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.

De Novo Transcriptome Sequencing and Analysis of the Cereal Cyst Nematode, Heterodera avenae

“…Genetic homogeneity of the population was determined by PCR-RFLP and sequencing of ITS (Internal Transcribed Spacer) regions of rDNA (ribosomal DNA). A genetically homogenous population was then multiplied on wheat plants grown in pots kept in a greenhouse [19] . Newly formed cysts were collected manually from the wheat roots using a dissecting microscope and kept for hatching.…”