An improved genome release (version Mt4.0) for the model legume Medicago truncatula

Abstract

Background: Medicago truncatula, a close relative of alfalfa, is a preeminent model for studying nitrogen fixation, symbiosis, and legume genomics. The Medicago sequencing project began in 2003 with the goal to decipher sequences originated from the euchromatic portion of the genome. The initial sequencing approach was based on a BAC tiling path, culminating in a BAC-based assembly (Mt3.5) as well as an in-depth analysis of the genome published in 2011.

Results: Here we describe a further improved and refined version of the M. truncatula genome (Mt4.0) based on de novo whole genome shotgun assembly of a majority of Illumina and 454 reads using ALLPATHS-LG. The ALLPATHS-LG scaffolds were anchored onto the pseudomolecules on the basis of alignments to both the optical map and the genotyping-by-sequencing (GBS) map. The Mt4.0 pseudomolecules encompass ~360 Mb of actual sequences spanning 390 Mb of which ~330 Mb align perfectly with the optical map, presenting a drastic improvement over the BAC-based Mt3.5 which only contained 70% sequences (~250 Mb) of the current version. Most of the sequences and genes that previously resided on the unanchored portion of Mt3.5 have now been incorporated into the Mt4.0 pseudomolecules, with the exception of ~28 Mb of unplaced sequences. With regard to gene annotation, the genome has been re-annotated through our gene prediction pipeline, which integrates EST, RNA-seq, protein and gene prediction evidences. A total of 50,894 genes (31,661 high confidence and 19,233 low confidence) are included in Mt4.0 which overlapped with ~82% of the gene loci annotated in Mt3.5. Of the remaining genes, 14% of the Mt3.5 genes have been deprecated to an "unsupported" status and 4% are absent from the Mt4.0 predictions.

Conclusions: Mt4.0 and its associated resources, such as genome browsers, BLAST-able datasets and gene information pages, can be found on the JCVI Medicago web site (http://www.jcvi.org/medicago). The assembly and annotation has been deposited in GenBank (BioProject: PRJNA10791). The heavily curated chromosomal sequences and associated gene models of Medicago will serve as a better reference for legume biology and comparative genomics.

Figure 1

Overview of (A) assembly and (B) annotation strategies used in the Mt4.0 genome release.

Figure 2

Example of breakpoint identification using (A) GBS map and (B) optical map alignment. Red arrows indicate the same breakpoint on Scaffold0004 indicated by GBS map and optical map alignment.

Figure 3

Increased amount of chromosome-anchored sequences in Medicago Mt4.0 compared to Mt3.5. Red-colored portion of the chromosomes represent BAC sequences used in Mt3.5, while the white regions on the chromosomes represent newly anchored sequences in Mt4.0.

Figure 4

Heatmap of linkage disequilibrium between pairwise SNP markers in the Mt4.0 assemblies. Pairwise linkage disequilibrium (LD) between markers was calculated as r² value based on segregations of individuals within LR4 mapping population.

Figure 5

Syntenic dot plot of Medicago genome versus itself, showing blocks derived from papilionoid whole genome duplication event. Contrasting (A) Mt3.5 and (B) Mt4.0 with the same synteny block chaining settings (see Methods).

Figure 6

Ks analyses of comparisons between legume species with whole genome sequences. Percentage of gene pairs is taken as the counts of syntenic homologs within a Ks range (with bin sizes of 0.05) divided by all the syntenic homologs identified.

Figure 7

JCVI website for Medicago genome resources, showing a number of services and tools to interact with the Mt4.0 datasets (A) JBrowse shows the alignment of annotation evidence to the genome; (B) Keyword search supports extraction of gene lists; (C) TAIR-style gene information page of Mt4.0 gene models; (D) Textpresso for mining Medicago-related literature.