Variation in Cytosine Methylation among Pecan Cultivars at Different Developmental Stages

in Journal of the American Society for Horticultural Science

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.

Contributor Notes

This study was supported by the SanXin project of Jiangsu province (LYSX[2016]44), the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD) and the Doctorate Fellowship Foundation of Nanjing Forestry University.

Corresponding author. E-mail:

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    Methylation levels of pecan cultivars among four stages are as follows: inflorescence emergence (IE), 30% of final fruit size (30%-FFS), 70% of final fruit size (70%-FFS), and fruit maturity (FM). The total methylation level is the sum of the hemimethylation level, full methylation level, and hypermethylation level.

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    Variation in methylation patterns among the pecan cultivars across the four stages: inflorescence emergence (IE), 30% of final fruit size (30%-FFS), 70% of final fruit size (70%-FFS), and fruit maturity (FM). (A) The methylation polymorphism rates of the sites amplified by eight primer sets among seven pecan cultivars. The polymorphic sites are defined as the sites with different methylation patterns among the cultivars. The polymorphic sites of the four stages are defined as the sites showing polymorphism among cultivars at any stage, and the polymorphism rates of the four stages are indicated with a dotted line. S (P) represents the polymorphism rates of the eight primer sets. (B) The specific polymorphism rates of the sites detected among seven pecan cultivars. The specific sites are the sites at which the methylation pattern of a cultivar is different from that belonging to the other six cultivars, whereas the methylation patterns of the other six cultivars are the same.

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    Changes in the methylation patterns between stages for the seven pecan cultivars. The values represent the ratios of CCGG sites with altered methylation patterns compared with those at the same sites from the previous stage. 30%-FFS = 30% of final fruit size; 70%-FFS = 70% of final fruit size; FM = fruit maturity; DM = demethylation; DNM = de novo methylation; NMSP = nonmethylation state preservation; CMS = changes in pattern of methylation state; NCMS = no change in pattern of methylation state; and MSP = methylation state preservation.

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    Dendrogram of the seven pecan cultivars established using Dice coefficients based on the methylation patterns. The matrix correlation (r) obtained from the cophenetic correlation analysis = 0.876. The line corresponding to the coefficient axis with a scale value of 0.75 was obtained by the NTSYS software automatically and was treated as the clustering standard of the pecan cultivars.

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    Principal coordinate analysis (PCoA) of the seven pecan cultivars based on the methylation patterns. (A) Two-dimensional diagram of the previous two principal coordinates from PCoA in the seven pecan cultivars. (B) Three-dimensional diagram of the previous three principal coordinates from PCoA in the seven pecan cultivars.

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