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First report on genome size and ploidy determination of five indigenous coffee species using flow cytometry and stomatal analysis

  • Genetics & Evolutionary Biology - Original Article
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Abstract

The nuclear 2C DNA content and ploidy level determination of five indigenous coffee species from India was carried out using flow cytometry and by assessing the stomatal characteristics. The nuclear DNA content (2C/pg) analyzed varied from 1.29 pg in Coffea wightiana Wall. ex Wight & Arn. to 1.48 pg in Coffea jenkinsii Hook.f. Based on the genome size, all five species were divided into two groups. The first group comprises Coffea travancorensis Wight & Arn and C. wightiana with lower genome size (2C DNA 1.29–1.30 pg), and the second group consists of Coffea bengalensis Roxb.ex Schult, Coffea khasiana (Korth.) Hook.f and C. jenkinsii with larger genome size (2C DNA 1.45–1.48 pg). The species included in the first group were inhabited in dry environmental conditions in contrast to the species included in the second group which was predominantly from mesic habitat. Among the stomatal characteristics, no significant variation in stomatal density was observed among the species, although stomatal guard cell length and stomatal chloroplast number recorded significant variation among some species. Based on flow cytometry and stomatal characteristics, all five endangered coffee species analyzed in the study were identified as diploids. The study revealed that stomatal guard cell length and stomatal chloroplast number could be fast, inexpensive, and reliable methods for determining the ploidy status of indigenous coffee species.

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References

  • Abdoli M, Moieni A, Naghdi Badi H (2013) Morphological, physiological, cytological and phytochemical studies in diploid and colchicine-induced tetraploid plants of Echinacea purpurea (L.). Acta Physiol Plant 35:2075–2083

    CAS  Google Scholar 

  • Andreasen K, Bremer B (2000) Combined phylogenetic analysis in the Rubiaceae-Ixoroideae: morphology, nuclear and chloroplast DNA data. Am J Bot 87:1731–1748

    CAS  PubMed  Google Scholar 

  • Alan AR, Zeng H, Assani A, Shi WL, McRae HE, Murch SJ, Saxena PK (2007) Assessment of genetic stability of the germplasm lines of medicinal plant Scutellaria baicalensis Georgi (Huang-qin) in long term, in vitro maintained cultures. Plant Cell Rep 26:1345–1355

    CAS  PubMed  Google Scholar 

  • Asif MJ, Chai M, Othman RY (2001) Characterization of indigenous Musa species based on Flow Cytometric analysis of ploidy and nuclear DNA content. Caryologia 54:161–168

    Google Scholar 

  • Beaulieu JM, Leitch IJ, Patel S, Pendharkar A, Knight CA (2008) Genome size is a strong predictor of cell size and stomatal density in angiosperms. New Phytol 179:975–986

    PubMed  Google Scholar 

  • Beck SL, Visser G, Dunlop RW, Hare PD (2005) A comparison of direct (flow cytometry) and indirect (stomatal guard cell lengths and chloroplast numbers) techniques as a measure of ploidy in black wattle, Acacia mearnsii (de Wild). S Afr J Bot 71:354–358

    CAS  Google Scholar 

  • Bennett MD, Leitch IJ (2011) Nuclear DNA amounts in angiosperms: targets, trends and tomorrow. Ann Bot 107:467–590

    CAS  PubMed  PubMed Central  Google Scholar 

  • Bennetzen JL, Ma J, Devos KM (2005) Mechanisms of recent genome size variation in flowering plants. Ann Bot 95:127–132

    CAS  PubMed  PubMed Central  Google Scholar 

  • Clarindo WR, Carvalho CR (2011) Flow cytometric analysis using SYBR Green I for genome size estimation in coffee. Acta Histochem 113:221–225

    CAS  Google Scholar 

  • Clarindo WR, Carvalho CR, Mendonça MAC (2012) Cytogenetic and flow cytometry data expand knowledge of genome evolution in three Coffea species. Plant Syst Evol 298:835–844

    Google Scholar 

  • Contreras RN, Shearer K (2018) Genome size, ploidy, and base composition of wild and cultivated acer. J Amer Soc Hort Sci 143:470–485

    CAS  Google Scholar 

  • Cros J, Combes MC, Chabrillange N, Hamon S, Duperray C, des Angles AM (1995) Nuclear DNA content in the subgenus Coffea (Rubiaceae): inter and intra specific variation in African species. Can J Bot 73:14–20

    Google Scholar 

  • da Silva HA, Scapim CA, Vivas JMS, Junior ATDA, Pinto RJB, Socorro K, Mourao M, Rossi RM, Baleroni AG (2020) Effect of ploidy level on guard cell length and use of stomata to discard diploids among putative haploids in maize. Crop Sci 60:1199–1209

    Google Scholar 

  • Davis AP (2011) Psilanthusmannii, the type species of Psilanthus, transferred to Coffea. Nord J Bot 29:471–472

    Google Scholar 

  • Davis AP, Chadburn H, Moat J, O’Sullivan R, Hargreaves S, Lughadha EN (2019) High extinction risk for wild coffee species and implications for coffee sector sustainability. Sci Adv. https://doi.org/10.1126/sciadv.aav3473

    Article  PubMed  PubMed Central  Google Scholar 

  • Dkhar J, Pareek A (2014) What determines a leaf’s shape? EvoDevo 5:47. https://doi.org/10.1186/2041-9139-5-47

    Article  PubMed  PubMed Central  Google Scholar 

  • Dolezel J, Gohde W (1995) Sex determination in dioecious plants Melandrium album and M. rubrum using high-resolution flow cytometry. Cytometry 19:103–106

    CAS  PubMed  Google Scholar 

  • Dolezel J, Bartos J, Voglmayr H, Greilhuber J (2003) Nuclear DNA content and genome size of trout and human. Cytometry 51:127–128

    CAS  PubMed  Google Scholar 

  • Dolezel J, Bartos J (2005) Plant DNA flow cytometry and estimation of nuclear genome size. Ann Bot 95:99–110

    CAS  PubMed  PubMed Central  Google Scholar 

  • Dulloo ME, Owadally AW (1991) Conservation and utilization of wild coffee. In: Attere F, Zedan H, Ng NQ, Perrino P (eds) Crop genetic resources of Africa, vol 1. IBPGR, UNEP, IITA & CNR, pp 231–238

    Google Scholar 

  • Hamon P, Siljak-Yakovlev S, Srisuwan S, Robin O, Poncet V, Hamon S, de Kochko A (2009) Physical mapping of rDNA and heterochromatin in chromosomes of 16 Coffea species: a revised view of species differentiation. Chromosome Res 17:291–304

    CAS  PubMed  Google Scholar 

  • He P, Li L, Cheng L, Wang H, Chang Y (2018) Variation in ploidy level and morphological traits in the progeny of the triploid apple variety Jonagold. Czech J Genet Plant Breed 54:135–142

    CAS  Google Scholar 

  • Hickey IJ (1973) Classification of architecture of dicotyledonous leaves. Am J Bot 60:17–33

    Google Scholar 

  • Hodgson JG, Sharafi M, Jalili A, Diaz S, Montserrat-Marti G, Palmer C, Cerabolini B, Pierce S, Hamzehee B, Asri Y, Jamzad Z, Wilson P, Raven JA, Band SR, Basconcelo S, Bogard A, Carter G, Charles M, Castro-Diez P, Cornelissen JH, Funes G, Jones G, Khoshnevis M, Perez-Harguindeguy N, Perez-Rontome MC, Shirvany FA, Vendramini F, Yazdani S, Abbas-Azimi R, Boustani S, Dehghan M, Guerrero-Campo J, Hynd A, Kowsary E, Kazemi-Saeed F, Siavash B, Villar-Salvador P, Craigie R, Naqinezhad A, Romo-Diez A, de Torres Espuny L, Simmons E (2010) Stomatal vs. genome size in angiosperms: the somatic tail wagging the genomic dog? Ann Bot 105:573–584

    CAS  PubMed  PubMed Central  Google Scholar 

  • Inceer H, Ayaz HS (2010) Chromosome numbers in Tripleurospermum Sch. Bip. (Asteraceae) and closely related genera: Relationships between ploidy level and stomatal length. Plant Syst Evol 285:149–157

    PubMed  PubMed Central  Google Scholar 

  • Jatt T, Lee MS, Rayburn AL, Jatoi MA, Mirani AA (2019) Determination of genome size variations among different date palm cultivars (Phoenix dactylifera L.) by flow cytometry. 3 Biotech 9:457. https://doi.org/10.1007/s13205-019-1987-y

    Article  PubMed  PubMed Central  Google Scholar 

  • Lashermes P, Combes MC, Trouslot P, Charrier A (1997) Phylogenetic relationships of coffee tree species (Coffea L.) as inferred from ITS sequences of nuclear ribosomal DNA. Theor Appl Genet 94:947–955

    CAS  Google Scholar 

  • Lomax BH, Woodward FI, Leitch IJ, Knight CA, Lake JA (2009) Genome size as a predictor of guard cell length in Arabidopsis thaliana is independent of environmental conditions. New Phytol 181:311–314

    CAS  PubMed  Google Scholar 

  • Lombello RA, Pinto-Maglio CAF (2004) Cytogenetic studies in Coffea L. and Psilanthus Hook.f. using CMA/DAPI and FISH. Cytologia 69:85–91

    Google Scholar 

  • Loureiro J, Rodriguez E, Dolezel J, Santos C (2006a) Comparison of four nuclear isolation buffers for plant DNA flow cytometry. Ann Bot 98:679–689

    CAS  PubMed  PubMed Central  Google Scholar 

  • Loureiro J, Rodriguez E, Dolezel J, Santos C (2006b) Flow cytometric and microscopic analysis of the effect of tannic acid on plant nuclei and estimation of DNA content. Ann Bot 98:515–527

    CAS  PubMed  PubMed Central  Google Scholar 

  • Lysak MA, Dolezel J (1998) Estimation of nuclear DNA content in Sesleria (Poaceae). Caryologia 51:123–132

    Google Scholar 

  • Lysak MA, Cihalikova J, Kubalakova M, Simkova H, Kunzel G, Dolezel J (1999) Flow karyotyping ahead sorting of mitotic chromosomes of barley (Hordeum vulgare L.). Chromosome Res 7:431–444

    CAS  PubMed  Google Scholar 

  • Maurin O, Davis AP, Chester M, Mvungi EF, Jaufeerally-Fakim Y, Fay MF (2007) Towards a Phylogeny for Coffea (Rubiaceae): identifying well-supported lineages based on nuclear and plastid DNA sequences. Ann Bot 100:1565–1583

    CAS  PubMed  PubMed Central  Google Scholar 

  • Mehra PN, Bawa KS (1969) Chromosomal evolution in tropical hardwoods. Evolution 23:466–481

    CAS  PubMed  Google Scholar 

  • Mishra MK, Prakash NS, Sreenivasan MS (1991) Relation of stomatal length and frequency to ploidy levels in Coffea L. J Coffee Res 21:32–41

    Google Scholar 

  • Mishra MK (1997) Stomatal characteristics at different ploidy levels of Coffea L. Ann Bot 80:689–692

    Google Scholar 

  • Mishra MK, Ram AS, Jyothi DP, Prakash NS, Srinivasan CS (2003) Stomatal characteristics of different species of Coffea L. J Plant Crops 31:35–39

    Google Scholar 

  • Mishra MK, Tornincasa P, De-Nardi B, Asquini E, Dreos R, Terra LD, Rathinavelu R, Rovelli P, Pallavicini A, Graziosi G (2011a) Genome organization in coffee as revealed by EST PCR-RFLP, SNPs and SSR Analysis. J Crop Sci Biotechnol 14:25–37

    Google Scholar 

  • Mishra MK, Padmajyothi D, Surya Prakash N, Sreenivasan MS, Srinivasan CS (2011b) Variability in stomatal features and leaf venation pattern in Indian coffee (Coffea arabica L.) cultivars and their functional significance. Bot Serb 35:111–119

    Google Scholar 

  • Mishra MK (2019) Genetic Resources and Breeding of Coffee (Coffea spp.). In: Al-Khayri JM, Jain M, Johnson DV (eds) Advances in Plant Breeding Strategies: Nut and Beverage Crops. Springer, Cham. https://doi.org/10.1007/978-3-030-23112-5_12

    Chapter  Google Scholar 

  • Murray BG, De Lange PJ, Ferguson AR (2005) Nuclear DNA variation, chromosome numbers and polyploidy in the endemic and indigenous grass flora of New Zealand. Ann Bot 96:1293–1305

    CAS  PubMed  PubMed Central  Google Scholar 

  • Narasimhaswamy RL, Vishweshwara S (1963) A note on the occurrence of three species of Coffea indigenous to India. Turrialba 5:65–71

    Google Scholar 

  • Noirot M, Barre P, Louarn J, Duperray C, Hamon S (2002) Consequences of stoichiometric error for nuclear DNA content evaluation in Coffea liberica var. dewevrei using DAPI and propidium iodide. Ann Bot 89:385–389

    CAS  PubMed  PubMed Central  Google Scholar 

  • Noirot M, Poncet V, Barre P, Hamon P, Hamon S, De Kochko A (2003) Genome size variation in diploid African Coffea species. Ann Bot 92:709–714

    CAS  PubMed  PubMed Central  Google Scholar 

  • Noirot M, Barre P, Duperray C, Hamon S, De Kochko A (2005) Investigation on the causes of stoichiometric error in genome size estimation using heat experiments: consequences on data interpretation. Ann Bot 95:111–118

    CAS  PubMed  PubMed Central  Google Scholar 

  • Nosrati H (2014) Relationship between ploidy level and genome size in strawberries. Plant Biosyst 49:1036–1041

    Google Scholar 

  • Ortega JO, Ramirez-OrtegFA R-M, Xoconostle-Cazares B (2019) Analysis of Genome Size of Sixteen Coffea arabica Cultivars Using Flow Cytometry. Hortic Sci 54:998–1004

    Google Scholar 

  • Otto F (1990) DAPI staining of fixed cells for high-resolution flow cytometry of nuclear DNA. In: Darzynkiewicz Z, Crissman HA (eds) Methods cell boil, vol 33. Academic Press, New York, pp 105–110

    Google Scholar 

  • Pinto G, Loureiro J, Lopes T, Santos C (2004) Analysis of the genetic stability of Eucalyptus globulus Labill. somatic embryos by flow cytometry. Theor Appl Genet 109:580–587

    CAS  PubMed  Google Scholar 

  • Price HJ, Chambers KL, Bachmann K (1981) Genome size variation in diploid Microseris bigelovii (Asteraceae). Bot Gaz 142:156–159

    Google Scholar 

  • Razafinarivo NJ, Rakotomalala JJ, Brown SC, Bourge M, Hamon S, de Kochko A, Poncet V, Dubreuil-Tranchant C, Couturon E, Guyot R, Hamon P (2012) Geographical gradients in the genome size variation of wild coffee trees (Coffea) native to Africa and Indian Ocean islands. Tree Genet Genomes 8:1345–1358

    Google Scholar 

  • Rego MM, Rego ER, Bruckner CH, Finger FL, Otoni WC (2011) In vitro induction of autotetraploids from diploid yellow passion fruit mediated by colchicine and oryzalin. Plant Cell Tiss Organ 107:451–459

    CAS  Google Scholar 

  • Sabzehzari M, Hoveidamanesh S, Modarresi M, Mohammadi V (2019) Morphological, anatomical, physiological, and cytological studies in diploid and tetraploid plants of Plantago psyllium. Plant Cell Tiss Organ 139:131–137

    CAS  Google Scholar 

  • Sakiroglu M, Brummer CE (2011) Clarifying the ploidy of some accessions in the USDA alfalfa germplasm collection. Turk J Bot 35:1–12

    Google Scholar 

  • Scalabrin S, Toniutti L, Di Gaspero G, Scaglione D, Magris G, Vidotto M, Pinosio S, Cattonaro F, Magni F, Jurman I, Cerutti M, Liverani FS, Navarini L, Del Terra L, Pellegrino G, Ruosi MR, Vitulo N, Valle G, Pallavicini A, Graziosi G, Klein PE, Bentley N, Murray S, Solano W, Al Hakimi A, Schilling T, Montagnon C, Morgante M, Bertrand B (2020) A single polyploidization event at the origin of the tetraploid genome of Coffea arabica is responsible for the extremely low genetic variation in wild and cultivated germplasm. Sci Rep 10:4642. https://doi.org/10.1038/s41598-020-61216-7

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Seidler-Lozykowska K (2003) Determination of the ploidy level in chamomile (Chamomilla recutita (L.) Rausch.) strains rich in alpha-bisabolol. J Appl Genet 44:151–155

    PubMed  Google Scholar 

  • Sharma S, Kaushik S, Raina SN (2019) Estimation of nuclear DNA content and its variation among Indian Tea accessions by flow cytometry. Physiol Mol Biol Plants 25:339–346

    CAS  PubMed  Google Scholar 

  • Sivarajan VV, Biju SD, Mathew P (1992) Revision of the genus Psilanthus Hook.f. (Rubiaceae, tribe Coffeeae), in India. Bot Bull Academia Sin 33:209–224

    Google Scholar 

  • Smarda P, Bures P (2010) Understanding intraspecific variation in genome size in plants. Preslia 82:41–61

    Google Scholar 

  • Sreenivasan MS, Prakash NS, Mishra MK (1992) Evaluation of some indirect ploidy indicators in Coffea L. Cafe Cacao The (France) 36:199–205

    Google Scholar 

  • Sybenga J (1960) Genetics and cytology of coffee. A literature review. Bibliographia Genetica (Wageningen) 19:217–316

    Google Scholar 

  • Temsch EM, Greilhuber J (2001) Genome size in Arachis duranensis: a critical study. Genome 44:826–830

    CAS  PubMed  Google Scholar 

  • Tuna M, Vogel KP, Arumuganathan K, Gill KS (2001) DNA content and ploidy determination of bromegrass germplasm accessions by flow cytometry. Crop Sci 41:1629–1634

    Google Scholar 

  • Zabka M, Durisova L, Elias P, Baranec T (2018) Genome size and ploidy level among wild and cultivated Prunus taxa in Slovakia. Biologia 73:121–128

    CAS  Google Scholar 

  • Zoldos V, Papes D, Brown SC, Panaud O, Siljak-Yakovlev S (1998) Genome size and base composition of seven Quercus species: Inter- and intra-population variation. Genome 41:162–168

    CAS  Google Scholar 

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The corresponding author designed and collected the material for the experiment and wrote the manuscript. AKH and PJ carried out the experiments and participated in manuscript preparation. All authors read and approved the manuscript.

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Correspondence to Manoj Kumar Mishra.

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Jingade, P., Huded, A.K.C. & Mishra, M.K. First report on genome size and ploidy determination of five indigenous coffee species using flow cytometry and stomatal analysis. Braz. J. Bot 44, 381–389 (2021). https://doi.org/10.1007/s40415-021-00714-y

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