High Resolution Melt (HRM) analysis is an efficient tool to genotype EMS mutants in complex crop genomes

Plant Methods

Martin R Broadley⁵  Phillip J White³  Graham J King²  Lars Østergaard¹  John P Hammond⁵  Andrew Stoute⁴  Smita Kurup⁴  Juan J Rios⁵  Khalid Alamer⁵  Neil S Graham⁵  Stephen Amoah⁴  Seosamh Ó Lochlainn⁵ 

[1] Department of Crop Genetics, John Innes Centre, Norwich, NR4 7UH, UK;Southern Cross Plant Science, Southern Cross University, PO Box 157Lismore NSW 2480, Australia;The James Hutton Institute, Invergowrie, Dundee, DD2 5DA, UK;Rothamsted Research, Harpenden AL5 2JQ, UK;School of Biosciences, University of Nottingham, Sutton Bonington Campus, Loughborough LE12 5RD, UK

关键词: genotyping;    Brassica;    HRM;    TILLING;   

Others  :  822834
DOI  :  10.1186/1746-4811-7-43

 received in 2011-09-06, accepted in 2011-12-08,  发布年份 2011

【 摘 要 】

Background

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.

Results

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.

Conclusions

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.

【 授权许可】

   
2011 Lochlainn et al; licensee BioMed Central Ltd.

【 图 表 】

Figure 3.

Figure 2.

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