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Title: Transcript Profiling of Two Alfalfa Genotypes with Contrasting Cell Wall Composition in Stems Using a Cross-Species Platform: Optimizing Analysis by Masking Biased Probes

item Yang, Suk
item XU, WAYNE WENZHONG - University Of Minnesota
item TESFAYE, MESFIN - University Of Minnesota
item Lamb, Joann
item Jung, Hans Joachim
item VANDENBOSCH, KATHRYN - University Of Minnesota
item Vance, Carroll
item Gronwald, John

Submitted to: BMC Genomics
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/24/2010
Publication Date: 5/24/2010
Citation: Yang, S.H., Xu, W., Tesfaye, M., Lamb, J.F., Jung, H.G., VandenBosch, K.A., Vance, C.P., Gronwald, J.W. 2010. Transcript Profiling of Two Alfalfa Genotypes with Contrasting Cell Wall Composition in Stems Using a Cross-Species Platform: Optimizing Analysis by Masking Biased Probes. Biomed Central (BMC) Genomics. 11:323. Available:

Interpretive Summary: There is a need for the U.S. to reduce its dependency on foreign sources of energy. Developing cellulosic feedstocks to be used for ethanol production addresses this need. Alfalfa, a nitrogen-fixing perennial forage widely grown in the U.S., offers considerable potential as a bioenergy crop. The model for the development of alfalfa as a bioenergy crop involves separating leaves from stems at harvest. The leaves would be used as a high-value livestock feed and the stems would be used for ethanol production. Modifying cell wall composition of alfalfa stems (increasing cellulose, decreasing lignin) would increase ethanol production. To advance the development of alfalfa as a biofuel feedstock, genes that control stem cell wall composition need to be identified. GeneChips are effective tools for identifying genes controlling plant traits, such as cell wall composition. However, an alfalfa GeneChip is not available. To overcome this limitation, we used the GeneChip developed for barrel medic, a close relative of alfalfa, to investigate gene expression in stems of two alfalfa varieties that differ in stem cell wall composition. A protocol was developed that increased the number of alfalfa genes detected on the barrel medic GeneChip. Numerous genes that regulate cell wall composition in alfalfa stems were identified. The research demonstrated that the barrel medic GeneChip can be used to identify genes for improving alfalfa as a bioenergy crop. The genes identified can be used as markers in a breeding program to develop alfalfa varieties that yield more ethanol. The new knowledge discovered in this study advances the development of alfalfa as a bioenergy crop.

Technical Abstract: The stem cell walls of alfalfa [Medicago sativa (L.) ssp. sativa] genotype 252 have high cellulose and lignin concentrations, while stem cell walls of genotype 1283 have low cellulose and lignin concentrations. The GeneChip® Medicago Genome Array, developed for Medicago truncatula, is a suitable platform for transcript profiling in tetraploid alfalfa. However, previous research involving cross-species hybridization (CSH) has shown that sequence variation between two species can bias transcript profiling resulting in decreased sensitivity and specificity. Transcript profiling using the Medicago GeneChip was conducted with elongating stem (ES) and post-elongation stem (PES) internodes from alfalfa genotypes 252 and 1283. A protocol was developed that masked probes targeting inter-species variable (ISV) regions of alfalfa transcripts. A probe signal intensity threshold was selected that optimized both sensitivity and specificity. After masking for both ISV regions and previously identified single-feature polymorphisms (SFPs), the number of differentially expressed genes between the two genotypes in both ES and PES internodes was approximately 2-fold greater than the number detected prior to masking. After masking, a total of 639 and 1129 genes were differentially expressed between the two genotypes in ES and PES internodes, respectively. Regulatory genes, including transcription factor and receptor kinase genes, that may play a role in development of secondary xylem were significantly over-represented among genes up-regulated in 252 PES internodes compared to 1283 PES Internodes. Several cell wall-related genes were also up-regulated in genotype 252 PES internodes. Real-time RT-PCR of differentially expressed regulatory and cell wall-related genes demonstrated increased specificity after masking. More than 1,000 genes that were differentially expressed in stem internodes between genotypes 252 and 1283 were mapped onto putative orthologous loci on M. truncatula chromosomes. Clustering simulation analysis of differentially expressed genes suggested co-expression of neighbouring genes on Medicago chromosomes. The problems associated with transcript profiling in alfalfa using the Medicago GeneChip as a CSH platform were mitigated by masking probes targeting ISV regions and SFPs. Differences in gene expression between stem internodes of two alfalfa genotypes reflect their phenotypic differences. Numerous candidate genes that may contribute to differences in cell wall concentration and composition of the two genotypes were identified.