Dot-blot Analysis for Identification of japonica Rice Cultivars and Genotyping of Recombinant Inbred Lines
Dot-blot Analysis for Identification of japonica Rice Cultivars and Genotyping of Recombinant Inbred Lines
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DOI：
10.1270/jsbbs.55.187
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发表时间：
2005-06
期刊：
影响因子：
2.4
通讯作者：
K. Shirasawa;S. Kishitani;T. Nishio
中科院分区：
文献类型：
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作者：
K. Shirasawa;S. Kishitani;T. Nishio
利用AFLP和PCR-RF-SSCP分析鉴定的14个水稻品种特异性序列作为探针，对水稻品种进行了斑点杂交分析。用一种不使用有机溶剂的简单方法从叶片中制备的DNA样品被发现可用于斑点印迹分析。虽然需要不止一粒水稻作为基因组DNA的直接斑点印迹的材料，但DNA的PCR扩增使得能够通过斑点印迹分析检测来自单个谷粒的DNA样品中的品种特异性信号。所有31个水稻品种检测可以区分彼此的分析，使用10个品种特异性探针。斑点杂交分析也被应用于重组自交系（RILs）的基因分型，其结果对应于那些通过特异引物序列扩增的PCR产物的电泳分析。利用Kasalath BAC-末端序列的BlastN搜索，通过RILs和染色体片段置换系（CSSLs）分析，将“日本晴”中缺失的探针序列定位在水稻染色体上。利用水稻品种特异性序列进行斑点杂交分析，可用于水稻品种鉴定和植株基因分型。
Dot-blot analysis of rice cultivars was performed using 14 cultivar-specific sequences identified previously by AFLP and PCR-RF-SSCP analyses as probes. DNA samples prepared from leaves by a simple method without the use of organic solvent were found to be usable for dot-blot analysis. Although more than a single grain of rice was needed as material for direct dot-blotting of genomic DNA, PCR amplification of DNA enabled detection of cultivar-specific signals in a DNA sample from a single grain by dot-blot analysis. All 31 rice cultivars examined could be distinguished from each other by analysis using ten cultivar-specific probes. Dot-blot analysis was also applied to genotyping of recombinant inbred lines (RILs), the results of which corresponded to those by electrophoretic analysis of PCR products amplified with specific primer sequences. The probe sequences deleted in ‘Nipponbare’ were mapped on the rice chromosome by BlastN search using all the Kasalath BAC-end sequences and by analysis of RILs and chromosome segment substitution lines (CSSLs). Dot-blot analysis using cultivar-specific sequences was found to be applicable for the identification of cultivars and genotyping of plants in rice.