Chromosome-specific painting provides insights into the karyotype evolutionary direction and trajectory in the genus Medicago

Abstract

Divergence in basic chromosome numbers among closely related species is widespread in plants, yet a fundamental question regarding the evolutionary direction of karyotype—whether descending (from higher to lower numbers) or ascending (from lower to higher)—remains contentious. Alfalfa (Medicago sativa L.), a key forage crop, displays two basic chromosome numbers (x = 8 and x = 7) within the genus, and whether this divergence arose through descending evolution from 8 to 7 or the reverse remains unclear. Here, we developed a set of chromosome-specific painting markers capable of tracing chromosomal evolutionary trajectories among Medicago species. Comparative cytological analysis of seven accessions (x = 8) from the M. sativa L. complex revealed conserved chromosomal synteny in both diploid and autotetraploid species, with no detectable interchromosomal rearrangements. In Medicago polymorpha (x = 7), we discovered that the divergence in basic chromosome numbers (x = 7 vs. x = 8) resulted from large-scale fission-fusion events involving chromosomes 3, 5, and 6, rather than the simple fusion of chromosomes 3 and 7 as previously published genomic hypotheses. Further supporting evidence from rDNA remodeling and phylogenetic analysis indicates a descending evolutionary pathway, with the ancestral x = 8 transitioning to x = 7 approximately Mid-Miocene (~12 million years ago). Our results offer new insights into Medicago speciation and evolutionary origins, and instantiate a strategy for studying karyotypic evolutionary direction in other plant taxa with similar chromosomal dynamics.

Acknowledgements

We thank Professor Quanwen Dou, the Northwest Institute of Plateau Biology, Chinese Academy of Sciences, for providing alfalfa accessions. The research was supported by the Natural Science Foundation of China (grant number 32400496), Tianchi Talent Project of Xinjiang (grant number CZ001604), High-level Talents Scientific Startup Project of Shihezi University (grant number RCZK202362), Basic Research General Project under the Science and Technology Program of Xinjiang Production and Construction Corps (grant number 2024DA021, 2024DA030), Shihezi University Youth Top-notch Training Project (grant number BJZK202403), Science and Technology Research Project of Bingtuan (grant number 2023AB070, 2025AB084), and Science and Technology Innovation Talent Project of Bingtuan (grant number 23CB008-01).

Authors contributions

Z.M. and H.L. conceived the project. W.W. and X.W. performed the FISH experiments. Y.Z., S.Z., and Q.X. conducted bioinformatics analysis. Z.M. designed the oligo-FISH probes. Z.G., Q.Z., G.S., Y.L., W.L., F.W., and H.S. participated in the data analysis. Z.M. and S.Z. wrote the manuscript. All authors read and approved the final manuscript.

Data availability

Data supporting this study are available in the Supporting Information files. The oligo sequences for alfalfa eight chromosome-specific probes are included in Data S1.

Conflicts of interest statement

The authors declare no competing interests.

Supplementary material

Supplementary material is available at Horticulture Research online.

References

[1] Mayrose I, Lysak MA. The evolution of chromosome numbers: mechanistic models and experimental approaches. Genome Biol Evol. 2021;13:evaa220

[2] Mayrose I, Barker MS, Otto SP. Probabilistic models of chro-mosome number evolution and the inference of polyploidy. Syst Biol. 2010; 59:132-44

[3] Lysak MA, Berr A, Pecinka A. et al. Mechanisms of chromosome number reduction in Arabidopsis thaliana and related Brassi-caceae species. Proc Natl Acad SciUSA. 2006; 103:5224-9

[4] Piperidis N, D’hont A. Sugarcane genome architecture decrypted with chromosome-specific oligo probes. Plant J. 2020; 103:2039-51

[5] Lysak MA, Fransz PF, Ali HB. et al. Chromosome painting in Arabidopsis thaliana. Plant J. 2001; 28:689-97

[6] Speicher MR, Gwyn Ballard S, Ward DC. Karyotyping human chromosomes by combinatorial multi-fluor FISH. Nat Genet. 1996; 12:368-75

[7] Schröck E, Du Manoir S, Veldman T. et al. Multicolor spectral karyotyping of human chromosomes. Science. 1996; 273:494-7

[8] Albert PS, Zhang T, Semrau K. et al. Whole-chromosome paints in maize reveal rearrangements, nuclear domains, and chromosomal relationships. Proc Natl Acad Sci U S A. 2019; 116:1679-85

[9] Hershberg EA, Camplisson CK, Close JL. et al. PaintSHOP enables the interactive design of transcriptome- and genome-scale oligonucleotide FISH experiments. Nat Methods. 2021; 18:937-44

[10] Beliveau BJ, Kishi JY, Nir G. et al. OligoMiner provides a rapid, flexible environment for the design of genome-scale oligonu-cleotide in situ hybridization probes. Proc Natl Acad SciUSA. 2018;115:E2183-e2192

[11] Jiang J. Fluorescence in situ hybridization in plants: recent developments and future applications. Chromosome Res. 2019; 27:153-65

[12] Zhang T, Liu G, Zhao H. et al. Chorus2: design of genome-scale oligonucleotide-based probes for fluorescence in situ hybridization. Plant Biotechnol J. 2021; 19:1967-78

[13] Yu F, Zhao X, Chai J. et al. Chromosome-specific painting unveils chromosomal fusions and distinct allopolyploid species in the Saccharum complex. New Phytol. 2022; 233:1953-65

[14] Yan H, Han J, Jin S. et al. Post-polyploidization centromere evolution in cotton. Nat Genet. 2025; 57:1021-30

[15] Li G, Zhang T, Yu Z. et al. An efficient oligo-FISH painting system for revealing chromosome rearrangements and polyploidization in Triticeae. Plant J. 2021; 105: 978-93

[16] Zhao Q, Meng Y, Wang P. et al. Reconstruction of ancestral karyotype illuminates chromosome evolution in the genus Cucumis. Plant J. 2021; 107:1243-59

[17] Li A, Liu A, Du X. et al. A chromosome-scale genome assembly of a diploid alfalfa, the progenitor of autotetraploid alfalfa. Hortic Res. 2020; 7:194

[18] Ye Q, Zhou C, Lin H. et al. Medicago2035: genomes, functional genomics, and molecular breeding. Mol Plant. 2025; 18:219-44

[19] Lu X. Analysis of research progress on classification of Med-icago plants. Acta Agrestia Sin. 2009; 17:680-5

[20] Pecrix Y, Staton SE, Sallet E. et al. Whole-genome landscape of Medicago truncatula symbiotic genes. Nat Plants. 2018; 4: 1017-25

[21] Wang T, Ren L, Li C. et al. The genome of a wild Medicago species provides insights into the tolerant mechanisms of legume forage to environmental stress. BMC Biol. 2021; 19:96

[22] Shi K, Dong H, Du H. et al. The chromosome-level assembly of the wild diploid alfalfa genome provides insights into the full landscape of genomic variations between cultivated and wild alfalfa. Plant Biotechnol J. 2024; 22:1757-72

[23] Chen H, Zeng Y, Yang Y. et al. Allele-aware chromosome-level genome assembly and efficient transgene-free genome editing for the autotetraploid cultivated alfalfa. Nat Commun. 2020; 11:2494

[24] Li W, Li X, Li W. et al. A haplotype-resolved genome assembly of tetraploid Medicago sativa ssp.falcata. Sci China Life Sci. 2025; 68:1186-9

[25] CuiJ, LuZ, Wang T. et al. The genome of Medicago polymorpha provides insights into its edibility and nutritional value as a vegetable and forage legume. Hortic Res. 2021; 8:47

[26] Meng Z, Zheng Q, Wang W. et al. Oligo-FISH barcode chromo-some identification system provides novel insights into the natural chromosome aberrations propensity in the autote-traploid cultivated alfalfa. Hortic Res. 2025;12:uhae266

[27] Xin H, Zhang T, Wu Y. et al. An extraordinarily stable karyotype of the woody Populus species revealed by chromosome paint-ing. Plant J. 2020; 101:253-64

[28] Meng Z, Zheng Q, Shi S. et al. Whole-chromosome oligo-painting in licorice unveils interspecific chromosomal evolu-tionary relationships and possible origin of triploid genome species. Plant J. 2024; 120:2089-100

[29] Braz GT, He L, Zhao H. et al. Comparative oligo-FISH mapping: an efficient and powerful methodology to reveal karyotypic and chromosomal evolution. Genetics. 2018; 208:513-23

[30] Chen C, Zhang X, Li Y. et al. Chromosome-specific paint-ing reveals the Y genome origin and chromosome rear-rangements of the St genome in Triticeae. Plant Physiol. 2024; 196:870-82

[31] Guo X, Han F. Asymmetric epigenetic modification and elimi-nation of rDNA sequences by polyploidization in wheat. Plant Cell. 2014; 26:4311-27

[32] Wang K, Xiang D, Xia K. et al. Characterization of repetitive DNA in Saccharum officinarum and Saccharum spontaneum by genome sequencing and cytological assays. Front Plant Sci. 2022; 13:814620

[33] Shen C, Du H, Chen Z. et al. The chromosome-level genome sequence of the autotetraploid alfalfa and resequencing of core germplasms provide genomic resources for alfalfa research. Mol Plant. 2020; 13:1250-61

[34] Cui J, Wang X, Wei Z. et al. Medicago truncatula (model legume), Medicago sativa (alfalfa), Medicago polymorpha (bur clover), and Medicago ruthenica. Trends Genet. 2022; 38:782-3

[35] Lesins K. Interspecific crosses involving alfalfa. V. Medicago saxatilis × M. sativa with reference to M. cancellata and M. rhodopaea. Can J Genet Cytol. 1970; 12:80-6

[36] Falistocco E. Insight into the chromosome structure of the cul-tivated tetraploid alfalfa (Medicago sativa subsp. sativa L.) by a combined use of GISH and FISH techniques. Plants (Basel). 2020; 9:542

[37] YuF LeiY,LiY. et al. Cloning and characterization of chro-mosomal markers in alfalfa (Medicago sativa L.). Theor Appl Genet. 2013; 126:1885-96

[38] Kulikova O, Gualtieri G, Geurts R. et al. Integration of the FISH pachytene and genetic maps of Medicago truncatula. Plant J. 2001; 27:49-58

[39] Yu F, Wang H, Zhao Y. et al. Karyotypic evolution of the Med-icago complex: sativa-caerulea-falcata inferred from com-parative cytogenetic analysis. BMC Evol Biol. 2017; 17:104

[40] He F, Chen S, Zhang Y. et al. Pan-genomic analysis highlights genes associated with agronomic traits and enhances genomics-assisted breeding in alfalfa. Nat Genet. 2025; 57:1262-73

[41] Rieseberg LH. Chromosomal rearrangements and speciation. Trends Ecol Evol. 2001; 16:351-8

[42] Lucek K, Giménez MD, Joron M. et al. The impact of chromoso-mal rearrangements in speciation: from micro- to macroevo-lution. Cold Spring Harb Perspect Biol. 2023;15:a041447

[43] Rajpal VR, Raina SN, Rao SR. et al. Chromosome comple-ments and karyological studies in Medicago species. Cytolo-gia. 2022; 87:113-8

[44] Zhou B, Ren H, Zhang Y. et al. Characteristics and analysis of simple sequence repeats (SSR) in Medicago polymorpha and main Medicago species genome. Xinjiang Agricultural Sciences 2022; 59:2217-23

[45] Meng Z, Han J, Lin Y. et al. Characterization of a Saccharum spontaneum with a basic chromosome number of x = 10 provides new insights on genome evolution in genus Saccharum. Theor Appl Genet. 2020; 133: 187-99

[46] Xuan Y, Ma B, Li D. et al. Chromosome restructuring and num-ber change during the evolution of Morus notabilis and Morus alba. Hortic Res. 2022; 9:9

[47] Koch M, Bishop J, Mitchell-Olds T. et al. Molecular systematics and evolution of Arabidopsis and Arabis. Plant Biol. 1999; 1: 529-37

[48] Yang W, Zhang L, Mandáková T. et al. The chromosome-level genome sequence and karyotypic evolution of Megadenia pygmaea (Brassicaceae). Mol Ecol Resour. 2021; 21:871-9

[49] Falistocco E. Chromosome investigations in annual Med-icago species (Fabaceae) with emphasis on the origin of the polyploid Medicago rugosa and M.scutellata. Plant Biosyst. 2019; 153:235-41

[50] Zhang Q, Qi Y, Pan H. et al. Genomic insights into the recent chromosome reduction of autopolyploid sugarcane Saccha-rum spontaneum. Nat Genet. 2022; 54:885-96

[51] Hong F. Alfalfa Science. Beijing, China: China Agriculture Press, 2009:186-90

[52] Minh BQ, Schmidt HA, Chernomor O. et al. IQ-TREE 2: new models and efficient methods for phylogenetic inference in the genomic era. Mol Biol Evol. 2020; 37:1530-4

[53] Yang Z. PAML 4: phylogenetic analysis by maximum likeli-hood. Mol Biol Evol. 2007; 24:1586-91

[54] Jiao YX, He XF, Song R. et al. Recent structural variations in the Medicago chloroplast genomes and their horizontal transfer into nuclear chromosomes. J Syst Evol. 2023; 61:627-42