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Title:   Author  item
                                            Yang, Sukitem
                                            XU, WAYNE - UNIVERSITY OF MINNESOTAitem
                                            TESFAYE, MESFIN - UNIVERSITY OF MINNESOTAitem
                                            Lamb, Joannitem
                                            Jung, Hans Joachimitem
                                            Samac, Deborah

                                                         - *Debby*item
                                            Vance, Carrollitem
                                            Gronwald, John
  Submitted to: The Plant Genome
                                    Publication Type: Peer Reviewed Journal
                                    Publication Acceptance Date: 9/13/2009
                                    Publication  Date: 11/5/2009
                                    Citation: Yang, S.H., Xu, W.W., Tesfaye, M., Lamb, J.F., Jung, H.G., Samac, D.A., Vance, C.P., Gronwald, J.W. 2009. Single-Feature Polymorphism Discovery in the Transcriptome of Tetraploid Alfalfa. The Plant Genome. 2(3):224-232.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 has considerable potential for development as a sustainable cellulosic feedstock. The model for 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. Cellulosic feedstocks that have high cellulose and low lignin yield more ethanol. Developing alfalfa varieties that have high cellulose and low lignin would increase the value of this crop as a cellulosic feedstock. However, because alfalfa has a large, complex genome, progress in improving traits by breeding has been slow. Faster progress could be made if molecular markers for traits of interest could be identified. Molecular markers are pieces of DNA that serve as signposts for genes that contribute to desirable traits. To date, very few molecular markers have been developed for alfalfa. We used a new approach to identify molecular markers that is very useful in crops like alfalfa where information about the genome is limited. This approach involves scanning genechips to identify single-feature polymorphisms (SFP). SFPs indicate small differences in DNA structure between two varieties. In this study, we scanned the genechip data from two alfalfa varieties that have different levels of cellulose and lignin. We identified more than 10,000 SFPs. Many of these SFPs were associated with genes that regulate cellulose and lignin synthesis. These SFPs will serve as useful molecular markers that will decrease the time required to develop new alfalfa varieties that yield more ethanol. The many SFPs identified in this study will also serve as useful tools for alfalfa breeders to select for other traits that improve this crop as a livestock feed and biofuel feedstock.
                                Technical Abstract: Alfalfa (Medicago sativa (L.) ssp. sativa ) has economic importance as a forage crop for livestock and potential for development as a biofuel feedstock. Advances in alfalfa breeding, 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. The stems of alfalfa genotype 252 have high cellulose and lignin while the stems of genotype 1283 have low levels of both cell wall components. In this study, we conducted a genome-wide evaluation of polymorphisms in genotypes 252 and 1283 by measuring single-feature polymorphisms (SFPs) using Medicago GeneChip expression data. Analysis of GeneChip expression data files of alfalfa stem internodes of genotypes 1283 and 252 at two growth stages (elongating, post-elongation) revealed 10,887 SFPs in 8230 probe sets. Of these SFPs, 5,807 were discovered in genotype 252 and 5,080 in genotype 1283. Seventeen SFPs were PCR-sequenced and 15 SFPs exhibited polymorphisms (false positive rate, FPR = 12%). We identified gene functional classes that were over- or under-represented among the SFP-harboring genes in each genotype. Cell wall genes were highly enriched among SFP-harboring genes in both genotypes. We mapped 5,833 of the 8,230 alfalfa SFP-harboring genes onto putative orthologous loci on Medicago truncatula chromosomes. Although SFPs were distributed over all chromosomes, several SFP hotspots were detected. Clustering of SFP-harboring genes within the same functional class was observed on some chromosomes. The Medicago GeneChip is a suitable platform for detecting SFPs in alfalfa. The SFPs discovered in this study provide a list of candidate genes that could be used as molecular markers in a breeding program to improve alfalfa as a livestock feed and biofuel feedstock. SFPs may also be useful for advancing understanding of genome organization in cultivated tetraploid alfalfa and for comparative genomics research with other legume species.


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