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High-resolution melting (HRM) analysis relies on the use of fluorescent dyes, such as LCGreen, ResoLight, and SYTO9, which bind in a saturated manner to the double-stranded DNAs. These dyes are expensive in use and may not be affordable when dealing with a large quantity of samples. EvaGreen is a much cheaper DNA helix intercalating dye and has been used in quantitative real-time polymerase chain reaction (PCR) and post-PCR DNA melt curve analysis. Here we report on the development of an EvaGreen-based HRM analysis and its performance, in comparison with the popular LCGreen-based HRM analysis, in detection of DNA polymorphism in plants. We found that various polymorphisms ranged from single nucleotide polymorphisms (SNPs) to Indels were equally detected by using EvaGreen- or LCGreen-based HRM. EvaGreen dye was sensitive enough in discovery of SNPs in fivefold pooled samples. Using this economical dye we successfully identified multiple novel mutant alleles of Gln1-3 gene, which produces a cytosolic glutamine synthetase isoenzyme (GS1), in a maize ethyl methanesulfonate (EMS)-mutagenized library, and genotyped rice mapping populations with SNP markers. The current results suggest that EvaGreen is a promising dye for HRM analysis for its ease to use and cost effectiveness., (© 2010 Institute of Botany, Chinese Academy of Sciences.)

Key message: 5,511 genic small-size PAVs in sorghum were identified and examined, including the pattern and the function enrichment of PAV genes. 325 PAV markers were developed to construct a genetic map. Abstract: Presence/absence variants (PAVs) correlate closely to the phenotypic variation, by impacting plant genome sizes and the adaption to the environment. To shed more light on their genome-wide patterns, functions and the possibility of using them as molecular markers, we generated next generation genome sequencing data for four sorghum inbred lines and used associated bioinformatic pipelines to identify small-size PAVs (40-10 kb). Five thousand five hundreds and eleven genic PAVs (40-10 kb) were identified and found to affect 3,238 genes. These PAVs were mainly distributed on the sub-telomeric regions, but the highest proportions occurred in the vicinity of the centromeric regions. One of the prominent features of the PAVs is the high occurrence of long terminal repeats retrotransposons and DNA transposons. PAVs caused various alterations to gene structure, primarily including the coding sequence variants, intron variants, transcript ablation, and initiator codon changes. The genes affected by PAVs were significantly enriched in those involved in stress responses and protein modification. We used 325 PAVs polymorphic between two sorghum inbred lines Ji2731 and E-Tian, together with 49 SSR markers, and constructed a genetic map, which consisted of 10 linkage groups corresponding to the 10 chromosomes of sorghum and spanned 1,430.3 cM in length covering 97 % of the physical genome. The resources reported here should be useful for genetic study and breeding of sorghum and related species. [ABSTRACT FROM AUTHOR]

The presence/absence variants (PAVs) are a major source of genome structural variation and have profound effects on phenotypic and genomic variation in animals and humans. However, little is understood about PAVs in plant genomes. Our previous resequencing effort on three sorghum ( Sorghum bicolour L.) genomes, each 12× coverage, uncovered 5 364 PAVs. Here, we report a detailed characterization of 51 large-size (>30 kb) PAVs. These PAVs spanned a total size of 2.92 Mb of the sorghum genome containing 202 known and predicted genes, including 38 genes annotated to encode cell death and stress response genes. The PAVs varied considerably for repeat sequences and mobile elements with DNA transposons as the major components. The frequency and distribution of these PAVs differed substantially across 96 sorghum inbred lines, and the low- and high frequency PAVs differed in their gene categories. This report shed new light on the occurrence and diversity of PAVs in sorghum genomes. Our research exemplifies a new perspective to explore genome structural variation for genetic improvement in plant breeding. [ABSTRACT FROM AUTHOR]