Molecular Studies on Nuclear Gene(s) Interacting with Rye Cytoplasm

在具有黑麦细胞质的普通小麦品系中，至少有一个被称为“侏儒”的微小染色体始终存在。减数分裂时，矮小染色体不与任何小麦染色体配对，没有该染色体的该品系的种子由于胚乳败育而不能存活。这些结果表明，小染色体来源于黑麦染色体(S)的某一片段(S)，具有黑麦细胞质功能所必需的基因(S)。为了分析该基因(S)，本研究试图用基因组消减的方法分离出小黑麦染色体特异的DNA序列，分离出3个不同的黑麦特异序列。斑点杂交和Southern杂交分析表明，其中一个克隆(pScm-6.39)的插入序列来源于黑麦5R染色体，另一个克隆(pScm-6.44)含有黑麦1R、2R、3R、5R和7R染色体的共同序列。结果表明，pScm-2.3插入片段来源于黑麦叶绿体DNA，并用原位杂交方法研究了矮小染色体的来源和分子结构。以生物素化黑麦基因组DNA为探针的原位杂交表明，侏儒染色体起源于黑麦染色体(S)的某一部分(S)。该侏儒染色体不具有与小麦rDNA相似的序列，也不具有与黑麦350bp重复单位端粒相似的序列。然而，另一个重复序列(120个碱基的黑麦家族)被发现出现在小染色体的一端。来自拟南芥的端粒重复序列与小麦染色体和侏儒染色体的两端杂交。

In a common wheat strain with rye cytoplasm, at least one diminutive chromosome, called "midget" always exists. The midget chromosome does not pair with any of the wheat chromosomes at meiosis, and the seeds of this strain without this chromosome are inviable because of their endosperm abortion. These results indicate that the midget chromosome was derived from certain segment(s) of rye chromosome(s), having the gene(s)essential for rye cytoplasmic function. To analyze the gene(s), in this study, we attempted to isolate the midget chromosome-specific DNA sequences by using a genomic subtraction method.Three different rye-specific sequences were isolated. Dot and Southern blot analyses showed that the inserted sequence of one clone(pScm-6.39)was derived from chromosome 5R of rye, and that clone pScm-6.44 contained a common sequence among rye chromosomes 1R, 2R, 3R, 5R and 7R. The insert of pScm-2.3 was shown to be derived from chloroplast DNA of rye.The origin and molecular structure of the midget chromosome were studied by using in situ hybridization. In situ hybridization with biotinylated rye genomic DNA as a probe clearly showed that the midget chromosome had originated from certain part(s)of rye chromosome(s). The midget chromosome did not possess sequences similar to wheat rDNA nor to rye telomeric sequence with 350-bp repeat unit. However, another repetitive sequence(120-bp family)of rye was found to occur in one distal end of the midget chromosome. The telomere repeat sequences from Arabidopsis thaliana cross-hybridized to both ends of the midget chromosome as well as wheat chromosomes.