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Analysis of Diverse Plant Species using Mass Spectrometric Cleaved Amplified Polymorphic Sequences (MS-CAPS) and Improvement of PCR Efficiency by Addition of Cysteine

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Abstract

The applicability of mass spectrometric cleaved amplified polymorphic sequences (MS-CAPS) was evaluated in several plant species. This method consists of genomic DNA extraction from plant tissues, polymerase chain reaction (PCR) amplification of a specific genetic region, enzymatic digestion of amplicons, and followed by rapid analysis of single nucleotide polymorphisms (SNPs) using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Crude extracts obtained by homogenizing plant tissues in water were used as templates for short PCR amplifications for MS-CAPS analysis. For most plant species tested, these crude extracts could be used directly as templates for PCR. However, extracts from lettuce leaves and stems showed enzymatic browning as a result of polyphenol oxidase (PPO) activity, and were not suitable PCR templates. The addition of cysteine to the homogenizing solution inhibited enzymatic browning and did not affect the other MS-CAPS procedures, including PCR amplification, uracil-DNA glycosylase treatments, or MALDI-TOF MS analysis. Thus, this method inhibits PPO in crude extracts, allowing them to be used directly for MS-CAPS analysis.

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Abbreviations

CAPS:

Cleaved amplified polymorphic sequences

EF1:

Elongation factor 1

gDNA:

Genomic DNA

HSP90:

Heat-shock protein 90

MALDI-TOF MS:

Matrix assisted laser desorption ionization time-of-flight mass spectrometry

MS-CAPS:

Mass spectrometric cleaved amplified polymorphic sequence

PCR:

Polymerase chain reaction

PPO:

Polyphenol oxidase

psbA:

Chloroplast D1 protein of photosystem II reaction center

SNPs:

Single nucleotide polymorphisms

UDG:

Uracil-DNA glycosylase

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Acknowledgements

I acknowledge Ms. Kaori Nakane and Ms. Jianping Yang for their contributions.

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  1. National Institute of Agrobiological Sciences, 2-1-2 Kannondai, Tsukuba, Ibaraki, 305-8602, Japan

    H. Kajiwara

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Kajiwara, H. Analysis of Diverse Plant Species using Mass Spectrometric Cleaved Amplified Polymorphic Sequences (MS-CAPS) and Improvement of PCR Efficiency by Addition of Cysteine. Plant Mol Biol Rep 30, 1507–1512 (2012). https://doi.org/10.1007/s11105-012-0448-0

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