Strategies for optimizing BioNano and Dovetail explored through a second reference quality assembly for the legume model, Medicago truncatula.

Third generation sequencing technologies, with sequencing reads in the tens- of kilo-bases, facilitate genome assembly by spanning ambiguous regions and improving continuity. This has been critical for plant genomes, which are difficult to assemble due to high repeat content, gene family expansions, segmental and tandem duplications, and polyploidy. Recently, high-throughput mapping and scaffolding strategies have further improved continuity. Together, these long-range technologies enable quality draft assemblies of complex genomes in a cost-effective and timely manner. Here, we present high quality genome assemblies of the model legume plant, Medicago truncatula (R108) using PacBio, Dovetail Chicago (hereafter, Dovetail) and BioNano technologies. To test these technologies for plant genome assembly, we generated five assemblies using all possible combinations and ordering of these three technologies in the R108 assembly. While the BioNano and Dovetail joins overlapped, they also showed complementary gains in continuity and join numbers. Both technologies spanned repetitive regions that PacBio alone was unable to bridge. Combining technologies, particularly Dovetail followed by BioNano, resulted in notable improvements compared to Dovetail or BioNano alone. A combination of PacBio, Dovetail, and BioNano was used to generate a high quality draft assembly of R108, a M. truncatula accession widely used in studies of functional genomics. As a test for the usefulness of the resulting genome sequence, the new R108 assembly was used to pinpoint breakpoints and characterize flanking sequence of a previously identified translocation between chromosomes 4 and 8, identifying more than 22.7 Mb of novel sequence not present in the earlier A17 reference assembly. Adding Dovetail followed by BioNano data yielded complementary improvements in continuity over the original PacBio assembly. This strategy proved efficient and cost-effective for developing a quality draft assembly compared to traditional reference assemblies.

Moll KM ，Zhou P ，Ramaraj T ，Fajardo D ，Devitt NP ，Sadowsky MJ ，Stupar RM ，Tiffin P ，Miller JR ，Young ND ，Silverstein KAT ，Mudge J ... -  《BMC GENOMICS》

Delineating the Tnt1 Insertion Landscape of the Model Legume Medicago truncatula cv. R108 at the Hi-C Resolution Using a Chromosome-Length Genome Assembly.

Legumes are of great interest for sustainable agricultural production as they fix atmospheric nitrogen to improve the soil. is a well-established model legume, and extensive studies in fundamental molecular, physiological, and developmental biology have been undertaken to translate into trait improvements in economically important legume crops worldwide. However, reference genome was generated in the accession Jemalong A17, which is highly recalcitrant to transformation. R108 is more attractive for genetic studies due to its high transformation efficiency and -insertion population resource for functional genomics. The need to perform accurate synteny analysis and comprehensive genome-scale comparisons necessitates a chromosome-length genome assembly for cv. R108. Here, we performed in situ Hi-C (48×) to anchor, order, orient scaffolds, and correct misjoins of contigs in a previously published genome assembly (R108 v1.0), resulting in an improved genome assembly containing eight chromosome-length scaffolds that span 97.62% of the sequenced bases in the input assembly. The long-range physical information data generated using Hi-C allowed us to obtain a chromosome-length ordering of the genome assembly, better validate previous draft misjoins, and provide further insights accurately predicting synteny between A17 and R108 regions corresponding to the known chromosome 4/8 translocation. Furthermore, mapping the insertion landscape on this reference assembly presents an important resource for functional genomics by supporting efficient mutant gene identification in insertion lines. Our data provide a much-needed foundational resource that supports functional and molecular research into the Leguminosae for sustainable agriculture and feeding the future.

Kaur P ，Lui C ，Dudchenko O ，Nandety RS ，Hurgobin B ，Pham M ，Lieberman Aiden E ，Wen J ，Mysore K ... -  《INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES》

Improving and correcting the contiguity of long-read genome assemblies of three plant species using optical mapping and chromosome conformation capture data.

Long-read sequencing can overcome the weaknesses of short reads in the assembly of eukaryotic genomes; however, at present additional scaffolding is needed to achieve chromosome-level assemblies. We generated Pacific Biosciences (PacBio) long-read data of the genomes of three relatives of the model plant and assembled all three genomes into only a few hundred contigs. To improve the contiguities of these assemblies, we generated BioNano Genomics optical mapping and Dovetail Genomics chromosome conformation capture data for genome scaffolding. Despite their technical differences, optical mapping and chromosome conformation capture performed similarly and doubled N50 values. After improving both integration methods, assembly contiguity reached chromosome-arm-levels. We rigorously assessed the quality of contigs and scaffolds using Illumina mate-pair libraries and genetic map information. This showed that PacBio assemblies have high sequence accuracy but can contain several misassemblies, which join unlinked regions of the genome. Most, but not all, of these misjoints were removed during the integration of the optical mapping and chromosome conformation capture data. Even though none of the centromeres were fully assembled, the scaffolds revealed large parts of some centromeric regions, even including some of the heterochromatic regions, which are not present in gold standard reference sequences.

Jiao WB ，Accinelli GG ，Hartwig B ，Kiefer C ，Baker D ，Severing E ，Willing EM ，Piednoel M ，Woetzel S ，Madrid-Herrero E ，Huettel B ，Hümann U ，Reinhard R ，Koch MA ，Swan D ，Clavijo B ，Coupland G ，Schneeberger K ... -  《-》

Comparison of structural variants in the whole genome sequences of two Medicago truncatula ecotypes: Jemalong A17 and R108.

Structural variants (SVs) constitute a large proportion of the genomic variation that results in phenotypic variation in plants. However, they are still a largely unexplored feature in most plant genomes. Here, we present the whole-genome landscape of SVs between two model legume Medicago truncatula ecotypes-Jemalong A17 and R108- that have been extensively used in various legume biology studies. To catalogue SVs, we first resolved the previously published R108 genome assembly (R108 v1.0) to chromosome-scale using 124 × Hi-C data, resulting in a high-quality genome assembly. The inter-chromosomal reciprocal translocations between chromosomes 4 and 8 were confirmed by performing syntenic analysis between the two genomes. Combined with the Hi-C data, it appears that these translocation events had a significant effect on chromatin organization. Using both whole-genome and short-read alignments, we identified the genomic landscape of SVs between the two genomes, some of which may account for several phenotypic differences, including their differential responses to aluminum toxicity and iron deficiency, and the development of different anthocyanin leaf markings. We also found extensive SVs within the nodule-specific cysteine-rich gene family which encodes antimicrobial peptides essential for terminal bacteroid differentiation during nitrogen-fixing symbiosis. Our results provide a near-complete R108 genome assembly and the first genomic landscape of SVs obtained by comparing two M. truncatula ecotypes. This may provide valuable genomic resources for the functional and molecular research of legume biology in the future.

Li A ，Liu A ，Wu S ，Qu K ，Hu H ，Yang J ，Shrestha N ，Liu J ，Ren G ... -  《BMC PLANT BIOLOGY》

Satellite repeats in the functional centromere and pericentromeric heterochromatin of Medicago truncatula.

Most eukaryotic centromeres contain long arrays of tandem repeats, with unit lengths of 150-300 bp. We searched for such repeats in the functional centromeres of the model legume Medicago truncatula (Medicago) accession Jemalong A17. To this end three repeats, MtR1, MtR2 and MtR3, were identified in 20 Mb of a low-pass, whole genome sequencing data set generated by a random shotgun approach. The nucleotide sequence composition, genomic organization and abundance of these repeats were characterized. Fluorescent in situ hybridization of these repeats on chromosomes at meiosis I showed that only the MtR3 repeat, encompassing stretches of 450 kb to more than 1.0 Mb, is located in the functional portion of all eight centromeres. MtR1 and MtR2 occupy distinct regions in pericentromeric heterochromatin. We also studied the presence and distribution of MtRs in Medicago accession R108-1, a genotype with a genome that is 20% smaller than that of Jemalong A17. We determined that while MtR3 is also centromeric on all pachytene bivalents in R108-1, MtR1 and MtR2 are not present in the R108 genome.

Kulikova O ，Geurts R ，Lamine M ，Kim DJ ，Cook DR ，Leunissen J ，de Jong H ，Roe BA ，Bisseling T ... -  《CHROMOSOMA》