Advertisement

Identification of Postharvest Senescence Regulators Through Map-Based Cloning Using Detached Arabidopsis Inflorescences as a Model Tissue

Protocol
First Online:
Part of the Methods in Molecular Biology book series (MIMB, volume 1744)

Abstract

Postharvest deterioration of fruits and vegetables can be accelerated by biological, environmental, and physiological stresses. Fully understanding tissue response to harvest will provide new opportunities for limiting postharvest losses during handling and storage. The model plant Arabidopsis thaliana (Arabidopsis) has many attributes that make it excellent for studying the underlying control of postharvest responses. It is also one of the best resourced plants with numerous web-based bioinformatic programs and large numbers of mutant collections. Here we introduce a novel assay system called AIDA (the Arabidopsis Inflorescence Degreening Assay) that we developed for understanding postharvest response of immature tissues. We also demonstrate how the high-throughput screening capability of AIDA can be used with mapping technologies (high-resolution melting [HRM] and needle in the k-stack [NIKS]) to identify regulators of postharvest senescence in ethyl methanesulfonate (EMS) mutagenized plant populations. Whether it is best to use HRM or NIKS or both technologies will depend on your laboratory facilities and computing capabilities.

Key words

Postharvest Senescence Whole genome sequencing WGS High-resolution melting HRM Ethyl methanesulfonate EMS 
This is a preview of subscription content, log in to check access.

Notes

Acknowledgments

The authors wish to thank Plant and Food Research staff Lyn Watson and Zoe Erridge for technical assistance, Elena Hilario for comments on the gDNA isolation protocol, and David Brummell and Elena Hilario for critically reading the manuscript. We also wish to thank Kris Tham (SlipStream Automation) for technical assistance and Korbinian Schneeberger for use of the NIKS method his team developed. We thank the Massey Doctoral Fund for providing a scholarship to RJ. The work was supported by Plant and Food Research CORE research funding program 12059 Leafy Crops.

References

  1. 1.
    Trivellini A, Jibran R, Watson LM et al (2012) Carbon deprivation-driven transcriptome reprogramming in detached developmentally arresting Arabidopsis inflorescences. Plant Physiol 160:1357–1372CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Gundry CN, Vandersteen JG, Reed GH et al (2003) Amplicon melting analysis with labeled primers: a closed-tube method for differentiating homozygotes and heterozygotes. Clin Chem 49:396–406CrossRefPubMedGoogle Scholar
  3. 3.
    Liew M, Pryor R, Palais R et al (2004) Genotyping of single-nucleotide polymorphisms by high-resolution melting of small amplicons. Clin Chem 50:1156–1164CrossRefPubMedGoogle Scholar
  4. 4.
    Chagné D, Gasic K, Crowhurst RN et al (2008) Development of a set of SNP markers present in expressed genes of the apple. Genomics 92:353–358CrossRefPubMedGoogle Scholar
  5. 5.
    Nordstrom KJV, Albani MC, James GV et al (2013) Mutation identification by direct comparison of whole-genome sequencing data from mutant and wild-type individuals using k-mers. Nat Biotechnol 31:325–330CrossRefPubMedGoogle Scholar
  6. 6.
    Marçais G, Kingsford C (2011) A fast, lock-free approach for efficient parallel counting of occurrences of k-mers. Bioinformatics 27:764–770CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Zerbino DR, Birney E (2008) Velvet: algorithms for de novo short read assembly using de Bruijn graphs. Genome Res 18:821–829CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Camacho C, Coulouris G, Avagyan V et al (2009) BLAST+: architecture and applications. BMC Bioinformatics 10:421CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Kim Y, Schumaker KS, Zhu JK (2006) EMS mutagenesis of Arabidopsis. In: Salinas J, Sanchez-Serrano JJ (eds) Arabidopsis protocols, 2nd edn. Humana Press Inc, Totowa, NJ, pp 101–103CrossRefGoogle Scholar
  10. 10.
    Wintermans JF, de Mots A (1965) Spectrophotometric characteristics of chlorophylls a and b and their pheophytins in ethanol. Biochim Biophys Acta 109:448–453CrossRefPubMedGoogle Scholar
  11. 11.
    Dellaporta SL, Wood J, Hicks JB (1983) A plant DNA minipreparation: version II. Plant Mol Biol Report 1:19–21CrossRefGoogle Scholar
  12. 12.
    Stepanova AN, Alonso JM (2006) PCR-based screening for insertional mutants. In: Salinas J, Sanchez-Serrano JJ (eds) Arabidopsis protocols, 2nd edn. Humana Press Inc, Totowa, NJ, pp 163–172CrossRefGoogle Scholar
  13. 13.
    Jander G (2006) Gene identification and cloning by molecular marker mapping. In: Salinas J, Sanchez-Serrano JJ (eds) Arabidopsis protocols, 2nd edn. Humana Press Inc, Totowa, NJ, pp 115–126CrossRefGoogle Scholar
  14. 14.
    Kearse M, Moir R, Wilson A et al (2012) Geneious basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data. Bioinformatics 28:1647–1649CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Boyes DC, Zayed AM, Ascenzi R et al (2001) Growth stage-based phenotypic analysis of arabidopsis: a model for high throughput functional genomics in plants. Plant Cell 13:1499–1510CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Salinas J, Sanchez-Serrano JJ (eds) (2006) Arabidopsis protocols. Methods in molecular biology, 2nd edn. Humana Press Inc, Totowa, NJGoogle Scholar
  17. 17.
    Rivero-Lepinckas L, Crist D, Scholl R (2006) Growth of plants and preservation of seeds. In: Salinas J, Sanchez-Serrano JJ (eds) Arabidopsis protocols, 2nd edn. Humana Press Inc, Totowa, NJ, pp 3–12CrossRefGoogle Scholar
  18. 18.
    Koornneef M, Alonso-Blanco C, Stam P (2006) Genetic analysis. In: Salinas J, Sanchez-Serrano JJ (eds) Arabidopsis protocols, 2nd edn. Humana Press Inc, Totowa, NJ, pp 65–77CrossRefGoogle Scholar
  19. 19.
    Warthmann N, Fitz J, Weigel D (2007) MSQT for choosing SNP assays from multiple DNA alignments. Bioinformatics 23:2784–2787CrossRefPubMedGoogle Scholar
  20. 20.
    Untergasser A, Cutcutache I, Koressaar T, Ye J et al (2012) Primer3—new capabilities and interfaces. Nucleic Acids Res e11:40Google Scholar
  21. 21.
    Lutz KA (2011) Isolation and analysis of high quality nuclear DNA with reduced organellar DNA for plant genome sequencing and resequencing. BMC Biotechnol 11:54CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Lamesch P, Berardini TZ, Li D et al (2012) The Arabidopsis Information Resource (TAIR): improved gene annotation and new tools. Nucleic Acids Res 40:D1202–D1210CrossRefPubMedGoogle Scholar
  23. 23.
    Gan X, Stegle O, Behr J et al (2011) Multiple reference genomes and transcriptomes for Arabidopsis thaliana. Nature 477:419–423CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2018

Authors and Affiliations

  1. 1.The New Zealand Institute for Plant & Food Research LimitedFood Industry Science CentrePalmerston NorthNew Zealand
  2. 2.Institute of Fundamental SciencesMassey UniversityPalmerston NorthNew Zealand
  3. 3.The New Zealand Institute for Plant & Food Research LimitedMt. Albert Research CentreAucklandNew Zealand

Personalised recommendations

Cite protocol

Buy options