Single-Feature Polymorphism Discovery in the Transcriptome of Tetraploid Alfalfa

Advances in alfalfa [Medicago sativa (L.) subsp. sativa] breeding, molecular genetics, and genomics have been slow because this crop is an allogamous autotetraploid (2n = 4x = 32) with complex polysomic inheritance and few genomic resources. Increasing cellulose and decreasing lignin in alfalfa stem cell walls would improve this crop as a cellulosic ethanol feedstock. We conducted genome-wide analysis of single-feature polymorphisms (SFPs) of two alfalfa genotypes (252, 1283) that differ in stem cell wall lignin and cellulose concentrations. SFP analysis was conducted using the Medicago GeneChip (Affymetrix, Santa Clara, CA) as a cross-species platform. Analysis of GeneChip expression data files of alfalfa stem internodes of genotypes 252 and 1283 at two growth stages (elongating, post-elongation) revealed 10,890 SFPs in 8230 probe sets. Validation analysis by polymerase chain reaction (PCR)-sequencing of a random sample of SFPs indicated a 17% false discovery rate. Functional classification and over-representation analysis showed that genes involved in photosynthesis, stress response and cell wall biosynthesis were highly enriched among SFP-harboring genes. The Medicago GeneChip is a suitable cross-species platform for detecting SFPs in tetraploid alfalfa.