Development of PCR-based Fingerprinting Tool in Banana (Musa sp., AAA) and Conversion of Negative to Positive DNA Marker
Development of PCR-based Fingerprinting Tool in Banana (Musa sp., AAA) and Conversion of Negative to Positive DNA Marker
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香蕉（Musa sp.，AAA）基于 PCR 的指纹识别工具的开发以及阴性 DNA 标记物到阳性 DNA 标记物的转换
DOI：
10.21273/hortsci.37.7.1108
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发表时间：
2002
期刊：
影响因子：
1.9
通讯作者：
I. Nakamura
中科院分区：
文献类型：
--
作者：
R. P. Umali;N. Kameya;I. Nakamura
香蕉（Musa sp.， AAA）基因组由于体细胞无性系变异的高频率而不断扩增。由于这种不断增加的多样性，香蕉品种的分类和系统发育鉴定的数量和形态学方法变得费力、困难，而且经常是分歧的主题。因此，本研究的目的是开发能够区分Musa， AAA卡文迪什亚群品种的DNA指纹图谱分子工具。在本文中，我们发现除了单碱基替换和缺失外，质体DNA rpl16和rpl14基因之间的非编码区PS-ID序列是高度保守的。这些差异将无性系划分为3组（G1、G2和G3），表明组内无性系亲缘关系密切。利用任意引物A13，我们随后分别为S4（来自Giant Cavendish亚群，AAA）和S11（来自‘Red’和‘Green Red’亚群，AAA）鉴定了阴性RAPD标记A13 3.0和A13 1.3。对缺失条带对应的片段进行测序，并以此为模板设计具有重叠序列的新引物。其中两个引物Ba3.0A和Ba1.3A在S4和S11中成功生成了阳性标记，扩增结果一致，分别为Ba3.0A 0.8和Ba1.3A 0.6。有人提出，上述方法可以是一个更好的替代筛选更任意引物产生阳性标记的情况下，阴性的情况下已经确定。PS-ID亚型分析结果同样表明在鉴定多倍体香蕉的野生母系祖先方面具有潜在的应用价值。
The banana (Musa sp., AAA) genome is continuously expanding due to the high frequency of somaclonal variation. Because of this increasing diversity, numerical and morphological methods of taxonomic and phylogenetic identification of banana cultivars became laborious, difficult, and often the subject of disagreements. The aim of this study, therefore, is to develop molecular tools for DNA fingerprinting that can discriminate Musa, AAA Cavendish subgroup cultivars. In this paper, we showed that the plastid- subtype identity (PS-ID) sequence of the noncoding region between rpl16 and rpl14 genes of plastid DNA was highly conserved except for single-base substitution and deletion. These differences separated the clones into three groups (G1, G2, and G3) and suggested that clones within groups are closely related maternally. Using arbitrary primer A13, we later identified negative RAPD markers A13 3.0 and A13 1.3 specifically for S4 (selection from Giant Cavendish subgroup, AAA) and S11 ('Morado' from 'Red' and 'Green Red' subgroup, AAA), respectively. Fragments corresponding to the missing bands were sequenced and used as templates to design new primers with overlapping sequences. Two of these primers, Ba3.0A and Ba1.3A, successfully generated positive markers consistently amplified as Ba3.0A 0.8 and Ba1.3A 0.6 for S4 and S11, respectively. It is proposed that the method just described can be a better alternative over screening more arbitrary primers in generating positive markers in cases when negative ones were already identified. Results of PS-ID subtype analysis likewise suggested potential use in identifying wild maternal progenitor in polyploid bananas.