Mapping of LOH events by RFLP-SNP assay.

*The location and sequences of the SNP markers in YPH45 or YJM789 strain backgrounds are listed in Table S2. For URA3 and G418R, PCR and agarose gel electrophoresis is carried out to verify the presence or the absence of the corresponding sequences (N- PCR product absent; Y – PCR product present). For StyI, BsrBI, SpeI, NarI, and HindIII, PCR products are digested with the corresponding restriction enzyme and run on agarose gel to verify presence of YJM789-SNP or YPH45-SNP. For example, 700 nt region including the StyI-SNP site is amplified and digested with StyI. If both YJM789 and YPH45 are present, there will be three bands – 700 nt uncut band from YJM789 and 450 nt and 250 nt bands resulting from enzyme digest of the PCR product amplified from YPH45 sequence. If the region of YPH45 containing the StyI-SNP site is lost due to recombination, PCR/restriction digest will only result in the 700 nt uncut band from YJM789 sequence. YJM – YJM789 sequence present; YPH/YJM- both YPH45 and YJM789 sequences present. Graphical representation of LOH events are shown in Figure 3A and Figure S4.Mapping of LOH events by RFLP-SNP assay.YPH45或YJM789菌株背景中的单核苷酸多态性（Single Nucleotide Polymorphism, SNP）标记的位置与序列详见表S2。针对URA3与G418R，采用聚合酶链式反应（Polymerase Chain Reaction, PCR）与琼脂糖凝胶电泳验证对应序列的存在与否：N代表PCR产物缺失，Y代表PCR产物存在。针对StyI、BsrBI、SpeI、NarI及HindIII，先对PCR扩增产物使用对应限制性内切酶进行酶切，再通过琼脂糖凝胶电泳验证YJM789-SNP或YPH45-SNP的存在。例如，对包含StyI-SNP位点的700 nt区域进行扩增后，使用StyI进行酶切：若同时存在YJM789与YPH45的序列，则会出现三条条带——来自YJM789的700 nt未酶切条带，以及由YPH45序列扩增得到的PCR产物经酶切后产生的450 nt与250 nt条带；若因重组导致包含StyI-SNP位点的YPH45区域丢失，则PCR-限制性酶切实验仅会出现来自YJM789序列的700 nt未酶切条带。YJM代表存在YJM789序列；YPH/YJM代表同时存在YPH45与YJM789序列。杂合性缺失（Loss of Heterozygosity, LOH）事件的图形化展示详见图3A与补充图S4。通过限制性片段长度多态性-单核苷酸多态性（RFLP-SNP）分析定位LOH事件。