, Volume 175, Issue 1, pp 109–121 | Cite as

Identification and sequence characterisation of molecular markers polymorphic between male kiwifruit (Actinidia chinensis var. deliciosa (A. Chev.) A. Chev.) accessions exhibiting different flowering time

  • Marina Novo
  • Silvia Romo
  • Manuel Rey
  • María Jesús Prado
  • María Victoria González


Fruit set in kiwifruit is strongly dependent on pollination, which is limited by the lack of efficient male pollen donors, among other factors. We searched for molecular markers that could be polymorphic in relation to flowering time in order to classify male kiwifruit plants to discard those that are not likely to perform as efficient pollen donors. Random amplified polymorphic DNA (RAPD) and modified amplified fragment length polymorphism (AFLP) markers were generated using 41 male kiwifruit plants in two flowering groups, early- and late-flowering males (with respect to the female cultivar ‘Hayward’). One RAPD and nine modified-AFLP markers polymorphic between male plants exhibiting different flowering time were identified, sequenced and analysed in databases. Unweighted pair group method with arithmetic average (UPGMA) clustering and multidimensional scaling showed that these markers could be used to classify the male plants into flowering groups. Analysis of molecular variance (AMOVA) agreed with this classification, showing that most of the genetic variation is found between flowering groups. Sequence analysis based on a database search revealed that the polymorphism PolM contains a 7-nucleotide long element involved in the repression of the phytochrome A gene, that Pol4 is a partial sequence of a phytochrome B gene, and that sequences Pol3, Pol5, Pol7, and Pol9 show high identity with ESTs from kiwifruit buds treated with hydrogen cyanamide. Clustering analysis supported the previous classification of males into flowering groups, making it feasible to predict male plants’ flowering times with respect to the cultivar ‘Hayward’ based upon these molecular markers.


Kiwifruit Flowering time Molecular markers RAPD Modified-AFLP Marker assisted selection 



Marina Novo and Silvia Romo contributed equally to this work. This is a contribution of the Interuniversity Research Group in Biotechnology and Reproductive Biology of Woody Plants. This research was supported by the Xunta de Galicia (Regional Government of Galicia, project PGIDIT04RAG291002PR). We thank SERIDA (Regional Government of the Principado de Asturias) and Kiwi Atlantico S.A. for providing the plant material, Drs. E. Labrador and B. Dopico (Univ. Salamanca, Spain) for letting us use their laboratory facilities, and Verónica López for her technical assistance. We also wish to thank Dr. Pablo Presa (Univ. Vigo, Spain) for his help with the AMOVA analysis.


  1. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215:403–410PubMedGoogle Scholar
  2. Bañuelos GR, Argumedo R, Patel K, Ng V, Zhou F, Vellanoweth RL (2008) The developmental transition to flowering in Arabidopsis is associated with an increase in leaf chloroplastic lipoxygenase activity. Plant Sci 174:366–373. doi: 10.1016/j.plantsci.2007.12.009 CrossRefPubMedGoogle Scholar
  3. Böhlenius H, Huang T, Charbonnel-Campaa L, Brunner AM, Jansson S, Strauss SH, Nilsson O (2007) CO/FT regulatory module controls timing of flowering and seasonal growth cessation in trees. Science 312:1040–1043. doi: 10.1126/science.1126038 CrossRefGoogle Scholar
  4. Brendel V, Xing L, Zhu W (2004) Gene structure prediction from consensus spliced alignment of multiple ESTs matching the same genomic locus. Bioinformatics 20:1157–1169. doi: 10.1093/bioinformatics/bth058 CrossRefPubMedGoogle Scholar
  5. Bruce MB, Deng XW, Quail PH (1991) A negatively acting DNA sequence element mediates phytochrome-directed regression of phyA gene transcription. EMBO J 10:3015–3024PubMedGoogle Scholar
  6. Cipriani G, Di Bella R, Testolin R (1996) Screening RAPD primers for molecular taxonomy and cultivar fingerprinting in the genus Actinidia. Euphytica 90:169–174. doi: 10.1007/BF00023855 CrossRefGoogle Scholar
  7. Cipriani G, Testolin R, Gardner R (1998) Restriction-site variation of PCR-amplified chloroplast DNA regions and its implication for the evolution and taxonomy of Actinidia. Theor Appl Genet 96:389–396. doi: 10.1007/s001220050754 CrossRefGoogle Scholar
  8. Crowhurst RN, Gleave AP, MacRae EA et al (2008) Analysis of expressed sequence tags from Actinidia: applications of a cross species EST database for gene discovery in the areas of flavor, health, color and ripening. BMC Genomics 9:351. doi: 10.1186/1471-2164-9-351 CrossRefPubMedGoogle Scholar
  9. Cutler RW, Chundet R, Handa T, Anuntalabhochai S (2006) Development of sequence characterized DNA markers linked to a temperate dependence for flower induction in lychee (Litchi chinensis Sonn.) cultivars. Sci Hortic 107:264–270. doi: 10.1016/j.scienta.2005.08.005 CrossRefGoogle Scholar
  10. Excoffier L, Smouse PE, Quattro JM (1992) Analysis of molecular variance inferred from metric distances among DNA haplotypes: application to human mitochondrial DNA restriction data. Genetics 131:479–491PubMedGoogle Scholar
  11. Excoffier L, Laval G, Schneider S (2005) Arlequin ver. 3.0: an integrated software package for population genetics data analysis. Evol Bioinform Online 1:47–50PubMedGoogle Scholar
  12. Ferguson AR (2007) The need for characterisation and evaluation of germplasm: kiwifruit as an example. Euphytica 154:371–382. doi: 10.1007/s10681-006-9188-2 CrossRefGoogle Scholar
  13. Fraser LG, Harvey CF, Crowhurst RN, De Silva HN (2004) EST-derived microsatellites from Actinidia species and their potential for mapping. Theor Appl Genet 108:1010–1016. doi: 10.1007/s00122-003-1517-4 CrossRefPubMedGoogle Scholar
  14. Fraser LG, McNeilage MA, Tsang GK, Harvey CF, De Silva HN (2005) Cross-species amplification of microsatellite loci within the dioecious, polyploid genus Actinidia (Actinidiaceae). Theor Appl Genet 112:149–157. doi: 10.1007/s00122-005-0117-x CrossRefPubMedGoogle Scholar
  15. Fraser LG, Tsang GK, Datson PM, De Silva HN, Harvey CF, Gill GP, Crowhurst RN, McNeilage MA (2009) A gene-rich linkage map in the dioecious species Actinidia chinensis (kiwifruit) reveals putative X/Y sex-determining chromosomes. BMC Genomics 10:102. doi: 10.1186/1471-2164-10-102 CrossRefPubMedGoogle Scholar
  16. Gill GP, Harvey CF, Gardner RC, Fraser LG (1998) Development of sex-linked PCR markers for gender identification in Actinidia. Theor Appl Genet 97:439–445. doi: 10.1007/s001220050914 CrossRefGoogle Scholar
  17. González MV, Coque M, Herrero M (1994) Pollinator selection in kiwifruit (Actinidia deliciosa). J Hortic Sci 70:375–387Google Scholar
  18. González MV, Coque M, Herrero M (1995a) Stigmatic receptivity limits the effective pollination period in kiwifruit. J Am Soc Hortic Sci 120:199–202Google Scholar
  19. González MV, Coque M, Herrero M (1995b) Papillar integrity as an indicator of stigmatic receptivity in kiwifruit (Actinidia deliciosa). J Exp Bot 46:263–269CrossRefGoogle Scholar
  20. Harvey CF, Gill GP, Fraser LG, McNeilage MA (1997) Sex determination in Actinidia. 1. Sex-linked markers and progeny sex ratio in diploid A. chinensis. Sex Plant Reprod 10:149–154. doi: 10.1007/s004970050082 CrossRefGoogle Scholar
  21. Huang W-G, Cipriani G, Morgante M, Testolin R (1998) Microsatellite DNA in Actinidia chinensis: isolation, characterisation, and homology in related species. Theor Appl Genet 97:1269–1278. doi: 10.1007/s001220051019 CrossRefGoogle Scholar
  22. Huang H, Li Z, Li J, Kubisiak TL, Layne DR (2002) Phylogenetic relationships in Actinidia as revealed by RAPD analysis. J Am Soc Hortic Sci 127:759–766Google Scholar
  23. Kobayashi S, Goto-Yamamoto N, Hirochika H (2004) Retrotransposon-induced mutations in grape skin color. Science 304:982. doi: 10.1126/science.1095011 CrossRefPubMedGoogle Scholar
  24. Korkovelos AE, Mavromatis AG, Huang WG, Hagidimitriou M, Giakoundis A, Goulas CK (2008) Effectiveness of SSR molecular markers in evaluating the phylogenetic relationships among eight Actinidia species. Sci Hortic 116:305–310. doi: 10.1016/j.scienta.200801.011 CrossRefGoogle Scholar
  25. Kumar A, Hirochika H (2001) Applications of retrotransposons as genetic tools in plant biology. Trends Plant Sci 6:127–134. doi: 10.1016/S1360-1385(00)01860-4 CrossRefPubMedGoogle Scholar
  26. Lin C (2000) Photoreceptors and regulation of flowering time. Plant Physiol 123:39–50CrossRefPubMedGoogle Scholar
  27. McPherson HG, Richardson AC, Snelgar WP, Currie MB (2001) Effects of hydrogen cyanamide on budbreak and flowering in kiwifruit (Actinidia deliciosa ‘Hayward’). N Z J Crop Hortic Sci 29:277–285Google Scholar
  28. Mockler T, Yang H, Yu X, Parikh D, Cheng Y, Dolan S, Lin C (2003) Regulation of photoperiodic flowering by Arabidopsis photoreceptors. Proc Natl Acad Sci USA 100:2140–2145. doi: 10.1073/pnas.0437826100 CrossRefPubMedGoogle Scholar
  29. Negi MS, Devic M, Delseny M, Lakshmikumaran M (2000) Identification of AFLP fragments linked to seed coat colour in Brassica juncea and conversion to a SCAR marker for rapid selection. Theor Appl Genet 101:281–285. doi: 10.1007/s001220051463 CrossRefGoogle Scholar
  30. Noguera FJ, Capel J, Alvarez JI, Lozano R (2005) Development and mapping of a codominant SCAR marker linked to the andromonoecious gene of melon. Theor Appl Genet 110:714–720. doi: 10.1007/s00122-004-1897-0 CrossRefPubMedGoogle Scholar
  31. Nybom H, Hall HK (1991) Minisatellite DNA ‘fingerprints’ can distinguish Rubus cultivars and estimate their degree of relatedness. Euphytica 53:107–114. doi: 10.1007/BF00023790 CrossRefGoogle Scholar
  32. Palombi MA, Damiano C (2002) Comparison between RAPD and SSR molecular markers in detecting genetic variation in kiwifruit (Actinidia deliciosa A. Chev.). Plant Cell Rep 20:1061–1066. doi: 10.1007/s00299-001-0430-z CrossRefGoogle Scholar
  33. Pearson WR, Lipman DJ (1988) Improved tools for biological sequence analysis. Proc Natl Acad Sci USA 85:2444–2448CrossRefPubMedGoogle Scholar
  34. Prado MJ, Herrera MT, Vazquez RA, Romo S, González MV (2005) Micropropagation of two selected male kiwifruit and analysis of genetic variation with AFLP markers. HortScience 40:740–746Google Scholar
  35. Prado MJ, Romo S, Novo M, Rey M, Herrera MT, González MV (2006) Molecular characterization of three commercial cultivars and a new pollinator in kiwifruit. HortScience 41:90–95Google Scholar
  36. Prado MJ, González MV, Romo S, Herrera MT (2007) Adventitious plant regeneration on leaf explants from adult male kiwifruit and AFLP analysis of genetic variation. Plant Cell Tissue Organ Cult 88:1–10. doi: 10.1007/s11240-006-9116-0 CrossRefGoogle Scholar
  37. Ranamukhaarachchi DG, Kane ME, Guy CL, Li KB (2000) Modified AFLP technique for rapid genetic characterization in plants. BioTechniques 29:858–866PubMedGoogle Scholar
  38. Reed JW, Nagatani A, Elich TD, Fagan M, Chory J (1994) Phytochrome A and phytochrome B have overlapping but distinct functions in Arabidopsis development. Plant Physiol 104:1139–1149PubMedGoogle Scholar
  39. Shirkot P, Sharma DR, Mohapatra T (2002) Molecular identification of sex in Actinidia deliciosa var. deliciosa by RAPD markers. Sci Hortic 94:33–39. doi: 10.1016/S0304-4238(01)00357-0 CrossRefGoogle Scholar
  40. Sneath PHA, Sokal RR (1973) Numerical taxonomy. The principles and practice of numerical classification. WH Freeman, San FranciscoGoogle Scholar
  41. Testolin R, Cipriani G, Gottardo L, Costa G (1995) Valutazione de selezione maschili di actinidia come impollinatori per la cv. ‘Hayward’. Riv Fruttic Ortofloric 57:63–68Google Scholar
  42. Testolin R, Huang WG, Laín O, Messina R, Vecchione A, Cipriani G (2001) A kiwifruit (Actinidia spp.) linkage map based on microsatellites and integrated with AFLP markers. Theor Appl Genet 103:30–36. doi: 10.1007/s00122-001-0555-z CrossRefGoogle Scholar
  43. Thorp TG, Zhang J, Lay-Yee M (1990) Horticultural characteristics of seven pistillate and three staminate New Zealand cultivars of kiwifruit. N Z J Crop Hortic Sci 18:233–240Google Scholar
  44. Urusaki N, Tokumoto M, Tarora K, Ban Y, Kayano T, Tanaka H, Oku H, Chinen I, Terauchi R (2002) A male and hermaphrodite specific RAPD marker for papaya (Carica papaya L.). Theor Appl Genet 104:281–285. doi: 10.1007/s001220100693 CrossRefGoogle Scholar
  45. Valverde F, Mouradov A, Soppe W, Ravenscroft D, Samach A, Coupland G (2004) Photoreceptor regulation of CONSTANS protein in photoperiodic flowering. Science 303:1003–1006. doi: 10.1126/science.1091761 CrossRefPubMedGoogle Scholar
  46. Walton EF, Fowke PJ (1993) Effect of hydrogen cyanamide on kiwifruit shoot flower number and position. J Hortic Sci 68:529–534Google Scholar
  47. Walton EF, Podivinsky E, Wu R-M (2001) Bimodal patterns of floral gene expression over the two seasons that kiwifruit flowers develop. Physiol Plant 111:396–404. doi: 10.1111/j.1399-3054.2001.1110318.x CrossRefPubMedGoogle Scholar
  48. Weising K, Fung RWM, Keeling DJ, Atkinson RG, Gardner RC (1996) Characterisation of microsatellites from Actinidia chinensis. Mol Breed 2:117–131. doi: 10.1007/BF00441427 CrossRefGoogle Scholar
  49. Wingender E, Chen X, Hehl R, Karas H, Liebich I, Matys V, Meinhardt T, Prüß M, Reuter I, Schacherer F (2000) TRANSFAC: an integrated system for gene expression regulation. Nucleic Acids Res 28:316–319CrossRefPubMedGoogle Scholar
  50. Xu W-J, Wang B-W, Cui K-M (2004) RAPD and SCAR markers linked to sex determination in Eucommia ulmoides Oliv. Euphytica 136:233–238. doi: 10.1023/B:EUPH.0000032741.99885.c4 CrossRefGoogle Scholar
  51. Yakubov B, Barazani O, Golan-Goldhirsh A (2005) Combination of SCAR primers and touchdown-PCR for sex identification in Pistacia vera L. Sci Hortic 103:473–478. doi: 10.1016/j.scienta.2004.06.008 CrossRefGoogle Scholar
  52. Ye Z, Rodríguez R, Tran A, Hoang H, de los Santos D, Brown S, Vellanoweth RL (2000) The developmental transition to flowering represses ascorbate peroxidase activity and induces enzymatic lipid peroxidation in leaf tissue in Arabidopsis thaliana. Plant Sci 158:115–127. doi: 10.1016/S0168-9452(00)00316-2 CrossRefPubMedGoogle Scholar
  53. Yu K, Pauls KP (1994) Optimization of DNA-extraction and PCR procedures for random amplified polymorphic DNA (RAPD) analysis in plants. In: Griffin HG, Griffin AM (eds) PCR technology: current innovations. CRC Press Inc, Boca Raton, pp 93–100Google Scholar
  54. Zhang J, Thorp TG (1986) Morphology of nine pistillate and three staminate New Zealand clones of kiwifruit (Actinidia deliciosa (Chev.) Liang and Ferguson var. deliciosa). N Z J Bot 24:589–613Google Scholar
  55. Zhen Y, Li Z, Huang H (2004) Molecular characterization of kiwifruit (Actinidia) cultivars and selections using SSR markers. J Am Soc Hortic Sci 129:374–382Google Scholar

Copyright information

© Springer Science+Business Media B.V. 2010

Authors and Affiliations

  • Marina Novo
    • 1
  • Silvia Romo
    • 1
  • Manuel Rey
    • 2
  • María Jesús Prado
    • 2
  • María Victoria González
    • 1
  1. 1.Departamento de Fisiología VegetalUniversidad de Santiago de CompostelaSantiago de CompostelaSpain
  2. 2.Departamento de Biología Vegetal y Ciencia del SueloUniversidad de VigoVigoSpain

Personalised recommendations