APPLICATIONS OF ISSR MARKERS IN STUDIES OF GENETIC DIVERSITY OF Pityrocarpa moniliformis

ABSTRACT Pityrocarpa moniliformis (Benth.) Luckow & R. W. Jobson (Fabaceae) is a native brazilian species with high potential for economic development programs in semiarid regions, mainly related to the production of honey, animal food and firewood. Thus, the objective of this work was to select Inter-Simple Sequence Repeat (ISSR) molecular markers for genetic diversity studies, as well as to test the efficiency of this approach in quantifying the genetic diversity of a natural P. moniliformis population. For this, 28 ISSR molecular markers were tested, evaluating the total number of loci, polymorphism rate and the Polymorphism Information Content (PIC) for the selected primers, the “Marker Index”, and the “Resolving Power”. Genetic diversity parameters (Nei genetic distance and Shannon index) were evaluated for 30 individuals located in Macaíba, Rio Grande do Norte State, Brazil. Seven primers were selected, which provided 74 loci, with 82% being polymorphic, while the PIC value was 0.344. The Nei genetic distance was 0.244, and the Shannon index was 0.374. Therefore, ISSR molecular markers (UBC 827, 840, 844, 857, 859, 860 and 873) are considered efficient in studying the genetic diversity of populations for the selection of matrices and germplasm banks, and may contribute to the conservation and genetic improvement of P. moniliformis populations.


INTRODUCTION
Pityrocarpa moniliformis (Benth.) Luckow & R. W. Jobson (Fabaceae) is a tree species that predominantly occurs in dry forests in Northeast Brazil and is popularly known as catanduva. These native species have social, ecological and economic potential, mainly due to their role in supplying wood for firewood, forage for animal feed, honey production and the recovery of degraded areas (AZERÊDO; PAULA; VALERI, 2011).
The wood from P. moniliformis presents excellent mechanical properties, as it demonstrates high density in relation to other currently commercialized species (NASCIMENTO et al., 2015). It has recently been found that its leaves, bark and fruits present high level of antioxidants, which can be used as phytotherapy in fighting against cancer cells (ALVES et al., 2014), and its seeds have secondary metabolites with larvicidal properties against Aedes aegypti L. vectors, which transmit viral diseases (FARIAS et al., 2010).
There is a great wealth of honey producing plants in the natural areas of this species; P. moniliformis is one of seven in the genus that is frequented by bees and is considered to be a honey plant (ALVES et al., 2014). Thus, it is one of the tree species that contributes most to the honey supply, both in terms of quality and quantity, these being requirements that are important for the consumer market (JESUS et al., 2015).
Therefore, knowledge regarding the genetic variability of natural populations contributes to the development and incorporation of the species into productive systems (COSTA et al., 2011). Additionally, these studies can support conservation and forest improvement programs, aiding the socioeconomic development of farmers and enabling in situ conservation in order to prevent a decline in the natural occurrence of the species (SIMON, 2010).
Molecular markers are widely used to characterize genetic variability (KOUR et al., 2014) among individuals and populations (COSTA et al., 2011), in addition to being used in conservation and forest improvement programs (LORENZONI et al., 2014). Among such markers, we focus on the ISSR (Inter-Simple Sequence Repeat), which are dominant markers used in genetic diversity studies, with low development cost, a high polymorphism rate and high reproducibility (CHEN et al., 2017). In addition, ISSR molecular markers can be used for diverse plant species without the need for genetic sequencing and can be indicated for studies of diversity and genetic mapping of populations.
In the north-eastern semiarid region, recent studies using ISSR molecular markers have been conducted on Copernicia prunifera (Mill) H. E. Moore (FAJARDO et al., 2018) Thus, the objective of this study was to select ISSR molecular markers to be used in genetic diversity studies, as well as to test the efficiency of this approach in quantifying the genetic diversity of a natural P. moniliformis population.

Collection of leaf material and DNA extraction
Leaf samples were collected from 30 naturally occurring P. moniliformis individuals, with the distance between them being two and a half times their height, located near the Forest Experimentation Area (5° 54' 01" S and 35° 21' 28" W, radius of 1000 m) of the Unidade Acadêmica Especializada em Ciências Agrárias (Figure 1), Universidade Federal do Rio Grande do Norte, Macaíba City, Rio Grande do Norte State, Brazil.

DNA amplification with ISSR markers
Twenty-eight (28) ISSR molecular primers (University of British Columbia -UBC) (Table 1) were tested, with the selection criteria consisting of choosing the primers that presented the best amplification standards and loci resolution quality.
Amplification DNA reactions were performed in a thermocycler over a period of 1 h 40 min. The following steps were used: initial denaturation at 94 °C for 2 min, followed by 37 cycles of 15 s at 94 °C for denaturation, 30 sec at 47 °C for annealing the ISSR primers and 1 min at 72 °C for extension, with final extension at 72 °C for 7 min and subsequent cooling at 4 °C.  1 R = purine (A or G) and Y = pirimidine (C or T).

Electrophoresis
After the amplification reaction, 5 μL of the PCR product was stained with 4 μL of bromophenol blue and GelRed TM , and placed in an agarose gel (1.5 w.v -1 ) submitted to electrophoresis on a horizontal system. In this step, a negative PCR control sample was employed without DNA. A 1 KB molecular weight marker (Ladder Kasvi ® ) was used. The agarose gel was then immersed in 1X TAE buffer (Tris-acetate-EDTA) and maintained at 100 V for 2 h 30 min. The electrophoresis product was photographed under ultraviolet light in an E-Box TM VX2.

Data analysis
The total number of loci, total number of polymorphic loci, polymorphism rate and Polymorphism Information Content (PIC) value were evaluated according to the equation proposed by Anderson et al. (1993): in which P ij is the frequency of the allele "j" in the primer "i".
The Marker Index (MI) was calculated as described by Varshney et al. (2007), the Resolving Power (RP) calculated according to Prevost and Wilkinson (1999), and the optimal number of loci following Kruskal (1964), using the Genes program.
The allele frequencies were obtained with the presence (1) and absence (0) of amplified loci in each ISSR primer for the 30 P. moniliformis individuals. The genetic diversity parameters were established using the Nei genetic distance estimation and the Shannon index, as calculated by the Popgene program, version 1.3. The genetic identity dendrogram was produced by the UPGMA grouping method (Unweighted Pair Group Method with Arithmetic Mean), using the NTSYS v.2.11 program.

RESULTS AND DISCUSSION
Among the 28 primers tested, seven were selected (UBC 827,840,844,857,859,860 and 873), which provided a total of 74 loci, ranging from 6 (UBC 844) to 14 loci (UBC 827), with an average of 11 loci per primer ( Table 2). The tested ISSR molecular markers were efficient in DNA amplification and loci identification (Table 1). However, only those with the highest number of amplified loci and good fragment resolution were selected. The implemented markers are considered superior to other molecular techniques due to their low cost, high polymorphism rate and reproducibility, so they are fundamental for genetic studies aimed at the conservation and improvement of forest species (LORENZONI et al., 2014).
ISSR molecular markers were also efficient and suitable for genetic diversity studies of other forest species in semiarid regions of Northeast Brazil, which showed similarities in the mean number of loci found for P. moniliformis, such as E. velutina, in which the formation of 14 loci per primer was observed (GONÇALVES et al., 2014); M. caesalpiniifolia, in which the amplification of 78 loci was observed in seven primers and where the obtained average was 11 loci per primer (ARAÚJO et al., 2016); and M. urundeuva, in which selection seven primers were found, with the formation of 17 loci per primer (LOPES; COSTA; ARRIEL, 2020).
The MI ranged from 1.608 (UBC 860) to 4.875 (UBC 827), with the highest values for the UBC 827, 857 and 859 primers, whereas RP of the loci varied from 4.57 (UBC 840) to 10.50 (UBC 827) ( Table 2). These parameters provide important information to evaluate and assist in comparing ISSR primers (ROSA et al., 2017) and, in this context, the higher these parameters are, the more suitable the primers are for use in genetic diversity studies. PIC values ranged from 0.268 (UBC 860) to 0.423 (UBC 859), with a mean of 0.344 for the seven ISSR primers, which was considered moderately informative (Table 2). Values between 0.25 and 0.50 are considered moderately informative, whereas values higher than 0.50 are considered to be very informative, while those less than 0.25 are not very informative (BOTSTEIN et al., 1980). Similar results were found in M. caesalpiniifolia (0.397) occurring in the brazilian semiarid region (ARAÚJO et al., 2016).
Stress values below 0.05 indicate high precision in the estimates, and r values close to 1.0 suggest a high correlation between the original genetic distance matrix and the simulated genetic distance matrix (KRUSKAL, 1964). Thus, it was possible to estimate enough loci for genetic diversity studies in P. moniliformis, as we found an increase in Pearson correlation values and a reduction of Kruskal stress values (Figure 2). When performing resampling with 53 loci, we obtained 0.045 for stress and 0.935 for correlation and these results confirm that the number of loci obtained in this study (74 loci) was sufficient to estimate the genetic diversity of P. moniliformis. The Nei genetic diversity value for the study population was He = 0.244 ± 0.033, while the Shannon index was I = 0.374 ± 0.046. These values are considered low since they can vary from 0 to 1, with 1 being the maximum genetic diversity which may occur in a population; however, these results are similar to those obtained by Nybom (2004) in studying different molecular markers (RAPD, AFLP and ISSR) to estimate genetic diversity in wild plants (long-lived perennial and outcrossing species). The index is close to that found (using ISSR molecular markers) for populations of other widely-explored species of the semiarid region: C. prunifera Studies of the genetic diversity of trees, either within or between populations, are essential to the establishment of forest breeding programs, in order to capture maximum genetic variability (BALLESTA et al., 2015), for example, the development of active germplasm banks with several genotypes (NING et al., 2017).
From the Nei genetic identity grouping, it may be observed that there is high similarity (values between 0.65 and 0.98) among the P. moniliformis individuals of the study population. Six groups were formed at the cut-off point at 0.76 (Figure 3), in which Group 1 has 12 individuals, Group 2 consists of six individuals, Group 3 has eight individuals and Group 4 has two, while individuals 11 and 5 have each been designated as separate groups because they are genetically more distant from the other trees. The studied population presents a discontinuity of P. moniliformis individuals, a species that naturally tends to occur in clusters. A more significant presence of these individuals was also observed at the edges of the population fragments, which is an indication that the anthropization in the region where the Forest Experimentation Area is located (Macaíba/Rio Grande do Norte State, Brazil) has caused the current discontinuity. Another point to be considered is that there are individuals that are genetically close (Group 1) spread across the entire population ( Figure  1).
The selection of groups of parent trees with higher genetic divergence is a strategy to increase the variability of the species that has several potential uses. This selection aims to capture greater genetic diversity in the collection of seeds to produce seedlings used in the recovery of degraded areas, germplasm banks and the selection of matrices for the formation of seed orchards. Adding seed sources from other populations in the semiarid region of the northeast of Brazil is also an alternative which could be used to increase the genetic variability of the species.

CONCLUSIONS
The ISSR molecular markers (UBC 827,840,844,857,859,860 and 873) are considered to be efficient in genetic diversity studies of populations, selection of matrices and germplasm banks, and may contribute to the conservation and genetic improvement of P. moniliformis populations.