In A. fraxinifolium transcriptome data, a predominance of -tri, followed by dinucleotides motifs, with more than 79% of all identified contigs, which could not affect the protein structure, with non-perturbation of the reading frame [9, 10]. When analyzing all repeats number, we identified that repeats number greater than 10 corresponds with less than 7.8% of all SSRs (Table 4). The SSR frequency decreased with an increase in motif length, as reported for Magnolia wufengensis [11]. The frequency of motifs from AG/CT corresponds to more than 24.3%, being the most abundant motif in this species, followed by TCT/AGA repeats with less than 6%. (Fig. 3). These frequencies of AG/CT repeats are higher than found for other species such bamboo (17.11%) [12], but less than for Magnolia (37.8%) [11]. High frequencies of AG repeats are also reported for other plant species being suggested that could be related to mutation mechanism of generation of SSRs or selective pressure to particular sequences [9, 10, 11, 13, 14].
Table 4
Frequency distribution of SSRs identified in the A. fraxinifolium transcriptome.
Repeat number | Di- | Tri- | Tetra- | Penta- | Hexa- | Total |
4 | N/A | N/A | N/A | 6685 | 11008 | 17693 |
5 | N/A | N/A | 3003 | 1740 | 1644 | 6387 |
6 | N/A | 33155 | 2139 | 365 | 466 | 36125 |
7 | 13340 | 15352 | 217 | 56 | 55 | 29020 |
8 | 6167 | 7539 | 52 | 3 | 20 | 13781 |
9 | 5489 | 2413 | 10 | 2 | 4 | 7918 |
10 | 3511 | 1013 | 4 | 0 | 2 | 4530 |
> 10 | 8882 | 859 | 9 | 1 | 3 | 9754 |
Total | 37389 | 60331 | 5434 | 8852 | 13202 | 125208 |
Insert Table 4
Insert Fig. 3
Figure 3 Frequency distribution of the most representative SSR motifs types in the A. fraxinifolium transcriptome.
The microsatellite markers developed were efficient in the genetic differentiation among populations sampled. The average levels of heterozygosity observed and expected were above that reported for populations of Astronium graveolens [15] and in other tropical species, as in populations of Cedrela fissilis (Meliaceae) [16], Campomanesia xanthocarpa (Myrtaceae) [17], Myracrodruon urundeuva [18] and Eugenia uniflora L. (Myrtaceae) [19], which confirms the existence of high genetic variability in the populations studied here. Therefore, these genetic markers are reliable to be used in population genetics studies, as such in the investigation of the pollen and seeds dispersal patterns aid to understand the actual distribution of natural populations, with impacts in the evolutionary history of a species. Previous studies with other tree species show a great range of pollen dispersal, such in Hymenaea stignocarpa, showed long-distance pollen dispersal reaching values of more than 8 km between the populations analyzed [20] and even more in Ceiba pentandra¸ reaching 18 km [21, 22]. However, these long distances are mainly related to the dispersion by bats, which have a large feeding area. For A. fraxinifolium, which are pollinated by bees, distances of almost 6 km were reported of the insects feeding behavior [23, 24]. Such results indicate that further investigations of pollen/seed dispersion are necessary for the species. To date, few studies were conducted based in natural populations of A. fraxinifolium, focused on silvicultural traits [25]. Recently in the genus, A. graveolens SSR loci were described, but not tested in other Astronium species [15]. Given this, the microsatellite markers in this work developed may be useful in genetic studies such as diversity and genetic structure, gene flow and mating system, providing information for conservation, breeding and reforestation plans of the species. In addition, our study provides a database with more than 125 thousand of expressed SSR sequences in the genome that will serve as a basis for studies of consequences of forest fragmentation in tropical forest of Brazil, thus contributing to the development of adequate strategies for the conservation of A. fraxinifolium and related species from Anacardiaceae family.