Open Access Highly Accessed Research article

Genome-wide SNP discovery in tetraploid alfalfa using 454 sequencing and high resolution melting analysis

Yuanhong Han1, Yun Kang2, Ivone Torres-Jerez2, Foo Cheung34, Christopher D Town4, Patrick X Zhao2, Michael K Udvardi2 and Maria J Monteros1*

Author Affiliations

1 Forage Improvement Division, The Samuel Roberts Noble Foundation, 2510 Sam Noble Parkway, Ardmore, OK, 73401, USA

2 Plant Biology Division, The Samuel Roberts Noble Foundation, 2510 Sam Noble Parkway, Ardmore, OK, 73401, USA

3 Center for Human Immunology, Autoimmunity and Inflammation, National Institute of Health, 9000 Rockville Pike, Bethesda, MD 20892, USA

4 The J. Craig Venter Institute, 9704 Medical Center Drive, Rockville, MD 20850, USA

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BMC Genomics 2011, 12:350  doi:10.1186/1471-2164-12-350

Published: 6 July 2011

Abstract

Background

Single nucleotide polymorphisms (SNPs) are the most common type of sequence variation among plants and are often functionally important. We describe the use of 454 technology and high resolution melting analysis (HRM) for high throughput SNP discovery in tetraploid alfalfa (Medicago sativa L.), a species with high economic value but limited genomic resources.

Results

The alfalfa genotypes selected from M. sativa subsp. sativa var. 'Chilean' and M. sativa subsp. falcata var. 'Wisfal', which differ in water stress sensitivity, were used to prepare cDNA from tissue of clonally-propagated plants grown under either well-watered or water-stressed conditions, and then pooled for 454 sequencing. Based on 125.2 Mb of raw sequence, a total of 54,216 unique sequences were obtained including 24,144 tentative consensus (TCs) sequences and 30,072 singletons, ranging from 100 bp to 6,662 bp in length, with an average length of 541 bp. We identified 40,661 candidate SNPs distributed throughout the genome. A sample of candidate SNPs were evaluated and validated using high resolution melting (HRM) analysis. A total of 3,491 TCs harboring 20,270 candidate SNPs were located on the M. truncatula (MT 3.5.1) chromosomes. Gene Ontology assignments indicate that sequences obtained cover a broad range of GO categories.

Conclusions

We describe an efficient method to identify thousands of SNPs distributed throughout the alfalfa genome covering a broad range of GO categories. Validated SNPs represent valuable molecular marker resources that can be used to enhance marker density in linkage maps, identify potential factors involved in heterosis and genetic variation, and as tools for association mapping and genomic selection in alfalfa.