NRPS-like ATRR in Plant-Parasitic Nematodes Involved in Glycine Betaine Metabolism to Promote Parasitism.

Plant-parasitic nematodes (PPNs) are among the most serious phytopathogens and cause widespread and serious damage in major crops. In this study, using a genome mining method, we identified nonribosomal peptide synthetase (NRPS)-like enzymes in genomes of plant-parasitic nematodes, which are conserved with two consecutive reducing domains at the N-terminus (A-T-R1-R2) and homologous to fungal NRPS-like ATRR. We experimentally investigated the roles of the NRPS-like enzyme (MiATRR) in nematode (Meloidogyne incognita) parasitism. Heterologous expression of Miatrr in Saccharomyces cerevisiae can overcome the growth inhibition caused by high concentrations of glycine betaine. RT-qPCR detection shows that Miatrr is significantly upregulated at the early parasitic life stage (J2s in plants) of M. incognita. Host-derived Miatrr RNA interference (RNAi) in Arabidopsis thaliana can significantly decrease the number of galls and egg masses of M. incognita, as well as retard development and reduce the body size of the nematode. Although exogenous glycine betaine and choline have no obvious impact on the survival of free-living M. incognita J2s (pre-parasitic J2s), they impact the performance of the nematode in planta, especially in Miatrr-RNAi plants. Following application of exogenous glycine betaine and choline in the rhizosphere soil of A. thaliana, the numbers of galls and egg masses were obviously reduced by glycine betaine but increased by choline. Based on the knowledge about the function of fungal NRPS-like ATRR and the roles of glycine betaine in host plants and nematodes, we suggest that MiATRR is involved in nematode-plant interaction by acting as a glycine betaine reductase, converting glycine betaine to choline. This may be a universal strategy in plant-parasitic nematodes utilizing NRPS-like ATRR to promote their parasitism on host plants.

Acquisition and transmission of Grapevine fanleaf virus (GFLV) by Xiphinema index and Xiphinema italiae (Longidoridae).

Grapevine fanleaf virus (GFLV) is one of the most severe virus diseases of grapevines, causing fanleaf degeneration that is transmitted by Xiphinema index. This paper aims to isolate Xiphinema species from Tunisian vineyard soil samples and assess their ability to acquire and transmit GFLV under natural and controlled conditions. Based on morphological and morphometric analyses, Tunisian dagger nematodes were identified as X. index and Xiphinema italiae. These results were confirmed with molecular identification tools using species-specific polymerase chain reaction primers. The total RNA of GFLV was extracted from specimens of Xiphinema and amplified based on real-time polymerase chain reaction using virus-specific primers. Our results showed that X. index could acquire and transmit the viral particles of GFLV. This nepovirus was not detected in X. italiae, under natural conditions; however, under controlled conditions, this nematode was able to successfully acquire and transmit the viral particles of GFLV.

Spatial and temporal heterogeneity alter the cost of plasticity in Pristionchus pacificus.

Phenotypic plasticity, the ability of a single genotype to produce distinct phenotypes under different environmental conditions, has become a leading concept in ecology and evolutionary biology, with the most extreme examples being the formation of alternative phenotypes (polyphenisms). However, several aspects associated with phenotypic plasticity remain controversial, such as the existence of associated costs. While already predicted by some of the pioneers of plasticity research, i.e. Schmalhausen and Bradshaw, experimental and theoretical approaches have provided limited support for the costs of plasticity. In experimental studies, one common restriction is the measurement of all relevant parameters over long time periods. Similarly, theoretical studies rarely use modelling approaches that incorporate specific experimentally-derived fitness parameters. Therefore, the existence of the costs of plasticity remains disputed. Here, we provide an integrative approach to understand the cost of adaptive plasticity and its ecological ramifications, by combining laboratory data from the nematode plasticity model system Pristionchus pacificus with a stage-structured population model. Taking advantage of measurements of two isogenic strains grown on two distinct diets, we illustrate how spatial and temporal heterogeneity with regard to the distribution of resources on a metapopulation can alter the outcome of the competition and alleviate the realized cost of plasticity.

Transcriptomic changes in soybean underlying growth promotion and defense against cyst nematode after Bacillus simplex Sneb545 treatment.

Bacillus simplex Sneb45 is a plant-growth-promoting rhizobacterium that promotes soybean growth and systemic resistance to cyst nematode. To investigate transcriptional changes in soybean roots in response to B. simplex Sneb45 treatment, transcriptome analysis and quantitative real-time PCR were conducted to detect and validate the differentially expressed genes (DEGs). In total, 19,109 DEGs were obtained. After B. simplex Sneb545 treatment, 970 and 1265 genes were up- and down-regulated at 5 days post-inoculation (dpi), respectively, and 142 and 47 genes were up- and down-regulated at 10 dpi, respectively, compared with untreated soybean roots. Functional annotation of DEGs indicated that B. simplex Sneb545 regulated soybean growth and defense against cyst nematode possibly through genes related to auxin, gibberellin, and NB-LRR protein. In addition, GO and KEGG enrichment analyses indicated that the DEGs were enriched in metabolism, signal transduction, and plant-pathogen interaction pathways. Moreover, the auxin and gibberellin contents were lower in B. simplex Sneb545-treated soybean roots than in untreated roots at 5 dpi. B. simplex Sneb545 possibly altered the expression of wound-induced protein and NAC transcription factor to regulate soybean growth and defense against cyst nematode. Our study provided deep insights into the alterations in soybean transcriptome after exposure to B. simplex Sneb45 and a theoretical basis for further exploring molecular functions underlying the biological control activity of B. simplex Sneb545.

Genomic characterization of a nematode tolerance locus in sugar beet.

BACKGROUND: Infection by beet cyst nematodes (BCN, Heterodera schachtii) causes a serious disease of sugar beet, and climatic change is expected to improve the conditions for BCN infection. Yield and yield stability under adverse conditions are among the main breeding objectives. Breeding of BCN tolerant sugar beet cultivars offering high yield in the presence of the pathogen is therefore of high relevance. RESULTS: To identify causal genes providing tolerance against BCN infection, we combined several experimental and bioinformatic approaches. Relevant genomic regions were detected through mapping-by-sequencing using a segregating F2 population. DNA sequencing of contrasting F2 pools and analyses of allele frequencies for variant positions identified a single genomic region which confers nematode tolerance. The genomic interval was confirmed and narrowed down by genotyping with newly developed molecular markers. To pinpoint the causal genes within the potential nematode tolerance locus, we generated long read-based genome sequence assemblies of the tolerant parental breeding line Strube U2Bv and the susceptible reference line 2320Bv. We analyzed continuous sequences of the potential locus with regard to functional gene annotation and differential gene expression upon BCN infection. A cluster of genes with similarity to the Arabidopsis thaliana gene encoding nodule inception protein-like protein 7 (NLP7) was identified. Gene expression analyses confirmed transcriptional activity and revealed clear differences between susceptible and tolerant genotypes. CONCLUSIONS: Our findings provide new insights into the genomic basis of plant-nematode interactions that can be used to design and accelerate novel management strategies against BCN.

An ex vitro hairy root system from petioles of detached soybean leaves for in planta screening of target genes and CRISPR strategies associated with nematode bioassays.

MAIN CONCLUSION: The ex vitro hairy root system from petioles of detached soybean leaves allows the functional validation of genes using classical transgenesis and CRISPR strategies (e.g., sgRNA validation, gene activation) associated with nematode bioassays. Agrobacterium rhizogenes-mediated root transformation has been widely used in soybean for the functional validation of target genes in classical transgenesis and single-guide RNA (sgRNA) in CRISPR-based technologies. Initial data showed that in vitro hairy root induction from soybean cotyledons and hypocotyls were not the most suitable strategies for simultaneous performing genetic studies and nematode bioassays. Therefore, an ex vitro hairy root system was developed for in planta screening of target molecules during soybean parasitism by root-knot nematodes (RKNs). Applying this method, hairy roots were successfully induced by A. rhizogenes from petioles of detached soybean leaves. The soybean GmPR10 and GmGST genes were then constitutively overexpressed in both soybean hairy roots and tobacco plants, showing a reduction in the number of Meloidogyne incognita-induced galls of up to 41% and 39%, respectively. In addition, this system was evaluated for upregulation of the endogenous GmExpA and GmExpLB genes by CRISPR/dCas9, showing high levels of gene activation and reductions in gall number of up to 58.7% and 67.4%, respectively. Furthermore, morphological and histological analyses of the galls were successfully performed. These collective data validate the ex vitro hairy root system for screening target genes, using classical overexpression and CRISPR approaches, directly in soybean in a simple manner and associated with nematode bioassays. This system can also be used in other root pathosystems for analyses of gene function and studies of parasite interactions with plants, as well as for other purposes such as studies of root biology and promoter characterization.

Loss-of-function of an α-SNAP gene confers resistance to soybean cyst nematode.

Plant-parasitic nematodes are one of the most economically impactful pests in agriculture resulting in billions of dollars in realized annual losses worldwide. Soybean cyst nematode (SCN) is the number one biotic constraint on soybean production making it a priority for the discovery, validation and functional characterization of native plant resistance genes and genetic modes of action that can be deployed to improve soybean yield across the globe. Here, we present the discovery and functional characterization of a soybean resistance gene, GmSNAP02. We use unique bi-parental populations to fine-map the precise genomic location, and a combination of whole genome resequencing and gene fragment PCR amplifications to identify and confirm causal haplotypes. Lastly, we validate our candidate gene using CRISPR-Cas9 genome editing and observe a gain of resistance in edited plants. This demonstrates that the GmSNAP02 gene confers a unique mode of resistance to SCN through loss-of-function mutations that implicate GmSNAP02 as a nematode virulence target. We highlight the immediate impact of utilizing GmSNAP02 as a genome-editing-amenable target to diversify nematode resistance in commercially available cultivars.

Unzipped chromosome-level genomes reveal allopolyploid nematode origin pattern as unreduced gamete hybridization.

The formation and consequences of polyploidization in animals with clonal reproduction remain largely unknown. Clade I root-knot nematodes (RKNs), characterized by parthenogenesis and allopolyploidy, show a widespread geographical distribution and extensive agricultural destruction. Here, we generated 4 unzipped polyploid RKN genomes and identified a putative novel alternative telomeric element. Then we reconstructed 4 chromosome-level assemblies and resolved their genome structures as AAB for triploid and AABB for tetraploid. The phylogeny of subgenomes revealed polyploid RKN origin patterns as hybridization between haploid and unreduced gametes. We also observed extensive chromosomal fusions and homologous gene expression decrease after polyploidization, which might offset the disadvantages of clonal reproduction and increase fitness in polyploid RKNs. Our results reveal a rare pathway of polyploidization in parthenogenic polyploid animals and provide a large number of high-precision genetic resources that could be used for RKN prevention and control.

Species-specific microRNA discovery and target prediction in the soybean cyst nematode.

The soybean cyst nematode (SCN) is a devastating pathogen for economic and food security considerations. Although the SCN genome has recently been sequenced, the presence of any miRNA has not been systematically explored and reported. This paper describes the development of a species-specific SCN miRNA discovery pipeline and its application to the SCN genome. Experiments on well-documented model nematodes (Caenorhabditis elegans and Pristionchus pacificus) are used to tune the pipeline's hyperparameters and confirm its recall and precision. Application to the SCN genome identifies 3342 high-confidence putative SCN miRNA. Prediction specificity within SCN is confirmed by applying the pipeline to RNA hairpins from known exonic regions of the SCN genome (i.e., sequences known to not be miRNA). Prediction recall is confirmed by building a positive control set of SCN miRNA, based on a limited deep sequencing experiment. Interestingly, a number of novel miRNA are predicted to be encoded within the intronic regions of effector genes, known to be involved in SCN parasitism, suggesting that these miRNA may also be involved in the infection process or virulence. Beyond miRNA discovery, gene targets within SCN are predicted for all high-confidence novel miRNA using a miRNA:mRNA target prediction system. Lastly, cross-kingdom miRNA targeting is investigated, where putative soybean mRNA targets are identified for novel SCN miRNA. All predicted miRNA and gene targets are made available in appendix and through a Borealis DataVerse open repository ( https://borealisdata.ca/dataset.xhtml?persistentId=doi:10.5683/SP3/30DEXA ).

Characterization of microRNAs in the cyst nematode Heterodera glycines identifies possible candidates involved in cross-kingdom interactions with its host Glycine max.

The soybean cyst nematode (SCN - Heterodera glycines) is one of the most damaging pests to the cultivated soybean worldwide. Using a wide array of stylet-secreted effector proteins, this nematode can restructure its host cells into a complex and highly active feeding structure called the syncytium. Tight regulation of these proteins is thought to be essential to the successful formation of this syncytium. To date, multiple mechanisms have been proposed to regulate the expression of these proteins including through post-transcriptional regulation. MicroRNAs (miRNAs) are a class of small, roughly 22-nucleotide-long, non-coding RNA shown to regulate gene expression through its interaction with the 3' untranslated region of genes. These same small RNAs have also been hypothesized to be able to cross over kingdom barriers and regulate genes in other species in a process called cross-kingdom interactions. In this study, we characterized the miRNome of the SCN via sequencing of small-RNAs isolated from whole nematodes and exosomes representing all developmental stages. We identified 121 miRNA loci encoding 96 distinct miRNA families including multiple lineage- and species-specific candidates. Using a combination of plant- and animal-specific miRNA target predictors, we generated a unique repertoire of miRNA:mRNA interacting partners in the nematode and its host plant leading to the identification of a set of nine probable cross-kingdom miRNA candidates.

Single Nematode Transcriptomic Analysis, Using Long-Read Technology, Reveals Two Novel Virulence Gene Candidates in the Soybean Cyst Nematode, Heterodera glycines.

The soybean cyst nematode (Heterodera glycines, SCN), is the most damaging disease of soybean in North America. While management of this pest using resistant soybean is generally still effective, prolonged exposure to cultivars derived from the same source of resistance (PI 88788) has led to the emergence of virulence. Currently, the underlying mechanisms responsible for resistance breakdown remain unknown. In this study, we combined a single nematode transcriptomic profiling approach with long-read sequencing to reannotate the SCN genome. This resulted in the annotation of 1932 novel transcripts and 281 novel gene features. Using a transcript-level quantification approach, we identified eight novel effector candidates overexpressed in PI 88788 virulent nematodes in the late infection stage. Among these were the novel gene Hg-CPZ-1 and a pioneer effector transcript generated through the alternative splicing of the non-effector gene Hetgly21698. While our results demonstrate that alternative splicing in effectors does occur, we found limited evidence of direct involvement in the breakdown of resistance. However, our analysis highlighted a distinct pattern of effector upregulation in response to PI 88788 resistance indicative of a possible adaptation process by SCN to host resistance.

Molecular characterization and functional analysis of glutathione S-transferase genes of pine wood nematode (Bursaphelenchus xylophilus) for avermectin.

Pine wilt disease (PWD), caused by Bursaphelenchus xylophilus (pine wood nematodes, PWNs), is a forest disease that seriously threatens the health of Pinus forestry. Glutathione S-transferases (GSTs) play important roles in xenobiotic metabolism, lipophilic compound transport, antioxidative stress reactions, anti-mutagenesis, and antitumor activity. The analysis and investigation of the specific functions of GSTs in the metabolism of toxic substances in nematodes are important for identifying potential target genes to control the spread and transmission of B. xylophilus. In this study, 51 Bx-GSTs were found in the genome of B. xylophilus. Two key Bx-gsts (Bx-gst12 and Bx-gst40) were analyzed when B. xylophilus was exposed to avermectin. The expression of Bx-gst12 and Bx-gst40 was significantly increased when B. xylophilus was exposed to 1.6 and 3.0 mg/mL avermectin solutions. Notably, combined silencing of both Bx-gst12 and Bx-gst40 did not further increase the mortality rates under avermectin exposure. Mortality rates were significantly increased in nematodes treated with dsRNA compared to control nematodes (p < 0.05) after RNAi. The feeding ability of nematodes was also significantly reduced after treatment with dsRNA. These results suggested that Bx-gsts are associated with the detoxification process and feeding behavior of B. xylophilus. Silencing Bx-gsts leads to increased susceptibility to nematicides and reduces the feeding ability of B. xylophilus. Therefore, Bx-gsts will be a new control target of PWNs in the future.

Development of a Droplet Digital PCR Assay for Detection and Quantification of Stubby Root Nematode, Paratrichodorus allius, in Soil.

The stubby root nematode Paratrichodorus allius is an important plant-parasitic nematode species within the Trichodoridae family. It can directly harm the plants by feeding on the roots or indirectly by transmitting Tobacco rattle virus. These nematodes are mostly diagnosed either by traditional microscopic methods or a polymerase chain reaction (PCR)-based method. Droplet digital PCR (ddPCR) is a novel PCR technique which is sensitive and precise in quantifying DNA templates of the test samples. In this study, we developed a ddPCR assay to detect and quantify P. allius in soil. The specificity and sensitivity of the assay was first determined using P. allius nematode DNA or DNA from sterilized soil artificially inoculated with P. allius, and the assay was used to quantify P. allius populations in field soils. The assay did not detect nematodes other than P. allius, thus showing high specificity. It was able to detect P. allius equivalent to a 0.01 and 0.02 portion of a single nematode in soil DNA and nematode DNA extracts, respectively. Highly linear relationships between DNA copy numbers from ddPCR and serial dilutions of known concentrations were observed with DNA from P. allius nematodes (R2 = 0.9842) and from artificially infested soil (R2 = 0.9464). The P. allius populations from field soils determined by ddPCR were highly correlated with traditional microscopic counts (R2 = 0.7963). To our knowledge, this is the first report of applying ddPCR to detect and quantify stubby root nematode in soil. The results of this study support the potentiality of a ddPCR assay as a new research tool in diagnostics of plant-parasitic nematodes.

Identification of genomic regions associated with cereal cyst nematode (Heterodera avenae Woll.) resistance in spring and winter wheat.

Cereal cyst nematode (CCN) is a major threat to cereal crop production globally including wheat (Triticum aestivum L.). In the present study, single-locus and multi-locus models of Genome-Wide Association Study (GWAS) were used to find marker trait associations (MTAs) against CCN (Heterodera avenae) in wheat. In total, 180 wheat accessions (100 spring and 80 winter types) were screened against H. avenae in two independent years (2018/2019 "Environment 1" and 2019/2020 "Environment 2") under controlled conditions. A set of 12,908 SNP markers were used to perform the GWAS. Altogether, 11 significant MTAs, with threshold value of -log10 (p-values) ≥ 3.0, were detected using 180 wheat accessions under combined environment (CE). A novel MTA (wsnp_Ex_c53387_56641291) was detected under all environments (E1, E2 and CE) and considered to be stable MTA. Among the identified 11 MTAs, eight were novel and three were co-localized with previously known genes/QTLs/MTAs. In total, 13 putative candidate genes showing differential expression in roots, and known to be involved in plant defense mechanisms were reported. These MTAs could help us to identify resistance alleles from new sources, which could be used to identify wheat varieties with enhanced CCN resistance.

The FMRFamide-like peptide FLP-1 modulates larval development by regulating the production and secretion of the insulin-like peptide DAF-28 in Caenorhabditis elegans.

The FMRFamide-like peptides (FLPs) are conserved in both free-living and parasitic nematodes. This molecular genetic study verified the relevance of the flp-1 gene, which is conserved in many nematode species, to the larval development of the free-living soil nematode Caenorhabditis elegans. Using C. elegans as a model, we found that: (1) FLP-1 suppressed larval development, resulting in diapause; (2) the secretion of FLP-1, which is produced in AVK head neurons, was suppressed by the presence of food (Escherichia coli) as an environmental factor to continue larval development; (3) the FLP-1 reduced the production and secretion of DAF-28, which is produced in ASI head neurons and is the predominant insulin-like peptide (INS) present. FLP-1 is conserved in many species of plant-parasitic root-knot nematodes that cause severe damage to crops. Therefore, our findings may provide insight into the development of new nematicides that can disturb their infection and development.

Cross-kingdom vitamin B5 biosynthesis and cyst nematode susceptibility.

Vitamin deficiencies are known to cause disorders in human beings. Siddique et al. discovered that vitamin B5 biosynthesis in cyst nematodes requires steps in their host plants. Disruption of an Arabidopsis thaliana 'susceptibility gene', which is involved in the production of vitamin B5 precursors, results in reduced parasitism.

Chitin contributes to the formation of a feeding structure in a predatory nematode.

Some nematode predators and parasites form teeth-like denticles that are histologically different from vertebrate teeth, but their biochemical composition remains elusive. Here, we show a role of chitin in the formation of teeth-like denticles in Pristionchus pacificus, a model system for studying predation and feeding structure plasticity. Pristionchus forms two alternative mouth morphs with one tooth or two teeth, respectively. The P. pacificus genome encodes two chitin synthases, with the highly conserved chs-2 gene being composed of 60 exons forming at least four isoforms. Generating CRISPR-Cas9-based gene knockouts, we found that Ppa-chs-2 mutations that eliminate the chitin-synthase domain are lethal. However, mutations in the C terminus result in viable but teethless worms, with severe malformation of the mouth. Similarly, treatment with the chitin-synthase inhibitor Nikkomycin Z also results in teethless animals. Teethless worms can feed on various bacterial food sources but are incapable of predation. High-resolution transcriptomics revealed that Ppa-chs-2 expression is controlled by the sulfatase-encoding developmental switch Ppa-eud-1. This study indicates a key role of chitin in the formation of teeth-like denticles and the complex feeding apparatus in nematodes.

Morphological and molecular characterization of Desmicola ryukyuensis n. sp. (Nematoda: Oxyuridomorpha: Thelastomatidae) from the wood-feeding cockroach Panesthia angustipennis yayeyamensis Asahina, 1988 (Blattaria: Blaberidae) in the Ryukyu Archipelago, Japan.

Desmicola ryukyuensis n. sp. (Nematoda: Oxyuridomorpha: Thelastomatidae) is described from the wood-feeding cockroach Panesthia angustipennis yayeyamensis Asahina, 1988 (Blattaria: Blaberidae) in Iriomote Island, Japan. The males of D. ryukyuensis n. sp. are similar to D. lamdongensis Sokolova, 2019 but differ by the length of the spicule and the extension of the lateral alae. The females of D. ryukyuensis n. sp. resemble those of D. ornata Jex, Schneider, Rose & Cribb, 2005, but can be differentiated by the shape of the sensilla in the interlabial space and the presence of lateral alae in D. ryukyuensis n. sp. that are absent in D. ornata. The females of D. ryukyuensis n. sp. are similar to D. lamdongensis. However, they differ in the morphology of the lips and the size of the eggs. The phylogeny of D. ryukyuensis n. sp. is inferred by the D2-D3 domains of the 28S rDNA. The new species forms a clade with another sequence of an unidentified Desmicola species from a Vietnamese wood-feeding cockroach.

A New Hope: A Hermaphroditic Nematode Enables Analysis of a Recent Whole Genome Duplication Event.

Whole genome duplication (WGD) is often considered a major driver of evolution that leads to phenotypic novelties. However, the importance of WGD for evolution is still controversial because most documented WGD events occurred anciently and few experimental systems amenable to genetic analysis are available. Here, we report a recent WGD event in the hermaphroditic nematode Allodiplogaster sudhausi and present a comparison with a gonochoristic (male/female) sister species that did not undergo WGD. Self-fertilizing reproduction of A. sudhausi makes it amenable to functional analysis and an ideal system to study WGD events. We document WGD in A. sudhausi through karyotype analysis and whole genome sequencing, the latter of which allowed us to 1) identify functional bias in retention of protein domains and metabolic pathways, 2) show most duplicate genes are under evolutionary constraint, 3) show a link between sequence and expression divergence, and 4) characterize differentially expressed duplicates. We additionally show WGD is associated with increased body size and an abundance of repeat elements (36% of the genome), including a recent expansion of the DNA-hAT/Ac transposon family. Finally, we demonstrate the use of CRISPR/Cas9 to generate mutant knockouts, whereby two WGD-derived duplicate genes display functional redundancy in that they both need to be knocked out to generate a phenotype. Together, we present a novel experimental system that is convenient for examining and characterizing WGD-derived genes both computationally and functionally.

Satellitome analyses in nematodes illuminate complex species history and show conserved features in satellite DNAs.

BACKGROUND: Satellite DNAs (satDNAs) are tandemly repeated non-coding DNA sequences that belong to the most abundant and the fastest evolving parts of the eukaryotic genome. A satellitome represents the collection of different satDNAs in a genome. Due to extreme diversity and methodological difficulties to characterize and compare satDNA collection in complex genomes, knowledge on their putative functional constraints and capacity to participate in genome evolution remains rather elusive. SatDNA transcripts have been detected in many species, however comparative studies of satDNA transcriptome between species are extremely rare. RESULTS: We conducted a genome-wide survey and comparative analyses of satellitomes among different closely related Meloidogyne spp. nematodes. The evolutionary trends of satDNAs suggest that each round of proposed polyploidization in the evolutionary history is concomitant with the addition of a new set of satDNAs in the satellitome of any particular Meloidogyne species. Successive incorporation of new sets of satDNAs in the genome along the process of polyploidization supports multiple hybridization events as the main factor responsible for the formation of these species. Through comparative analyses of 83 distinct satDNAs, we found a CENP-B box-like sequence motif conserved among 11 divergent satDNAs (similarity ranges from 36 to 74%). We also found satDNAs that harbor a splice leader (SL) sequence which, in spite of overall divergence, shows conservation across species in two putative functional regions, the 25-nt SL exon and the Sm binding site. Intra- and interspecific comparative expression analyses of the complete satDNA set in the analyzed Meloidogyne species revealed transcription profiles including a subset of 14 actively transcribed satDNAs. Among those, 9 show active transcription in every species where they are found in the genome and throughout developmental stages. CONCLUSIONS: Our results demonstrate the feasibility and power of comparative analysis of the non-coding repetitive genome for elucidation of the origin of species with a complex history. Although satDNAs generally evolve extremely quickly, the comparative analyses of 83 satDNAs detected in the analyzed Meloidogyne species revealed conserved sequence features in some satDNAs suggesting sequence evolution under selective pressure. SatDNAs that are actively transcribed in related genomes and throughout nematode development support the view that their expression is not stochastic.