Insights into the evolution of symbiosis gene copy number and distribution from a chromosome-scale Lotus japonicus Gifu genome sequence

Abstract

Lotus japonicus is a herbaceous perennial legume that has been used extensively as a genetically tractable model system for deciphering the molecular genetics of symbiotic nitrogen fixation. So far, the L. japonicus reference genome assembly has been based on Sanger and Illumina sequencing reads from the L. japonicus accession MG-20 and contained a large fraction of unanchored contigs.Here, we use long PacBio reads from L. japonicus Gifu combined with Hi-C data and new highdensity genetic maps to generate a high-quality chromosome-scale reference genome assembly for L. japonicus. The assembly comprises 554 megabases of which 549 were assigned to six pseudomolecules that appear complete with telomeric repeats at their extremes and large centromeric regions with low gene density. The new L. japonicus Gifu reference genome and associated expression data represent valuable resources for legume functional and comparative genomics. Here, we provide a first example by showing that the symbiotic islands recently described in Medicago truncatula do not appear to be conserved in L. japonicus.Lotus japonicus (Lotus) is a well-characterized perennial legume widely used because of its short generation time, abundant flowers, small diploid genome, amenability to tissue culture and Agrobacterium transformation, self-compatibility, and inter-and intra-specific fertility enabling genetic mapping 1-4 . Due to its ability to participate in both nitrogen fixing symbiosis with rhizobia and symbiosis with arbuscular mycorrhiza 5 , Lotus has played a key role in developing our current understanding of the molecular mechanisms behind root nodule development and symbiotic nitrogen-fixation 6-8 and arbuscular mycorrhizal symbiosis 9-11 .The genetic tools available in Lotus include EMS mutants 12 , sequenced sets of wild accessions 13 and recombinant inbred lines 4,14,15 as well as extensive populations of TILLING lines 16 and LORE1 insertion mutants 17-19 . Last, but not least, large volumes of biological data generated using Lotus, such as expression studies that describe responses to environmental cues and symbionts and pathogens 20-30 , and data from more than 700,000 unique LORE1 insertions found in more than 130,000 mutant lines 18 , have been integrated in an online portal known as Lotus Base 31 , available from https://lotus.au.dk. This online portal provides researchers a one-stop resource for Lotus data retrieval, visualization, interrogation, and analysis.Lotus has a relatively small genome size estimated to ~500 Mb 32 . Three major sequencing and assembly efforts have been performed using the Lotus accession MG-20. MG-20 was selected due to its early, abundant flowering phenotype, and ability to sustain robust growth under both growth chamber and greenhouse conditions 33 . For clone-by-clone sequencing of the Lotus genome, genomic clones, constructed mainly using a TAC (Transformation-competent Artificial Chromosome) vector, were selected based on expressed sequence tag (EST) sequence information as seed points of the gene-rich...

Widespread and transgenerational retrotransposon activation in inter‐ and intraspecies recombinant inbred populations of Lotus japonicus

“…Third, accurate de novo identification of LTR retrotransposons is easier than that of the other classes of TEs because of its characteristic LTRs. We identified LTR retrotransposons in the Gifu reference genome sequence LjGifu1.2 (Kamal et al., 2020) using ltrharvest (Ellinghaus et al., 2008) and ltrdigest (Steinbiss et al., 2009), and clustered them using cd‐hit‐est (Li & Godzik, 2006). According to the cluster information, the hierarchical list of LTR retrotransposons that was composed of 12 423 copies of 9379 families, including LORE1 and LORE2 , was generated.…”

Aromatic amino acid biosynthesis by aLotusAldolase impacts root hair development and symbiotic associations

“…1b). To determine whether Aldolase1 belongs to a gene family, a BLAST was performed in the Lotus genome (Kamal et al ., 2020). Two homolog genes were identified, LotjaGi1g1v0239000 and LotjaGi6g1v0351200, which were named LjAldolase2 and LjAldolase3 , respectively (Fig.…”

“…The predicted promoter (2 kb upstream from the start codon) and coding sequence (1,617 bp) of LjAldolase1 were obtained from the L. japonicus Gifu genome (Kamal et al ., 2020) in the Lotus database (Mun et al ., 2016), and synthetized with 5′overghangs for goldengate cloning with compatible vectors. Promoter, CDS, fluorescent reporters and terminator modules were assembled and cloned into a pIV10 vector (Stougaard, 1995), suitable for goldengate technology.…”

Genome sequence of 12 Vigna species as a knowledge base of stress tolerance and resistance

“…Greene (Griesmann et al, 2018), Cicer arientinum L. kabuli (Varshney et al, 2013), Glycine soja Siebold & Zucc. (Valliyodan et al, 2019), Lotus japonicus (Regel) K.Larsen (Kamal et al, 2020), Lupinus albus L. (Hufnagel et al, 2020), Medicago truncatula Gaertn. (Tang et al, 2014), P. vulgaris (Schmutz et al, 2014), Pisum sativum L. (Kreplak et al, 2019), and Trifolium pratense L. (De Vega et al, 2015).…”