The floral homeotic C-function gene AGAMOUS (AG) has been shown to be critical in the determination of stamen and carpel identity in Arabidopsis. In the present study, a new homologue of AGAMOUS gene from pecan [Carya illinoinensis (Wangenh.) K. Koch], denoted by CiAG, was isolated and its function was characterized. The complementary DNA (cDNA) of CiAG contains an open reading frame of 687 base pairs (bp) encoding 227 amino acids. Multiple sequence comparisons revealed that CiAG had the typical MIKC structure. Phylogenetic analysis indicated that CiAG is closely related to C-lineage AG. The expression of CiAG was highly accumulated in the reproductive tissues (staminate flowers, pistillate flowers, and fruitlets) than in vegetative tissues (leaves and current-growth branches). Arabidopsis overexpressing CiAG exhibited earlier flowering. The homeotic transformations of petals into stamen organs were observed in 35S::CiAG transgenic plants. All these results indicated that CiAG plays a key role in the process of flower development of pecan.
Cytosine methylation plays important roles in regulating gene expression and modulating agronomic traits. In this study, the fluorescence-labeled methylation-sensitive amplified polymorphism (F-MSAP) technique was used to study variation in cytosine methylation among seven pecan (Carya illinoinensis) cultivars at four developmental stages. In addition, phenotypic variations in the leaves of these seven cultivars were investigated. Using eight primer sets, 22,796 bands and 950 sites were detected in the pecan cultivars at four stages. Variation in cytosine methylation was observed among the pecan cultivars, with total methylation levels ranging from 51.18% to 56.58% and polymorphism rates of 82.29%, 81.73%, 78.64%, and 79.09% being recorded at the four stages. Sufficiently accompanying the polymorphism data, significant differences in phenotypic traits were also observed among the pecan cultivars, suggesting that cytosine methylation may be an important factor underlying phenotypic variation. Hypermethylation was the dominant type of methylation among the four types observed, and full methylation occurred at higher levels than did hemimethylation in the pecan genomes. Cluster analysis and principal coordinate analysis (PCoA) identified Dice coefficients ranging from 0.698 to 0.778, with an average coefficient of 0.735, and the variance contribution rates of the previous three principal coordinates were 19.6%, 19.0%, and 18.2%, respectively. Among the seven pecan cultivars, four groups were clearly classified based on a Dice coefficient of 0.75 and the previous three principal coordinates. Tracing dynamic changes in methylation status across stages revealed that methylation patterns changed at a larger proportion of CCGG sites from the 30% of final fruit-size (30%-FFS) stage to the 70%-FFS stage, with general decreases in the total methylation level, the rate of polymorphism, and specific sites being observed in each cultivar. These results demonstrated that the F-MSAP technique is a powerful tool for quantitatively detecting cytosine methylation in pecan genomes and provide a new perspective for studying many important life processes in pecan.