2012 Annual Report
1a.Objectives (from AD-416):
The collaboration with Jiming Jiang is to provide genomic in-situ hybridization expertise. The goal is to investigate the phylogenetic relationships of the wild and cultivated potato species, with a focus on the allopolyploid species. This line of investigation if very productive and has already resulted in a peer-reviewed publication.
1b.Approach (from AD-416):
Roots of potato allopolyploids will be collected from greenhouse-grown plants and pretreated in 0,002M 8-hydroxyquinoline at 20°C for 3 hrs. Root tips and flower buds will be fixed in a 3:1 (100% ethanol : glacial acetic acid) solution and stored in freezer (-20°C) until use. Root tips will be digested by 4% cellulase and 1% pectinase at 37°C for 80 min. The macerated root tips will be suspended by forceps in a drop of 45% acetic acid and squashed. Slides will be pretreated by pepsin solution (final concentration 0.1 mg/ml) for 45 min at 37°C and subsequently incubated in a RNase A solution (6 'l stock solution – 10 'g/'l + 24 'l 2xSSC per slide; 40 min at 37'C) and then – in formaldehyde solution (4% for 10 min). After each treatment slides will be washed in 2x SSC buffer 5 min for three times at room temperature. Finally slides will be incubated in 70%, 90%, 100% ethanol series, for 3 min each at room temperature.
DNA extraction and labeling:
Genomic DNA will be isolated from the putative diploid progenitor species of the allopolyploids as determined by results from Spooner’s DNA phylogenetic studies using young leaves of greenhouse-grown plants. The GISH technique will be done according to published protocols with minor modifications. DNA will be either labeled with DIG-UTP or Biotin-UTP by nick-translation (DIG- and Biotin-Nick Translation Mix, Roche, cat. No. 11745816910, cat. No. 11745824910).
Hybridization mix (40 'l per slide) for GISH will be prepared with differential labeled DNA from the putative parental species and included: sheared fish sperm DNA (20 µg), Probe DNA of one parent (100 ng), Probe DNA of the other parent (100 ng), 10% dextran sulfate, deionized formamide (50%). Hybridization will be performed over night at 37'C.
DIG-labeled DNA will be detected with rhodamine anti-DIG conjugate and biotin labeled probes detected with FITC conjugated avidin (Roche, cat. No. 11207750910, cat. No. 11975595910). 29 'l of blocking reagent (30 mg BSA solution in 999 'l 4x SSC) will be added to slides, followed by incubation for 30 min. at room temperature. The antibody solution composed of 1 'l Anti-DIG-rhodamine stock solution + 1 'l Avidin- fluorescein stock solution + 28 'l Detection buffer (DB: 0.1 g BSA dissolved in 9.9 ml 4 x SSC, pH=7.4) will be added to each slide; incubation for 45 min at 37'C. Slides will be washed three times in 4x SSC (pH=7,4) (5 min each) at 42'C. Chromosomes will be counterstained by 4’, 6-diamidino-2-phenylindole (DAPI) in Vectashield antifade solution (Vector Laboratories).
All images will be captured digitally using a SenSys CCD (charge coupled device) camera (Roper Scientific, Tucson, AZ) attached to an Olympus BX60 epifluorescence microscope. The CCD camera will be controlled using IPLab Spectrum v3.1 software (Signal Analytics, Vienna, VA) on a Macintosh computer.
This research uses a new technique, termed chromosome painting, or more technically genomic in situ hybridization, to see how the hexaploid species growing in North and Central America obtained these duplicated chromosome sets. In the past, ideas of how species obtained these different chromosome sets were determined by how chromosomes behaved when they paired during the sexual process to form sperms and eggs. Chromosome painting, in contrast, uses an entirely new and very powerful technique to investigate this problem by staining “painting” the chromosomes with the deoxyribonucleic acid (DNA) of different species that could be the parents of these species with duplicated chromosome sets. The results show two very different modes of formation of these hexaploid species. One mode, exemplified in the hexaploid species technically called Solanum demissum, has all three sets of chromosomes the same, while another hexaploid, technically called Solanum iopetalum, has three very different chromosome sets. We use these data to speculate what are the parents of these two species. These results are very useful for potato breeders in that it informs them of the genetic constitution of these wild species that helps them better plan their breeding programs.
This research relates to sub-objective 2.B., Develop and apply new and appropriate DNA markers for phylogenetic and genetic analyses of potato, tomato, and/or carrot genetic resources, and incorporate resultant characterization data into GRIN and/or other databases, such as SolGenes (for potato and tomato), GenBank, or on-line repositories of aligned DNA sequences of peer-reviewed scientific journals.