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

根据限制性内切酶EcoO1091的识别位点设计寡核苷酸引物，通过聚合酶链式反应(PCR)扩增出小麦育种有用的黑麦染色体特异DNA序列。(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利用EcoO1091识别位点作为聚合酶链式反应的引物来扩增遗传背景中的外源(黑麦)序列。从黑麦自交系IR27的幼叶中提取基因组DNA。中国春(CS)和黑麦(CS-Rye cv.)帝国染色体附加系。用基因组装仪(Pharmacia)合成了45种10聚体引物。用20 ng的基因组DNA、0.2mM的10聚体引物、100mM的脱氧核糖核酸、1单位Taq聚合酶和1×Taq聚合酶缓冲液，对50亩L进行聚合酶扩增。聚合酶链式反应在…中进行更正常的循环PC-700(ASTEC)45个循环，在97ﾟC下1分钟变性，在36ﾟC下1分钟退火，在72ﾟC下2分钟延伸。10亩L扩增产物经1.5%琼脂糖凝胶在1×TAE缓冲液中分离。在7个小麦-黑麦染色体附加系的扩增产物中发现了10种大小与黑麦1R、3R、4R、5R和7R染色体特异的片段。从琼脂糖凝胶中回收这些黑麦染色体特异扩增产物，作为Southern杂交的探针，作为小麦-黑麦染色体附加系的PCR产物。回收的DNA片段与每个片段本身杂交，而在其他扩增产物中没有杂交信号，特别是来自小麦的DNA片段，表明黑麦染色体特异扩增是黑麦染色体所特有的。用PMOS Blue克隆了这些黑麦染色体特异序列，并用A.L.F DNA测序仪(Pharmacia)进行了测序。3R和5R染色体特异扩增呈现类似tRNA的结构，这种结构常见于动物将重复DNA序列“逆转录”和涂抹杂交模式分散到各种小麦-黑麦染色体附加系中。这一发现表明，黑麦染色体特异扩增是由EcoO1091识别位点在分散的重复序列中的内部变异产生的。EcoO1091引物可以识别小麦-黑麦附加系中的黑麦染色体，并在不同来源的黑麦中表现出显著的多态性。该方法利用从EcoO1091位点设计的DNA引物进行聚合酶链式反应，是鉴定小麦遗传背景和携带黑麦染色体的小麦品系中外源(黑麦)DNA的有效方法。较少

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