Novel Diversity Identified in a Wild Apple Population from the Kyrgyz Republic

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  • 1 National Center for Genetic Resources Preservation, U.S. Department of Agriculture, 1111 South Mason Street, Fort Collins, CO 80521
  • 2 Department of Horticulture and Crop Science, The Ohio State University, Wooster, OH 44691
  • 3 Plant Genetic Resources Unit, U.S. Department of Agriculture, Geneva, NY 14456-0462

The genetic diversity of a wild Malus population collected in the Kyrgyz Republic was compared with seedlings of Malus sieversii collected in Kazakhstan. Based on microsatellite marker results, we conclude that the population of 49 individuals collected in the Kyrgyz Republic includes private alleles and this population is assigned to a common genetic lineage with M. sieversii individuals found in the Karatau Mountain range of Kazakhstan. We recommend that a subset of these individuals be included in the National Plant Germplasm System Malus collection so they may be made available to breeders, physiologists, and other scientists for further examination.

Abstract

The genetic diversity of a wild Malus population collected in the Kyrgyz Republic was compared with seedlings of Malus sieversii collected in Kazakhstan. Based on microsatellite marker results, we conclude that the population of 49 individuals collected in the Kyrgyz Republic includes private alleles and this population is assigned to a common genetic lineage with M. sieversii individuals found in the Karatau Mountain range of Kazakhstan. We recommend that a subset of these individuals be included in the National Plant Germplasm System Malus collection so they may be made available to breeders, physiologists, and other scientists for further examination.

The wild apple species Malus sieversii (Ledeb.) M. Roem. is native to xeric regions and high mountain ranges of Kazakhstan, the Kyrgyz Republic, China, Tajikistan, Uzbekistan, and Turkmenistan (Yan et al., 2008). With a wide range of edible phenotypes observed in its natural habitat, M. sieversii is believed to be a primary progenitor species of the cultivated apple (M. ×domestica) (Harris et al., 2002; Luby et al., 2001; Watkins, 1995). Yan et al. (2008) suggested that the apple forests of Central Asia represent a derived form of M. sieversii, and the forests in the Xinyuan (Xinjiang) province in China represent a more primitive form.

In some texts, Malus kirghisorum is considered a unique Malus species, closely related to M. sieversii (Dzhangaliev, 2003). Fruits of both M. kirghisorum and M. sieversii are diverse in size, form, color, and flavor, ranging from sour–sweet to acid to bitter and astringent and have 10% to 12% sugar (Dzhangaliev et al., 2003). The habitats of M. sieversii and M. kirghisorum are overlapping and heterogenetic populations of these species exist in areas of close contact. Malus sieversii is found in many habitats, whereas M. kirghisorum is reported to be found specifically in rich, moist soils of northern slopes between elevations of 1200 and 1800 m. Malus kirghisorum is believed to have become more localized during periods of glaciation, whereas the more adaptable M. sieversii became more widely distributed (Dzhangaliev, 2003). Malus kirghisorum is considered mesophyllous and found adjacent to relict walnut, aspen, hawthorn, and maple forests (Dzhangaliev, 2003).

The USDA has sponsored four collection trips to Kazakhstan to collect Malus sieversii germplasm (Forsline et al., 2003; Hokanson et al., 1997). The genetic diversity of seedling apple trees originating from seeds collected in Kazakhstan has been previously described (Richards et al., 2009). Nine hundred forty-nine M. sieversii seedling individuals representing 88 half-sib families collected in the forests of Kazakhstan were compared using seven unlinked microsatellite markers (Richards et al., 2009). Classification of individuals into four clusters based on Bayesian assignment tests illustrated that genetic differentiation aligned with regional location. Individuals from the eastern Kazakhstan collection Sites 4, 5, and 9 (and half of the individuals from Sites 3 and 10) were predominantly localized to Cluster 1. Cluster 2 included individuals from across southern Kazakhstan, including Sites 3, 6, 10, 11, and 12. Sites 10, 11, and 12 also included individuals that were assigned to Clusters 3 and 4 (Richards et al., 2009).

In this work, we compared the genetic diversity of the M. sieversii forests of Kazakhstan with a set of Malus seedlings resulting from seeds collected in the forests of the Kyrgyz Republic. We identified genotypes based on neutral allele variation that could complement the USDA–National Plant Germplasm System germplasm collections of M. sieversii.

Materials and Methods

Plant materials.

In 2005, a collection expedition led by Dr. Diane Miller to the Kyrgyz Republic (sponsored by Winrock International Central Asia Farmer-to-Farmer program funded by the U.S. Agency for International Development) sought to identify new germplasm that represented novel diversity that could be useful to apple-breeding programs in the midwestern United States. This collection trip focused on the forests in the Arslanbob region (lat. 41.333, long. 72.933, elevation 1446 m) of the Kyrgyz Republic and resulted in the collection of 2500 seeds from a highly diverse site that was 25 km2 and contained hundreds of wild apple trees (Fig. 1). The collection site was characterized as predominantly wild Juglans with a lesser amount of wild Malus. The wild Malus was classified as Malus kirghisorum based on elevation and political boundary definitions of species delineations. All the Malus seeds from the Kyrgyz Republic were planted in Carrollton, OH, for evaluation purposes. Leaf samples from 50 randomly sampled seedlings were collected and sent to the USDA–ARS National Center for Genetic Resources Preservation in Ft. Collins, CO, for genotyping.

Fig. 1.
Fig. 1.

Map of Central Asia showing eight collection sites for Malus sieversii in Kazakhstan and the Arslanbob locality of the Kyrgyz Republic. Dominant mountain ranges are labeled.

Citation: HortScience horts 44, 2; 10.21273/HORTSCI.44.2.516

Molecular genotyping.

Genomic DNA from duplicate leaf samples from 50 M. sieversii trees was extracted using DNeasy plant kits (Qiagen, Valencia, CA) as previously reported (Volk et al., 2005). Malus microsatellites (simple sequence repeat) were amplified using unlinked primers (GD12, GD15, GD96, GD100, GD142, GD147, GD162) (Hemmat et al., 2003; Hokanson et al., 1998). Standard cultivar controls were Golden Delicious, Rome Beauty Law, and Cox Orange Pippin. Polymerase chain amplifications were performed as previously described (Volk et al., 2005). Unlabeled reverse primers were purchased from IDT (Coralville, IA) and forward primers, labeled with either IRD 700 or IRD 800, were obtained from MWG-Biotech (High Point, NC). Amplified products were multiplexed on a DNA sequencer (model 4200; LI-COR Inc., Lincoln, NE) using denatured acrylamide gels (Volk et al., 2005). Digital images were manually interpreted using LI-COR Saga™ Generation 2 software. Individuals were included in the analyses when they had missing data for no more than one marker.

Molecular data analysis.

The genetic diversity of M. sieversii genotypes collected from Kazakhstan has been previously described using the same set of microsatellite markers (Richards et al., 2009). The current analyses sought to compare the differentiation and diversity of the samples from the Kyrgyz Republic in the context of the 949 accessions assayed from eight locations in Kazakhstan (Fig. 1). Genotypic data were analyzed using the software package GDA (Lewis and Zaykin, 2001) and FSTAT (Goudet, 1995). Allelic richness measurements were normalized using the method of El Mousadik and Petit (1996).

In addition to estimating population genetic parameters from a priori-defined collection sites, we also used a nonhierarchical genotypic clustering method (Pritchard et al., 2000), which allows genotypes to form clusters without prior information about sampling structure. This method is useful for estimating regional patterns of differentiation that may otherwise go unrecognized. This Bayesian clustering was performed using the genotypes obtained for all 49 Kyrgyz Republic individuals (one genotype was discarded as a result of unacceptably high levels of missing data) as well as a combined data set that included 949 M. sieversii samples from Kazakhstan. Allelic richness statistics were calculated among genetic clusters. We graphically displayed the relative differentiation among collection sites and genetic clusters by using a minimum spanning network (Excoffier et al., 2005).

Results and Discussion

An evaluation of the genetic diversity of Malus seedling trees from the Kyrgyz Republic is most informative when placed in the context of the known diversity of the wild apple species M. sieversii in neighboring Kazakhstan. The Kyrgyz Republic site near Arslanbob is separated by ≈200 km from the nearest Kazakhstan sites by the rugged Karatau and Kyrgyz mountain ranges.

The sample size per collection site ranged from 24 individuals in Site 3 to 263 individuals in Site 9 (Table 1). Despite the discrepancy in population sizes, the effective heterozygosity, observed heterozygosity, and allelic richness were similar across the collection sites. A total of 106 alleles were amplified using seven markers in the seedlings from the Kyrgyz Republic site. In comparison, a total of 103 alleles were amplified in the set of 949 seedlings from the eight Kazakhstan sites (Richards et al., 2009). The set of individuals from the Kyrgyz Republic has six private alleles. These alleles were each found in more than one individual originating from the Kyrgyz Republic but not identified in any of the seedlings originating in Kazakhstan (Table 1).

Table 1.

Genetic diversity for each of the numbered eight Kazakhstan collection sites and the Kyrgyz Republic collection site (K) measured by effective heterozygosity (He), observed heterozygosity (Ho), allelic richness, and the number of private alleles based on microsatellite data.

Table 1.

Genotypes of the 949 Kazakhstan seedlings and the 49 Kyrgyz Republic seedlings were included in cluster analyses to identify which, if any, of the four Kazakhstan clusters shared lineages with the Kyrgyz Republic seedlings. These assignment tests were inconclusive because the 49 individuals were not stable across replicate Markov chain Monte Carlo runs. We determined that the optimal number of clusters (k) based on posterior likelihood increased from four to five when the 49 Kyrgyz Republic individuals were included in the data set. The 49 Kyrgyz Republic individuals shared a common lineage that comprised the fifth cluster (Table 2).

Table 2.

Site representation in each of the five clusters.

Table 2.

Measures of genetic differentiation across the five clusters revealed similar levels of effective heterozygosity, observed heterozygosity, and allelic richness (Table 3). When analyzed by cluster, the number of private alleles ranged from four private alleles in Clusters 3 and 4 to nine private alleles in Cluster 1 and five private alleles in Cluster 5 (Kyrgyz Republic) (Table 3).

Table 3.

Genetic diversity for each of five clusters measured by effective heterozygosity (He), observed heterozygosity (Ho), allelic richness, and the number of private alleles based on microsatellite data.z

Table 3.

Network diagrams were developed to illustrate the relationships among the individuals in the nine sites or among the five clusters (Fig. 2). The site network diagram demonstrates the relationship among the north and central eastern Kazakhstan Sites 9, 4, and 5 with Site 3 more closely aligned with Site 5 than the other sites. Site 10 is centrally positioned in the network, with Sites 6, 11, and 12 grouping together. The Kyrgyz Republic site (labeled K) shares most similar allelic composition with Site 11, one of the Kazakhstan sites that is south and centrally located.

Fig. 2.
Fig. 2.

Network diagrams illustrate the genetic relationships among (A) sites and (B) clusters of wild apple populations in Kazakhstan and in the Kyrgyz Republic.

Citation: HortScience horts 44, 2; 10.21273/HORTSCI.44.2.516

The relationship of the clusters reveals that Cluster 1 representing the eastern Kazakhstan sites and Cluster 2, representing many individuals in the southern Kazakhstan Sites 6, 11, 12, and 10, contain most of the M. sieversii individuals genotyped. Clusters 3 (primarily Sites 11 and 12) and 4 (primarily Sites 11 and 12) show signs of differentiation from Cluster 2, a widespread and common genetic lineage found in Kazakhstan. Cluster 5, comprised of the Kyrgyz Republic individuals, exhibits an alternative differentiation from the Cluster 2 set of individuals (Fig. 2B). This may have resulted in some novelty resulting from colonization of individuals from a common source lineage. As a result of the rugged mountainous terrain, subsequent gene flow among the Kyrgyz Republic individuals from the Arslanbob region could be limited.

Some taxonomists have described the apple populations within the Kyrgyz Republic to represent a novel Malus species, Malus kirghisorum. Despite the local levels of differentiation observed across the southern tier of Kazakhstan and into the Kyrgyz Republic, the diversity does not support the consideration of the Kyrgyz Republic individuals as a unique and independent species. Malus kirghisorum and M. sieversii are not reproductively isolated, because they cohabitate and hybrids between the species are reported to have particularly diverse phenotypes (Dzhangaliev, 2003). In addition, the lack of specific phenotypic traits distinguishing the species is another example supporting the lack of species differentiation between M. kirghisorum and M. sieversii.

In conclusion, we have identified novel forms of diversity within the M. sieversii individuals that have been recently collected in the Kyrgyz Republic. Because these individuals comprise an additional cluster, they likely share a common genetic lineage that may provide novel and useful phenotypic traits. We have recommended that 12 of the Kyrgyz individuals be included in the National Plant Germplasm System Malus collection so they may be made available to breeders, physiologists, and other scientists for further examination.

Literature Cited

  • Dzhangaliev, A.D. 2003 The wild apple tree of Kazakhstan Hort. Rev. (Amer. Soc. Hort. Sci.) 29 63 303

  • Dzhangaliev, A.D., Salova, T.N. & Turekhanova, P.M. 2003 The wild fruit and nut plants of Kazakhstan Hort. Rev. (Amer. Soc. Hort. Sci.) 29 305 371

  • El Mousadik, A. & Petit, R.H. 1996 High level of genetic differentiation for allelic richness among populations of the argan tree [Argania spinosa (L.) Skeels] endemic of Morocco Theor. Appl. Genet. 92 832 839

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  • Excoffier, L.G.L. & Schneider, S. 2005 Arlequin ver. 3.0: An integrated software package for population genetics data analysis Evol. Bioinform. Online 1 47 50 <http://lgb.unige.ch/arlequin/>

    • Search Google Scholar
    • Export Citation
  • Forsline, P.L., Aldwinckle, H.S., Dickson, E.E. & Hokanson, S.C. 2003 Collection, maintenance, characterization, and utilization of wild apples from central Asia Hort. Rev. (Amer. Soc. Hort. Sci.) 29 1 61

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  • Goudet, J. 1995 FSTAT, a program for IBM PC compatibles to calculate Weir and Cockerham's (1984) estimators of F-statistics J. Hered. 86 485 486

    • Search Google Scholar
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  • Harris, S.A., Robinson, J.P. & Juniper, B.E. 2002 Genetic clues to the origin of the apple Trends Genet. 18 426 430

  • Hemmat, M., Weeden, N.F. & Brown, S.K. 2003 Mapping and evaluation of Malus × domestica microsatellites in apple and pear J. Amer. Soc. Hort. Sci. 128 515 520

    • Search Google Scholar
    • Export Citation
  • Hokanson, S.C., McFerson, J.R., Forsline, P.L., Lamboy, W.F., Djangaliev, A.D. & Aldwinckle, H.S. 1997 Collecting and managing wild Malus germplasm in its center of diversity HortScience 32 173 176

    • Search Google Scholar
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  • Hokanson, S.C., Szewc-McFadden, A.K., Lamboy, W.F. & McFerson, J.R. 1998 Microsatellite (SSR) markers reveal genetic identities, genetic diversity and relationships in a Malus × domestica borkh. core collection Theor. Appl. Genet. 97 671 683

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  • Lewis, P.O. & Zaykin, D. 2001 GDA user's manual Department of Ecology and Evolutionary Biology, University of Connecticut 27 July 2004 <http://lewis.eeb.uconn.edu/lewishome>.

    • Export Citation
  • Luby, J., Forsline, P., Aldwinckle, H., Bus, V. & Giebel, M. 2001 Silk road apples—Collection, evaluation, and utilization of Malus sieversii from Central Asia HortScience 36 225 231

    • Search Google Scholar
    • Export Citation
  • Pritchard, J.K., Stephens, M. & Donnelly, P. 2000 Inference of population structure using multilocus genotype data Genetics 155 945 959

  • Richards, C.M., Volk, G.M., Reilley, A.A., Henk, A.D., Lockwood, D.R., Reeves, P.A. & Forsline, P.L. 2009 Genetic diversity and population structure in Malus sieversii, a wild progenitor species of domesticated apple Tree Genet. Genomes <http://www.springerlink.com/content/48741v0122104gu1/fulltext.pdf>

    • Search Google Scholar
    • Export Citation
  • Volk, G.M., Richards, C.M., Reilley, A.A., Henk, A.D., Forsline, P.L. & Aldwinckle, H.S. 2005 Ex situ conservation of vegetatively-propagated species: Development of a seed-based core collection for Malus sieversii J. Amer. Soc. Hort. Sci. 130 203 210

    • Search Google Scholar
    • Export Citation
  • Watkins, R. 1995 Apple and pear 418 422 Smartt J. & Simmonds N.W. Evolution of crop plants Longman Scientific and Technical Essex, UK

  • Yan, G.R., Long, H., Song, W.Q. & Chen, R.Y. 2008 Genetic polymorphism of Malus sieversii populations in Xinjiang, China Genet. Resources Crop Evol. 55 171 181

    • Search Google Scholar
    • Export Citation

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Contributor Notes

We acknowledge the support of Winrock International Central Asia Farmer-to-Farmer program funded by the U.S. Agency for International Development. This project was partially supported by the National Research Initiative of the USDA Cooperative State Research, Education and Extension Service (grant no. 2005-00751). The collection trip to the Kyrgyz Republic was supported by the Winrock International Central Asia Farmer-to-Farmer program funded by the U.S. Agency for International Development.

We thank Angela Baldo for reviewing the manuscript before submission.

Any mention of trade names or commercial products in this article is solely for the purpose of providing speciffic information and does not imply recommendation or endorsement by the U.S. Department of Agriculture.

To whom reprint requests should be addressed; e-mail Gayle.Volk@ars.usda.gov.

  • View in gallery

    Map of Central Asia showing eight collection sites for Malus sieversii in Kazakhstan and the Arslanbob locality of the Kyrgyz Republic. Dominant mountain ranges are labeled.

  • View in gallery

    Network diagrams illustrate the genetic relationships among (A) sites and (B) clusters of wild apple populations in Kazakhstan and in the Kyrgyz Republic.

  • Dzhangaliev, A.D. 2003 The wild apple tree of Kazakhstan Hort. Rev. (Amer. Soc. Hort. Sci.) 29 63 303

  • Dzhangaliev, A.D., Salova, T.N. & Turekhanova, P.M. 2003 The wild fruit and nut plants of Kazakhstan Hort. Rev. (Amer. Soc. Hort. Sci.) 29 305 371

  • El Mousadik, A. & Petit, R.H. 1996 High level of genetic differentiation for allelic richness among populations of the argan tree [Argania spinosa (L.) Skeels] endemic of Morocco Theor. Appl. Genet. 92 832 839

    • Search Google Scholar
    • Export Citation
  • Excoffier, L.G.L. & Schneider, S. 2005 Arlequin ver. 3.0: An integrated software package for population genetics data analysis Evol. Bioinform. Online 1 47 50 <http://lgb.unige.ch/arlequin/>

    • Search Google Scholar
    • Export Citation
  • Forsline, P.L., Aldwinckle, H.S., Dickson, E.E. & Hokanson, S.C. 2003 Collection, maintenance, characterization, and utilization of wild apples from central Asia Hort. Rev. (Amer. Soc. Hort. Sci.) 29 1 61

    • Search Google Scholar
    • Export Citation
  • Goudet, J. 1995 FSTAT, a program for IBM PC compatibles to calculate Weir and Cockerham's (1984) estimators of F-statistics J. Hered. 86 485 486

    • Search Google Scholar
    • Export Citation
  • Harris, S.A., Robinson, J.P. & Juniper, B.E. 2002 Genetic clues to the origin of the apple Trends Genet. 18 426 430

  • Hemmat, M., Weeden, N.F. & Brown, S.K. 2003 Mapping and evaluation of Malus × domestica microsatellites in apple and pear J. Amer. Soc. Hort. Sci. 128 515 520

    • Search Google Scholar
    • Export Citation
  • Hokanson, S.C., McFerson, J.R., Forsline, P.L., Lamboy, W.F., Djangaliev, A.D. & Aldwinckle, H.S. 1997 Collecting and managing wild Malus germplasm in its center of diversity HortScience 32 173 176

    • Search Google Scholar
    • Export Citation
  • Hokanson, S.C., Szewc-McFadden, A.K., Lamboy, W.F. & McFerson, J.R. 1998 Microsatellite (SSR) markers reveal genetic identities, genetic diversity and relationships in a Malus × domestica borkh. core collection Theor. Appl. Genet. 97 671 683

    • Search Google Scholar
    • Export Citation
  • Lewis, P.O. & Zaykin, D. 2001 GDA user's manual Department of Ecology and Evolutionary Biology, University of Connecticut 27 July 2004 <http://lewis.eeb.uconn.edu/lewishome>.

    • Export Citation
  • Luby, J., Forsline, P., Aldwinckle, H., Bus, V. & Giebel, M. 2001 Silk road apples—Collection, evaluation, and utilization of Malus sieversii from Central Asia HortScience 36 225 231

    • Search Google Scholar
    • Export Citation
  • Pritchard, J.K., Stephens, M. & Donnelly, P. 2000 Inference of population structure using multilocus genotype data Genetics 155 945 959

  • Richards, C.M., Volk, G.M., Reilley, A.A., Henk, A.D., Lockwood, D.R., Reeves, P.A. & Forsline, P.L. 2009 Genetic diversity and population structure in Malus sieversii, a wild progenitor species of domesticated apple Tree Genet. Genomes <http://www.springerlink.com/content/48741v0122104gu1/fulltext.pdf>

    • Search Google Scholar
    • Export Citation
  • Volk, G.M., Richards, C.M., Reilley, A.A., Henk, A.D., Forsline, P.L. & Aldwinckle, H.S. 2005 Ex situ conservation of vegetatively-propagated species: Development of a seed-based core collection for Malus sieversii J. Amer. Soc. Hort. Sci. 130 203 210

    • Search Google Scholar
    • Export Citation
  • Watkins, R. 1995 Apple and pear 418 422 Smartt J. & Simmonds N.W. Evolution of crop plants Longman Scientific and Technical Essex, UK

  • Yan, G.R., Long, H., Song, W.Q. & Chen, R.Y. 2008 Genetic polymorphism of Malus sieversii populations in Xinjiang, China Genet. Resources Crop Evol. 55 171 181

    • Search Google Scholar
    • Export Citation
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