Abstract
(Englisch)
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The goal of our participation in Project SESA was the establishment of molecular markers distinguishing spelt wheat (Triticum aestivum speka) from common bread wheat (T. ciestivum vulgare) through the analysis of gliadin proteins and their genes. Two experimental approches were to be used: One, the gliadin patterns obtained with various electrophoretic methods were to be analyzed, spelt- or wheat-specific proteins identified, sequenced and through reverse genetics the underlying genes found. They in turn would have allowed the design of specific PCR Systems. And two, published gliadin gene sequences of wheat were to be compared, PCR primers designed on them, applied to both spelt and wheat cultivars, genetic differences identified and again used to define specific PCR primers. In the course of the project the first approach was found to be unfeasible with our means. Our efforts were thus largely devoted to the gene-analytical strategy.
Using various stringency criteria, a total of five and four PCR primer pairs were designed on a- and y-gliadin genes, respectively. Primer pair (3AG5 and 0A06 directed at y-gliadin genes proved to be very fruifflil. It amplified two fragments from hexaploid wheat originating from two genes GAGS6D and GAGS6B on chromosomes ID and lB, respectively. Of GAG56D, two alleles ok andfo were found, differing in only a 9-bp duplication/deletion and a restrictionetectable l-bp polymorphism. Testing a large panel of cultivars revealed that all spelts carried the ok allele while all bread wheat cultivars contained the fo counterpart. Shorter GAGS6D-specific PCR systems were designed to allow, coupled with diagnostic restriction, fast qualitative detection of the alleles. Furthermore, a wheat-specific PCR system was defined by exploiting the allele-specific 9-bp difference to enable detection of wheat contamination in spelt flour. An internal standard was constructed to quantify the wheat-specific PCR system.
Analysis of GAGS6D was extended to the other four recognized subspecies of 7: aestivum using sequencing and PCR-RFLP. Curiously, we found that all sequenced fragments of GAGS6D were identical to either the ok or thefo allele. Considering published botanical and archaeological evidence, we concluded from our results that hexaploid wheat must have been formed more than once (about 1O'OOO years ago). Accessions of Aegilops squarrosa (diploid progenitor of the D-genome) collected in the Caspian region carried GAGS6D alleles closely related to ok andfo thus cotifiring the geographical origin of hexaploid wheat. When the GAGS6B pseudogene was analyzed, four distinct allelic lineages were found among cultivated wheats: a, p-aes, p-dur and p-tim typical of European spelt, bread wheat, durum wheat of gliadin 42-type and Timopheev's wheat, respectively. The a allele of spelt differs from the p-aes allele of bread wheat by eight consistent l-bp differences, three of which were used for PCR-RFLP differentiation of the two alleles. Most interestingly the a allele found in spelt was shared by durum cultivars of gliadin 45-type, which strongly supported the (old) hypothesis that European spelt arose through hybridization between a tetraploid and a hexaploid wheat in Europe itself. More than one distinct allelic lineage in the B-genome furthermore suggested multiple domestications of tetraploid emmer. Examining GAGS6 genes in putative Bgenome donors excluded all five modern species of the Sitopsis section of Aegilops as immediate pr9genitors, but instead supported a monophyletic origin of all tetraploid wheats.
In conclusion, we found that the okia-genetic constitution in y-gliadin genes GAGS6D and GAGS6B distiguishes spelt from bread wheat. Morever, we provided molecular evidence concerning the origin of spelt. And finally, a wheat-specific PCR system to detect wheat adulteration of spelt flour was developed.
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