DiscussionTop

Diversity, frequency and distribution of S-alleles

In the present study, 18 different S-alleles were identif ied in three apple populations.

Among the identified S-alleles in this study, S1 and S6 had the highest frequency at 12.3% and 10.5%, respectively. S19 and S23 were the second most abundant S-alleles, with 8.7% of the studied samples. Rahemi et al. (2010) suggested that geographical positions and altitudinal differences of wild almond and other Prunus populations influence the diversity and differentiation at these loci. The S-alleles identified in M. orientalis are consistent with the S-alleles in cultivated M. domestica cultivars, which suggests cross-compatibility between the two species. The diversity maintained by wild populations of M. orientalis might therefore be relevant in future breeding programs to increase the genetic diversity in M. domestica.

Based on Vekemans et al., (1998), approximately 8-10 different S-alleles in each population are sufficient for successful pollination (mate availability index= 1) and production of viable seeds. Accordingly, populations with less than five different S-alleles exhibit successful mating rates lower than 90% (Vekemans et al., 1998).

Loss of genetic diversity and consequent loss of the S-allele in populations, especially in species with small size and patchy distribution, such as wild M. orientalis, can significantly increase the risk of those populations becoming endangered in a short time (Frankham, 2005). In our study, although the number of trees per population was less than 10, at least 8 S-allele types per population were detected. According to the predictions of Gutfitti balanced models (Vekemans et al., 1998; Wright, 1939) and what has been reported about wild populations of wildflowers with the Gutfitti self-incompatibility system in previous studies (e.g., Hoebee et al., 2011; Holderegger et al., 2008), mates were available for more than 90% for the studied populations. Despite the results of other studies that found reduced fertilization success in small populations of SI species (e.g., Fischer et al., 2003; Willi et al., 2005), in line with Vekemans et al., (1998), for the populations under study, more than 80% successful seed germination has been reported (Karimi, 2015).

In fact, the high frequency and diversity of the S-allele, even in small populations, can increase the chance of successful pollination and reduce concern about mate availability (Holderegger et al., 2008; Hoebee et al., 2011). Hence, based on this research, there is no concern about mate availability in Malus orientalis populations and, consequently, in production of viable seeds in Hyrcanian forests. Furthermore, seed germination of these same populations was previously studied by Karimi (2015), who indicated that seed germination was over 80%.

 

Figure 3.(a) Correlation between observed heterozygosity and S-allele frequency with the altitude of the population; (b) He and Ho trends along altitudinal gradients of studied populations; (c) comparison of Shannon index values with SSR marker and S-allele frequency results.

 

Table 2.. Genetic diversity in three Malus orientalis populations based on SSR markers

Ho = observed heterozygosity; He = expected heterozygosity; F = F-values; PPL =Percentage of Polymorphism; AR = Allelic Richness; PAR = Private Allelic Richness

 

Table 3. Analyses of molecular variance (AMOVA) for the studied populations

**- Significant at the 0.01 level. Squared deviations (SS); mean squared deviations (MS), variance component estimates (Est. Var.), the percentage of the total variance contributed by each component and gene flow (Nm).

 

Genetic diversity and structure

Here, we studied the S-alleles and genetic diversity in three populations of M. orientalis in the Hyrcanian forest of Iran. The number of trees included here is too low to give a true description of the actual diversity in a large and diverse forested area. However, it may contribute to an understanding of the diversity in M. orientalis populations of the area, and the S-allele diversity is especially important for the successful reproduction of this species in its natural populations. In this study, allelic richness (AR) showed a slight difference between the lower and upper Asalem populations, which showed AR values of 3.51 and 3.85, respectively, but in the middle Asalem population, the AR (8.28) was much higher. The high density of summer villages and the transfer and cultivation of domestic and improved cultivars for domestic use can be one of the reasons for the high allelic richness of Caucasian apples in the western parts of the Hyrcanian forest.

The observed heterozygosity for populations under study, west of Hyrcanian forest, varied from low (0.22 and 0.39 in lower and middle populations) to medium (0.52 in upper population). The observed heterozygosity showed an increasing trend with altitude (0.22, 0.39, and 0.52), in agreement with results from Goto et al. (2009), which revealed that there is a positive correlation between altitude and genetic diversity parameters, and as altitude increases, the observed heterozygosity increases.

High concentration of human populations, changes in land use and grazing in the low and middle elevations of Hyrcanian forests (Saeei, 1950; Tohidifar et al., 2016) are the main reasons for the sharp decrease in the genetic diversity of most plant species. Additionally, the loss of genetic diversity (gap between Ho and He) can be a consequence of repeated artificial selection of apple trees by indigenous people.

The habitats of the studied populations are under strong pressure from habitat degradation due to human activities (agriculture and urbanization). Land use change is much higher at low elevations than at middle elevations, although the main habitat of M. orientalis is above the timber line (>1800 m a.s.l) of Hyrcanian forest. The relatively low gene flow (Nm = 0.8) and consequently relatively high Fst (0.23) among the studied populations can be attributed to various reasons. A clearly distinct genetic structure was observed among populations under study, especially between high and low populations. An important factor in these findings is no doubt the phenological separation. There is approximately one-month interval between the flowering time of M. orientalis in highland and lowland populations in the Hyrcanian forest (Amirchakhmaghi et al., 2018). The studied populations are separated from each other by approximately 1000 meters in altitude, and their flowering times do not overlap. Thus, as noted by Alberto et al., (2018), the different flowering times of populations can increase their genetic differentiation. The most important bee for pollination of apple is Apis mellifera (Visscher & Seeley, 1982). The effective pollination distance of this bee is a maximum of 6 km (Visscher & Seeley, 1982). Since our study locations were separated horizontally by at least 20 km, no major gene f low among populations was expected.

Additionally, a positive trend was observed between genetic diversity (Ho and AR) and the altitude of the habitat. Similarly, a strong association between altitude and genetic differentiation (Yousefzadeh et al., 2018; Nazarzadeh et al., 2020) or genetic diversity (Mathiasen & Premoli 2013; Shirmohammadli et al., 2018; Meng et al., 2019) has been reported in many published works. Ohsawa & Ide (2008) expressed that altitudinal climatic gradients can influence the distribution of genetic variation within and among plant populations. In fact, knowing the genetic diversity of a plant species across elevation gradients is important to obtain a suitable strategy for conservation (Hahn et al., 2012).

 

Figure 4. Principal coordinate analysis of three populations of Caucasian apple along an altitudinal gradient; (b) Discriminant analysis of principal components (DAPC) with populations used as clusters: 1: AS _L; 2: AS_M; 3: AS_H populations.