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 crossspecies platform. Analysis of GeneChip expression data fi les 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 classifi cation 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. ALFALFA [Medicago sativa (L.) subsp. sativa], a member of the Fabaceae, is the most widely cultivated forage legume in the world (Michaud et al., 1988) and the fourth most widely grown crop in the United States (Samac et al., 2006). In 2008, over 60 million Mg of alfalfa dry hay with a value of over $10 billion were harvested from over 8.5 million hectares (NASS, 2008). In addition to being a valuable forage crop for livestock, alfalfa has considerable potential as a sustainable, cellulosic feedstock for ethanol production (Samac et al., 2006). Th e strategy for development of alfalfa as a bioenergy feedstock involves separating stems and leaves. Th e leaves would be used as a protein supplement for livestock while the stems would be used to produce ethanol. Th e effi ciency of ethanol production from cellulosic biomass is positively correlated with cellulose content but negatively correlated with lignin content (Chapple et al., 2007; Chen and Dixon, 2007). Th e value of alfalfa as a cellulosic feedstock would be enhanced by developing new alfalfa varieties that have increased cellulose and decreased lignin in stem cell walls. Published in The Plant Genome 2:224–232. Published 5 Nov. 2009. doi: 10.3835/plantgenome2009.03.0014 © Crop Science Society of America 677 S. Segoe Rd., Madison, WI 53711 USA An open-access publication All rights reserved. No part of this periodical may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Permission for printing and for reprinting the material contained herein has been obtained by the publisher. W.W. Xu, Supercomputing Institute for Advanced Computational Research, Univ. of Minnesota, 117 Pleasant St. SE, Minneapolis, MN 55455; S.S. Yang and M. Tesfaye, USDA-ARS, Plant Science Research Unit, 1991 Upper Buford Cir., St. Paul, MN 55108; J.F.S. Lamb, H.-J.G. Jung, C.P. Vance, and J.W. Gronwald, USDA-ARS, Plant Science Research Unit, St. Paul, MN 55108 and Dep. of Agronomy and Plant Genetics, Univ. of Minnesota, 1991 Upper Buford Cir., St. Paul, MN 55108; D.A. Samac, USDA-ARS, Plant Science Research Unit, St. Paul, MN 55108 and Dep. of Plant Pathology, 1991 Upper Buford Cir., Univ. of Minnesota, St. Paul, MN 55108. Present address of M. Tesfaye: Dep. of Plant Biology, 1445 Gortner Ave, Univ. of Minnesota, St. Paul, MN 55108. S. Yang and W. Xu contributed equally to this work. Received 18 Mar. 2009. *Corresponding authors ([email protected]), (John. [email protected]). Abbreviations: ES, elongating stem internodes; EST, expressed sequence tag; FDR, false discovery rate; MAS, marker-assisted selection; PES, post-elongation stem internodes; PCR, polymerase chain reaction; PM, perfect match; SAM, signifi cance analysis of microarrays; SFP, single-feature polymorphism; SNP, single nucleotide polymorphism.