Comparative analysis of butternut (Juglans cinerea) and Japanese walnut (Juglans ailantifolia) chloroplast genomes.

Juglans cinerea L. (2n = 16), commonly known as butternut, is a diploid species native to the eastern forests of the United States and Canada. It plays a crucial ecological role as a food source for wildlife and is traditionally used as a medicine by tribal communities. Butternut populations have severely declined over 50 years due to butternut canker disease (BCD). The ability of butternut to hybridize with Japanese walnut, which exhibits resistance to BCD, complicates hybrid identification based on morphological traits alone. In this study, we assembled the chloroplast genome of butternut using next-generation sequencing, resulting in a genome size of 160,289 bp. The genome comprises 112 genes, including 78 protein-coding genes, 30 tRNA genes, and four rRNA genes, with a GC content of 36%. Comparative analysis revealed 1,156 SNPs, including 500 species-specific SNPs between 13 butternut and 6 Japanese walnut accessions. Of these, 367 SNPs were in coding regions, 32 of which exhibited unique variations causing codon and amino acid changes between the two species. We developed and validated 12 species-specific CAPS markers in matK and ycf1 genes for assessing the genetic background of Juglans species. The ycf1 gene displayed the highest variability, harboring 37 unique SNPs, many resulting in non-synonymous mutations. The newly sequenced chloroplast genome of J. cinerea and the identified SNP resources provide valuable genetic tools for characterizing butternut’s genetic diversity and distinguishing it from Japanese walnut. These findings enhance our understanding of Juglans species and contribute significantly to plant genetics and genomics by comprehensively analyzing chloroplast genomes and SNP variation, addressing a critical gap in previous studies.