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 strate- gies 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 plas- ticity (Lafitte and Courtois 2002; Zhe et al. 2010). Most Plant material and DNA extraction molecular markers are comparatively independent of environ- mental 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