Progress 10/01/12 to 09/30/13
Outputs
Progress Report 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. 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 �g), 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. This project was renumbered from 3655-21000-050-03S to 3655-21000-058-01S. This is the final report, project terminated 7/31/2013. This research uses a new technique, termed chromosome painting, 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 (or �painting�) the chromosomes with the deoxyribonucleic acid (DNA), colored differently from different parents, responsible for these duplicated chromosome sets. The results show three very different modes of formation of these polyploid wild potato species. One mode, exemplified in the tetraploid species, had two different chromosome sets that clearly matched prior ideas of their parents. A second mode, from the hexaploid species, has all three sets of chromosomes the same, again matching prior hypotheses of its origin. A third mode, from the hexaploid species, has three very different chromosome sets. These results were published in two separate peer-reviewed scientific journals. The results provide biologists with data supporting the origin of these species from other sources, including studies of DNA sequences and how the chromosome pair together when they form sperms and eggs in the reproductive process. We use these data to speculate on the parentage 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 contributes to the progress of sub-objective 2.B by demonstrating the genomes that contributed, through hybridization, to these plants with more than one compliment of chromosomes. This is not only of theoretical interest to evolutionists, but also of use to plant breeders who can use this information to design their sexual crossing programs.
Impacts
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Publications
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Progress 10/01/11 to 09/30/12
Outputs
Progress Report 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. 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 �g), 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. 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.
Impacts
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Publications
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Progress 10/01/10 to 09/30/11
Outputs
Progress Report 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. 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 �g), 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. This project forms a cooperation with an expert in genomic in-situ hybridization (GISH). In this particular study we used this staining method to determine the parents of wild potato �allopolyploids.� Allopolyploids are plants that have duplicated sets of chromosomes from different parents, forming hybrids, where the new species doubles their number of chromosomes, with one chromosome set from each parent. What makes this technique so powerful and useful is that after this hybrid plant is formed the chromosome sets can exchange their deoxyribonucleic acid (DNA) or lose their DNA, and GISH analyses provide a very easy and effective way to see these changes under a microscope. In this study we stained chromosomes of two polyploid species from Mexico with DNA linked to these stains that are potential parents of the polyploids. The results showed very different results in these two species. Wild potato species A, (Solanum demissum), was found to be formed by parents that were very similar genetically. Species B, (Solanum iopetalum) in contrast, was formed by parents that were very different genetically. Species A has been very easy to use to produce wild species hybrids with cultivated potato, while species B has been very difficult to use. These results inform potato breeders why this has occurred, in that the genes of Solanum demissum and Solanum tuberosum (the cultivated potato), are very similar, much more similar than either is to Solanum iopetalum. These results give clues to potato breeders on the potential ease to use other related wild potato species in their breeding programs. This project has resulted in a publication: Pendinen, G., T. Gavrilenko, J. Jiang, and D.M. Spooner. 2008. Allopolyploid speciation of the tetraploid Mexican potato species S. stoloniferum and S. hjertingii revealed by genomic in situ hybridization. Genome 51: 714-720. The project is being monitored by monthly meetings.
Impacts
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Publications
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Progress 10/01/09 to 09/30/10
Outputs
Progress Report 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. 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 �g), 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. 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
Impacts
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Publications
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Progress 10/01/08 to 09/30/09
Outputs
Progress Report 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. 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 �g), 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. Significant Activities that Support Special Target Populations The purpose of this project is to use Deoxyribonucleic acid (DNA) sequences from single-copy orthologous genic regions (COSII regions) to investigate polyploid evolution in potato. To date, 17 accessions of 14 diploid species and 54 accessions of 11 polyploid species (71 accessions in total) have been sequenced with 4 conserved orthologous sequence (COS) markers, for a total of .2805 bp concatenated DNA alignment length. One- six alleles per accession were generated. Phylogenetic analyses of these data are at present not producing concordant phylogenetic results. This work is continuing with additional diploid species for comparison and with additional COS markers. Monitoring of this project is being accomplished by bi-weekly meetings with cooperator at the University of Wisconsin-Madison.
Impacts
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Publications
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