Barley is one of major crops in the UK and is cultivated for malting and animal feed and forms an important part of the ecologically friendly crop rotation. The use of high quality barley for malting underpins other important UK industrys such as brewing, food and distilling. The importance of the crop means that there are several companies that are breeding improved barley varieties for growing in the UK .Barley breeders have a difficult task in improving barley as the characters or traits that are important for the crop’s environmental and industry acceptance are usually controlled genetically by more than one gene. These characters include yield, how tall the plants are, how resistant they are to diseases (and so how much the crop will need to be sprayed) and many others.
We propose to address this difficulty by using information we have gathered over the last couple of years to look at the variation at the DNA level at sites across the whole barley genome. New technology used in human genome studies will allow us to determine the particular variation shown across the barley genome, quickly and cheaply. Once established, we will can then take a retrospective look at the particular variants of genes that exist within UK varieties and investigate how they have been assembled in the sucessful varieties in order to locate the genes that control the important characters. This will be difficult but this project brings new experimental and analytical tools of genomics and genetics to bear on the problem and also takes advantage of new technologies, statistical expertise and very extensive plant material and data sets from past trials.
Having derived a genetic understanding of what makes a good barley variety, breeders will have a better idea of what combinations will make even better varieties that will meet the industries’ exact requirements and help improve the impact the crop has on the environment. So why barley? In short this project is possible now for barley, but not for other UK crops such as wheat, barley’s more commercially important relative. Wheat is genetically more complex and the information required to develop the necessary technology has not yet been derived. This project will however generate information, data and data handling expertise that will underpin any future study on wheat.
The Illumina GoldenGate Assay queries genomic DNA with three oligonucleotide probes for each locus and creates DNA fragments that can be amplified by standard PCR methods using universal primers. For each of the 1536 loci interrogated in each assay, the oligo mix contains 2 allele specific and one locus specific probe (ie 3 x 1536 oligos). The 3′ ends of the two alternative allele specific probes are complementary to two universal primers, U1 and U2, with the 5′ end complementary to the 3′ end of the locus. Each probe sequence terminates at the SNP that is to be assayed with an allele specific base. The third probe, the locus specific probe, is complementary to the genomic DNA that starts 5 to 20 bases 3′ of the locus in question. As well as a locus specific sequence, this probe also contains a specific Illumicode sequence that is used to identify the locus (on the BeadArray) as well as the sequence for universal primer sequence U3. All 1536 probes are annealed to the genomic DNA at the same time, DNA polymerase is added to close the gap between the allele specific (including either U1 OR U2) and the locus specific (including U3) probes and the paired fragments are ligated together. The probe fragments are then separated from the genomic DNA and used to inoculate a PCR reaction. The primer mix for this PCR reaction consists of primers U1 and U2 labeled with Cy3 and Cy5 respectively and biotinylated primer U3. As a result of this labeling scheme, the PCR product consists of double stranded DNA of which one strand, containing the complement to the Illumicode, is labeled with either Cy3 or Cy5 in an allele specific manner, and a complementary strand labeled with biotin. The biotinylated strand is removed and the single, florescently labeled strand hybridized to the BeadArray.
For further information on this project please contact Bill Thomas (firstname.lastname@example.org) from the James Hutton Institute.