Chromosome-scale assembly of the highly heterozygous genome of red clover (Trifolium pratense L.), an allogamous forage crop species

Abstract

Relative to other crops, red clover (Trifolium pratense L.) has various favorable traits making it an ideal forage crop. Conventional breeding has improved varieties, but modern genomic methods could accelerate progress and facilitate gene discovery. Existing short-read-based genome assemblies of the ∼420 megabase pair (Mbp) genome are fragmented into >135,000 contigs, with numerous order and orientation errors within scaffolds, probably associated with the plant's biology, which displays gametophytic self-incompatibility resulting in inherent high heterozygosity. Here, we present a high-quality long-read-based assembly of red clover with a more than 500-fold reduction in contigs, improved per-base quality, and increased contig N50 by three orders of magnitude. The 413.5 Mbp assembly is nearly 20% longer than the 350 Mbp short-read assembly, closer to the predicted genome size. We also present quality measures and full-length isoform RNA transcript sequences for assessing accuracy and future genome annotation. The assembly accurately represents the seven main linkage groups in an allogamous (outcrossing), highly heterozygous plant genome. Subjects Genetics and Genomics, Bioinformatics, Plant Genetics DATA DESCRIPTION BackgroundThe species Trifolium pratense L. (red clover, NCBI:txid57577) is an important legume forage crop grown on approximately 4 million hectares worldwide [1]. Red clover is a versatile crop grown as animal feed and/or as a green manure in pure and mixed stands for hay, haylage, silage, and grazing. Red clover is known for its ease of establishment and shade tolerance, and its ability to grow in poorly drained and low pH soils. The reduced need for exogenous nitrogen application owing to its nitrogen-fixing ability and the relatively high protein content of this plant compared with other forage crops provide potential for reducing the environmental footprint of livestock production. Compared to alfalfa, another common legume forage crop, red clover varieties have higher forage yields, are a better source of magnesium to avoid grass tetany in grazing cattle, and may have improved post-harvest protein preservation [2] and bypass protein content in ruminant production

An improved reference genome for Trifolium subterraneum L. provides insight into molecular diversity and intra-specific phylogeny

“…The graphics were performed using Rscript. Note that a recent high-quality assembly of the T. pratense genome (ARS_RCv1.1; Bickhart et al., 2022 ) was not used in this analysis as it has yet to be annotated.…”

“…Macrosynteny between T. subterraneum , and T. pratense , T. repens , M. truncatula , and L. japonicas was investigated based on homologous translated protein sequences using BLAST searches with an E-value cut-off of 1E-100. For this macrosyntenic analysis, a recent high-quality T. pratense assembly (ARS_RCv1.1; Bickhart et al., 2022 ) was used. As T. repens is an allotetraploid, the two subgenomes were merged into single consensus homoeologous groups, hence eight chromosomes rather than 16.…”

Mabs, a suite of tools for gene-informed genome assembly

“…To test Mabs, I performed a literature analysis and compiled a set of 5 species for which authors reported a high number of haplotypic duplications: Trifolium pratense (Bickhart et al , 2022), Manihot esculenta (Qi et al , 2022), Myripristis murdjan (Guan et al , 2020; Rhie et al , 2021), Adineta vaga (Guiglielmoni et al , 2021), Mytilus coruscus (Li et al , 2020; Sun et al , 2021). In addition, I used Heracleum sosnowskyi , which I studied personally and which also had a lot of haplotypic duplications (preprint to be uploaded to BioRxiv in 2022).…”

Expression and Variation of the Genes Involved in Rhizobium Nodulation in Red Clover

“…Raw reads were filtered to remove reads less than 30 bases and then mapped to the red clover genome assembly (version ARS_RC1.1; GenBank Accession No. GCA_020283565.1) [ 25 ] using the RNA-Seq tool, CLC Genomics Workbench (v22.0; CLC Genomics Workbench, Qiagen Aarhus A/S, Denmark). Minimum length fraction was set to 0.5 and minimum similarity fraction set to 0.8.…”

“…Molecular work on red clover has lagged behind many other cultivated crop species despite its relatively small genome [ 20 ], primarily due to the fact that it is an outcrossing species making genetic gains more difficult [ 21 ]. Genomic resources have been developed that now allow for molecular characterization and analysis [ 22 , 23 , 24 , 25 , 26 ]. The high heterozygosity in red clover, while complicating the assembly of contiguous chromosome molecules, has allowed for the identification of chromosomal regions and genes involved in various functions using single nucleotide polymorphic loci (SNPs) mapping [ 26 , 27 , 28 , 29 , 30 ], including identification of red clover genotypes differing in their nitrogen fixation potential [ 31 ].…”