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, 5 (8), 1663-73

Evidence for a Common Origin of Homomorphic and Heteromorphic Sex Chromosomes in Distinct Spinacia Species


Evidence for a Common Origin of Homomorphic and Heteromorphic Sex Chromosomes in Distinct Spinacia Species

Satoshi Fujito et al. G3 (Bethesda).


The dioecious genus Spinacia is thought to include two wild relatives (S. turkestanica Ilj. and S. tetrandra Stev.) of cultivated spinach (S. oleracea L.). In this study, nuclear and chloroplast sequences from 21 accessions of Spinacia germplasm and six spinach cultivars or lines were subjected to phylogenetic analysis to define the relationships among the three species. Maximum-likelihood sequence analysis suggested that the Spinacia plant samples could be classified into two monophyletic groups (Group 1 and Group 2): Group 1 consisted of all accessions, cultivars, and lines of S. oleracea L. and S. turkestanica Ilj. and two of five S. tetrandra Stev. accessions, whereas Group 2 was composed of the three remaining S. tetrandra Stev. accessions. By using flow cytometry, we detected a distinct difference in nuclear genome size between the groups. Group 2 also was characterized by a sexual dimorphism in inflorescence structure, which was not observed in Group 1. Interspecific crosses between the groups produced hybrids with drastically reduced pollen fertility and showed that the male is the heterogametic sex (XY) in Group 2, as is the case in S. oleracea L. (Group 1). Cytogenetic and DNA marker analyses suggested that Group 1 and Group 2 have homomorphic and heteromorphic sex chromosome pairs (XY), respectively, and that the sex chromosome pairs of the two groups evolved from a common ancestral pair. Our data suggest that the Spinacia genus may serve as a good model for investigation of evolutionary mechanisms underlying the emergence of heteromorphic sex chromosome pairs from ancestral homomorphic pairs.

Keywords: Spinacia; dioecy; genetics of sex; nuclear DNA amount; sex chromosome; spinach.


Figure 1
Figure 1
Maximum-likelihood (ML) tree of concatenated DNA sequences consisting of five chloroplast intergenic spacers and nuclear ITS regions from 27 Spinacia samples and three outgroup taxa in the genus Beta. This tree was constructed using the general time reversible model. Bootstrap percentages (>80%) are listed below branches.
Figure 2
Figure 2
Flow cytometry estimates of relative nuclear DNA amounts in Spinacia species. The vertical axis represents relative fluorescence intensity against an internal reference standard, Beta vulgaris L. TK81-MS. Error bars represent standard deviations (N = 5).
Figure 3
Figure 3
Mitotic metaphase chromosomes in S. oleracea L. Mazeran and S. tetrandra Stev. PI 647859. (A–D) 4′,6-diamidino-2-phenylindole (DAPI)-stained metaphase chromosomes of S. oleracea L. Mazeran (A, male; B, female) and S. tetrandra Stev. PI 647859 (C, male; D, female). The Arabic numerals next to chromosomes represent the chromosome number. The sex chromosomes of S. tetrandra Stev. are denoted by X and Y. Positions of satellites (SAT) are indicated by arrows. (A′–D′) Fluorescence in situ hybridization mapping of 45S (FITC, green) and 5S (Cy3, red) rDNA loci on metaphase chromosomes of S. oleracea L. Mazeran (A′, male; B′, female) and S. tetrandra Stev. PI 647859 (C′, male; D′, female). Triangles and arrows show the locations of 45S and 5S rDNA loci. Bars = 5 µm.
Figure 4
Figure 4
Polymerase chain reaction−based genotyping of male and female plants from S. tetrandra Stev. PI 647859, PI 647860, and PI 647861. (A) Genotyping of microsatellite SO4 and sequenced-characterized amplified region (SCAR) marker SP_0008. (B) Single-nucleotide polymorphism typing for the ketohexokinase (khk) locus in parental and progeny plants of a cross between a male plant and a female plant in PI 647859, using dCAPS marker SP_0048. DNA bands representing the “T” and “A” alleles are indicated by arrows. SNP genotypes (TT or TA) at the khk locus are shown below the gel image.

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