|
|
|
| Establishment and Application of a Method for Chromosome Ploidy Identification and Genome Size Estimation Using Flow Cytometry in Ziziphus jujuba |
|
|
|
|
Abstract Flow cytometry has been widely used in chromosome ploidy identification and genome size estimation in plants. This research is to establish a flow cytometry method suitable for jujube (Ziziphus jujuba) in order to provide technological supports for jujube ploidy breeding and genome analysis. Chinese jujube 'Linyilizao' (diploid) and its autotetraploid 'Chenguang' were employed as materials to systematically compare the ploidy detection effects of cell lysis solution types, maturity level of leaves, preserved way of leaves, and dosage of propidium iodide (PI). The results showed that cell lysis solution of Tris-MgCl2, woody plant buffer (WPB) and general purpose buffer (GPB) had clear peak when the 'Linyilizao' was as the test material. The GPB and Tris-MgCl2 had clear peak, while WPB not showed peak when the 'Chenguang' was as the test material. In general, cell lysis solution of Tris-MgCl2 showed better results than cell lysis solution of WPB and GPB. So the cell lysis solution of Tris-MgCl2 was the best for cell lysis and WPB was the worst. Young leaves showed better result than mature leaves, and senescent leaves could not make clear peak. With the increase of preserved period of leaves, the testing effects slightly decreased under 4 ℃ and -20 ℃. There was no significant difference among the test effects of leaves preserved 1~3 d at 4 ℃ or 3~6 months at -20 ℃. And the leaves preserved 7 d at 4 ℃ or 12 months at -20 ℃ could still be used for ploidy identification. In consideration of cost and detection effect of different dosages of PI, 30 μL PI was optimal. By using the above established method for ploidy identification, the ratio of fluorescence intensity of triploid to diploid in Chinese jujube and sour jujube was in the range of 1.43~1.60. In addition, the ratio of fluorescence intensity of tetraploid to diploid was in the range of 1.70~2.11, while the ratio of hexaploid to diploid was 2.90~3.27. The genome size for Chinese jujube 'Dongzao' was estimated as 449.94±3.60 Mb by this method, which just has 1.33% deviation compared with the result of actual genome sequencing. Meanwhile, the genome size of sour jujube 'Xianxiansuanzao' (Z. acidojujuba), Indian jujube 'Tainan1' (Z. mauritiana) and wild diploid Indian jujube were 404.25±2.33, 1 349.73±8.22 and 462.97±8.72 Mb, respectively. In this study, a method of flow cytometry suitable for Ziziphus plants ploidy identification and genome size estimation was established. The new method is simple, accurate, labor-saving and very fast, and a detection of over 200 samples could be completed by 1 people within 1 day. It could also be used for other plants including pear (Pyrus bretschneideri), grape (Vitis vinifera), poplar (Populus trichocarpa) and cabbage (Brassica rapa). The results of this study can provide technical support for Zizyphus plant on ploidy breeding and omics study, and also a reference for other plants.
|
|
Received: 14 July 2017
Published: 14 February 2018
|
|
|
|
|
|
|
