Research Highlights: For the first time, the complete chloroplast (cp) genome of Carya illinoinensis cv. ‘Pawnee’ was de novo assembled. Comprehensive analysis the cp genome of C. illinoinensis revealed potential cpDNA markers for intraspecies identification, genes involved in adaptation, and its phylogenetic position. Background and Objectives: C. illinoinensis is an economically important nut tree in the family Juglandaceae. Cp-derived markers are helpful for genetic research, but they still need to be developed in C. illinoinensis. Additionally, the adaptation and phylogenetic relationships of C. illinoinensis have not been revealed based on the complete cp genome. Materials and Methods: Chloroplast genomic DNA of C. illinoinensis cv. ‘Pawnee’ was extracted and subjected to Illumina sequencing. Results: The cp genome is 160,819 bp in size, exhibiting a typical quadripartite structure with a large single copy (LSC) of 90,022 bp, a small single copy (SSC) of 18,791 bp, and a pair of inverted repeats (IRA and IRB) regions of 26,003 bp each. The genome was predicted to encode 112 unique genes, including 79 protein-coding genes, 29 tRNAs, and four rRNAs, with 19 duplicates in the IR regions. In total, 213 SSRs and 44 long repeats were identified in the cp genome. A comparison of two different C. illinoinensis genotypes, ‘Pawnee’ and 87MX3-2.11, obtained 143 SNPs and 74 indels. The highly variable regions such as atpF, clpP, and ndhA genes, and matK-rps16, trnS-trnG, and trnT-psbD intergenic spacers might be helpful for future intraspecific identification. Positive selection was acting on the ccsA and rps12 cp genes based on the Ka/Ks ratios. Phylogenetic analysis indicated that C. illinoinensis forms a sister clade to Asian Carya species, represented by C. kweichowensis and Annamocarya sinensis. Conclusions: The genome information in our study will have significance for further research on the intraspecies identification and genetic improvement of C. illinoinensis.
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