Effector gene vap1 based DGGE fingerprinting to assess variation within and among Heterodera schachtii populations

Abstract

Populations of beet cyst nematodes Heterodera schachtii vary in aggressiveness and virulence toward sugar beet varieties, but also in traits like host range, or decline rate in the field. Diversity of their essential pathogenicity gene vap1 is shaped by diversifying selection and gene flow. The authors developed a technique to study interpopulation variation and intra-population diversity and dynamics of H. schachtii based on the gene vap1. Degenerate primers were designed to amplify, clone, and sequence this gene from diverse species and populations of cyst nematodes. This resulted in a high diversity of sequences for H. schachtii, and allowed to design non-degenerated primers to amplify a fragment suitable for sequence dependent separation of gene variants in denaturing gradient gel electrophoresis (DGGE). The developed primers span a highly variable intron and part of a slightly variable exon. A marker comprised of the 14 mostly detected gene variants was established for gel-to-gel comparisons. For individual juveniles up to six gene variants were resolved and substantial variation within and among cysts was observed. A fast and easy DNA extraction procedure for 20 pooled cysts was established, which provided DGGE patterns with high similarity among replicate samples from field populations. Permutation tests on pairwise similarities within and among populations showed significant differences among vap1 patterns of field populations of H. schachtii. Similarly, gene diversity as expressed by the Shannon index was statistically different among field populations. In conclusion, the DGGE technique is a fast andcompared to sequencing approaches-inexpensive tool to compare populations of H. schachtii and link observed biological characteristics to genetic pattern.

Biological gene extraction path based on knowledge graph and natural language processing

“…Cai J L pointed out that a new multi-layer convolutional network model can be used in the model construction and connection prediction of large-scale knowledge graphs, which can build highly complex knowledge graphs based on its high parameter efficiency (Cai et al, 2019). Nuaima R H applied a new learning framework in the study of reasoning large-scale knowledge graphs (KG), which can construct knowledge graphs with continuous states in the KG vector space more accurately, diversely and efficiently (Nuaima et al, 2018). Shi J found that existing self-symptom detectors are far from meeting the needs of clinical decision support systems.…”

Effects of Cover Cropping on Microbial Communities Associated with Heterodera schachtii and Nematode Virulence

“…DNA was extracted from the pooled cysts using the lysis buffer method [23]. In brief, the volume of the lysis buffer was added to one volume of the prepared nematode suspension, and then incubated at 66 • C and 100 • C for two hours and 5 min, respectively.…”

“…The PCR-amplification of the effector gene vap1 from the nematode DNA was conducted using the primer pair HSvap244f/HSvap548r designed to amplify vap1 fragments of approximately 365 bp from the genomic DNA of H. schachtii populations [23]. The conditions for PCR reaction used the published protocol.…”

“…The denaturing gradient gel electrophoresis (DGGE) analysis of the vap1 gene was performed using polyacrylamide gels containing a gradient of 23 to 58%. By applying the INGENY PhorU2 system (Ingeny, Goes, The Netherlands), the DGGE gels were casted [23]. The DGGE profiles were visualized by silver staining by consecutively fixing (the main components: acetic acid and ethanol), staining (silver nitrate), developing (sodium hydroxide and formaldehyde), stopping development (sodium carbonate), and conserving (glycerin and ethanol) [30].…”

“…Resistant cultivars of cover crops may alter gene frequencies of the beet cyst nematode H. schachtii, thereby jeopardizing the longevity of this technique [22]. H. schachtii has a high genetic diversity and the ability to overcome resistance in sugar beet after a limited number of selection cycles [6,23,24].…”