Development and Identification of Expressed Sequence Tag-based SSR Markers Associated with the Heterostyly Trait in Primula forbesii

in Journal of the American Society for Horticultural Science

Heterostylous Primula forbesii is an important ornamental flower in China because of its long-lasting flowers and winter bloom. This study aimed to develop markers of expressed sequence tag–simple sequence repeats (EST-SSRs) that are associated with heterostyly and that can be used for molecular-assisted selective breeding in P. forbesii. We investigated 114,474 unigenes and identified 25,095 SSRs in P. forbesii. Dinucleotide repeats (46.14%), mononucleotide repeats (44.65%), and trinucleotide repeats (8.27%) were the most abundant SSRs. Among the 25,095 SSRs, 10,645 SSR primer pairs were successfully designed, of which 130 primer pairs were randomly selected for further amplification validation using eight accessions of P. forbesii; 98 pairs produced clear and stable polymerase chain reaction (PCR) products, and 28 pairs showed polymorphism. Bulked segregant analysis (BSA) was conducted for the F1 population with respect to thrum style and pin style by scanning 28 polymorphic SSR primer combinations. One SSR marker, c64326, linked to the heterostyly trait at a genetic distance of ≈3.70 cM was identified. The marker c64326 was further validated in two populations with an accuracy of 97.92% and 90.63%. The novel and linked EST-SSR markers can be valuable resources for genetic diversity analysis, mapping, and marker-assisted breeding in P. forbesii.

Contributor Notes

This work was supported by the 12th Five Years Key Programs for Science and Technology Development of China and the Special Fund for Beijing Common Construction Project.

These authors contributed equally.

Corresponding author. E-mail: zqxbjfu@126.com.

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    Length distribution of simple sequence repeat loci among the transcriptome of Primula forbesii. bp = base pairs.

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    Partial image of the polymerase chain reaction amplification products of simple sequence repeat primer c64326 in each gene pool and their parents in Primula forbesii; P2 = pin-style-type parent, T2 = thrum-style-type parent, M = marker, BT = thrum-style gene pool, BP = pin-style gene pool. Each gene pool was mixed with eight pin-style and eight thrum-style offspring of F1 individuals from the T2 × P2 population. bp = base pairs.

  • View in gallery

    Partial image of the polymerase chain reaction amplification products of simple sequence repeat primer c64326 in F1 offspring, thrum-style gene pool, pin-style gene pool, and their parents of Primula forbesii; P2 = pin-style-type parent, T2 = thrum-style-type parent, M = marker, BT = thrum-style gene pool, BP = pin-style gene pool. Each gene pool was mixed with eight pin-style and eight thrum-style offspring of F1 individuals from the T2 × P2 population. “P” represents pin-style; “T” represents thrum-style. “▲” performed differently from the bands of parents. bp = base pairs.

  • View in gallery

    Polymerase chain reaction amplification products of the simple sequence repeat primer c64326 that was used for sequencing in Primula forbesii. Lanes 1 to 3 refer to the amplification products in three randomly selected pin-style individuals of the F1 population; Lanes 4 to 6 refer to the amplification products in three randomly selected thrum-style individuals of the F1 population. Lane M is the DNA size marker. Each band was recovered and sequenced. bp = base pairs.

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