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Litchi trees flower at the apex of terminal shoots. Flowering is affected by the maturity of terminal shoots before growth cessation occurs during the winter. In this study, we focused on changes of flowering in three important cultivars, Guiwei, Feizixiao, and Huaizhi, from Dec. 2012 to Mar. 2013 under natural winter conditions. Flowering rate, carbohydrate accumulation, and expression of the flowering-related genes were determined at three different developmental stages of terminal shoots with dark green, yellowish green and yellowish red leaves, respectively. The results showed that the total soluble sugar and starch contents in the dark green leaves were the highest, whereas those in the yellowish red leaves were the lowest. Trees with dark green terminal shoots had the highest flowering rates, whereas those with yellowish green or yellowish red shoots had relatively lower flowering rates. SPAD was highest in dark green leaves and lowest in yellowish red leaves at the start of the trial. The SPAD value of yellowish red leaves slightly increased but did not reach the levels of the dark green leaves, whereas levels of the other leaf stages remained fairly constant. Expression level of the litchi homolog FLOWERING LOCUS C (LcFLC), the floral inhibitor in yellowish red leaves, increased from 16 Jan., whereas that in dark green leaves declined to a level lower than the yellowish red leaves on 4 Feb. Expression level of the litchi homolog CONSTANTS (LcCO), the floral promoter in dark green leaves, was higher than that of yellowish red leaves before 26 Jan. Expression level of the litchi homolog FLOWERING LOCUS T 2 (LcFT2), encoding florigen, was higher in dark green leaves than in the other two leaf types. Our results suggest that terminal shoots should be matured and leaves should turn green for successful flowering. Mature leaves had higher expression levels of the floral promoter and florigen. In litchi production, leaves of the terminal shoots (potential flowering branches) should be dark green during floral induction and differentiation stages, and winter flushes should be removed or killed.

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.

Scion wood of ‘Caddo’ and ‘Desirable’ pecan (Carya illinoinensis) was grafted onto the epicotyl of 1-month-old, open-pollinated ‘Shaoxing’ pecan seedlings for evaluation as a grafting technique to reduce the time to produce grafted trees. The results showed that seedlings grafted with “base scions” had higher survival than those grafted with “terminal scions” for both ‘Caddo’ and ‘Desirable’. Also, grafting with paraffinic tape could achieve greater success rate than that with medical tape. The most ideal time to perform this grafting was late April in Nanjing, China, when pecan seedlings were about 35 days old. This study demonstrated that the technique yielded successful epicotyl grafting of >70%, and it could thus be applied in practice.