Plant Parasitic Nematode Identification in Complex Samples with Deep Learning.

Plant parasitic nematodes are significant contributors to yield loss worldwide, causing devastating losses to every crop species, in every climate. Mitigating these losses requires swift and informed management strategies, centered on identification and quantification of field populations. Current plant parasitic nematode identification methods rely heavily on manual analyses of microscope images by a highly trained nematologist. This mode is not only expensive and time consuming, but often excludes the possibility of widely sharing and disseminating results to inform regional trends and potential emergent issues. This work presents a new public dataset containing annotated images of plant parasitic nematodes from heterologous soil extractions. This dataset serves to propagate new automated methodologies or speedier plant parasitic nematode identification using multiple deep learning object detection models and offers a path towards widely shared tools, results, and meta-analyses.

Root-Knot-Nematode-Encoded CEPs Increase Nitrogen Assimilation.

C-terminally encoded peptides (CEPs) are plant developmental signals that regulate growth and adaptive responses to nitrogen stress conditions. These small signal peptides are common to all vascular plants, and intriguingly have been characterized in some plant parasitic nematodes. Here, we sought to discover the breadth of root-knot nematode (RKN)-encoded CEP-like peptides and define the potential roles of these signals in the plant-nematode interaction, focusing on peptide activity altering plant root phenotypes and nitrogen uptake and assimilation. A comprehensive bioinformatic screen identified 61 CEP-like sequences encoded within the genomes of six root-knot nematode (RKN; Meloidogyne spp.) species. Exogenous application of an RKN CEP-like peptide altered A. thaliana and M. truncatula root phenotypes including reduced lateral root number in M. truncatula and inhibited primary root length in A. thaliana. To define the role of RKN CEP-like peptides, we applied exogenous RKN CEP and demonstrated increases in plant nitrogen uptake through the upregulation of nitrate transporter gene expression in roots and increased 15N/14N in nematode-formed root galls. Further, we also identified enhanced nematode metabolic processes following CEP application. These results support a model of parasite-induced changes in host metabolism and inform endogenous pathways to regulate plant nitrogen assimilation.

First Description of the Nuclear and Mitochondrial Genomes and Associated Host Preference of Trichopoda pennipes, a Parasitoid of Nezara viridula.

Trichopoda pennipes is a tachinid parasitoid of several significant heteropteran agricultural pests, including the southern green stink bug, Nezara viridula, and leaf-footed bug, Leptoglossus phyllopus. To be used successfully as a biological control agent, the fly must selectively parasitize the target host species. Differences in the host preference of T. pennipes were assessed by assembling the nuclear and mitochondrial genomes of 38 flies reared from field-collected N. viridula and L. phyllopus. High-quality de novo draft genomes of T. pennipes were assembled using long-read sequencing. The assembly totaled 672 MB distributed among 561 contigs, having an N50 of 11.9 MB and a GC of 31.7%, with the longest contig at 28 MB. The genome was assessed for completeness using BUSCO in the Insecta dataset, resulting in a score of 99.4%, and 97.4% of the genes were single copy-loci. The mitochondrial genomes of the 38 T. pennipes flies were sequenced and compared to identify possible host-determined sibling species. The assembled circular genomes ranged from 15,345 bp to 16,390 bp and encode 22 tRNAs, two rRNAs, and 13 protein-coding genes (PCGs). There were no differences in the architecture of these genomes. Phylogenetic analyses using sequence information from 13 PCGs and the two rRNAs individually or as a combined dataset resolved the parasitoids into two distinct lineages: T. pennipes that parasitized both N. viridula and L. phyllopus, and others that parasitized only L. phyllopus.

Temporally Regulated Plant-Nematode Gene Networks Implicate Metabolic Pathways.

Root-knot nematodes (RKN) (Meloidogyne spp.) constantly communicate with their host to establish and maintain specialized feeding cells. They likely regulate this interaction by monitoring host biology. As plant host biology is influenced by light and gene expression varies correspondingly, RKN gene transcription and biology likely follow similar patterns. We profiled RKN transcripts over a period of 24 h and identified approximately 1,000 differentially expressed genes (DEG) in nematode and model host Medicago truncatula, with the majority of DEG occurring in the middle of the dark period. Many of the plant DEG are involved in defense-response pathways, while the nematode DEG are involved in establishing infection, suggesting a strong host-nematode interaction occurring during the dark. To identify interacting genes, we developed a plant-nematode gene network based on DEG signals. The phenylpropanoid pathway was identified as a significant plant-nematode interacting pathway, representing four of 33 genes in the network. We further examined if this pathway interacts similarly in another host, tomato, by quantifying phenolic and flavonoid compounds produced by this pathway. Phenolic compounds showed a significant increase in production during the day in uninoculated plants as compared with during the night. However, during the dark period, there was an increase in flavonoid content in infected plants when compared with uninfected controls, indicating potential host defense mechanisms active during the height of nematode activity at night. This study elucidated cross-species interacting pathways that could be targeted to develop novel management strategies to these important pests.[Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Potential of nicotinamide adenine dinucleotide (NAD) for management of root-knot nematode in tomato.

Nicotinamide adenine dinucleotide (NAD) has been shown to induce plant defense responses to different plant pathogens, including reducing northern root-knot nematode, Meloidogyne hapla, penetration and increasing plant mass in tomato. We wanted to further evaluate NAD that are effective against the more economically important species, M. incognita and whether NAD treatments of tomato seedlings in transplant trays can protect plants in the field. Different NAD concentrations (1 mM, 0.1 mM and 0.01 mM) and three application timings (pre; post; pre and post inoculation) were evaluated in growth room and greenhouse trials. The highest tested NAD concentration (1 mM) suppressed second-stage juveniles (J2) infection for all three application methods. Root gall ratings at 30 days after inoculation (DAI) were also suppressed by 1 mM NAD compared to the other two concentrations, and egg mass number was significantly suppressed for all concentrations and application timings compared to the non-treated control. The rate of 1 mM NAD for all three application timings also improved plant growth at 30 DAI. Long-term effects of 1 mM NAD (pre, pre + post, or post applications) on nematode infection, growth and yield of tomato were evaluated in two additional experiments. All NAD applications suppressed root galls after 60 days, but only the pre + post 1 mM NAD application suppressed gall severity at 105 days, as well as suppressed egg counts by 50% at 60 DAT. No significant difference in plant biomass and fruit yield after 105 days was observed among the treatments. Two field trials were conducted in spring and fall 2020 using tomato seedlings (cv. HM 1823) treated with two different NAD concentrations (1 mM and 5 mM in spring; 5 mM and 10 mM in fall) and transplanting seedlings in fumigated (chloropicrin ± 1,3-dichloropropene) and non-fumigated plastic-mulch beds. No significant impact of NAD in terms of reducing RKN severity or overall tomato growth and production was seen in fumigated beds, but in non-fumigated beds 5 mM NAD slightly increased early fruit yield in spring, and 10 mM NAD reduced root-knot soil populations in fall.

Morphometric and Molecular Diversity among Seven European Isolates of Pratylenchus penetrans.

Pratylenchus penetrans is an economically important root-lesion nematode species that affects agronomic and ornamental plants. Understanding its diversity is of paramount importance to develop effective control and management strategies. This study aimed to characterize the morphological and genetic diversity among seven European isolates. An isolate from the USA was included in the molecular analyses for comparative purposes. Morphometrics of the European P. penetrans isolates generally were within the range of the original descriptions for this species. However, multiple morphometric characteristics, including body length, maximum body width, tail length and length of the post-vulval uterine sac showed discrepancies when compared to other populations. Nucleotide sequence-based analyses revealed a high level of intraspecific diversity among the isolates. We observed no correlation between D2-D3 rDNA- and COXI-based phylogenetic similarities and geographic origin. Our phylogenetic analyses including selected GenBank sequences also suggest that the controversy surrounding the distinction between P. penetrans and P. fallax remains.

Genome announcement of Steinernema khuongi and its associated symbiont from Florida.

Citrus root weevil (Diaprepes abbreviates) causes significant yield loss in citrus, especially in Florida. A promising source of control for this pest is biological control agents, namely, native entomopathogenic nematodes (EPNs) within the genus Steinernema. Two species of endemic EPN in Florida are S. diaparepesi, abundant within the central ridge, and S. khuongi, dominating the flatwood regions of the state. These citrus-growing regions differ significantly in their soil habitats, which impacts the potential success of biological control measures. Although the genome sequence of S. diaprepesi is currently available, the genome sequence of S. khuongi and identity of the symbiotic bacteria is still unknown. Understanding the genomic differences between these two nematodes and their favored habitats can inform successful biological control practices. Here, MiSeq libraries were used to simultaneously sequence and assemble the draft genome of S. khuongi and its associated symbionts. The final draft genome for S. khuongi has 8,794 contigs with a total length of ∼82 Mb, a largest contig of 428,226 bp, and N50 of 46 kb; its BUSCO scores indicate that it is > 86% complete. An associated bacterial genome was assembled with a total length of ∼3.5 Mb, a largest contig at 116,532 bp, and N50 of 17,487 bp. The bacterial genome encoded 3,721 genes, similar to other Xenorhabdus genomes. Comparative genomics identified the symbiotic bacteria of S. khuongi as Xenorhabdus poinarii. These new draft genomes of a host and symbiont can be used as a valuable tool for comparative genomics with other EPNs and its symbionts to understand host range and habitat suitability.

Collection and DNA Detection of Dirofilaria immitis (Rhabditida Onchocercidae), Using a Novel Primer Set, in Wild-Caught Mosquitoes From Gainesville, FL.

Dirofilaria immitis, the causative agent of dog heartworm disease, is an important cause of canine morbidity and mortality, expensive to treat, and severe infections are often fatal. Much is known about the pathogen in the canine host, yet little is known on the basic ecology of the nematode in the mosquito vector. Thus, to evaluate the effectiveness of collection techniques on ability to capture dog heartworm-infected mosquitoes (Diptera Culicidae), we conducted a field study spanning 111 wk. Four methods were used: two aspirators types, sweep netting, and a CDC trap. All sites had canines present in either residential yards (n = 4) or dog kennel facilities (n = 3). Collected mosquitoes were sorted by site, trap, species, and date, then pooled into groups of up to 25 individuals. Mosquito head and thorax pools were extracted for DNA, that was screened using currently available protocols. These protocols were found unreliable; thus, we developed a novel qPCR primer and probe set. Using this method, the original samples were re-assayed and provided 494 positive pools. Approximately 10% of positive samples were confirmed by Sanger sequencing. Twenty-two mosquito species tested positive for dog heartworm DNA, including a new association with Wyeomyia mitchellii (Theobald). Although Aedes atlanticus (Dyar and Knab), Anopheles crucians Wiedemann, and Culiseta melanura (Coquillett) composed nearly 36% of the total collection, these species represented 42% of the qPCR positive pools. Infection rates within commonly collected mosquitoes ranged up to 2.5%, with more rarely collected species ranging up to 14%. The CDC trap was the most effective collection method at trapping infected mosquitoes.

Wireworm (Coleoptera: Elateridae) Species Composition and Management in Sweet Potato Grown in North Florida Using Chemical Insecticides and Entomopathogenic Nematodes.

Wireworms are immature stages of click beetles (Coleoptera: Elateridae) and are considered a serious threat to sweet potato production in the southern United States. The major wireworm species collected in North Florida sweet potato fields in 2017 and 2018 were Conoderus scissus, C. rudis, C. amplicollis, and C. falli. These species vary in their behavior and biology. During a 2-yr study period, we conducted two insecticide field trials using eleven insecticides belonging to organophosphates, neonicotinoids, pyrethroids, and botanical classes, and three field trials using entomopathogenic nematode (EPN) species to control wireworms. In 2017, all insecticide treatments significantly reduced new feeding holes and total holes (old + new + other) as compared to the untreated control. In 2018, the result was similar with a few variations. In both years, all insecticides showed a percentage reduction in wireworm damage holes (2017: 34.88-96.19%; 2018: 12.38-97.02%) with the highest by Regent. In the EPN field study, one application of EPN near planting significantly reduced soil insects. In a laboratory study conducted at the Tropical Research and Education Center, UF-IFAS, chlorpyrifos caused higher percentage mortality of C. rudis (55.5%) than C. scissus (22.2%). At the present experiment rates, none of the insecticides caused the mortality of C. amplicollis. Heterorhabditids strain 'FL-2122' was more susceptible to chlorpyrifos than other strains of EPN.

A Comprehensive Transcriptional Profiling of Pepper Responses to Root-Knot Nematode.

Genetic resistance remains a key component in integrated pest management systems. The cosmopolitan root-knot nematode (RKN; Meloidogyne spp.) proves a significant management challenge as virulence and pathogenicity vary among and within species. RKN greatly reduces commercial bell pepper yield, and breeding programs continuously develop cultivars to emerging nematode threats. However, there is a lack of knowledge concerning the nature and forms of nematode resistance. Defining how resistant and susceptible pepper cultivars mount defenses against RKN attacks can help inform breeding programs. Here, we characterized the transcriptional responses of the highly related resistant (Charleston Belle) and susceptible (Keystone Resistance Giant) pepper cultivars throughout early nematode infection stages. Comprehensive transcriptomic sequencing of resistant and susceptible cultivar roots with or without Meloidogyneincognita infection over three-time points; covering early penetration (1-day), through feeding site maintenance (7-days post-inoculation), produced > 300 million high quality reads. Close examination of chromosome P9, on which nematode resistance hotspots are located, showed more differentially expressed genes were upregulated in resistant cultivar at day 1 when compared to the susceptible cultivar. Our comprehensive approach to transcriptomic profiling of pepper resistance revealed novel insights into how RKN causes disease and the plant responses mounted to counter nematode attack. This work broadens the definition of resistance from a single loci concept to a more complex array of interrelated pathways. Focus on these pathways in breeding programs may provide more sustainable and enduring forms of resistance.

Nematode Identification Techniques and Recent Advances.

Nematodes are among the most diverse but least studied organisms. The classic morphology-based identification has proved insufficient to the study of nematode identification and diversity, mainly for lack of sufficient morphological variations among closely related taxa. Different molecular methods have been used to supplement morphology-based methods and/or circumvent these problems with various degrees of success. These methods range from fingerprint to sequence analyses of DNA- and/or protein-based information. Image analyses techniques have also contributed towards this success. In this review, we highlight what each of these methods entail and provide examples where more recent advances of these techniques have been employed in nematode identification. Wherever possible, emphasis has been given to nematodes of agricultural significance. We show that these alternative methods have aided nematode identification and raised our understanding of nematode diversity and phylogeny. We discuss the pros and cons of these methods and conclude that no one method by itself provides all the answers; the choice of method depends on the question at hand, the nature of the samples, and the availability of resources.

A draft genome of Steinernema diaprepesi.

Entomopathogenic nematodes within the genus Steinernema are used as biological control agents against significant agricultural pests. Steinernema diaprepesi is native to Florida and very effective in controlling citrus root weevil, a devastating pest of citrus, ornamental plants, and vegetables. Here, we present the draft genome of Steinernema diaprepesi, which is a valuable tool for understanding the efficacy of this nematode as a biological control agent.Entomopathogenic nematodes within the genus Steinernema are used as biological control agents against significant agricultural pests. Steinernema diaprepesi is native to Florida and very effective in controlling citrus root weevil, a devastating pest of citrus, ornamental plants, and vegetables. Here, we present the draft genome of Steinernema diaprepesi, which is a valuable tool for understanding the efficacy of this nematode as a biological control agent.

Root-knot nematodes demonstrate temporal variation in host penetration.

Root-knot nematodes (RKN; Meloidogyne spp.) are obligate plant parasites that require constant communication with their host to establish and maintain specialized feeding cells. The intimacy of this interaction likely requires constant monitoring of host biology and behavior. As plant processes follow tightly regulated circadian and diurnal patterns, RKN may use similar cues to regulate aspects of this symbiosis. We interrogated RKN biology within the context of host diurnal rhythms throughout nematode development. At 24-hr post-inoculation, RKN penetrated host roots significantly more when inoculated during the night compared to the day. We excluded the possibility that this phenomenon is due to nematode perception of light penetrating the soil, as an identical phenomenon is observed under inverted light conditions. Additionally, when plants were allowed to equilibrate and adjust their light-driven clock under constant light conditions, the temporal variation in nematode penetration was abolished. This phenomenon is not present during earlier nematode developmental stages as egg hatch and infective juvenile mobility did not follow rhythmic patterns and are not affected by light. Taken together, it appears nematode host seeking and penetration are at least partially influenced by daily changes in plant root signaling and light does not have a direct effect on RKN developmental stages. Understanding the role and origin of circadian and diurnal rhythms in the plant-nematode interaction underscores the importance of exploiting basal plant biology to develop novel control methods for these pathogens.Root-knot nematodes (RKN; Meloidogyne spp.) are obligate plant parasites that require constant communication with their host to establish and maintain specialized feeding cells. The intimacy of this interaction likely requires constant monitoring of host biology and behavior. As plant processes follow tightly regulated circadian and diurnal patterns, RKN may use similar cues to regulate aspects of this symbiosis. We interrogated RKN biology within the context of host diurnal rhythms throughout nematode development. At 24-hr post-inoculation, RKN penetrated host roots significantly more when inoculated during the night compared to the day. We excluded the possibility that this phenomenon is due to nematode perception of light penetrating the soil, as an identical phenomenon is observed under inverted light conditions. Additionally, when plants were allowed to equilibrate and adjust their light-driven clock under constant light conditions, the temporal variation in nematode penetration was abolished. This phenomenon is not present during earlier nematode developmental stages as egg hatch and infective juvenile mobility did not follow rhythmic patterns and are not affected by light. Taken together, it appears nematode host seeking and penetration are at least partially influenced by daily changes in plant root signaling and light does not have a direct effect on RKN developmental stages. Understanding the role and origin of circadian and diurnal rhythms in the plant­nematode interaction underscores the importance of exploiting basal plant biology to develop novel control methods for these pathogens.

Identification of Suitable Meloidogyne spp. Housekeeping Genes.

Gene expression studies often require reliable housekeeping (HK) genes to accurately capture gene expression levels under given conditions. This is especially true for root-knot nematodes (RKN, Meloidogyne spp.), whose drastic developmental changes are strongly dependent upon their environment. Here we utilized a publicly available M. hapla RNAseq database to identify putative HK genes throughout the nematode lifecycle. We then validated these candidate HK genes on M. incognita in order to develop a small library of suitable HK genes for RKN. Seven putative HK genes were selected for validation based on high expression level and ease of primer design. The expression of these genes was quantified by qPCR at different developmental stages to capture the entire life cycle of M. incognita which included eggs and naive infective juveniles through 3-wk post inoculation. Two algorithms, geNorm and Normfinder, identified three genes (Disu, Poly, and Skinase) constitutively and uniformly expressed throughout the entire life cycle of RKN. We believe these genes are superior HK genes suitable to be used as internal reference genes at all stages of RKN. Importantly, while we identified Actin, a commonly used HK gene, as a candidate gene within our RNAseq analyses, our qPCR results did not demonstrate stable expression throughout the nematode life cycle of this gene. This study successfully validated suitable HK genes utilizing both RNAseq data and standard qPCR methods across two species of RKN; suitable HK genes are likely applicable to other species of RKN, or even plant-parasitic nematodes. Additional lists of potential HK genes are also provided if the nematode of interest does not have homologues of the three superior reference genes described here. Gene expression studies on RKN should use validated HK genes to ensure accurate representation of transcript abundance.

Temporal expression patterns of Pasteuria spp. sporulation genes.

Endospore-forming bacterium in the genus Pasteuria spp. infect multiple agriculturally significant plant parasitic nematodes and has potential as a potent biological control. Success as a biological control requires not only spore attachment to the cuticle, but sporulation and reproduction within the nematode host. Tracking and identifying Pasteuria spp. development is then critical to demonstrating efficacy as a biocontrol. Microscopic observations suggest Pasteuria spp. follows the model bacterium, Bacillus subtilis, sporulation. Here, we identified B. subtilis homologs of sporulation regulators in Pasteuria spp. and characterized the temporal expression of these genes throughout the bacterium's ∼30-d lifecycle in Meloidogyne arenaria as a means of tracking sporulation development. Detectable levels of transcripts of Spo0F were present as early as 5 d after the nematodes were exposes to Pasteuria spp. and were relatively constant throughout the 30-d lifecycle. Transcripts to Sigma-F were significantly higher in the middle of the lifecycle, while the transcripts of Sigma-G were detectable between 15 and 25 d, nearing the end of the lifecycle. These three markers can be used to track the process of sporulation in the nematode and augment microscopic observations. Tracking sporulation of Pasteuria spp. is important to fully realize its potential as a biological control method as it can more readily identify successful parasitism, define host ranges, and inform in vitro growth progress.

Peptide signaling molecules CLE5 and CLE6 affect Arabidopsis leaf shape downstream of leaf patterning transcription factors and auxin.

Intercellular signaling mediated by small peptides is critical to coordinate organ formation in animals, but whether extracellular polypeptides play similar roles in plants is unknown. Here we describe a role in Arabidopsis leaf development for two members of the CLAVATA3/ESR-RELATED peptide family, CLE5 and CLE6, which lie adjacent to each other on chromosome 2. Uniquely among the CLE genes, CLE5 and CLE6 are expressed specifically at the base of developing leaves and floral organs, adjacent to the boundary with the shoot apical meristem. During vegetative development CLE5 and CLE6 transcription is regulated by the leaf patterning transcription factors BLADE-ON-PETIOLE1 (BOP1) and ASYMMETRIC LEAVES2 (AS2), as well as by the WUSCHEL-RELATED HOMEOBOX (WOX) transcription factors WOX1 and PRESSED FLOWER (PRS). Moreover, CLE5 and CLE6 transcript levels are differentially regulated in various genetic backgrounds by the phytohormone auxin. Analysis of loss-of-function mutations generated by genome engineering reveals that CLE5 and CLE6 independently and together have subtle effects on rosette leaf shape. Our study indicates that the CLE5 and CLE6 peptides function downstream of leaf patterning factors and phytohormones to modulate the final leaf morphology.

Networks Underpinning Symbiosis Revealed Through Cross-Species eQTL Mapping.

Organisms engage in extensive cross-species molecular dialog, yet the underlying molecular actors are known for only a few interactions. Many techniques have been designed to uncover genes involved in signaling between organisms. Typically, these focus on only one of the partners. We developed an expression quantitative trait locus (eQTL) mapping-based approach to identify cause-and-effect relationships between genes from two partners engaged in an interspecific interaction. We demonstrated the approach by assaying expression of 98 isogenic plants (Medicago truncatula), each inoculated with a genetically distinct line of the diploid parasitic nematode Meloidogyne hapla With this design, systematic differences in gene expression across host plants could be mapped to genetic polymorphisms of their infecting parasites. The effects of parasite genotypes on plant gene expression were often substantial, with up to 90-fold (P = 3.2 × 10-52) changes in expression levels caused by individual parasite loci. Mapped loci included a number of pleiotropic sites, including one 87-kb parasite locus that modulated expression of >60 host genes. The 213 host genes identified were substantially enriched for transcription factors. We distilled higher-order connections between polymorphisms and genes from both species via network inference. To replicate our results and test whether effects were conserved across a broader host range, we performed a confirmatory experiment using M. hapla-infected tomato. This revealed that homologous genes were similarly affected. Finally, to validate the broader utility of cross-species eQTL mapping, we applied the strategy to data from a Salmonella infection study, successfully identifying polymorphisms in the human genome affecting bacterial expression.

Solution NMR studies of the plant peptide hormone CEP inform function.

The C-terminally Encoded Peptide (CEP) family of regulatory peptides controls root development in vascular plants. Here, we present the first NMR structures of CEP. We show that root-knot nematode (RKN: Meloidogyne spp.) also encodes CEP, presumably to mimic plant CEP as part of their stereotypic, parasitic interaction with vascular plants. Molecular dynamics simulations of plant- and nematode-encoded CEP displaying known posttranslational modifications (PTM) provided insight into the structural effects of PTM and the conformational plasticity and rigidity of CEP. Potential mechanisms of action are discussed with respect to the structure and sampling of conformational space.

PCR cloning of partial nbs sequences from grape (Vitis aestivalis Michx).

Plants defend themselves against pathogens via the expressions of disease resistance (R) genes. Many plant R gene products contain the characteristic nucleotide-binding site (NBS) and leucine-rich repeat (LRR) domains. There are highly conserved motifs within the NBS domain which could be targeted for polymerase chain reaction (PCR) cloning of R genes. Here, we report a 4-week undergraduate laboratory exercise that mimics the research environment to PCR-clone partial nbs sequences using degenerate primers corresponding to the phosphate-binding loop (P-loop) and GLPL motifs within the NBS domain of potential R gene products from the North American grape, Vitis aestivalis Michx. Students were able to complete the laboratory procedures successfully and obtained four different clones, among which three are new. Through the laboratory exercise, students learned a variety of important molecular techniques including genomic DNA isolation, DNA quantification, PCR, agarose gel electrophoresis, DNA extraction from agarose gel, ligation, bacterial transformation, and plasmid DNA isolation and purification. They also used currently available web-based bioinformatic programs for sequence analysis. The laboratory exercise provides students the hands-on experience on PCR cloning and shows them how it is done in a research environment. The clones obtained may be further tested for their potential use as markers to differentiate resistant cultivars from the susceptible ones, a useful tool in breeding programs.