Pecan (Carya illinoinensis), as a popular nut tree, has been widely planted in China in recent years. Grafting is an important technique for its cultivation. For a successful grafting, graft union development generally involves the formation of callus and vascular bundles at the graft union. To explore the molecular mechanism of graft union development, we applied high throughput RNA sequencing to investigate the transcriptomic profiles of graft union at four timepoints (0 days, 8 days, 15 days, and 30 days) during the pecan grafting process. After de novo assembly, 83,693 unigenes were obtained, and 40,069 of them were annotated. A total of 12,180 differentially expressed genes were identified between by grafting. Genes involved in hormone signaling, cell proliferation, xylem differentiation, cell elongation, secondary cell wall deposition, programmed cell death, and reactive oxygen species (ROS) scavenging showed significant differential expression during the graft union developmental process. In addition, we found that the content of auxin, cytokinin, and gibberellin were accumulated at the graft unions during the grafting process. These results will aid in our understanding of successful grafting in the future.

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.

Background: Calcium (Ca²⁺) serves as a ubiquitous second messenger and plays a pivotal role in signal transduction. Calcineurin B-like proteins (CBLs) are plant-specific Ca²⁺ sensors that interact with CBL-interacting protein kinases (CIPKs) to transmit Ca²⁺ signals. CBL-CIPK complexes have been reported to play pivotal roles in plant development and response to drought stress; however, limited information is available about the CBL and CIPK genes in pecan, an important nut crop. Results: In the present study, a total of 9 CBL and 30 CIPK genes were identified from the pecan genome and divided into four and five clades based on phylogeny, respectively. Gene structure and distribution of conserved sequence motif analysis suggested that family members in the same clade commonly exhibited similar exon-intron structures and motif compositions. The segmental duplication events contributed largely to the expansion of pecan CBL and CIPK gene families, and Ka/Ks values revealed that all of them experienced strong negative selection. Phylogenetic analysis of CIPK proteins from 14 plant species revealed that CIPKs in the intron-poor clade originated in seed plants. Tissue-specific expression profiles of CiCBLs and CiCIPKs were analysed, presenting functional diversity. Expression profiles derived from RNA-Seq revealed distinct expression patterns of CiCBLs and CiCIPKs under drought treatment in pecan. Moreover, coexpression network analysis helped to elucidate the relationships between these genes and identify potential candidates for the regulation of drought response, which were verified by qRT–PCR analysis. Conclusions: The characterization and analysis of CBL and CIPK genes in pecan genome could provide a basis for further functional analysis of CiCBLs and CiCIPKs in the drought stress response of pecan.

Pecan [Carya illinoinensis (Wangenh.) K. Koch] is a high-value fruit tree with a long juvenile period. The fruiting process of pecan seedlings can be largely accelerated through grafting. As non-coding small RNAs, plant miRNAs participate in various biological processes through negative regulation of gene expression. To reveal the roles of miRNAs in the graft union development of pecan, four small RNA libraries were constructed from the graft union at days 0, 8, 15, and 30 after grafting. A total of 47 conserved miRNAs belonging to 31 families and 39 novel miRNAs were identified. For identified miRNAs, 584 target genes were bioinformatically predicted, and 266 of them were annotated; 29 miRNAs (including 16 conserved and 13 novel miRNAs) were differentially expressed during the graft process. The expression profiles of 12 miRNA were further validated by quantitative reverse transcription PCR (qRT-PCR). In addition, qRT-PCR revealed that the expression levels of 3 target genes were negatively correlated with their corresponding miRNAs. We found that miRS26 might be involved in callus formation; miR156, miR160, miR164, miR166, and miRS10 might be associated with vascular bundle formation. These results indicate that the miRNA-mediated gene regulations play important roles in the graft union development of pecan.