De novo assembly and characterization of the leaf, bud, and fruit transcriptome from the vulnerable tree Juglans mandshurica for the development of 20 new microsatellite markers using Illumina sequencing.

Abstract

Manchurian walnut (Juglans mandshurica Maxim.) is a vulnerable, temperate deciduous tree valued for its wood and nut, but transcriptomic and genomic data for the species are very limited. Next generation sequencing (NGS) has made it possible to develop molecular markers for this species rapidly and efficiently. Our goal is to use transcriptome information from RNA-Seq to understand development in J. mandshurica and develop polymorphic simple sequence repeats (SSRs, microsatellites) to understand the species' population genetics. In this study, more than 47.7 million clean reads were generated using Illumina sequencing technology. De novo assembly yielded 99,869 unigenes with an average length of 747 bp. Based on sequence similarity search with known proteins, a total of 39,708 (42.32 %) genes were identified. Searching against the Kyoto Encyclopedia of Genes and Genomes Pathway database (KEGG) identified 15,903 (16.9 %) unigenes. Further, we identified and characterized 63 new transcriptome-derived microsatellite markers. By testing the markers on 4 to 14 individuals from four populations, we found that 20 were polymorphic and easily amplified. The number of alleles per locus ranged from 2 to 8. The observed and expected heterozygosity per locus ranged from 0.209 to 0.813 and 0.335 to 0.842, respectively. These twenty microsatellite markers will be useful for studies of population genetics, diversity, and genetic structure, and they will undoubtedly benefit future breeding studies of this walnut species. Moreover, the information uncovered in this research will also serve as a useful genetic resource for understanding the transcriptome and development of J. mandshurica and other Juglans species.