SSR marker mining and its strategy under low-coverage shotgun sequencing
Date Issued
2012
Date
2012
Author(s)
Lin, Yen-Yu
Abstract
Simple sequence repeat (SSR) markers have been widely used in many areas of genetic research due to their codominance, ease of use, multiallelic nature, and abundance characteristics in the genome. Though various SSR markers in pepper have been published, it is still not sufficient to create a highly saturated linkage map. Here we utilized the 454 pyrosequencing technique to perform whole genome shotgun sequencing for pepper (Capsicum annuum). Tandem Repeats Finder (TRF) was applied to identify SSRs followed by primer design using Primer3. A Perl script was written to connect these processes. A total of 120 designed primers were verified by 15 varieties of C. annuum and 1 variety of C. baccatum. Verified results show that 104 primer sets were successfully amplified (87%), among them, 65 primer sets possess high polymorphism between the 15 varieties of C. annuum. The success rate and polymorphism of these designed primers were higher than the previously published primers. In order to determine an optimum strategy for SSR marker development, simulated shotgun sequence reads were generated using Flowsim based on rice reference genome sequences to compare the effects of assembly, mining tools, and primer selected methods on SSR primer development. Simulation results revealed that assembling low coverage genomic shotgun sequences not only did not elongate the sequence effectively to facilitate primer design, but also may introduce false sequence information. Compared with commonly used mining tools such as Msatcommander and QDD, we obtained the highest number of SSRs by TRF. We also found that using TRF, coupling with re-PCR or BLAST against original sequence reads may effectively eliminate potentially redundant markers while maintaining higher marker yield. Our findings may provide researchers an efficient SSR marker development strategy and decrease further primer verification cost.
Subjects
SSR
Microsatellite
Marker mining
Pyrosequencing
Shotgun sequencing
Low-coverage
Type
thesis
File(s)![Thumbnail Image]()
Loading...
Name
ntu-101-R98621109-1.pdf
Size
23.32 KB
Format
Adobe PDF
Checksum
(MD5):f026d8009ea5c214fc5d9a8d1ba8d612