Gynoecy instability in cucumber (Cucumis sativus L.) is due to unequal crossover at the copy number variation-dependent Femaleness (F) locus

Abstract

Cucumber, Cucumis sativus is an important vegetable crop, and gynoecy has played a critical role in yield increase of hybrid cucumber production. Cucumber has a unique genetic system for gynoecious sex expression, which is determined by the copy number variation (CNV)-based, dominant, and dosage-dependent femaleness (F) locus. However, this gynoecy expression system seems unstable since monecious plants could often be found in F-dependent gynoecious cucumber inbreds. We hypothesized that gynoecy instability (gynoecy loss) may be due to unequal crossing over (UCO) during meiosis among repeat units of the CNV. In this study, using high throughput genome resequencing, fiber-FISH and genomic qPCR analyses, we first confirmed and refined the structure of the F locus, which was a CNV of a 30.2-kb tandem repeat. Gynoecious plants contained three genes: CsACS1, CsACS1G, and CsMYB, of which CsACS1G is a duplication of CsACS1 but with a recombinant distal promoter that may contribute to gynoecy sex expression. In two large populations from self-pollinated gynoecious inbred lines, ‘gynoecy loss’ mutants were identified with similar mutation rates (~0.12%). We show that these monecious mutants have lost CsACS1G. In addition, we identified gynoecious lines in natural populations that carry two copies of CSACS1G. We proposed a model to explain gynoecy instability in F-dependent cucumbers, which is caused by UCO among CSACS1/G units during meiosis. The findings present a convincing case that the phenotypic variation of an economically important trait is associated with the dynamic changes of copy numbers at the F locus. This work also has important implications in cucumber breeding.

References

[1] Cronquist, A . The Evolution and Classification of Flowering Plants (New York Botanical Gardens, Bronx, New York, 1988).

[2] Chuck, G. Molecular mechanisms of sex determination in monoecious and dioecious plants. Adv. Bot. Res. 54, 54-83 (2010).

[3] Schaefer, H. & Renner, S. S. Cucurbitaceae. In Families and Genera of Flowering Plants, Vol. 10 (ed. Kubitzki, K.) 112-174 (Springer Verlag, Berlin, Germany, 2011).

[4] Renner, S. S. Pathways for making unisexual flowers and unisexual plants: moving beyond the “two mutations linked on one chromosome” model. Am. J. Bot. 103, 587-589 (2016).

[5] Correns, C. Bestimmung. Vererbung und Verteilung des Geschlechtes bei den hoheren Pflanzen. Handbuch der Vererbungswissenschaft, 3(II, C.) Band II (English: Determination, Inheritance and Distribution of Sex in Higher Plants. Handbook of Inheritance Science, 3 (II, C) Vol. II (Borntraeger, 1928).

[6] Galun, E. Study of the inheritance of sex expression in the cucumber: the interaction of major genes with modifying genetic and non-genetic factors. Genetica 32, 134-163 (1961).

[7] Kater, M. M., Franken, J., Carney, K. J., Colombo, L. & Angenent, G. C. Sex determination in the monoecious species cucumber is confined to specific floral whorls. Plant Cell 12, 481-494 (2001).

[8] Hao, Y. et al. DNA damage in the early primordial anther is closely correlated with stamen arrest in the female flower of cucumber (Cucumis sativus L.) . Planta 217, 888-895 (2003).

[9] Bai, S. et al. Developmental analyses reveal early arrests of the spore-bearing parts of reproductive organs in unisexual flowers of cucumber (Cucumis sativus L.) . Planta 220, 230-240 (2004).

[10] Rosa, J. T. The inheritance of flower types in Cucumis and Citrullus . Hilgardia 3, 235-250 (1928).

[11] Whitaker, T. W. Sex ratio and sex expression in the cultivated cucurbits. Am. J. Bot. 18, 359-366 (1930).

[12] Tkachenko, N. N. Preliminary results of a genetic investigation of the cucumber, Cucumis sativus L. Bull. Appl. Plant Breed. 9, 311-356 (1935).

[13] Shifriss, O. Sex control in cucumber. J. Hered. 52, 5-12 (1961).

[14] Kubicki, B. Investigations of sex determination in cucumber (Cucumis sativus L.). IV. Multiple alleles of locus . Genet. Pol. 10, 23-68 (1969).

[15] Kubicki, B. Investigations of sex determination in cucumber (Cucumis sativus L.). V. Genes controlling intensity of femaleness . Genet. Pol. 10, 69-86 (1969).

[16] Kubicki, B. Investigations on sex determination in cucumbers (Cucumis sativus L.). VI. Androecism . Genet. Pol. 10, 87-99 (1969).

[17] Malepszy, S. & Niemirowicz-Szczytt, K. Sex determination in cucumber (Cucumis sativus) as a model system for molecular biology . Plant Sci. 80, 39-47 (1991).

[18] Chen, H. et al. An ACC oxidase gene essential for cucumber carpel development. Mol. Plant 9, 1315-1327 (2016).

[19] Bu, F. et al. A major quantitative trait locus conferring subgynoecy in cucumber. Theor. Appl. Genet. 129, 97-104 (2016).

[20] Win, K. T. et al. Identification of quantitative trait loci governing subgynoecy in cucumber. Theor. Appl. Genet. 132, 1505-1521 (2019).

[21] Pawełkowicz, M. E., Skarzyńska, A., Pląder, W. & Przybecki, Z. Genetic and molecular bases of cucumber (Cucumis sativus L.) sex determination . Mol. Breed. 39, 50 (2019).

[22] Li, D., Sheng, Y., Niu, H. & Li, Z. Gene interactions regulating sex determination in Cucurbits. Front. Plant Sci. 10, 1231 (2019).

[23] Shifriss, O., George, W. L. & Quinones, J. A. Gynodioecism in cucumber. Genetics 49, 285-291 (1964).

[24] Ito, H. & Kato, T. Factors responsible for the sex expression of Japanese cucumbers. Hort. Assoc. Jpn. J. 22, 138-144 (1953).

[25] Peterson, C. E. A gynoecious inbred line of cucumber. Quart. Bull. Mich. Agric. Exp. Stat. 3, 40-42 (1960).

[26] Peterson, C. E. & Anhder, L. D. Induction of staminate flowers on gynoecious cucumbers with gibberellin A3. Science 131, 1673-1674 (1960).

[27] Wehner, T. C. & Miller, C. H. Effect of gynoecious expression on yield and earliness of a fresh-market cucumber hybrid. J. Am. Soc. Hort. Sci. 110, 464-466 (1985).

[28] Staub, J. E., Robbins, M. D. & Wehner, T. C. Cucumber. in Vegetables I. Handbook of Plant Breeding, Vol. 1 (eds. Prohens, J. & Nuez, F.) (Springer, New York, NY, 2008).

[29] Rudich, J., Halevy, A. & Kedar, N. Ethylene evolution from cucumber plants as related to sex expression. Plant Physiol. 49, 998-999 (1972).

[30] Takahashi, H. & Jaffe, M. Further studies of auxin and ACC induced feminization in the cucumber plant using ethylene inhibitors. Phyton 44, 81-86 (1984).

[31] Yin, T. J. & Quinn, J. A. Tests of a mechanistic model of one hormone regulating both Sexes in Cucumis sativus (Cucurbitaceae) . Am. J. Bot. 82, 1537-1546 (1995).

[32] Yamasaki, S., Fujii, N. & Takahashi, H. Characterization of ethylene effects on sex determination in cucumber plants. Sex. Plant Reprod. 16, 103-111 (2003).

[33] Atsmon, D. & Tabbak, C. Comparative effects of gibberellin, silver nitrate and aminoethoxyvinyl glycine on sexual tendency and ethylene evolution in cucumber plant (Cucumis sativus L.) . Plant Cell Physiol. 20, 1547-1555 (1979).

[34] Trebitsh, T., Staub, J. E. & O’Neill, S. D. Identification of a 1-aminocyclopropane-1-carboxylic acid synthase gene linked to the female (F) locus that enhances female sex expression in cucumber . Plant Physiol. 113, 987-995 (1997).

[35] Mibus, H. & Tatlioglu, E. T. Molecular characterization and isolation of the F/f gene for femaleness in cucumber (Cucumis sativus L.) . Theor. Appl. Genet. 109, 1669-1676 (2004).

[36] Li, Z. et al. Molecular isolation of the M gene suggests that a conserved-residue conversion induces the formation of bisexual flowers in cucumber plants . Genetics 182, 1381-1385 (2009).

[37] Boualem, A. et al. A cucurbit androecy gene reveals how unisexual flowers develop and dioecy emerges. Science 350, 688-691 (2015).

[38] Knopf, R. R. & Trebitsh, T. The female-specific CS-ACS1G gene of cucumber. A case of gene duplication and recombination between the non-sex-specific 1-aminocyclopropane-1-carboxylate synthase gene and a branched-chain amino acid transaminase gene . Plant Cell Physiol. 47, 1217-1228 (2006).

[39] Zhang, Z. et al. Genome-wide mapping of structural variations reveals a copy number variant that determines reproductive morphology in cucumber. Plant Cell 27, 1595-1604 (2015).

[40] Zmienko, A., Samelak, A., Kozlowski, P. & Figlerowicz, M. Copy number polymorphism in plant genomes. Theor. Appl. Genet. 127, 1-18 (2014).

[41] Hollox, E. J. et al. Psoriasis is associated with increased beta-defensin genomic copy number. Nat. Genet. 40, 23-25 (2008).

[42] Conrad, D. F. et al. Origins and functional impact of copy number variation in the human genome. Nature 464, 704-712 (2010).

[43] Weischenfeldt, J., Symmons, O., Spitz, F . & Korbel, J. O. Phenotypic impact of genomic structural variation: insights from and for human disease. Nat. Rev. Genet. 14, 125-138 (2013).

[44] Díaz, A., Zikhali, M., Turner, A. S., Isaac, P. & Laurie, D. A. Copy number variation affecting the photoperiod-B1 and vernalization-A1 genes is associated with altered flowering time in wheat (Triticum aestivum) . PLoS ONE 7, e33234 (2012).

[45] Würschum, T., Boeven, P. H., Langer, S. M., Longin, C. F. & Leiser, W. L. Multiply to conquer: copy number variations at Ppd-B1 and Vrn-A1 facilitate global adaptation in wheat . BMC Genet. 16, 96 (2015).

[46] Theißen, G., Rümpler, F. & Gramzow, L. Array of MADS-Box genes: facilitator for rapid adaptation? Trends Plant Sci. 23, 563-576 (2018).

[47] Cook, D. E. et al. Copy number variation of multiple genes at Rhg1 mediates nematode resistance in soybean . Science 338, 1206-1209 (2012).

[48] Cook, D. E. et al. Distinct copy number, coding sequence and locus methylation patterns underlie Rhg1-mediated soybean resistance to soybean cyst nematode . Plant Physiol. 165, 630-647 (2014).

[49] Lee, T. G., Kumar, I., Diers, B. W. & Hudson, M. E. Evolution and selection of Rhg1, a copy-number variant nematode-resistance locus . Mol. Ecol. 24, 1774-1791 (2015).

[50] Lee, T. G., Diers, B. W. & Hudson, M. E. An efficient method for measuring copy number variation applied to improvement of nematode resistance in soybean. Plant J. 88, 143-153 (2016).

[51] Wang, Y. et al. Copy number variation at the GL7 locus contributes to grain size diversity in rice . Nat. Genet. 47, 944-948 (2015).

[52] Sutton, T. et al. Boron-toxicity tolerance in barley arising from efflux transporter amplification. Science 318, 1446-1449 (2007).

[53] Maron, L. G. et al. Aluminum tolerance in maize is associated with higher MATE1 gene copy number . Proc. Natl Acad. Sci. USA 110, 5241-5246 (2013).

[54] Gaines, T. A. et al. Mechanism of resistance of evolved glyphosate-resistant palmer amaranth (Amaranthus palmeri) . J. Agric. Food Chem. 59, 5886-5889 (2011).

[55] Gaines, T. A. et al. Gene amplification confers glyphosate resistance in Amaranthus palmeri . Proc. Natl Acad. Sci. USA 107, 1029-1034 (2010).

[56] Caicedo, A. L., Richards, C., Ehrenreich, I. M. & Purugganan, M. D. Complex rearrangements lead to novel chimeric gene fusion polymorphisms at the Arabidopsis thaliana MAF2-5 flowering time gene cluster . Mol. Biol. Evol. 26, 699-711 (2009).

[57] Magadum, S., Banerjee, U., Murugan, P., Gangapur, D. & Ravikesavan, R. Gene duplication as a major force in evolution. J. Genet. 92, 155-161 (2013).

[58] Lye, Z. N. & Purugganan, M. D. Copy number variation in domestication. Trends Plant Sci. 24, 352-366 (2019).

[59] de Bolt, S. Copy number variation shapes genome diversity in Arabidopsis over immediate family generational scales . Genome Biol. Evol. 2, 441-453 (2010).

[60] Kondrashov, F. A. Gene duplication as a mechanism of genomic adaptation to a changing environment. Proc. R. Soc. B 279, 5048-5057 (2012).

[61] Lu, P. et al. Analysis of Arabidopsis genome-wide variations before and after meiosis and meiotic recombination by resequencing Landsberg erecta and all four products of a single meiosis . Genome Res. 22, 508-518 (2012).

[62] Osipowski, P. et al. A high-quality cucumber genome assembly enhances computational comparative genomics. Mol. Genet. Genomics https://doi.org/10.1007/s00438-019-01614-3 (2019).

[63] Hastings, P. J., Lupski, J. R., Rosenberg, S. M. & Ira, G. Mechanisms of change in gene copy number. Nat. Rev. Genet. 10, 551-564 (2009).

[64] Li, Q. et al. A chromosome-scale genome assembly of cucumber (Cucumis sativus L.) . Gigascience 8, 1-10 (2019).

[65] Tao, Q. et al. Ethylene responsive factor ERF110 mediates ethylene-regulated transcription of a sex determination-related orthologous gene in two Cucumis species . J. Exp. Bot. 69, 2953-2965 (2018).

[66] Martin, A. et al. A transposon-induced epigenetic change leads to sex determination in melon. Nature 461, 1135-1138 (2009).

[67] Zhang, J. et al. A unique chromosome translocation disrupting ClWIP1 leads to gynoecy in watermelon . Plant J. https://doi.org/10.1111/tpj.14537 (2019).

[68] Hu, B. et al. Engineering non-transgenic gynoecious cucumber using an improved transformation protocol and optimized CRISPR/Cas9 system. Mol. Plant 10, 1575-1578 (2017).

[69] Shiomi, S. et al. cDNA cloning of ACC synthase and ACC oxidase genes in cucumber fruit and their differential expression by wounding and auxin. J. Jpn. Soc. Hort. Sci. 67, 685-692 (1998).

[70] Mathooko, F. M. et al. Expression characteristics of CSACS1, CS-ACS2 and CS-ACS3, three members of the 1-aminocyclopropane-1-carboxylate synthase gene family in cucumber (Cucumis sativus L.) fruit under carbon dioxide stress . Plant Cell Physiol. 40, 164-172 (1999).

[71] Shiber et al. The origin and mode of function of the Female locus in cucumber. In Cucurbitaceae 2008, Proc. 9th EUCARPIA Meeting on Genetics and Breeding of Cucurbitaceae, May 21-24, 2008, France (ed. Pitrat, M.) 263-270 (INRA, Avignon, 2008).

[72] Kooistra, K. Femaleness in breeding glasshouse cucumbers. Euphytica 6, 1-17 (1967).

[73] Davidi, H. A historical survey of cucumber breeding in israel. Proc. 4th Balkan Symposium on Vegetables and Potatoes (eds. Krasteva, L. and Panayotov, N.) Acta Hortic. 830, 33-36 (2009).

[74] Lv, J. et al. Genetic diversity and population structure of cucumber (Cucumis sativus L.) . PLoS ONE 7, e46919 (2012).

[75] Wang, X. et al. The USDA cucumber (Cucumis sativus L.) collection: genetic diversity, population structure, genome-wide association studies and core collection development . Hortic. Res. 5, 64 (2018).

[76] Zmienko, A., Samelak-Czajka, A., Kozlowski, P., Szymanska, M. & Figlerowicz, M. Arabidopsis thaliana population analysis reveals high plasticity of the genomic region spanning MSH2, AT3G18530 and AT3G18535 genes and provides evidence for NAHR-driven recurrent CNV events occurring in this location . BMC Genomics 17, 893 (2016).

[77] Cho, Y. B., Jones, I. & Vodkin, L. O. Nonallelic homologous recombination events responsible for copy number variation within an RNA silencing locus. Plant Direct 3, e00162 (2019).

[78] Leister, D. Tandem and segmental gene duplication and recombination in the evolution of plant disease resistance genes. Trends Genet. 20, 116-122 (2004).

[79] Oling, D., Masoom, R. & Kvint, K. Loss of Ubp3 increases silencing, decreases unequal recombination in rDNA, and shortens the replicative life span in Saccharomyces cerevisiae . Mol. Biol. Cell 25, 1916-1924 (2014).

[80] Pettersson, M., Eisfeldt, J., Syk Lundberg, E., Lundin, J. & Lindstrand, A. Flanking complex copy number variants in the same family formed through unequal crossing-over during meiosis. Mutat. Res. 812, 1-4 (2018).

[81] Li, Z. et al. Development and fine mapping of three co-dominant SCAR markers linked to the M/m gene in the cucumber plant (Cucumis sativus L.) . Theor. Appl. Genet. 117, 1253-1260 (2008).

[82] Jackson, S. A., Wang, M. L., Goodman, H. M. & Jiang, J. Application of fiber-FISH in genome analysis of Arabidopsis thaliana . Genome 41, 566-572 (1998).

[83] Han, Y. et al. Distribution of the tandem repeat sequences and karyotyping in cucumber (Cucumis sativus L.) by fluorescence in situ hybridization . Cytogenet. Genome Res. 122, 80-88 (2008).

[84] Jiang, J., Gill, B. S., Wang, G., Ronald, P. C. & Ward, D. C. Metaphase and interphase fluorescence in situ hybridization mapping of the rice genome with bacterial artificial chromosomes. Proc. Natl Acad. Sci. USA 92, 4487-4491 (1995).

[85] Pan, Y., Bo, K., Cheng, Z. & Weng, Y. The loss-of-function GLABROUS 3 mutation in cucumber is due to LTR-retrotransposon insertion in a class IV HD-ZIP transcription factor gene CsGL3 that is epistatic over CsGL1 . BMC Plant Biol. 15, 302 (2015).

[86] Yang, L. et al. Chromosome rearrangements during domestication of cucumber as revealed by high-density genetic mapping and draft genome assembly. Plant J. 71, 895-906 (2012).

[87] Qi, J. et al. A genomic variation map provides insights into the genetic basis of cucumber domestication and diversity. Nat. Genet. 45, 1510-1515 (2013).

[88] Wang, Y. et al. STAYGREEN, STAY HEALTHY: a loss-of-susceptibility mutation in the STAYGREEN gene provides durable, broad-spectrum disease resistances for over 50 years of US cucumber production . New Phytol. 221, 415-430 (2019).

[89] Li, Z. et al. A putative positive feedback regulation mechanism in CsACS2 expression suggests a modified model for sex determination in cucumber (Cucumis sativus L.) . J. Exp. Bot. 63, 4475-4484 (2012).