JoAnn F. S. Lamb

Using RNA-Seq for gene identification, polymorphism detection and transcript profiling in two alfalfa genotypes with divergent cell wall composition in stems

BackgroundAlfalfa, [Medicago sativa (L.) sativa], a widely-grown perennial forage has potential for development as a cellulosic ethanol feedstock. However, the genomics of alfalfa, a non-model species, is still in its infancy. The recent advent of RNA-Seq, a massively parallel sequencing method for transcriptome analysis, provides an opportunity to expand the identification of alfalfa genes and polymorphisms, and conduct in-depth transcript profiling.ResultsCell walls in stems of alfalfa genotype 708 have higher cellulose and lower lignin concentrations compared to cell walls in stems of genotype 773. Using the Illumina GA-II platform, a total of 198,861,304 expression sequence tags (ESTs, 76 bp in length) were generated from cDNA libraries derived from elongating stem (ES) and post-elongation stem (PES) internodes of 708 and 773. In addition, 341,984 ESTs were generated from ES and PES internodes of genotype 773 using the GS FLX Titanium platform. The first alfalfa (Medicago sativa) gene index (MSGI 1.0) was assembled using the Sanger ESTs available from GenBank, the GS FLX Titanium EST sequences, and the de novo assembled Illumina sequences. MSGI 1.0 contains 124,025 unique sequences including 22,729 tentative consensus sequences (TCs), 22,315 singletons and 78,981 pseudo-singletons. We identified a total of 1,294 simple sequence repeats (SSR) among the sequences in MSGI 1.0. In addition, a total of 10,826 single nucleotide polymorphisms (SNPs) were predicted between the two genotypes. Out of 55 SNPs randomly selected for experimental validation, 47 (85%) were polymorphic between the two genotypes. We also identified numerous allelic variations within each genotype. Digital gene expression analysis identified numerous candidate genes that may play a role in stem development as well as candidate genes that may contribute to the differences in cell wall composition in stems of the two genotypes.ConclusionsOur results demonstrate that RNA-Seq can be successfully used for gene identification, polymorphism detection and transcript profiling in alfalfa, a non-model, allogamous, autotetraploid species. The alfalfa gene index assembled in this study, and the SNPs, SSRs and candidate genes identified can be used to improve alfalfa as a forage crop and cellulosic feedstock.

The Medicago sativa gene index 1.2: a web-accessible gene expression atlas for investigating expression differences between Medicago sativa subspecies

BackgroundAlfalfa (Medicago sativa L.) is the primary forage legume crop species in the United States and plays essential economic and ecological roles in agricultural systems across the country. Modern alfalfa is the result of hybridization between tetraploid M. sativa ssp. sativa and M. sativa ssp. falcata. Due to its large and complex genome, there are few genomic resources available for alfalfa improvement.ResultsA de novo transcriptome assembly from two alfalfa subspecies, M. sativa ssp. sativa (B47) and M. sativa ssp. falcata (F56) was developed using Illumina RNA-seq technology. Transcripts from roots, nitrogen-fixing root nodules, leaves, flowers, elongating stem internodes, and post-elongation stem internodes were assembled into the Medicago sativa Gene Index 1.2 (MSGI 1.2) representing 112,626 unique transcript sequences. Nodule-specific and transcripts involved in cell wall biosynthesis were identified. Statistical analyses identified 20,447 transcripts differentially expressed between the two subspecies. Pair-wise comparisons of each tissue combination identified 58,932 sequences differentially expressed in B47 and 69,143 sequences differentially expressed in F56. Comparing transcript abundance in floral tissues of B47 and F56 identified expression differences in sequences involved in anthocyanin and carotenoid synthesis, which determine flower pigmentation. Single nucleotide polymorphisms (SNPs) unique to each M. sativa subspecies (110,241) were identified.ConclusionsThe Medicago sativa Gene Index 1.2 increases the expressed sequence data available for alfalfa by ninefold and can be expanded as additional experiments are performed. The MSGI 1.2 transcriptome sequences, annotations, expression profiles, and SNPs were assembled into the Alfalfa Gene Index and Expression Database (AGED) at http://plantgrn.noble.org/AGED/, a publicly available genomic resource for alfalfa improvement and legume research.

Medicago truncatula as a Model for Dicot Cell Wall Development

We have initiated a genome-wide transcript profiling study using the model legume Medicago truncatula to identify putative genes related to cell wall biosynthesis and regulatory function in legumes. We used the GeneChip® Medicago Genome Array to compare transcript abundance in elongating versus postelongation stem internode segments of two M. truncatula accessions and two Medicago sativa (alfalfa) clones with contrasting stem cell wall concentration and composition. Hundreds of differentially expressed probe sets between elongating and postelongation stem segments showed similar patterns of gene expression in the model legume and cultivated alfalfa. Differentially expressed genes included genes with putative functions associated with primary and secondary cell wall biosynthesis and growth. Mining of public microarray data for coexpressed genes with two marker genes for secondary cell wall synthesis identified additional candidate secondary cell wall-related genes. Coexpressed genes included protein kinases, transcription factors, and unclassified groups that were not previously reported with secondary cell wall-associated genes. M. truncatula has been recognized as an excellent model plant for legume genomics. The stem tissue transcriptome analysis, described here, indicates that M. truncatula has utility as a model plant for cell wall genomics in legumes in general and shows excellent potential for translating gene discoveries to its close relative, cultivated alfalfa, in particular. The natural variation for stem cell wall traits in Medicago may offer a new tool to study an expanded repertoire of valuable agronomic traits in related species, including woody dicots in the eurosid I clade.

Transcript profiling of two alfalfa genotypes with contrasting cell wall composition in stems using a cross-species platform: optimizing analysis by masking biased probes

BackgroundThe GeneChip®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.ResultsTranscript profiling using the Medicago GeneChip® 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.ConclusionsThe 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.

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 cross‐species platform. Analysis of GeneChip expression data files 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 classification 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.

Field sampling strategies for studies of alfalfa forage quality

Information is scarce on sampling techniques for field studies of alfalfa forage quality. Standard formulas are available for estimating the number of samples needed for reducing error in a study, but little is known about the impact of plot sampling on forage quality. Our objectives were to compare the strategy of manual harvesting from small areas within plots with that of grab sampling mechanically harvested forage, and to determine whether the within-plot location of sampling affected forage quality in any systematic way. Alfalfa forage was sampled from swaths of mechanically clipped forage (grab samples) and from hand-clipped areas within field plots (area samples). Systematic sample location within a plot had no discernable effect on forage quality. Calculations of predicted standard errors and required sample numbers indicated that one area or one grab sample per plot with three replicates would provide an acceptable standard error for comparison of alfalfa entries for protein and fiber concentrati...