Changes in chromosome number are common in plants, but the direction of those changes is often difficult to reconstruct because ancestral forms are extinct or unknown. In Medicago, this question has remained unresolved for years: did species with seven basic chromosomes evolve from ancestors with eight, or did the reverse occur? Although genome sequencing has improved evolutionary analysis, chromosome-level evidence has remained limited, especially in plants with repeat-rich and polyploid genomes. Traditional cytogenetic tools often lack the precision needed to track entire chromosomes across related species. Based on these challenges, further in-depth research is needed on the evolutionary direction and chromosomal trajectory of genome change in Medicago.
Researchers from Shihezi University, Northwest A&F University, and Anhui Normal University reported (DOI: 10.1093/hr/uhaf313) in Horticulture Research on November 14, 2025 that chromosome-specific painting can resolve the evolutionary history of karyotype change in Medicago. Their study shows that the lineage with seven basic chromosomes most likely descended from an ancestral eight-chromosome form through complex chromosomal fission–fusion events, while also revealing strong chromosomal conservation across major alfalfa relatives.
The team built a whole-chromosome painting system for all eight chromosomes of alfalfa using more than 736,000 chromosome-specific oligos, then applied sequential fluorescence in situ hybridization to compare multiple Medicago accessions. This allowed the researchers to visualize each chromosome directly and compare species with basic chromosome numbers of eight and seven. Across the M. sativa complex, including both diploid and autotetraploid materials, chromosome structure proved remarkably conserved, with no detectable interchromosomal rearrangements. But in M. polymorpha, the story changed dramatically. Instead of confirming an earlier hypothesis that chromosome number reduction resulted from a simple fusion of chromosomes 3 and 7, the new cytological evidence showed that chromosomes 3, 5, and 6 each broke into segments and were reassembled into two new chromosomes.
The researchers then examined ribosomal DNA patterns. In species with x = 8, 5S rDNA was located on chromosomes 2 and 4, while 45S rDNA was found on chromosome 6. In M. polymorpha with x = 7, 5S rDNA was retained only on chromosome 2 and 45S rDNA shifted to chromosome 4. Together with the structural data, these patterns strongly supported a descending model, from x = 8 to x = 7. Phylogenetic analysis further suggested that this split occurred about 12 million years ago, with autotetraploidization in alfalfa taking place around 6 million years ago.
The study shows that chromosome evolution in Medicago was not a simple one-step event, but a more dynamic process involving multiple coordinated rearrangements. By combining direct cytogenetic observation with phylogenetic analysis, the researchers provide stronger evidence for how chromosome-number change contributed to diversification in this important crop lineage. Their findings also demonstrate why genome assemblies alone may not fully capture complex chromosomal histories, especially in repeat-rich regions.
Beyond solving an evolutionary puzzle, the new chromosome-painting toolkit could become highly useful for breeding and germplasm research. The authors note that it can help distinguish chromosomal composition among Medicago resources, monitor karyotype stability across generations, and verify chromosome inheritance in distant hybrids. That makes the method valuable not only for evolutionary biology, but also for crop improvement and genetic resource management in alfalfa. More broadly, the study offers a workable framework for examining chromosome-number evolution in other plant groups where similar genomic transitions remain unclear.
###
References
DOI
10.1093/hr/uhaf313
Original Source URL
https://doi.org/10.1093/hr/uhaf313
Funding information
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).
About Horticulture Research
Horticulture Research is an open access journal of Nanjing Agricultural University and ranked number one in the Horticulture category of the Journal Citation Reports ™ from Clarivate, 2023. The journal is committed to publishing original research articles, reviews, perspectives, comments, correspondence articles and letters to the editor related to all major horticultural plants and disciplines, including biotechnology, breeding, cellular and molecular biology, evolution, genetics, inter-species interactions, physiology, and the origination and domestication of crops.