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Planta

, Volume 237, Issue 1, pp 43–54 | Cite as

Anti-microbial peptide (AMP): nucleotide variation, gene expression, and host resistance in the white pine blister rust (WPBR) pathosystem

  • Jun-Jun LiuEmail author
  • Arezoo Zamany
  • Richard A. Sniezko
Original Article

Abstract

Pinus monticola antimicrobial peptide (PmAMP1) inhibits growth of Cronartium ribicola and other fungal pathogens. C. ribicola causes white pine blister rust and has resulted in a dramatic reduction of native white pines across North America. Quantitative disease resistance (QDR) is a highly desirable trait screened in breeding programs for durable resistance against C. ribicola. Along with phenotyping on a collection of germplasms, we analyzed PmAMP1 transcript and protein expression and re-sequenced the full-length gene including its promoter region. A mixed linear model was used to identify the association of single nucleotide polymorphisms (SNPs) with accumulated protein and stem QDR levels. Among 16 PmAMP1 SNPs identified in the present study, we found an association of protein levels with 6 SNPs (P < 0.05), including 2 in the 5′-untranslated region (UTR), 3 in the open reading frame (ORF) region with 2 nonsynonymous SNPs, and 1 SNP in the 3′-UTR. Another set of six SNPs was associated with stem QDR levels (P < 0.05), with one localized in the promoter region and the other five in the ORF region with four nonsynonymous changes, suggesting that multiple isoforms may have antifungal activity to differing degrees. Of three common PmAMP1 haplotypes, the trees with haplotype 2 showed high QDR levels with moderate protein abundance while those trees with haplotype 3 exhibited low QDR levels in the susceptible range and the lowest level of protein accumulation. Thus, an association of gene variations with protein abundance and resistance-related traits may facilitate elucidation of physiological contribution of PmAMP1 to host resistance.

Keywords

Association analysis Protein expression Quantitative disease resistance Transcript regulation White pine blister rust 

Abbreviations

AMP

Anti-microbial peptide

ORF

Open reading frame

PR

Pathogenesis-related

QDR

Quantitative disease resistance

qRT-PCR

Quantitative reverse transcription-polymerase chain reaction

SNP

Single nucleotide polymorphism

UTR

Untranslated region

WPBR

White pine blister rust

Notes

Acknowledgments

This research was supported in part by the Canadian Forest Service (CFS) and the CFS-Genomics R&D Initiative Fund awarded to J.-J.L. We thank J. Hutchinson and L. Baerg at CFS for support on DNA sequencing; J. Hill and A. Kegley at Dorena Genetic Resource Center (DGRC) for support on tree resistance assessment.

Supplementary material

425_2012_1747_MOESM1_ESM.pdf (160 kb)
Supplementary Fig. S1 Nucleotide sequence comparison of the PmAMP1 genomic sequences. Multiple sequence alignment analysis was performed using an on-line ClustalW2 program provided by the European Bioinformatics Institute (http://www.ebi.ac.uk/Tools/msa/clustalw2/). Genomic DNA fragment was amplified using gene-specific primers from each of 150 individual trees for direct DNA sequencing. Amplified fragments showed only one unique sequence for each of 128 trees as an indicator of homozygous genotypes. Genomic DNA sequences derived from other 22 trees showed SNPs at multiple sites, suggesting a heterozygous genotype for the PmAMP1 locus in each of these trees (PDF 160 kb)
425_2012_1747_MOESM2_ESM.tif (1.6 mb)
Supplementary Fig. S2 Phylogenetic analysis of the PmAMP1 genomic sequences. Based on PmAMP1 nucleotide sequence alignment result as shown in Suppl. Fig. 1, a representative of phylogenetic trees was generated by MEGA 4.0 software using the neighbor-joining (NJ) method (TIFF 1,662 kb)
425_2012_1747_MOESM3_ESM.bmp (1 mb)
Supplementary Fig. S3 Linkage disequilibrium (LD) analysis of the PmAMP1 single nucleotide polymorphism (SNP) sites. Plot of intra-locus linkage disequilibrium between 16 polymorphic DNA variation sites in the PmAMP1 gene estimated using the TASSEL software package. Graphic r 2 is presented in the upper right and P-values are indicated in the lower left (BMP 1,040 kb)
425_2012_1747_MOESM4_ESM.ppt (58 kb)
Supplementary Fig. S4 The LD scatter-plots of squared correlation coefficient (r 2) as a function of DNA distance in base-pairs between informative polymorphic sites (f > 0.1 and P < 0.01) in the PmAMP1 gene (PPT 58 kb)
425_2012_1747_MOESM5_ESM.doc (31 kb)
Supplementary material 5 (DOC 31 kb)

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Copyright information

© Her Majesty the Queen in Right of Canada  2012

Authors and Affiliations

  • Jun-Jun Liu
    • 1
    Email author
  • Arezoo Zamany
    • 1
  • Richard A. Sniezko
    • 2
  1. 1.Natural Resources Canada, Pacific Forestry Centre, Canadian Forest ServiceVictoriaCanada
  2. 2.Dorena Genetic Resource CenterUSDA Forest ServiceCottage GroveUSA

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