Efficiency of PCR-RF-SSCP marker production in Brassica oleracea using Brassica EST sequences

Hisashi Inoue, Takeshi Nishio

    Research output: Contribution to journalArticlepeer-review

    7 Citations (Scopus)

    Abstract

    Efficiencies of SCAR, CAPS and PCR-RF-SSCP marker production were investigated using two combinations of breeding lines in Brassica oleracea. Published EST sequences of B. oleracea, Brassica rapa, Brassica napus, and Arabidopsis thaliana and newly determined nucleotide sequences of anther cDNA clones from B. oleracea were used for designing primer pairs to amplify genes. The percentage of primer pairs yielding DNA amplification of a single gene was higher in primer pairs of B. oleracea (91%) than those of B. rapa (56%) and A. thaliana (17%). Single DNA fragments amplified by 9% of the primer pairs showed polymorphism as SCAR markers between a broccoli line and a Chinese kale line by agarose-gel electrophoresis. CAPS analysis showed different band patterns in 32% of the same-sized DNA fragments, and PCR-RF-SSCP analysis revealed DNA polymorphism in 52% of those showing no DNA polymorphism by CAPS. In total, 71% of the single DNA fragments were converted to DNA markers. The frequency of DNA polymorphism between parental lines of a cabbage F 1 hybrid was lower, 5% by SCAR and 12% by CAPS. However PCR-RF-SSCP analysis revealed DNA polymorphism in 21% of the DNA fragments showing no polymorphism by CAPS. These results suggest that PCR-RF-SSCP analysis enables highly efficient DNA marker production for mapping of genes in Brassica using progeny, even progeny of closely related parents. Analysis of selfed seeds of broccoli F 1 cultivars using PCR-RF-SSCP markers indicated that PCR-RF-SSCP analysis is also applicable to seed purity tests.

    Original languageEnglish
    Pages (from-to)233-242
    Number of pages10
    JournalEuphytica
    Volume137
    Issue number2
    DOIs
    Publication statusPublished - 2004 May

    Keywords

    • Brassica
    • CAPS
    • DNA marker
    • PCR-RF-SSCP
    • SSCP
    • seed purity test

    ASJC Scopus subject areas

    • Agronomy and Crop Science
    • Genetics
    • Plant Science
    • Horticulture

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