Development of polymorphic genic-SSR markers by cDNA library sequencing in boxwood, Buxus spp. (Buxaceae)

Chandra S Thammina; Richard T Olsen; Martha Malapi-Wight; Jo Anne Crouch; Margaret R Pooler

doi:10.3732/apps.1400095

Development of polymorphic genic-SSR markers by cDNA library sequencing in boxwood, Buxus spp. (Buxaceae)

Appl Plant Sci. 2014 Nov 28;2(12):apps.1400095. doi: 10.3732/apps.1400095. eCollection 2014 Dec.

Authors

Chandra S Thammina¹, Richard T Olsen², Martha Malapi-Wight³, Jo Anne Crouch³, Margaret R Pooler²

Affiliations

¹ United States Department of Agriculture, Agricultural Research Service (USDA-ARS), U.S. National Arboretum, Floral and Nursery Plants Research Unit, 10300 Baltimore Avenue, Building 010A, Beltsville, Maryland 20705 USA ; Department of Plant Biology and Pathology, Rutgers University, 59 Dudley Road, New Brunswick, New Jersey 08901 USA.
² United States Department of Agriculture, Agricultural Research Service (USDA-ARS), U.S. National Arboretum, Floral and Nursery Plants Research Unit, 10300 Baltimore Avenue, Building 010A, Beltsville, Maryland 20705 USA.
³ USDA-ARS, Systematic Mycology and Microbiology Laboratory, 10300 Baltimore Avenue, Building 010A, Beltsville, Maryland 20705 USA.

Abstract

Premise of the study: Genic microsatellites or simple sequence repeat (genic-SSR) markers were developed in boxwood (Buxus taxa) for genetic diversity analysis, identification of taxa, and to facilitate breeding. •

Methods and results: cDNA libraries were developed from mRNA extracted from leaves of Buxus sempervirens 'Vardar Valley' and sequenced using the Illumina MiSeq system. Approximately 11.9 million base pairs of sequence data were examined and 845 genic-SSRs were identified, including 469 dinucleotide, 360 trinucleotide, seven tetranucleotide, one pentanucleotide, and eight hexanucleotide repeats. Primer pairs were designed for 71 selectively chosen genic-SSRs containing trinucleotide repeat motifs and were used to amplify the corresponding loci in 18 diverse boxwood accessions. Twenty-three primer pairs amplified polymorphic loci, with two to 10 alleles per locus. •

Conclusions: These novel polymorphic genic-SSR markers will aid in evaluating genetic diversity of boxwood germplasm and allow verification of hybrids and cultivars for breeding programs.

Keywords: Buxaceae; Buxus sempervirens; boxwood; cDNA library sequencing; genetic diversity; genic simple sequence repeat (genic-SSR) markers; microsatellites; polymorphism.