Abstract
Modern sugarcane is an unusually complex heteroploid crop, and its genome comprises two or three subgenomes. To reduce the complexity of sugarcane genome research, the ploidy level and number of chromosomes can be reduced using flow chromosome sorting. However, a cell cycle synchronization (CCS) protocol for Saccharum spp. is needed that maximizes the accumulation of metaphase chromosomes. For flow cytometry analysis in this study, we optimized the lysis buffer, hydroxyurea(HU) concentration, HU treatment time and recovery time for sugarcane. We determined the mitotic index by microscopic observation and calculation. We found that WPB buffer was superior to other buffers for preparation of sugarcane nuclei suspensions. The optimal HU treatment was 2 mM for 18 h at 25 °C, 28 °C and 30 °C. Higher recovery treatment temperatures were associated with shorter recovery times (3.5 h, 2.5 h and 1.5 h at 25 °C, 28 °C and 30 °C, respectively). The optimal conditions for treatment with the inhibitor of microtubule polymerization, amiprophos-methyl (APM), were 2.5 μM for 3 h at 25 °C, 28 °C and 30 °C. Meanwhile, preliminary screening of CCS protocols for Badila were used for some main species of genus Saccharum at 25 °C, 28 °C and 30 °C, which showed that the average mitotic index decreased from 25 °C to 30 °C. The optimal sugarcane CCS protocol that yielded a mitotic index of >50% in sugarcane root tips was: 2 mM HU for 18 h, 0.1 X Hoagland’s Solution without HU for 3.5 h, and 2.5 μM APM for 3.0 h at 25 °C. The CCS protocol defined in this study should accelerate the development of genomic research and cytobiology research in sugarcane.
Highlights
Modern sugarcane is an unusually complex heteroploid crop, and its genome comprises two or three subgenomes
Preliminary screening of cycle synchronization (CCS) protocols for Badila were used for some main species of genus Saccharum at 25 °C, 28 °C and 30 °C, which showed that the average mitotic index decreased from 25 °C to 30 °C
The G2/M peak was broader for suspensions prepared with LB01 buffer relative to those prepared with WPB buffer
Summary
Modern sugarcane is an unusually complex heteroploid crop, and its genome comprises two or three subgenomes. A cell cycle synchronization (CCS) protocol for Saccharum spp. is needed that maximizes the accumulation of metaphase chromosomes. Preliminary screening of CCS protocols for Badila were used for some main species of genus Saccharum at 25 °C, 28 °C and 30 °C, which showed that the average mitotic index decreased from 25 °C to 30 °C. Genome sequencing on the tetraploid S. spontaneum has been performed, the assembly accuracy was not high and many gene sequences were absent[4] To reduce this complexity, the sugarcane genome can be dissected into single chromosomes using flow cytometry. The sugarcane genome can be dissected into single chromosomes using flow cytometry Since this approach requires sufficient numbers of metaphase chromosomes, a stable and efficient cell cycle synchronization (CCS) method for sugarcane is needed. Name 14NG124 51NG-103 Badila Guan A LA Purple 12–165 2007–11 87–20 Laos-2 SES-208 82–63 82–110 82–114 51NG3 51NG63 51NG155 NG77–004 57NG208 Songxizhe Nagans Pansahi ROC22 Guitang[30] Mintang[01–77] Yuetang[96–86] Yunzhe0551 ROC10 Funong[40]
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