Register      Login
Crop and Pasture Science Crop and Pasture Science Society
Plant sciences, sustainable farming systems and food quality
RESEARCH ARTICLE (Open Access)

Effects of cyanogenesis on morphology and estimated leaf flavonoid content in 51 white clover accessions

Jennifer Gabriel https://orcid.org/0000-0002-1836-1301 A B * , Nicole M. van Dam https://orcid.org/0000-0003-2622-5446 A B C and Henriette Uthe https://orcid.org/0000-0002-2523-196X A B
+ Author Affiliations
- Author Affiliations

A German Centre for Integrative Biodiversity Research (iDiv) Halle-Jena-Leipzig, Leipzig 04103, Germany.

B Institute of Biodiversity, Friedrich Schiller University, Jena 07743, Germany.

C Leibniz Institute for Vegetable and Ornamental Crops (IGZ) e.V., Großbeeren 14979, Germany.

* Correspondence to: jennifer.gabriel@div.de

Handling Editor: Marta Santalla

Crop & Pasture Science 74(5) 494-506 https://doi.org/10.1071/CP22140
Submitted: 19 April 2022  Accepted: 28 November 2022   Published: 30 January 2023

© 2023 The Author(s) (or their employer(s)). Published by CSIRO Publishing. This is an open access article distributed under the Creative Commons Attribution 4.0 International License (CC BY)

Abstract

Context: Plant secondary metabolites are of increasing interest for agriculture due to their diverse beneficial ecological functions. The forage crop white clover (Trifolim repens L.) has been intensively studied for its heritable polymorphism in the production of hydrogen cyanide (HCN), a toxic defense phytochemical. In fodder production, white clover accessions are selected for biomass production, whereby HCN production is an unwanted trait.

Aim: Although white clover is a legume crop species of global importance, little is known about the linkage between cyanogenesis and growth traits, in particular in combination with resistance-related phytochemicals, such as flavonoids. We aimed to identify differences in biomass production, estimated leaf flavonoid content, and trait correlations in cyanogenic (HCN-producing) and acyanogenic (not HCN-producing) individuals and accessions of white clover.

Methods: We analysed 51 white clover accessions from a German germplasm collection for variability in selected traits: cyanogenesis as equivalent electrode potential, estimated leaf flavonoid content, root and shoot production, leaf area, specific leaf area, and number of leaves produced.

Key results: Most accessions considered as cyanogenic were heterogeneous for HCN production. Chemical–morphological trait correlations differed between cyanogenic and acyanogenic plants. Acyanogenic individuals and accessions produced more and larger leaves compared to cyanogenic ones. Within cyanogenic accessions, the higher the HCN level of a plant, the fewer but larger leaves were produced.

Conclusions: Our results highlight the variation in HCN production within the selected accessions, which calls for a consistent approach for cyanogenesis-based categorisation.

Implication: This study demonstrates the potential of combining phytochemical traits with biomass production in white clover when selecting material in a breeding program.

Keywords: cyanogenesis, forage legume, genotypic diversity, leaf flavonoids, legume chemistry, morphological traits, plant breeding, Trifolium repens, white clover germplasm.


References

Aasmo Finne M, Rognli OA, Schjelderup I (2000) Genetic variation in a Norwegian germplasm collection of white clover (Trifolium repens L.). Euphytica 112, 45–56.
Genetic variation in a Norwegian germplasm collection of white clover (Trifolium repens L.).Crossref | GoogleScholarGoogle Scholar |

Annicchiarico P (2003) Breeding white clover for increased ability to compete with associated grasses. The Journal of Agricultural Science 140, 255–266.
Breeding white clover for increased ability to compete with associated grasses.Crossref | GoogleScholarGoogle Scholar |

Ballhorn DJ, Elias JD (2014) Salinity-mediated cyanogenesis in white clover (Trifolium repens) affects trophic interactions. Annals of Botany 114, 357–366.
Salinity-mediated cyanogenesis in white clover (Trifolium repens) affects trophic interactions.Crossref | GoogleScholarGoogle Scholar |

Ballhorn DJ, Lieberei R, Ganzhorn JU (2005) Plant cyanogenesis of Phaseolus lunatus and its relevance for herbivore-plant interaction: the importance of quantitative data. Journal of Chemical Ecology 31, 1445–1473.
Plant cyanogenesis of Phaseolus lunatus and its relevance for herbivore-plant interaction: the importance of quantitative data.Crossref | GoogleScholarGoogle Scholar |

Ballhorn DJ, Kautz S, Lieberei R (2010a) Comparing responses of generalist and specialist herbivores to various cyanogenic plant features. Entomologia Experimentalis et Applicata 134, 245–259.
Comparing responses of generalist and specialist herbivores to various cyanogenic plant features.Crossref | GoogleScholarGoogle Scholar |

Ballhorn DJ, Pietrowski A, Lieberei R (2010b) Direct trade-off between cyanogenesis and resistance to a fungal pathogen in lima bean (Phaseolus lunatus L.). Journal of Ecology 98, 226–236.
Direct trade-off between cyanogenesis and resistance to a fungal pathogen in lima bean (Phaseolus lunatus L.).Crossref | GoogleScholarGoogle Scholar |

Bjarnholt N, Laegdsmand M, Hansen HCB, Jacobsen OH, Møller BL (2008) Leaching of cyanogenic glucosides and cyanide from white clover green manure. Chemosphere 72, 897–904.
Leaching of cyanogenic glucosides and cyanide from white clover green manure.Crossref | GoogleScholarGoogle Scholar |

Blaedal WJ, Easty DB, Anderson L, Farrell TR (1971) Potentiometric determination of cyanide with an ion selective electrode. Application to cyanogenic glycosides in Sudan grasses. Analytical Chemistry 43, 890–894.
Potentiometric determination of cyanide with an ion selective electrode. Application to cyanogenic glycosides in Sudan grasses.Crossref | GoogleScholarGoogle Scholar |

Bradbury JH (2009) Development of a sensitive picrate method to determine total cyanide and acetone cyanohydrin contents of gari from cassava. Food Chemistry 113, 1329–1333.
Development of a sensitive picrate method to determine total cyanide and acetone cyanohydrin contents of gari from cassava.Crossref | GoogleScholarGoogle Scholar |

Caradus JR (1986) World checklist of white clover varieties. New Zealand Journal of Experimental Agriculture 14, 119–164.
World checklist of white clover varieties.Crossref | GoogleScholarGoogle Scholar |

Caradus JR, Woodfield DR (1997) Review: World checklist of white clover varieties II. New Zealand Journal of Agricultural Research 40, 115–206.
Review: World checklist of white clover varieties II.Crossref | GoogleScholarGoogle Scholar |

Caradus JR, MacKay AC, Woodfield DR, van den Bosch J, Wewala S (1989) Classification of a world collection of white clover cultivars. Euphytica 42, 183–196.
Classification of a world collection of white clover cultivars.Crossref | GoogleScholarGoogle Scholar |

Caradus JR, Hay RJM, Woodfield DR (1996) The positioning of white clover cultivars in New Zealand. Agronomy Society of New Zealand Special Publication 11/Grassland Research and Practice Series 6, 45–50.
The positioning of white clover cultivars in New Zealand.Crossref | GoogleScholarGoogle Scholar |

Chang C-C, Yang M-H, Wen H-M, Chern J-C (2002) Estimation of total flavonoid content in propolis by two complementary colometric methods. Journal of Food and Drug Analysis 10, 3
Estimation of total flavonoid content in propolis by two complementary colometric methods.Crossref | GoogleScholarGoogle Scholar |

Cheeke PR (1995) Endogenous toxins and mycotoxins in forage grasses and their effects on livestock. Journal of Animal Science 73, 909–918.
Endogenous toxins and mycotoxins in forage grasses and their effects on livestock.Crossref | GoogleScholarGoogle Scholar |

Chen Y, Lübberstedt T (2010) Molecular basis of trait correlations. Trends in Plant Science 15, 454–461.
Molecular basis of trait correlations.Crossref | GoogleScholarGoogle Scholar |

Conn EE (1981) Cyanogenic glycosides. In ‘The biochemistry of plants. A comprehensive treatise, Vol. 7’. Secondary Plant Products. (Ed. EE Conn) pp. 479–499. (Academic Press: New York)

Coop IE (1940) Cyanogenesis in white clover (Trifolium repens L.). III. A study of linamarase, the enzyme which hydrolyses lotaustralin. New Zealand Journal of Science and Technology 22, 71B–83B.

Corkill L (1942) Cyanogenesis in white clover (Trifolium repens L.). V. The inheritance of cyanogenesis. New Zealand Journal of Science and Technology 23, 178–193.

Crush JR, Caradus JR (1995) Cyanogenesis potential and iodine concentration in white clover (Trifolium repens L.) cultivars. New Zealand Journal of Agricultural Research 38, 309–316.
Cyanogenesis potential and iodine concentration in white clover (Trifolium repens L.) cultivars.Crossref | GoogleScholarGoogle Scholar |

Cunningham SA, Summerhayes B, Westoby M (1999) Evolutionary divergences in leaf structure and chemistry, comparing rainfall and soil nutrient gradients. Ecological Monographs 69, 569–588.
Evolutionary divergences in leaf structure and chemistry, comparing rainfall and soil nutrient gradients.Crossref | GoogleScholarGoogle Scholar |

Daday H (1954) Gene frequencies in wild populations of Trifolium repens. Heredity 8, 61–78.
Gene frequencies in wild populations of Trifolium repens.Crossref | GoogleScholarGoogle Scholar |

Daday H (1965) Gene frequencies in wild populations of Trifolium repens L. IV Mechanisms of natural selection. Heredity 20, 355–365.
Gene frequencies in wild populations of Trifolium repens L. IV Mechanisms of natural selection.Crossref | GoogleScholarGoogle Scholar |

de Araújo AM (1976) The relationship between altitude and cyanogenesis in white clover (Trifolium repens, L.). Heredity 37, 291–293.
The relationship between altitude and cyanogenesis in white clover (Trifolium repens, L.).Crossref | GoogleScholarGoogle Scholar |

Dolph GE, Dilcher DL (1980) Variation in leaf size with respect to climate in Costa Rica. Biotropica 12, 91–99.
Variation in leaf size with respect to climate in Costa Rica.Crossref | GoogleScholarGoogle Scholar |

D’Amelia V, Aversano R, Chiaiese P, Carputo D (2018) The antioxidant properties of plant flavonoids: their exploitation by molecular plant breeding. Phytochemistry Reviews 17, 611–625.
The antioxidant properties of plant flavonoids: their exploitation by molecular plant breeding.Crossref | GoogleScholarGoogle Scholar |

Eckardt NA (2006) The role of flavonoids in root nodule development and auxin transport in Medicago truncatula. The Plant Cell 18, 1539–1540.
The role of flavonoids in root nodule development and auxin transport in Medicago truncatula.Crossref | GoogleScholarGoogle Scholar |

Egan SV, Yeoh HH, Bradbury JH (1998) Simple picrate paper kit for determination of the cyanogenic potential of cassava flour. Journal of the Science of Food and Agriculture 76, 39–48.
Simple picrate paper kit for determination of the cyanogenic potential of cassava flour.Crossref | GoogleScholarGoogle Scholar |

Egan LM, Hofmann RW, Ghamkhar K, Hoyos-Villegas V (2021) Prospects for Trifolium improvement through germplasm characterisation and pre-breeding in New Zealand and beyond. Frontiers in Plant Science 12, 653191
Prospects for Trifolium improvement through germplasm characterisation and pre-breeding in New Zealand and beyond.Crossref | GoogleScholarGoogle Scholar |

Ennos RA (1981) Detection of selection in populations of white clover (Trifolium repens L.). Biological Journal of the Linnean Society 15, 75–82.
Detection of selection in populations of white clover (Trifolium repens L.).Crossref | GoogleScholarGoogle Scholar |

Feigl F, Anger V (1966) Replacement of benzidine by copper ethylacetoacetate and tetra base as spot-test reagent for hydrogen cyanide and cyanogen. Analyst 91, 282–284.
Replacement of benzidine by copper ethylacetoacetate and tetra base as spot-test reagent for hydrogen cyanide and cyanogen.Crossref | GoogleScholarGoogle Scholar |

Fonseca CR, Overton JM, Collins B, Westoby M (2000) Shifts in trait-combinations along rainfall and phosphorus gradients. Journal of Ecology 88, 964–977.
Shifts in trait-combinations along rainfall and phosphorus gradients.Crossref | GoogleScholarGoogle Scholar |

Foulds W, Grime JP (1972) The response of cyanogenic and acyanogenic phenotypes of Trifolium repens to soil moisture supply. Heredity 28, 181–187.
The response of cyanogenic and acyanogenic phenotypes of Trifolium repens to soil moisture supply.Crossref | GoogleScholarGoogle Scholar |

Fox J, Weisberg S (2019) An R companion to applied regression, Third Edition. Thousand Oaks CA. Available at https://socialsciences.mcmaster.ca/jfox/Books/Companion/

Frame J, Newbould P (1986) Agronomy of white clover. Advances in Agronomy 40, 1–88.
Agronomy of white clover.Crossref | GoogleScholarGoogle Scholar |

Gillingham JT, Shirer MM, Page NR (1969) Evaluation of the orion cyanide electrode for estimating the cyanide content of forage samples. Agronomy Journal 61, 717–718.
Evaluation of the orion cyanide electrode for estimating the cyanide content of forage samples.Crossref | GoogleScholarGoogle Scholar |

Gleadow RM, Møller BL (2014) Cyanogenic glycosides: synthesis, physiology, and phenotypic plasticity. Annual Review of Plant Biology 65, 155–185.
Cyanogenic glycosides: synthesis, physiology, and phenotypic plasticity.Crossref | GoogleScholarGoogle Scholar |

Gleadow RM, Woodrow IE (2002) Constraints on effectiveness of cyanogenic glycosides in herbivore defense. Journal of Chemical Ecology 28, 1301–1313.
Constraints on effectiveness of cyanogenic glycosides in herbivore defense.Crossref | GoogleScholarGoogle Scholar |

Gong H, Gao J (2019) Soil and climatic drivers of plant SLA (specific leaf area). Global Ecology and Conservation 20, e00696
Soil and climatic drivers of plant SLA (specific leaf area).Crossref | GoogleScholarGoogle Scholar |

Hamel J (2011) A Review of acute cyanide poisoning with a treatment update. Critical Care Nurse 31, 72–82.
A Review of acute cyanide poisoning with a treatment update.Crossref | GoogleScholarGoogle Scholar |

Hawkins RP (1959) Botanical characters for the classification and identification of varieties of white clover. National Institute of Agricultural Botany Journal 8, 675–682.

Hayden KJ, Parker IM (2002) Plasticity in cyanogenesis of Trifolium repens L.: inducibility, fitness costs and variable expression. Evolutionary Ecology Research 4, 155–168.

Hoagland DR, Arnon DI (1938) The water culture method for growing plants without soil. California Agricultural Experiment Station Publications C347, 36–39.

Hofmann RW, Jahufer MZZ (2011) Tradeoff between biomass and flavonoid accumulation in white clover reflects contrasting plant strategies. PLoS ONE 6, e18949
Tradeoff between biomass and flavonoid accumulation in white clover reflects contrasting plant strategies.Crossref | GoogleScholarGoogle Scholar |

Hope RM (2022) Rmisc: Ryan Miscellaneous. R package version 1.5.1. Available at https://CRAN.R-project.org/package=Rmisc

Hothorn T, Bretz F, Westfall P (2008) Simultaneous inference in general parametric models. Biometrical Journal 50, 346–363.

Hughes MA (1991) The cyanogenic polymorphism in Trifolium repens L. (white clover). Heredity 66, 105–115.
The cyanogenic polymorphism in Trifolium repens L. (white clover).Crossref | GoogleScholarGoogle Scholar |

Hughes MA, Brown K, Pancoro A, Murray BS, Oxtoby E, Hughes J (1992) A molecular and biochemical analysis of the structure of the cyanogenic β-glucosidase (linamarase) from cassava (Manihot esculenta Cranz). Archives of Biochemistry and Biophysics 295, 273–279.
A molecular and biochemical analysis of the structure of the cyanogenic β-glucosidase (linamarase) from cassava (Manihot esculenta Cranz).Crossref | GoogleScholarGoogle Scholar |

Jahufer MZZ, Ford JL, Widdup KH, Harris C, Cousins G, Ayres JF, Lane LA, Hofmann RW, Ballizany WL, Mercer CF, Crush JR, Williams WM, Woodfield DR, Barrett BA (2012) Improving white clover for Australasia. Crop & Pasture Science 63, 739–745.
Improving white clover for Australasia.Crossref | GoogleScholarGoogle Scholar |

Jones DA (1998) Why are so many food plants cyanogenic? Phytochemistry 47, 155–162.
Why are so many food plants cyanogenic?Crossref | GoogleScholarGoogle Scholar |

Kakes P (1989) An analysis of the costs and benefits of the cyanogenic system in Trifolium repens L. Theoretical and Applied Genetics 77, 111–118.
An analysis of the costs and benefits of the cyanogenic system in Trifolium repens L.Crossref | GoogleScholarGoogle Scholar |

Kooyers NJ, Gage LR, Al-Lozi A, Olsen KM (2014) Aridity shapes cyanogenesis cline evolution in white clover (Trifolium repens L.). Molecular Ecology 23, 1053–1070.
Aridity shapes cyanogenesis cline evolution in white clover (Trifolium repens L.).Crossref | GoogleScholarGoogle Scholar |

Larose G, Chênevert R, Moutoglis P, Gagne S, Piché Y, Vierheilig H (2002) Flavonoid levels in roots of Medicago sativa are modulated by the developmental stage of the symbiosis and the root colonizing arbuscular mycorrhizal fungus. Journal of Plant Physiology 159, 1329–1339.
Flavonoid levels in roots of Medicago sativa are modulated by the developmental stage of the symbiosis and the root colonizing arbuscular mycorrhizal fungus.Crossref | GoogleScholarGoogle Scholar |

Leavesley HB, Li L, Prabhakaran K, Borowitz JL, Isom GE (2008) Interaction of cyanide and nitric oxide with cytochrome c oxidase: implications for acute cyanide toxicity. Toxicological Sciences 101, 101–111.
Interaction of cyanide and nitric oxide with cytochrome c oxidase: implications for acute cyanide toxicity.Crossref | GoogleScholarGoogle Scholar |

Li B, Fan R, Sun G, Sun T, Fan Y, Bai S, Guo S, Huang S, Liu J, Zhang H, Wang P, Zhu X, Song C-P (2021) Flavonoids improve drought tolerance of maize seedlings by regulating the homeostasis of reactive oxygen species. Plant and Soil 461, 389–405.
Flavonoids improve drought tolerance of maize seedlings by regulating the homeostasis of reactive oxygen species.Crossref | GoogleScholarGoogle Scholar |

Lieberei R (1988) Relationship of cyanogenic capacity (HCN-c) of the rubber tree Hevea brasiliensis to susceptibility to Microcyclus ulei, the agent causing South American leaf blight. Journal of Phytopathology 122, 54–67.
Relationship of cyanogenic capacity (HCN-c) of the rubber tree Hevea brasiliensis to susceptibility to Microcyclus ulei, the agent causing South American leaf blight.Crossref | GoogleScholarGoogle Scholar |

Mather RDJ, Melhuish DT, Herlihy M (1995) Trends in the global marketing of white clover cultivars. Agronomy Society of New Zealand 6, 7–14.
Trends in the global marketing of white clover cultivars.Crossref | GoogleScholarGoogle Scholar |

Mierziak J, Kostyn K, Kulma A (2014) Flavonoids as important molecules of plant interactions with the environment. Molecules 19, 16240–16265.
Flavonoids as important molecules of plant interactions with the environment.Crossref | GoogleScholarGoogle Scholar |

Nahrstedt A (1985) Cyanogenic compounds as protecting agents for organisms. Plant Systematics and Evolution 150, 35–47.
Cyanogenic compounds as protecting agents for organisms.Crossref | GoogleScholarGoogle Scholar |

Nakabayashi R, Yonekura-Sakakibara K, Urano K, Suzuki M, Yamada Y, Nishizawa T, Matsuda F, Kojima M, Sakakibara H, Shinozaki K, Michael AJ, Tohge T, Yamazaki M, Saito K (2014) Enhancement of oxidative and drought tolerance in Arabidopsis by overaccumulation of antioxidant flavonoids. The Plant Journal 77, 367–379.
Enhancement of oxidative and drought tolerance in Arabidopsis by overaccumulation of antioxidant flavonoids.Crossref | GoogleScholarGoogle Scholar |

Noitsakis B, Jacquard P (1992) Competition between cyanogenic and acyanogenic morphs of Trifolium repens. Theoretical and Applied Genetics 83, 443–450.
Competition between cyanogenic and acyanogenic morphs of Trifolium repens.Crossref | GoogleScholarGoogle Scholar |

Oliveira JA, López JE, Palencia P (2013) Agromorphological characterization, cyanogenesis and productivity of accessions of White Clover (Trifolium repens L.) collected in Northern Spain. Czech Journal of Genetics and Plant Breeding 49, 24–35.
Agromorphological characterization, cyanogenesis and productivity of accessions of White Clover (Trifolium repens L.) collected in Northern Spain.Crossref | GoogleScholarGoogle Scholar |

Olsen KM, Sutherland BL, Small LL (2007) Molecular evolution of the Li/li chemical defence polymorphism in white clover (Trifolium repens L.). Molecular Ecology 16, 4180–4193.
Molecular evolution of the Li/li chemical defence polymorphism in white clover (Trifolium repens L.).Crossref | GoogleScholarGoogle Scholar |

Pinheiro J, Bates D, DebRoy S, Sarkar D, R Core Team (2018) nlme: Linear and nonlinear mixed effects models. R Package Version 3.1-137. http://CRAN.R-project.org/package=nlme

Provenza FD, Pfister JA, Cheney CD (1992) Mechanisms of learning in diet selection with reference to phytotoxicosis in herbivores. Journal of Range Management 45, 36–45.
Mechanisms of learning in diet selection with reference to phytotoxicosis in herbivores.Crossref | GoogleScholarGoogle Scholar |

R Core Team (2020) R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. Available at https://www.R-project.org/

Seigler DS (1998) Cyanogenic glycosides and cyanolipids. In ‘Plant secondary metabolism’. (Ed. DS Seigler) pp. 273–299. (Springer: Boston) https://doi.org/10.1007/978-1-4615-4913-0_16

Siqueira JO, Nair MG, Hammerschmidt R, Safir GR, Putnam AR (1991) Significance of phenolic compounds in plant-soil-microbial systems. Critical Reviews in Plant Sciences 10, 63–121.
Significance of phenolic compounds in plant-soil-microbial systems.Crossref | GoogleScholarGoogle Scholar |

Stochmal A, Oleszek W (1997) Changes of cyanogenic glucosides in white clover (Trifolium repens L.) during the growing season. Journal of Agricultural and Food Chemistry 45, 4333–4336.
Changes of cyanogenic glucosides in white clover (Trifolium repens L.) during the growing season.Crossref | GoogleScholarGoogle Scholar |

Thomas H (1984) Effects of drought on growth and competitive ability of perennial ryegrass and white clover. Journal of Applied Ecology 21, 591–602.
Effects of drought on growth and competitive ability of perennial ryegrass and white clover.Crossref | GoogleScholarGoogle Scholar |

Till-Bottraud I, Kakes P, Dommee B (1988) Variable phenotypes and stable distribution of the cyanotypes of Trifolium repens L. in southern France. Acta Oecologica 9, 393–404.

Vickery PJ, Wheeler JL, Mulcahy C (1987) Factors affecting the hydrogen cyanide potential of white clover (Trifolium repens L.). Australian Journal of Agricultural Research 38, 1053–1059.
Factors affecting the hydrogen cyanide potential of white clover (Trifolium repens L.).Crossref | GoogleScholarGoogle Scholar |

Viette M, Tettamanti C, Saucy F (2000) Preference for acyanogenic white clover (Trifolium Repens) in the vole Arvicola Terrestris. II. Generalization and further investigations. Journal of Chemical Ecology 26, 101–122.
Preference for acyanogenic white clover (Trifolium Repens) in the vole Arvicola Terrestris. II. Generalization and further investigations.Crossref | GoogleScholarGoogle Scholar |

Vu VQ (2011) ggbiplot: A ggplot2 based biplot. R Package version 0.55.

Wei T, Simko V (2017) R package ‘corrplot’: Visualization of a correlation matrix. (Version 0.84), Available at https://github.com/taiyun/corrplot

Whitehead SR, Bass E, Corrigan A, Kessler A, Poveda K (2021) Interaction diversity explains the maintenance of phytochemical diversity. Ecology Letters 24, 1205–1214.
Interaction diversity explains the maintenance of phytochemical diversity.Crossref | GoogleScholarGoogle Scholar |

Wickham H, Chang W, Henry L, Pedersen TL, Takahashi K, Wilke K, Woo K, Yutani H, Dunnington D (2016) Ggplot2: Elegant graphics for data analysis. 2nd ed. Cham, Switzerland: Springer International Publishing; 2016. 260 p.

Widdup KH, Barrett BA (2011) Achieving persistence and productivity in white clover. NZGA: Research and Practice Series 15, 173–180.
Achieving persistence and productivity in white clover.Crossref | GoogleScholarGoogle Scholar |

Wu F, Ma S, Zhou J, Han C, Hu R, Yang X, Nie G, Zhang X (2021) Genetic diversity and population structure analysis in a large collection of white clover (Trifolium repens L.) germplasm worldwide. PeerJ 9, e11325
Genetic diversity and population structure analysis in a large collection of white clover (Trifolium repens L.) germplasm worldwide.Crossref | GoogleScholarGoogle Scholar |

Yan W, Frégeau-Reid J (2008) Breeding line selection based on multiple traits. Crop Science 48, 417–423.
Breeding line selection based on multiple traits.Crossref | GoogleScholarGoogle Scholar |

Yan W, Wallace DH (1995) Breeding for negatively associated traits. In ‘Plant breeding reviews. Vol. 13’. (Ed. J Janick) pp. 141–177. (John Wiley & Sons, Inc.) https://doi.org/10.1002/9780470650059.ch4

Zhang X, Zhang Y, Yan R, Han J, Fuzeng-Hong F, Cao K (2010) Genetic variation of white clover (Trifolium repens L.) collections from China detected by morphological traits, RAPD and SSR. African Journal of Biotechnology 9, 3032–3041.