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Isozyme Analysis of Entire and Core Collections of Solanum tuberosum subsp. andigena Potato Cultivars.

THE POTATO CROP was domesticated by the early Andean farmers of South America after sexual polyploization of diploid tuber-bearing Solanum species occurred (Ortiz and Ehlenfeldt, 1992). The latest comprehensive taxonomic treatment of potatoes recognizes seven cultivated and 216 tuber-bearing wild potato species (Hawkes, 1990). Among the cultivated species, two are tetraploid: the white or Irish long-day adapted potato cultivars of the northern and southern hemispheres (Solanum tuberosum subsp, tuberosum Hawkes) and short-day adapted native Solanum tuberosum subsp, andigena cultivars from the Andes from Venezuela to Northern Argentina (Huaman and Ross, 1985). These farmer-selected andigena potato cultivars possess resistance to many of the pests and diseases that affect the potato crop elsewhere (Huaman et al., 1997).

The International Potato Center (CIP) maintains one of the largest collections of andigena cultivars (Huaman, 1998) in a field genebank near Huancayo, Peru (3200 m). Duplicate sets of tubers are stored at 4 [degrees] C at La Molina (near Lima). A duplicate set of the whole collection is also conserved in in vitro culture at CIP at La Molina and Quito, Ecuador.

The collection at CIP originally consisted of more than 15 000 accessions of Andean diploid, triploid, tetraploid, and pentaploid cultivars. However, after a process of duplicate identification with morphological descriptors and electrophoretic analysis only 3527 accessions remain in the collection (Huaman and Stegemann, 1989; Huaman, 1994), of which 2379 are tetraploid cultivars. CIP has selected a core collection (Brown, 1989) of farmer selected andigena cultivars collected from the highlands of Mexico, Guatemala, Venezuela, and southwards along the Andes as far as northwestern Argentina. The Central American cultivars were most likely introduced from South America, while the cultivars from Colombia to Argentina were likely native. This core subset was selected by cluster analysis of morphological characters of the entire andigena collection and consists of 306 unique potato cultivars. Other core subsets are being defined by CIP from the collection of diploid cultivars of S. phureja Juz. et Bukasov by cluster analysis of DNA markers (Ghislain et al., 1997).

The objective of our research was to investigate whether the chosen sampling strategy to select the core andigena collection was adequate to capture a representative sample of isozyme loci. Such an analysis provides a means to validate the sampling strategy of the core collection using an independent set of genetic markers. For this purpose, the reserve collection and the core subset were characterized with the aid of nine isozyme markers that have been genetically studied and used to characterize other potato gene pools (Douches and Quiros, 1988; Douches and Ludlam, 1991; Douches et al., 1991; Zimmerer and Douches, 1991).

MATERIALS AND METHODS

A total of 2379 andigena cultivars from eight countries in Latin America comprising the entire potato collection (including the 306 accessions of the core collection) held at CIP were included in this investigation (Table 1). Accessions for the core collection were chosen considering one representative accession from the most different morphological clusters, which were determined by a dendrogram generated by cluster analysis of 25 characteristics. Priority was given to select accessions from geographical areas with a very low frequency. From those clusters containing many accessions, the representative accession was selected on the basis of data on host response to diseases and pests and dry matter content. Likewise, cultivars with widespread cultivation, as determined by their number of duplicates in original collection, were also favored while sampling accessions for the core collection.

Table 1. Number of accessions per country in the entire and core collections of S. tuberosum subsp. andigena conserved in the genebank at CIP.
                     Entire                      Core

Country        No. of accessions      %     No. of accessions     %

Guatemala             27             1.1            9            2.9
Mexico                19             0.8            7            2.3
Venezuela             35             1.5            9            2.9
Colombia             126             5.3           26            8.5
Ecuador              193             8.1           39           12.7
Peril               1581            66.5          147           48.0
Bolivia              314            13.2           54           17.7
Argentina             84             3.5           15            4.9


Allozyme diversity in the entire collection was determined with horizontal gel electrophoresis using two buffer systems (Histidine-citrate at pH 5.7 and Lithium-borate at pH 8.3) on tissue from newly expanded leaflets from each cultivar. The procedures for tissue processing, electrophoresis, staining, and nomenclature were those of Douches and Quiros (1988). Nine isozyme loci of six enzyme systems were resolved without progeny testing following the protocol of Douches and Ludlam (1991). The five isozyme loci assayed at pH 8.3 were diaphorase-1 (Dia-1), glutamate oxaloacetate transaminase-1 (Got-1), Got-2, phosphoglucomutase-1 (Pgm-1), and Pgm-2, whereas isocitric acid dehydrogenase-1 (Idh-1), malate dehydrogenase-1 (Mdh-1), Mdh-2, and phosphoglucoisomerase-1 (Pgi-1) were assayed at pH 5.7. Eight of these isozyme loci (except Mdh-1) have been mapped to seven distinct (out of 12) chromosomes (Pillen et al., 1996) and used to analyze phenotypic variation in potato haploids (2n = 2x = 24) of tuberosum (Ortiz et al., 1993).

Once all cultivars were analyzed, summary gels for each isozyme locus were prepared containing representative accessions of all different banding patterns obtained for each locus. In total we identified 100 banding patterns for the nine isozymes. Those patterns that appeared similar were compared side by side to identify only those that were different. Dr. D.S. Douches (Michigan State Univ., East Lansing) kindly reviewed the final summary gels and provided the standard genetic scoring corresponding to the different banding patterns found in this study. New allozymes with bands migrating distinctly different to those previously reported for a specific locus were recorded as "new". Their genetic designation awaits a review by potato geneticists according to accepted standards.

A tetrasomic polyploid crop species such as potato could have genotypes with four distinct alleles per locus (tetra-allelic) and up to five distinct genotypes for a di-allelic locus. Di-allelic heterozygotes for a dimeric enzyme locus (e.g., Mdh-1) were differentiated according to their band dosage (or banding intensity). A duplex (or balanced di-allelic) genotype showed a balanced three-banded phenotype in a 1:2:1 ratio (Douches and Ludlam, 1991). Similarly, the simplex (or unbalanced di-allelic) genotype had an unbalanced three-banded pattern with a dosage relationship favoring the most frequent allozyme in a 9:6:1 ratio. For a monomeric enzyme (e.g., Pgm), the dosage pattern at a locus was directly proportional to the allelic dosage (Douches and Ludlam, 1991).

Allozyme frequencies (q) were calculated for each isozyme locus in both the entire and core collections by counts of respective alleles (Weir, 1996). Standard errors (s.e.) for each allozyme frequency were calculated by s.e. = [[q - (1 - q)/ 4N].sup.0.5], where N is the number of accessions scored (Agresti, 1990). Statistical differences between q were determined by non-overlapping confidence intervals at P = 0.05 (Angers, 1989). The allozyme frequency distribution for each locus was tested for homogeneity between the entire and core collections by [chi square] tests. The average locus heterozygosity (ALH) (%) was determined by ALH = 100 - [[Sigma](1 - {[m.sub.i]/N})], where [m.sub.i] was the number of accessions with mono-allelic genotypes for isozyme locus i.

RESULTS AND DISCUSSION

The core collection was biased towards accessions collected in countries other than Peru (Table 1) because the number of Peruvian accessions was several fold higher than from the other countries. A total of 38 allozymes were scored in the entire collection (Table 1), but two (Idh-[1.sup.3] and Pgi-[1.sup.5]) were missing in the core collection. The two missing allozymes were rare variants with q = 0.0002 (or 0.02%). For example, Idh-[1.sup.3] was only scored in one Bolivian accession (HHCH 4980), while Pgi-[1.sup.5] was observed in two Peruvian accessions (OCH 4740 and OCH 4933). New allozymes not previously reported in potato were found for the Mdh-1 and Pgm-1 loci.

The allozyme frequency distributions between the entire and core collections were statistically homogeneous for seven of nine isozyme loci (Table 2). The core had a significantly higher frequency of Pgi-[1.sup.3] and a significantly lower frequency of Pgi-[1.sup.4]. Similarly the entire collection had a higher frequency of Got-[1.sup.3] and correspondingly lower q for Got-[1.sup.1] and Got-[1.sup.4] than the core collection. The reduction of Got-[1.sup.3] frequency in the core collection may not be considered as an important bias because nearly all the clones in either set probably possess this allele. On the contrary, such bias sampling for Got-[1.sup.3] in the core collection should be regarded as a very desirable practical outcome because other allozymes with a lower frequency in the entire collection have greater than expected representation in the core subset.

Table 2. Frequencies of isozyme alleles in nine loci in the entire and core collections of Solanum tuberosum subsp. andigena conserved in the genebank at CIP. Linkage group indicated in parentheses after isozyme locus if known.

                      Entire
Isozyme                       No. of alleles
locus             Allele        ([dagger])

Idh-1 (I)           1              8002
                    2              1381
                    3                 2
                    4               131

Pgm-1 (III)        new              410
                    1               828
                    2              1986
                    3              6292

Pgm-2 (IV)          1               158
                    2              9244
                    3                96
                    4                18

Dia-1 (V)           0               154
                    1              6159
                    2              3170
                    3                33

Got-2 (VII)         3               598
                    5              7900
                    7              1018

Mdh-1 (VII)        new               12
                    1              3148
                    2              5883
                    3               245
                    4               228

Got-1 (VIII)        1               765
                    2                91
                    3              7185
                    4               561
                    5               285
                    6                 9

Pgi-1 (XII)         1                41
                    2              7624
                    3                78
                    4              1771
                    5                 2

Mdh-2               1              1761
                    2              7696
                    3                59

Isozyme                             Core
locus                 %         No. of alleles        %
                              ([double dagger])
Idh-1 (I)             84.08          1019           83.25
                      14.51           179           14.62
                      <0.1              0            0.00
                       1.38            26            2.12

                 [chi square] = 4.503; P = 0.212

Pgm-1 (III)            4.31            52            4.25
                       8.70           115            9.10
                      20.87           285           23.28
                      66.12           772           63.07

                 [chi square] = 5.126; P = 0.163

Pgm-2 (IV)             1.66            24            1.96
                      97.14          1189           97.14
                       1.01             9            0.73
                       0.19             2            0.16

                 [chi square] = 1.446; P = 0.695

Dia-1 (V)              1.62            19            1.55
                      64.72           797           65.11
                      33.31           404           33.03
                       0.35             4            0.33

                 [chi square] = 0.098; P = 0.992

Got-2 (VII)            6.28            94            7.69
                      83.02          1004           82.03
                      10.70           126           10.29

                 [chi square] = 3.573; P = 0.168

Mdh-1 (VII)            0.13             3            0.24
                      33.08           374           30.56
                      61.82           775           63.32
                       2.57            39            3.19
                       2.40            33            2.70

                 [chi square] = 5.536; P = 0.237

Got-1 (VIII)           8.60           115           10.16
                       1.02            18            1.59
                      80.77           863           76.24
                       6.31            95            8.39
                       3.20            39            3.14
                       0.10             2            0.18

                 [chi square] = 15.706; P = 0.008

Pgi-1 (XII)            0.43             7            0.57
                      80.12          1006           82.19
                       0.82            22            1.80
                      18.61           189           15.44
                       0.02             0            0.00

                 [chi square] = 18.424; P = 0.001

Mdh-2                 18.51           218           17.31
                      80.87           996           81.37
                       0.62            10            0.82

                 [chi square] = 0.973; P = 0.615


([dagger]) Of a maximum of 9516 possible in 2379 tetraploid genotypes.

([double dagger]) Of a maximum of 1224 possible in 306 tetraploid genotypes.

The core collection was meticulously developed by cluster analysis of morphological data to capture most of the genetic diversity available in the entire collection. The allozyme data, however, provide a more reliable estimate of the amount of genetic diversity than morphological descriptors. Furthermore, this work also confirms that useful morphological diversity may be associated with allozyme diversity in tetraploid andigena cultivars, as was reported by Ortiz et al. (1993) in tuberosum haploids.

Our results validate the sampling strategy of the core collection because only two allozymes (of 38) were missed. Frankel et al. (1995) indicate that according to the theory of selectively neutral alleles, a core subset consisting of 10% of the entire collection would retain at least 70% of the alleles of the core collection. However, rare localized alleles may be missed in the sampling procedure. In our study, the two rare allozymes missed in the core collection have a q = 0.0002 or 0.02% (Table 2). The single Bolivian accession with Idh-[1.sup.3] and one of the two Peruvian accessions bearing Pgi-[1.sup.5] may be added to the original core collection to avoid losing these two allozymes from the core subset. Furthermore, Pgi-[1.sup.5] has been observed among North American potato cultivars at a higher q (0.005 or 0.5%) (Douches and Ludlam, 1991). However, the role of rare localized alleles in the evolution of adaptedness in plants is questionable (Allard, 1996), and such alleles cannot be maintained in populations of a size practical for genebanks (Lawrence et al., 1995).

The locus heterozygosity between the entire and core collections were statistically similar (Table 3). Mdh-1, which has five allozymes, was the locus with the highest heterozygosity, while Pgm-2, with 4 allozymes, showed the lowest heterozygosity. The Andean potato cultivars have always two copies of Pgm-[2.sup.2], which explains its high q (97.14%) in this gene pool. Both allozyme frequence and average locus heterozygosity are important because they interact to determine the probability of selecting an individual genotype with a given allele. The results shown in Tables 2 and 3 suggest that this probability may be not very different for either set.

Table 3. Number of mono-allelic genotypes and average locus heterozygosity (ALH) in the entire and core collections of Solanum tuberosum subsp. andigena conserved in genebank at CIP. Linkage group indicated in parentheses after isozyme locus if known.
                             Entire
                     Mono-
Isozyme locus       allelic        Heterozygotes

                      no.                 %

Idh-1 (I)            1402               41.1
Pgm-1 (III)           867               63.6
Pgm-2 (IV)           2186                8.1
Dia-1 (V)             449               81.1
Got-2 (VII)          1173               50.7
Mdh-1 (VII)           453               81.0
Got-1 (VI)           1513               36.4
Pgi-1 (XII)          1438               39.6
Mdh-2                1398               41.2
ALH                                     49.2

                         Core
                                                      Most
                    Mono-                            common
Isozyme locus      allelic     Heterozygotes        allozyme

                     no.             %
Idh-1 (I)            176           42.5          Idh-[1.sup.1]
Pgm-1 (III)          101           67.0          Pgm-[1.sup.3]
Pgm-2 (IV)           283            7.5          Pgm-[2.sup.2]
Dia-1 (V)             63           79.4          Dia-[1.sup.1]
Got-2 (VII)          142           53.6          Got-[2.sup.5]
Mdh-1 (VII)           63           79.4          Mdh-[1.sup.2]
Got-1 (VI)           169           44.8          Got-[1.sup.3]
Pgi-1 (XII)          198           35.3          Pgi-[1.sup.2]
Mdh-2                179           40.9          Mdh-[2.sup.2]
ALH                                50.0
                       [chi square] = 5.729; P  = 0.678


Brown (1989) suggested three genetic criteria for a good core collection. First, the major subspecific taxa and geographic regions must be included in the core subset. Second, emphasis should be given to broadly adapted rather than intensely specialized alleles. Finally, within the above criteria, genetic diversity, especially as determined by number of alleles per locus, should be maximized in the core collection. The sampling of the original core collection of Andean tetraploid potato was defined by the cluster analysis of morphological diversity and the use of available data on eco-geographic provenance of each accession, and evaluation of reaction to diseases, pests and other desirable traits. Moreover, a newly defined core subset may meet the third criteria by adding to the core collection the two accessions bearing rare allozymes. A core collection can be used to identify accessions with genes controlling characteristics that have never before been assessed, or that are not visually obvious. For this purpose, the core collection should contain the maximum number of alleles in the species.

The Andean cultivars contained in this core collection are undergoing pathogen eradication to produce healthy planting materials for further worldwide distribution to potato breeders and researchers. This core collection of Andean tetraploid cultivars also offers a new tool to potato breeders looking for broadening their gene pools. The core collection may be included in a dynamic management program (Goldringer et al., 1994) for adapting exotic germplasm to specific local environments. Seed bulks of open pollinated half-sib offspring of selected accessions, derived from the original core collection, will be harvested after each selection cycle. This dynamic management may preserve genetic diversity between different populations evolving in distinct environments. These changes associated with the adaptedness of the population(s) derived from the core subset to the new breeder's environment(s) could also be monitored with the allozymes already investigated in the core collection or with other available co-dominant DNA markers.

As indicated by Brown (1995), the breeding of clonal species such as potato capitalizes on heterosis. Breeding gains for tuber yield in potato depend on identifying the right combinations of parents with high specific combining ability owing to non-additive intra- and interlocus interactions (Ortiz, 1998). This core collection should be seen as a preliminary genetic source for such combining ability tests in a gene pool that still lacks well defined heterotic groups.

ACKNOWLEDGMENTS

The authors thank Dr. David S. Douches (Michigan State Univ., East Lansing) for providing the standard genetic scoring of the isozyme markers to the different banding patterns found in this study, to Biol. Maria del Rosario Herrera (CIP, Lima, Peru) for establishing the methodology at CIP, and to Dr. David Spooner (Univ. of Wisconsin, Madison) for helpful comments on this paper.

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Zosimo Huaman, Rodomiro Ortiz,(*) Dapeng Zhang, and Flor Rodriguez

Z. Huaman, D. Zhang, and F. Rodriguez, Dep. of Crop Improvement and Genetic Resources, Centro Internacional de la Papa, Apartado 1558, Lima 100, Peril; R. Ortiz, ICRISAT, Patancheru 502 324, Andhra Pradesh, India. Received 24 Feb. 1999.

(*) Corresponding author (R.ORTIZ@CGIAR.ORG).
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Date:Jan 1, 2000
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