Screening the Best Material for Ploidy Identification of Sugarcane-related Genera

Lianying MAO Yiyun GUI Jinju WEI Haibi LI Ronghua ZHANG Hui ZHOU Xihui LIU

Abstract [Objectives]Flow cytometry is widely used to identify plant chromosome ploidy because of its simplicity， rapidity and accuracy. Chromosome ploidy identification is an important part of sugarcane ploidy breeding and application research. It is particularly important to find out the best detection part for ploidy identification in sugarcane. [Methods]The cell suspensions of sugarcane stem tips and leaves were prepared by blade chopping method. The cell suspensions were detected by flow cytometry. The best position for ploidy identification was determined by comparing the cell suspension prepared from stem tips and cell suspension prepared from leaves. [Results]The results showed that the cell suspension dissociated from stem tips was more clear than that from leaf cell suspension; the proportion of non-adherent cells in the suspension prepared from stem tips was larger than that from the leaf cell suspension; the main peak of the stem tip cell suspension was single and the number of cells was more than that of the stem tip cell suspension by flow cytometry. Using the known ploidy ‘Badilar as the internal reference， the ploidy of cyathomi 87-16 was detected to be 8.37. [Conclusions]Sugarcane shoot tips are an ideal material for ploidy identification. This study provides a theoretical basis for selecting the best detection site for ploidy identification of sugarcane.

Key words Flow cytometry; Chromosome ploidy; Sugarcane; Cell suspension

Sugarcane （Saccharum spp. hybrids） is a C4 plant in the Gramineae family. It is a sugar and important energy crop， and can also be used as a high-quality feed and edible fungus culture material. At present， Miscanthus Anderss， Erianthus Michx. and Saccharum Linn. are the sugarcane relatives with breeding value[1]. Sugarcane is a highly heterogeneous allopolyploid plant， and the identification of chromosome ploidy is an important part of sugarcane ploidy breeding and application research. The traditional ploidy identification method is based on the morphological identification. This method is influenced greatly by subjective factors， and the ploidy accuracy of breeding materials needs further verification[2]. The identification of ploidy based on physiological and biochemical indicators and physiological conditions can only judge the ploidy level of plants from a macroscopic perspective， but there are still certain differences between varieties. The chromosome counting method is the most direct and common method using conventional tableting and wall degradation hypotonic method， but it has high technical requirements， complicated operations， and the results are not necessarily accurate[3-4]. Karyotype analysis is based on the karyotype formula to determine chromosome ploidy， but requires in-depth study on karyotypes of plants[5]. Comparing plant guard cells， the number of chloroplasts and the size， number， and ratio of stomata from the aspect of cytology has relatively higher accuracy than methods such as morphological identification and physiological and biochemical identification[6]. Flow cytometry （FCM） developed after the 1960s can detect single cell suspensions to obtain distribution curves of DNA content， from which the ploidy of plants can be directly observed. The sample preparation required by the flow cytometry takes a short time and the steps are simple and convenient; it can sort a large number of cells at one time; the DNA content can be visually displayed on the automatically generated distribution diagrams; and a variety of parameters can be measured with relatively good repeatability[7]. At present， there are many successful applications of plant identification based on flow cytometry， such as banana[8]， grape[9]， cranberry[10]， mulberry[11]， and Forsythia suspensa[12]. Ploidy identification based on flow cytometry has been used in sugarcane breeding. Liu et al.[13]used the stem tip tissue inside sugarcane shoots as the material to identify the ploidy of 46 sugarcane and their relatives. Sun et al.[5]used the young leaves of Erianthus Michx. As the material to identify 55 materials from seven provinces for ploidy. At present， the materials used to identify the ploidy of sugarcane relatives in previous researches are not the same. It is particularly important to select the detection materials of sugarcane correctly， while research on this aspect has not been reported. In this study， cell suspensions were prepared from different parts of sugarcane （stem tips and young leaves）， and comprehensively evaluated by flow cytometry， so as to screening out the most suitable part for identifying sugarcane ploidy by flow cytometry. This study provides a theoretical basis for selecting the best detection part for identifying sugarcane ploidy by flow cytometry.

Materials and Methods

Materials

Materials and reagents

The test sugarcane materials were ‘Badilar （CK） and Saccharum spontaneum 87-16， which were planted in the Sugarcane Research Institute， Guangxi Academy of Agricultural Sciences.

Preparation of reagents： ① MgSO4 dissociation solution （100 ml）： MgSO4·7H2O 0.246 g， KCl 0.370 g， Hepes 0.120 g， adjust the pH to 8.0; ② buffer A： 14.3 ml of MgSO4 was added and thoroughly mixed with 375 μl Triton X-100， and then added with 300 μl of PI and 15 mg of DTT （DTT should be dissolved after weighing immediately， because it absorbed water easily when exposed to the air）; ③ Buffer B： 3 ml of buffer A was added into and mixed well with 7.5 ml of RNase， followed by storage at 4 ℃ for later use.

Instruments and equipment

The flow cytometer used was BD AccuriTMC6 PLUS. The setting of related parameters referred to the numerical setting of Liu et al.[13]. Specifically， the threshold was 5000; FSC-SSC was selected to show a scatter diagram， in which the main cell cluster was circled; FL2-A-FL2-H was selected to show a scatter diagram， in which the PI-positive and non-adhesive cells in the upper right corner was circled; and FL2-A was used to draw a histogram.

Methods

The young leaves and stems of sugarcane were collected， placed in an ice box， washed and stored in a refrigerator at 4 ℃ for later use. An ice box was prepared in advance， and small petri dishes were placed on the ice. About 50 mg of fresh sample was weighed， added with 1 ml of pre-cooled buffer A， and quickly cut with scissors or blades to pieces<0.5 mm. The cut tissue and dissociation solution were filtered together with a 400-mesh nylon membrane into a 1.5 ml centrifuge tube （when using a pipette tip to absorb the liquid in the petri dish， the pipette tip was cut flat）. Centrifugation was performed at 8 000 r/min for 4 min， and the supernatant was discarded. The precipitate was re-suspended with 200 μl buffer B， incubated at 37 ℃ for 15 min， and tested.

Data analysis

The images obtained by flow cytometry were analyzed by Cytomics Expo32 software， and the corresponding G1 phase fluorescence intensity peak values were obtained.

Results and Analysis

The effect of different parts of sugarcane material on main cell cluster

When preparing samples with the 4 parts of materials from the 2 sugarcane varieties by cutting with blades， we found with naked eyes that the cell suspensions dissociated with leaves as the materials were relatively turbid， while the cell suspensions dissociated with stems as the materials were relatively clear. It can be seen from Fig. 1A that the non-adherent cells accounted for 2.4% of the main cell cluster of CK leaves. It can be seen from Fig. 1B that the non-adherent cells accounted for 5.3% of the main cell cluster of CK stems. It can be seen from Fig. 1C that the non-adherent cells accounted for 1.2% of the main cell cluster of S. spontaneum 87-16 leaves. It can be seen from Fig. 1D that the non-adherent cells accounted for 1.7% of the main cell cluster of S. spontaneum 87-16 stems. The above results indicated that and the proportion of non-adherent cells obtained from the cell suspension prepared with stems as the material was greater than that of the cell suspension prepared with leaves.

The effect of different parts of sugarcane material on fluorescence intensity

After the suspensions prepared from the above two kinds of materials were tested by flow cytometry， it was found that no matter whether the stems were used as the material or the leaves were used as the material， a clear peak map could be obtained （Fig. 2）. The CK leaves showed a single main peak， with slight impurity peaks on both sides of the main peak （Fig. 2A）; compared with the main peak of the leaves， the main peak of the CK stems was shifted to the right， and showed increased impurity peaks on both sides， while the cell contents were not much different （Fig. 2B）; and the fluorescence intensity of the CK leaves was 429 280 （2n=80）， and that of the stems was 655 217. Based on the above results， the stem tip of sugarcane is an ideal material for ploidy identification.

Identification of ploidy of sugarcane sibling species S. spontaneum 87-16

In this study， the tropical species ‘Badilar （2n=80， octoploid） was used as the internal reference. It can be seen from Fig. 3 that the number of cells detected by the internal reference ‘Badilar was 5 511， and the number of cells detected by S. spontaneum 87-16 was 9 126. The ploidy of the tested material was calculated according to Sample ploidy=（Reference ploidy×Sample peak fluorescence intensity）/Internal reference peak fluorescence intensity[14-15]. The ploidy of S. spontaneum 87-16 was 8.37 （Fig. 3）.

Discussion

The main steps of using flow cytometry to identify plant ploidy include the selection of dissociation fluids， the preparation of cell suspensions， and on-machine inspection. Due to the diversity of plant tissues， the identification of ploidy of different plants is not only different in the dissociation fluid and detection voltage， but also in ideal material positions for ploidy determination. Zhao et al.[16]used Jerusalem artichoke tubers， roots， old leaves， and young leaves as materials and found that the fresh and young leaves of J. artichoke were ideal materials for ploidy determination. Wang et al.[17]tested the ploidy of the young， mature and old leaves of Linyi Lizao and found that the young leaves were more suitable as the material for jujube chromosome ploidy identification， and mature leaves that had not got old could also be used for ploidy identification when there were no young leaves. PICHOT and MAATAOUI[18]selected plant endosperms of Abies， Cedar and Pinus， and successfully determined their ploidy. Plant root tip meristems can also be used to prepare chromosome cell suspensions for the study of the DNA content and ploidy of plant tissues and cells[19]. In this study， the shoot tips and young leaves of sugarcane were selected to prepare cell suspensions for comparison. According to the clarity of the suspensions， the proportion of non-adherent cells in the main cell cluster and the number of nuclei corresponding to the peak of the histogram were measured by flow cytometry， and it was found that stems were a better material for ploidy detection. When using flow cytometry to determine plant ploidy， the more the number of cells analyzed， the more accurate the results[20]. In this study， the number of cells detected by the internal reference ‘Badilar was 5 511， and the number of cells detected by S. spontaneum 87-16 was 9 126. The cells of the test materials detected were both more than 5 000， so the results obtained in this study were more accurate. This study provides a theoretical basis for selecting stem tips as the best detection part for the detection of sugarcane ploidy by flow cytometry.

Conclusions

In this study， sugarcane stem tips and leaves were selected as the materials for identifying sugarcane ploidy. Comprehensively from the clarity of the suspensions prepared， and the proportion of non-adherent cells that were positive for the PE channel and the number of relative cell nuclei corresponding to the peak of the histogram detected by flow cytometry， the best choice of the material for ploidy identification was sugarcane stem tips.

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