Isolation and Characterization of Microsatellite Markers for Passiflora contracta (original) (raw)
Applications in plant sciences, 2014
The genus Passifl ora L. (Passifl oraceae) comprises approximately 400 species, of which at least 30% are distributed within Brazilian forests (Cervi et al., 2010). Species such as P. edulis Sims are important because of the economic value of their fruit (Faleiro et al., 2005). Certain wild species, including P. setacea DC. and P. cincinnata Mast., are of interest because of their potential use in genetic breeding. However, the limited number of molecular genetic diversity studies of this genus (Faleiro et al., 2005 ; Cerqueira-Silva et al., 2012) attests to the need for and relevance of novel molecular tools for studies of its populations and mating system. Although diversity studies of passion fruit began in the late 1990s, efforts to use microsatellites only began in 2005 (Oliveira et al., 2005 ; Pádua et al., 2005), and studies related to the development of microsatellites have been published for P. cincinnata (Cerqueira-Silva et al., 2012) and P. contracta Vitta (Cazé et al., 2012) only recently. The markers available are still insuffi cient for performing consistent genetic studies of most Passifl ora species because the evaluated populations exhibit low variability and percentages of polymorphic loci (between 0% and 26%) (Pereira, 2010 ; Ortiz et al., 2012 ; Cerqueira-Silva et al., 2012). Thus, considering the diffi culty in obtaining informative microsatellites for Passifl ora and to enhance the genetic investigation of both wild and commercial populations, we isolated, characterized, and evaluated the cross-amplifi cations of microsatellites for P. edulis , P. setacea , and P. cincinnata. METHODS AND RESULTS Two microsatellite-enriched genomic libraries were developed using genotypes from the germplasm collection of P. edulis (Pe-UESB01) and P. setacea (Ps-UESB01) from the Universidade Estadual do Sudoeste da Bahia (UESB; Itapetinga, Bahia, Brazil). Genomic DNA was isolated from fresh leaves using the cetyltrimethylammonium bromide (CTAB) method, and libraries were constructed following Billote et al. (1999). DNA samples (5 μ g) were digested with Afa I and ligated to the double-stranded adapters 5 ′-CTCTTGCTTA-CGCGTGGACTA-3 ′ and 5 ′-TAGTCCACGCGTAAGCAAGAGCACA-3 ′. Enrichment was performed using a hybridization-based capture with (GT) 8 and (CT) 8 biotin-linked probes and streptavidin-coated magnetic beads (Streptavidin