Cloning of ribosomal ITS PCR products creates frequent, non-random chimeric sequences – a test involving heterozygotes between Gymnopus dichrous taxa I and II
Gymnopus dichrous exists in the southern Appalachians (USA) as two distinct entities with essentially identical nuclear ribosomal ITS1 sequences but differing ITS2 and LSU sequences (for convenience, called G. dichrous I and II). F1 ITS heterozygotes between the two are routinely collected from natu...
| Main Authors: | , , |
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| Format: | Article |
| Language: | English |
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Pensoft Publishers
2015-06-01
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| Series: | MycoKeys |
| Online Access: | http://mycokeys.pensoft.net/lib/ajax_srv/article_elements_srv.php?action=download_pdf&item_id=5126 |
| _version_ | 1811272089338904576 |
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| author | Karen W. Hughes Samuel D. Morris Ana Reboredo Segovia |
| author_facet | Karen W. Hughes Samuel D. Morris Ana Reboredo Segovia |
| author_sort | Karen W. Hughes |
| collection | DOAJ |
| description | Gymnopus dichrous exists in the southern Appalachians (USA) as two distinct entities with essentially identical nuclear ribosomal ITS1 sequences but differing ITS2 and LSU sequences (for convenience, called G. dichrous I and II). F1 ITS heterozygotes between the two are routinely collected from nature. Cloning of ITS PCR products from F1 heterozygotes produced sequences of both parental haplotypes but also numerous chimeric sequences (21.9%). The location of template switching was non-random leading to recovery of the same chimera several times and the chimeric region varied from 45bp to 300bp. By comparison, single-basidiospore isolates from heterozygote F1 fruitbodies showed no recombinant haplotypes within the ITS + LSU span and clones derived from P1 homozygotes were identical to the P1 parent. Thus, chimeric sequences are likely an artifact of the PCR-cloning process and not a consequence of natural recombination events found in nature, nor are they due to hidden existing variation within the ribosomal repeat. Chimeras and PCR-induced mutations are common in cloned PCR products and may result in incorrect sequence information in public databases. |
| first_indexed | 2024-04-12T22:33:01Z |
| format | Article |
| id | doaj.art-8ac601634ed048a785c0c143b5b754be |
| institution | Directory Open Access Journal |
| issn | 1314-4057 1314-4049 |
| language | English |
| last_indexed | 2024-04-12T22:33:01Z |
| publishDate | 2015-06-01 |
| publisher | Pensoft Publishers |
| record_format | Article |
| series | MycoKeys |
| spelling | doaj.art-8ac601634ed048a785c0c143b5b754be2022-12-22T03:13:55ZengPensoft PublishersMycoKeys1314-40571314-40492015-06-0110455610.3897/mycokeys.10.51265126Cloning of ribosomal ITS PCR products creates frequent, non-random chimeric sequences – a test involving heterozygotes between Gymnopus dichrous taxa I and IIKaren W. HughesSamuel D. MorrisAna Reboredo SegoviaGymnopus dichrous exists in the southern Appalachians (USA) as two distinct entities with essentially identical nuclear ribosomal ITS1 sequences but differing ITS2 and LSU sequences (for convenience, called G. dichrous I and II). F1 ITS heterozygotes between the two are routinely collected from nature. Cloning of ITS PCR products from F1 heterozygotes produced sequences of both parental haplotypes but also numerous chimeric sequences (21.9%). The location of template switching was non-random leading to recovery of the same chimera several times and the chimeric region varied from 45bp to 300bp. By comparison, single-basidiospore isolates from heterozygote F1 fruitbodies showed no recombinant haplotypes within the ITS + LSU span and clones derived from P1 homozygotes were identical to the P1 parent. Thus, chimeric sequences are likely an artifact of the PCR-cloning process and not a consequence of natural recombination events found in nature, nor are they due to hidden existing variation within the ribosomal repeat. Chimeras and PCR-induced mutations are common in cloned PCR products and may result in incorrect sequence information in public databases.http://mycokeys.pensoft.net/lib/ajax_srv/article_elements_srv.php?action=download_pdf&item_id=5126 |
| spellingShingle | Karen W. Hughes Samuel D. Morris Ana Reboredo Segovia Cloning of ribosomal ITS PCR products creates frequent, non-random chimeric sequences – a test involving heterozygotes between Gymnopus dichrous taxa I and II MycoKeys |
| title | Cloning of ribosomal ITS PCR products creates frequent, non-random chimeric sequences – a test involving heterozygotes between Gymnopus dichrous taxa I and II |
| title_full | Cloning of ribosomal ITS PCR products creates frequent, non-random chimeric sequences – a test involving heterozygotes between Gymnopus dichrous taxa I and II |
| title_fullStr | Cloning of ribosomal ITS PCR products creates frequent, non-random chimeric sequences – a test involving heterozygotes between Gymnopus dichrous taxa I and II |
| title_full_unstemmed | Cloning of ribosomal ITS PCR products creates frequent, non-random chimeric sequences – a test involving heterozygotes between Gymnopus dichrous taxa I and II |
| title_short | Cloning of ribosomal ITS PCR products creates frequent, non-random chimeric sequences – a test involving heterozygotes between Gymnopus dichrous taxa I and II |
| title_sort | cloning of ribosomal its pcr products creates frequent non random chimeric sequences a test involving heterozygotes between gymnopus dichrous taxa i and ii |
| url | http://mycokeys.pensoft.net/lib/ajax_srv/article_elements_srv.php?action=download_pdf&item_id=5126 |
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