Mol Breeding (2012) 29:489–501 DOI 10.1007/s11032-011-9566-x High-resolution melting analysis for SNP genotyping and mapping in tetraploid alfalfa (Medicago sativa L.) Yuanhong Han • Dong-Man Khu • Maria J. Monteros Received: 16 December 2010 / Accepted: 12 March 2011 / Published online: 29 March 2011 Ó The Author(s) 2011. This article is published with open access at Springerlink.com Abstract Single nucleotide polymorphisms (SNPs) a 384-well format is a fast, consistent, and efficient represent the most abundant type of genetic polymor- approach for SNP discovery and genotyping, useful in phism in plant genomes. SNP markers are valuable polyploid species with uncharacterized genomes. tools for genetic analysis of complex traits of agro- Possible applications of this method include variation nomic importance, linkage and association mapping, discovery, analysis of candidate genes, genotyping for genome-wide selection, map-based cloning, and comparative and association mapping, and integration marker-assisted selection. Current challenges for of genome-wide selection in breeding programs. SNP genotyping in polyploid outcrossing species include multiple alleles per loci and lack of high- Keywords Alfalfa Á Medicago sativa Á throughput methods suitable for variant detection. In Mapping Á SNP Á HRM this study, we report on a high-resolution melting (HRM) analysis system for SNP genotyping and mapping in outcrossing tetraploid genotypes. The Introduction sensitivity and utility of this technology is demon- strated by identification of the parental genotypes and Single base changes in sequence, including single segregating progeny in six alfalfa populations based on nucleotide polymorphisms (SNPs) and small insertions unique melting curve profiles due to differences in and deletions (InDels), are the most abundant source of allelic composition at one or multiple loci. HRM using variation across genomes (Kwok 2001). Compared to simple sequence repeats (SSRs), SNPs are more amenable to high-throughput automated genotyping Electronic supplementary material The online version of assays that allow samples to be genotyped faster and this article (doi:10.1007/s11032-011-9566-x) contains supplementary material, which is available to authorized users. more economically (Hurley et al. 2005; Ha and Boerma 2008). SNP detection technologies have evolved from Y. Han Á D.-M. Khu Á M. J. Monteros (&) expensive, time-consuming, and labor-intensive pro- Forage Improvement Division, The Samuel Roberts Noble cesses to some of the most highly automated, efficient, Foundation, 2510 Sam Noble Parkway, Ardmore, OK 73401, USA and relatively inexpensive methods of DNA marker e-mail: [email protected] detection (Kwok and Chen 2003). Y. Han Polyploidy is widespread in plants and of greater e-mail: [email protected] evolutionary importance than originally supposed D.-M. Khu (Soltis and Soltis 1995). Autopolyploid species have e-mail: [email protected] more than two homologous chromosomes such that 123 490 Mol Breeding (2012) 29:489–501 pairing during meiosis can occur either between monitoring fluorescence during an increase in temper- randomly chosen pairs of homologous chromosomes ature and discriminated by their characteristic melting or between more than two homologous chromo- curves, visualized by a loss of fluorescence as the DNA somes, resulting in a polysomic pattern of inheritance duplex melts (Zhou et al. 2004). HRM can be (Gallais 2003). Autopolyploid species are generally performed with or without unlabeled probes. Duplexes outcrossing, which maintains a high level of heter- may be formed from the two strands of a PCR ozygosity and hence greater genetic diversity com- amplicon or from an oligonucleotide probe and an pared to their diploid progenitors. Examples of amplicon strand (Taylor 2009). When probes are not autopolyploid species include alfalfa (Medicago used, the sensitivity of genotyping can be increased by sativa L.), sugarcane (Sacharum officinarum L.), using internal temperature standards and reducing the and potato (Solanum tuberosum L.). Alfalfa is a amplicon size (Seipp et al. 2007). Previous knowledge perennial species that requires little or no nitrogen of the SNP variation present in a genotype is not fertilizer because of its ability to perform symbiotic required; thus HRM analysis offers unique opportuni- nitrogen fixation. Most commercially grown alfalfa ties for genotyping species with limited genomic species are tetraploid (2n = 4x = 32); however, resources. HRM has been used in genotyping in many diploid species including M. sativa subsp. caerulea plant species including apple (Malus domestica are known (McCoy and Bingham 1988). Alfalfa is Borkh.) (Chagne´ et al. 2008), white lupin (Lupinus partially self-incompatible and populations are quite albus L.) (Croxford et al. 2008), almond (Prunus dulcis heterozygous due to their high degree of outcrossing. (Mill) D.A.Webb) (Wu et al. 2009), perennial ryegrass Although the possible presence at many loci of more (Lolium perenne L.) (Studer et al. 2009), and potato than two alleles (heterozygosity) for each individual (S. tuberosum) (De Koeyer et al. 2010). In this study, gives perennial plants a buffering advantage to we developed a HRM analysis system to screen and survive unstable environments (Soltis and Soltis genotype SNP markers in diploid and tetraploid alfalfa 1995), it offers unique challenges for SNP genotyping and to map SNP markers in tetraploid F1 and backcross due to the number of possible genotypes based on the populations. number of alleles. A number of SNP genotyping methods emerged from the availability of the Human Genome Project Materials and methods and vary in terms of their mechanisms for allelic discrimination, reaction formats, and method of Plant materials detection (Kwok 2001). Homogenous, closed-tube methods for SNP genotyping that do not require a Parents and subsets of progeny from two diploid and separation step are attractive for their simplicity and four tetraploid alfalfa mapping populations were containment of amplified products. Melting curve evaluated in this study (Electronic Supplementary analysis using fluorescent dyes has proven to be a Material Table 1). The diploid population DIP1 was highly sensitive method for mutation discovery and derived from a cross between M. sativa subsp. caerulea SNP genotyping (Liew et al. 2004; Cho et al. 2008; genotypes MS-13 and MS-186. The second diploid Smith et al. 2008). High-resolution melting (HRM) population (DIP2) was obtained from a cross between analysis is a closed-tube method for rapid analysis of P1-4, a M. sativa subsp. caerulea genotype, and P6–1, genetic variation within PCR amplicons (Reed and which is a genotype from M. prostrata, a diploid Wittwer 2004). Upon completion of PCR in the species closely related to alfalfa (Wang and Brummer presence of a saturating intercalating dye such as 2010). Clonally propagated samples from a single F1 LCGreenÒ, which binds to double-stranded but not plant (F1-192) resulting from a cross between the single-stranded DNA, the PCR product is heated while tetraploid genotypes M. sativa subsp. sativa var. the level of fluorescence is measured. As the temper- Chilean and M. sativa subsp. falcata var. Wisfal ature rises and the duplex melts, dye is released and (Sledge et al. 2005) were backcrossed to the Chilean fluorescence intensity is reduced. Genetic variants and the Wisfal parent to generate the backcross with differences in base composition result in differ- populations CHBC and MFBC, respectively. The ences in melting temperature, which are detected by tetraploid genotype Altet-4 was crossed to the breeding 123 Mol Breeding (2012) 29:489–501 491 lines NECS-141 and 95–608 to generate the two defined on the negative first derivative plot for data tetraploid F1 populations AL608 and ALNECS (Khu calibration. The range of target melting region was et al. 2010). Young leaf samples from all plants were defined to normalize the data. After data calibration harvested and processed for DNA extraction using the and normalization, the samples were assigned to DNeasy plant kit (Qiagen, Valencia, CA, USA) as different genotyping groups by the LightScanner previously described (Zhang et al. 2008). Software using hierarchical clustering based on the shape of their normalized melting curves and peaks. Primer design and HRM analysis The sensitivity level was adjusted to discriminate all visually distinguishable groups. The clustering of Twenty primer pairs targeting SNP sites were eval- genotypes into distinct groups was visualized as both uated using the HRM assay (Supplementary Table 2). normalized melting curve plots and normalized Three primers were developed from M. truncatula melting peaks that correspond to the normalized putative transcription factor sequences. Criteria for negative first derivative plot. PCR primer design includes a predicted annealing temperature (Tm) of 58–61°C, limited self-comple- Sequencing PCR amplicons mentarity and poly-X, and PCR amplicon lengths of 100–150 base pairs (bp). Secondary hits were min- The PCR products from the three transcription factor imized by aligning primer candidates to all known primers were directly sequenced using a BigDye Medicago sequences and eliminating primer pairs Terminator v3.1 Cycle Sequencing Kit (Applied with multiple potential hits, as previously described Biosystems) using both forward and reverse primers. (Kakar et al. 2008). The remaining 17 primers were The amplicons were analyzed using an ABI 3730 developed from