Caetano-Anollés G, Bassam B J, Gresshoff P M
Institute of Agriculture and Center for Legume Research, University of Tennessee, Knoxville 38901-1071.
Mol Gen Genet. 1993 Oct;241(1-2):57-64. doi: 10.1007/BF00280201.
Multiple endonuclease digestion of template DNA or amplification products can increase significantly the detection of polymorphic DNA in fingerprints generated by multiple arbitrary amplicon profiling (MAAP). This coupling of endonuclease cleavage and amplification of arbitrary stretches of DNA, directed by short oligonucleotide primers, readily allowed distinction of closely related fungal and bacterial isolates and plant cultivars. MAAP analysis of cleaved template DNA enabled the identification of molecular markers linked to a developmental locus of soybean (Glycine max L. Merrill). Ethyl methane sulfonate (EMS)-induced supernodulating, near-isogenic lines altered in the nts locus, which controls nodule formation, could be distinguished from each other and from the parent cultivar by amplification of template pre-digested with 2-3 restriction enzymes. A total of 42 DNA polymorphisms were detected using only 19 octamer primers. In the absence of digestion, 25 primers failed to differentiate these soybean genotypes. Several polymorphic products co-segregated tightly with the nts locus in F2 families from crosses between the allelic mutants nts382 and nts1007 and the ancestral G. soja Sieb. & Succ. PI468.397. Our results suggest that EMS is capable of inducing extensive DNA alterations, probably around discrete mutational hot-spots. EMS-induced DNA polymorphisms may constitute sequence-tagged markers diagnostic of specific genomic regions.
对模板DNA或扩增产物进行多次核酸内切酶消化，可显著提高通过多任意扩增子谱分析（MAAP）生成的指纹图谱中多态性DNA的检测率。核酸内切酶切割与由短寡核苷酸引物引导的任意DNA片段的扩增相结合，能够轻松区分密切相关的真菌和细菌分离株以及植物品种。对切割后的模板DNA进行MAAP分析，能够鉴定与大豆（Glycine max L. Merrill）一个发育位点连锁的分子标记。通过对用2 - 3种限制酶预消化的模板进行扩增，可以区分在nts位点发生改变的、由甲基磺酸乙酯（EMS）诱导的超结瘤近等基因系，这些系控制着根瘤形成，它们彼此之间以及与亲本品种都能区分开来。仅使用19个八聚体引物就检测到了总共42个DNA多态性。在没有消化的情况下，25个引物无法区分这些大豆基因型。在等位基因突变体nts382和nts1007与祖先大豆G. soja Sieb. & Succ. PI468.397杂交产生的F2家族中，几个多态性产物与nts位点紧密共分离。我们的结果表明，EMS能够诱导广泛的DNA改变，可能是在离散的突变热点周围。EMS诱导的DNA多态性可能构成特定基因组区域诊断性的序列标签标记。
