Location: Vegetable Crops Research2010 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)
Slide preparation: 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: 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. Detection: 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.
3. Progress Report
Chromosome evaluations were made for representative polyploid potato species for Fluorescent and genomic in situ hybridization techniques (GISH and FISH) to investigate their origins relative to extant diploids. We probed mitotic chromosomes of the polyploid species with deoxyribonucleic acid (DNA) of their proposed diploid progenitors, hybridized meiotic chromosomes of some of the polyploid species with DNA of their proposed diploid progenitors, and some of polyploid species were analyzed by FISH using 5S and 45S rDNA probes. Genomic in situ hybridization (GISH) analysis support an AABB genome constitution for S. stoloniferum and S. hjertingii, with S. verrucosum (or its progenitor) supported as the A genome donor and other North or Central American diploid species to include S. cardiophyllum, S. ehrenbergii, or S. jamesii as the B genome donor. GISH analysis of chromosome pairing of S. stoloniferum also confirms the strict allopolyploid nature of this species. GISH analysis of S. colombianum did not reveal differentiation of parental sub-genomes. This result was confirmed in several independent replications of each series of GISH experiments. Our results indicate: (a) high level of homology between two component genomes of tetraploid species S. colombianum and (b) high homology between genome of S. colombianum and genomes of diploid species of the series Conicibaccata (S. violaceimarmoratum, S. santolallae). GISH detected that the 1st subgenome of hexaploid species S. hougasii, S. schenckii, S. iopetalum, S. demissum is very similar to the A genome of S. verrucosum. The 2nd subgenome of S. hougasii and S. schenckii is closed to the B genome of S. jamesii and S. ehrenbergii. The 3rd subgenome of S. hougasii, S. schenckii, S. iopetalum was not differentiated in these GISH experiments and it is homeological both to the A genome of S. verrucosum and to the B genome of S. jamesii and S. ehrenbergii. All subgenomes of S. demissum have very close homology to the basic A genome of potato species. Preliminary results of GISH analysis of tetraploid species S. acaule showed that it has two homological genomes. The genomes of S. acaule are all close to the basic A genome species of section Petota. GISH of hexaploid species of the Demissa series with differentially labeled DNA of S. verrucosum and S. andreanum (or S. piurae, S. pascoense) revealed that one of genomes of S. hougasii, S. schenckii, S. iopetalum includes chromosomes and chromosomal fragments that are homological to the genome of S. andreanum, S. piurae and S. pascoense (from 4 to 6 chromosome pairs depend on hexaploid species). GISH was also performed on mitotic chromosomes of tetraploid species of the series Conicibaccata - S. colombianum with differentially labeled DNA of diploid species in the following four experiments: (1) S. santolallae and S. andreanum, (2) S. violaceimarmoratum and S. andreanum, (3) S. violaceimarmoratum and S. pascoense, (4) S. violaceimarmoratum and S. piurae. Our results indicate high homology between genome of S. colombianum and genomes of diploid species of the series Piurana (S. andreanum, S. pascoense). Project is monitored by monthly meetings