning had the lowest level of genetic diversity.

Cluster analysis

The unweighted pair group with arithmetic mean (UPGAM) method was used to cluster 108 isolates of V. inaequalis from 6 regions of Gansu Province. A tree graph (Fig. 3) was obtained, which was proven to be accurate via the cophenetic correlation test (r = 0.8969). As can be seen from Fig. 3, all the isolates tested were concentrated together when the similarity coefficient was 0.67. The isolates could be divided into two clusters when the similarity coefficient was 0.69. 104 isolates were contained in cluster I and 4 isolates were contained in Cluster II. Cluster I can be further divided into two sub-clusters, with Jingning isolates included in subcluster 1. Maiji, Lingtai, Jingchuan, Ning, Xifeng and several Jingning isolates were included in subcluster 2. Only Jingning isolates were included in Cluster II. The results show that there is no significant correlation between the isolates and their geographical locations in Gansu Province.

Analysis of genetic distance

Popgene1.32 was used to analyze Nei’s genetic identity and genetic distance (Table 3). These were obtained for six isolates from different geographical regions, in which isolates of Ning and Jingchuan have the highest level of genetic identity (0.9906) and the closest genetic distance (0.0095). This indicates that Ning and Jingchuan isolates have the smallest genetic difference. Xifeng and Jingning isolates have the lowest level of genetic identity (0.9267) and the furthest genetic distance (0.0762), which indicates that the genetic difference between Xifeng and Jingning isolates is the largest.

Analysis of molecular variance (AMOVA)

AMOVA analysis of population variance (Table 4) showed that the coefficient of population differentiation (PHiPt) in the six geographical regions is 0.027 (P < 0.05). This indicates that there are some genetic variations for V. inaequalis in different regions of Gansu Province, but the genetic variations are not statistically significant (PHiPt <0.25). There is molecular variance in 108 isolates, among which the frequency of molecular variance is 98% within the same region and 2% across different regions. Therefore, it can be concluded that there is greater molecular variance in the same region and less gene exchange between different regions.

The results of PCoA analysis (Fig. 4) showed that Jingning had more isolates in the fourth quadrant with several isolates independently distributed in the first quadrant. The genetic structure of the tested isolates was analyzed, with results showing that the six regions in Gansu Province could be divided into 2 populations (K = 2, Fig. 5). As can be seen from Fig. 6, the genetic background of the Jingning isolates was mainly derived from ancestors indicated using a red color, with few green components. The genetic background of the other isolates involved different extents of contribution by both red and green ancestors with a high level of genetic similarity.