Abstract
BackgroundSingle 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.ResultsThe 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.ConclusionsWe 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.
Highlights
Single nucleotide polymorphisms (SNPs) are the most common type of sequence variation among plants and are often functionally important
For the purposes of annotation, the combined alfalfa transcript assemblies were located on the Medicago truncatula Gene Index (MtGI) Release 10.0 maintained by the Dana Farber Cancer Institute
We focused on validating Category 2 single nucleotide polymorphisms (SNP) due to their suitability for mapping in tetraploid alfalfa based on their segregation patterns in this population
Summary
Single nucleotide polymorphisms (SNPs) are the most common type of sequence variation among plants and are often functionally important. DNA pyrosequencing using 454 sequencing technology enables sequencing millions of high-quality DNA bases per sequencing run [1,2] This method has been used successfully for transcriptome sequencing and identification of single nucleotide polymorphisms (SNP) in many plant species including maize (Zea mays L.) [3], sugarcane (Saccarum spp.) [4], eucalyptus (Eucalyptus grandis) [5], and the model legume and close relative of alfalfa, M. truncatula [6]. Alfalfa is a high yielding perennial species that requires little or no nitrogen fertilizer because of its ability to carry out symbiotic nitrogen fixation and can be harvested multiple times during the growing season It has been an important component of sustainable agricultural systems for many years and has recently been promoted as a potential bioenergy crop [13]. Alfalfa like many other crop species, lacks validated SNP markers which are required in large numbers for map-based gene isolation, association genetics and genomic selection approaches [10]
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