Comparative Transcriptome Analysis of Saccharum

Two wild type Saccharum species, Molokai6081 (S. robustum, SR 2n = 8× = 80), and SES208 (S. spontaneum, SS, 2n = 8× = 64), one cultivated species, LA Purple (S. officinarum, SO, 2n = 8× = 80), and one hybrid cultivar ROC-22 (SH 2n = 8× = 100–130) were used in this study [43 (link)]. Plants were grown in the field on the campus of Fujian Agricultural and Forestry University (Fuzhou, China) in the February of 2015. Tissue samples from leaf roll, leaf, top internode (i.e., internode number 3), maturing internode (i.e., internode number 9 for ‘LA Purple and Roc-22, internode number 8 for Molokai6081, and internode number 6 for SES208) and mature internode (i.e., internode number 15 for ‘LA Purple’ and Roc-22), internode number 13 for Molokai6081, internode number 9 for SES208) were collected from premature 7-month-old sugarcane plants and 11-month-old mature sugarcane plants from different branches of the same individuals (as replicates). Internodes were numbered from the top, as previously described [44 (link)], and the corresponding internode number for the different Saccharum species, due to the variation in number of stems, was also established according to the previously described approach [45 (link)].
The plants for PEG and hormone treatment were grown in a growth chamber at 30 °C, 70% RH, and a 14 h:10 h L:D photoperiod. Seedlings were treated with PEG6000 (30%) for 48 h, and the leaf tissue was collected for RNA isolation. Seedlings were treated with gibberellin (GA,200 μM), abscisic acid(ABA, 200 μM), indole-3-acetic acid(IAA, 200 μM), or ethephon (Et, 200 μM) for 24, 48, and 96 h. Stem and leaf tissues from the seedlings of the four sugarcane species were collected from 35-day-old plants. Harvested tissue was immediately frozen in liquid nitrogen and stored at −80 °C prior to RNA isolation.