Title page for ETD etd-112199-204735

Type of Document Dissertation
Author Boluarte, Tatiana
Author's Email Address tboluar@vt.edu
URN etd-112199-204735
Title Bulk segregant analysis for anther culture response and leptine content in backcross families of diploid potato
Degree PhD
Department Horticulture
Advisory Committee
Advisor Name Title
Veilleux, Richard E. Committee Chair
Beers, Eric P. Committee Member
Grabau, Elizabeth A. Committee Member
Griffey, Carl A. Committee Member
Maroof, M. A. Saghai Committee Member
  • RAPDs
  • SSRs
  • unreduced pollen
  • potato
  • leptines
  • solanum
  • anther culture
  • segregation distortion
  • bulk segregant analysis
Date of Defense 1999-09-15
Availability unrestricted
Diploid potato populations between a primitive cultivated species, Solanum phureja, and a weedy species, S. chacoense, were used to examine the segregation of microsatellite markers and three traits in backcrosses. Two of the traits, anther culture competence and 2n pollen production, originated from S. phureja whereas the third, leptine production (a specific glycoalkaloid known to convey resistance to the Colorado potato beetle) originated from S. chacoense. Using CP2, a self-incompatible F1 hybrid originating from a cross between S. chacoense clone 80-1 and S. phureja clone 1-3, three populations were developed: 1-3 x CP2 (PBCp), CP2 x 1-3 (PBCc), and CP2 x 80-1 (CBC).

For the microsatellite study, four simple sequence repeat (SSR) primer pairs that amplified fragments within potato sequences found in the GenBank were used to look at segregation ratios in our backcross populations and to eliminate possible spurious genotypes bearing non-parental alleles in these populations. Seventeen spurious genotypes were discarded from PBCp; none was found in PBCc or CBC. Two SSR loci showed skewed segregation in PBCp (favoring transmissnion of the allele originally found in 80-1), PBCc showed normal segregation at all loci, and CBC showed distorted segregation at one locus (revealing a deficiency of homozygotes).

In the study of anther culture, three components of ACR were investigated in a preliminary study: 1) embryos produced per anther (EPA), 2) embryo regeneration rate and 3) percentage of monoploids (2n=1x=12) among regenerants. CP2 was intermediate, 80-1 was low, and 1-3 was high for ACR. Only EPA was selected for further characterization in our populations. PBCp (78 genotypes) and CBC (57 genotypes), were characterized for anther culture response ACR/EPA in a series of studies. Nine high and ten low selections were identified in CBC, and ten high and ten low selections were identified in PBCp. EPA selections were used for bulk segregant analysis (BSA) using 214 RAPD primers. Two bands, one amplified by OPQ-10 and another by OPZ-4 were linked in coupling and in repulsion, respectively, to ACR in PBCp. One band amplified by OPW-14 primer was linked in coupling to ACR in CBC. One-way ANOVAs for data from remaining genotypes of the populations verified linkage of the markers to ACR/EPA.

For 2n pollen production, a total of 77 PBCp genotypes was characterized; 80-1 produces low % 2n pollen, and 1-3 produces high % 2n pollen. Pollen samples were stained with propidium iodide and examined by flow cytometry. The frequency of 2n pollen varied continuously from 1.7 % to 40.6 % among the 41 genotypes that flowered sufficiently to allow three separate pollen collections. Variation due to the environment was observed where the frequency of 2n pollen appeared greater over a range of genotypes on single collection days. BSA could not be used due to limited population size and a low number of selections at the extremes of the distribution of phenotypes. The continuous variation for 2n pollen production suggests multigenic control of the trait.

In the study of leptine content in reciprocal backcross populations, 87 genotypes within PBCp, and 42 genotypes within PBCc were characterized using gas chromatography of leaf samples. CP2 was intermediate, 1-3 had zero, and 80-1 was high for leptine content in the foliage. Leptines were present in low levels in 43 of 87 genotypes in PBCp, indicating simple genetic control. In PBCc, only 7 of 42 genotypes expressed leptines, generally at a higher level than in PBCp, indicating cytoplasmic inheritance. Ten high and ten nil selections within PBCp, and seven high and eight nil selections within PBCc were used for BSA using 214 RAPD primers. Three primers OPQ-2, OPT-16 and OPT-20 amplified bands segregating with high bulks in both populations. These markers were linked in coupling to leptine content in PBCp. Linkage was verified by ANOVAs for leptine content in the entire population.

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