High Resolution Melt (HRM) analysis is an efficient tool to genotype EMS mutants in complex crop genomes.
High Resolution Melt (HRM) analysis is an efficient tool to genotype EMS mutants in complex crop genomes.
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DOI：
10.1186/1746-4811-7-43
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发表时间：
2011-12-08
期刊：
影响因子：
5.1
通讯作者：
Broadley MR
中科院分区：
文献类型：
--
作者：
Lochlainn SÓ;Amoah S;Graham NS;Alamer K;Rios JJ;Kurup S;Stoute A;Hammond JP;Østergaard L;King GJ;White PJ;Broadley MR
基因组中靶向诱导位点损伤（TILLING）越来越多地用于产生和鉴定作物基因组的靶基因中的突变。在包括芜菁在内的许多作物物种中已经产生了数千个品系的TILLING群体。通过TILLING鉴定的突变体的遗传分析需要一种高效、高通量和成本有效的基因分型方法来跟踪多代突变。高分辨率解链（HRM）分析已用于许多系统中以鉴定单核苷酸多态性（SNP）和插入/缺失（IN/DEL），从而能够对不同类型的样品进行基因分型。HRM非常适合复杂作物基因组中多个TILLING突变体的高通量基因分型。迄今为止，它已被用于鉴定突变体和基因型单突变。本研究的目的是确定HRM是否可以促进通过TILLING鉴定的多个突变系的下游分析，以便在复杂作物基因组中跨多个世代检测EMS诱导的靶基因突变的等位基因系列。我们证明，HRM可用于基因型突变的两个基因，BraA.CAX1a和BraA.MET1.a在芜菁的等位基因系列。我们分析了BraA.CAX1.a中的12个突变和BraA. MET 1.a中的5个突变，包括与野生型的回交。使用市售的HRM试剂盒和Lightscanner™系统，我们能够检测两种基因的杂合和纯合状态的突变。对TILLING衍生的突变体使用HRM基因分型，可以在多个靶基因内快速产生等位基因系列突变。从接受来自RevGenUK TILLING服务的芜菁M3种子起大约8-9个月（3代）可以分离适合于表型分析的品系。
Targeted Induced Loci Lesions IN Genomes (TILLING) is increasingly being used to generate and identify mutations in target genes of crop genomes. TILLING populations of several thousand lines have been generated in a number of crop species including Brassica rapa. Genetic analysis of mutants identified by TILLING requires an efficient, high-throughput and cost effective genotyping method to track the mutations through numerous generations. High resolution melt (HRM) analysis has been used in a number of systems to identify single nucleotide polymorphisms (SNPs) and insertion/deletions (IN/DELs) enabling the genotyping of different types of samples. HRM is ideally suited to high-throughput genotyping of multiple TILLING mutants in complex crop genomes. To date it has been used to identify mutants and genotype single mutations. The aim of this study was to determine if HRM can facilitate downstream analysis of multiple mutant lines identified by TILLING in order to characterise allelic series of EMS induced mutations in target genes across a number of generations in complex crop genomes. We demonstrate that HRM can be used to genotype allelic series of mutations in two genes, BraA.CAX1a and BraA.MET1.a in Brassica rapa. We analysed 12 mutations in BraA.CAX1.a and five in BraA.MET1.a over two generations including a back-cross to the wild-type. Using a commercially available HRM kit and the Lightscanner™ system we were able to detect mutations in heterozygous and homozygous states for both genes. Using HRM genotyping on TILLING derived mutants, it is possible to generate an allelic series of mutations within multiple target genes rapidly. Lines suitable for phenotypic analysis can be isolated approximately 8-9 months (3 generations) from receiving M3 seed of Brassica rapa from the RevGenUK TILLING service.
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影响因子：
5.3
作者：
Knoll JE;Ramos ML;Zeng Y;Holbrook CC;Chow M;Chen S;Maleki S;Bhattacharya A;Ozias-Akins P
通讯作者：
Ozias-Akins P
影响因子：
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作者：
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通讯作者：
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影响因子：
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通讯作者：
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影响因子：
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作者：
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通讯作者：
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影响因子：
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作者：
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通讯作者：
Rossi, Anna Maria