A genetic linkage map of dioecious garden asparagus (Asparagus officinalis L., 2n = 2x = 20) was constructed using F1 population, simple sequence repeat (SSR) and single nucleotide polymorphism (SNP) markers. In total, 1376 SNPs and 27 SSRs were used for genetic mapping. Two resulting parental maps contained 907 and 678 markers spanning 1947 and 1814 cM, for female and male parent, respectively, over ten linkage groups representing ten haploid chromosomes of the species. With the aim to anchor the ten genetic linkage groups to individual chromosomes and develop a tool to facilitate genome analysis and gene cloning, we have optimized a protocol for flow cytometric chromosome analysis and sorting in asparagus. The analysis of DAPI-stained suspensions of intact mitotic chromosomes by flow cytometry resulted in histograms of relative fluorescence intensity (flow karyotypes) comprising eight major peaks. The analysis of chromosome morphology and localization of 5S and 45S rDNA by FISH on flow-sorted chromosomes, revealed that four chromosomes (IV, V, VI, VIII) could be discriminated and sorted. Seventy-two SSR markers were used to characterize chromosome content of individual peaks on the flow karyotype. Out of them, 27 were included in the genetic linkage map and anchored genetic linkage groups to chromosomes. The sex determining locus was located on LG5, which was associated with peak V representing a chromosome with 5S rDNA locus. The results obtained in this study will support asparagus improvement by facilitating targeted marker development and gene isolation using flow-sorted chromosomes.

Department of Genetics ETSIAM University of Córdoba Córdoba Spain

Institute of Experimental Botany Centre of the Region Haná for Biotechnological and Agricultural Research Olomouc Czechia

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Amaro-Lopez M. A., Zurera-Cosano G., Moreno-Rojas R., Garcia-Gimeno R. M. (1995). Influence of vegetative cycle of asparagus (Asparagus officinalis L.) on copper, iron, zinc and manganese content. Plant Foods Hum. Nutr. 47 349–355. 10.1007/BF01088273 PubMed DOI

Anido F. L., Cointry E. (2008). “Asparagus,” in Vegetables II Vegetables II: Fabaceae, Liliaceae, Solanaceae, and Umbelliferae eds Prohens J., Nuez F. (New York, NY: Springer; ) 87–119. 10.1007/978-0-387-74110-9_3 DOI

Arumuganathan K., Earle E. D. (1991). Nuclear DNA content of some important plant species. Plant Mol. Biol. Rep. 9 208–218. 10.1007/BF02672069 DOI

Benson B. L. (1982). Sex influences on foliar trait morphology in asparagus. HortScience 17 625–627.

Brown C. T., Howe A., Zhang Q., Pyrkosz A. B., Brom T. H. (2012). A Reference-Free algorithm for Computational Normalization of Shotgun Sequencing Data. Available at: https://arxiv.org/abs/1203.4802

Cápal P., Blavet N., Vrána J., Kubaláková M., Doležel J. (2015). Multiple displacement amplification of the DNA from single flow–sorted plant chromosome. Plant J. 84 838–844. 10.1111/tpj.13035 PubMed DOI

Caruso M., Federici C. T., Roose M. L. (2008). EST–SSR markers for asparagus genetic diversity evaluation and cultivar identification. Mol. Breed. 21 195–204. 10.1007/s11032-007-9120-z DOI

Castro P., Gil J., Cabrera A., Moreno R. (2013). Assessment of genetic diversity and phylogenetic relationships in Asparagus species related to Asparagus officinalis. Genet. Resour. Crop Evol. 60 1275–1288. 10.1007/s10722-012-9918-3 DOI

Castro P., Rubio J., Gil J., Moreno R. (2014). Introgression of new germplasm in current diploid cultivars of garden asparagus from a tetraploid Spanish landrace “Morado de Huétor.”. Sci. Hortic. 168 157–160. 10.1016/j.scienta.2014.01.007 DOI

Deng C.-L., Qin R.-Y., Wang N.-N., Cao Y., Gao J., Gao W.-J., et al. (2012). Karyotype of asparagus by physical mapping of 45S and 5S rDNA by FISH. J. Genet. 91 209–212. 10.1007/s12041-012-0159-1 PubMed DOI

Doležel J., Binarová P., Lucretti S. (1989). Analysis of Nuclear DNA content in plant cells by Flow cytometry. Biol. Plant. 31 113–120. 10.1007/BF02907241 DOI

Doležel J., Číhalíková J., Lucretti S. (1992). A high-yield procedure for isolation of metaphase chromosomes from root tips of Vicia faba L. Planta 188 93–98. 10.1007/BF00198944 PubMed DOI

Doležel J., Kubaláková M., Cíhalíková J., Suchánková P., Šimková H. (2011). “Chromosome analysis and sorting using flow cytometry,” in Plant Chromosome Engineering ed. Birchler J. (Clifton, NJ: Methods in molecular biology; ) 221–238. 10.1007/978-1-61737-957-4_12 PubMed DOI

Doležel J., Vrána J., Cápal P., Kubaláková M., Burešová V., Šimková H. (2014). Advances in plant chromosome genomics. Biotechnol. Adv. 32 122–136. 10.1016/j.biotechadv.2013.12.011 PubMed DOI

Ellison J. H. (1986). “Asparagus breeding,” in Breeding Vegetable Crops. 521–569.

Ewing B., Green P. (1998). Base-calling of automated sequencer traces using phred. II. Error probabilities. Genome Res. 8 186–194. 10.1101/gr.8.3.186 PubMed DOI

Ewing B., Hillier L., Wendl M. C., Green P. (1998). Base-calling of automated sequencer traces using phred. I. Accuracy assessment. Genome Res. 8 175–185. 10.1101/gr.8.3.175 PubMed DOI

Falavigna A., Casali P. E. (2002). Practical aspects of a breeding program of asparagus based on in vitro anther culture. Acta Hortic. 1 201–210. 10.17660/ActaHortic.2002.589.28 DOI

FAOSTAT (2016). Available at: http://faostat.fao.org

Fu L., Niu B., Zhu Z., Wu S., Li W. (2012). CD-HIT: accelerated for clustering the next-generation sequencing data. Bioinformatics 28 3150–3152. 10.1093/bioinformatics/bts565 PubMed DOI PMC

Fuentes-Alventosa J. M., Rodríguez G., Cermeño P., Jiménez A., Guillén R., Fernández-Bolaños J., et al. (2007). Identification of flavonoid diglycosides in several genotypes of asparagus from the Huétor-Tájar population variety. J. Agric. Food Chem. 55 10028–10035. 10.1021/jf071976z PubMed DOI

Fukui K., Ohmido N., Khush G. S. (1994). Variability in rDNA loci in the genus Oryza detected through fluorescence in situ hybridization. Theor. Appl. Genet. 87 893–899. 10.1007/BF00225782 PubMed DOI

Gao W. J., Li R. L., Li S. F., Deng C. L., Li S. P. (2007). Identification of two markers linked to the sex locus in dioecious Asparagus officinalis plants. Russ. J. Plant Physiol. 54 816–821. 10.1134/S1021443707060143 DOI

Gerlach W. L., Bedbrook J. R. (1979). Cloning and characterization of ribosomal RNA genes from wheat and barley. Nucleic Acids Res. 7 1869–1885. 10.1093/nar/7.7.1869 PubMed DOI PMC

Giorgi D., Farina A., Grosso V., Gennaro A., Ceoloni C., Lucretti S. (2013). FISHIS: fluorescence in situ hybridization in suspension and chromosome flow sorting made easy. PLoS One 8:e57994. 10.1371/journal.pone.0057994 PubMed DOI PMC

Guillén R., Rodríguez R., Jaramillo S., Rodríguez G., Espejo J. A., Fernández-Bolaños J., et al. (2008). Antioxidants from asparagus spears: phenolics. Acta Hortic. 776 247–253. 10.17660/ActaHortic.2008.776.31 DOI

Hoagland D. R., Arnon D. I. (1950). The water-culture method for growing plants without soil. Circ. Calif. Agric. Exp. Station 347:32.

Jamsari A., Nitz I., Reamon-Büttner S. M., Jung C. (2004). BAC-derived diagnostic markers for sex determination in asparagus. Theor. Appl. Genet. 108 1140–1146. 10.1007/s00122-003-1529-0 PubMed DOI

Jaramillo S., Muriana F. J., Guillen R., Jimenez-Araujo A., Rodriguez-Arcos R., Lopez S. (2016). Saponins from edible spears of wild asparagus inhibit AKT, p70S6K, and ERK signalling, and induce apoptosis through G0/G1 cell cycle arrest in human colon cancer HCT-116 cells. J. Funct. Foods 26 1–10. 10.1016/j.jff.2016.07.007 DOI

Jiang C., Sink K. C. (1997). RAPD and SCAR markers linked to the sex expression locus M in asparagus. Euphytica 94 329–333. 10.1023/A:1002958007407 DOI

Kanno A., Kubota S., Ishino K. (2014). Conversion of a male-specific RAPD marker into an STS marker in Asparagus officinalis L. Euphytica 197 39–46. 10.1007/s10681-013-1048-2 DOI

Kim S.-C., Jung Y.-H., Seong K.-C., Chun S.-J., Kim C. H., Lim C. K., et al. (2014). Development of a SCAR marker for sex identification in asparagus. Korean J. Plant Resour. 27 236–241. 10.7732/kjpr.2014.27.3.236 DOI

Kofler R., Bartoš J., Gong L., Stift G., Suchánková P., Šimková H., et al. (2008). Development of microsatellite markers specific for the short arm of rye (Secale cereale L.) chromosome 1. Theor. Appl. Genet. 117 915–926. 10.1007/s00122-008-0831-2 PubMed DOI

Kubaláková M., Valárik M., Barto J., Vrána J., Číhalíková J., Molnár-Láng M., et al. (2003). Analysis and sorting of rye (Secale cereale L.) chromosomes using flow cytometry. Genome 46 893–905. 10.1139/g03-054 PubMed DOI

Lewis M. E., Sink K. C. (1996). RFLP linkage map of asparagus. Genome 39 622–627. 10.1139/g96-079 PubMed DOI

Li S., Zhang G., Li X., Wang L., Yuan J., Deng C., et al. (2016). Genome-wide identification and validation of simple sequence repeats (SSRs) from Asparagus officinalis. Mol. Cell. Probes 30 153–160. 10.1016/j.mcp.2016.03.003 PubMed DOI

Löptien H. (1976). Giemsa-Banden auf mitosechromosomen dess- pargels (Asparagus officinalis L.) und des spinats (Spinacia oleracea L.). Z. Pflanzenzuecht. 76 225–230.

Löptien H. (1979). Identification of the sex chromosome pair in asparagus (Asparagus officinalis L.). Z. Pflanzenzücht. 82 162–173.

Losa A., Caporali E., Spada A., Martinelli S., Marziani G. (2004). AOM3 and AOM4: two MADS box genes expressed in reproductive structures of Asparagus officinalis. Sex. Plant Reprod. 16 215–221. 10.1007/s00497-003-0199-z DOI

Maestri E., Restivo F. M., Longo G. P. M., Falavigna A., Tassi F. (1991). Isozyme gene markers in the dioecious species Asparagus officinalis L. Theor. Appl. Genet. 81 613–618. 10.1007/BF00226726 PubMed DOI

Martis M. M., Zhou R., Haseneyer G., Schmutzer T., Vrána J., Kubaláková M., et al. (2013). Reticulate evolution of the rye genome. Plant Cell 25 3685–3698. 10.1105/tpc.113.114553 PubMed DOI PMC

Mayer K. F. X., Martis M., Hedley P. E., Šimková H., Liu H., Morris J. A., et al. (2011). Unlocking the barley genome by chromosomal and comparative genomics. Plant Cell 23 1249–1263. 10.1105/tpc.110.082537 PubMed DOI PMC

Mayer M., Tóth V., Kuti C., Vida G., Láng L., Bedodouble acute Z. (2014). “Identification of wheat-rye chromosome translocations with molecular markers in Martonvásár winter wheat cultivars,” in Növénynemesítés a Megújuló Mezögazdaságban. XX eds Veisz O. (Budapest: Növénynemesítési Tudományos Nap; ) 294–298.

Melo N. F., Guerra M. (2001). Karyotypic stability in asparagus (Asparagus officinalis L.) cultivars revealed by rDNA in situ hybridization. Citologia 66 127–131. 10.1508/cytologia.66.127 DOI

Mercati F., Riccardi P., Leebens-Mack J., Abenavoli M. R., Falavigna A., Sunseri F. (2013). Single nucleotide polymorphism isolated from a novel EST dataset in garden asparagus (Asparagus officinalis L.). Plant Sci. 20 115–123. 10.1016/j.plantsci.2013.01.002 PubMed DOI

Moreno R., Gil J., Cabrera A. (2005). “Characterization of cultivated asparagus and wild related species by FISH with ribosomal DNA,” in Proceedings of the First International Cytogenetics and Genome Society Congress Granada, Spain: 60.

Mousavizadeh S. J., Hassandokht M. R., Kashi A., Gil J., Cabrera A., Moreno R. (2016). Physical mapping of 5S and 45S rDNA genes and ploidy levels of Iranian Asparagus species. Sci. Hortic. 211 269–276. 10.1016/j.scienta.2016.09.011 DOI

Muñoz-Amatriaín M., Moscou M. J., Bhat P. R., Svensson J. T., Bartoš J., Suchánková P., et al. (2011). An improved consensus linkage map of barley based on flow-sorted chromosomes and single nucleotide polymorphism markers. Plant Genome 4 238–249. 10.3835/plantgenome2011.08.0023 DOI

Nakayama H., Ito T., Hayashi Y., Sonoda T., Fukuda T., Ochiai T., et al. (2006). Development of sex-linked primers in garden asparagus (Asparagus officinalis L.). Breed. Sci. 56 327–330. 10.1270/jsbbs.56.327 DOI

Neumann P., Pozárková D., Vrána J., Doležel J., Macas J. (2002). Chromosome sorting and PCR-based physical mapping in pea (Pisum Sativum L.). Chromosome Res. 10 63–71. 10.1023/A:1014274328269 PubMed DOI

Park J. H., Ishikawa Y., Ochiai T., Kanno A., Kameya T. (2004). Two GLOBOSA-like genes are expressed in second and third whorls of homochlamydeous flowers in Asparagus officinalis L. Plant Cell Physiol. 45 325–332. 10.1093/pcp/pch040 PubMed DOI

Park J. H., Ishikawa Y., Yoshida R., Kanno A., Kameya T. (2003). Expression of AODEF, a B-functional MADS-box gene, in stamens and inner tepals of the dioecious species Asparagus officinalis L. Plant Mol. Biol. 51 867–875. 10.1023/A:1023097202885 PubMed DOI

Požárková D., Koblížková A., Román B., Torres A. M., Lucretti S., Lysák M., et al. (2002). Development and characterization of microsatellite markers from chromosome 1-specific DNA libraries of Vicia faba. Biol. Plant. 45 337–345. 10.1023/A:1016253214182 DOI

Reamon-Büttner S. M., Schmidt T., Jung C. (1999). AFLPs represent highly repetitive sequences in Asparagus officinalis L. Chromosome Res. 7 297–304. 10.1023/A:1009231031667 PubMed DOI

Reamon-Büttner S. M., Schondelmaier J., Jung C. (1998). Aflp markers tightly linked to the sex locus in Asparagus officinalis L. Mol. Breed. 4 91–98. 10.1023/A:1009650221460 DOI

Restivo F. M., Tassi F., Biffi R., Falavigna A., Caporali E., Carboni A., et al. (1995). Linkage arrangement of RFLP loci in progenies from crosses between doubled haploid Asparagus officinalis L. clones. Theor. Appl. Genet. 90 124–128. 10.1007/BF00221005 PubMed DOI

Riccardi P., Casali P. E., Mercati F., Falavigna A., Sunseri F. (2011). Genetic characterization of asparagus doubled haploids collection and wild relatives. Sci. Hortic. 130 691–700. 10.1016/j.scienta.2011.08.028 DOI

Rick C. M., Hanna G. C. (1943). Determination of sex in Asparagus officinalis L. Am. J. Bot. 30 711–714. 10.2307/2437718 DOI

Sánchez-Martín J., Steuernagel B., Ghosh S., Herren G., Hurni S., Adamski N., et al. (2016). Rapid gene isolation in barley and wheat by mutant chromosome sequencing. Genome Biol. 17:221. 10.1186/s13059-016-1082-1 PubMed DOI PMC

Schnable P. S., Liu S., Wu W. (2013). Data2Bio genotyping by next-generation sequencing. U.S. Patent No 6090051. Washington, DC: U.S. Patent and Trademark Office.

Schuelke M. (2000). An economic method for the fluorescent labeling of PCR fragments. Nat. Biotechnol. 18 233–234. 10.1038/72708 PubMed DOI

Schulz M. H., Weese D., Holtgrewe M., Dimitrova V., Niu S., Reinert K., et al. (2014). Fiona: a parallel and automatic strategy for read error correction. Bioinformatics 30 i356–i363. 10.1093/bioinformatics/btu440 PubMed DOI PMC

Šimková H., Svensson J. T., Condamine P., Hřibová E., Suchánková P., Bhat P. R., et al. (2008). Coupling amplified DNA from flow-sorted chromosomes to high-density SNP mapping in barley. BMC Genomics 9:294. 10.1186/1471-2164-9-294 PubMed DOI PMC

Sneep J. (1953). The significance of andromonoecism for the breeding of Asparagus officinalis L. II. Euphytica 2 224–228. 10.1007/BF00053730 DOI

Spada A., Caporali E., Marziani G., Portaluppi P., Restivo F. M., Tassi F., et al. (1998). A genetic map of Asparagus officinalis based on integrated RFLP, RAPD and AFLP molecular markers. Theor. Appl. Genet. 97 1083–1089. 10.1007/s001220050995 DOI

Telgmann-Rauber A., Jamsari A., Kinney M. S., Pires J. C., Jung C. (2007). Genetic and physical maps around the sex-determining M-locus of the dioecious plant asparagus. Mol. Genet. Genomics 278 221–234. 10.1007/s00438-007-0235-z PubMed DOI

Thind A. K., Wicker T., Šimková H., Fossati D., Moullet O., Brabant C., et al. (2017). Rapid cloning of genes in hexaploid wheat using cultivar-specific long-range chromosome assembly. Nat. Biotechnol. 35 793–796. 10.1038/nbt.3877 PubMed DOI

Valárik M., Bartoš J., Kovářová P., Kubaláková M., De Jong J. H., Doležel J. (2004). High-resolution FISH on super-stretched flow-sorted plant chromosomes. Plant J. 37 940–950. 10.1111/j.1365-313X.2003.02010.x PubMed DOI

van Ooijen J. W. (2006). JoinMap 4 Software for the Calculation of Genetic Linkage Maps in Experimental Populations. Wageningen: Kyazma BV.

Vázquez-Castilla S., Jaramillo-Carmona S., Fuentes-Alventosa J. M., Jiménez-Araujo A., Rodríguez-Arcos R., Cermeño-Sacristán P., et al. (2013). Saponin profile of green asparagus genotypes. J. Agric. Food Chem. 61 11098–11108. 10.1021/jf403343a PubMed DOI

Voorrips R. E. (2002). MapChart: Software for the graphical presentation of linkage maps and QTLs. J. Hered. 93 77–78. 10.1093/jhered/93.1.77 PubMed DOI

Vrána J., Cápal P., Šimková H., Karafiátová M., Čížková J., Doležel J. (2016). Flow analysis and sorting of plant chromosomes. Curr. Protoc. Cytom. 78 5.3.1–5.3.43. 10.1002/cpcy.9 PubMed DOI

Wenzl P., Suchánková P., Carling J., Šimková H., Huttner E., Kubaláková M., et al. (2010). Isolated chromosomes as a new and efficient source of DArT markers for the saturation of genetic maps. Theor. Appl. Genet. 121 465–474. 10.1007/s00122-010-1323-8 PubMed DOI

Wu T. D., Nacu S. (2010). Fast and SNP-tolerant detection of complex variants and splicing in short reads. Bioinformatics 26 873–881. 10.1093/bioinformatics/btq057 PubMed DOI PMC

Zatloukalová P., Hřibová E., Kubaláková M., Suchánková P., Šimková H., Adoración C., et al. (2011). Integration of genetic and physical maps of the chickpea (Cicer arietinum L.) genome using flow-sorted chromosomes. Chromosome Res. 19 729–739. 10.1007/s10577-011-9235-2 PubMed DOI

Flow Cytometric Analysis and Sorting of Plant Chromosomes

Chromosome analysis and sorting