Development of Genic Microsatellite Markers for Genetic Diversity Analysis of Boxwood Accessions to Breed for Blight-tolerant Cultivars

Boxwood (Buxus spp., Buxaceae) are evergreen, deer-resistant and popular landscape plants in the nursery industry. There are numerous cultivars available for use in various landscape functions. Each year, more than 13 million boxwood plants are sold in the U.S., with an annual market value of $103 million. Boxwood blight caused by Calonectria pseudonaviculata was first reported in Connecticut and North Carolina in October 2011, and later identified in many other states and several Canadian provinces. This fungal pathogen causes severe defoliation leading to plant death in nurseries and established landscapes. Hence, there is an urgent need to develop blight tolerant boxwood cultivars. The U.S. National Arboretum’s (USNA) National Boxwood Collection with more than 700 accessions is one of the most complete collections in the world. However, genetic relationships and diversity among these accessions has not been determined. We developed genic simple sequence repeat markers (SSRs) to evaluate genetic diversity of the USNA boxwood collection. Total RNA was extracted from leaf samples of Buxus sempervirens ‘Vardar Valley’ and used to develop cDNA libraries using the Illumina TruSeq RNA sample preparation kit. The validated, normalized and pooled cDNA libraries were sequenced using the Illumina MiSeq system. 3.6 million paired-end reads containing 0.5 Gbp of data were generated from a single sequencing run. The sequence data was assembled and mapped into 12027 contigs which were mined for SSRs and associated PCR primer pairs using the PrimerPro integrated analysis pipeline. 845 SSRs were identified, including 469 dinucleotide, 360 trinucleotide, 7 tetranucleotide, 1 pentanucleotide, and 8 hexanucleotide repeats. The genic SSRs are being used to analyze the genetic diversity and relationships of the USNA boxwood accessions which will aid in our overall objective of developing blight tolerant varieties.