Studies on development of techniques for isolating virus resistance genes in tomato

1.离Tm-1和Tm-2位点最近的DNA标记与Tm-1位点相连的已知6个DNA标记（SCAR标记）的线性顺序无法确定，因为它们不能分离m。近等基因番茄系GCR26（+/+）与GC R237 （Tm-2/Tm-2）的杂交后代。在另一种方法中，我们检测了4个多毛番茄株系。因此，估计I101100是距离Tm-1位点最近的标记。同样，我们试图通过比较4个具有Tm-2或其中一个Tm-2等位基因的番茄品系来确定已知的9个DNA标记（RAPD或SCAR标记）的线性顺序。结果表明，Nl3_<1000>、I13_<1300>、E16900和G09700最接近Tm-2位点。在番茄近等基因系中寻找抗病基因样序列我们比较了近等基因系间的PCR产物模式和Southern杂交蛋白模式，检验了近等基因系间是否存在序列差异，但未发现它们之间有任何差异。另一方面，我们发现存在大量的抗性基因样序列，并且能够估计一个序列到一个序列的分化过程。两个不同的序列。我们分离GCR26（+/+）和GCR236 （Tm-2/Tm-2）的差异cDNA片段，并测定其序列。结果，我们发现了GCR236特有的两个片段，其中一个片段的序列与编码番茄木葡聚糖内转糖酵素的基因片段相似，另一个片段与编码蛋白激酶的基因片段相似。

1.DNA markers nearest the Tm-1 and Tm-2 lociThe linear order of the known six DNA markers (SCAR markers) linked to the Tm-1 locus was not able to be determined, since they did not segregate m . progeny of a hybrid between near-isogenic tomato lines, GCR26 (+/+) and GC R237 ( Tm-2/Tm-2) . In another approach, we checked four Lycopersicon hirsutum lines. As a result, I101100 was estimated to be the nearest marker of the Tm-1 locus.Similarly, we attempted to determine the linear order of the known nine DNA markers (RAPD or SCAR markers) by comparing four tomato lines which have Tm-2 or one of the alleles of Tm-2. As a result, Nl3_<1000>, I13_<1300>, E16900 and, G09700 was estimated to be nearest the Tm-2 locus.2. Search for disease resistance gene-like sequences in near-isogenic tomato linesComparing patterns of PCR products and Southern hybridizatin patterns betwee the near-isogenic lines, we examined whether there are sequences different between near-isogenic lines, but failed to find any difference betweenthem.On the other hand, we found that there are a great number of resistance gene-like sequences, and was able to estimate a differentiation process of one sequence to .two differnt sequences.3. Detection of cDNA sequences specific to a ToMV resistant lineWe isolated cDNA fragments differentially displayed between GCR26 (+/+) and GCR236 (Tm-2/Tm-2) and determined their sequences. As a result, we found two fragments specific to GCR236, one of which has a sequence similar to apart of a gene encoding xyloglucan endo-transglycolase of tomato, and the other to a part of a gene encoding a protein kinase.