title: An accurate and efficient method for large-scale SSR genotyping and applications.
abstract: Accurate and efficient genotyping of simple sequence repeats (SSRs) constitutes the basis of SSRs as an effective genetic marker with various applications. However, the existing methods for SSR genotyping suffer from low sensitivity, low accuracy, low efficiency and high cost. In order to fully exploit the potential of SSRs as genetic marker, we developed a novel method for SSR genotyping, named as AmpSeq-SSR, which combines multiplexing polymerase chain reaction (PCR), targeted deep sequencing and comprehensive analysis. AmpSeq-SSR is able to genotype potentially more than a million SSRs at once using the current sequencing techniques. In the current study, we simultaneously genotyped 3105 SSRs in eight rice varieties, which were further validated experimentally. The results showed that the accuracies of AmpSeq-SSR were nearly 100 and 94% with a single base resolution for homozygous and heterozygous samples, respectively. To demonstrate the power of AmpSeq-SSR, we adopted it in two applications. The first was to construct discriminative fingerprints of the rice varieties using 3105 SSRs, which offer much greater discriminative power than the 48 SSRs commonly used for rice. The second was to map Xa21, a gene that confers persistent resistance to rice bacterial blight. We demonstrated that genome-scale fingerprints of an organism can be efficiently constructed and candidate genes, such as Xa21 in rice, can be accurately and efficiently mapped using an innovative strategy consisting of multiplexing PCR, targeted sequencing and computational analysis. While the work we present focused on rice, AmpSeq-SSR can be readily extended to animals and micro-organisms.
authors.affiliation: Institute for Systems Biology, Jianghan University, Wuhan, Hubei 430056, China.
authors.forename: Lun
authors.initials: L
authors.lastname: Li
authors.affiliation: Institute for Systems Biology, Jianghan University, Wuhan, Hubei 430056, China.
authors.forename: Zhiwei
authors.initials: Z
authors.lastname: Fang
authors.affiliation: Institute for Systems Biology, Jianghan University, Wuhan, Hubei 430056, China.
authors.forename: Junfei
authors.initials: J
authors.lastname: Zhou
authors.affiliation: Center for Development of Science and Technology, Ministry of Agriculture, P.R. China, Beijing 100122, China.
authors.forename: Hong
authors.initials: H
authors.lastname: Chen
authors.affiliation: Institute for Systems Biology, Jianghan University, Wuhan, Hubei 430056, China.
authors.forename: Zhangfeng
authors.initials: Z
authors.lastname: Hu
authors.affiliation: Institute for Systems Biology, Jianghan University, Wuhan, Hubei 430056, China.
authors.forename: Lifen
authors.initials: L
authors.lastname: Gao
authors.affiliation: Institute for Systems Biology, Jianghan University, Wuhan, Hubei 430056, China.
authors.forename: Lihong
authors.initials: L
authors.lastname: Chen
authors.affiliation: Division of Biomedical Informatics, Cincinnati Children's Hospital Research Foundation, 3333 Burnet Avenue, Cincinnati, OH 45229-3026, USA.|Department of Mathematical Sciences, McMicken College of Arts & Sciences, University of Cincinnati, 2815 Commons Way, Cincinnati, OH 45221-0025, USA.
authors.forename: Sheng
authors.initials: S
authors.lastname: Ren
authors.affiliation: Thermo Fisher Scientific, Building 6, No. 27, Xin Jinqiao Rd., Pudong, Shanghai 201206, China.
authors.forename: Hongyu
authors.initials: H
authors.lastname: Ma
authors.affiliation: Institute for Systems Biology, Jianghan University, Wuhan, Hubei 430056, China.|Division of Biomedical Informatics, Cincinnati Children's Hospital Research Foundation, 3333 Burnet Avenue, Cincinnati, OH 45229-3026, USA.
authors.forename: Long
authors.initials: L
authors.lastname: Lu
authors.affiliation: Institute for Systems Biology, Jianghan University, Wuhan, Hubei 430056, China.|Department of Computer Science and Engineering, Washington University in St Louis, MO 63130, USA.|Department of Genetics, Washington University School of Medicine, St Louis, MO 63130, USA.
authors.forename: Weixiong
authors.initials: W
authors.lastname: Zhang
authors.affiliation: Institute for Systems Biology, Jianghan University, Wuhan, Hubei 430056, China.
authors.forename: Hai
authors.initials: H
authors.lastname: Peng
mesh_terms: D001483:Base Sequence; D005819:Genetic Markers; D018745:Genome, Plant; D005838:Genotype; D060005:Genotyping Techniques; D006579:Heterozygote; D059014:High-Throughput Nucleotide Sequencing; D006720:Homozygote; D018895:Microsatellite Repeats; D058977:Molecular Sequence Annotation; D060885:Multiplex Polymerase Chain Reaction; D012275:Oryza / Q000145:classification / Q000235:genetics*; D010940:Plant Proteins / Q000235:genetics; D017346:Protein Serine-Threonine Kinases / Q000235:genetics; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA
chemical_list: D005819:Genetic Markers; D010940:Plant Proteins; C097148:Xa21 protein, Oryza sativa; D017346:Protein Serine-Threonine Kinases