2.1. SSR Polymorphism and Genetic Diversity
All the SSR markers used in this study showed correct amplification and turned out to be polymorphic in the analysis of 115 accessions of interspecific plum × apricot hybrids and 27 reference genotypes. The genetic profiles of each accession obtained with the eight SSR markers are included in
Table S1 of the Supplementary Materials. The parameters of SSR genetic diversity are summarized in
Table 1. A total of 149 alleles were detected in the whole population, ranging from 12 (CPPCT033) to 21 (BBPCT007 and UDP96005) alleles per locus (
NA), with an average value of 19. These values were similar to those reported in previous works in apricot (48 accessions, 20 SSR,
NA = 4.1 [
24]; 890 accessions, 25 SSR,
NA = 24.36 [
25]) and Japanese plum (47 accessions, 8 SSR,
NA = 13 [
22]). However, the values reported herein were higher than those obtained in a previous study of commercial cultivars of apricot (202 accessions, 10 SSR,
NA = 6.3 in recent releases and
NA = 9.3 in commercial releases), which can be related to the genetic bottleneck as a consequence of the use of common parents in breeding programs of apricot [
26]. The smaller allele was obtained with the SSR marker UDP6005 (95 bp) and the larger one with CPSCT005 (229 bp). These results confirmed the high transferability among
Prunus species of the eight SSR markers used in this study and previously observed in other works [
15,
20,
24,
25] and the utility of these SSR markers in the interspecific plum × apricot hybrids and progenies.
The polymorphic information content (PIC) ranged from 0.61 (CPPCT033) to 0.90 (CPSCT005), with a mean value of 0.81; therefore, the alleles were considered to be highly polymorphic, showing their usefulness for the study of genetic diversity [
27]. The observed heterozygosity values (Ho) ranged from 0.48 (BPPCT039) to 0.85 (pchgms2), with an average value of 0.70, whereas the expected heterozygosity (He) ranged from 0.58 (CPPCT033) to 0.87 (CPSCT005), with a mean value of 0.76. These values were similar to those observed in several plum species such as
P. salicina (
Ho = 0.71;
He = 0.67),
P. domestica (
Ho = 0.73;
He = 0.69), and
P. insititia (
Ho = 0.74;
He = 0.70) [
28], indicating that the number of accessions analyzed in this work is a representative sample of the current genetic variability. The heterozygosity (
Ho and
He) of the analyzed accessions was lower than that observed in apricot cultivars, a crop in which a decrease in genetic diversity has been observed [
26]. The
FIS values ranged from −0.13 (CPPCT033) to 0.37 (CPSCT026), with a mean of 0.08, indicating that there is no inbreeding in the whole population since values were observed close to zero. The
FST ranged from 0.06 (BPPCT007) to 0.29 (BPPCT039), with a mean value of 0.13, showing low genetic differentiation due to the gene flow between accessions [
29] (
Table 1).
2.2. Genetic Relationships by UPGMA
The analysis for the detection of homonyms and synonyms allowed the identification of 141 unique genotypes and 2 synonyms: ‘IBG047’ and ‘IBG057’, both accessions from the Ibergen breeding program, which were grouped in the subgroup B6.
The genetic relationships between the interspecific hybrids and the reference genotypes were assessed using the unweighted pair group method based on arithmetic averages (UPGMA) to generate a dendrogram based on the Nei and Li similarity index. The dendrogram showed the first node with a high bootstrap value (100), separating the accessions into two groups: group A, formed by 12 accessions (8.5%); and group B, formed by 130 accessions (91.5%) (
Figure 1a). These two groups were divided into several internal secondary nodes.
Group A was divided into two subgroups. The subgroup A1 was formed by the ten reference genotypes of apricot. In subgroup A2, two advanced selections from different breeding programs were grouped, ‘IBG024’ and ‘Z029’, which could be aprium hybrids since they showed a greater genetic relationship with the apricot reference genotypes than with those of plum [
12].
Group B, the larger of the two groups, was made up of the rest of the interspecific hybrids and the reference genotypes of diploid plums (
P. cerasifera,
P. salicina, and
P. simonii). Plumcots, pluots, and the reference genotypes were allocated into different groups, mixing and grouping mainly according to their genealogical origin. This grouping trend has been found between pluots, plumcots, plums, and apricots in a previous study, suggesting that the grouping is mainly due to the relationship between the accessions and their parents [
20].
Subgroup B1 was separated a greater distance from the rest, grouping five Zaiger Genetics accessions. Two of them were reference genotypes: ‘Honey Top’, a nectarine (
P. persica) with yellow flesh [
30]; and ‘Honey Sun’, an apricot × peach hybrid [
12]. The accessions ‘Honey Queen’, a yellow-fleshed nectarine, and ‘Bella Gold’, a peacotum [
12], might have in their pedigree some genotype of
P. persica. Subgroup B2 was a small but diverse group, grouping the reference genotypes ‘Kelsey’ (
P. salicina), ‘Mitard’ (
P. cerasifera), and ‘Songold’ (‘Golden King’ (unknown origin) × ‘Wickson’ (
P. salicina)). ‘Songold’ could be a hybrid between
P. salicina and
P. cerasifera since it is grouped close to ‘Mitard’ (
P. cerasifera). ‘Songold’ was developed by ARC-Infruitec, a public breeding program of South Africa that frequently uses ‘Methley’ (
P. salicina ×
P. cerasifera) as a parent [
31]. In subgroup B3, ‘Methley’ was grouped with ‘Angeleno’ (‘Queen Ann’ × Unknown), ‘Abundance’ (
P. salicina), and the pluots of Zaiger Genetics: ‘Flavor Fusion’, ‘Flavor Finale’, and ‘Flavor Grenade’. These accessions were located near to ‘Red Beaut’ (‘Burmosa’ × ‘Eldorado’) [
31], which was allocated in subgroup B4 with ‘Simon’ (
P. simonii). Subgroup B5 contained some commercial cultivars from Zaiger Genetics. In subgroup B6, 16 accessions were allocated, including the reference genotype ‘Sweet Treat’ (pluerry).
‘Red Beaut’ is one of the cultivars most used by Zaiger Genetics as a parent [
32], which could explain why Zaiger accessions were allocated in the same group B to which ‘Red Beaut’ and other cultivars usually used as parents such as ‘Queen Ann’ (‘Gaviota’ × ‘Eldorado’) and ‘Mariposa’ (
P. salicina) [
33,
34,
35]. ‘Queen Ann’ and ‘Mariposa’ were grouped in subgroup B7 with three pluots (‘Emerald Drop’, ‘Fall Fiesta’, and ‘Flavor King’). The subgroup B8 was the most numerous of all (
n = 35), including four reference genotypes (‘Dapple Jack’, ‘Queen Rosa’, ‘Santa Rosa’, and ‘Splash’) and eight commercial cultivars. The genealogy of ‘Glory Red’ includes ‘Queen Ann’ [
32], which was used as a parent of ‘Queen Rosa’ in a cross with ‘Santa Rosa’ [
31]. The three cultivars ‘Glory Red’, ‘Queen Rosa’, and ‘Santa Rosa’ could therefore be genetically related. The pluot cultivars ‘Splash’ and ‘Dapple Jack’ were also genetically related, as ‘Splash’ is the ancestor of ‘Dapple Jack’ [
32]. Finally, six accessions were included in subgroup B9. Subgroups B6 and B9 were made up of only interspecific hybrids, which possibly shared parents among them.
The advanced selections were allocated in the subgroups A2 and B1–B9, showing the same diversity observed among the commercial cultivars.
2.3. Analysis of Population Structure by DAPC
To establish the pattern of the population structure, discriminant analysis of principal components (DAPC) was performed. Despite the high degree of introgression in this type of interspecific hybrids [
2,
7,
12,
34,
36], the DAPC analysis showed the formation of five groups (
K = 5) according to the lowest BIC value (166.17) (
Figure 2A). The cross-validation of the DAPC showed that the proportion of success for the prediction of the groups (
K = 5) would be obtained with 25 principal components (PCs) (
Figure 2b), so 25 PCs (
Figure 3—inset of PCA eigenvalues) and four eigenvalues of the discriminant analysis functions (DA) (
Figure 3—inset of DA eigenvalues) were retained for the DAPC analysis.
In the scatterplot of the DAPC analysis (
Figure 3), groups G2, G3, and G4 overlapped near the intersection of the first two linear discriminants (LD1 and LD2). Groups G1 and G5 differed from the rest of the groups through LD2 and LD1, respectively. The membership probabilities of each accession belonging to its assigned group are shown in
Figure 1b and were based on the retained discriminant functions of the DAPC analysis. The stacked bars indicate the proportions of successful reassignment of accessions to their original groups. This grouping corresponded to the UPGMA dendrogram (
Figure 1a), indicating that the accessions were grouped according to their genealogical origin.
The accessions were allocated into five groups (
K = 5) (
Table S2 of the Supplementary Materials). Group G1 contained 13 accessions (9% of the total population), including three Zaiger Genetics commercial cultivars (‘Bella Gold’, ‘Flavor Fall’, and ‘Honey Queen’). In group G2 (
n = 38, 27%), the reference genotypes ‘Dapple Jack’ (pluot) and ‘Mariposa’ (
P. salicina) were grouped with ‘Queen Rosa’ and’ Santa Rosa’, which are considered simple hybrids of
P. salicina [
2]. Group G3 (
n = 32, 23%) included reference genotypes of different species, such as ‘Abundance’ and ‘Kelsey’ (
P. salicina), ‘Honey Sun’ and ‘Honey Top’ (
P. persica), and ‘Mitard’ (
P. ceracifera), and some hybrids of
P. salicina, such as ‘Angeleno’, ‘Methley’ (
P. salicina ×
P. cerasifera), and ‘Red Beaut’. Group G4 included the largest number of accessions (
n = 49, 35%), including four reference genotypes: ‘Queen Ann’, ‘Simon’ (
P. simonii), ‘Songold’, and ‘Sweet Treat’ (pluerry). Finally, group G5 was formed exclusively by the 10 reference genotypes of
P. armeniaca (apricot).
The composition of groups G1, G2, G3, and G4 was homogeneous, probably due to their accessions being obtained from the selection of crosses and backcrosses in which common parents such as ‘Mariposa’, ‘Queen Ann’, ‘Queen Rosa’, ‘Friar’, and ‘Red Beaut’ were used [
20,
31,
37].
2.4. Genetic Diversity among Groups by AMOVA
The analysis of molecular variance (AMOVA) on genetic differentiation among the five groups based on DAPC and within accessions revealed that 80% of the total variation in the genetic structure (
K = 5) was attributed to the variability within accessions with significant differences (
p < 0.01) (
Table 2), a percentage similar to that observed in a population of Japanese plums (81.8%) in a previous study [
15]. The variance among the five groups inferred with the DAPC analysis represented 11% of the total, and the variance among accessions within the five inferred groups represented the remaining 9%. Previous studies on apricot [
25] and almond [
38] reported that the variance between groups contributed 8 and 29% of the total variance, respectively, being much lower than the variance due to differences between the accessions, which corresponds with the results obtained in this work.
The parameters of genetic diversity were calculated for each of the five groups (
K = 5) (
Table 3). The number of alleles per locus (
NA PER LOCUS) ranged from 6 (G5) to 12 (G3). The total number of alleles for each group ranged from 49 (G5) to 95 (G3). The same trend was observed in allelic richness (
AR), with values between 6.25 (G5) and 8.14 (G3). Alleles observed in only one group were considered private alleles (
PA), with the smallest value (4) in group G2 and the largest value (24) in group G5. These results showed moderate and relatively homogeneous levels of genetic diversity, except in group G3, which showed a greater number of alleles (
NA PER LOCUS and
NA TOTAL), greater allelic richness (
AR), and a value of the coefficient of inbreeding notably higher than the rest (
FIS = 0.21), revealing a heterozygosity deficit, as observed in a diverse group of apricots from different geographical origins [
25]. The values of observed heterozygosity (
Ho) ranged from 0.61 (G2) to 0.75 (G1 and G5), and the expected heterozygosity (
He), from 0.67 (G2) to 0.86 (G3).
Ho was slightly lower than
He in groups G1, G2, G3, and G4, which could be attributed to the exhaustive breeding activity developed in these hybrids. The heterozygosity (
Ho and
He) of G5, which includes the apricot cultivars, was similar to that observed in a previous work including traditional and new apricot cultivars [
26].
To validate the genetic differentiation between groups, the correlations of pairwise genetic differentiation values (
FST) were determined (
Figure 4). The mean value observed was 0.16 and ranged from 0.05 (between G3 and G4) to 0.28 (between G2 and G5), indicating a moderate differentiation between groups. The correlations between group G5, which was formed entirely by apricot cultivars, and the rest of the groups showed the highest values, revealing a restricted flow of genes from apricot cultivars towards the 132 accessions that were grouped in G1 to G4 (diploid plums, plumcots, pluots, and other hybrids). A moderate but significant genetic differentiation was observed in G1, G2, G3, and G4, except the correlation between groups G3 and G4, which showed slight genetic differentiation. All the correlations presented lower and upper limits different from zero within a 99% confidence interval (
Figure 4).
Since their introduction at the beginning of the 20th century, the interspecific plum × apricot hybrids have generated controversy due to the lack of clarity in classifying them as plum, plumcot, pluot, or aprium for their commercialization, due to the similarity in the appearance of their fruits, as well as the difficulty in determining the quality standards that must be applied [
7]. The DAPC analysis used to assess the population structure revealed the five groups in which the whole population was structured. This structure is related with the genealogical background of the accessions, which can be useful for inferring the real interspecific status of the complex interspecific plum × apricot hybrids analyzed. The ability to distinguish these hybrids is not only important to sellers and consumers but also to breeders [
8] and producers [
20], due to the different agronomic management. In certain markets, such as the USA, the classification as plums or pluots can greatly affect the price of the fruit [
9].
The four inferred groups including the interspecific hybrids showed a weaker genetic relationship to the apricot group than would be expected if they were simple plum × apricot hybrids, as suggested by the terms ‘plumcot’, ‘pluot’, and ‘aprium’ [
12]. A closer genetic relationship of pluots to Japanese plums than to apricots has also been found in a previous study [
20]. Previous analysis of the genetic structure and diversity between interspecific hybrids is limited to a study that included 29 Japanese plum cultivars, 4 interspecific hybrids, 2 cultivars of
P. domestica, a cultivar of
P. cerasifera, and another of
P. armeniaca, which showed a low genetic differentiation between the determined population structure [
35]. The unexpected distance found between interspecific hybrids and apricot cultivars may be due to several backcrosses, or new crosses with plum cultivars, of the first descendants of simple plum × apricot hybrids, to search for fruits more similar to glabrous-skinned plums than to apricots [
13].
Although many species are involved in the genealogy of the interspecific hybrids, our results suggest that the observed diversity is lower than expected, probably due to the use of the same parents, such as ‘Friar’, ‘Mariposa’, ‘Queen Ann’, ‘Queen Rosa’, and ‘Red Beaut’, in different breeding programs [
32]. In addition, the Japanese plum cultivars used as parents are complex hybrids that come directly or indirectly from the first hybridizations carried out by Luther Burbank, who used as parents a small number of Japanese plum cultivars (‘Abundance’, ‘Burbank’, ‘Kelsey’, and ‘Satsuma’) [
4], other diploid plums (‘Maritima’ (
P. maritima), ‘Simon’ (
P. simonii), and ‘Robinson’ (
P. munsoniana)), and the first simple hybrids (‘Gaviota’, ‘Santa Rosa’, and ‘Wickson’) [
5]. In previous works, low percentages of fruit set have been obtained in plum × apricot crosses, and very low or even null in apricot × plum crosses, which shows the difficulty of obtaining these hybrids [
10,
11]. This situation may have caused some of the hybrids to have been erroneously considered as plumcots, pluots, or apriums, being Japanese plum-type hybrids. However, it is difficult to determine the genealogy of the interspecific hybrids, since in most cases the parents are unknown.