The sequences of the three PDI cDNAs, wPDI1, wPDI2 and wPDI3 (Section 1.5.5), the published genomic sequences from T. turgidum (Section 1.5.5) and the sequences from T. turgidum and Ae. tauschii generated during this study (Section 2.9 and Chapter 3) were used to identify nucleotide positions unique to each of the three PDI homeoalleles. These positions were used to design primer sequences that were able to discriminate between the three PDI genes of T. aestivum, wherein the terminal 3’ base of both the forward and reverse primers was unique to the homeoalleles to be amplified (an example is shown in Figure 2.3). Wherever possible, these primers were designed to flank non-coding sequences, eg. introns and 5’ and 3’ UTRs. The details of these allele specific (AS)- primers are shown in Table 2.6 and their positions are illustrated in Figure 2.4.
2.10.2 Confirming the allele-specificity of primers
The primers that were based solely on cDNA sequences were first used to amplify the cDNA clones wPDI1 (PA1F/PA1R, PA3F/PA3RB and PA4F/PA4R), wPDI2 (PB1F/PB1R, PB2F/PB2R and PB3F/PB3R) and wPDI3 (PD2F/PD2R, PD4F/PD4R, PD5F/PD5R and PD6F/PD6R) to ensure that they amplified only the clone targeted by the PCR. As this strategy would not work for the primers based on intron sequences in
the PDI genes of T. turgidum and Ae. tauschii (PA2F/PA2R, PA3F/PA3R, PD1F/PD1R and PD3F/PD3R; Table 2.6), specificity of these primers pairs was confirmed by direct sequencing of these PCR products. As the different PDI genes have characteristic sequences (Johnson et al. 2001, Ciaffi et al. 2001 and Section 3.3.3), mixed populations of PCR products amplified from multiple loci, due to non-specificity of AS-primer annealing, would appear as areas of sequence ambiguity due to SNPs or overlapping, nonsense sequences due to insertion/deletions (indels) between the homeoalleles (Section 3.3.3). Thus, allele specificity was checked by ensuring unambiguous sequence data was generated from all AS-primer pairs during the initial amplification and sequencing of T. aestivum cv. Katepwa gDNA. Initial annealing temperatures (Table 2.6) were determined using the web-based software NetPrimer (www.premierbiosoft.com) and increased in 1°C increments, until allele specificity was achieved.
Figure 2.3 An example of the design of allele specific primers
The sequences of the homeoalleles from T. turgidum (TaPDI4A and TaPDI4B) and Ae. tauschii (AePDI4D) (Chapter 3)flanked by the allele specific primer pair PAF1/PAR1. This alignment illustrates the basis for primer allele-specificity, in this case for TtPDI4A. *The complementary sequence of PAR1 is shown. The GT/AG at the beginning and end of intron 3 are highlighted. The numbering system is based on TtPDI4A (Chapter 3).
Table 2.6 The allele-specific primers used to amplify the PDI genes of wheat
Primer pairs and sequences (5’-3’)a
Expected gDNA PCR productb (bp) Annealing temperatures Calculatedc/Actual (°C) Expected cDNA PCR productd (if applicable) (bp) PA1F: GCTTCCAAGGAGATAAAGGCG PA1R: GACCGCAGCTTCTCAGCAAG 227 61/64 134 PA2F: GAATTTACAAACTTCCTTGAGC
PA2Re: AAAAATATACAGCAGTAATCATCTG 650 54/57 N/A
PA3F: CAACCATCCTTACCTCTTGAAATA
PA3Re: TAAAGAAATAGAAAGGGAAACAAT 900 55/58 N/A
PA3F: CAACCATCCTTACCTCTTGAAATA PA3RB: GCCTGATCCTCTTTCAGGCCA 987 58/60 210 A Genome PA4F: GAGGCCAACAATGAGCCTGTG PA4R: ATACGAGACCTTCTTCCCGCTA 511 60/62 282 PB1F: CCTGAAGATGCCACTTACCTC PB1R: CCAATGGCCTCTCCACTGCG 291 57/61 185 PB2F: CTGCTTTGGAGAAATTCATTGAG PB2R: AGGAACTTGACATCCTTGCCG 888 58/63 214 B Genome PB3F: TTGCTTGGTTGAAGGATTACTTC PB3R: CCCTCGTAGGAGACCTTCTTT 653 57/62 352 PD1Fe: CCCTCGCCTCTGGTGTCT PD1Re: CATGGCTATATGACGATTGCTATT 824 58/62 N/A PD2F: GGGAGGCTGAGGGAATTGTC PD2R: AGTGCCGCTGAATTCAGTGAAA 244 61/65 140 PD3Fe: TTGTATAGGTTGGTGTTTTCACT PD3R: CATTGGTCTGGAAGAATTTCAG 488 57/58 N/A PD4F: CTGACAACCATCCTTACCTCC PD4R: GTCACTGTCTTGAATGAGGATC 960 53/60 242 PD5F: AAAGAGGATCAGGCACCACTG PD5R: GCGATCACAACGTCCTCTTCA 553 59/62 332 D Genome PD6F: GCTGCCACCCTTCAAAGT PD6R: TACAGTATTTCTCGCAACGGGA 519 55/56 434 a
The positions of these primers are shown in Figure 2.4. Nucleotides unique to the amplified gene are highlighted in yellow.
b
The expected size is predicted from the PDI genes from T. turgidum (A and B genomes) (Ciaffi et al., 2001 and Section 3.3) and Ae. tauschii (D genome) (Section 3.3).
c
Calculated annealing temperatures were determined with NetPrimer (www.premierbiosoft.com).
d
The expected sizes are from wPDI1 (A genome), wPDI2 (B genome) or wPDI3 (D genome) (Section 1.5.5).
e
C h ap te r 2 8 7
Figure 2.4 Allele-specific primer locations in the PDI genes of wheat.
The genomes (A, B and D), exons (boxes) and introns (lines joining boxes) are shown and are based on Ciaffi et al. (2001) and work generated in this study (Chapter 3). The sizes (bp) of the introns and exons are shown. Arrows pointing right or left indicate forward or reverse primers, respectively. Details of these primers are given in Table 2.6.
2.10.3 AS-PCR amplification and sequencing of T. aestivum PDI genes
Sections of the three PDI homeoalleles of T. aestivum were amplified from gDNA isolated from the parental cultivars of the mapping populations (Section 1.8.4.1), Cranbrook, Halberd, Egret, Sunstar, Sunco, Tasman, CD87 and Katepwa using standard PCR conditions (Section 2.3.2) with annealing temperatures specific for each primer pair (Table 2.6). Aliquots of the PCR reactions were electrophoresed on 1.5% agarose gels (Section 2.5.3) to test for successful amplifications. The PCR products were then purified using the Concert Rapid PCR Purification Kit (Invitrogen) (Section 2.4.2) and directly sequenced (Section 2.6.2) using the forward and reverse primers used in the amplification of the PCR product according. DNA sequences were aligned as described in Section 2.6.3.2 to identify any inter-cultivar polymorphisms, which were then confirmed by repetition of the PCR and sequencing of the appropriate section of the PDI gene from the appropriate cultivar(s).
2.11 CHARACTERISATION OF FURTHER PUTATIVE PDI cDNA