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 for 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. 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, with 367 SNPs located in coding regions, 32 of which had unique SNPs leading to codon and amino acid variations between the two species. We developed 12 species-specific markers using the matK and ycf1 genes because of their higher variability compared with other genes located in coding regions that can be used to assess the genetic background of Juglans species. The ycf1 gene displayed the highest variability, harboring 38 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.