In silico development and characterization of tri-nucleotide simple sequence repeat markers in hazelnut (Corylus avellana L.)

PloS One
Gehendra Bhattarai, Shawn A Mehlenbacher

Abstract

Plant genomes are now sequenced rapidly and inexpensively. In silico approaches allow efficient development of simple sequence repeat markers, also known as microsatellite markers, from these sequences. A search of the genome sequence of 'Jefferson' hazelnut (Corylus avellana L.) identified 8,708 tri-nucleotide simple sequence repeats with at least five repeat units, and stepwise removal of the less promising sequences led to the development of 150 polymorphic markers. Fragments in the 'Jefferson' sequence containing tri-nucleotide repeats were used as references and aligned with genomic sequences from seven other cultivars. Following in silico alignment, sequences that showed variation in number of repeat units were selected and primer pairs were designed for 243 of them. Screening on agarose gels identified 173 as polymorphic. Removal of duplicate and previously published sequences reduced the number to 150, for which fluorescent primers and capillary electrophoresis were used for amplicon sizing. These were characterized using 50 diverse hazelnut accessions. Of the 150, 132 generated the expected one or two alleles per accession while 18 amplified more than two amplicons in at least one accession. Diversity parameters of the...Continue Reading

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Methods Mentioned

BETA
PCR
electrophoresis
Illumina sequencing
genotyping

Software Mentioned

JoinMap
Gen5
MISA ( MIcroSAtellite ) Identification Tool
Cervus
Primer
BLASTx
BLAST
Websat
MAQ
GeneMapper

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