Transcriptome Profiles of Medicago Stem Internodes

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. Increasing cellulose and decreasing lignin in alfalfa stem cell walls would improve this crop as a cellulosic ethanol feedstock. We selected two alfalfa genotypes (252, 1283) that differ in cellulose and Klason lignin concentration in stem cell walls. Analysis of GeneChip expression data files of alfalfa stem internodes of genotypes 252 and 1283 at two growth stages (elongating, post-elongation) revealed 10,887 SFPs in 8,230 probe sets. Validation analysis by PCR-sequencing of a random sample of SFPs indicated a 12% false discovery rate. Functional classification and over-representation analysis showed that both genotypes were highly enriched in SFP-harboring cell wall genes. We mapped 5,833 of the 8,230 SFP-harboring genes onto putative orthologous loci on Medicago truncatula chromosomes. Clustering and over-representation of SFP-harboring genes within the same functional class (e.g. cell wall genes) was observed on some chromosomes. Prior to analysis of expression data for the two alfalfa genotypes, SFP probes were masked to reduce false positives and false negatives. The combination of SFP and gene expression analysis provide a list of candidate cell wall genes that can be used as molecular markers in a breeding program to improve alfalfa as a cellulosic feedstock. The results of this study will also be useful in advancing understanding of genome organization in alfalfa and for comparative genomics research with other legume species. Keywords: Stem development and genotype comparison Overall design: The alfalfa clonal lines 252 and 1283 were propagated from cuttings and grown in the greenhouse. The greenhouse experiments consisted of three replicates arranged in a randomized complete block design. There were eight plants of each clone, in individual pots, in each replicate. Plant material for analysis was composited within each replicate at harvest. Stem internode tissues were harvested at full bloom. Based on tissue pliability and coloration, the internode in transition from elongation to post-elongation cambial growth was identified. The internodes immediately above (elongating internodes) and below this transition internode (post-elongation internodes) were collected for RNA extraction.