Pecan [Carya illinoinensis (Wangenh.) K. Koch] is an economically important nut tree and grafting is often used for clonal propagation of cultivars. However, there is a lack of research on the effects of rootstocks on scions, which are meaningful targets for directed breeding of pecan grafts. MicroRNAs (miRNAs) play an important role in many biological processes, but the mechanism underlying the involvement of miRNAs in grafting-conferred physiological changes is unclear. To identify the grafting-responsive miRNAs that may be involved in the regulation of growth in grafted pecan, six small RNA libraries were constructed from the phloem of two groups of grafts with significantly different growth performance on short and tall rootstocks. A total of 441 conserved miRNAs belonging to 42 miRNA families and 603 novel miRNAs were identified. Among the identified miRNAs, 24 (seven conserved and 17 novel) were significantly differentially expressed by the different grafts, implying that they might be responsive to grafting and potentially involved in the regulation of graft growth. Ninety-five target genes were predicted for the differentially expressed miRNAs; gene annotation was available for 33 of these. Analysis of their targets suggested that the miRNAs may regulate auxin transport, cell activity, and inorganic phosphate (Pi) acquisition, and thereby, mediate pecan graft growth. Use of the recently-published pecan genome enabled identification of a substantial population of miRNAs, which are now available for further research. We also identified the grafting-responsive miRNAs and their potential roles in pecan graft growth, providing a basis for research on long-distance regulation in grafted pecan.

Rootstocks are well known to have important effects on scion growth performance. However, the involved mechanisms remain unclear. Recent studies provided some clues on the potential involvement of DNA methylation in grafting, which open up new horizons for exploring how rootstocks induce the growth changes. To better understand the involvement of DNA methylation in rootstock-induced growth alterations, whole-genome bisulfite sequencing (WGBS) was used to evaluate the methylation profiles of two sets of pecan grafts with different growth performances on different sizes of rootstocks. The results showed that methylated cytosines accounted for 24.52%–25.60% of all cytosines in pecan. Methylation levels in CG were the highest, with the lowest levels being in CHH (C= cytosine; G= guanine; H = adenine, thymine, or cytosine). Rootstocks induced extensive methylation alterations in scions with 934, 2864, and 15,789 differentially methylated regions (DMRs) determined in CG, CHG, and CHH contexts, respectively. DMR-related genes (DMGs) were found to participate in various processes associated with plant growth, among which 17 DMGs were found, most likely related to hormone response, that may play particularly important roles in graft growth regulation. This study revealed DNA methylomes throughout the pecan genome for the first time, and obtained abundant genes with methylation alterations that were potentially involved in rootstock-induced growth changes in pecan scions, which lays a good basis for further epigenetic studies on pecan and deeper understanding of grafting mechanisms in pecan grafts.

This study examined the ability to vegetatively propagate 1-year-old pecan ( Carya illinoinensis ) through the rooting of hardwood cuttings. Cuttings were treated with varying concentrations of different auxins and different combinations of media and ambient temperatures. Under different temperature conditions, all auxin treatments induced the rooting of cuttings but did not promote sprouting. The effectiveness of the induction of adventitious roots was as follows: 1-naphthalene acetic acid (NAA) > indole 3-butyric acid > indole 3-acetic acid. The base of the parent shoot treated by NAA at a concentration of 0.09%, planted in substrate with bottom heat was the most effective, with 82% rooting, 8.3 roots/cutting and root lengths of 7.3 cm. These findings suggested that auxin and substrate/air temperature differences are both indispensable in the process of adventitious roots formation in pecan. This study revealed that the propagation of hardwood cuttings derived from branches of 1-year-old pecan is possible.

Drought severely affects the growth and production of pecan (Carya illinoinensis), while genes conferred drought adaptation are yet to be fully elucidated. Here, an in-depth exploration of the two different RNA-seq projects regarding drought stress (designated as P1 and P2) was performed via weighted gene co-expression network analysis. For the two projects, there existed one pair of modules (P1 turquoise module and P2 blue module) that was probably associated with drought resistance, as the paired modules both exhibited an increased expression profile with increasing water shortage stress and were annotated to be involved in oxidative stress response and the signaling pathways of abscisic acid and jasmonic acid. There were 441 and 1258 hub genes in the P1 turquoise module and P2 blue module, respectively, among which, 140 were overlapped and thus were recognized as core drought responsive genes. An additional drought stress experiment was conducted for RT-qPCR validation, and the results showed that the 20 core genes selected for detection were highly responsive to water deficit. Together, our results will be helpful for understanding the molecular mechanism of drought response and improving drought resistance in pecan.