Mutation scanning of peach floral genes

HRM analysis of PpAG and PpTFL1 coding regions in 36 peach cultivars found one polymorphic site in each gene. PpTFL1 and PpAG SNPs were used to examine approaches to increase HRM throughput. Cultivars with SNPs could be reliably detected in pools of twelve genotypes. COLD-PCR was found to increase the sensitivity of HRM analysis of pooled samples, but worked best with small amplicons. Examination of QMC-PCR demonstrated that primary PCR products for further analysis could be produced from variable levels of genomic DNA.Natural SNPs in exons of target peach genes were discovered by HRM analysis of cultivars from a southeastern US breeding program. For detecting natural or induced SNPs in larger populations, HRM efficiency can be improved by increasing sample pooling and template production through approaches such as COLD-PCR and QMC-PCR. Technical advances developed to improve clinical diagnostics can play a role in the targeted mutation breeding of crops.Crops with improved traits are being developed by screening for mutations induced in candidate genes [1-5]. Several methods have been used to screen plant populations mutagenized by chemicals such as ethyl methanesulfonate (EMS). EMS-mutagenized tobacco lines, for example, were screened by SSCP analysis [1]. Tobacco genotypes with induced mutations in the nicotine N-demethylase gene (NtabCYP82E4) were identified that had dramatically reduced levels of nornicotine. TILLING was used to screen EMS-mutagenized lines of a wheat variety null for Wx-B1, one of three waxy homeologs involved in starch biosynthesis [2]. Wheat genotypes with induced Wx-A1 and Wx-D1 mutations were detected and later crossed to produce wx-a1/wx-b1/wx-d1 grain with low amylose starch. A third mutation scanning method, high resolution melting (HRM), was used to identify tomato lines with EMS-induced mutations in candidate genes regulating fruit quality and drought tolerance [3].Modifications that improve screening throughput and sensitivity would