PCR identification of rpgip1 transgene in Pisum sativum L.
Recent efforts to increase Ascochyta blight resistance of pea have focused on the introduction of foreign genes by genetic engineering. The rpgip1 gene from Rubus idaeus was introduced by Agrobacterium-mediated transformation into Pisum sativum, cv. Baroness with the aim to increase pea resistance...
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| Format: | Article |
| Language: | English |
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University of Ljubljana Press (Založba Univerze v Ljubljani)
2011-11-01
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| Series: | Acta Agriculturae Slovenica |
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| Online Access: | https://journals.uni-lj.si/aas/article/view/14601 |
| _version_ | 1797390801111613440 |
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| author | Kornelia POLOK Hans-Jörg JACOBSEN |
| author_facet | Kornelia POLOK Hans-Jörg JACOBSEN |
| author_sort | Kornelia POLOK |
| collection | DOAJ |
| description |
Recent efforts to increase Ascochyta blight resistance of pea have focused on the introduction of foreign genes by genetic engineering. The rpgip1 gene from Rubus idaeus was introduced by Agrobacterium-mediated transformation into Pisum sativum, cv. Baroness with the aim to increase pea resistance to fungal diseases. Notwithstanding this success, practical applications have to be preceded by the development of analytical methods for screening. Singleplex and multiplex PCR assays were employed to test primer efficiency in identifying the rpgip1 transgene in 11 pea genotypes. Five from ten primer combinations were effective in identifying transgene or insert sequences. PCR amplification using five other primer pairs revealed unspecific amplicons. According to in silico analyses, they arose from retrotransposons and pea genes including homologues of rpgip1. Two sets of primers were prepared with the aim of simultaneous amplification of different rpgip1 fragments. Fingerprints were sums of bands observed from individual pairs so the utility of multiplex assays was demonstrated. An additional advantage of multiplex PCR was clear differentiation between the transgene and endogenous pgip genes present in the donor species, R. idaeus. Sequencing of two PCR products confirms that no substantial rearrangements at the rpgip1 transgene arose during development of transgenic plants. However, a deletion occurred at 59 bp in the PGIP+VST line and a substitution at 392 bp in the PGIP line. The frequency of point mutations was not high (1.1 x 10-3) and comparable with the frequency expected for host genes based on the neutral theory of molecular evolution.
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| first_indexed | 2024-03-08T23:23:34Z |
| format | Article |
| id | doaj.art-3907d4ad4b764e4a916d78be6d6a730b |
| institution | Directory Open Access Journal |
| issn | 1854-1941 |
| language | English |
| last_indexed | 2024-03-08T23:23:34Z |
| publishDate | 2011-11-01 |
| publisher | University of Ljubljana Press (Založba Univerze v Ljubljani) |
| record_format | Article |
| series | Acta Agriculturae Slovenica |
| spelling | doaj.art-3907d4ad4b764e4a916d78be6d6a730b2023-12-14T22:14:34ZengUniversity of Ljubljana Press (Založba Univerze v Ljubljani)Acta Agriculturae Slovenica1854-19412011-11-01973PCR identification of rpgip1 transgene in Pisum sativum L.Kornelia POLOK0Hans-Jörg JACOBSEN1Department of Genetics, University of Warmia and Mazury in Olsztyn, Plac Lodzki 3, 10-967 OlsztynPlant Biotech Unit, Gottfried Wilhelm Leibniz Universität Hannover, Herrenhauserstr 2, D30-419 Hannover Recent efforts to increase Ascochyta blight resistance of pea have focused on the introduction of foreign genes by genetic engineering. The rpgip1 gene from Rubus idaeus was introduced by Agrobacterium-mediated transformation into Pisum sativum, cv. Baroness with the aim to increase pea resistance to fungal diseases. Notwithstanding this success, practical applications have to be preceded by the development of analytical methods for screening. Singleplex and multiplex PCR assays were employed to test primer efficiency in identifying the rpgip1 transgene in 11 pea genotypes. Five from ten primer combinations were effective in identifying transgene or insert sequences. PCR amplification using five other primer pairs revealed unspecific amplicons. According to in silico analyses, they arose from retrotransposons and pea genes including homologues of rpgip1. Two sets of primers were prepared with the aim of simultaneous amplification of different rpgip1 fragments. Fingerprints were sums of bands observed from individual pairs so the utility of multiplex assays was demonstrated. An additional advantage of multiplex PCR was clear differentiation between the transgene and endogenous pgip genes present in the donor species, R. idaeus. Sequencing of two PCR products confirms that no substantial rearrangements at the rpgip1 transgene arose during development of transgenic plants. However, a deletion occurred at 59 bp in the PGIP+VST line and a substitution at 392 bp in the PGIP line. The frequency of point mutations was not high (1.1 x 10-3) and comparable with the frequency expected for host genes based on the neutral theory of molecular evolution. https://journals.uni-lj.si/aas/article/view/14601Transgenic peafungal diseasesRubus idaeuspgip homologuesmultiplex PCR |
| spellingShingle | Kornelia POLOK Hans-Jörg JACOBSEN PCR identification of rpgip1 transgene in Pisum sativum L. Acta Agriculturae Slovenica Transgenic pea fungal diseases Rubus idaeus pgip homologues multiplex PCR |
| title | PCR identification of rpgip1 transgene in Pisum sativum L. |
| title_full | PCR identification of rpgip1 transgene in Pisum sativum L. |
| title_fullStr | PCR identification of rpgip1 transgene in Pisum sativum L. |
| title_full_unstemmed | PCR identification of rpgip1 transgene in Pisum sativum L. |
| title_short | PCR identification of rpgip1 transgene in Pisum sativum L. |
| title_sort | pcr identification of rpgip1 transgene in pisum sativum l |
| topic | Transgenic pea fungal diseases Rubus idaeus pgip homologues multiplex PCR |
| url | https://journals.uni-lj.si/aas/article/view/14601 |
| work_keys_str_mv | AT korneliapolok pcridentificationofrpgip1transgeneinpisumsativuml AT hansjorgjacobsen pcridentificationofrpgip1transgeneinpisumsativuml |