RESUMO
Simple sequence repeats (SSRs), one of the most powerful molecular markers, can be used for DNA fingerprinting, variety identification, genetic mapping, and marker-assisted selection. Using the pear's (Pyrus pyrifolia Nakai) 75,764 unigenes (55,676,271 bp) obtained by deep transcriptome sequencing, a total of 10,622 novel SSRs were identified in 9154 unigenes, accounting for 14.02% of all unigenes. The average length and distribution of these SSRs was about 16 bp and 5.24 kb, respectively. Dinucleotide repeat motifs were the main type, with a frequency of 55.87%, followed by trinucleotides (24.45%). There were 159 kinds of repeat motifs existing in the pear transcriptome. AG/CT was the most frequent motif, accounting for 49.64%. All 9154 SSR-containing unigenes were functionally annotated using Nr (NCBI non-redundant protein database), Nt (NCBI non-redundant nucleotide database), and the Swiss-Prot database, and were classified further by Gene Ontology and Clusters of Orthologous Groups. In addition, a total of 4300 primer pairs were designed from all SSR loci obtained. Of these, 40 primers were randomly selected for PCR amplification and polyacrylamide gel (PAGE) analysis. Among the 40 primer pairs, 31 were successfully separated via PAGE. These findings also confirm that mining SSRs using next-generating sequencing technologies is a fast, effective, and reliable approach.
Assuntos
Repetições de Microssatélites/genética , Anotação de Sequência Molecular , Pyrus/genética , Análise de Sequência de RNA/métodos , Transcriptoma/genética , Sequência de Bases , Primers do DNA/metabolismo , Eletroforese em Gel de Poliacrilamida , Motivos de Nucleotídeos/genética , Reprodutibilidade dos TestesRESUMO
The use of simple sequence repeats (SSRs), or microsatellites, as genetic markers has become popular due to their abundance and variation in length among individuals. In this study, we investigated linkage groups (LGs) in the woodland strawberry (Fragaria vesca) and demonstrated variation in the abundances, densities, and relative densities of mononucleotide, dinucleotide, and trinucleotide repeats. Mononucleotide, dinucleotide, and trinucleotide repeats were more common than longer repeats in all LGs examined. Perfect SSRs were the predominant SSR type found and their abundance was extremely stable among LGs and chloroplasts. Abundances of mononucleotide, dinucleotide, and trinucleotide repeats were positively correlated with LG size, whereas those of tetranucleotide and hexanucleotide SSRs were not. Generally, in each LG, the abundance, relative abundance, relative density, and the proportion of each unique SSR all declined rapidly as the repeated unit increased. Furthermore, the lengths and frequencies of SSRs varied among different LGs.