Elucidation of the genetic basis underlying rooting ability in vegetatively propagated chrysanthemum

Abstract

Chrysanthemum, a globally renowned economic crop, primarily relies on vegetative propagation methods such as cutting for commercial cultivation. However, certain varieties with exceptional ornamental qualities often encounter difficulties in widespread adoption due to poor rooting ability and suboptimal root quality. The genetic underpinnings of rooting ability in chrysanthemum cuttings have remained largely unexplored. This study marks a significant advancement in this field. By evaluating 11 rooting traits across a diverse panel of 188 chrysanthemum genotypes, we found that spray cut chrysanthemums exhibit superior rooting ability compared to other cultivated types and wild species. Selective sweep analysis identified 534 selected genomic regions potentially linked to rooting traits during the domestication and improvement of chrysanthemums. Genome-wide association studies (GWAS) conducted on four key rooting traits - total root length, root surface area, average root diameter, and number of roots, using multiple models discovered 71 significant SNPs and 98 candidate genes, including 21 differentially expressed genes identified via transcriptomic sequencing. A weighted gene co-expression network analysis further revealed two key modules (yellow and lightyellow) related to rooting traits. By integrating GWAS, transcriptomic data, and functional verification, we pinpointed the candidate gene CmNRAMP3 as a negative regulator of rooting ability. These findings substantially enrich our understanding of the genetic mechanisms underlying rooting ability in chrysanthemum cuttings and provide a promising gene pool for improving rooting traits in future breeding programs.

Acknowledgements

This work was financially supported by the National Natural Science Foundation of China (31870691, 32171857), the China Agriculture Research System (CARS-23-A18), the Fundamental Research Funds for the Central Universities (QTPY2025005), and a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions. We thank the Bioinformatics Center of Nanjing Agricultural University for providing data analysis platform services.

Authors contributions

Z.F. and F.W. conceived and supervised the study. Z.X. and S.W. conducted the experiments and data analysis. S.J. and L.Y. assisted with data analysis. Z.X. drafted the manuscript. Z.X. and Z.F. revised the manuscript. J.J., G.Z., and C.F. supervised the study. Z.F. has given final approval of the version to be published. Z.X. and S.W. contributed equally to this work.

Data availability

The GBS data utilized in this study are publicly available in the National Center for Biotechnology Information (NCBI) Sequence Read Archive (SRA) under the BioProject accession number PRJNA1004079. All other data generated or analyzed in this study are provided within the article and its supplementary materials.

Conflicts of interest statement

The authors declare no conflict of interest.

Supplementary material

Supplementary material is available at Horticulture Research online.

References

[1] Alexander DH, Novembre J, Lange K. Fast model-based esti-mation of ancestry in unrelated individuals. Genome Res. 2009; 19:1655-64

[2] Bradbury PJ, Zhang Z, Kroon DE. et al. TASSEL: software for association mapping of complex traits in diverse samples. Bioinformatics. 2007; 23:2633-5

[3] Busov V, Meilan R, Pearce DW. et al. Transgenic modifica-tion of gai or rgl1 causes dwarfing and alters gibberellins, root growth, and metabolite profiles in Populus. Planta. 2006; 224:288-99

[4] Bvindi C, Tang L, Lee SH. et al. Histone methyltransferases SDG33 and SDG34 regulate organ-specific nitrogen responses in tomato. Front Plant Sci. 2022; 13:1005077

[5] Chen CF, Zhang Y, Cai JF. et al. Multi-copper oxidases SKU5 and SKS1 coordinate cell wall formation using apoplastic redox-based reactions in roots. Plant Physiol. 2023; 192:2243-60

[6] Chen JX, Yang YL, Feng HY. et al. Novel phosphatase PvPAP1 from the As-hyperaccumulator Pteris vittata promotes organic P utilization and plant growth: extracellular exudation and phytate hydrolysis. J Hazard Mater. 2024; 474:134867

[7] Cheng BX, Du WT, Bourke PM. et al. Population genetics of horticultural crops aided by multi-omics technology and its implications for ornamental plants. Ornam Plant Res. 2024; 4:e024

[8] Chong XR, Su JS, Wang F. et al. Identification of favorable SNP alleles and candidate genes responsible for inflorescence-related traits via GWAS in chrysanthemum. Plant Mol Biol. 2019; 99:407-20

[9] Chong XR, Zhang F, Wu YY. et al. A SNP-enabled assessment of genetic diversity, evolutionary relationships and the iden-tification of candidate genes in chrysanthemum. Genome Biol Evol. 2016; 8:3661-71

[10] Danecek P, Auton A, Abecasis G. et al. The variant call format and VCFtools. Bioinformatics. 2011; 27:2156-8

[11] Du LD, Guan ZJ, Liu YH. et al. The BTB/TAZ domain-containing protein CmBT1-mediated CmANR1 ubiquitination negatively regulates root development in chrysanthemum. J Integr Plant Biol. 2024; 66:285-99

[12] Eisa EA, Tilly-Mándy A, Honfi P. et al. Chrysanthemum: a com-prehensive review on recent developments on in vitro regen-eration. Biology (Basel). 2022; 11:1774

[13] Hirano T, Matsuzawa T, Takegawa K. et al. Loss-of-function and gain-of-function mutations in FAB1A/B impair endomem-brane homeostasis, conferring pleiotropic developmental abnormalities in Arabidopsis. Plant Physiol. 2011; 155:797-807

[14] Hu DW, He SP, Sun GF. et al. A genome-wide association study of lateral root number for Asian cotton (Gossypium arboreum L.). J Cotton Res. 2022; 5:1-10

[15] Hu SH, Lin SF, Huang YC. et al. Significance of AtMTM1 and AtMTM2 for mitochondrial MnSOD activation in Arabidopsis. Front Plant Sci. 2021; 12:690064

[16] Huang CF, Yamaji N, Chen ZC. et al. A tonoplast-localized half-size ABC transporter is required for internal detoxification of aluminum in rice. Plant J. 2012; 69:857-67

[17] Jammes F, Song C, Shin DJ. et al. MAP kinases MPK9 and MPK12 are preferentially expressed in guard cells and posi-tively regulate ROS-mediated ABA signaling. Proc Natl Acad Sci USA. 2009; 106:20520-5

[18] Kaler AS, Gillman JD, Beissinger T. et al. Comparing differ-ent statistical models and multiple testing corrections for association mapping in soybean and maize. Front Plant Sci. 2019; 10:1794

[19] Kang HM, Sul JH, Service SK. et al. Variance component model to account for sample structure in genome-wide association studies. Nat Genet. 2010; 42:348-54

[20] Kim D, Paggi JM, Park C. et al. Graph-based genome align-ment and genotyping with HISAT2 and HISAT-genotype. Nat Biotechnol. 2019; 37:907-15

[21] Koevoets IT, Venema JH, Elzenga JTM. et al. Roots withstand-ing their environment: exploiting root system architecture responses to abiotic stress to improve crop tolerance. Front Plant Sci. 2016; 07:1335

[22] Langfelder P, Horvath S. WGCNA: an R package for weighted correlation network analysis. BMC Bioinf. 2008; 9:559

[23] Lanquar V, Ramos MS, Lelièvre F. et al. Export of vacuolar manganese by AtNRAMP3 and AtNRAMP4 is required for opti-mal photosynthesis and growth under manganese deficiency. Plant Physiol. 2010; 152:1986-99

[24] Larsen PB, Cancel J, Rounds M. et al. Arabidopsis ALS1 encodes a root tip and stele localized half type ABC transporter required for root growth in an aluminum toxic environment. Planta. 2007; 225:1447-58

[25] Li MX, Yeung JMY, Cherny SS. et al. Evaluating the effective numbers of independent tests and significant p-value thresh-olds in commercial genotyping arrays and public imputation reference datasets. Human Genet. 2012; 131:747-56

[26] Livak KJ, Schmittgen TD. Analysis of relative gene expres-sion data using real-time quantitative PCR and the 2^-△△CT method. Methods. 2001; 25:402-8

[27] Mary V, Ramos MS, Gillet C. et al. Bypassing iron stor-age in endodermal vacuoles rescues the iron mobiliza-tion defect in the natural resistance associated-macrophage protein3natural resistance associated-macrophage protein4 double mutant. Plant Physiol. 2015; 169:748-59

[28] Pacheco JM, Song LM, Kubenová L. et al. Cell surface receptor kinase FERONIA linked to nutrient sensor TORC signaling con-trols root hair growth at low temperature linked to low nitrate in Arabidopsis thaliana. New Phytol. 2023; 238:169-85

[29] Putri GH, Anders S, Pyl PT. et al. Analysing high-throughput sequencing data in python with HTSeq 2.0. Bioinformatics. 2022; 38:2943-5

[30] Richter J, Watson JM, Stasnik P. et al. Multiplex mutagenesis of four clustered CrRLK1L with CRISPR/Cas9 exposes their growth regulatory roles in response to metal ions. Sci Rep. 2018; 8:12182

[31] Ruzicka K, Ljung K, Vanneste S. et al. Ethylene regulates root growth through effects on auxin biosynthesis and transport-dependent auxin distribution. Plant Cell. 2007; 19:2197-212

[32] Sanchez DL, Liu SS, Ibrahim R. et al. Genome-wide association studies of doubled haploid exotic introgression lines for root system architecture traits in maize (Zea mays L.). Plant Sci. 2018; 268:30-8

[33] Shu MJ, Yates TB, John C. et al. Providing biological context for GWAS results using eQTL regulatory and co-expression networks in Populus. New Phytol. 2024; 244:603-17

[34] Song AP, Su JS, Wang HB. et al. Analyses of a chromosome-scale genome assembly reveal the origin and evolution of cultivated chrysanthemum. Nat Commun. 2023; 14:2021

[35] Su JS, Zeng JW, Wang SY. et al. Multi-locus genome-wide association studies reveal the dynamic genetic architecture of flowering time in chrysanthemum. Plant Cell Rep. 2024; 43:84

[36] Sun CH, Yu JQ, Duan X. et al. The MADS transcription fac-tor CmANR1 positively modulates root system development by directly regulating CmPIN2 in chrysanthemum. Hortic Res. 2018; 5:52

[37] Sun HW, Guo XL, Qi XJ. et al. SPL14/17 act downstream of strigolactone signalling to modulate rice root elongation in response to nitrate supply. Plant J. 2021; 106:649-60

[38] TamV, PatelN, TurcotteM. et al. Benefits and limitations of 605 genome-wide association studies. Nat Rev Genet. 2019; 20:467-84

[39] Tamura K, Stecher G, Kumar S. MEGA11: molecular evolution-ary genetics analysis version 11. Mol Biol Evol. 2021; 38:3022-7

[40] Teixeira da Silva JA, Shinoyama H, Aida R. et al. Chrysanthemum biotechnology: quo vadis? Crit Rev Plant Sci. 2013; 32:21-52

[41] Villacorta-Martín C, Sánchez-García AB, Villanova J. et al. Gene expression profiling during adventitious root formation in car-nation stem cuttings. BMC Genomics. 2015; 16:789

[42] Wamhoff D, Patzer L, Schulz DF. et al. GWAS of adventitious root formation in roses identifies a putative phosphoinositide phosphatase (SAC9) for marker-assisted selection. PLoS One. 2023; 18:e0287452

[43] Wang Q, De Gernier H, Duan XL. et al. GH3-mediated auxin inactivation attenuates multiple stages of lateral root devel-opment. New Phytol. 2023a; 240:1900-12

[44] Wang YT, Yan X, Xu M. et al. Transmembrane kinase 1-mediated auxin signal regulates membrane-associated clathrin in Arabidopsis roots. J Integr Plant Biol. 2023b; 65: 82-99

[45] Wu XM, Qiao Z, Liu HP. et al. CML20, an Arabidopsis calmodulin-like protein, negatively regulates guard cell ABA signaling and drought stress tolerance. Front Plant Sci. 2017; 8:824

[46] Wu YY, Chang Y, Luo LM. et al. Abscisic acid employs NRP-dependent PIN2 vacuolar degradation to suppress auxin-mediated primary root elongation in Arabidopsis. New Phytol. 2022; 233:297-312

[47] Xu X, Ye JH, Yang YY. et al. Genome-wide association study of rice rooting ability at the seedling stage. Rice. 2020; 13:1-15

[48] Yang JA, Lee SH, Goddard ME. et al. GCTA: a tool for genome-wide complex trait analysis. Am J Hum Genet. 2011; 88:76-82

[49] Yin LL, Zhang HH, Tang ZS. et al. rMVP: a memory-efficient, visualization-enhanced, and parallel-accelerated tool for genome-wide association study. Genom Proteom Bioinf. 2021; 19:619-28

[50] Yuan TT, Xu HH, Li J. et al. Auxin abolishes SHI-RELATED SEQUENCE5-mediated inhibition of lateral root development in Arabidopsis. New Phytol. 2020; 225:297-309

[51] Zhai XZ, Yan XC, Zenda T. et al. Overexpression of the perox-idase gene ZmPRX1 increases maize seedling drought toler-ance by promoting root development and lignification. Crop J. 2024; 12:753-65

[52] Zhang T, Ge YC, Cai G. et al. WOX-ARF modules initiate differ-ent types of roots. Cell Rep. 2023a; 42:112966

[53] Zhang XF, Su JS, Jia FF. et al. Genetic architecture and genomic prediction of plant height-related traits in chrysan-themum. Hortic Res. 2023b;11:uhad236

[54] Zhu LY, Zhou L, Li JW. et al. Regeneration of ornamen-tal plants: current status and prospects. Ornam Plant Res. 2024; 4:e022

[55] Zhuo MN, Sakuraba Y, Yanagisawa S.A jasmonate-activated MYC2-Dof2.1-MYC 2 transcriptional loop promotes leaf senes-cence in Arabidopsis. Plant Cell. 2020; 32:242-62