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Zhuang-Zhuang Liu, Tao Chen, Fang-Ren Peng, You-Wang Liang, Peng-Peng Tan, Zheng-Hai Mo, Fan Cao, Yang-Juan Shang, Rui Zhang, and Yong-Rong Li

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.

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Li-Xiao Yao, Yong-Rui He, Hai-Fang Fan, Lan-Zhen Xu, Tian-Gang Lei, Xiu-Ping Zou, Ai-Hong Peng, Qiang Li, and Shan-Chun Chen

Ferric chelate reductase (FRO) is a critical enzyme for iron absorption in strategy I plants, reducing Fe3+ to Fe2+. To identify FRO family genes in the local Citrus junos cultivar Ziyang Xiangcheng and to reveal their expression model, the citrus (Citrus sp.) genome was searched for homologies of the published sequence CjFRO1. Five FROs were found, including CjFRO1; these were named CjFRO2, CjFRO3, CjFRO4, and CjFRO5, respectively, and cloned via reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) PCR. The deduced amino acid sequences of five CjFROs contained flavin adenine dinucleotide (FAD)-binding motifs, nicotinamide adenine dinucleotide (NAD)-binding motifs, and 6–10 transmembrane domains, with isoelectric points between 6.73 and 9.46, and molecular weights between 67.2 and 79.9 kD. CjFRO1 and CjFRO2 were predominantly found in the aboveground parts of C. junos, with CjFRO1 highly expressed in leaves, and CjFRO2 largely expressed in stems and leaves. CjFRO3 was less expressed in roots, stems, and leaves. CjFRO4 and CjFRO5 were predominately found in roots. Under iron-deficient conditions, CjFRO4 was significantly and specifically increased in the roots of C. junos, whereas CjFRO1 was upregulated in the roots and leaves.