DNA content estimation of Fig and Black Mulberry using flow cytometry

Copyright: All the contents of this journal, except where otherwise noted, is licensed under a Creative Commons Attribution License. AbstractIn this study, fig and black mulberry DNA contents were estimated using DAPI fluorescence stain in flow cytometry. The 2C DNA contents of the fig and black mulberry were found as 0.82 pg and 8.34 pg, respectively. The calculated 1C value of genome size of fig is 401.8 Mbp and that of black mulberry is 4086.6 Mbp. The ratio of 2C DNA content and 1C genome of the black mulberry was 10.17 times that of the fig although fig is diploid and black mulberry is decosaploid.


Material and Methods
The nuclear DNA content was determined from leafs of barley, common vetch, fig, and black mulberry plants.The methods were used from TUNA et al. (2001) andTAVARES et al. (2014) with some modifications.For intact nuclei isolation, leaf tissues of sample and standard species were used alone and then simultaneously in flow cytometry.Each of 1.0 cm 2 (20-50 mg) of leaf tissues of H. vulgare, V. sativa, F. carica, and M. nigra analyzed one by one to observe corresponding G1 and G2 peaks.Then, leaf tissues of fig or black mulberry were mixed together with 1.0 cm 2 (20-50 mg) of leaf tissues of diploid common vetch and barley as internal reference standards.While fig leaf sample was used with common vetch leaf standard (1:1), black mulberry leaf sample was used with barley leaf standard (1:1) since their peak sizes were comparable.Leaf samples from both plants of each species pair were simultaneously chopped with a sharp razor blade for approximately 30 s in a plexiglass Petri dish containing 400 μl of ice-cold extraction buffer (stock: 24 ml MgSO 4 , 25 mg dithiothreitol, 625 μl Triton X-100 stock (1.0 g Triton X-100 in 10 ml ddH 2 O)).The nuclear suspension was filtered through a 30 μm nylon filter (Partec CellTrics®, Görlitz, Germany) to remove cell fragments and large leaf debris.Then, nuclei were stained using the kit CyStain® UV Precise P (Partec GmbH., Münster, Germany) with 1600 μl of DAPI (1×) solution.After a 2-3 min incubation period in darkness, the relative fluorescence intensity (FI) of nuclei per G1 peak was analyzed in a CyFlow® Space flow cytometer (Partec GmbH., Münster, Germany) equipped with a green solid state laser for monitoring DAPI fluorescent dye excitation (>435 nm, YAMANOUCHI et al., 2008, or 365 nm YAMANOUCHI et al., 2010).It is recommended to10-15 particles per second during the excitation recording.DNA content was calculated as follows: 2C or Nuclear DNA content (pg) = (mean position of unknown sample peak)/(mean position of known standard peak) × DNA content of known standard.1C= (nuclear DNA content (pg) × 980 Mbp) / 2.

Results and discussion
In the beginning of the experiment, barley, common vetch, fig, and black mulberry leaf samples were analyzed alone (data not presented).Their corresponding G1 and G2 peaks were observed and compared.When fig and common vetch leaf samples were analyzed, ıt was seen that G1 peak of fig ( 115.94) was 4.5 folds smaller than common vetch (517.96)(Fig  LOUREIRO et al. (2007b).The reason for this discrepancy can be attributed to a fluorochrome difference used in this study.While DAPI was used in the current study, PI was used in LOUREIRO et al. (2007b).The CV values of the current study were deviated from 2.53 to 4.67.PI-stained F. elastica samples had higher CVs than DAPI-stained samples (KOLÁŘ et al. 2012).The 2C DNA content of black mulberry was much higher than in the work done by HORJALES et al. (2003).In their work, HORJALES et al. ( 2003 their work which can be in accordance with a decosaploid M. nigra (2C=8.34pg) in the current study.The results obtained from the current study after nuclei staining with DAPI (binding preferentially to AT-rich regions) did not agree with those obtained using FD (OHRI; KHOSHOO 1987, DOLEŽEL et al. 1998).Higher FI was obtained using laser FC compared to FD (DOLEŽEL et al. 1998).
The present work showed that samples of unknown DNA content and the standards of known DNA content should be first prepared one by one and then, simultaneously with each other in order to see the peak positions.DAPI can give a better resolution of DNA content histograms using with lamp-based FC (BUITENDIJK et al. 1997).Due to preferential binding of DAPI to AT-rich regions of DNA, the estimation of DNA content observed after DAPI staining might be expected to differ from that of PI staining.It was stated that DAPI may be preferred as the fluorochrome for genome size estimation and PI was found to be less reliable (RAYBURN et al. 1992) ) used intercalating EB stain.They used diploid Morus spp.whose 2C DNA content is very similar to that of diploid F. carica in the current study.In other words, the 2C DNA contents of diploid Morus spp.and diploid F. carica are similar in number.On the other hand, YAMANOUCHI et al. (2010) found 2C DNA content (0.704-0.746 pg) close to that obtained by HORJALES et al. (2003) (0.79 pg).It can be said that the results obtained from decosaploid M. nigra in the current study is in agreement with both YAMANOUCHI et al. (2010) and HORJALES et al. (2003) since they used diploid Morus spp.Nevertheless, OHRI; KUMAR (1986) used probably a tetraploid Morus sp.(2C=1.70 pg) in . It would be interesting to compare DAPI with PI in fig and black mulberry.PI dye could be used to make DNA content estimation for fig and black mulberry in the future.This is a preliminary study to estimate the DNA content and genome size of black mulberry.The DNA content and genome size of diploid M. alba and M. rubra, and decosaploid M. nigra (AGAEV; FEDOROVA 1970) need to be determined in the same study.Previous knowledge from the published results can help to choose reliable internal standards before start to analyze the sample plant.Both common vetch and barley can be used to estimate DNA contents of fig and black mulberry, respectively.In the following experiments, other standards could be used for F. carica such as Arabidopsis thaliana (L.) Heynh.accession Stoc (0.9 pg, 435 Mbp), Raphanus sativus L. (1.11 pg, 544 Mbp), Lycopersicon esculentum (syn.Solanum lycopersicum L.) (1.96 pg, 1C=960 Mbp), and that for M. nigra, such as Zea mays L. (5.43 pg, 1C=2,261 Mbp), Pisum sativum L. (9.09 pg, 1C=4,454 Mbp), Secale cereale L. (16.19 pg, 1C=7,933 Mbp) (DOLEŽEL; GREILHUBER 2010).The peaks for these suggested standards appear less than three orders of the DNA content of the sample need of fig and mulberry.

Figure 1 .
Figure 1.Fluorescence intensity (relative DNA content) of Ficus carica (peak 2) and Vicia sativa (peak 3) in flow cytometry.The first peak was a default for the DNA or cell noise signal.

Figure 2 .
Figure 2. Fluorescence intensity (relative DNA content) of Morus nigra (peak 2) and Hordeum vulgare (peak 3) in flow cytometry.The first peak was a default for the DNA or cell noise signal.
1, Table 1).G1 peak of black mulberry (163.53) was 1.2 fold smaller than that of barley (208.77)(Fig 2, Table 1).The estimated 2C DNA contents of fig and black mulberry were 0.82 pg and 8.34 pg, respectively (Table 1).The ratio of 2C DNA content of the black mulberry was 10.17 times of the fig.The calculated 1C values (1C=980 Mbp) of fig and black mulberry genomes were 401.8 Mbp and 4086.6 Mbp, respectively.The ratio of 1C genome of the black mulberry was 10.17 times of the fig.The estimated 2C DNA content and calculated 1C genome values of fig were more than those of

Table 1 .
Expected DNA amount of fig (Ficus carica) and black mulberry (Morus nigra) compared to that of standards, barley (Hordeum vulgare) and common vetch (Vicia sativa)