Genetic diversity in sugar apple ( Annona squamosa L . ) by using RAPD markers

A diversidade genética da coleção de 64 acessos de pinheira, coletados em diferentes cidades, no norte do Estado de Minas Gerais, foi avaliada por meio do uso de marcadores RAPD. Foram selecionados 20 primers RAPD que geraram 167 fragmentos, dos quais 48 foram polimórficos (28,7%), produzindo uma média de 2,4 fragmentos polimórficos por primer. Baixa percentagem de polimorfismo foi obtida com o conjunto de primers (< 29%), indicando baixa variação genética entre os 64 acessos avaliados. As distâncias genéticas foram estimadas, utilizando-se o coeficiente de similaridade de Jaccard. Acessos de diferentes cidades foram agrupados em um mesmo grupo, indicando que não há correlação entre os agrupamentos moleculares e origem geográfica. O dendrograma revelou cinco grupos. O primeiro grupo reuniu os acessos C19 e G29, coletados nas cidades de Verdelândia e Monte Azul, respectivamente. O segundo grupo reuniu os acessos G16 e B1 1, coletados nas cidades de Monte Azul e Coração de Jesus, respectivamente. Os acessos remanescentes foram agrupados em três grupos, com oito, 15 e 37 acessos, respectivamente. O marcador RAPD apresentou baixo nível de polimorfismo entre os acessos avaliados.


INTRODUCTION
In Brazil, sugar apple (Annona squamosa L.) is an important fruit crop grown commercially in semiarid regions.There are few well-established breeding programs for sugar apple around the world.India, China and Taiwan have produced a few named cultivars that are propagated vegetatively (Nakasone & Paul, 1998).Annonaceae family presents a large intra and interspecific variability offering an ample scope for studying genetic variation.Despite this great variability, germplasm banks that contain Annona spp., mainly Annona squamosa, are rare throughout the world, which is a limiting factor for selecting and crossing among elite cultivars (Pinto et al., 2005).Brazil has the largest number of sugar apple accessions (92); therefore a systematic effort to evaluate these accessions will be fundamental to improve the species (IPGRI, 2000).In the past few years, morphological traits have been used as tools to characterize germplasm resources.Unfortunately, morphological characters tend to be influenced by environmental factors and problems with ambiguity are frequent.Molecular markers have provided a powerful tool for proper characterization of germplasm diversity (Williams et al., 1990).PCR-based molecular techniques provide a variety of DNA markers for diversity analysis (Newbury & Ford-Lloyd, 1993), identification of cultivars (Matsumoto & Fukui, 1996), gene localization, and construction of linkage maps (Giovanni et al., 2004).RAPD markers have been used for diversity analysis in cacao (Leal et al., 2008), banana (Souza et al., 2008) and, sugar apple (Bharad et al., 2009, Ahmad et al., 2010).
The objective of the present study was to assess the genetic diversity in 64 accessions of sugar apple germplasm by using RAPD markers.

MATERIALS AND METHODS
The plant material used in this study is listed in Table 1.The collection is located at experimental farm of State University of Montes Claros, Janaúba, Brazil.The germplasm collection included accessions that were collected from different municipalities located in the semiarid region.
The pair wise comparisons and dissimilarity matrix (data not shown) among the 64 accessions showed genetic dissimilarities ranging from a minimum of 0.0000 to a maximum of 0.17884.The most genetically diverse genotype was E4, collected from Montes Claros, which was 15.20% genetically related to all genotypes, while C2, collected from Verdelândia, was the most genetically similar (0.59%).
The similarity coefficients generated from RAPD data were used to construct a dendrogram.The dendrogram grouped the genotypes in five major clusters (I, II, III, IV and V) (Figure 1), based on a relative genetic distance of 40%, determined by Mojena (1977).The first cluster was formed by genotypes C19 and G29 (collected from Verdelândia and Monte Azul, respectively); the second cluster was formed by genotypes B11 and G16 (collected from Coração de Jesus and Monte Azul, respectively), and the clusters III, IV and V were formed by the remaining 60 accessions, with cluster V grouping most of accessions analyzed (57.8%).Accessions from different sites of collection were found to cluster together indicating no correlation between molecular groupings and their geographical origin.Dissimilarity values of 0% were observed between several accessions grouped in the III, IV and V clusters.In evaluating the values of dissimilarity of accessions by origin, it was observed that accessions from Verdelândia showed the smallest genetic distance.
The percentage of polymorphism obtained in this study (28.7%) was very low compared to other studies in Annona species.Ronning et al. (1995) used 15 RAPD primers to evaluate the genetic variability of atemoya, sugar apple and cherimoya and observed highly distinct and polymorphic patterns for the genotypes studied.Bharad et al. (2009) analyzed 11 sugar apple genotypes using five RAPD primers, and found 73% polymorphism, with all genotypes clustered in three groups.A recent study using 20 RAPD and 30 ISSR primers to assess the genetic diversity in four Annona species found levels of polymorphisms of 52% and 58% and divided them into two and three major clusters, respectively (Ahmad et al., 2010).
Although sugar apple is open-pollinated species and the plants evaluated in this study were propagated by seedlings and collected from different locations, low values of genetic dissimilarity were observed.The greatest genetic distance observed in this study (17.88%) was between accessions collected from Montes Claros and Verdelândia, distanced 250 km from each other.However, values of dissimilarity of 17.00% were obtained from accessions collected in Montes Claros city from the same orchard.Telles et al. (2003) evaluated the genetic diversity of Annona crassiflora Mart and found a smaller genetic distance among individuals of the same location due to high rate of ancestry in common.These authors suggest that a greater genetic variability is necessary and can be achieved by collection of few individuals from a number of different locations rather than a large number of individuals from the same location.In the present study 45.3% of the accessions were collected from the city of Verdelândia.
Montes Claros is the convergent center of agricultural activities in the region of northern Minas Gerais.Most farmers purchase agricultural inputs, seedlings and seeds in the Central Market located in the city of Montes Claros.Although the collection of accessions are originated from eight different cities, representing an area of about of about 16.720 Km 2 , the sugar apple orchards might have been established from fruits purchased in the Central Market.This fact could be increasing the probability of common ancestry and may explain the high similarity observed between the accessions evaluated.
Furthermore the low genetic diversity in the present study may be associated with the number and type of primers tested.Therefore, future studies should test additional primers and in larger numbers aiming at additional loci for the potential differentiation of accessions and validation of genetic variability, if any exists.

CONCLUSION
Amplification of genomic DNA of 64 sugar apple accessions from Northern Minas Gerais, Brazil, by using analysis of 20 RAPD showed a low percentage of polymorphism.

Table 1 .
Accession identification (AI) and collection site of 64 sugar apple germplasm located in Janaúba, Minas Gerais, Brazil, used for RAPD analysis Figure 1.Dendrogram depicting 64 sugar apple accessions from a germplasm collection in North Minas Gerais, Brazil.Similarities were estimated based on the UPGMA method.The values refer to bootstrap values greater than 50%.