HORTSCIENCE 48(12):1440–1444. 2013. M. dasyphylla, and M. kirghisorum. In the Genetic Resources Information Network (GRIN; USDA, ARS, National Genetic Re- Malus sieversii: A Diverse Central sources Program, 2013), these are synony- mous with M. pumila var. niedzwetzkyana, Asian Apple Species in the USDA-ARS M. pumila, and M. sieversii var. kirghisorum, respectively. Dzhangaliev (2003) summa- National Plant Germplasm System rized the Malus species native to Kazakhstan. M. sieversii and M. niedzwetazkyana are Gayle M. Volk2, Adam D. Henk, and Christopher M. Richards most similar to cultivated apple with USDA-ARS National Center for Genetic Resources Preservation, 1111 South M. niedzwetzkyana as a botanical variety of Mason Street, Fort Collins, CO 80521 M. dasyphylla. Furthermore, M. kirghisorum is described as distinct from M. sieversii Philip L. Forsline because it grows adjacent to relic Juglans USDA-ARS Plant Genetic Resources Unit, Geneva, NY 14456 regia forests as part of a mesophilous Turgai forest flora with a limited range in C. Thomas Chao1 Dzhungareskei and Zailijskei Alatau USDA-ARS National Center for Genetic Resources Preservation, 1111 South (Dzhangaliev, 2003). Based on molecular and phenotypic evi- Mason Street, Fort Collins, CO 80521 dence, M. sieversii is likely a progenitor species Additional index words. genepool, genetic diversity, Malus pumila, subspecies, taxonomy of the domesticated apple, Malus ·domestica (Harris et al., 2002; Velasco et al., 2010; Abstract. There are several Central Asian Malus species and varieties in the USDA-ARS Zhou and Li, 2000). In fact, M. sieversii and National Plant Germplasm System (NPGS) apple collection. Malus sieversii is the most M. ·domestica have sometimes been coclas- comprehensively collected species native to Central Asia. Other taxa such as M. sieversii sified as M. pumila (Harris et al., 2002; var. kirghisorum, M. sieversii var. turkmenorum, M. pumila, and M. pumila var. Juniper and Mabberley, 2006; Phipps et al., niedzwetzkyana have primarily been donated to the collection by other institutions and 1990, 1991). Although there are data suggest- arboreta. We sought to determine if genetic and/or phenotypic differences among the ing similarities between these species, ge- individuals that make up the gene pools of these taxa in the NPGS exhibit unique netic evidence suggests that they can be characteristics. Genetic data, based on microsatellite analyses, suggested that the genetically distinguished based on microsa- diversity within each taxa is significantly greater than that among taxa. Trait data also tellite markers (Gross et al., 2012). In fact, revealed very few differences among taxa, the primary characteristic being the dark red putative hybrids between M. sieversii and M. fruit coloration and tinted flesh color of the accessions assigned to M. pumila var. ·domestica can also be identified, thus fur- niedzwetzkyana resulting from a known single-gene mutation in anthocyanin production. ther supporting the differentiation of these We found that M. sieversii is a highly diverse species with a range in genetic and species (Gross et al., 2012). Other species phenotypic trait variation that includes the characteristics of the other Central Asian such as M. orientalis, M. sylvestris, and taxa of interest. We conclude that the gene pools that comprise the accessions within the M. prunifolia likely played a role in Malus NPGS Central Asian Malus collection are highly overlapping with respect to both domestication (Coart et al., 2006; Cornille phenotypic traits and genotypic characters. et al., 2012; Forte et al., 2002; Gross et al., 2012; Luby, 2003; Robinson et al., 2001; Velasco et al., 2010). Single nucleotide poly- The mountains of Central Asia have forest drought tolerance, and disease resistance morphism transferability was 26% from species that include Malus sieversii (Ledeb.) (Dzhangaliev, 2003; Yan et al., 2008; Zhou, M. ·domestica to either M. sieversii or M. Roem., a highly diverse apple crop wild 1999). M. sylvestris, supporting the evidence that relative. The Russian scientist Vavilov ex- The U.S. Department of Agriculture there are significant genomic differences plored the forests of Central Asia in the 1920s (USDA) sponsored four plant exploration among M. ·domestica and its likely pro- and made note of the wide range of M. sieversii trips in Central Asia to collect wild apples genitor species (Micheletti et al., 2011). phenotypes, suggesting that this region, includ- between 1989 and 1996. Participants on those Previously, we assessed the diversity of ing Kazakhstan, Kyrgyzstan, Tajikistan, and trips sought clones with unusual phenotypes the M. sieversii collection in the USDA-ARS western China, is a center of origin for the and collected seeds from trees growing in NPGS using microsatellite markers. Collec- domesticated apple (Luby et al., 2001; Wan diverse habitats, primarily in Kazakhstan. tion sites were significantly differentiated et al., 2011; Yan et al., 2008). Malus sieversii Over 1000 seedlings from the wild-collected and had unique alleles; however, differenti- grows in a wide range of habitats, including seeds have been grown in Geneva, NY, in the ation within individual families was more areas with hot summers and short winters as USDA-ARS NPGS apple collection. Most of than three times the level of that among sites. well as those with long, severe winters in the the seedlots were assigned to the M. sieversii Differentiation was congruent with geo- Tien Shan Mountains (Dzhangaliev, 2003). species, although some have been labeled as graphical location with southwestern collec- Wild populations are found in montane, scrub, Malus sieversii var. kirghisorum (Al. Fed. & tion sites being more admixed and more humid as well as dry continental forests and Fed.) Ponomar. The repository also has a diverse than the northern sites (Richards in diverse stream habitats in xeric areas number of accessions that have been classi- et al., 2009b). These data supported results (Forsline et al., 2003). Malus sieversii trees fied as Central Asian taxa, namely Malus published previously that used isozymes to are phenotypically diverse, offering variation sieversii var. turkmenorum (Juz. & Popov) determine within- and among-population dif- in the time of flowering, quantity and quality Ponomar. and Malus pumila Mill. Malus ferences in M. sieversii collections (Lamboy of fruit, ripening period, fruit biochemical pumila var. niedzwetzkyana Dieck. was for- et al., 1996). composition, tree architecture, winter and merly listed as a variety but in 2010 was Core collections were also proposed using reclassified as M. pumila.Manyofthese available microsatellite and quantitative trait accessions were donated to the repository from data for the M. sieversii trees from Kazakh- Received for publication 18 June 2013. Accepted other gene banks or arboreta. In addition to stan in the NPGS. Three sets of 35 trees, for publication 17 Sept. 2013. these species/varieties, the literature describes representative of Site 6, Site 9, and ‘‘other’’ 1Retired. a number of variations in nomenclature for collection sites, were identified as core col- 2To whom reprint requests should be addressed; Central Asian Malus species, subspecies, lections that capture greater than 95% of the e-mail [email protected]. and varieties including: M. niedzwetzkyana, measured allelic and phenotypic diversity
1440 HORTSCIENCE VOL. 48(12) DECEMBER 2013 (Forsline et al., 2003; Richards et al., 2009a; volatile components (Bai et al., 2012; Fazio 1990s (Forsline et al., 2003; Luby et al., Volk et al., 2005). et al., 2009; Forsline and Aldwinckle, 2004; 2001). Seeds were germinated and planted Malus sieversii accessions in the NPGS Forsline et al., 2003; Luby et al., 2001, 2002). as own-root seedlings within an orchard set- have been evaluated for a number of fruit, Some traits even differed by collection site ting. Clones representing individuals of the biotic, and abiotic stress resistance traits both such as the later harvest time and an increased other Central Asian taxa were provided to the on-site in Geneva, NY, and by collaborators. level of fireblight resistance in Site 6 and NPGS by donors and are maintained as Physiological and phenological traits such as larger, red fruit more common in Site 9 grafted trees within the collection (Table 1). high chilling requirement, early fruit matu- (Forsline and Aldwinckle, 2004). A total of 99 M. sieversii, nine M. sieversii rity, short juvenility, dwarf growth habit, late The large M. sieversii collection in the var. kirghisorum,threeM. sieversii var. flowering, stooling ability, and vigor were of NPGS provided us with an opportunity to turkmenorum,eightM. pumila,andthree particular interest to breeding programs assess the relationship among accessions M. pumila var. niedzwetzkyana trees were (Bassett et al., 2011; Fazio et al., 2009; Forsline collected and labeled as M. sieversii to those available for inclusion in this research. and Aldwinckle, 2004; Forsline et al., 2003; that have been classified as related species/ Genetic comparisons among accessions Luby et al., 2001, 2002). In addition, sources varieties: M. sieversii var. kirghisorum, assigned to the five Malus species/varieties of disease and insect resistance have been M. sieversii var. turkmenorum, M. pumila, were performed using microsatellite markers. identified for apple scab (Venturia inaequalis), and M. pumila var. niedzwetzkyana. We use DNA extraction, polymerase chain reaction fireblight (Erwinia amylovora), cedar apple genotypic and phenotypic data to determine amplifications, and allele identifications have rust (Gymnosporangium juniper-virginianae), if the gene pools in the NPGS that comprise been described previously (Volk et al., 2005). Rosellinia necatrix, Helicobasidium mompa, these taxa can be distinguished from one Two sets of data were available. A total of 18 woolly apple aphid (Eriosoma lanigerum), another. simple sequence repeat (SSR) loci (GD12, apple leaf curling midge (Dasineura mali), GD15, GD96, GD142, GD147, GD162, soilborne apple replant pathogens, and apple Materials and Methods CH01h01, CH01f02, CH01d08, CH01f07a, maggot (Rhagoletis pomonella)(Fazioetal., CH05e03, CH02d12, CH02b10, COL, 2009; Forsline and Aldwinckle, 2004; Forsline Malus sieversii and several accessions of NH009b, CH01d09, NZ28f4, NH015a; et al., 2003; Luby et al., 2001, 2002). Charac- M. sieversii var. kirghisorum seeds were Guilford et al., 1997; Hokanson et al., 1998; terized fruit traits include aroma, flavor, size, collected from forests in the Tien Shan Moun- Liebhard et al., 2002; Yamamoto et al., 2002) color, weight, texture, soluble solids, pH, and tains in Kazakhstan and Kyrgyzstan in the were available to compare 111 accessions
Table 1. National Plant Germplasm System accessions of Malus pumila, M. pumila var. niedzwetzkyana, M. sieversii var. kirghisorum, and M. sieversii var. turkmenorum for which phenotypic and/or genotypic data were available. SSR loci available Admixture Original collection Taxon Accession Local number (no.) (Gross et al., 2012) country Donor Malus pumila PI 323617 GMAL 3163 5 Afghanistan Arnold Arboretum Malus pumila PI 588827 GMAL 140 5 Finland (through Agriculture Canada) Malus pumila PI 588916 GMAL 244 5 Morton Arboretum Malus pumila PI 589166 GMAL 1016 5 Cornell University Malus pumila PI 590096 GMAL 3370 5 Arnold Arboretum Malus pumila PI 590097 GMAL 3378 5 Arnold Arboretum Malus pumila PI 590098 GMAL 3379 5 Arnold Arboretum Malus pumila PI 594106 GMAL 3223 5 Beijing Botanical Garden Malus pumila PI 589225 GMAL 1223 5 Turkestan/southwest Arnold Arboretum var. niedzwetzkyana Siberia (through Cornell University) Malus pumila PI 589287 GMAL 1389 5 District of Columbia var. niedzwetzkyana Malus pumila PI 589857 GMAL 2613 5 University of Maine var. niedzwetzkyana Malus sieversii PI 589373 GMAL 1740 18 Morton Arboretum var. kirghisorum Malus sieversii PI 589380 GMAL 1750 18 Morton Arboretum var. kirghisorum Malus sieversii PI 590043 GMAL 3158 18 Arnold Arboretum var. kirghisorum Malus sieversii PI 590094 GMAL 3367 18 x Arnold Arboretum var. kirghisorum Malus sieversii PI 613854 GMAL 2288.a1 18 x Vavilov Research Institute var. kirghisorum of Plant Industry Malus sieversii PI 613855 GMAL 2288.b1 x Vavilov Research Institute var. kirghisorum of Plant Industry Malus sieversii PI 613968 GMAL 4445 18 x Kazakhstan USDA collection trip var. kirghisorum Malus sieversii PI 633798 GMAL 3574 18 Kyrgyzstan USDA collection trip var. kirghisorum Malus sieversii PI 657001 GMAL 3583 18 Kyrgyzstan USDA collection trip var. kirghisorum Malus sieversii PI 594104 GMAL 2251 5 Vavilov Research Institute var. turkmenorum of Plant Industry Malus sieversii PI 613851 GMAL 2251 5 Vavilov Research Institute var. turkmenorum of Plant Industry Malus sieversii PI 613852 GMAL 2251 5 Vavilov Research Institute var. turkmenorum of Plant Industry SSR = simple sequence repeat; USDA = U.S. Department of Agriculture.
HORTSCIENCE VOL. 48(12) DECEMBER 2013 1441 assigned to M. sieversii with nine accessions Table 2. Comparison of Malus sieversii and Malus sieversii var. kirghisorum apple accessions in the assigned to M. sieversii var. kirghisorum. National Plant Germplasm System using data for 18 microsatellites. Another data set with five SSR loci (GS12, Sum Percentage GD15, GD142, GD147, GD162) was used to Source of variation df of squares Variance components of variation compare 61 M. sieversii accessions with three Among species/varieties 1 11.3 0.14 1.91 M. sieversii var. turkmenorum, eight M. Within species/varieties 232 1662 7.16 98.09 pumila, and three M. pumila var. niedzwetz- kyana accessions. Analyses of multivariance (AMOVA) determined the among- and within- population variation for each data set. DARwin Table 3. Comparison of Malus sieversii, M. sieversii var. turkmenorum, Malus pumila, and M. pumila var. niedzwetzkyana accessions within the National Plant Germplasm System using data for five software was used to graphically display the microsatellites. interindividual differences among accessions within each data set. Genetic distances were Sum Percentage calculated using a simple matching coefficient. Source of variation df of squares Variance components of variation Among 3 14 0.18 8.91 Bayesian structure analyses were per- species/varieties formed to determine if individuals could Within 140 254.7 1.82 91.09 be assigned to groups that corresponded to species/varieties species, subspecies, or varieties without a pri- ori taxa definitions (Pritchard et al., 2000). Individuals in the five SSR and 18 SSR data sets were analyzed independently. Phenotypic data were primarily collected for a single season for each accession between 1996 and 2003, when the trees were mature. Phenotypic data were collected according to descriptors that are described in the GRIN database (USDA, ARS, National Genetic Resources Program, 2013). Trait data for each descriptor were compared across the five Malus species/varieties. Categorical data were analyzed using c2 analyses and likeli- hood ratio tests to determine significance. Quantitative data were compared using anal- yses of variance and Tukey-Kramer honestly significant difference means comparisons. Fig. 1. Graphical representation from DARwin analyses. Each sample is represented as a terminal node in Results the neighbor-joining tree for (A) Malus sieversii (unlabeled) and M. sieversii var. kirghisorum (K) in the 18 simple sequence repeat (SSR) data set and (B) M. sieversii (unlabeled) and M. pumila (P), M. Malus sieversii is a broadly defined species pumila var. niedzwetzkyana (N), and M. sieversii var. turkmenorum (T) for the five SSR data sets. native to Kazakhstan, Kyrgyzstan, Tajikistan, Uzbekistan, and China. Many accessions of this species are accessible through the NPGS the 18 SSR and five SSR sets of data (Fig. 1). exhibit some degree of red coloration in the as a result of collection trips to Central Asia Individuals from each species/variety were flesh. in the 1990s. In contrast, fewer individuals identified. For both data sets, the non-M. Many of the phenotypic data for the represent M. sieversii var. kirghisorum, M. sieversii individuals were dispersed through- NPGS apple collection are categorical; how- sieversii var. turkmenorum, M. pumila, and out the trees rather than occurring together. ever, data for fruit length, fruit width, over- M. pumila var. niedzwetzkyana in the NPGS This suggests that the genetic diversity color, and soluble solids were collected (Table 1). Interindividual distances, genetic encompassed by M. sieversii is broader than quantitatively. Malus sieversii had the small- assignments, and genetic differentiation anal- that of the other species and varieties. est average fruit length (39.7 ± 0.6 mm) and yses were used to identify differences among Results from structure analysis showed width (44.2 ± 0.7 mm) among the species and the designated gene pools. no significant discontinuity. Using either the varieties compared (Table 4). Statistically, All three of the M. pumila var. niedzwetz- 18 locus data set to separate M. sieversii var. the fruit length was the same for all the kyana accessions in the data set had identical kirghisorum variety for M. sieversii or using species/varieties except M. pumila var. niedz- scores for the five SSRs tested. It is likely that the five locus data to partition the M. pumila wetzkyana, which had larger length and width. these are clones of the same individual. accessions, results indicated no supportable In addition, the M. sieversii var. kirghisorum AMOVA results for the 18 SSR data sets partition among the named taxa. Admixture fruit were wider than the M. sieversii fruit. The revealed no statistical differences between models with or without taxon identity as overcolor percentage of the M. sieversii fruit the microsatellite scores for M. sieversii and priors resulted in the same lack of separation. was lower than that of the M. pumila and the M. sieversii var. kirghisorum. Using AMOVA, Admixture was evenly distributed among solid-colored M. pumila var. niedzwetzkyana. we found 2% of the variation explained differ- each of the named taxa indicating that link- M. sieversii had the same soluble solids as the ences among varieties (Table 2). There were age disequilibrium has not built up among accessions representing other species/varieties also no differences among M. sieversii ac- these accessions (data not shown). (Table 4). cessions and M. sieversii var. turkmenorum, Image data are available for many of the Chi square analyses among NPGS acces- M. pumila, and M. pumila var. niedzwetz- accessions included in these analyses (Sup- sions assigned to M. sieversii, M. sieversii kyana accessions in the NPGS. Like in the plementary Fig. 1). Overall, the images of var. kirghisorum, M. sieversii var. turkmeno- 18 SSR data sets, most of the variation (91%) M. sieversii, M. sieversii var. kirghisorum, rum, M. pumila, and M. pumila var. niedz- is attributed to within-taxa and a small por- M. sieversii var. turkmenorum, and M. pumila wetzkyana revealed no significant differences tion (9%) is among the taxa (Table 3). are similar and overlapping in the range of for the following traits: calyx persistence, A dissimilarity index as determined by variation observed for each taxon. In con- calyx basin, fruit flesh flavor, fruit flesh using DARwin software was used to con- trast. M. pumila var. niedzwetzkyana is dis- firmness, fruit juiciness, fruit firmness, fruit struct an unrooted neighbor joining tree with tinct. These apples are dark red to purple and flesh oxidation, fruit weight, fruit ground
1442 HORTSCIENCE VOL. 48(12) DECEMBER 2013 Table 4. Quantitative phenotypic trait values among Central Asian Malus species/varieties. MYB transcription factors is responsible for Fruit length Fruit width Overcolor Soluble novel red fruit-flesh phenotypes. Plant Physiol. (mm) (mm) (%) solids (%) 161:225–239. Coart, E., S. Van Glabeke, M. De Loose, A.S. Malus sieversii 39.7 ± 0.6 b 44.2 ± 0.7 b 28 ± 3 c 11.9 ± 0.1 ab Larsen, and I. Roldan-Ruiz. 2006. Chloroplast Malus sieversii 46.0 ± 5.3 b 53.3 ± 4.9 a 51 ± 10 bc 13.1 ± 0.5 a diversity in the genus Malus: New insights into var. kirghisorum Malus sieversii 45.8 ± 11.8 ab 54.0 ± 12.0 ab 70 ± 5 abc 11.9 ± 1.2 ab the relationship between the European wild var. turkmenorum apple [Malus sylvestris (L.) Mill.] and the Malus pumila 44.3 ± 2.8 b 51.6 ± 3.6 ab 56 ± 8 ab 12.4 ± 0.8 ab domesticated apple (Malus domestica Borkh.). Malus pumila 59.0 ± 1.3 a 62.5 ± 2.4 a 94 ± 1 a 10.2 ± 0.4 b Mol. Ecol. 15:2171–2182. var. niedzwetzkyana Cornille, A., P. Gladieux, M.J.M. Smulders, I. Roldan-Ruiz, F. Laurens, B. Le Cam, A. Means, standard errors, and significant differences between Malus species/varieties were determined using Nersesyan, J. Clavel, M. Olonova, L. Feugey, Tukey means separation tests (P < 0.05). I. Gabrielyan, X.-G. Zhang, M.I. Tenaillon, and T. Giraud. 2012. New insight into the history of color, harvest season, overcolor pattern, top a mutation in a transcriptional regulator of domesticated apple: Secondary contribution of the European wild apple to the genome of fruit shape, stem cavity, fruit russet location, anthocyanin biosynthesis. This specific mu- cultivated varieties. PLoS Genet. 8:e1002703. and fruit bloom. tation confers the red-fleshed trait and the Dzhangaliev, A.D. 2003. The wild apple tree of Some traits differed among the Central presence alone of this allele. Kazakhstan. Hort. Rev. 29:65–304. Asian species/varieties. Several fruit color Malus pumila is a taxon that includes Fazio, G., H.S. Aldwinckle, G.M. Volk, C.M. traits, including fruit flesh color and fruit M. ·domestica and M. sieversii (Harris Richards, W. Janisiewicz, and P.L. Forsline. overcolor, were significantly different for et al., 2002; Juniper and Mabberley, 2006). 2009. Progress in evaluating Malus sieversii for M. pumila var. niedzwetzkyana than the four Although these species do exhibit similari- disease resistance and horticultural traits. Acta other species/varieties. The fruit flesh color ties, genetic data suggest that they are distinct Hort. 814:59–66. of M. pumila var. niedzwetzkyana was cream (Gross et al., 2012). Our analyses do not Forsline, P.L. and H.S. Aldwinckle. 2004. Evalu- and red, red, or rose, whereas most of the differentiate the accessions of M. pumila in ation of Malus sieversii seedling populations for disease resistance and horticultural traits. other fruit flesh colors were shades of white the NPGS from those of M. sieversii. Acta Hort. 663:529–534. and cream. Likewise, the fruit overcolor of The taxonomy of the Central Asian wild Forsline, P.L., H.S. Aldwinckle, E.E. Dickson, J.J. M. pumila var. niedzwetzkyana accessions apple species is complex. Many species have Luby, and S.C. Hokanson. 2003. Collection, was dark red or purple–red and the other been identified in the literature and then maintenance, characterization, and utilization of fruits were mostly green, yellow, orange, subsequently renamed (Korban and Skirvin, wild apples of Central Asia. Hort. Rev. 29:1–62. brown, or red. In addition, the fruit of 1984). In some cases, accessions that are Forte, A.V., A.N. Ignatov, V.V. Ponomarenko, M. pumila var. niedzwetzkyana were classi- donated to the NPGS from other institutions D.B. Dorokhov, and N.I. Savelyev. 2002. fied as conical, whereas those of the other have retained their original species classifi- Phylogeny of the Malus (apple tree) species, species/varieties were mostly globose or flat. cations. As a result, the few numbers of inferred from the morphological traits and mo- With respect to fruit weight, a higher accessions of these taxa can lead curators to lecular DNA analysis. Russ. J. Genet. 38:1357– 1369. proportion of M. sieversii had small fruit believe that they have some species that may Gross, B.L., A.D. Henk, P.L. Forsline, C.M. (less than 50 g) than the accessions of the be underrepresented in their collections. Richards, and G.M. Volk. 2012. Identification other four taxa. M. sieversii also had more Careful assessments of the validity of the of interspecific hybrids among domesticated variable fruit shape and size uniformity than assignments to taxa may clear up these mis- apple and its wild relatives. Tree Genet. Ge- the other types. This might be indicative of conceptions. Accessions that may be viewed nomes 8:1223–1235. the large sample size available for M. siever- as particularly novel based on their taxo- Guilford, P., S. Prakash, J.M. Zhu, E. Rikkerink, sii phenotypic measurements and that this nomic identities may in fact be quite similar S. Gardiner, H. Bassett, and R. Forster. 1997. species is generally diverse for many traits. to those already represented in the collection. Microsatellites in Malus · domestica (apple): The results from this research assert that Abundance, polymorphism and cultivar identi- the M. sieversii collection in the NPGS fication. Theor. Appl. Genet. 94:249–254. Discussion Harris, S.A., J.P. Robinson, and B.E. 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1444 HORTSCIENCE VOL. 48(12) DECEMBER 2013 Supplementary Fig. 1. Fruit images from Genetic Resources Information Network for Malus pumila, M. pumila var. niedzwetzkyana, M. sieversii var. kirghisorum, M. sieversii var. turkmenorum, and M. sieversii.
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