DIVERSITY AND GENETIC STRUCTURE OF NATURAL POPULATIONS OF ARAÇÁ ( Psidium guineense Sw . ) 1

ABSTRAT Psidium guineense Sw, popularly known as araçá, is a fruit tree there is widely distributed in Brazil and belongs to the Myrtaceae family. In northeastern Brazil, araçá occurs along coast and in the Zona da Mata; its fruit looks like guava but is more acidic and has a stronger smell. There is a little information about this species, which increases the difficulty of conserving its genetic resources and exploiting araçá as an economic resource. The objective of this research was the evaluation of the genetic diversity and genetic structure of P. guineense from Pernambuco’s Zona da Mata. One hundred and fourteen individuals and 18 isozyme loci were evaluated, showing 28 alleles. The percentage of polymorphic loci ( ) and the average number of alleles per locus ( ) were 0.5 and 1.5, respectively, in this population. The expected heterozygosity , which corresponds to the genetic diversity, ranged from 0.22 to 0.23, a high value when considering that isozymes mark access from the functional genome. The differentiation index among the population was ( ) = 0.015; therefore, the populations were not different among the sampled places. The inbreeding values


INTRODUCTION
Psidium guineense SW., popularly known as araçá, belongs to the Myrtaceae family, which includes approximately 100 genera and 3000 species, including trees and shrubs, and is distributed on all continents except Antarctica; it occurs more often in tropical and subtropical regions (PEREIRA;NACHTIGAL, 2003).
This specie is originally from South America (BRANDÃO et al., 2002), and in Brazil occurs spontaneously from the Rio Grande do Sul to the Amazon, mainly in coastal areas (BEZERRA et al., 2006).In northeastern Brazil, P. guineense occurs along the seaside and Zona da Mata, principally in coastal board areas (DEMATTÊ, 1997;BALOCH et al., 2006) and the Semi-arid region of Bahia (QUEIROZ, 2011;SANTOS et al., 2014).
P. guineense is a shrub with stature from 2.0 to 2.5 m and stem with smooth bark that peels off.Their fruits are similar to those of the guava but are smaller, more acidic, stronger smelling, globular (sometimes ovoid), pedunculated, and clear-yellow when ripe with a cream-colored pulp and many seeds (GONZÁLEZ et al., 2005).P. guineense fruit is accepted by consumers, who appreciate its exotic flavor and high C vitamin content (RASEIRA and RASEIRA, 1996); however, exploration performed through the extractivism (BEZERRA et al., 2006).
P. guineense has great importance as a genetic resource to be exploited directly in the prebreeding process or as a source of genes to transfer to the guava, Psidium guajava L., by conventional crossing.Nematode gall-resistant genotypes of P. guineense reportedly showed promise as grafts to guava (CASTRO et al., 2012;MARTINS et al., 2013).
The conserved genetic resources of P. guineense are little, especially considering the wide geographic distribution of the species.The accessions of this species are maintained in collections with other species of the Psidium genus, including cultivated species of P. guajava, according to Bezerra (2006), Santos et al. (2008), and Queiroz (2011).There is little information about the diversity and genetic structure of natural populations of P. guineense; however, severe genetic erosion is assumed due to the devastation of ecosystems where the species occurs, as registered with other fruit native species.
Therefore, studies of the diversity and genetic structure of natural populations are very important because they could provide fundamental parameters for pre-breeding and define conservation strategies, especially those that guarantee the continuity of the evolutionary process of populations over time (RAU;HODGKIM, 2002).
The objective of this work was to study the diversity and genetic structure of four populations of araçá in Zona da Mata region of Pernambuco State using isoenzymatic markers, in order to provide information for use in conservation and pre-breeding programs.

Sampling
Four natural populations of P. guineense, named Itamaracá, Marieta, Arariba, and Palmares, located in the municipalities of Itamaracá, Moreno, Escada, and Palmares, respectively, corresponding to the coastal lowland of the State of Pernambuco, were studied (Table 1).The Itamaracá population occurred in clearings of the remnant vegetation of the Atlantic Forest and the individuals showed different ages.The other three populations occurred in areas where sugar cane was previously cultivated, where soil conditions seemed to favor the development of the plants.The plants seemed to have similar ages, and according to reports from local people, the populations were established for around seven to 11 years.
Random sampling was conducted to accurately represent plant density and population sizes.The young leaves of sampled individuals were collected, placed in plastic bags, kept in an ice cooler, and carried to the Genetics Population Laboratory of the Biology Department of the Federal Rural University of Pernambuco, where they were stored in at -80⁰C until isoenzyme extraction. 1 n* = number of individuals sampled Twenty-eight to 30 individuals in each population were sampled, according to the abundance of plants and population size, in the period of January to April 2013.The minimum distance between sampled individuals was 50 meters to minimize the probability of collecting sister plants.Each tree was identified by a numbered aluminum label and georeferenced using a Global Positioning System (GPS) to facilitate if necessary, resampling or the collection of propagative parts.

Extraction and revelation of enzymes
The enzymes were extracted using the buffer no. 1 of Alfenas et al. (1998), and PVP40 (Polyvinylpyrrolidone) was added during leaf maceration.The obtained samples were stored at -80⁰C until the isozymes were separated by horizontal electrophoresis on 13% starch gels, according to the method of Alfenas et al. (1998).The gel/electrode buffer systems used were: TCP (Tris-Borate, Citrate, pH 7.5) and LB (Lithium Borate, pH 8.5).

Data analysis
Zymograms were interpreted using allele frequencies and diversity indices, such as: percentage of polymorphic loci ( ), estimated by the ratio of the average of number of polymorphic loci and the total number of loci, considering loci whose frequency of the most common polymorphic alleles did not exceed 95%; average number of alleles by locus ( ), obtained by dividing the total number of alleles by the total number of loci; observed heterozygosity ( ), which was obtained by the equation , where = frequency of homozygous genotypes; expected heterozygosity ( ), obtained by the equation , where = allele frequency estimated the ith allele; and index ( ), estimated by the equation .The effective size (N e ) was estimated as reported by Vencovsky (1992), i.e., , where n is the number of sampled plants and is the population inbreeding coefficient.
The estimated parameters of the genetic structure of the population were: unbiased genetic divergence among populations (F ST ), according to Nei (1978); genetic identity (GI); and estimates of the gene flow among populations, according to the method of Crow and Aoki (1984), which followed the equation: , where , and N m = number of migrants per generation, n the number of populations, and F ST is genetic divergence among populations.The coancestry coefficients were estimated using the method of Cockerham (1969), i.e., using confidence intervals with 95% probability and the bootstrap resampling method with 10000 repetitions per locus.All analyses were performed using the GDA software program (LEWIS; ZAYKIN, 2000).

RESULTS AND DISCUSSION
From the 18 isoenzymatic systems tested, nine revealed band patterns on all individuals of the four studied populations, which were used for analysis (Table 2).The nine systems showed 18 loci and 28 alleles.The ACP1, EST1, GOT1, GOT2, PGI1, SKDH1, 6-PGDH1, and AKP1 loci revealed only one allele that was present in the four studied populations.The other loci (LAP1, LAP2, ACP2, EST2, PGI2, 6PGDH2, AKP2, ASK2, ADH1, and ADH2) revealed two alleles each, with frequencies ranging from 0.017 to 1,000, showing variation among and within populations.
Changes in allele frequencies in finite populations can indicate genetic drift resulting from gamete sampling in each generation (HEDRINK, 2005).Analyses of allele frequencies are of great importance because they reflect the stochastic effects better than most of the other parameters used in studies of population genetics (BOTREL and CARVALHO, 2004).In the Arariba, Marieta, and Palmares populations, the changes in allele frequencies may be more associated with genetic drift because the populations are new, formed after the interruption of sugar cane cultivation in areas of steep topography; in contrast, in the Itamaracá population, the glades are open and booked sporadically because of anthropic action and due to the regeneration of the vegetation by secondary and tertiary plants that restrict the permanence of P. guineense, and therefore young plants are the most prevalent.

Table 2 .
Frequency of alleles observed at each locus in the four populations of araçá (P.guineense) studied.

Table 4 .
Elltsrad (2003)y (GI), estimated genetic differentiation (F ST ) according toNei (1978), geographical distance, and gene flow (Nm) for each pair of population of araçá (P.guineense) in the Zona da Mata of Pernambuco.ST ) and genetic identity (GI) in pairs of populations (Table4) showed that there is no difference between populations.Indirect estimates of gene flow (Nm) showed values that ranged from 3.23 to 20.77 (Table4), considered high and sufficient to avoid differentiation among populations; according toElltsrad (2003), values higher than 1.0 prevent genetic differentiation among the four studied populations.These results indicate that all the studied populations are representatives to ex situ or in situ conservation, as well as to perform in situ conservation because the four have the same representative genetic potential.

Table 5 .
Average inbreeding for each locus in all populations ( ), average of inbreeding for each locus within populations ( ), and coefficient of coancestrality ( ) in four natural populations of P. guineense on 18 allozyme loci.