Analysis of Genetic Diversity and Relatedness of Boxwood Accessions Using Genic-simple Sequence Repeat Markers

Boxwood (Buxus L. spp., Buxaceae) are popular woody landscape shrubs grown for their diverse forms and broad-leaved evergreen foliage. Each year, more than 13 million boxwood plants are sold in the United States, with an annual market value of $103 million. Boxwood plants grown in temperate zones are now threatened by a destructive new blight disease caused by the ascomycete fungus Calonectria pseudonaviculata Henricot (syn. Cylindrocladium pseudonaviculatum, Cylindrocladium buxicola). The disease was first identified from the United Kingdom in 1994 and it has spread throughout continental Europe, parts of western Asia, New Zealand, and into North America. This fungal pathogen causes dark brown to black lesions on leaves and severe defoliation leading to plant death in nurseries and established landscapes, hence the need to develop blight-tolerant boxwood cultivars. The National Boxwood Collection at the U.S. National Arboretum (USNA) contains more than 700 Buxus accessions, making it one of the most complete collections in the world and a valuable genetic resource for developing blight-tolerant varieties. However, genetic relationships and diversity among these accessions have not been determined. We developed genic simple sequence repeat markers (genic-SSRs) by cDNA library sequencing in Buxus sempervirens ‘Vardar Valley’. Twenty-three genic-SSR primer pairs that amplified polymorphic loci in preliminary tests were used to estimate genetic diversity and relatedness of 268 boxwood accessions. Genic-SSR loci were highly variable among the accessions, detecting an average of 5.7 alleles (amplicons) per locus. However, six of the primer pairs amplified allele numbers that were inconsistent with known ploidy in some accessions, so were not used in the analysis. Amplified allele data from the remaining 17 primer pairs were analyzed using the R polysat package, with a distance matrix based on Jaccard’s similarity index, followed by UPGMA clustering. Five major clusters were identified consisting of individual accessions from B. balearica, B. harlandii, B. microphylla, B. sempervirens, B. sinica, and their interspecific hybrids. The accessions generally clustered by cultivar, provenance, or species. This information will be used for breeding programs and collection management, and for identifying possible sources of disease tolerance in boxwood.