Table 1. Primers used to isolate/analyze GL1, GL2, GL3, TTG1 and TRY genes from B. villosa.
Primers | NCBI accession | Sequence (5′ to 3′) |
Coding Sequences from B. napus 1 | ||
GL1-F | HQ162473.1 | GTCAGGATCCATGAGAACGAGGAGAAGAACAGA |
GL1-R | GTCACTGCAGCTAGAGACAGTAGCCAGTATCA | |
GL2-F | EU826520.1 | GTCAGGATCCATGTCAATGGCCGTCGAGATGTCA |
GL2-R | ATATCTGCAGTGTTGTGCAGCGTGACAGAGACGA | |
TRY-F | EE451172.1 | AGCTGGATCCGCTTGCATTCTCCAACT |
TRY-R | CGCACTGCAGGCAATTTCGTTATGCTATATG | |
Coding Sequences from B. rapa 1 | ||
EGL3-F | HM208589.1 | GTCAGGATCCATGGCTGCTGTAGAAAACAG |
EGL3-R | GCAGCTGCAGAGTGCATCTTGAATCATTCCT | |
TTG1-F | HM208590.1 | TAGAGGATCCATGGACAACGCAGCTCCGGACT |
TTG1-R | AGTCGGTACCTCAAACTCTAAGGAGCTGCA | |
Conserved Regions (for Q-PCR) 2 | ||
GL1-F | ACTGGGCTGAAGAGGTGTGGA | |
GL1-R | GAGATGAGTGTTCCAGTGA | |
GL2-F | CGCTGGCCGGGAGAAAGAGC | |
GL2-R | GGAGGTTTTTTCTGGATGAA | |
EGL3-F | ACATTCAATGGAGTTACGGA | |
EGL3-R | AGAGATTCGTAAAGCTCTCT | |
TTG1-F | CTCTGGGAGGTCAACGAA | |
TTG1-R | ATGCTGCACGTGCCTAAC | |
TRY-F | CATCACTCCTCTTCTCACA | |
TRY-R | TGTGGTGGGGAAGAAAACAGA | |
BnEF1F (B. napus) | CCCATTCGTCCCCATCTCTGGA | |
BnEF1R (B. napus) | ACGGAGGGGCTTGTCCGAGG |
Forward (F) primers were designed at the 5′ coding end and reverse (R) primers to the 3′ end of cDNA sequences from B. napus for GL1 and GL2 and from B. rapa for EGL3 and TTG1. Forward primers for TRY were designed 72 nucleotides before coding region and reverse primer at 3′ end of the cDNA from B. rapa. Underlined sequences indicate incorporated restriction enzyme sites; BamHI and PstI sites in the GL1, GL2, EGL3 and TRY forward and reverse primers, respectively; BamHI and KpnI sites in the TTG1 forward and reverse primers, respectively.
Primers for Q-PCR were designed to conserved regions based on alignments between the A. thaliana homologue and the homologues from four Brassica species. B nEF1F/BnEF1R are endogenous reference gene primers.