Table 1.
Base sequence and characteristics of the two primers used for genotyping the pecan trees [13].
Table 2.
The sequence of primers/probes used for qPCR analysis.
Fig 1.
Catkin bloom patterns for ‘Western’ and ‘Wichita’.
The number of catkins on a given ‘Wichita’ shoot was negatively correlated with the presence of pistillate flowers on the same shoot (p-value < 0.0001). The presence of fruit the previous year also had a positive impact, and a significant increase of catkins was observed than compared to shoots that were not fruiting the year before (p-value < 0.01 for the previous year). (A) This graph indicates the number of catkins on ‘Western’ and ‘Wichita’ on shoots that had produced fruit (Fr) the previous year and those shoots with no fruit production the previous year (NFr). In ‘Wichita’, the non-fruiting shoots in 2018 had fewer catkins produced on the same shoots in 2019. (B) This graph indicates the number of catkins on ‘Western’ and ‘Wichita’ on shoots that had pistillate flowers (Fl) during the current season year and those shoots with no pistillate flowers (NFl) during the current season. The ‘Wichita’ shoots with pistillate flowers had less catkins produced during the same year (2019) while no significant effect was observed on ‘Western’ catkin production.
Fig 2.
Microscopic images of the dormant buds of ‘Wichita’ and ‘Western’ pecan cultivars.
Dormant buds were collected in February 2019. Buds were fixed with 2.5% glutaraldehyde and hand sectioned prior to confocal microscopy. (A) Overview of the entire ‘Wichita’ bud on a stereoscope. Catkin structures are observed on the right and left side of the bud (designated with a *). (B) Stereoscope overview of a ‘Western’ bud, more developed catkin structures are observed on the right and left side of the bud (designated with a *). (C) Confocal microscopy of partially formed catkin on ‘Wichita’ bud. (D) Confocal microscopy of fully formed catkin showing anthers on the ‘Western’ bud.
Fig 3.
PCA plot of the transcriptomes of fruiting and non-fruiting samples of ‘Western’ and ‘Wichita’ cultivars.
Samples were clustered separately in the PCA plot showing the first and second PCs which together explained 52.8% of variances. Fruiting and non-fruiting samples from Wichita clearly clustered separately revealing more differences between them in comparison to Western samples.
Table 3.
Selected DEGs between non-fruiting (NFr) and fruiting (Fr) samples collected in June 2017 and September 2016 from ‘Wichita’(WI) and ‘Western’ (WE) cultivars (‘Wichita’ non-fruiting vs. ‘Wichita’ fruiting / ‘Western’ non-fruiting vs. ‘Western’ fruiting).
The samples in second column had the higher expression of the genes. The cut-off for the DEGs were set to a minimum log2fold change of 1.5 with adjusted p-value of less than 0.05.
Table 4.
Selected DEGs between samples collected from ‘Western’ (WE) and ‘Wichita’ (WI) in June 2017 and September 2016 (‘Wichita’ fruiting (Fr) vs. ‘Western’ fruiting / ‘Wichita’ non-fruiting (NFr) vs. ‘Western’ non-fruiting).
Second column is the sample that had higher expression. The cut-off for the DEGs were set to a minimum log2fold change of 1.5 with adjusted p-value of less than 0.05.
Table 5.
Selected DEGs between non-fruiting and fruiting sample (March 5 and March 22, 2018) from ‘Wichita’ (WI) and ‘Western’ (WE) cultivars.
Second column indicates the sample in which the given gene was significantly higher expressed compared to the other fruiting (Fr)/non-fruiting (NFr) status within the same genotype. The cut-off for the DEGs were set to a minimum log2fold change of 1.5 with adjusted p-value of less than 0.05.
Fig 4.
The expression of some of the canonical flowering genes in our RNA-Seq data.
The RPKM of individual flowering genes is shown in regard to ‘Western’ (WE) and ‘Wichita’ (WI) throughout time on both fruiting (Fr) and non-fruiting (NFr) shoots. These samples were collected in June 2017 and March 2018. Error bars indicate standard error.
Fig 5.
Validation of RNA-Seq through qPCR.
Five genes were selected to verify RNA-Seq results using qRT-PCR. Graphs on the left (A, C, E, G, I, K) are 2^ddCQ values from qPCR and graphs on the right (B, D, F, H, J, L) show normalized gene expression (RPKM) values from RNA-Seq. Pearson correlations analyzes was performed to determine correlation between RNA-Seq normalized gene expression (RPKM) and qPCR (2^ddCQ).
Fig 6.
Schematic of the proposed flowering “clocks” in North American grown pecan.