Cross hybridization breeding of sake yeasts is hampered by difficulty in acquisition of haploid cells through sporulation. We previously demonstrated that typical sake yeast strains were defective in meiotic chromosome recombination, which caused poor sporulation and loss of spore viability. In this study, we screened a single copy plasmid genomic DNA library of the laboratory Saccharomyces cerevisiae GRF88 for genes that might complement the meiotic recombination defect of UTCAH-3, a strain derived from the sake yeast Kyokai no. 7 (K7). We identified the SPO11 gene of the laboratory strain (ScSPO11), encoding a meiosis-specific endonuclease that catalyzes DNA double-strand breaks required for meiotic recombination, as a gene that restored meiotic recombination and spore viability of UTCAH-3. K7SPO11 could not restore sporulation efficiency and spore viability of UTCAH-3 and a laboratory strain BY4743 spo11Δ/spo11Δ, indicating that K7SPO11 is not functional. Sequence analysis of the SPO11 genes of various Kyokai sake yeasts (K1, and K3–K10) revealed that the K7 group of sake yeasts (K6, K7, K9, and K10) had a mutual missense mutation (C73T) in addition to other three common mutations present in all Kyokai yeasts tested. ScSPO11^C73T created through in vitro mutagenesis could not restore spore viability of BY4743 spo11Δ/spo11Δ. On the other hand, K8SPO11, which have the three common mutations except for C73T could restore spore viability of BY4743 spo11Δ/spo11Δ. These results suggest that C73T might be a causative mutation of recombination defect in K7SPO11. Moreover, we found that the introduction of ScRIM15 restored sporulation efficiency but not spore viability.

清酒酵母的交叉杂交育种由于难以通过孢子形成获得单倍体细胞而受到阻碍。我们先前证明典型的清酒酵母菌株在减数分裂染色体重组中存在缺陷，这会导致孢子形成能力差和孢子活力丧失。在这项研究中，我们筛选了实验室酿酒酵母GRF88的单拷贝质粒基因组DNA文库，寻找可能与UTCAH-3减数分裂重组缺陷互补的基因，该菌株是缘故酵母Kyokai No. 7（K7）。我们鉴定了实验室菌株（ScSPO11）的SPO11基因），它编码一种减数分裂特异的核酸内切酶，可催化减数分裂重组所需的DNA双链断裂，作为恢复UTCAH-3减数分裂重组和孢子活力的基因。K7SPO11无法恢复UTCAH-3和实验室菌株BY4743 spo11Δ / spo11Δ的孢子形成效率和孢子活力，表明K7SPO11无法正常工作。各种Kyokai清酒酵母（K1和K3-K10）的SPO11基因的序列分析表明，除其他三个酵母外，K7组清酒酵母（K6，K7，K9和K10）具有相互错义突变（C73T）所有经测试的酵母中存在的常见突变。通过创建ScSPO11 ^C73T在VITR Ø突变无法恢复BY4743的孢子生存能力SPO11 Δ/ SPO11 Δ。在另一方面，K8SPO11，其中有除C73T三个常见的突变能够恢复BY4743的孢子生存能力SPO11 Δ/ SPO11 Δ 。这些结果表明，C73T可能是K7SPO11中重组缺陷的致病突变。此外，我们发现ScRIM15的引入可恢复孢子形成效率，但不能恢复孢子活力。