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Assessment of genetic and epigenetic changes in virus-free garlic (Allium sativum L.) plants obtained by meristem culture followed by in vitro propagation

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This is the first report assessing epigenetic variation in garlic. High genetic and epigenetic polymorphism during in vitro culture was detected. Sequencing of MSAP fragments revealed homology with ESTs.

Abstract

Garlic (Allium sativum) is a worldwide crop of economic importance susceptible to viral infections that can cause significant yield losses. Meristem tissue culture is the most employed method to sanitize elite cultivars. Often the virus-free garlic plants obtained are multiplied in vitro (micro propagation). However, it was reported that micro-propagation frequently produces somaclonal variation at the phenotypic level, which is an undesirable trait when breeders are seeking to maintain varietal stability. We employed amplification fragment length polymorphism and methylation sensitive amplified polymorphism (MSAP) methodologies to assess genetic and epigenetic modifications in two culture systems: virus-free plants obtained by meristem culture followed by in vitro multiplication and field culture. Our results suggest that garlic exhibits genetic and epigenetic polymorphism under field growing conditions. However, during in vitro culture system both kinds of polymorphisms intensify indicating that this system induces somaclonal variation. Furthermore, while genetic changes accumulated along the time of in vitro culture, epigenetic polymorphism reached the major variation at 6 months and then stabilize, being demethylation and CG methylation the principal conversions. Cloning and sequencing differentially methylated MSAP fragments allowed us to identify coding and unknown sequences of A. sativum, including sequences belonging to LTR Gypsy retrotransposons. Together, our results highlight that main changes occur in the initial 6 months of micro propagation. For the best of our knowledge, this is the first report on epigenetic assessment in garlic.

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Acknowledgments

The authors would like to thank M.Sc. José Luis Burba for kindly supplying vegetal materials and making suggestions, Dr. Marcos Celli for its collaboration in plant virus analysis, Dr. Ricardo W. Masuelli for generously providing equipment and for his valuable comments, to Dr. Nicolás Cara for technical support and to PhD María Virginia Sánchez Puerta for her careful revision of this manuscript and valuable comments. The present work was funded by grants from INTA (National Institute for Agricultural Technology) through the project “Generación de tecnologías alternativas frente a los nuevos escenarios para la sustentabilidad de la cadena de valor de ajo diferenciado” (INTA-PNHFA061251) and by the grants from SECTyP (Science, Technique and Postgraduate Secretariat) through the project “Estrés y cambios asociados a la metilación de ADN en plantas de ajo (Allium sativum L.) cultivadas in vitro: influencia del genotipo (Parte II)” (SECTyP UNCuyo 06/A49).

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Authors and Affiliations

  1. Laboratorio de Biología Molecular, Instituto de Biología Agrícola de Mendoza, Consejo Nacional de Investigaciones Científicas y Técnicas (IBAM-CONICET), Facultad de Ciencias Agrarias, Universidad Nacional de Cuyo, Almirante Brown 500, Chacras de Coria, Mendoza, Argentina

    Magalí Diana Gimenez, Carlos Federico Marfil & Sandra Claudia García-Lampasona

  2. CIGEOBIO (FCEFyN, UNSJ/CONICET), Av. Ignacio de la Roza 590 (Oeste), J5402DCS, Rivadavia, San Juan, Argentina

    Anahí Mara Yañez-Santos & Rosalía Cristina Paz

  3. Instituto de Fisiología y Recursos Genéticos Vegetales (IFRGV), Centro de Investigaciones Agropecuarias (CIAP), Instituto Nacional de Tecnología Agropecuaria (INTA), Camino 60 cuadras km 5.5 (Av. 11 de setiembre 4755), Córdoba, Córdoba, Argentina

    Mariana Paola Quiroga

  4. Instituto de Patología Vegetal (IPAVE), Centro de Investigaciones Agropecuarias (CIAP), Instituto Nacional de Tecnología Agropecuaria (INTA), Camino 60 cuadras km 5.5 (Av. 11 de setiembre 4755), Córdoba, Córdoba, Argentina

    Vilma Cecilia Conci

  5. Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Córdoba, Córdoba, Argentina

    Vilma Cecilia Conci

  6. EEA Mendoza, Instituto Nacional de Tecnología Agropecuaria (INTA), San Martín 3853, (5507), Luján de Cuyo, Mendoza, Argentina

    Sandra Claudia García-Lampasona

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  1. Magalí Diana Gimenez
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  2. Anahí Mara Yañez-Santos
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Communicated by Z.-Y. Wang.

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299_2015_1874_MOESM4_ESM.tif

Suppl. Figure 1 Diagram of vegetal material showing the two culture systems used in this work: field culture and meristem regeneration followed by in vitro propagation. The original material, Perla INTA accession was cultured in soil and in vitro by meristem plus micro propagation culture. Three linages of virus-free plants were selected. For DNA extraction leaf samples from three replicates were excised (FA, FB, FC) from meristem field-grown plants (MA, MB, MC), 6 (IV6A, IV6B, IV6C) and 12 (IV12A, IV12B, IV12C) months micropropagated plants in vitro. (TIFF 6750 kb)

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Gimenez, M.D., Yañez-Santos, A.M., Paz, R.C. et al. Assessment of genetic and epigenetic changes in virus-free garlic (Allium sativum L.) plants obtained by meristem culture followed by in vitro propagation. Plant Cell Rep 35, 129–141 (2016). https://doi.org/10.1007/s00299-015-1874-x

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