ISSR分子标记在甘蔗及其近缘属分类上的应用

Applicability of inter-simple sequence repeat polymorphisms in sugarcane and its related genera as DNA markers

对11个甘蔗及其近缘属供试材料,以锚定简单重复序列为引物,通过PCR扩增和凝胶电泳,从100个引物(来自UBC#9)中得到32个引物的清晰特异图谱.出现清晰条带的二核苷酸重复序列引物19个,三核苷酸重复序列引物3个,四、五核苷酸重复序列引物分别为2、3个,5′简并基序引物4个.引物(AC)n产生的多态性条带最多,其次是5′简并基序引物和(GT)n.所有(AG)n引物和6个(TC)n引物中的5个产生多态性条带,说明甘蔗基因组中含有丰富的AG/TC重复序列.总的来看,不同属间平均出现11.6条多态性条带,属内9.5条,种内有6.6条.对32个引物的特征图谱进行聚类分析,树状图与Irvine的分类结果非常相似.由此可见ISSR分子标记在甘蔗分类、基因多态性分析和品种特征图谱的建立中有一定潜力.

One hunderd anchored SSR primers from UBC#9 were used for PCR amplification from a sample set of 11 genotypes including individuals of S.spontaneum, S.sinense, S.barberi, S.robustom, S.officinarum, and other wild relatives, Erianthus rockii, E. arundinaceus and Miscanthus floridulus. Amplification products were resolved in 1.5% agarose gels and banding patterns were compared among the 11 genotypes. Of the 100 primers tested, 32 produced clear banding patterns that could be easily scored. Nineteen of these 32 primers were designed to dinucleotide repeats, three to trinucleotide repeats, two to tetranucleotide repeats, three to pentannucleotide repeats. Four primers designed to 5′ degenerate motifs also yielded discrete repeats. All primers designed to (AG)n motifs and 5 of six (TC)n motifs gave polymorphic bands indicating that in sugarcane AG/TC is the most abundant. Primers based on (AC)n produced the highest level of polymorphism, followed by 5′ degenerate motifs and (GT)n motifs. PCR amplification using ISSR primers produced 9.5 bands, on average, for species amongst genus Saccharum, 11.6 for related genera and 6.6 within species. A similarity matrix and a dendrogram of 11 genotypes were generated, which were consistent with the result of Irvine. The characteristic features of the ISSR markers suggest their potential applicability to classification, phylogenetic analysis and construction of PCRbased genome maps in sugarcane.