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

寡核苷酸引物是根据限制酶 Eco1091 的识别位点设计的，并在聚合酶链式反应 (PCR) 中扩增黑麦、黑麦的染色体特异性 DNA 序列，黑麦是小麦育种的有用遗传材料。 TOMITA 等人 (1993) ）发现EcoO1091识别位点在黑麦基因组中频繁分布。本研究的目的是探索EcoO1091识别位点作为PCR引物的用途。从黑麦近交系 IR27、小麦 cv. Chiness Spring (CS) 和 CS-rye Imperial 染色体附加系的 yong 叶中分离出基因组 DNA。通过Gene Assembler (Pharmacia)合成10mer引物，在总体积50μl中用20ng基因组进行PCR。 DNA、0.2 μM 10 mer 引物、100 μM 每种脱氧核糖核苷酸、1 单位 Taq 聚合酶和 1*Taq PCR 缓冲液在 The … More rmal Cycler PC-700 (ASTEC) 中进行 45 个循环。 97°C 1 分钟用于变性，36°C 1 分钟用于退火，2 分钟72℃延伸，10μl PCR产物在1×TAE缓冲液中的1.5％琼脂糖凝胶上分离，发现10种大小为黑麦染色体1R、3R、4R、5R和7R特异的片段。来自七种小麦-黑麦染色体附加系的PCR产物从琼脂糖凝胶中回收这些黑麦染色体特异性扩增产物并用作Southern杂交的探针。小麦-黑麦染色体附加系。回收的 DNA 片段与每个片段本身杂交，并且在其他 PCR 产物（尤其是小麦）中没有表现出杂交信号，表明黑麦染色体特异性扩增是黑麦染色体所独有的。用 pMOS Blue 克隆并使用 A.L.F DNA 测序仪 (Pharmacia) 测序。3R 和 5R 染色体特异性扩增显示 tRNA 样结构。经常在动物中发现分散的重复DNA序列“反座子”和与各种小麦-黑麦染色体附加系的涂片杂交模式，这一发现表明黑麦染色体特异性扩增是由分散的重复序列中EcoO1091识别位点的内部变异产生的。 EcoO1091 引物允许鉴定小麦-黑麦附加系中的黑麦染色体，并且还证明了不同来源的黑麦之间的显着多态性。这种方法使用 DNA 引物进行 PCR。从 EcoO1091 位点设计的，被证明是一种在小麦遗传背景和携带黑麦染色体较少的小麦品系中鉴定外来（黑麦）DNA 的有用方法。