Introduction

Almost all major crops surrender yield losses to parasitic nematodes with annual damages exceeding US one billion worldwide [1]. The most well-known and complex group of these plant parasites comprises root-knot (Meloidogyne spp) and cyst nematodes (Globoderaspp. and Heterodera spp), which manipulate the host to develop a long-term feeding site. The soybean cyst nematode (Heterodera glycines ) is of particularly great economic importance due to prominent role in reducing soybean yields worldwide [1-4]. Overcoming this crop pest requires scrutinizing the H. glycines lifestyle and the molecular exchange at the core of this problem.
H. glycine s’ lifecycle begins as an egg that is queued to hatch. The emerged juvenile nematode migrates to the host root zone, where it penetrates the outer layers of roots using a combination of mechanical and enzymatic processes, and eventually induces a single root cell near the vascular cylinder to form a feeding site, known as a syncytium [5]. The syncytium becomes metabolically active and expands to incorporate hundreds of adjacent cells through cell wall breakdown and protoplast fusion. The syncytium matures to an efficient nutrient sink with enlarged host nuclei and pronounced cytoplasmic streaming [6, 7].
Successful feeding site development depends upon the parasite’s ability to manipulate a complex interaction with its host via the transfer of nematode gland cell-produced effector proteins into or around host root cells [8-10]. During juvenile nematode migration within the root, plant cell walls are digested by an abundance of secreted enzymes including cellulases, pectate lyases and other hydrolases [11-13]. In later parasitic stages, the nematode manipulates plant metabolism [14], development [15-18], and elicits a dramatic and long-term suppression of host defenses (reviewed by [9, 10, 19, 20]). While the functional mechanisms of many effector proteins remain elusive, a variety of functions have been attributed to previously characterized effector proteins secreted from the esophageal gland cells of root-knot and cyst nematodes [11, 19-33]. For example, a chorismate mutase protein, typically absent in animals, is secreted by root-knot and cyst nematodes to manipulate the plant host’s shikimate pathway, a pathway involved in producing aromatic amino acids, plant hormones, cell wall components, and plant defense metabolites [14, 34-36]. Signaling peptides, like CLAVATA3 plant peptide mimics, can affect plant developmental pathways [16, 17, 33, 37-39]. While these effectors have led to a better understanding of plant-nematode interactions, only a small portion have been functionally characterized.
Understanding the totality of effector proteins in the nematode genome and how they manipulate the host will shed light on this molecular interplay, inspiring the development of novel mechanisms to defend plants from these important pests. To accomplish this goal for the soybean cyst nematode, two annotated genome assemblies were published from two different nematode strains: a partially virulent TN10 line [40] and a highly virulent X12 line [41]. Here we improve upon the current genomes by reassembling the TN10 PacBio reads and scaffolding with Chicago and Hi-C reads to obtain the highest quality plant-parasitic nematode genome assembly to date with nine complete pseudomolecule chromosomes and zero unscaffolded contigs. We went to great lengths to ensure the integrity of the assembly, as shown by 97% of input reads mapping back to the assembly and by a high degree of synteny to related species. Though 30-39% of the genome is repetitive, 28 and 58% of the newly assembled genome is syntenic to the X12 assembly and TN10 draft, respectively. While large rearrangements exist between the TN10 and X12 pseudomolecule assemblies, technological improvements in Hi-C scaffolding software (Lachesis vs Juicer) revealed that these differences can be attributed to many small and a few large chromosomal misjoins in the X12 assembly. Though the X12 and this latest TN10 assembly have similar assembly metrics and size, 141 vs 158Mb, choices in gene prediction created a large disparity in gene frequency between annotations. Here we have attempted to bridge this gap with an extensive gene annotation that uses multiple prediction pipelines and lines of evidence to generate an annotation that is complete and comparable to other parasitic nematode species. We also limited our gene homology input to include only genes of related Tylenchida species to prevent the homology-driven over-simplification of gene structure when using more distant and nonparasitic relatives. Fortuitously, this resulted in gene counts (22,465) that were neatly positioned between the two previous assemblies’ gene counts [29,769 (TN10) and 11,882 (X12)]. Even with catering to bias in gene structure from parasitism, the evaluation of universal single copy orthologs with BUSCO is still higher in our latest TN10 assembly than in previous assemblies at 83% (Table 1). Using this vastly improved genomic resource, we explore the nature of previously published effectors and other secreted proteins to address the heart of H. glycines genomics, to understand the adaptive evolution involved in the constant battle between host resistance and parasite virulence.