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Rapid sex identification method of spinach (Spinacia oleracea L.) in the vegetative stage using loop-mediated isothermal amplification

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Abstract

Main conclusion

A LAMP-mediated, simple and rapid method for sex identification in spinach was developed. Nutrient compositional analysis showed a higher iron content in male than female plants.

Spinach (Spinacia oleracea L.) is a dioecious plant with its sex determined by the XY system. Male and female floral organs differ morphologically, but plants do not differ in the vegetative stage before flowering. PCR with Y chromosome markers has been used to determine the sex of dioecious plants before flowering. In this study, we developed a genotype-specific loop-mediated isothermal amplification (LAMP) for sex identification of individual vegetative-stage spinach plants, using primers designed for the genomic region flanked by male-specific markers. LAMP could specifically detect spinach males. The method was further modified to omit DNA purification and use just an aliquot of crude leaf extract homogenized in water. We compared the nutrient composition of males and females, finding higher amounts of iron in the males. Our method could therefore be used for rapidly discriminating male plants in the field, which is useful for efficient hybrid breeding.

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References

  • Akamatsu T, Suzuki R, Uchimiya H (1998) Determination of male or female of spinach by using DNA marker. Japanese patent JPH1052284. Sakata No Tane KK, Japan

  • Centeno-Cuadros A, Abbasi I, Nathan R (2016) Sex determination in the wild: a field application of loop-mediated isothermal amplification successfully determines sex across three raptor species. Mol Ecol Resour. doi:10.1111/1755-0998.12540

    PubMed  Google Scholar 

  • Chang AC, Yang TY, Riskowski GL (2013) Ascorbic acid, nitrate, and nitrite concentration relationship to the 24 hour light/dark cycle for spinach grown in different conditions. Food Chem 138:382–388

    Article  CAS  PubMed  Google Scholar 

  • Ellis JR, Janick J (1960) The chromosomes of Spinacia oleracea. Am J Bot 47:210–214

    Article  Google Scholar 

  • Hsu TH, Gwo JC, Lin KH (2012) Rapid sex identification of papaya (Carica papaya) using multiplex loop-mediated isothermal amplification (mLAMP). Planta 236:1239–1246

    Article  CAS  PubMed  Google Scholar 

  • Janick J, Stevenson E (1954) A genetic study of the heterogametic nature of the staminate plant in spinach. Proc Am Soc Hortic 63:444–446

    Google Scholar 

  • Janick J, Stevenson E (1955) The effects of polyploidy on sex expression in spinach. J Hered 46:150–156

    Google Scholar 

  • Komatsu K, Maejima K, Fujita N, Netsu O, Tomomitsu T, Arie T, Teraoka T, Namba S (2015) Development of a detection method based on reverse transcription loop-mediated isothermal amplification for a genetically heterogeneous plantago asiatica mosaic virus. J Gen Plant Pathol 81:297–303

    Article  CAS  Google Scholar 

  • Li F, Yan W, Long L, Qi X, Li C, Zhang S (2014) Development and application of loop-mediated isothermal amplification assays for rapid visual detection of cry2Ab and cry3A genes in genetically-modified crops. Int J Mol Sci 15:15109–15121

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Maejima K, Hoshi H, Hashimoto M, Himeno M, Kawanishi T, Komatsu K, Yamaji Y, Hamamoto H, Namba S (2010) First report of plum pox virus infecting Japanese apricot (Prunus mume Sieb. et Zucc.) in Japan. J Gen Plant Pathol 76(3):229

    Article  Google Scholar 

  • Maejima K, Himeno M, Komatsu K, Takinami Y, Hashimoto M, Takahashi S, Yamaji Y, Oshima K, Namba S (2011) Molecular epidemiology of plum pox virus in Japan. Phytopathology 101:567

    Article  PubMed  Google Scholar 

  • Milewicz M, Sawicki J (2013) Sex-linked markers in dioecious plants. Plant Omics J 6:144–149

    Google Scholar 

  • Ogiwara I, Arie T (2010) Development on year round production method of blueberry fruits in plant factory with artificial four seasons. In: Nikkei BP Cleantech Institute, Nikkei Monozukuri (eds) Plant factory encyclopedia (In Japanese). Nikkei Business Publications Inc, Tokyo, pp 40–46

    Google Scholar 

  • Okuse I, Saga K (1995) Comparative studies on the contents of some inorganic constituents in the leaves within cultivars and among sexuality in spinach (Spinacia oleracea L.). Bull Fac Agric Hirosaki Univ 59:62–70

    Google Scholar 

  • Saini HS, Shepherd M, Henry RJ (1999) Microwave extraction of total genomic DNA from barley grains for use in PCR. J Inst Brew 105:185–190

    Article  CAS  Google Scholar 

  • Sather DN, Jovanovic M, Golenberg EM (2010) Functional analysis of B and C class floral organ genes in spinach demonstrates their role in sexual dimorphism. BMC Plant Biol 10:46

    Article  PubMed  PubMed Central  Google Scholar 

  • Shiobara Y, Yoshino M, Uragami A, Widiastuti Omori A, Kuba K, Saito H, Hirata Y, Sonoda T, Koizumi T, Sato T (2011) Sex distinction of asparagus by loop-mediated isothermal amplification and observation of seedling phenotypes. Euphytica 177:91–97

    Article  Google Scholar 

  • Sugiyama S, Suto C (1964) Studies on the artificial control of sex expression in spinach. Bull Nat Inst Agri Sci Jpn Ser D11:211–329 (in Japanese)

    Google Scholar 

  • Tendulkar SR, Gupta A, Chattoo BB (2003) A simple protocol for isolation of fungal DNA. Biotechnol Lett 25:1941–1944

    Article  CAS  PubMed  Google Scholar 

  • Wang L, Wang R, Yu Y, Zhang F, Wang X, Ying Y, Wu J, Xu J (2016) A powerless on-the-spot detection protocol for transgenic crops within 30 min, from leaf sampling up to results. Anal Bioanal Chem 408:657–662

    Article  CAS  PubMed  Google Scholar 

  • Yamamoto K, Oda Y, Haseda A, Fujito S, Mikami T, Onodera Y (2014) Molecular evidence that the genes for dioecism and monoecism in Spinacia oleracea L. are located at different loci in a chromosomal region. Heredity 112:317–324

    Article  CAS  PubMed  Google Scholar 

  • Yoon YE, Kuppusamy S, Cho KM, Kim PJ, Kwack YB, Lee YB (2017) Influence of cold stress on contents of soluble sugars, vitamin C and free amino acids including gamma-aminobutyric acid (GABA) in spinach (Spinacia oleracea). Food Chem 215:185–192

    Article  CAS  PubMed  Google Scholar 

  • Zhou D, Guo J, Xu L, Gao S, Lin Q, Wu Q, Wu L, Que Y (2014) Establishment and application of a loop-mediated isothermal amplification (LAMP) system for detection of cry1Ac transgenic sugarcane. Sci Rep 4:4912. doi:10.1038/srep04912

    CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

This work was supported by a grant from the Toyo Institute of Food Technology (Hyogo, Japan). We would like to thank Kodera Masaaki at Yamayoshi Farm in Kiyose, Tokyo, for providing a set of spinach samples for the nutrient component analysis.

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Authors and Affiliations

  1. Laboratory of Plant Pathology, Graduate School of Agriculture, Tokyo University of Agriculture and Technology, 3-5-8 Saiwaicho, Fuchu, Tokyo, 183-8509, Japan

    Naoko Fujita, Yu Ayukawa, Mitsutoshi Fuke, Tohru Teraoka, Tsutomu Arie & Ken Komatsu

  2. United Graduate School of Agricultural Science, Tokyo University of Agriculture and Technology, Fuchu, 183-8509, Japan

    Yu Ayukawa

  3. Graduate School of Agriculture, Tamagawa University, Machida, 194-8610, Japan

    Kyoko Watanabe

  4. Mycology and Metabolic Diversity Research Center, Tamagawa University Research Institute, Machida, 194-8610, Japan

    Kyoko Watanabe

Authors
  1. Naoko Fujita
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  2. Yu Ayukawa
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  3. Mitsutoshi Fuke
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  4. Tohru Teraoka
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  5. Kyoko Watanabe
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  6. Tsutomu Arie
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  7. Ken Komatsu
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Corresponding author

Correspondence to Ken Komatsu.

Electronic supplementary material

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425_2016_2618_MOESM1_ESM.pptx

Supplementary Figure S1 Positions of primers designed for sex determination in Spinacia oleracea L. using LAMP. LAMP primer positions for V20A, the male-specific region used in this study are shown in brackets. LAMP primers are boxed with an arrow above the sequence (PPTX 40 kb)

Supplementary material 2 (DOCX 12 kb)

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Fujita, N., Ayukawa, Y., Fuke, M. et al. Rapid sex identification method of spinach (Spinacia oleracea L.) in the vegetative stage using loop-mediated isothermal amplification. Planta 245, 221–226 (2017). https://doi.org/10.1007/s00425-016-2618-z

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  • DOI: https://doi.org/10.1007/s00425-016-2618-z

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