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. 2020 Apr;70(2):231-240.
doi: 10.1270/jsbbs.19116. Epub 2020 Feb 26.

DNA markers based on retrotransposon insertion polymorphisms can detect short DNA fragments for strawberry cultivar identification

Chiharu Hirata  1 , Takamitsu Waki  2 , Katsumi Shimomura  1 , Takuya Wada  1 , Seiya Tanaka  3 , Hidetoshi Ikegami  1 , Yousuke Uchimura  1 , Keita Hirashima  1 , Yoshiko Nakazawa  2 , Kaori Okada  2 , Kiyoshi Namai  2 , Makoto Tahara  4 , Yuki Monden  4
Affiliations

DNA markers based on retrotransposon insertion polymorphisms can detect short DNA fragments for strawberry cultivar identification

Chiharu Hirata et al. Breed Sci. 2020 Apr.

Abstract

In this study, DNA markers were developed for discrimination of strawberry (Fragaria × ananassa L.) cultivars based on retrotransposon insertion polymorphisms. We performed a comprehensive genomic search to identify retrotransposon insertion sites and subsequently selected one retrotransposon family, designated CL3, which provided reliable discrimination among strawberry cultivars. Through analyses of 75 strawberry cultivars, we developed eight cultivar-specific markers based on CL3 retrotransposon insertion sites. Used in combination with 10 additional polymorphic markers, we differentiated 35 strawberry cultivars commonly cultivated in Japan. In addition, we demonstrated that the retrotransposon-based markers were effective for PCR detection of DNA extracted from processed food materials, whereas a SSR marker was ineffective. These results indicated that the retrotransposon-based markers are useful for cultivar discrimination for processed food products, such as jams, in which DNA may be fragmented or degraded.

Keywords: Fragaria × ananassa; PCR product; high-throughput sequencing; processed foods; retrotransposon insertion polymorphisms.

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Figures

Fig. 1.
Fig. 1.
Schematic illustration of the genomic structure of the CL3 retrotransposon and the location of PCR primers designed to detect putative cultivar-specific sites. The LTR sequence is shown in Supplemental Fig. 1. PPT, polypurine tract; PBS, primer-binding site.
Fig. 2.
Fig. 2.
PCR amplification of cultivar-specific CL3 retrotransposon-based markers. MultiNA gel image showing the markers developed in this study. CL3_Cl321, CL3_P386, CL3_P1155, CL3_P508, CL3_P364, CL3_P495, CL3_P418, and CL3_P619 are cultivar-specific markers developed to discriminate the strawberry cultivars ‘Harunoka’, ‘Fukuba’, ‘Ookimi’, ‘Miyazakinatsuharuka’, ‘Elsanta’, ‘Deco-Rouge’, ’Summer Tiara’, and ‘Otomegokoro’, respectively. Lane numbers correspond to the cultivar numbers listed in Table 1 and NTC indicates no template control. Primer sequences for each marker are listed in Supplemental Table 3.
Fig. 3.
Fig. 3.
DNA signal detection of retrotransposon-based and SSR markers for strawberry jam and leaf samples. MultiNA gel image showing markers developed in this study. CL3_Cl19, CL3_P524, CL3_Cl124, CL3_Cl322, CL3_Cl261, CL3_P320, CL3_Cl115, CL3_Cl242, CL3_Cl258, and CL3_Cl76 are a set of markers for discrimination of 35 commercial strawberry cultivars grown in Japan. Retrotransposon-based marker CL3_Cl214 and SSR marker FVES3384 are positive control markers. Jams A and B were labeled ‘Made of “Fukuoka S6 (Amaou)” fruits’ in the marketplace. NTC indicates no template control. M: 100 bp DNA ladder marker. Primer sequences for the markers are listed in Supplemental Table 3.
Fig. 4.
Fig. 4.
DNA signal detection of control markers (retrotransposon-based and SSR) in strawberry jam and leaf samples. MultiNA gel image showing the markers developed in this study. Retrotransposon-based markers Cl214_1, Cl214_2 and Cl214_3 were designed as markers to yield PCR products of different sizes. Jams C and D were derived from an unknown strawberry cultivar. A PCR band for the SSR marker (FVES3384) was only detected in leaf samples, while a PCR band for the retrotransposon-based marker (CL3_Cl214) was detected in leaf and jam samples. NTC indicates no template control. M: 100 bp DNA ladder marker. Primer sequences for the markers are listed in Supplemental Table 3.
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References

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