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Molecular Biotechnology

, Volume 56, Issue 11, pp 1049–1059 | Cite as

Identification and Characterization of Microsatellite from Alternaria brassicicola to Assess Cross-Species Transferability and Utility as a Diagnostic Marker

  • Ruchi Singh
  • Sudheer KumarEmail author
  • Prem Lal Kashyap
  • Alok Kumar Srivastava
  • Sanjay Mishra
  • Arun Kumar Sharma
Research

Abstract

Alternaria blight caused by Alternaria brassicicola (Schwein.) Wiltshire and A. brassicae (Berk.) Sacc., is one of the most important disease of rapeseed–mustard, characterized by the formation of spots on leaves, stem, and siliquae with premature defoliation and stunting of growth. These two species are very difficult to differentiate based on disease symptoms or spore morphology. Therefore, the aim of present investigation was to identify and characterize transferable microsatellite loci from A. brassicicola to A. brassicae for the development of diagnostic marker. A total of 8,457 microsatellites were identified from transcript sequences of A. brassicicola. The average density of microsatellites was one microsatellite per 1.94 kb of transcript sequence screened. The most frequent repeat was tri-nucleotide (74.03 %), whereas penta-nucleotide (1.14 %) was least frequent. Among amino acids, arginine (13.11 %) showed maximum abundance followed by lysine (10.11 %). A total of 32 alleles were obtained across the 31 microsatellite loci for the ten isolates of A. brassicicola. In cross-species amplifications, 5 of the 31 markers amplified the corresponding microsatellite regions in twenty isolates of A. brassicae and showed monomorphic banding pattern. Microsatellite locus ABS28 was highly specific for A. brassicicola, as no amplification was observed from twenty-nine other closely related taxa. Primer set, ABS28F/ABS28R, amplified a specific amplicon of 380 bp from all A. brassicicola isolates. Standard curves were generated for A. brassicicola isolate using SYBR Green I fluorescent dye for detection of amplification in real-time PCR assay. The lowest detection limit of assay was 0.01 ng. Thus, the primer set can be used as diagnostic marker to discriminate and diagnose A. brassicicola from synchronously occurring fungus, A. brassicae associated with rapeseed and mustard.

Keywords

Alternaria Microsatellite Rapeseed–mustard Transferability 

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Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Ruchi Singh
    • 1
  • Sudheer Kumar
    • 1
    Email author
  • Prem Lal Kashyap
    • 1
  • Alok Kumar Srivastava
    • 1
  • Sanjay Mishra
    • 2
  • Arun Kumar Sharma
    • 1
  1. 1.National Bureau of Agriculturally Important Microorganisms (NBAIM)MauIndia
  2. 2.IFTM UniversityMoradabadIndia

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