Degradome sequencing analysis for the young spike by cold stress at the pistal and stamen differentiation stage in wheat
For the common wheat, spring frost injury frequently occurs especially when low temperatures coincide with plant from pistil and stamen differentiation stage to anther connective formation stage, which lead to the decrease of production. Degradome sequencing analysis were done for the young spike by cold stress at the pistal and stamen differentiation stage in wheat. Two Degradome libraries for the control and cold treatment samples were constructed as previously described by German et al. In brief, ploy (A+) RNA molecules were isolated from 100 ug of total RNA using the Oligotex mRNA Mini kit (Qiagen), and then a 5’- RNA adaptor containing an Mme? recognition site was ligated to the 5’- monophosphorylate product, which contain mRNA fragments generated through mRNA-induced cleavage. The ligated products were followed by purification using theOligotex mRNA Mini kit (Qiagen). Subsequently, six PCR cycles were performed on the products of a reverse transcription reaction which were then digested with Mme?and ligated to a 3’double DNA adapter. Finally, the ligation products were amplified with 21 PCR cycles, gel-purified, and subjected to SBS sequencing by the Illumina Genome analyzer.