Articles
DEVELOPMENT OF POLYMORPHIC SIMPLE SEQUENCE REPEAT MARKERS IN LILIUM REGALE BY MAGNESPHERE METHOD
Article number
977_39
Pages
327 – 333
Language
English
Abstract
Simple sequence repeat (SSR) is one of the most useful molecular markers based on DNA length polymorphism and an efficient tool for population genetic studies and primary genetic linkage maps construction.
In this study, we selected lily Lilium regale as a model material.
Genomic DNA was extracted and digested with restriction enzyme Mse1, and ligated with adapters.
After PCR, the fragments were hybridized with biotin-labeled simple sequence repeat probes (AG)15 and (GT) 15, the nonspecific adsorptions were washed away by using 0.1×SSR, then the enriched product of (AG)n and (GT)n were obtained.
The SSR containing sequences were amplified using primers designed complementary to linkers, cloned into pMD18-T vectors and transformed into E. coli, two SSR-enriched libraries (AG and GT) were constructed.
Screening positive clones by PCR cloning method, 192 and 48 clones were screened in AG-enriched and GT-enriched library respectively. 109 and 9 SSR-containing sequences were obtained, the concentration rates were 56.8 and 16.7%. The result showed the concentration rate of AG-enriched library is higher than GT-enriched library.
After sequence analysis, 36 pairs of SSR primers were designed successfully, then the primers were used to amplify the genomic DNA of 15 wide lilies genotypes with diversified origin, 18 SSR primers had amplified polymorphism, but only 2 primers had amplification in all the 15 lilies, cross of SSR primers among lily species was low, suggesting the genetic relationship between wild species of lily was far, more in-depth research is needed in the future.
In this study, we selected lily Lilium regale as a model material.
Genomic DNA was extracted and digested with restriction enzyme Mse1, and ligated with adapters.
After PCR, the fragments were hybridized with biotin-labeled simple sequence repeat probes (AG)15 and (GT) 15, the nonspecific adsorptions were washed away by using 0.1×SSR, then the enriched product of (AG)n and (GT)n were obtained.
The SSR containing sequences were amplified using primers designed complementary to linkers, cloned into pMD18-T vectors and transformed into E. coli, two SSR-enriched libraries (AG and GT) were constructed.
Screening positive clones by PCR cloning method, 192 and 48 clones were screened in AG-enriched and GT-enriched library respectively. 109 and 9 SSR-containing sequences were obtained, the concentration rates were 56.8 and 16.7%. The result showed the concentration rate of AG-enriched library is higher than GT-enriched library.
After sequence analysis, 36 pairs of SSR primers were designed successfully, then the primers were used to amplify the genomic DNA of 15 wide lilies genotypes with diversified origin, 18 SSR primers had amplified polymorphism, but only 2 primers had amplification in all the 15 lilies, cross of SSR primers among lily species was low, suggesting the genetic relationship between wild species of lily was far, more in-depth research is needed in the future.
Authors
Y.P. Zhao, W.H. Wang, G.S. Liu, X.Y. Zhao, S.D. Wang, T.F. Qin
Keywords
L. regale, magnesphere method, library screening, SSR markers
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