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Microdissection and painting of the Y chromosome in spinach (Spinacia oleracea)

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Abstract

Spinach has long been used as a model for genetic and physiological studies of sex determination and expression. Although trisomic analysis from a cross between diploid and triploid plants identified the XY chromosome as the largest chromosome, no direct evidence has been provided to support this at the molecular level. In this study, the largest chromosomes of spinach from mitotic metaphase spreads were microdissected using glass needles. Degenerate oligonucleotide primed polymerase chain reaction was used to amplify the dissected chromosomes. The amplified products from the Y chromosome were identified using the male-specific marker T11A. For the first time, the largest spinach chromosome was confirmed to be a sex chromosome at the molecular level. PCR products from the isolated chromosomes were used in an in situ probe mixture for painting the Y chromosome. The fluorescence signals were mainly distributed on all chromosomes and four pair of weaker punctate fluorescence signal sites were observed on the terminal region of two pair of autosomes. These findings provide a foundation for the study of sex chromosome evolution in spinach.

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Abbreviations

DOP-PCR:

Degenerate oligonucleotide primed-polymerase chain reaction

AFLP:

Amplified fragment length polymorphism

EST:

Expressed sequence tag

SSC:

Saline-sodium citrate

TE:

Tris-ethylenediaminetetraacetic acid

DAPI:

4′-6-Diamidino-2-phenylindole

FISH:

Fluorescence in situ hybridization

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Acknowledgments

This work was supported by the National Natural Science Funds (31000165) and Foundation of He’nan Education Committee (2007180028).

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Authors and Affiliations

  1. College of Life Science, Henan Normal University, Xinxiang, 453007, People’s Republic of China

    Chuan-liang Deng, Rui-yun Qin, Ying Cao, Jun Gao, Shu-fen Li, Wu-jun Gao & Long-dou Lu

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  1. Chuan-liang Deng
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  2. Rui-yun Qin
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Correspondence to Chuan-liang Deng.

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10265_2013_549_MOESM1_ESM.tif

S1 Chromosome painting of root-tip cells at mitotic metaphase in female spinach. The DOP-PCR products that were amplified from the Y chromosome were labeled with fluorescein-12-dUTP by nick translation and hybridized to the chromosomes of female spinach plants. Uniform fluorescence signals were also observed on all chromosomes (a. DAPI staining, b. fluorescein-12-dUTP staining, c. merged panels; Arrows showed the weaker punctate fluorescence signal sites on the terminal part of two pair of autosome; Asterisks showed the largest chromosomes). Bar = 10 μm. (TIFF 1294 kb)

10265_2013_549_MOESM2_ESM.tif

S2 Chromosome painting of root-tip cells at mitotic metaphase in female spinach. The DOP-PCR products that were amplified from the Y chromosome were labeled with fluorescein-12-dUTP by nick translation and hybridized to the chromosomes of female spinach plants. Uniform fluorescence signals were also observed on all chromosomes (a. DAPI staining, b. fluorescein-12-dUTP staining, c. merged panels; Arrows showed the weaker punctate fluorescence signal sites on the terminal part of two pair of autosome; Asterisks showed the largest chromosomes). Bar = 10 μm. (TIFF 1191 kb)

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Deng, Cl., Qin, Ry., Cao, Y. et al. Microdissection and painting of the Y chromosome in spinach (Spinacia oleracea). J Plant Res 126, 549–556 (2013). https://doi.org/10.1007/s10265-013-0549-3

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