Genomic and Transcriptomic Analysis of the Polyploidy Cyst Nematode, Heterodera trifolii, and Heterodera schachtii

Abstract

Cyst nematodes remain a major threat to global agricultural production, causing huge losses. To understand the parasitism of the cyst nematodes Heterodera trifolii (HT) and Heterodera schachtii (HS), we constructed whole-genome assemblies using short- and long-read sequencing technologies. The nematode genomes were 379 Mb and 183 Mb in size, with the integrated gene models predicting 40,186 and 18,227 genes in HT and HS, respectively. We found more than half of the genes predicted in HT (64.7%) and HS (53.2%) were collinear to their nearest neighbor H. glycines (HG). Large-scale duplication patterns in HT and segmental duplications of more than half of the orthologous genes indicate that the genome of HT is polyploid in nature. Functional analysis of the genes indicated that 65.6% of the HG genes existed within the HT genome. Most abundant genes in HT and HS were involved in gene regulation, DNA integration, and chemotaxis. Differentially expressed genes showed upregulation of cuticle structural constituent genes during egg and female stages and cytoskeletal motor activity-related genes in juvenile stage 2 (J2). Horizontal gene transfer analyses identified four new vitamin biosynthesis genes, pdxK, pdxH, pdxS, and fabG, of bacterial origin, to be first reported in HT and HS. Mitogenomes of HT, HS, and HG showed similar structure, composition, and codon usage. However, rates of substitution of bases in the gene nad4l were significantly different between HT and HS. The described genomes, transcriptomes, and mitogenomes of plant-parasitic nematodes HT and HS are potential bio-resources used to identify several strategies of control of the nematode.

Keywords: Heterodera; cyst nematode; genome assembly; transcriptome.

Figure 1

Genome structure of cyst nematode Heterodera trifolii and H. schachtii (A) Statistics of repeats observed in the assemblies of HT and HS genomes (B) A phylogenetic tree derived from horizontal gene transfer data indicating genome sizes of the studied nematodes and their nearest neighbors along with the number of genes predicted in each assembly. (C) Synteny of HT and HS genomes along with soybean cyst nematode H. glycines genome assembly. (D) Duplication patterns of genes observed in HT and HS. (E) Overview of duplications observed in the HT and HS genome assemblies. The labels are contigs identified in respective nematodes. (F) Orthogroups of genes identified in HT, HS, and HG genome assemblies. The inset chart shows the extent of whole-genome or segmental duplications (WGD/SD) observed within the orthologous genes.

Figure 2

Transcriptome sequencing of HT and HS. (A) Classification of isoforms identified at various stages of growth in the nematodes HT and HS. Splice variants included full splice match (FSM), where all splice junctions perfectly matched with the reference transcript; incomplete splice match (ISM), with partial splice junction matches; novel in catalog (NIC), which are novel isoforms with a new combination of splice sites; novel not in catalog (NNC), which were also novel isoforms and contained at least one new splice site; genic intron (GI), which were within an intron; and genic genomic (GG), which overlapped introns and exons. (B) Types and statistics of different splice sites identified in HT and HS (C) Nucleotide bias in positions that precede the start codon in Kozak sequences identified in the transcripts of HT and HS. The bit at each position means the information content and the heights of individual bases are in proportion to their frequencies. (D) Statistics of isoforms and novel isoforms identified at various stages of the life cycle of the nematodes HT and HS. (E) Isoform gene statistics and stage-specificity observed in nematodes HT and HS.

Figure 3

Differential gene expression at various stages of the life cycle of HT and HS. (A) A heatmap displaying differentially expressed genes based on different life stages of HT, along with highly upregulated as well as downregulated genes at specific stages of the HT life cycle. (B) Heatmap displaying differentially expressed genes based on different life stages of HS along with highly upregulated as well as downregulated genes at specific stages of the HS life cycle.

Figure 4

Functional analysis of genes identified in the genome assemblies of HT and HS. (A) Statistics of genes with functions matched in the analyzed databases for HT and HS. (B) Overview of major metabolism/functions in the identified genes based on the number of hits in the databases. Numbers given in parentheses indicate the number of genes with similar function identified during the database search. (C) Identification of transcription factor genes and major families of effector genes in HT, HS, and HG genome assemblies.

Figure 5

Evidence of horizontal gene transfer (HGT) observed in genome assemblies of HT and HS. (A,B) Horizontally transferred genes were identified in HT and HS using the AvP program by a survey in various databases. AI—alien values and numbers indicate the number of gene hits in respective databases. (C) Identification of vitamin biosynthesis genes among the HGT genes involved in the synthesis of vitamins B1, B5, B6, and B7 in HT and HS. The genes in red font indicate genes identified in HT and HS that have been already reported in soybean cyst nematode HG, and genes mentioned in green (pdxK, pdxH, pdxS, and fabG) are first reported in Heterodera in this study. Line graphs adjacent to the pathway show the expression levels of the genes at various stages of the nematode life cycle.

Figure 6

Mitochondrial genomes of nematodes HT and HS. (A) A phylogenetic tree derived from comparing the mitochondrial genome sequences of the nematodes. On the right are various genes identified and indicative sizes of the mitochondrial sequences. (B) Molecular evolution and selection in mitochondrial genes interpreted through the ratio of nonsynonymous substitutions (Ka) to synonymous substitutions (Ks). The genes, values observed, and compared nematodes are given in the graph. A threshold value of 1 was taken to identify positive selection. (C) Relative synonymous codon usage (RSCU) statistics among the nematodes HT, HS, and HG. The codons with RSCU values of >1 are highlighted in the table below and were found to be consistent among the three nematodes. (D) Comparison of the GC % in various protein-coding genes (PCGs) identified in the mitochondrial sequence of HT, HS, and HG. The PCGs ND—NADH dehydrogenase; CytB—cytochrome B oxidase; COX—cytochrome C oxidase; ATP—ATP synthase.