Genetic diversity indices using URP + IRAP, SRAP, and CDDP markers
The current work used 41 primers (11 URP + IRAP, 18 SRAP, and 12 CDDP) to predict the molecular genetic diversity of melon accessions. The capacity to amplify melon DNA and the quality of the primer product were criteria in the selection of these primers. Table 2 highlight the informativeness criteria used to compute the informativeness of 41 primers. Following URP + IRAP analysis of the accessions studied, the 11 URP + IRAP primers tested amplified a total of 154 diverse fragments ranging in size from 100 to 3000 bp, with 143 of them revealing polymorphisms. The number of polymorphic bands (TPB) ranged from 8 (URP-2F and LTR6149-3'LTR) to 18 (URP-1F), with an average of 13.00. URP + IRAP makers had a PIC value per primer of 0.29 on average, URP-13R and URP-25F had the highest polymorphism information content (PIC) value (0.34), and the LTR6149-3'LTR combination had the lowest (0.20). The average marker index (MI) score was 3.93, with URP-13R having the maximum (5.79) and LTR6149-3'LTR combination having the least (1.62). Shannon's information index (I) varied between 0.27 (LTR6149-3'LTR) and 0.49 (URP-4R), with an average value of 0.39. Additionally, gene diversity, or expected heterozygosity (He), varied between 0.16 (URP-2F) and 0.33 (URP-4R), with an average of 0.25.
The genetic diversity of 57 melon accessions was evaluated using 18 different SRAP primer combinations. The SRAP primer pairs demonstrated good amplification for all accessions and produced polymorphic bands ranging in size from 0.08 to 1.20 kb. By using PCR, the 18 SRAP amplified a total of 143 different and reproducible bands. The Me2-Em11 primers combination obtained the greatest number of amplified bands (16) and polymorphic fragments (15), while the Me1-Em7 primer pair computed the minimum number of amplified (4) and polymorphic bands (3). The PIC value ranged from 0.14 to 0.38, with Me2-Em2 having the highest value of 0.38, followed by Me1-Em12 and Me10-Em2 (0.34), and Me1-Em7 and Me1-Em8 combinations having the lowest value of 0.14. Each primer set produced a marker index in the range of 0.43 (Me1-Em7) to 3.94 (Me2-Em11), with an average of 1.88. Shannon's information index (I) spanned from 0.22 at Me7-Em12 to 0.54 at Me1-Em2, with an average of 0.39. Furthermore, the gene diversity or predicted heterozygosity (He) ranged from 0.12 at Me7-Em12 to 0.36 at Me1-Em2, with an average of 0.25. The 12 CDDP primers generated variable and reliable bands in 57 melon individuals. These CDDP primers amplified 157 reliable bands in total, with the number of reliable bands per primer set ranging from 9 (MADS-1 and WRKY-R3B) to 19 (MADS-4). The size of the scorable band varied from 100 to 1100 base pairs. Polymorphism was found in 131 of the 157 scorable bands. The polymorphic bands formed by 12 CDDP primers ranged from 5 to 18, with a mean of 10.92 per primer. KNOX-2 has the least polymorphic bands, while MADS-4 had the maximum. The PIC and MI values of the CDDP primers showed a high level of variance. The PIC of the 12 CDDP primers ranged from 0.20 to 0.37, with the KNOX-3 primer having the highest value. The MI values ranged from 0.62 (WRKY-R3B) to 5.55 (KNOX-3) per primer, with a mean value of 3.50. The Shannon’s information index (I) ranged from 0.32 at WRKY-R3B to 0.58 at MYB-1 with an average 0.45. Moreover, the gene diversity or expected heterozygosity (He) ranged from 0.18 at WRKY-R3B to 0.40 at MYB-1 with an average of 0.30.
Table 2
Description of URP + IRAP, SRAP, and CDDP primers, their amplification, and the degree of polymorphism obtained in melon accessions.
| URP markers | | | | SRAP markers | | | | CDDP markers | | |
Marker | TAB | TPB | PIC | MI | I | He | Marker | TAB | TPB | PIC | MI | I | He | Marker | TAB | TPB | PIC | MI | I | He |
URP-1F | 18.00 | 18.00 | 0.31 | 5.52 | 0.43 | 0.29 | Me1-Em2 | 9.00 | 9.00 | 0.25 | 2.28 | 0.54 | 0.36 | ABP1-1 | 12.00 | 11.00 | 0.31 | 3.41 | 0.34 | 0.22 |
URP-2F | 10.00 | 8.00 | 0.22 | 1.77 | 0.28 | 0.16 | Me1-Em7 | 4.00 | 3.00 | 0.14 | 0.43 | 0.23 | 0.12 | ERF-1 | 12.00 | 12.00 | 0.31 | 3.72 | 0.48 | 0.32 |
URP-4R | 13.00 | 11.00 | 0.30 | 3.29 | 0.49 | 0.33 | Me1-Em8 | 6.00 | 5.00 | 0.14 | 0.72 | 0.39 | 0.26 | ERF-2 | 13.00 | 10.00 | 0.34 | 3.40 | 0.45 | 0.30 |
URP-9F | 14.00 | 13.00 | 0.33 | 4.29 | 0.48 | 0.32 | Me1-Em11 | 6.00 | 5.00 | 0.18 | 0.91 | 0.32 | 0.19 | KNOX-2 | 9.00 | 5.00 | 0.20 | 1.00 | 0.35 | 0.23 |
URP-13R | 19.00 | 17.00 | 0.34 | 5.79 | 0.41 | 0.27 | Me1-Em12 | 9.00 | 8.00 | 0.34 | 2.70 | 0.37 | 0.22 | KNOX-3 | 16.00 | 15.00 | 0.37 | 5.55 | 0.49 | 0.33 |
URP-17R | 14.00 | 13.00 | 0.32 | 4.11 | 0.46 | 0.31 | Me1-Em13 | 8.00 | 8.00 | 0.17 | 1.33 | 0.35 | 0.23 | MADS-1 | 10.00 | 9.00 | 0.36 | 3.24 | 0.45 | 0.30 |
URP-25F | 16.00 | 15.00 | 0.34 | 5.16 | 0.40 | 0.25 | Me2-Em2 | 9.00 | 8.00 | 0.38 | 3.02 | 0.46 | 0.30 | MADS-4 | 19.00 | 18.00 | 0.29 | 5.22 | 0.44 | 0.29 |
URP-30F | 12.00 | 11.00 | 0.29 | 3.23 | 0.35 | 0.20 | Me2-Em11 | 16.00 | 15.00 | 0.26 | 3.94 | 0.35 | 0.23 | MYB-1 | 12.00 | 9.00 | 0.34 | 3.06 | 0.58 | 0.40 |
URP-38F | 16.00 | 16.00 | 0.32 | 5.19 | 0.39 | 0.24 | Me2-Em12 | 10.00 | 8.00 | 0.31 | 2.45 | 0.44 | 0.29 | WRKY-F1 | 13.00 | 11.00 | 0.34 | 3.74 | 0.53 | 0.36 |
LTR6149-3'LTR | 9.00 | 8.00 | 0.20 | 1.62 | 0.27 | 0.17 | Me4-Em7 | 7.00 | 6.00 | 0.28 | 1.69 | 0.41 | 0.27 | WRKY-R1 | 13.00 | 12.00 | 0.30 | 3.60 | 0.44 | 0.29 |
LTR6150-3'LTR | 13.00 | 13.00 | 0.25 | 3.24 | 0.36 | 0.23 | Me5-Em7 | 12.00 | 10.00 | 0.37 | 3.72 | 0.46 | 0.30 | WRKY-R3 | 19.00 | 17.00 | 0.32 | 5.44 | 0.50 | 0.34 |
Mean | 14.00 | 13.00 | 0.29 | 3.93 | 0.39 | 0.25 | Me5-Em12 | 7.00 | 6.00 | 0.21 | 1.25 | 0.26 | 0.15 | WRKY-R3B | 9.00 | 2.00 | 0.31 | 0.62 | 0.32 | 0.18 |
Total | 154.00 | 143.00 | 3.22 | 43.21 | 4.32 | 2.77 | Me5-Em13 | 7.00 | 6.00 | 0.31 | 1.86 | 0.39 | 0.26 | Mean | 13.08 | 10.92 | 0.32 | 3.50 | 0.45 | 0.30 |
| | | | | | | Me6-Em7 | 5.00 | 4.00 | 0.25 | 0.98 | 0.44 | 0.28 | Total | 157.00 | 131.00 | 3.79 | 42.00 | 5.37 | 3.56 |
| | | | | | | Me6-Em13 | 9.00 | 8.00 | 0.27 | 2.20 | 0.52 | 0.36 | | | | | | | |
| | | | | | | Me7-Em12 | 6.00 | 5.00 | 0.21 | 1.04 | 0.22 | 0.12 | | | | | | | |
| | | | | | | Me10-Em2 | 10.00 | 7.00 | 0.34 | 2.36 | 0.45 | 0.29 | | | | | | | |
| | | | | | | Me10-Em13 | 6.00 | 4.00 | 0.24 | 0.96 | 0.34 | 0.21 | | | | | | | |
| | | | | | | Mean | 8.11 | 6.94 | 0.26 | 1.88 | 0.39 | 0.25 | | | | | | | |
| | | | | | | Total | 146.00 | 125.00 | 4.65 | 33.84 | 6.95 | 4.45 | | | | | | | |
TNB: total number of amplified bands; TPB: total number of polymorphic bands; PIC: polymorphism information content; MI: marker index; ; I: Shannon’s information index; He: expected heterozygosity or gene diversity
Clustering and structure analysis of melon accessions
The dendrogram built by Ward analysis revealed that URP + IRAP results classified 57 accessions of melon into two primary categories (G-1 and G-2) (Fig. 1A). Twenty-three accessions were included in the first. This group was subdivided into two subgroups (SG-1 and SG-2). The AN1 accession was included in the first subgroup (SG-1). Twenty-two accessions were included in the second subgroup (SG-2). The second major group was further subdivided into two subgroups, the first of which included 7 accessions and the second of which included 27 accessions. The Jaccard coefficient was used to calculate genetic dissimilarity, which ranged from 0.32 to 0.84. The accessions AN37 and AN38 had the highest genetic dissimilarity (84%), whereas the accessions AN13 and AN26 had the lowest genetic distance (32%). The STRUCTURE software was used to assess the marker information with a Bayesian-based model in order to better understand the association between the analyzed accessions. The proportions of membership ranged from K = 1 to K = 9. According to Evanno's approach, Delta K had the largest ad hoc value at K = 2, as shown in Fig. 2A, demonstrating that the 57 accessions are better separated into two populations using URP + IRAP data. Population 1 consisted of 27 accessions, while population 2 included 30 accessions. Assuming that accessions with a membership coefficient (Q value) of 0.79 or higher were considered pure (Ahmed et al. 2021), 49.12% of the accessions tested belonged to corresponding pure groups, while the remaining 50.88% belonged to an admixed group (Fig. 2B).
Based on the SRAP data, the Ward method was used to perform hierarchical clustering, which divided 57 melon accessions into two primary groups (SG-1 and SG-2) (Fig. 1B). The initial group (G-1) included twenty-one different accessions. This was divided into two subgroups (SG-1 and SG-2) with the first (SG-1) having one accession (AN50) and the second (SG-2) having twenty accessions. The second largest group (G-2) consisted of 36 accessions. The genetic distance was calculated using the Jaccard coefficient, which ranged from 0.20 to 0.78. The accessions AN6 and AN46 had the smallest genetic dissimilarity (22%), whereas AN12 and AN36 had the largest genetic difference (78%). To infer population structure (K > 1) based on SRAP data, the STRUCTURE software employed a model-based Bayesian approach. The ad hoc statistic K was used to calculate the actual number of clusters (K) based on the log probability of data with regard to K values. STRUCTURE analysis of the 18 SRAP primer pairs suggests that K = 2 is the greatest value (Fig. 2C). This number denotes the presence of two informative populations among all melon accessions. Population 1 included 29 accessions, while population 2 comprised 28 accessions. Thirty-one of the accessions investigated corresponded to pure populations, whereas the other 26 belonged to an admixed population (Fig. 2D).
The CDDP dendrogram classified the accessions into three principal groupings (G-1, G-2, and G-3) (Fig. 1C). The largest cluster (G-1) has 42 accessions, which were further subdivided into two sub-clusters. There were nine accessions in the first sub-cluster (SG-1). The second sub-cluster included thirty-three accessions. The second (G-2) and third (G-3) main CDDP groups were made up of eleven and four accessions, respectively. The genetic disparity was determined using the Jaccard coefficient, which varied from 0.25 to 0.73. The accessions AN8 and AN24 exhibited the least genetic distance (25%), while AN4 and AN20 had the highest genetic variation (73%). Using the STRUCTURE software, the CDDP genotyping findings were utilized to perform population structure analysis on 57 accessions under an admixture model. The Evano method determined K = 3 as the best number of clusters (Fig. 2E). An accession was deemed a pure member of a cluster if the likelihood of membership in that cluster was greater than 79%. For K = 3, the resulting clusters had 15, 18, and 24 accessions, respectively, for clusters 1, 2, and 3. Thirty accessions were classified as pure, while the remaining accessions were designated as admixed (Fig. 2F).
Analysis of molecular variance and diversity indices
Based on the results of STRUCTURE clustering, the analysis of molecular variance (AMOVA) approach approximates population divergence directly from the three types of markers. AMOVA revealed 5.64, 9.80, and 7.55% variation among populations for the URP + IRAP, SRAP, and CDDP markers, respectively, as well as significant AMOVA variance within populations of 94.36, 91.20, and 92.45% for the URP + IRAP, SRAP, and CDDP markers, respectively. Phi-statistics provides a summary of the degree of differentiation between clusters. According to the phi-statistics, there is little differentiation between STRUCTURE clusters (0.056, 0.098, and 0.076 for URP + IRAP, SRAP, and CDDP markers, respectively), but there were high and significant variance (p < 0.001) within STRUCTURE groupings for three types of markers (Table 3). Heterozygosity can be estimated using expected heterozygosity (He), which provides information about the likelihood of an individual's fraction of heterozygosity for all studied loci. Using URP + IRAP primers, the number of effective allele (Ne), Shannon’s information index (I), expected heterozygosity (He), and polymorphic loci percentage (PP) ranged from 1.42–1.43, 0.40–0.41, 0.25–0.26, and 90.21–95.80, respectively (Table 4). The highest fixation index (Fst) value was recorded by population 1. The gene flow (GF), based on the PhiPT value, between both populations was 4.2. Based on SRAP primers, the Ne, I, He, and PP were between 1.40–1.46, 0.38–0.42, 0.24–0.28, and 92.89–94.40, respectively. The highest Ne, I, He, and PP were registered by population 1. Population 1 had the greatest Fst value (0.44). The gene flow (GF) between the two populations was 2.30 based on the PhiPT value (Table 4). According to the CDDP data, population 3 had the highest Ne (1.55), I (0.48), He (0.32), and PP (93.89%) values. These populations likewise had a high Fst value (0.30), which was followed by population 1. Based on the PhiPT value, the gene flow (GF) between the two populations was 3.04 (Table 4).
Table 3
Analysis of molecular variance (AMOVA) in melon populations using URP + IRAP, SRAP, and CDDP data.
| URP + IRAP markers |
Source | df | SS | MS | Est. Var. | Var (%) |
Among Pops | 1.00 | 59.37 | 59.37 | 1.32 | 5.64 |
Within Pops | 55.00 | 1209.46 | 21.99 | 21.99 | 94.36 |
Total | 56.00 | 1268.82 | | 23.31 | 100.00 |
PhiPT | 0.056** | | | | |
P-value | 0.001 | | | | |
| SRAP markers |
Source | df | SS | MS | Est. Var. | Var (%) |
Among Pops | 1.00 | 69.74 | 69.74 | 1.85 | 9.80 |
Within Pops | 55.00 | 936.59 | 17.03 | 17.03 | 90.20 |
Total | 56.00 | 1006.33 | | 18.88 | 100.00 |
PhiPT | 0.098** | | | | |
P-value | 0.001 | | | | |
| CDDP markers |
Source | df | SS | MS | Est. Var. | Var (%) |
Among Pops | 2.00 | 102.91 | 51.46 | 1.67 | 7.55 |
Within Pops | 54.00 | 1101.95 | 20.41 | 20.41 | 92.45 |
Total | 56.00 | 1204.86 | | 22.07 | 100.00 |
PhiPT | 0.076** | | | | |
P-value | 0.001 | | | | |
Df: degree of freedom; SS: sum of squared observations; MS: mean of the squared observations; Est Var: estimated variance; Var: variance; PhiPT: proportion of the total genetic variance among the individuals within a population; p-value: probability value.
Table 4
Diversity indices, genetic differentiation, and gene flow detected in melon populations based on the data of URP + IRAP, SRAP, and CDDP data.
| URP + IRAP markers |
Population | N | Ne | I | He | PP | Fst | GF |
Pop-1 | 27.00 | 1.43 | 0.40 | 0.26 | 90.21 | 0.26 | 4.21 |
Pop-2 | 30.00 | 1.42 | 0.41 | 0.25 | 95.80 | 0.25 |
Mean | 28.50 | 1.42 | 0.40 | 0.26 | 93.01 | 0.26 | |
| SRAP marker |
Population | N | Ne | I | He | PP | Fst | GF |
Pop-1 | 29.00 | 1.46 | 0.42 | 0.28 | 94.40 | 0.44 | 2.30 |
Pop-2 | 28.00 | 1.40 | 0.38 | 0.24 | 92.80 | 0.28 |
Mean | 28.50 | 1.43 | 0.40 | 0.26 | 93.60 | 0.36 | |
| CDDP marker |
Population | N | Ne | I | He | PP | Fst | GF |
Pop-1 | 15.00 | 1.53 | 0.46 | 0.31 | 88.55 | 0.30 | 3.04 |
Pop-2 | 18.00 | 1.51 | 0.45 | 0.30 | 88.55 | 0.25 |
Pop-3 | 24.00 | 1.55 | 0.48 | 0.32 | 93.89 | 0.30 |
Mean | 19.00 | 1.53 | 0.46 | 0.31 | 90.33 | 0.28 | |
N: number of accessions; Ne: number of effective alleles; I: Shannon’s information index; He: expected heterozygosity or gene diversity; PP: percentage of polymorphism, populations; Fst: fixation index; GF: gene flow; Pop: population.