1School of Molecular Sciences, Victoria University, Werribee, VIC 3030, Australia.
2Molecular Plant Breeding CRC, Murdoch University and Department of Agriculture Western Australia, Murdoch, WA 6150, Australia.
3Environment and Biotechnology Centre, Faculty of Life and Social Sciences, Swinburne University of Technology, PO Box 218, Hawthorn, VIC 3122, Australia.
The folding, association and deposition of storage proteins is an important process influencing the elastic and extensible properties of the wheat dough (Shewry et al., 2003). However, little is known regarding the cellular factors that may coordinate these processes and ensure the proper formation of the protein bodies. A strong candidate for these activities is the ‘foldase’ enzyme, protein disulfide isomerase (PDI) which is localised in the endoplasmic reticulum (ER) and catalyses the formation, cleavage and isomerization of disulfide bonds in nascent proteins (Freedman et al., 1994). Direct evidence for the role of PDI in storage protein deposition was provided through analysis of the rice mutant, esp2, which exhibits abnormal storage protein deposition in conjunction with an absence of PDI expression (Takemoto et al., 2002). We recently conducted molecular analyses of the wheat PDI gene family (Johnson and Bhave, 2004) providing the basis for development of markers for these genes which could be used in determining their more precise locations and the genetic dissection of the role of PDI. The present work reports the physical mapping of the three PDI genes of wheat, as well as their genetic mapping using the mapping populations established as part of Australia’s National Wheat Molecular Marker Program (NWMMP) (Kammholz et al., 2001). The synteny between the PDI loci of wheat and the esp2 locus of rice is described also.
Materials and methods
Plant material and genomic DNA preparation
Seeds of Triticum aestivum L. cvs. Cranbrook (Cr), Halberd (Ha), Egret (Eg), Sunstar (Ss), Sunco (Sc), Tasman (Ta) and Katepwa (Ka), were obtained from the Australian Winter Cereals Collection (Tamworth, NSW), grown under 16 hour day lengths at 18°C and genomic DNA (gDNA) was isolated using Plant DNAzol (Invitrogen). gDNAs of T. aestivum L. cv. CD87, the doubled haploid (DH) mapping populations of Cr X Ha, Eg X Ss, Sc X Ta and CD X Ka (Kammholz et al., 2001), all 42 nullisomic-tetrasomic lines and 15 selected deletion lines of the group 4 chromosomes of Chinese Spring (CS) were kindly provided by L. Rampling (CSIRO-Plant Industry, Canberra).
Design of CAPS markers and linkage mapping of the PDI genes
Sections of the three individual PDI homoealleles were amplified and sequenced for the assessment of any polymorphisms between the parent cultivars of the four mapping crosses mentioned above, using the allele-specific (AS) primers developed earlier (Johnson and Bhave, 2004). PCRs, PCR purifications and sequencing of the PCR products with the primers used for their amplification were conducted according to Johnson and Bhave (2004). The TaPD4A CAPS marker was amplified from the gDNA of 160 doubled haploid (DH) lines of the Cr x Hb cross and 180 DH lines each of the Sc x Ta and CD x Ka crosses (Kammholz et al., 2001), with the PA3F/PA3RB primer pair followed by digestion of the products with SmlI for 4 hours at 65°C. The TaPDI4B CAPS marker was amplified from the gDNA of 180 DH lines of CD x Ka, followed by digestion of the PCR products with Bsu36I at 37°C. Amplifications were performed with the HotStar Taq DNA Polymerase kit (Qiagen) at annealing temperatures of 60°C (PA3F/PA3RB) or 64°C (PB3F/PB3R). Determination of linkage groups was assessed using the Map Manager QT software package.
Investigation of synteny between the PDI loci of wheat and esp2 locus of rice
The putative PDI gene of rice was identified by BLASTn search of the Gramene TIGR genome assembly 2004 in the “Rice_genome_japonica_TIGR” database on the Gramene website (www.gramene.org) using the wheat PDI cDNA wPDI1 as a query sequence. An investigation of whether the PDI gene of rice is present at the esp2 locus was conducted by search of the marker database on Gramene (www.gramene.org/cmap/feature_search), using the query “esp2” to identify the genetic position of the latter. Construction of comparative genetic/sequence maps of the putative PDI locus and the position of the esp2 marker was conducted using the CMap interface of the Gramene website (www.gramene.org/cmap/). The “Genome Browser” interface of the Gramene website (version 16; accessed 1/05) was used to investigate putative wheat orthologues at the PDI locus of rice. This multistep-process involved: (i) centring the genome browser on the rice PDI gene on chromosome 11 and zooming to view a 1 Mb section; (ii) selecting the “Wheat_ESTCluster_TGI” feature track to identify the wheat tentative consensus (TC) sequences in the TIGR TaGI database (www.tigr.org) putatively orthologous to this region of rice genome; and (iii) comparing the ESTs used to assemble these TC sequences with those sequenced from probes used in wheat physical mapping experiments available from the wEST database (http://wheat.pw.usda.gov/cgi-bin/westsql/map_locus.cgi; accessed 10/04). Discrepancies identified in this mapping data were clarified by the physical mapping of the PDI genes using the AS-primers PA1F/PA1R, PB3F/PB3R and PD2F/PD2R and the gDNA of all 42 nullisomic/tetrasomic lines and select ditelosomic and deletion lines of T. aestivum cv. Chinese Spring. PCR amplifications were performed as described earlier for linkage mapping with annealing temperatures of 70°C, 64°C and 69°C, respectively.
Results and discussion
Development and genetic mapping of CAPS markers for two PDI genes
The homoeoallele-specific primers used earlier to characterize sections of the three PDI gene of T. aestivum cv. Katepwa (Johnson and Bhave, 2004) were used to characterise four sections of the TaPDI4A gene, three sections of TaPDI4B gene and six sections of TaPDI4D gene, revealing PCR products of the predicted sizes in all cases (data not shown). Direct sequencing of these PCR products, representing 50-53% of the TaPDI4A gene, 43-50% of the TaPDI4B gene and 83-92% of the TaPDI4D gene revealed, (i) a single G→T substitution at position 312 of the 684bp long intron 5 of the TaPDI4A genes in Hb, Sc, Eg, Ss and Ka. This SNP would introduce a SmlI restriction site, which would digest a 987bp PCR product from their gDNA, obtained with the primer pair PA3F-PA3RB, into 630bp and 357bp bands; (ii) a single A→T SNP in the TaPDI4B genes, at position 83 of intron 9 that was polymorphic only in the CD x Ka cross. This SNP would introduce a second Bsu36I restriction site in Ka, which would digest a 653bp PCR product, obtained with the primer pair PB3F-PB3R, into 169, 382 and 102bp bands in Ka; and (iii) complete sequence conservation of the TaPDI4D gene amongst all cultivars, indicating inadequacy of at least these sections for designing a suitable marker for linkage analysis and in agreement with reports of higher sequence conservation in the D genome of common wheat (Chalmers et al. 2001). Scoring of the TaPDI4A CAPS marker was carried out successfully for 154/160 lines generated from the Cr x Hb cross, 174/180 lines from Sc x Ta and 84/180 lines from CD x Ka. Analysis of the marker data with Map Manager QT allowed the integration of this gene-specific marker (designated XvutPDI) into the genetic maps generated from these crosses. In agreement with Ciaffi et al. (1999), the gene mapped to chromosome 4A in all crosses. The assay of the TaPDI4B CAPS marker was carried out successfully in 118/180 lines from the CD x Ka cross; the lower success rate of this assay appears to be attributable to the quality of the template gDNAs, as the TaPDI4A marker from this cross also had a lower success rate (see above). The data allowed the integration of this marker (designated as XvutPDIb) into the chromosome 4B genetic map generated from this cross (these genetic maps available at http://rye.pw.usda.gov/cmap/). The genetic mapping of the two PDI genes has thus opened the possibility of determining their association with QTLs of agronomic importance. To date, no quality based QTL has been identified in the PDI containing regions of the group 4 chromosomes using standard analyses. However, a significant epistatic interaction has been identified by Ma et al. (in press) between the Glu B1 locus on chromosome 1B and the PDI locus on 4AL, consistent with the predicted significance of the PDI locus.
Investigation of synteny between the PDI loci of rice and wheat
The BLASTn search of the Gramene TIGR pseudomolecule assembly release 2 (accessed 1/05), using the wheat PDI cDNA wPDI1 as the query sequence identified a ~173kb BAC, OSJNBa0058p12 (referred to by its GenBank accession# AC139170), containing a putative rice PDI gene between 4,960,745 and 4,964,540bp on chromosome 11 of rice genome. Comparison of this with the durum wheat PDI gene (TtPDI4A, Johnson and Bhave, 2004) revealed a conserved 10 exon structure, ~77% identity over the exons and ~40% identity over introns suggesting this gene represents the rice orthologue of the wheat PDI gene.
The search of the marker database of Gramene (www.gramene.org) revealed the esp2 marker to be present on the Hokkaido Morphological 2000 (Morph 2000) map on chromosome 11. A direct comparison of map positions between the Rice Gramene TIGR Assembly sequence map (containing the PDI gene) and the Morph 2000 map (containing the esp2 marker) was not possible due to absence of marker correspondences, thus a third map with correspondence to markers on both maps, the JRGP RFLP 2000 genetic map (www.gramene.org) was used. This comparison revealed that the PDI gene and the esp2 marker were located at similar map positions, distal to sp, S20163S and C827S; however, absence of markers distal to esp2 with correspondence to any of the available maps prevented the assessment of whether the esp2 marker and PDI gene are flanked by similar markers. However, presence of the PDI gene and esp2 marker on chromosome 11 at similar map positions, together with the observation of lack of PDI expression in the esp2 mutant (Takemoto et al., 2002), suggests that the esp2 marker in rice is closely linked to the PDI structural gene or its cis-acting regulatory sequences. The Genome Browser of Gramene was used to identify a 1Mb region (4,462,642 - 5,462,642 bp) of rice chromosome 11, flanking the PDI gene. Wheat tentative consensus (TC) sequences putatively orthologous to this area of the rice genome that were present in the TIGR TaGI database (v9.0; accessed 1/05) were then identified, revealing 42 EST singletons and 34 TCs compiled from 745 individual EST and cDNA sequences, including 17 EST singletons and 7 TCs that aligned with the rice PDI locus. To determine whether any of the 745 ESTs were sequenced from cDNA probes that had been used for physical mapping experiments, the EST accession numbers were queried in the GrainGenes-SQL database. This analysis identified 9 ESTs that had been sequenced from 5 cDNA probes (two aligning with the PDI locus) physically mapped to wheat chromosomal ‘bins’, all of which were located on the group 4 chromosomes. The discrepancy noted between probe 2 and 3 (the PDI probes) in their chromosome assignment (Fig. 1) is most likely due to ambiguities in interpreting the complex RFLP analyses (Miftahudin et al., 2004). The position of PDI genes was resolved using the allele-specific PCRs on the nullisomic/tetrasomic, ditelosomic and deletion stocks of T. aestivum cv. Chinese Spring, revealing the expected products in all lines except the null-4A lines with the primer pair PA1F/PA1R, the null-4B and the 4BS4 and 4BS8 deletion lines with PB3F/PB3R, and the null-4D lines, the DT4DL ditelosomic line and the 4DS1 and 4DS3 deletion lines with PD2F/PD2R. These results localise the PDI genes in bins on the group 4 chromosomes, in agreement with probe 3 (above) and Ciaffi et al. (1999).
Figure 1. Locations of the PDI genes and probes BF200779 (1), BE398523 (2), BQ280847 (3), BE499456 (4) and BM135436 (5) on the group 4 chromosomes of wheat.
The data generated here establishes firmly that several genes are present in the chromosome 11 region of the rice genome carrying the PDI locus that are also present on the group 4 chromosomes of wheat, in agreement with the findings of La Rota and Sorrells (2004). The results provide a basis for predicting a QTL involved in storage protein deposition at the PDI loci in wheat and the means for testing this hypothesis using the PDI markers described.
JCJ was supported by a Grains Research and Development Corporation PhD scholarship. We are grateful to Dr Bryan Clarke for extending the use of lab facilities at CSIRO-Plant Industry (Canberra) and Lynette Rampling at CSIRO-PI (Canberra) for technical assistance.’
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