Microsatellite marker development by partial sequencing of the sour passion fruit genome (Passiflora edulis Sims)

Abstract

BackgroundThe Passiflora genus comprises hundreds of wild and cultivated species of passion fruit used for food, industrial, ornamental and medicinal purposes. Efforts to develop genomic tools for genetic analysis of P. edulis, the most important commercial Passiflora species, are still incipient. In spite of many recognized applications of microsatellite markers in genetics and breeding, their availability for passion fruit research remains restricted. Microsatellite markers in P. edulis are usually limited in number, show reduced polymorphism, and are mostly based on compound or imperfect repeats. Furthermore, they are confined to only a few Passiflora species. We describe the use of NGS technology to partially assemble the P. edulis genome in order to develop hundreds of new microsatellite markers.ResultsA total of 14.11 Gbp of Illumina paired-end sequence reads were analyzed to detect simple sequence repeat sites in the sour passion fruit genome. A sample of 1300 contigs containing perfect repeat microsatellite sequences was selected for PCR primer development. Panels of di- and tri-nucleotide repeat markers were then tested in P. edulis germplasm accessions for validation. DNA polymorphism was detected in 74% of the markers (PIC = 0.16 to 0.77; number of alleles/locus = 2 to 7). A core panel of highly polymorphic markers (PIC = 0.46 to 0.77) was used to cross-amplify PCR products in 79 species of Passiflora (including P. edulis), belonging to four subgenera (Astrophea, Decaloba, Distephana and Passiflora). Approximately 71% of the marker/species combinations resulted in positive amplicons in all species tested. DNA polymorphism was detected in germplasm accessions of six closely related Passiflora species (P. edulis, P. alata, P. maliformis, P. nitida, P. quadrangularis and P. setacea) and the data used for accession discrimination and species assignment.ConclusionA database of P. edulis DNA sequences obtained by NGS technology was examined to identify microsatellite repeats in the sour passion fruit genome. Markers were submitted to evaluation using accessions of cultivated and wild Passiflora species. The new microsatellite markers detected high levels of DNA polymorphism in sour passion fruit and can potentially be used in genetic analysis of P. edulis and other Passiflora species.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-017-3881-5) contains supplementary material, which is available to authorized users.

The genomic sequence and comparative genomic analysis of cultivated passion fruit(Passiflora edulis L.)

“…The SSR Search software [16] and homologous annotation were used to annotate the repeated sequences in passion fruit genome. The results showed that there were 428,294 full-type SSR and 1,544,549 incomplete- and composite-type SSR [6]. For transposons, there were 59 Mutator transposons, 41 EnSpm transposons, 49 hAT transposons, 221 PIF transposons, and 2 MLE transposons.…”

“…Santos et al [5] used BAC end sequencing method to obtain 6,194,248 bp of passion fruit genome data, in which 669 microsatellite sequences were found, with an average of one SSR per 9.25 kb genome sequence. Later, Araya et al [6] developed 816 pairs of SSR primers in the structural and functional regions using parts of the passion fruit genome sequence. The results showed that 53.2% of SSR primers were polymorphic.…”

Transferibilidade de marcadores microssatélites para espécies silvestres de maracujazeiro

“…A taxa de transferibilidade variou de 14,28 a 33,33%, estudos anteriores relataram essa variação na taxa de transferibilidade de primers SSR em Passifloraceae (Oliveira et al, 2013;Paiva et al, 2014b;Pereira et al, 2015;Araya et al, 2017).…”

“…Estudos como este que avaliam a transferibilidade de primers microssatélites em Passifloracea tornam-se extremamente úteis, pois são ferramentas que antecedem estudos na área da genética vegetal, possibilitando medir os efeitos evolutivos e de seleção natural das espécies, e realizar análises filogenéticas, construções de mapas gênicos, análise de diversidade genética, e estudos de preservação da população durante programas de melhoramento genético (Gonçalves-Vidigal & Rubiano, 2011;Pereira et al, 2015). Além de serem capazes de acompanhar relações intra e interespécies (Araya et al, 2017), e serem utilizados na confirmação de paternidade de híbridos (Belo et al, 2018), otimizando assim os estudos de caracterização molecular das espécies silvestres de maracujazeiro, uma vez que através da análise de transferibilidade é possível otimizar os custos onerosos e longos períodos de desenvolvimento de novos marcadores SSR.…”

“…A transferência de microssatélites entre espécies vem sendo estudada e realizada com sucesso no gênero Passiflora (Oliveira et al, 2013;Silva et al, 2014;Araya et al, 2017), contudo fazemse necessários novos estudos, uma vez que existem inúmeras espécies que ainda não possuem marcadores microssatélites desenvolvidos. Portanto, o objetivo deste trabalho foi avaliar a transferibilidade de 21 loci microssatélites das espécies comerciais para quatro espécies silvestres de Passiflora.…”