In this study, an （AG） n microsatellite-enriched library of Rhododendron simsii Planch. was constructed with FIASCO （ fast Isolation by AFLP sequences containing repeats） method. Among 356 positive colonies verifi...In this study, an （AG） n microsatellite-enriched library of Rhododendron simsii Planch. was constructed with FIASCO （ fast Isolation by AFLP sequences containing repeats） method. Among 356 positive colonies verified by colony PCR of the （AG）n microsatellite-enriched library, 233 colonies were selected for further sequencing. According to the results, 202 colonies containing microsatellites were obtained, accounting for 86.7%. Discarding sequences with miscella- neous peaks, overlapping peaks and weak signals, 39 sequences were screened finally for subsequent primer exploitation, including 22 perfect type SSR loci, 5 im- perfect type SSR loci and 12 compound type SSR loci. The FIASCO method is efficient and feasible for constructing microsatellite-enriched libraries of Rhododendron spp. , which laid a solid foundation for isolation of microsatellite loci and investigation of genetic diversity and genetic structure of Rhododendron spp.展开更多
A microsatellite-enriched library of plateau pika （Ochotona curzoniae） was constructed according to the strong affinity between biotin and streptavidin. Firstly, genomic DNA was fragmented by ultrasonication, which ...A microsatellite-enriched library of plateau pika （Ochotona curzoniae） was constructed according to the strong affinity between biotin and streptavidin. Firstly, genomic DNA was fragmented by ultrasonication, which is a major improvement over traditional methods. Linker-ligated DNA fragments were hybridized with biotinylated microsatellite probes, and then were subjected to streptavidin-coated magnetic beads. PCR amplification was performed to obtain double-stranded DNA fragments containing microsatellites. Ligation and transformation were carried out by using the pGEM-T Vector System I and Escherichia coli DH10B competent cells. Sequencing results showed that 80.2% of clones contained microsatellite repeat motif. Several modifications make this protocol time-efficient and technically easier than the traditional ones; particularly, composition and relative abundance of microsatellite repeats in plateau pika genome were truly represented through the optimized PCR conditions. This method has also been successfully applied to construct microsatellite-enriched genomic libraries of Chinese hamster （Cricetulus griseus） and small abalone [Haliotis diversicolor （Reeve）] with high rates of positive clones, demonstrating its feasibility and stability.展开更多
基金Supported by Doctoral Starting up Foundation of Huanggang Normal University(2013030903)Opening Fund of Hubei Key Laboratory of Economic Forest Germplasm Improvement and Resource Comprehensive Utilization(2013000403)
文摘In this study, an （AG） n microsatellite-enriched library of Rhododendron simsii Planch. was constructed with FIASCO （ fast Isolation by AFLP sequences containing repeats） method. Among 356 positive colonies verified by colony PCR of the （AG）n microsatellite-enriched library, 233 colonies were selected for further sequencing. According to the results, 202 colonies containing microsatellites were obtained, accounting for 86.7%. Discarding sequences with miscella- neous peaks, overlapping peaks and weak signals, 39 sequences were screened finally for subsequent primer exploitation, including 22 perfect type SSR loci, 5 im- perfect type SSR loci and 12 compound type SSR loci. The FIASCO method is efficient and feasible for constructing microsatellite-enriched libraries of Rhododendron spp. , which laid a solid foundation for isolation of microsatellite loci and investigation of genetic diversity and genetic structure of Rhododendron spp.
文摘A microsatellite-enriched library of plateau pika （Ochotona curzoniae） was constructed according to the strong affinity between biotin and streptavidin. Firstly, genomic DNA was fragmented by ultrasonication, which is a major improvement over traditional methods. Linker-ligated DNA fragments were hybridized with biotinylated microsatellite probes, and then were subjected to streptavidin-coated magnetic beads. PCR amplification was performed to obtain double-stranded DNA fragments containing microsatellites. Ligation and transformation were carried out by using the pGEM-T Vector System I and Escherichia coli DH10B competent cells. Sequencing results showed that 80.2% of clones contained microsatellite repeat motif. Several modifications make this protocol time-efficient and technically easier than the traditional ones; particularly, composition and relative abundance of microsatellite repeats in plateau pika genome were truly represented through the optimized PCR conditions. This method has also been successfully applied to construct microsatellite-enriched genomic libraries of Chinese hamster （Cricetulus griseus） and small abalone [Haliotis diversicolor （Reeve）] with high rates of positive clones, demonstrating its feasibility and stability.
