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compare the total in-situ population of native cultivars maintained by farmers in Huancavelica
against a geographically restricted subset of accessions from CIP’s core ex-situ population as
maintained in the genebank.
2.2 Materials and methods
2.2.1 In-situ collections for characterization
Eight communities following a north-south transect through the department of Huancavelica
were selected on the basis of distribution and distance along the transect, tradition of potato
cultivation, ethnicity, and relative distance from major markets or cities (see chapter 1). During
three subsequent agricultural seasons, farmer and communal potato collections of native potato
cultivars were characterized using different tools: ploidy counts, morphological descriptor lists
and molecular markers. Families or communal groups with appreciable diversity were identified
and seed-tubers of each farmer-recognized cultivar stored in net-bags. Some farmer families
maintained very large cultivar collections and subsets of their total collection were installed in
subsequent agricultural seasons. Accessions were labeled and on-farm trials with a minimum of
five and a maximum of ten plants per accession installed. Field space was provided by the
participating families or groups with prior informed consent and with authorization obtained
during communal assemblies. The in-situ collections were used as the basis for ploidy counts,
botanical species identification, morphological and molecular (SSR) characterization (table 2.2).
Additionally, two collection trips were undertaken in 2005 to search for Solanum phureja in
inter-Andean valleys below 3,400 m altitude. One trip was undertaken to the remote eastern
districts of Huachocolpa and Tintay Puncu (Tayacaja province) and one to the low inter-Andean
valley of Lircay following the Totora watershed (Angaraes province).
2.2.2 Ploidy counts and species identification
Ploidy levels in combination with morphological keys were used to determine the botanical
species to which accessions belonged. Species identification was double checked in open field
and under greenhouse conditions using the botanical keys developed by Huamán (1983). Ploidy
was determined by both microscopy and flow cytometry. The microscopy method followed
standard procedures (Chen and Hai, 2005; Watanabe and Orrillo, 1993): a. collection of 0.5-1.0 cm
long root tips between 11:20 and 12:00 a.m., b. treatment of root tips with a solution of Ambush
50EC for the detention of mitosis in metaphase (incubate root tips in solution with 15 µl stock of
Ambush 50EC in 100 ml H 2
O pH 5.8 for 24 hours at 4ºC; wash 15 minutes with H 2
O pH 5.8), c.
optional fixation (incubate in 3 parts ethanol 96% and 1 part acetic acid at 20ºC for 24 hours;
rinse with ethanol 70% and air dry; store at 4ºC for not longer than 7-10 days), d. hydrolysis
(incubate in 1N HCl at 60ºC for 8-10 minutes, wash for 15 minutes with distilled water), e. staining
(incubate in lacto-propionic orcein for 3 minutes), f. preparation of squash slides, g. microscopy
(actual counting). Flow cytometry was conducted with a Partec® ploidy analyzer through
measurement of the total DNA content of nuclei from leaf samples. Ploidy levels were read from
real time DNA histograms.
2.2.3 Morphological characterization
The morphological diversity of native cultivar collections was characterized using CIP’s formal
descriptor list and color card (Gómez, 2000; Huamán and Gómez, 1994). This formal list consists
of 17 morphological descriptors with a total of 32 morphological character states, 18 of which
are considered to be environmentally stable (appendix I). Similarity analysis, dendrogram
construction, cophenetic analysis and matrix correlations calculations (Mantel tests) were
conducted. A standardized similarity analysis with an average taxonomic distance coefficient
38 Potato diversity at height: Multiple dimensions of farmer-driven in-situ conservation in the Andes