Detection and quantification of in vitro-culture induced chimerism using simple sequence repeat (SSR) analysis in Theobroma cacao (L.)
Detection and quantification of in vitro-culture induced chimerism using simple sequence repeat (SSR) analysis in Theobroma cacao (L.)
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DOI：
10.1007/s00122-004-1823-5
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发表时间：
2004-10
影响因子：
5.4
通讯作者：
C. R. Rodríguez López;A. Wetten;M. Wilkinson
中科院分区：
文献类型：
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作者：
C. R. Rodríguez López;A. Wetten;M. Wilkinson
从体外培养再生的植物中，突变率通常会升高，从而产生所谓的“体细胞克隆变异”。对培养过程中出现的突变谱的详细表征应该可以提高我们对影响突变的过程的理解，并允许选择产生最少/最不严重变化的方案。体细胞突变通常会产生遗传嵌合体，其中一些细胞保留未改变的等位基因。这种嵌合体很难检测到，但可能在任何再生群体中占很大比例。我们提出了一个简单的方案，可以临时诊断大型再生群体中的同质突变体和嵌合突变体，以及估计突变细胞比例的半定量方法。该测定利用杂合基因座上交替简单序列重复等位基因的一致差异扩增。对两种基因型等位基因的相对扩增以及由它们产生的合成嵌合体的校准揭示了毛细管电泳后代表交替等位基因的“峰高度”之间存在很强的线性关系。只要不常见等位基因超过模板的 15%，该测定就能以合理的置信度 (±5%) 预测嵌合组成。该系统应用于 233 个可可体细胞胚胎发生再生体，并鉴定了 72 个（31%）两个基因座滑移突变或等位基因丢失的推定嵌合突变体。
Mutation rates are often elevated in plants regenerated from in vitro culture, giving rise to so-called ‘somaclonal variation’. Detailed characterisation of mutation profiles that arise during culture should improve our understanding of processes influencing mutation and allow the selection of protocols yielding the fewest/least severe changes. Somatic mutations will usually produce genetic chimeras where unchanged alleles are retained by some cells. Such chimeras are difficult to detect but likely to form a significant proportion of any regenerant population. We present a simple protocol that enables the provisional diagnosis of both homogenous and chimeric mutants among large regenerant populations, together with a semi-quantitative means of estimating the proportion of mutant cells. The assay exploits consistent differential amplification of alternate simple sequence repeat alleles at heterozygous loci. Calibration of the relative amplification of alleles from two genotypes—and the synthetic chimeras created from them—revealed a strong linear relationship between ‘peak heights’ representing alternate alleles following capillary electrophoresis. The assay predicts chimeric composition to a reasonable level of confidence (±5%) so long as the infrequent allele exceeds 15% of the template. The system was applied to 233 regenerants of cocoa somatic embryogenesis and identified 72 (31%) putative chimeric mutants for slippage mutation or allele loss across two loci.