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Characterization of genetic homogeneity of an Indian population of cereal cyst nematode, Heterodera avenae using sequencing and PCR-RFLP of ribosomal DNA.

ABSTRACT: The cereal cyst nematodes belonging to Heterodera avenae group is a complex species consisting of 12 valid species and overlapping morphological characters make them difficult to be distinguished from one another. The non coding internal transcribed spacer sequences, ITS1 and ITS2 including 5.8S region of ribosomal DNA (rDNA) has been very useful for the accurate identification of the species and characterization of molecular genetic variation within the species of plant parasitic nematodes. In the present study, sequencing and PCR-RFLP of rDNA has been used to confirm the species identity. Further it was used to determine the genetic homogeneity of an Indian population used for whole genome and transcriptome sequencing. The Sequence of ITS1 and ITS2 including 5.8S showed approximately 99% similarity with the existing sequences of H. avenae from different countries and confirmed the species identity. Secondary structure of ITS2 region shows that the isolates from India, China, Israel and Australian possess more stable conformational energy than the German strain. Further to characterize the genetic variation within the population, about 200 individual cysts or females were analyzed separately by PCR-RFLP of rDNA with five restriction enzymes that could distinguish H. avenae from other closely related species within the group. This analysis did not reveal any variation within the population indicating it is genetically homogeneous and suitable for next generation sequencing using Illumina platform.

SUBMITTER: Rao U

PROVIDER: S-EPMC3563400 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Characterization of genetic homogeneity of an Indian population of cereal cyst nematode, Heterodera avenae using sequencing and PCR-RFLP of ribosomal DNA.

Rao Uma U Sharma Amita A Tyagi Nidhi N Banakar Prakash P Kumar Mukesh M

Bioinformation 20130118 2

The cereal cyst nematodes belonging to Heterodera avenae group is a complex species consisting of 12 valid species and overlapping morphological characters make them difficult to be distinguished from one another. The non coding internal transcribed spacer sequences, ITS1 and ITS2 including 5.8S region of ribosomal DNA (rDNA) has been very useful for the accurate identification of the species and characterization of molecular genetic variation within the species of plant parasitic nematodes. In ...[more]

PMID: 23390348

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Project description:BACKGROUND:Cereal cyst nematode Heterodera avenae, an important soil-borne pathogen in wheat, causes numerous annual yield losses worldwide, and use of resistant cultivars is the best strategy for control. However, target genes are not readily available for breeding resistant cultivars. Therefore, comparative transcriptomic analyses were performed to identify more applicable resistance genes for cultivar breeding. METHODS:The developing nematodes within roots were stained with acid fuchsin solution. Transcriptome assemblies and redundancy filteration were obtained by Trinity, TGI Clustering Tool and BLASTN, respectively. Gene Ontology annotation was yielded by Blast2GO program, and metabolic pathways of transcripts were analyzed by Path_finder. The ROS levels were determined by luminol-chemiluminescence assay. The transcriptional gene expression profiles were obtained by quantitative RT-PCR. RESULTS:The RNA-sequencing was performed using an incompatible wheat cultivar VP1620 and a compatible control cultivar WEN19 infected with H. avenae at 24 h, 3 d and 8 d. Infection assays showed that VP1620 failed to block penetration of H. avenae but disturbed the transition of developmental stages, leading to a significant reduction in cyst formation. Two types of expression profiles were established to predict candidate resistance genes after developing a novel strategy to generate clean RNA-seq data by removing the transcripts of H. avenae within the raw data before assembly. Using the uncoordinated expression profiles with transcript abundance as a standard, 424 candidate resistance genes were identified, including 302 overlapping genes and 122 VP1620-specific genes. Genes with similar expression patterns were further classified according to the scales of changed transcript abundances, and 182 genes were rescued as supplementary candidate resistance genes. Functional characterizations revealed that diverse defense-related pathways were responsible for wheat resistance against H. avenae. Moreover, phospholipase was involved in many defense-related pathways and localized in the connection position. Furthermore, strong bursts of reactive oxygen species (ROS) within VP1620 roots infected with H. avenae were induced at 24 h and 3 d, and eight ROS-producing genes were significantly upregulated, including three class III peroxidase and five lipoxygenase genes. CONCLUSIONS:Large-scale identification of wheat resistance genes were processed by comparative transcriptomic analysis. Functional characterization showed that phospholipases associated with ROS production played vital roles in early defense responses to H. avenae via involvement in diverse defense-related pathways as a hub switch. This study is the first to investigate the early defense responses of wheat against H. avenae, not only provides applicable candidate resistance genes for breeding novel wheat cultivars, but also enables a better understanding of the defense mechanisms of wheat against H. avenae.

Project description:Cereal cyst nematode (CCN, Heterodera avenae) presents severe challenges to wheat (Triticum aestivum L.) production worldwide. An investigation of the interaction between wheat and CCN can greatly improve our understanding of how nematodes alter wheat root metabolic pathways for their development and could contribute to new control strategies against CCN. In this study, we conducted transcriptome analyses of wheat cv. Wen 19 (Wen19) by using RNA-Seq during the compatible interaction with CCN at 1, 3 and 8 days past inoculation (dpi). In total, 71,569 transcripts were identified, and 10,929 of them were examined as differentially expressed genes (DEGs) in response to CCN infection. Based on the functional annotation and orthologous findings, the protein phosphorylation, oxidation-reduction process, regulation of transcription, metabolic process, transport, and response process as well as many other pathways previously reported were enriched at the transcriptional level. Plant cell wall hydrolysis and modifying proteins, auxin biosynthesis, signalling and transporter genes were up-regulated by CCN infection to facilitate penetration, migration and syncytium establishment. Genes responding to wounding and jasmonic acid stimuli were enriched at 1 dpi. We found 16 NBS-LRR genes, 12 of which were down-regulated, indicating the repression of resistance. The expression of genes encoding antioxidant enzymes, glutathione S-transferases and UDP-glucosyltransferase was significantly up-regulated during CCN infection, indicating that they may play key roles in the compatible interaction of wheat with CCN. Taken together, the results obtained from the transcriptome analyses indicate that the genes involved in oxidation-reduction processes, induction and suppression of resistance, metabolism, transport and syncytium establishment may be involved in the compatible interaction of Wen 19 with CCN. This study provides new insights into the responses of wheat to CCN infection. These insights could facilitate the elucidation of the potential mechanisms of wheat responses to CCN.

Project description:One of the reasons for the progressive yield decline observed in cereals production is the rapid build-up of populations of the cereal cyst nematode (CCN, Heterodera avenae). These nematodes secrete so-call effectors into their host plant to suppress the plant defense responses, alter plant signaling pathways and then induce the formation of syncytium after infection. However, little is known about its molecular mechanism and parasitism during incompatible infection. To gain insight into its repertoire of parasitism genes, we investigated the transcriptome of the early parasitic second-stage (30 hours, 3 days and 9 days post infection) juveniles of the CCN as well as the CCN infected tissue of the host Aegilops variabilis by Illumina sequencing. Among all assembled unigenes, 681 putative genes of parasitic nematode were found, in which 56 putative effectors were identified, including novel pioneer genes and genes corresponding to previously reported effectors. All the 681 CCN unigenes were mapped to 229 GO terms and 200 KEGG pathways, including growth, development and several stimulus-related signaling pathways. Sixteen clusters were involved in the CCN unigene expression atlas at the early stages during infection process, and three of which were significantly gene-enriched. Besides, the protein-protein interaction network analysis revealed 35 node unigenes which may play an important role in the plant-CCN interaction. Moreover, in a comparison of differentially expressed genes between the pre-parasitic juveniles and the early parasitic juveniles, we found that hydrolase activity was up-regulated in pre J2s whereas binding activity was upregulated in infective J2s. RT-qPCR analysis on some selected genes showed detectable expression, indicating possible secretion of the proteins and putative role in infection. This study provided better insights into the incompatible interaction between H. avenae and the host plant Ae. varabilis. Moreover, RNAi targets with potential lethality were screened out and primarily validated, which provide candidates for engineering-based control of cereal cyst nematode in crops breeding.

Dataset Information

Characterization of genetic homogeneity of an Indian population of cereal cyst nematode, Heterodera avenae using sequencing and PCR-RFLP of ribosomal DNA.

Publications

Characterization of genetic homogeneity of an Indian population of cereal cyst nematode, Heterodera avenae using sequencing and PCR-RFLP of ribosomal DNA.

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