Genetic Diversity in Kiwifruit Polyploid Complexes: Insights Into Cultivar Evaluation, Conservation, and Utilization

Tree Genetics & Genomes (2014) 10:1451–1463 DOI 10.1007/s11295-014-0773-6

ORIGINAL PAPER

Genetic diversity in kiwifruit polyploid complexes: insights into cultivar evaluation, conservation, and utilization

Dawei Li & Yifei Liu & Xinwei Li & Jingyun Rao & Xiaohong Yao & Caihong Zhong

Received: 14 November 2013 /Revised: 22 June 2014 /Accepted: 30 June 2014 /Published online: 6 July 2014 # Springer-Verlag Berlin Heidelberg 2014

Abstract Understanding the extent and partitioning of crop morphology and genetic backgrounds. Based on these find- genetic diversity is necessary for conserving and utilizing their ings, strategies were proposed for the conservation and utili- genetic potentials for breeding. In the present study, zation of the current kiwifruit genetic resources for future fluorescence-labeled amplified fragment length polymor- breeding programs. phism markers were used to characterize the genetic diversity and relationships of 79 cultivars and also of 122 F1 hybrids Keywords Kiwifruit cultivars . Genetic diversity . which resulted from six kiwifruit interploid crosses. A high Polyploidy . Interploid cross . Conservation level of mean genetic diversity was detected (Hj > 0.22) for all cultivars investigated, without significant differences among diploids (2x), tetraploids (4x), and hexaploids (6x). This sug- Introduction gested that no significant genetic erosion occurred in these cultivars, which were directly selected from natural resources Crop genetic diversity is the raw material for breeding new or created from crosses. The Unweighted Pair Group Method crop varieties in response to the needs of diverse agricultural with Arithmetic Mean analysis of the genetic dissimilarity systems (Brussaard et al. 2010). Domestication or plant breed- between cultivars showed three main groups mostly based ing per se can be harmful for maintaining crop diversity on their three ploidy levels. Among these, the red-fleshed (Esquinas-Alcázar 2005). However, reduction in genetic di- cultivars which were originally derived from ‘Hongyang’ versity during crop breeding is variable, mostly depending on clustered into one subgroup of group I, suggesting their the biological nature of plant and also the differences in unique genetic background despite they were marked as dif- domestication activities (Zhao et al. 2014). Assessing the level ferent cultivars used in the current kiwifruit industry. By and pattern of the genetic variation for crop cultivars or the analyzing the genetic variation of hybrids with variable ploidy conserved genetic resources is thus crucial, in particular, for levels, our genetic analyses further revealed that interploid determining and constructing the core or mini-core collections crosses can increase the genetic diversity of F1 offsprings, (Zhang et al. 2011), assisting the selection of parental combi- especially from the parental combinations of 6x–2x and 6x–4x, nations to create hybrids with superior agronomic characters in which both parents showed significant differences in (Glaszmann et al. 2010), and developing conservation strategies to impede genetic erosion during domestication and breeding programs (Gepts 2006;Frankham2010). Communicated by R. Burdon : : : : The genus Actinidia Lindl., well known as kiwifruit, con- D. Li X. Li J. Rao X. Yao C. Zhong (*) tains 54 species which are generally dioecious, deciduous, and Key Laboratory of Plant Germplasm Enhancement and Specialty scrambling vines (Li et al. 2007). Currently, kiwifruit cultivars Agriculture, Wuhan Botanical Garden, Chinese Academy of Sciences, Wuhan 430074, Hubei, People’sRepublicofChina were mainly developed based on Actinidia chinensis var. e-mail: [email protected] chinensis and A. chinensis var. deliciosa (Li et al. 2007)in different breeding programs launched in China, New Zealand, Y. Li u and Italy during the last two decades (Ferguson and Huang Key Laboratory of Plant Resources Conservation and Sustainable Utilization, South China Botanical Garden, Chinese Academy of 2007). Most cultivars are direct selections from the wild or Sciences, Guangzhou, Guangdong 510650, China seedling populations of the two varieties with expected flavor, 1452 Tree Genetics & Genomes (2014) 10:1451–1463 flesh color, storage life, and ecological adaptation (Seal 2003; candidates for creating seedless cultivars (Zhu et al. 2009). Ferguson and Seal 2008). Although the existing kiwifruit Kiwifruit is known to have complex ploidy variations, includ- cultivars are deemed to be highly heterozygous, they still have ing diploids (2x), tetraploids (4x), and hexaploids (6x) a fairly narrow genetic base, especially for the cultivars de- (Ferguson and Huang 2007). In particular, many kiwifruit veloped outside China (Datson and Ferguson 2011). cultivars are polyploids (Li et al. 2010a). Details of genetic Moreover, the widespread adoption of the economically im- variation in relation to ploidy levels and interploidy hybrids portant kiwifruit cultivars replacing local landraces is further are thus needed to improve our understanding of polyploidy narrowing the genetic base of kiwifruit industries. This is contributing to the diversity of kiwifruit germplasm. dangerous in the face of climatic fluctuations and spread of In the present study, we used fluorescent AFLP markers to diseases such as Pseudomonas syringae pv. Actinidiae perform genetic analysis of 79 kiwifruit cultivars (selections) (Vanneste 2012). Several major Actinidia repositories have and 122 F1 hybrids derived from six interploid crosses. been constructed in and outside China in terms of conserva- Together with the investigation of ploidy levels of these sam- tion of core Actinidia germplasm. However, the basic genetic ples, we aimed to (1) analyze the genetic variations in both the information on these collections is unclear, which is of major diploid and polyploid cultivars, (2) identify the genetic rela- concern for future kiwifruit industry developments (Ferguson tionships of these cultivars, (3) and evaluate the genetic var- 2007). iations of F1 hybrids derived from interploid crosses, in terms Characterizing germplasms diversity is the first step for of selecting good parental combinations for kiwifruit cross future management and utilization of genetic resources breeding. (Naval et al. 2010). Traditionally, phenotypic characters were used for genetic germplasm identification (e.g., UPOV for fruits). However, many vegetative characteristics are highly Materials and methods influenced by environmental conditions or phenotypic plasticity (Lafitte and Courtois 2002; Zhe et al. 2010). Most Plant material and DNA extraction molecular markers are comparatively independent of environmental and of pleiotropic and epistatic effects, providing Seventy-nine kiwifruit cultivars and selections (Table 3 of the efficient tools for identifying quantitative effects on traits Appendix), including 36 cultivars directly developed from the (Collard et al. 2005; Kalia et al. 2011). So far, few DNA- natural germplasm, were sampled from the National Actinidia based marker techniques, such as random amplification of Germplasm Repository of China, Wuhan, Hubei Province, for polymorphic DNA (RAPD), simple sequence repeat (SSR), genetic analysis. These cultivars represent more than 95 % of and amplified fragment length polymorphism (AFLP) the world kiwifruit production. Moreover, 3,250 F1 seedlings markers, have been applied to determine the molecular char- generated from six interploid crosses in the Wuhan Botanical acterization (Messina et al. 1991;Zhenetal.2004;Novoetal. Garden, Chinese Academy of Science since 2006 were also 2010), genetic variability (Palombi and Damiano 2002), and involved in our analysis. After examining the ploidy levels of phylogenetic relationships (Kokudo et al. 2003; Korkovelos these seedlings, 122 F1 progenies were randomly selected to et al. 2008)ofActinidia species or cultivars. analyze their genomic-level genetic variation (Table 2). Total Most polyploid species are polyphyletic, having recurrent- genomic DNA materials were extracted from fresh leaf mate- ly formed from genetically distinct diploid parents, resulting rial following a modified CTAB procedure (Doyle and Doyle in a relatively high level of genetic diversity (Soltis and Soltis 1987). 2000). Polyploid crops generally perform better than diploid ancestors in major agronomic traits, which can be attributed to Investigation of ploidy levels the genomic “buffering” effects, the increased allelic diversity, the increased or “fixed” heterozygosity, and the novel pheno- The ploidy levels of all analyzed materials were determined typic variation which occur after genomic duplications by flow cytometry (FCM). The newly expanding leaves of (Stebbins 1950; Udall and Wendel 2006; Leitch and Leitch both parents and progenies were collected in spring of 2012. 2008). Based on ploidy races, creating interploid hybrids is a To release individual nuclei for FCM measurements, the powerful approach for producing new genetic variability use- leaves were chopped in 0.5 ml of nuclear extraction buffer ful for genetic breeding. It allows bidirectional introgression (solution A of High Resolution Kit, Partec, Germany) and of noteworthy genes/alleles from crossing parents with differ- incubated for 2 min, and then filtered through a nylon sieve ent ploidy levels into novel ploidy hybrid genotypes and with a mesh diameter of 30 μm (CellTrics™, Partec, phenotypes (Soltis and Soltis 2009). In maize, reciprocal Germany). Two milliliters of a solution of 6-diamidino-2- interploid crosses can result in infertile seeds with defective phenylindole (DAPI, solution B of the kit) held at 4 °C was endosperms (Pennington et al. 2008), and in citrus, triploids then added. Two minutes later, the samples were automatical- selecting from interploid crosses are the most important ly analyzed for the estimation of their ploidy levels on a Tree Genetics & Genomes (2014) 10:1451–1463 1453

CyFlow Space (Partec, Germany). Generally, only samples Both the “band-based” and “fragment-frequency-based” producing two histogram peaks (sample and standard) with a approaches (Bonin et al. 2007) were used for the next analy- low coefficient of variation (below 5 %) were retained. The ses. In the latter approach, statistical analysis were set equal to previously cytologically studied diploid (2n=58) cultivar of fragment frequency and under the assumption of “Hardy– var. chinensis ‘Hongyang’ wasusedasaninternalreference Weinberg equilibrium.” In this study, the Shannon’sindex standard for the relative DNA content measurements. was employed because it is more insensitive to the bias related to the inability to differentiate all heterozygotes from homo- zygotes at all loci when using a dominant marker system like AFLP analysis AFLP (Dawson et al. 1995; Adoukonou-Sagbadja et al. 2007). Genotypic diversity was then estimated using the AFLP analysis was performed using EcoRI/Mse ItypeAFLP Shannon’s index (Shannon and Weaver 1949)inprogramR kit (Beijing Dingguo Biotechnology Co. Ltd., China). The (R Development Core Team 2010) according to Yeh et al. DNA adaptors and 11 fluorescence-labeled primer pairs were (1995)andLacerdaetal.(2001): H = −∑Pi log Pi/N, where shown in Table 4 of the Appendix. Of that, the primer com- 2 Pi is the frequency of a particular AFLP fragment and N is the binations No1–No8 were chosen for the final selective ampli- total number of loci. Secondly, allelic frequencies of AFLP fication of 79 cultivars, and the primer combinations No4– loci were estimated using the Bayesian method (Zhivotovsky No11 were chosen for the final selective amplification of 1999) with a non-uniform prior distribution of allele frequen- 122 F1 hybrids (Tables 4 and 5 of the Appendix). In addition, cies assuming no deviation from Hardy–Weinberg genotypic six samples from 79 cultivars were selected for replicated proportions. For each ploidy level, after estimating allele experiments to assess the accuracy and repeatability of the frequencies and the proportion of polymorphic loci at the AFLP data. The amplified fragments were separated by 4 % 5 % level, Nei’s gene diversity (Hj) was calculated following denaturing polyacrylamide gel electrophoresis on the ABI Lynch and Milligan (1994) in AFLP-SURV (Vekemans Prism 377 DNA sequencer (Applied Biosystems, Foster 2002). City, CA, USA). The size of the AFLP fragments was deter- Unweighted Pair Group Method with Arithmetic Mean mined by the software packages GeneScan 3.1 and Genotyper (UPGMA) analyses for both the 79 cultivars and the subset 3.7 (Applied Biosystems). of 36 cultivars derived from natural germplasm were performed in NTSYS-pc version 2.1e (Rohlf 2000), respectively. Data analyses The dendrogram was based on pairwise similarity between samples according to the Dice index (Dice 1945) (Dalirsefat It is impossible to unambiguously estimate the allele frequen- et al. 2009): Sij =2a/(2a + b + c), where Sij is the similarity cies of AFLP markers, in particular when samples have dif- between two individuals i and j, a is the number of bands ferent ploidy levels. Therefore, we assumed that each ampli- present in both i and j, b is the number of bands present in i and fied fragment (peak) corresponded to a dominant allele at a absent in j,andc is the number of bands present in j and absent given polymorphic locus. We scored the amplification prod- in i. On the other hand, Principal Coordinate (PCO) analysis ucts as a binary matrix encoding with 1 (homozygous and utilizing Nei and Li's coefficient (Nei and Li 1979)werealso heterozygous states) or 0 (null alleles) for presence or absence carried out to calculate genetic similarities. Analysis of mo- of each peak, respectively. To increase homology of AFLP lecular variance (AMOVA) was performed to quantify the bands (Althoff et al. 2007), only fragments between the size distribution of genetic variation within and among cultivar range of 125–501 bp and the fluorescence intensity above 80 of different ploidy levels. The last two analyses were per- relative fluorescence units were considered for further formed in Genalex 6 (Peakall and Smouse 2006). analysis. The AFLP data and marker combinations were assessed by means of the polymorphism information content (PIC) and the marker index (MI). The polymorphism information content Results was calculated though the formula PICi =1−∑Pi2,wherePi is the band frequency of the ith allele. The marker index was The assessments of marker polymorphism and AFLP calculated for each AFLP primer combination: MI = PIC × fragments βη, where PIC is the mean value of the total PIC, β is the proportion of the polymorphic bands, and η is the number of The results of AFLP analysis are presented in Table 5 of the bands (Powell et al. 1996). In addition, the proportions Appendix. The AFLP primer pairs used for the 79 cultivars of polymorphic loci (PPL) and of the shared and private yielded a total of 1,007 polymorphic bands, with an average of bands among different ploidy races were also calculated 126 bands per primer pair (Table 5 of the Appendix). The in Excel 2007. percentage of the polymorphic bands varied from 94 to 98 %, 1454 Tree Genetics & Genomes (2014) 10:1451–1463 with an average of 96 % per primer combination. The PIC observed between hexaploids and tetraploids (Table 1(a)). values were also variable, ranged between 0.21 and 0.33 with Thirty-six cultivars, which were directly selected from the an average of 0.28 per fragment, while the MI ranged between natural resources, were further analyzed separately. The ge- 27.91 and 37.33 with an average of 33.03. Similarly, a high netic diversity of hexaploid and tetraploid cultivars (PPL= polymorphism was observed by using the eight primer com- 84.2 %, Hj=0.239) in this subset was significantly higher than binations performing on 122 samples of six interploid crosses those of diploids (PPL=51.9 %, Hj=0.228) (Table 1(b)). (Table 5 of the Appendix). In addition, repeated measurements Moreover, more private fragments (>5 %) and less shared based on six testing samples demonstrated that 95.3–98.8 % fragments (2 %≤) of AFLPs were found in the 36 cultivars AFLP fragments were identical to the fragments studied pre- (Fig. 1). For both datasets of 79 and 36 cultivars, AMOVA viously. Our data thus confirmed that the primer pairs gener- analysis showed that majority of the genetic marker diversity ated strong and reproducible amplification products, all of to be within the same ploidy level (>88 %), rather than among which displayed high polymorphism and are suitable for ploidy levels (<12 %). further study. The genetic similarity coefficients of the 79 cultivars ranged from 0.52 to 0.79. The UPGMA dendrogram clustered the 79 genotypes into three main groups (Fig. 2). Group I Ploidy levels of the cultivars and F1 hybrids contained only diploid cultivars. Within group I, it was noteworthy that the female cultivars with red flesh (‘Hongyang,’ The ploidy levels of the 79 cultivars were presented in Table 3 ‘Wanhong,’‘Honghua,’‘Oriental red,’‘Wzred 8, 10, 11’) of the Appendix, with diploids, tetraploids, and hexaploids were genetically very similar and clustered into a subgroup accounting for 36.7, 35.4, and 27.8 %, respectively. All the (Fig. 2). Except for hexaploid ‘Jinkui M2,’ group II was diploids belong to var. chinensis and all the hexaploids to var. composed of all tetraploids. Group III was predominately deliciosa, whereas the tetraploids were predominantly classi- composed of the hexaploid cultivars, except for ‘Chuhong.’ fied into var. chinensis, except for ‘Xiangma No6’ and The UPGMA dendrogram based on 36 cultivars from natural ‘Chuhong’ which belong to var. deliciosa. The ploidy levels resources, likewise, clustered into three groups (2x,4x,6x; of F1 hybrids studied were highly diverse due to the hybrid- Fig. 2) unambiguously. The clustering pattern in the dendro- ization across ploidy levels, including triploid, tetraploid, gram was strongly supported by PCO analysis, which also pentaploid, hexaploid, and heptaploid, and the triploid and showed a clear differentiation of three clusters according to pentaploid constitute the majority in those progenies the ploidy levels of cultivars (Fig. 3). (Table 2).

Genetic diversity and relationship of kiwifruit cultivars Genetic variations between parents and F1 hybrids

Within the 79 cultivars, the proportions of polymorphic loci Estimates of genetic diversity for both parents and F1 hybrids were 65.5, 68.5, and 66.1 % in diploid, tetraploid, and hexa- are presented in Table 2. The number of different bands ploid cultivars, respectively. Moreover, three ploidy clusters amplified from eight AFLP primers ranged from 267 to 423 (2x,4x,and6x) had 37, 29, and 41 private AFLP bands in turn. among six interploid crosses. The progenies were observed to The values of genetic diversity parameters calculated (Hj and harbor higher genetic diversity than their parents, according to I) were shown in Table 1. Obviously, hexaploid and tetraploid significantly higher PPL, specific bands (Sb), and gene diver- cultivars exhibited a slightly higher level of genetic diversity sity values (Hj and I; Table 2). In addition, the genetic vari- than diploid cultivars, whereas no significant difference was ability was significantly different among six interploid crosses

Table 1 Comparison of genetic diversity among diploid-, tetraploid- and hexaploid cultivars

Provenances Code Sampling numbers Nb PrB PPL (%) Hj I a. 79 cultviars selected from natural resources, seedlings or hybrids Diploid cultivars 29 827 37 65.5 0.222 0.559 Tetraploid cultivars 28 872 29 68.5 0.228 0.563 Hexaploid cultivars 22 840 41 66.1 0.241 0.561 b. 36 cultivars directly selected from natural resources Diploid cultivars 6 537 29 51.9 0.228 0.555 Tetraploid cultivars 20 832 118 84.2 0.239 0.564 Hexaploid cultivars 10 733 79 71.5 0.255 0.574

Nb number of different bands, PrB number of private bands, PPL proportion of polymorphic loci, Hj unbiased Nei’s gene diversity, I Shannon’s information index Tree Genetics & Genomes (2014) 10:1451–1463 1455

Fig. 1 The proportion of the shared (red) and private (blue) a b AFLP fragments among diploids, 5% 4% 1% tetraploids, and hexaploids in 79 4% cultivars (a) and 36 cultivars (b), respectively

94% 92% Diploid

3% 3% 14% 2%

94% Tetraploid 84%

5% 3% 11% 1%

Hexaploid 92% 88%

Shared fragments of diploid, tetraploid and hexaploid

Private fragments of diploid, tetraploid and hexaploid, respectively

Other polymorphic fragments

in that hybrids from 6x–2x and 6x–4x crosses exhibited higher as high variability in flesh color, fruit size, nutritional content, levels of genetic diversity than hybrids from 4x–2x crosses flavor, and storage life (Huang 2013;Fig.4). For example, the (Table 2). However, there was no significant differentiation red- (‘Hongyang’) and yellow-fleshed (‘Jinyan’) cultivars within the 4x–2x combinations (Table 2, codes 4, 5, and 6). have already accounted for 20 % of planting areas and have changed the cultivar compositions in China. Our statistical results demonstrated that the majority of current kiwifruit cultivars were directly obtained (45.6 %) from natural re- Discussion sources or were only a few generations (e.g., 31.6 % cultivars selected from seedlings, in Table 3 of the Appendix) removed Cultivar diversity and genetic variability from the wild, implying that the present kiwifruit industry is still benefitting from the natural resources of Actinidia (Huang Commercial kiwifruit cultivars can be divided into three 2009). groups based on their green-, yellow-, or red flesh color. Our AFLP analysis found a high level of genetic diversity Currently, the greens are still the predominant cultivars (over (PPL=0.655–0.685) present in all 79 cultivars investigated, 90 %) planted around the world (Belrose Inc 2013). However, which was consistent with the previous results which used in China, the recent expanding of kiwifruit industry was both SSR (PPL=0.778–1.000, Zhen et al. 2004) and RFLP characterized by planting cultivars with diversified traits such markers (PPL=0.5505, Palombi and Damiano 2002). 1456 Tree Genetics & Genomes (2014) 10:1451–1463

Fig. 2 UPGMA dendrograms showing clustering patterns of 79 (a) and 36 (b) kiwifruit cultivars based on Dice similarity coefficients obtained from the AFLP data. The red-, blue-, and green color presented diploid, tetraploid, and hexaploid, respectively

Fig. 3 Two-dimensional graphs based on the ordination scores of the principal coordinate analyses according to Nei’s genetic distances. Pooling 79 (a) and 36 (b) cultivars in three group according to the ploidy levels, respectively Tree Genetics & Genomes (2014) 10:1451–1463 1457

Table 2 The ploidy level and genetic variations of six interploid crosses in this study

Code Parental combinations(♀ × ♂) no.F1 no.S Ploidy levels f F1 hybrids Nb PrB PPL He I

Pa F1b PF1P F1 PF1PF1 (%) (%)

1 Xinguan No2 (6x)×Guihai 60 56 3x(20), 4x(22),5x(1), 6x(1), 303 402 0 99 41.1 93.3 0. 308 0.430 0.531 0.552 No4-M♂ (2x) 7x(12), 2 Xinguan No2 (6x) × Hongyang- 26 26 3x(1), 4x(20), 5x(2), 6x(3) 315 423 0 108 46.1 92.2 0.346 0.439 0.551 0.572 M ♂ (2x) 3Jintao(4x) × Jinkui-M♂ (6x)1623105x(10) 383 267 0 116 47.9 97.1 0.359 0.464 0.521 0.549 4Jintao(4x) × Hongyang-M ♂ (2x) 288 10 3x(10) 300 389 0 89 44.2 79.2 0.331 0.404 0.542 0.544 5Jintao(4x) × Guihai No4-M ♂ (2x)1239103x(10) 305 375 0 66 43.4 79.5 0.325 0.396 0.538 0.543 6Jinyi(2x)×MoshanNo4♂ (4x)14103x(10) 278 354 0 76 47.7 75.9 0.358 0.416 0.52 0.532 no.F1 the total F1 hybrids from the interploid cross, no.S the hybrids numbers sampled for the genetic analysis, Nb number of different bands, PrB number of private bands, PPL proportion of polymorphic loci, Hj unbiased Nei’s gene diversity, I Shannon's information index a Parents b F1 hybrids

Moreover, no significant differences in genetic diversity were selected directly from the wild can be diverse without genetic observed between ploidy groups, although diploid cultivars erosion due to the biological nature of Actinidia species such were slightly less diverse than tetraploids and hexaploids as perennial habit and outcrossing; (2) the selections (e.g., (Table 1). However, the genetic diversity was significantly diploid ‘Lvzhu,’‘Mantianhong’) that originated from inter- different between polyploids and diploids in the subset of the specific crosses resulting in novel fruit traits (Beatson et al. 36 cultivars (Table 1). Higher genetic variation can be attrib- 2007) have high genetic diversity because of hybridization uted to both polyploidy (Li 2010) and the widespread natural (Zhang et al. 2010); and (3) the hybrids (e.g., Jinkui M2) hybridization (Liu et al. 2010). Taken together, the high level generated by interploid crosses can be productive with im- of genetic diversity observed, both in diploids and polyploids, proved genetic variation by breaking the interploid fertility can be indicative that (1) most kiwifruit cultivars that were block (Yan et al. 1997; Seal et al. 2012) and altering the

Fig. 4 The morphological diversity of fruit characters in diploid-, tetraploid-, and hexaploid cultivars 1458 Tree Genetics & Genomes (2014) 10:1451–1463 genetic compositions of ploidy offspring (Table 2, Fig. 2). deliciosa and the 4x and 2x parents were var. chinensis,and Therefore, our study showed a case of a fruit crop conserving these two taxonomic varieties show significant differences in high level of genetic diversity in cultivars/selections with low both morphology and genetic backgrounds. Similarly, the selection and domestication pressures. pentaploids, which were hybrids from the cross of tetraploid and hexaploid parents, obtained higher genetic diversity Genetic relationships and compositions of kiwifruit cultivars (PPL=0.971) than parents (PPL=0.479). However, the genetic variability of hybrids from 2x–4x crosses seemed to be Our UPGMA (Fig. 2)andPCO(Fig.3)analysesofboththe indistinct from that of their parents (Table 2), including in 79 and 36 cultivar datasets showed three distinct groups reciprocal crosses (4x–2x). These findings suggested that mostly according to their ploidy levels (Fig. 2). interploid crossing could be an innovation for kiwifruit breed- Consequently, our study revealed the relationships of the ing and enhance the genetic variability of progenies. In cultivars investigated: (1) cultivars clustered mainly based particular, the genetic diversity of F1 hybrids among on their ploidy levels, suggesting a high level of genetic crosses in our study suggested that the genetic distance similarities of cultivars within the same ploidy level; (2) between parents may be related to genetic variations of tetraploid cultivars preferentially clustered with the diploids their hybrid progeny, as was found in alfalfa (Medicago rather than the hexaploids, which is consistent with the tradi- sativa L.; Kidwell et al. 1994). tional taxonomic treatment of the var. chinensis variety (Li et al. 2007); (3) together with the provenances, three main Conservation and utilization of kiwifruit resource pedigrees based on flesh color were detected, including diploid red-fleshed pedigree (‘Hongyang’ and its progenies Although our study has confirmed that most kiwifruit cultivars ‘Honghua,’‘Wanhong,’‘Oriental red,’‘WZred-8, 10,11,’ harbor a high level of genetic diversity, it is only a small ‘Hongyang-M’ ), tetraploid yellow-fleshed pedigree fraction of that present in the entire gene pools of natural (‘Jinyan’ and its progenies ‘Jinyuan’ and ‘Jinmei’), and hexa- resources. Current kiwifruit breeding, with its emphasis on ploid green-fleshed pedigree (‘Hayward’ and its progenies elite × elite crosses in the incessant pursuit of higher perfor- ‘Shixuan No-2’ and ‘Xuguan’); (4) the clear subgroup of the mance and close adherence to customer preferences imposed new red-fleshed cultivars (2x), which were originally derived by the market (Cheng et al. 2004;Gepts2006), is a strong from the seedlings or F1 hybrids of the ‘Hongyang’ cultivar, force in the reduction in genetic diversity. Improved cultivars was genetically uniform. This uniformity might be risky when also lead to a reduction in genetic diversity through displace- they were used in the current kiwifruit industry in face of ment of native landrace cultivars. Effective protection and diseases (e.g., P. syringae pv. Actinidiae)orclimaticfluctua- germplasm innovation are expected to ensure the sustainable tions. Collecting or creation of red-fleshed germplasms with and healthy development of world kiwifruit industry. Firstly, variable ploidy levels and focused resistance would be useful ex situ and in situ conservation should be conducted to con- for future breeding (Sui et al. 2013). serve kiwifruit wild resources and make them available to scientists and breeders. In China, the National Actinidia The genetic variations generated by interploid crosses Germplasm Repository funded by the Ministry of Agriculture has been constructed. A total of about 40 For the hybrids derived from the six interploid crosses, we Actinidia species, 80 cultivars, and hundreds of germplasms found a diverse ploidy variation pattern including 3x,4x,5x, were ex situ conserved. In addition, the natural kiwifruit core 6x,and7x (Table 2). The abundant ploidy races might be collections, including the natural hybrids of both var. attributed to the prevalence of 2n gametes within the Actinidia chinensis and var. deliciosa, the special red-fleshed kiwifruit genus (Chat and Dumoulin 1997; Mizugami et al. 2007;Seal genotypes in Xuefeng-Wuling mountains, Mufu mountains, et al. 2012), and interploid crosses could be deemed to be a Qingling mountains, Daba mountains, and Funiu mountains successful strategy for transferring the genomes of elite (Sui et al. 2013;Huang2009;Lietal.2010a, b; Liu et al. fruiting cultivars to higher ploidy levels by unilateral sexual 2010), have been in situ conserved effectively. Secondly, more polyploidization. Likewise, ploidy manipulation (chro- genome-wide molecular marker sets, such as the next- mosome doubling) could have considerable potential as generation genome sequencing derived SNPs (Huang et al. such in exploring the polyploid advantage of fruit char- 2013), should be applied to help us identify the core germ- acters (e.g., the fruit size of autotetraploid var. chinensis plasm of Actinidia and manage the gene bank, in particular, increased significantly comparing to the diploid ances- deepening our understanding of the genetic basis for and tor, Wu et al. 2011, 2012). quantitative relationships involving specific traits. Thirdly, In our results, higher levels of genetic variation in proge- interploid crosses in our study and interspecific hybridization nies were found from the hybridization between 6x–4x and seem to be a good strategy to expand the gene pool and resist 6x–2x parents. In these hybrid crosses, the 6x parents were var. genetic erosion of kiwifruit. Tree Genetics & Genomes (2014) 10:1451–1463 1459

Conclusions diseases or climatic fluctuations. A different strategy such as interploid cross breeding confirmed in the present study is a Our AFLP analysis found a high level of genetic diversity promising way to enhance the genetic base of these kiwifruit (PPL=0.5822–0.6105) in all 79 cultivars investigated, includ- breeding pedigrees. Moreover, the conservation and utiliza- ing variable ploidy levels with diploids, tetraploids, and hexa- tion of Actinidia natural resources is critical for future kiwi- ploids. This suggested that the current kiwifruit industry has fruit industry development. already benefited from the abundant Actinidia wild resources and the direct selection strategies of kiwifruit breeding con- Acknowledgments We would like to thank Fei Han and Xiaoli Liu for help with crossing experiments and cultivation, and Siping Li for exper- ducted in the recent past. However, the clear genetic similar- iments. This project was funded by NSF of China grant (Y211121N01). ities based on ploidy levels and the uniformity of some sub- groups such as the red-fleshed kiwifruit pedigree, which were ‘ ’ Data archiving statement We followed the standard Tree Genetics and originally derived from the Hongyang cultivar suggest that Genomes policy. The marker information has been listed in Table 4 of the the current kiwifruit industry may be at risk in the face of Appendix

Appendix

Table 3 List of name, ploidy level, and the description of 79cultivars (selections) analyzed in this study

Cultivar name Ploidy Description levels

Hongyanga Diploid Female, red fleshb, selected from the natural resources of A. chinensis in Xixia County, Henan Province, China Wanhong Diploid Female, red fleshb, selected from the grafting plants of ‘Hongyang’ in Shanxi Province, China Oriental red Diploid Female, red fleshb, selected from the seedlings of ‘Hongyang’ in Wuhan Botanical Garden, Hubei Province, China Honghua Diploid Female, red fleshb, selected from the hybrids of red-fleshed germplasm in Sichuan Province, China Jinnonga Diploid Female, yellow flesh, selected from the natural resources of A. chinensis in Fang County, Hubei Province, China Jinyi Diploid Female, yellow green flesh, selected by Institute of Fruit and Tea, Hubei Academy of Agricultural Sciences, Hubei Province, China Chuanmi No3a Diploid Female, light yellow flesh, selected from the natural resources of A. chinensis in Henan Province, China Guihai No4a Diploid Female, green yellow flesh, selected from the natural resources of A. chinensis in Longsheng County, Guangxi Province, China Huangguang No2a Diploid Female, light yellow flesh, selected from the natural resources of A. chinensis in Xixia County, Henan Province, China Jinyu Diploid Female, golden yellow flesh, selected from the seedlings of ‘Hongyang’ in Wuhan Botanical Garden, Hubei Province, China Fengyuea Diploid Female, yellow flesh, selected from the natural resources of A. chinensis in Hunan Province, China Hort16A Diploid Female, yellow flesh, selected by Plant & Food Research, New Zealand Mantianhong Diploid Female, yellow flesh, selected from the seedlings of the open-pollinated fruit of A. eriantha in Wuhan Botanical Garden, Hubei Province, China Lvzhu Diploid Female, green flesh, selected from the interspecific hybrids in Wuhan Botanical Garden, Hubei Province, China Wuzhi No7 Diploid Female, yellow flesh, selected from the seedlings of A. chinensis in Wuhan Botanical Garden, Hubei Province, China Wzred-8 Diploid Female, red fleshb, selected from the red-fleshed germplasm in Wuhan Botanical Garden, Hubei Province, China Wzred-10 Diploid Female, red fleshb, selected from the red-fleshed germplasm in Wuhan Botanical Garden, Hubei Province, China Wzred-11 Diploid Female, red fleshb, selected from the red-fleshed germplasm in Wuhan Botanical Garden, Hubei Province, China Wuzhi No7-1 Diploid Female, yellow flesh, selected from the yellow-fleshed germplasm in Wuhan Botanical Garden, Hubei Province, China Guihai-M Diploid Male, pollinator, selected from the seedlings of ‘Guihai No4’ in Wuhan Botanical Garden, Hubei Province, China 1460 Tree Genetics & Genomes (2014) 10:1451–1463

Table 3 (continued)

Cultivar name Ploidy Description levels

Huangguang No2- Diploid Male, pollinator, selected from the seedlings of ‘Huangguang No2’in Wuhan Botanical Garden, Hubei M Province, China Hongyang-M Diploid Male, pollinator, selected from the seedlings of ‘Hongyang’ in Wuhan Botanical Garden, Hubei Province, China Wzp-M1 Diploid Male, pollinator, selected from the seedlings of A. chinensis in Wuhan Botanical Garden, Hubei Province, China Wzp-M2 Diploid Male, pollinator, selected from the seedlings of A. chinensis in Wuhan Botanical Garden, Hubei Province, China Hort16A-M1 Diploid Male, pollinator, selected from the seedlings of ‘Hort16A’ in Wuhan Botanical Garden, Hubei Province, China Hort16A-M2 Diploid Male, pollinator, selected from the seedlings of ‘Hort16A’ in Wuhan Botanical Garden, Hubei Province, China Hort16A-M3 Diploid Male, pollinator, selected from the seedlings of ‘Hort16A’ in Wuhan Botanical Garden, Hubei Province, China Hort16A-M4 Diploid Male, pollinator, selected from the seedlings of ‘Hort16A’ in Wuhan Botanical Garden, Hubei Province, China Hort16A-M5 Diploid Male, pollinator, selected from the seedlings of ‘Hort16A’ in Wuhan Botanical Garden, Hubei Province, China Jinzaoa Tetraploid Female, yellow flesh, selected from the natural resources in Wuning County, Jiangxi Province, China Wuzhi No3a Tetraploid Female, green flesh, selected from the natural resources in Wuning County, Jiangxi Province, China Jinxiaa Tetraploid Female, yellow or yellow-green flesh, selected from the natural resources in Wuning County, Jiangxi Province, China Lushanxianga Tetraploid Female, yellow flesh, selected from the natural resources in Wuning County, Jiangxi Province, China Jinyan Tetraploid Female, yellow flesh, selected from the hybrids of A. eriantha and A. chinensis in Wuhan Botanical Garden, Hubei Province, China Jinmei Tetraploid Female, yellow or yellow green flesh, selected from the hybrids of ‘Jinyan’ in Wuhan Botanical Garden, Hubei Province, China Jinyuan Tetraploid Female, golden yellow flesh, selected from the hybrids of ‘Jinyan’ in Wuhan Botanical Garden, Hubei Province, China Tongshan No5a Tetraploid Female, green-yellow flesh, selected from the natural resources in Tongshan County, Hubei Province, China Jinfenga Tetraploid Female, yellow flesh, selected from the natural resources in Fengxin County, Jiangxi Province, China Huaguang No3a Tetraploid Female, yellow flesh, selected from the natural resources in Henan Province, China Luoyang No1a Tetraploid Female, yellow flesh, selected from the natural resources of A. chinensis in Song County, Henan Province, China Zaoxiana Tetraploid Female, yellow or green-yellow flesh, selected from the natural resources in Jiangxi Province, China Kuimia Tetraploid Female, yellow or green-yellow flesh, selected from the natural resources in Fengxin County, Jiangxi Province, China Xiaya No15a Tetraploid Female, yellow-green flesh, selected from the natural resources of A. chinensis in Fujian Province, China Xiaya No1a Tetraploid Female, yellow-green flesh, selected from the natural resources of A. chinensis in Fujian Province, China Jintaoa Tetraploid Female, yellow flesh, selected from the natural resources of A. chinensis in Wuning County, Jiangxi Province, China Jianke No1a Tetraploid Female, light yellow flesh, selected from the natural resources of A. chinensis in Fujian Province, China Jinyanga Tetraploid Female, yellow flesh, selected from the natural resources of A. chinensis in Chongyang County, Hubei Province, China Cuiyua Tetraploid Female, green flesh, selected from the natural resources in Xupu County, Hunan Province, China Xiangma No6a Tetraploid Female, green flesh, selected from the natural resources in Mayang County, Hunan Province, China Chuhonga Tetraploid Female, red fleshc, selected from the natural resources in Mayang County, Hunan Province, China Qinyuanqiucuia Tetraploid Female, light yellow flesh, selected from the natural resources in Qingyuan County, Zhejiang Province, China Moshan N04a Tetraploid Male, pollinator, selected from the natural resources in Wuning County, Jiangxi Province, China Wzp-M5 Tetraploid Male, pollinator, selected from the seedlings of A. chinensis in Wuhan Botanical Garden, Hubei Province, China Sanxia-M Tetraploid Male, pollinator, selected from the seedlings of A. chinensis in Wuhan Botanical Garden, Hubei Province, China Qinyuanqiucui-M Tetraploid Male, pollinator, selected from the seedlings of A. chinensis in Wuhan Botanical Garden, Hubei Province, China Wzpred-M Tetraploid Male, pollinator, selected from the seedlings of the red-fleshed germplasm in Wuhan Botanical Garden, Hubei Province, China Wzpred-M2 Tetraploid Male, pollinator, selected from the seedlings of the red-fleshed germplasm in Wuhan Botanical Garden, Hubei Province, China Miliang No1a Hexaploid Female, green flesh, selected from the natural resources in Hunan Province, China KH-1 Hexaploid Female, green flesh, selected by Wuhan Botanical Garden, Hubei Province, China Qinmeia Hexaploid Female, light green flesh, selected from the natural resources of A. deliciosa in Zhouzhi County, Shannxi Province, China Xianglv Hexaploid Female, green flesh, selected from the seedlings of A. deliciosa in Japan Bruno Hexaploid Female, green flesh, selected from the seedlings of A. deliciosa in New Zealand Tree Genetics & Genomes (2014) 10:1451–1463 1461

Table 3 (continued)

Cultivar name Ploidy Description levels

Sanxia No1a Hexaploid Female, green flesh, selected from the natural resources of A. deliciosa in Xingshan County, Hubei Province, China Xixuan No1a Hexaploid Female, light yellow flesh, selected from the natural resources of A. deliciosa in Qinling Mountain, Shannxi Province, China Jinkuia Hexaploid Female, green flesh, selected from the natural resources of A. deliciosa in Zhuxi County, Hubei Province, China Chuanmi No1a Hexaploid Female, green flesh, selected from the natural resources of A. deliciosa by Sichuan Province Natural Resources Science Academy and Cangxi County Bureau of Agriculture, China Chuanmi No2a Hexaploid Female, green flesh, selected from the natural resources of A. deliciosa in Henan Province, China Xinguan No2a Hexaploid Female, green flesh, selected from the natural resources of A. deliciosa in Cangxi County, Sichuan Province, China Xuxiang Hexaploid Female, green flesh, selected from the seedlings of A. deliciosa in Jiangsu Province, China Changan No1 Hexaploid Female, green flesh, selected by Institute of Agricultural Sciences of Xian city, Shannxi Province, China Shixuan No2 Hexaploid Female, green flesh, selected from the seedlings of ‘Hayward’ in Jiangsu Province, China Hayward Hexaploid Female, green flesh, selected from the seedlings of A. deliciosa in New Zealand Huamei No1a Hexaploid Female, green flesh, selected from the natural resources of A. deliciosa in Henan Province, China Xuguan Hexaploid Female, green flesh, selected from the seedlings of ‘Hayward’ in Jiangsu Province, China Qinxiang# Hexaploid Female, green flesh, selected from the natural resources of A. deliciosa in Hunan Province, China Xuxiang-M Hexaploid Male, pollinator, selected by Wuhan Botanical Garden, Hubei Province, China Jinkui-M Hexaploid Male, pollinator, selected by Wuhan Botanical Garden, Hubei Province, China Jinkui-M2 Hexaploid Male, pollinator, selected by Wuhan Botanical Garden, Hubei Province, China Jinkui-M3 Hexaploid Male, pollinator, selected by Wuhan Botanical Garden, Hubei Province, China a Cultivars directly selected from the natural resources b The fruit flesh color is yellow or yellow-green with brilliant red around the central core c The fruit flesh color is green with brilliant red around the central core

Table 4 Sequences of adapters and primers used for the AFLP Code Sequence reactions Adaptors: EcoRI Adaptors: 5′-CTCGTAGACTGCGTACC-3′ MseI adaptors 5′-GACGATGAGTCCTGAG-3′ Preselective amplification primers: EcoRI primer 5′-GACTGCGTACCAATTC-3′ MseI primer 5′-GATGAGTCCTGAGTAA-3′ Selective amplification primer pairs: No.1 5′-GAC TGC GTA CCA ATTC AAC-3′ 5′-GAT GAG TCC TGA GTAA CTG-3′ No.2 5′-GAC TGC GTA CCA ATTC AGC-3′ 5′-GAT GAG TCC TGA GTAACTG-3′ No.3 5′-GAC TGC GTA CCA ATTC AGG-3′ 5′-GAT GAG TCC TGA GTAA CAA-3′ No.4 5′-GAC TGC GTA CCA ATTC AGC-3′ 5′-GAT GAG TCC TGA GTAA CAC-3′ No.5 5′-GAC TGC GTA CCA ATTC ACC-3′ 5′-GAT GAG TCC TGA GTAA CTA-3′ No.6 5′-GAC TGC GTA CCA ATTC AGG-3′ 5′-GAT GAG TCC TGA GTAA CTA-3′ No.7 5′-GAC TGC GTA CCA ATTC ACT-3′ 5′-GAT GAG TCC TGA GTAA CAT-3′ No.8 5′-GAC TGC GTA CCA ATTC AAC-3′ 5′-GAT GAG TCC TGA GTAA CCA-3′ No.9 5′-GAC TGC GTA CCA ATTC ACA-3′ 5′-GAT GAG TCC TGA GTAA CGA-3′ No.10 5′-GAC TGC GTA CCA ATTC AGC-3′ 5′-GAT GAG TCC TGA GTAA CGA-3′ No.11 5′-GAC TGC GTA CCA ATTC AAC-3′ 5′-GAT GAG TCC TGA GTAA CGA-3′ 1462 Tree Genetics & Genomes (2014) 10:1451–1463

Table 5 Degree of polymorphism and quality of AFLP data generated from combinations of 11 primers

AFLP analysis Primer combinations applied to 79 cultivars Primer combinations applied to 122 samples from 6 interploidy crosses

No.1a No.2 No.3 No.4 No.5 No.6 No.7 No.8 No.4 No.5 No.6 No.7 No.8 No.9 No.10 No.11

TNB 124.00 108.00 136.00 116.00 137.00 125.00 137.00 124.00 85.00 98.00 88.00 103.00 94.00 96.00 95.00 99.00 PPB 0.97 0.94 0.95 0.95 0.98 0.96 0.97 0.96 0.93 0.90 0.96 0.94 0.90 0.94 0.92 0.95 PIC 0.24 0.36 0.28 0.33 0.21 0.31 0.24 0.24 0.36 0.44 0.44 0.48 0.26 0.52 0.44 0.45 MI 29.24 36.55 35.85 36.10 27.91 37.33 32.31 28.94 28.46 38.59 36.96 46.63 22.03 47.28 38.24 42.55

TNB total number of bands, PPB percentage of polymorphic bands, PIC polymorphic information content, MI marker index a Please see Table 4 of the Appendix for code of 11 primer pairs

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