Assessment of genetic diversity
Figure 1 shows the results of the AFLP profile of the 39 Date palm genotypes obtained with the primer combination E-ACT X M-CG. A total of 360 bands were generated by six primer combinations, with a mean number of 60 amplicons per primer combination. The number of polymorphic amplicons were 127 (34.35%), and the one polymorphic bands per primer varied between 4 for E-AT x M-GT to 34 for E-AG x M-CG with an average of 22 fragments primer of 22 (Table 2). The maximum genetic similarity (Jc = 0.931) was between accessions ABD2 and ABD3, which were collected from the same experimental plot, CRRAO Degache in Tunisia, noting that significant morphological similarities have also been recorded between these two cultivars, mainly concerning the characters of the inflorescence. On the other hand, the minimum genetic similarity (Jc = 0.161) was found between accessions P171 collected from the experimental plot of CRRAO, Tunisia, and PD5 collected from Madhya Pradesh state, India. The mean genetic distance between the germplasm was 0.568, suggesting a very high degree of genetic diversity between the Tunisian and Indian accessions.
Marker attributes
The EMR, PIC, MI, and RP with polymorphic bands were calculated to evaluate the discriminatory power of each AFLP primer combination. The EMR value ranged from 4 (E-AT x M-GT) to 34 (E-AG x M-CG), PIC values ranged from 0.31 (E-AG x M-CG and E-AT X M-GT) to 0.43 (E-AG x M-GA) and MI value from 1.23 (E-AT x M-GT) to 10.90 (E-ACT x M-GA). RP values varied from 1.59 (E-AT x M-GT) to 16.15 (E-ACT x M-CG). The mean values for all marker attributes like effective multiplex ratio, polymorphic information content, marker index, and resolving power were 21.16, 0.36, 7.28, and 10.91, respectively (Table 2).
Genetic relationships
The accession cluster analysis was performed using the neighbor-joining clustering technique, and based on data obtained from AFLP markers, all germplasm accessions were color-coded based on the sex of the plant in the dendrogram (Fig. 2). based on the genotypic data, five major clusters were identified. Cluster 1 showed the largest group, which includes fifteen genotypes from Tunisia, composed of three sub-clusters. The first sub-group comprised two females, "Menaker" and "Ammari," and four males, P169, PS2, P8, and P165. The second sub-cluster was formed only by the female cultivar Gondi. Finally, the last sub-cluster was itself divided into two subgroups, and the first was composed of three male cultivars, P90, P105, and P106. The second was formed female cultivar "Deglet Nour" and five pollinators by female cultivar "Deglet Nour" and four pollinators, P7, P13, P175, and P4. Eleven are pollinizers out of fifteen samples in cluster one. Cluster 2 is the smallest cluster which only includes three cultivars, "Tezerzit Safra," "Mejhool," and "Lagou," all are female. Cluster 3 includes seven samples which divide into three clades, the first and second sub-cluster were composed only of P197 and "Zohdi," respectively. The third clade includes four female cultivars, "Khssab," "Rhimiya," Khadraoui," and "Boumaan," and one pollinizer P202. Cluster 4 includes eight cultivars, and all are pollinizers; this group was divided into three subgroups, P195 and P171 formed the first clade, the second subgroup was composed of ABD1, ABD2, and ABD3, whereas the third clade was formed by 162 and 174. Cluster five is very diverse (outlier) from other clusters, including all accessions from India PD1, PD2, PD3, PD4, PD5, and PD6. Indian date palm accessions are grouped into a separate cluster from Tunisian germplasm, whereas one of the Tunisian cultivars, P120 found to be closely related to Indian germplasm. In order to confirm this conclusion, the results of pairwise similarity indices and dendrograms constructed for different cultivars from the AFLP were analyzed by multivariate PCA, and the results obtained are summarized in (Fig. 3). The first three axes contributed 41.46% of the total variability. Considering the distribution of cultivars according to the PCA (axis 1-axis 2-axis 3), four clusters were obtained, similar to those reported in the UPGMA dendrogram. However, this confirmed the significant divergence of Indian cultivars from others. In addition, this observed grouping is achieved depending on the geographical origin of the cultivars studied. This distribution shows that the female foreign varieties introduced diverged significantly from the native affiliations. Likewise, the male trees are grouped in the same subgroup and differ significantly from the female trees (Fig. 3). The projection of the cultivars on the Cartesian plane formed by the three axes X, Y, and Z, representing 39.07% of the total variability, allowed us to distinguish 3 large groups, A, B, and C (Fig. 3). Group A defines the X axis, which is made up of nine cultivars and which is subdivided into two subgroups; the first subgroup is formed only by Indian cultivars and, which correlate negatively with this axis, the second subgroup is formed by the Tunisian pollinators P90, P171 and P197 belonging to the plot of Tozeur. Group B defines the Y axis, which is divided into two clusters; the first cluster is composed of the cultivars which negatively correlate with this axis Mnaker, Ammari, P8, P120, P162, and P174, the second cluster is formed by the cultivars indigenous females, Tazerzitsafra, Rhimiya and introduced Medjhol, Khsab, Khadraoui, Boumaan, Zohdi and the pollinator P202. The second cluster is composed only of the autochthonous cultivars that positively define this axis, Mnaker Ammari, P8, P120, P162, and P174. Group C is defined by the Z axis and subdivided into two subgroups; the first comprises the cultivars Deglet Nour, Gondi, P4, P7, P13, P105, P106, and P175, which correlate negatively with this axis. However, the second has only pollinators, P165, P169, PS2,ABD1, ABD2,ABD3 and P196 which positively define the Z axis.
AFLP data showed a significant genetic difference (P = 0.001) among the studied populations, and the total genetic diversity was 52%. Pairwise comparisons of populations (Fig. 4) showed that the Tunisian population and the Indian one were significantly different, and the PhiPT was 0.518 (P = 0.001).
Heat map graphic
The heat map (Fig. 5) identified 10 AFLP loci specific to Indian cultivars, which are defined as follows: rr112, rr118, rr7, rr110, rr111, rr95, rr103, rr68, rr77, and rr36. 7 specific AFLP markers identify Tunisian cultivars: rr107, rr114, rr21, rr55, rr105, rr34, and rr41. The other markers were common to all the cultivars analyzed relatively. On the other hand, no specific sex-linked markers were defined. However, rr99, rr98, and rr29 bands were specific to PD5, PD6, and Khadrawi cultivars.