Cloning of random amplified polymorphic DNA molecular markers useful to introduce alien genes for wheat breeding.

随机扩增多态性 DNA 分子标记的克隆可用于引入外来基因用于小麦育种。

基本信息

批准号：
05660005
负责人：
TOMITA Motonori
金额：
$ 1.41万
依托单位：
Tottori University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1993
资助国家：
日本
起止时间：
1993 至 1994
项目状态：
已结题

项目摘要

Oligonucleotide primers were designed from the recognition sites of the restriction enzyme EcoO1091 and enabled in polymerase chain reaction (PCR) to amplify chromosome-specific DNA sequences of rye, Secale cereale, being an useful genetic stock for wheat breeding.TOMITA et al. (1993) found that EcoO1091 recognition site were frequently distributed in the rye genome. The objective of the present study is to explore the use of the EcoO1091 recognition site as PCR primers to amplify alien (rye) sequences in whert genetic backgrounds. Genomic DNAs were isolated from yong leaves of a rye inbred line IR27, a wheat cv. Chiness Spring (CS) and CS-rye cv. Imperial chromosome addition lines. Forty-five kinds of 10 mer primers were synthesized by a Gene Assembler (Pharmacia). PCR were conducted in a total volume of 50 mu l with 20ng genomic DNA,0.2 mu M of 10 mer primer, 100 mu M of each deoxyribonucleotide, 1 unit of Taq polymerase and 1*Taq PCR buffer. The PCR reactions were performed in a The … More rmal Cycler PC-700 (ASTEC) for 45 cycles of 1 min at 97ﾟC for denaturation, 1 min at 36ﾟC for annealing and 2 min at 72ﾟC for extension. Ten mu l of the PCR products were separated on 1.5% agarose gels in 1*TAE buffer. Ten kinds of fragments whose size is specific to rye chromosome 1R,3R,4R,5R and 7R were found in the PCR products from seven kinds of Wheat-Rye chromosome addition lines. These rye chromosome-specific amplification were recovered from agarose gel and used as probes for Southern Hybridization to be PCR products from Wheat-Rye chromosome addition lines. Recovered DNA fragments hybridized to each fragment itself and did not exhibit hybridization signal in the other PCR products especially from wheat, indicating that rye chromosome-specific amplifications were unique to rye chromosomes. These rye chromosome-specific sequences were cloned with pMOS Blue and sequenced using by an A.L.F DNA sequencer (Pharmacia). The 3R and 5R chromosome-specific amplifications showed tRNA-like structure which is often discovered in animal dispersed repeated DNA sequences "retroposon" and smear hybridization patterns to all kinds of Wheat-Rye chromosome addition lines. This finding indicated that rye chromosome-specific amplifications were produced by internal variations of the EcoO1091 recognition sites in dispersed repeated sequences.EcoO1091 primers allowed identification of rye chromosomes in wheat-rye addition lines and also demonstrated significant polymorphism among diverse sources of rye. This approach, PCR using DNA primers designed from the EcoO1091 site, was shown to be an useful method for the identification of alien (rye) DNA in wheat genetic backgrounds and wheat lines carrying rye chromosomes. Less

从限制酶Ecoo1091的识别位点设计寡核苷酸引物，并在聚合酶链反应（PCR）中启用，以扩增黑麦的染色体特异性DNA序列，Secale Cereale，是小麦育种的有用遗传库存。（1993）发现Ecoo1091识别位点经常分布在黑麦基因组中。本研究的目的是探索ECOO1091识别位点作为PCR引物的使用，以扩大遗传背景的外星人（RYE）序列。从黑麦叶植物IR27（小麦CV）的Yong叶子中分离出基因组DNA。中国春季（CS）和CS-RYE CV。帝国染色体添加线。由基因组件（Pharmacia）合成了45种10种MER引物。在总体积为50 MU L中，具有20Ng基因组DNA，10 MU M的10 mu Mu L，10 MER底漆，每种脱氧核糖核苷酸的100 MU M，1单位TAQ聚合酶和1*TAQ PCR缓冲液。 PCR反应是在…更多的RMAL Cycler PC-700（ASTEC）中进行的45个循环，在97°C时进行1分钟，以进行变性，在36°C下进行1分钟进行退火，在72°C下进行2分钟，以进行延伸。在1*TAE缓冲液中，将10 Mu L的PCR产物分离为1.5％琼脂糖凝胶。从七种小麦叶染色体添加线中发现了十种大小特异性的尺寸，其大小特异于黑麦染色体1R，3R，4R，5R和7R。从琼脂糖凝胶中回收了这些黑麦染色体特异性扩增，并用作南部杂交的探针，是小麦染色体添加线的PCR产物。回收的DNA片段与每个片段本身杂交，并未在其他PCR产物中尤其是小麦中表现出杂交信号，这表明黑麦染色体特异性的扩增是黑麦染色体所特有的。将这些黑性染色体特异性序列用PMOS蓝色克隆，并使用A.L.F DNA Sequencer（Pharmacia）进行测序。 3R和5R染色体特异性扩增显示了类似tRNA的结构，在动物分散的重复的DNA序列“ retroposon”中经常发现，并将其涂抹在各种小麦 - rye-rye染色体添加线上。这一发现表明，黑麦染色体特异性扩增是通过分散重复序列中的Ecoo1091识别位点的内部变化产生的。ECOO1091引物允许在小麦加成线中鉴定出小麦染色体的鉴定，并在Rye的Rye中也表现出显着的聚构象。这种方法是使用来自Ecoo1091位点设计的DNA引物的PCR，被证明是在小麦遗传背景和带有黑麦染色体的小麦背景和小麦系中鉴定出外星（黑麦）DNA的有用方法。较少的