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.
abstract: BACKGROUND
  The GeneChip(R) Medicago Genome Array, developed for Medicago truncatula, is a suitable platform for transcript profiling in tetraploid alfalfa [Medicago sativa (L.) subsp. sativa]. However, previous research involving cross-species hybridization (CSH) has shown that sequence variation between two species can bias transcript profiling by decreasing sensitivity (number of expressed genes detected) and the accuracy of measuring fold-differences in gene expression.
  
  
  RESULTS
  Transcript profiling using the Medicago GeneChip(R) was conducted with elongating stem (ES) and post-elongation stem (PES) internodes from alfalfa genotypes 252 and 1283 that differ in stem cell wall concentrations of cellulose and lignin. 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 accuracy. 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. 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 quantitative RT-PCR of differentially expressed regulatory and cell wall-related genes demonstrated increased sensitivity and accuracy after masking for both ISV regions and SFPs. Over 1,000 genes that were differentially expressed in ES and PES internodes of genotypes 252 and 1283 were mapped onto putative orthologous loci on M. truncatula chromosomes. Clustering simulation analysis of the differentially expressed genes suggested co-expression of some neighbouring genes on Medicago chromosomes.
  
  
  CONCLUSIONS
  The problems associated with transcript profiling in alfalfa stems using the Medicago GeneChip as a CSH platform were mitigated by masking probes targeting ISV regions and SFPs. Using this masking protocol resulted in the identification of numerous candidate genes that may contribute to differences in cell wall concentration and composition of stems of two alfalfa genotypes.
authors.affiliation: USDA-Agricultural Research Service, Plant Science Research Unit, St, Paul, MN 55108, USA. sam.yang@ars.usda.gov
authors.forename: S Samuel
authors.initials: SS
authors.lastname: Yang
authors.forename: Wayne Wenzhong
authors.initials: WW
authors.lastname: Xu
authors.forename: Mesfin
authors.initials: M
authors.lastname: Tesfaye
authors.forename: JoAnn F S
authors.initials: JF
authors.lastname: Lamb
authors.forename: Hans-Joachim G
authors.initials: HJ
authors.lastname: Jung
authors.forename: Kathryn A
authors.initials: KA
authors.lastname: VandenBosch
authors.forename: Carroll P
authors.initials: CP
authors.lastname: Vance
authors.forename: John W
authors.initials: JW
authors.lastname: Gronwald
mesh_terms: D002473:Cell Wall / Q000737:chemistry*; D020869:Gene Expression Profiling / Q000379:methods*; D005838:Genotype; D000455:Medicago sativa / Q000166:cytology* / Q000235:genetics* / Q000254:growth & development; D020411:Oligonucleotide Array Sequence Analysis; D020161:Physical Chromosome Mapping; D018547:Plant Stems / Q000166:cytology* / Q000235:genetics* / Q000254:growth & development; D012333:RNA, Messenger / Q000235:genetics; D015203:Reproducibility of Results; D013045:Species Specificity
chemical_list: D012333:RNA, Messenger