3.3 Approval of anther timeline based on specific genes quantification
We approved the timeline by analyzing the TA developmental and TA program cell death genes by using in qRT-PCR. We divided the anthers into five range i.e. 200-300 µm, 400-600 µm, 700-900 µm, 1100-1300 µm and 1400-2300 µm. RAFTIN1 and TaMYB80 have a role in TA development and TA program cell death, respectively. According to the timeline, TA appear in 400 µm long anthers and disappear in 1300 µm anthers (Figure 1 ). Relative expression of both genes in 200-300 µm anthers can show the presence of genes. The expression ofRAFTIN1 reached at its peak in 400-600 µm anthers whileTaMYB80 showed little expression. This observation showed that TA development has been start in this size of anthers. Similar response was observed in 700-900 µm anthers. On the other hand, the highest expression of TaMYB80 was noted in 1100-1300 µm anthers and vice versa in RAFTIN1 . This is because of that TA started to degrade after 1100 µm anthers and disappear in 1300 µm anthers. In the case of 1400-2300 µm anthers, the expression of both genes not enough to prove the presence of TA in the anther length. Such expression level therefore provides information to approve the timeline ( Figure