流式细胞仪测定荔枝倍性和基因组大小的细胞核提取液筛选与应用

Nuclear isolation buffer optimization and the determination of ploidy level and genome size of litchi using flow cytometry

【目的】流式细胞术是目前测定植物倍性和基因组大小差异的最快、最有效的方法。建立适合荔枝的流式细胞术的方法,对荔枝倍性育种以及基因组大小的确定是必不可少的。【方法】笔者以荔枝的幼嫩叶片为材料,筛选适合荔枝的细胞核提取液配方,建立利用流式细胞仪测定荔枝倍性和基因组大小的方法。用改进的标准两步法,比较了6 种常用细胞核提取液提取细胞核的效果。【结果】利用WPB(Woody Plant Buffer)提取的大部分荔枝品种(品系)叶片细胞核稳定、分辨率高、细胞碎片少且细胞G0/G1峰的变异系数(CV)较低,平均CV值为5.12%,说明该配方适用于酚类物质丰富的荔枝细胞核的提取。同时以已知染色体数量的‘无核荔’(2n=30)为外标,检测了18 个品种(品系)的倍性,发现参测样品的G0/G1峰与‘无核荔’G0/G1峰的荧光均值的比值为0.78~1.24,说明所测荔枝品种(品系)均为二倍体。以已知基因组大小的‘Stupické polní rané’番茄为内标测定了14 个品种(品系)的基因组大小,结果表明荔枝基因大小约为550~620 Mb,平均602 Mb,不同荔枝品种(品系)间基因组大小存在一定差异。【结论】WPB细胞核提取液提取的荔枝幼嫩的叶片的细胞核质量好,可用于流式细胞术荔枝倍性和基因组大小的测定,测定的结果显示参测荔枝品种(品系)均为二倍体,无单倍或多倍的情况,不同荔枝品种基因组大小有一定的差异。

【Objective】Flow cytometry is particularly applicable in the determination of ploidy and genome size of diverse plant samples because it is convenient, fast and reliable. The sample preparation in flow-cytometry determination is relatively simple and less costly. The nuclei can be counted in a short time using a sample volume of only a few milligrams of plant tissues. Although chromosome counting has been used in ploidy determination in plants, in many cases representative picture of a cell population is difficult to obtain and the determination is inefficient. DNA flow cytometry requires preparation of the suspensions of intact nuclei, and suitable nuclei isolation buffer is a bottleneck for litchi ploidy and genome size estimation. The main problem is that it is not easy to prepare suspensions of intact nuclei from the leaves of woody plants, which contain cells with a rigid cell wall and secondary metabolites. This study aimed to choose the best nuclei isolation buffer and establish an appropriate flow cytometry method for litchi ploidy and genome size estimation.【Methods】In this study, young litchi leaves were selected as material to compare the nuclei isolation efficiency and extraction quality among six frequently-used buffers, including Otto’s, MgSO4, Tris-MgCl2, Galbraith’s, WPB and GPB buffer, using modified two step procedures. First, place a small amount of young litchi leaf (typically 50-100 mg) in the center of a glass petri dish, which was placed on ice to keep the sample cold. Then add 2 mL ice-cold nuclei isolation buffer to the petri dish and chop the tissue immediately in the buffer with a razor blade (use new one for each sample). Filter the chopped solution through a 53 mm nylon mesh into a new a 2 mL Eppendorf tube, which was centrifuged (1 000 r·min^-1) for 5 minutes at 4 ℃ and the supernatant was carefully remove. The nuclei in the sediment was re-suspended by gentle shaking in 400 μL new nuclei isolation buffer supplemented with 50 μg·mL^-1 PI (Propidium iodide) and 50 μg·mL-1 RNase, and filtered through a 42 mm nylon mesh. Incubate the sample for a few minutes, with occasional shaking before testing. The isolated nuclei were analyzed by Beckman Cell Lab Quanta SC FL3 fluorescence channel. The G0/G1 fluorescence value and CV (Coefficient of variation) were analyzed by Cell Lab Quanta SC software. CV of DNA peaks, presence of debris background and the number of nuclei released from sample tissue were the key parameters to evaluate those buffers.【Results】Clear differences among buffers were observed. After chopping the young leaf tissue in Tris-MgCl2, Galbraith’s or GPB isolation buffer, the isolated nuclei solution turned brown quickly, indicating that they are not suitable for litchi leaf nuclei isolation. In contrast, Otto’s Buffer, MgSO4 and WPB could isolate the nuclei and the nuclei solution stayed green. The CV of G0/G1 fluorescence peak obtained using MgSO4 buffer and Otto’s was 8.35% and 5.64%, respectively. A much better nuclei stability, higher integrity and resolution ratio, but lower amount of cell debris and CV of G0/G1 peak (3.25%) were obtained in most of samples tested using Woody Plant Buffer (WPB). These results indicated that WPB buffer was the most suitable nuclei isolation buffer for young litchi leaf and can be used for further study like ploidy and genome size estimation. Subsequently ploidy level of 18 tested cultivars/lines were determined by flow cytometry using known chromosome number cultivar‘Wuheli’(2n=30) as an external standard. The results showed that the fluorescence ratio of the G0/G1 peak among tested 18 samples compare to‘Wuheli’range from 0.78 to 1.24, which suggested that the tested cultivars/lines should belong to diploid. Genome size of 14 cultivars/lines was also determined by flow cytometry using‘Stupické polní rané’, a known genome size (958 Mb) tomato, as an internal standard control. The genome size of litchi ranged from 550 to 620 Mb with an average size of 602 Mb. Flow cytometry revealed the great variation in genome sizes among different litchi cultivars/lines.【Conclusion】Among the six frequently- used buffers, WPB nuclei isolation buffer was found to be the most effective for litchi young leaf nuclei isolation. The isolated nuclei can be used for litchi ploidy level, genome size determination and maybe other studies. The results also indicated that all the tested cultivars/lines are diploid. The genome size of litchi varies among different cultivars/lines. Hopefully, these results will provide useful information in the further studies of litchi ploidy and genome size and also new clue for other woody plant crops.